Serotonin: Actions, Receptors, Pathophysiology

Serotonin: Actions, Receptors, Pathophysiology

The following titles of satellite symposia of the IUPHAR lOth International Congress of Pharmacology are published by The Palgrave Macmillan:

Pharmacology and Functional Regulation of Dopaminergic Neurons, edited byP. M. Beart,G.N. Woodruff and D. M.Jackson

Peripheral Actions of Dopamine, edited by C. Bell and B. McGrath

Serotonin: Actions, Receptors, Pathophysiology, edited by E. J. Mylecharane, J. A. Angus, I. S. de la Lande and P. P. A. Humphrey

SATELLITESYMPOSIAOFTHEIUPHARlOthiNTERNATIONAL CONGRESS OF PHARMACOLOGY

SEROTONIN ACTIONS, RECEPTORS, PATHOPHYSIOLOGY

Edited by

Ewan J. Mylecharane Department of Pha171Ulcology

University of Sydney, Australia

James A. Angus Baker Medical Research Institute

Prahran, Australia

Ivan S. de Ia Lande Department of Clinical and Experimental Pha171UlCology

University of Adelaide, Australia

and

Patrick P. A. Humphrey Glaxo Group Research Ltd

Ware, England

M MACMILLAN

PRESS Scientific & Medical

© The editors and contributors 1989 Softcover reprint of the hardcover 1st edition 1989 978-0-333-46149-5

All rights reserved. No reproduction, copy or transmission of this publication may be made without written permission.

No paragraph of this publication may be reproduced, copied or transmitted save with written permission or in accordance with the provisions of the Copyright, Design and Patents Act 1988, or under the terms of any licence permitting limited copying issued by the Copyright Licensing Agency, 33-4 Alfred Place, London WClE 7DP.

Any person who does any unauthorised act in relation to this publication may be liable to criminal prosecution and civil claims for damages.

First published 1989

Published by TilE MACMILLAN PRESS LID Houndmills, Basingstoke, Hampshire RG21 2XS and London Companies and representatives throughout the world

Typeset by Vine & Gorfin Ltd Exmouth, Devon

British Library Cataloguing in Publication Data Serotonin 1. Serotonin I. Mylecharane II. Series 612'.01575 ISBN 978-1-349-10116-0 ISBN 978-1-349-10114-6 (eBook) DOI 10.1007/978-1-349-10114-6

Contents

Preface Abbreviations and Drug Code Names

PARTI INVITED LECTURES

1 The Development of 5-HT 2 Receptor Antagonists J. M. VanNueten, P. A. J. Janssen, W. J. Janssens and P.M. Vanhoutte

2 The Development of 5-HT 3 Receptor Antagonists J.R. Fozard

PART II NEURONAL ACTIONS

3 5-HT Receptors on Afferent Neurones B. P. Richardson, G. Engel, P. Donatsch and K. -H. Buchheit

4 Electrophysiological Investigation of the Actions of 5-Hydroxytryptamine on Sympathetic Ganglionic Neurones D. I. Wallis and N.J. Dun

5 Multiple 5-HT Receptors in the Enteric Nervous System M.D. Gershon, G. M. Maweand T. A. Branchek

6 Pre-synaptic 5-HT Receptors Mediating Inhibition of Transmitter Release from Peripheral Cholinergic and Noradrenergic Nerves D. E. Clarke, R. A. Bond, K. G. Charlton and D. R. Blue

7 5-HT Receptors Mediating Pre-synaptic Autoinhibition in Central Serotoninergic Nerve Terminals M.Gothert

8 Need the Autoregulation of Raphe Neurones Involve 5-Hydroxytryptamine? J. S. Kelly, N.J. Penington and D. G. Rainnie

ix xiii

3

12

21

31

37

48

56

64

Vl Contents

9 In vivo Electrophysiology of Receptors Mediating the Central Nervous System Actions of 5-Hydroxytryptamine M. H. T. Roberts and M. Davies 70

10 Neuronal Actions of 5-Hydroxytryptamine: An Overview I. S. de Ia Lande and E. J. Mylecharane 77

PART III BEHAVIOURAL ACTIONS

11 Behavioural Correlates of the Activation of 5-HT Receptors M.D. Tricklebank 87

12 5-HT 3 Receptors in the Central Nervous System M. B. Tyers, B. Costal/ and R. J. Naylor 95

13 Serotoninergic Function and Aggression in Animals P. Bevan, B. Olivier, J. Schipper and]. Mos 101

14 Behavioural Actions of 5-Hydroxytryptamine: An Overview S. Z. Langer 109

PARTIV CARDIAC, VASCULARANDOT!IERSMOOTHMUSCLE ACTIONS

15 Cardiac Actions of 5-Hydroxytryptamine P. R. Saxena 115

16 Amplifying Action of5-Hydroxytryptamine in the Rabbit Ear Artery I. S. de IaLande, J. A. Kennedy and B. J. Stanton 123

17 Serotonin-induced Vasoconstriction and Contractile Synergism with Noradrenaline: Role of a-Adrenoceptors R. E. Purdy and D. L. Murray 130

18 Pre-synaptic Sympathetic Inhibition and 5-Hydroxytryptamineinduced Vasodilatation E. J. Mylecharane and C. A. Phillips 136

19 5-HT lA Receptors and Cardiovascular Control J. R. Fozard,A. K. MirandA. G. Ramage 146

Contents vn

20 Cardiac, Vascular and Other Smooth-muscle Actions of 5-Hydroxytryptamine: An Overview R. E. Purdy and P. R. Saxena 152

PARTV CLASSIFICATIONOFS-HTRECEPTORSANDBINDING SITES

21 The Subclassification of Functional5-HT rlike Receptors P. P. A. Humphrey and W. Feniuk 159

22 New Pharmacological Tools for Studies of Central5-HT lA

Binding Sites M. Hamon, M. B. Emerit, M. PfJnchant, J. M. Cossery, S. E/Mestikawy, D. Verge, G. DavalandH. Gozlan 169

23 Serotonin 5-HT1c Receptors: What Do They Do? P. R. Hartig 180

24 Characterization of 5-HT 1 Binding Site Subtypes Labelled by [3H]-5-Hydroxytryptamine S. J. Peroutka 188

25 The Classification of 5-HT Receptors Using Tryptamine Analogues P. LeffandG. R. Martin 195

26 Classification of 5-HT Receptors and Binding Sites: An Overview P. P. A. Humphrey and B. P. Richardson 204

PARTVI PATHOPHYSIOLOGY

27 Vascular Actions of Serotonin in Large and Small Arteries are Amplified by Loss of Endothelium, Atheroma and Hypertension J. A. Angus, C. E. Wright and T. M. Cocks 225

28 Sympathetic Nerves Associated with Brain Vessels Store and Release Serotonin which Interacts with Noradrenaline in Cerebrovascular Contraction J. E. Hardebo, J.-Y. Chang and Ch. Owman

29 5-HT 3 Receptors in the Gastrointestinal Tract G. Engel, K.-H. BuchheitandB. P. Richardson

233

241

viii Contents

30 Different Recognition Sites for Serotonin: The Neuronal Na +dependent Transporter and the Release-modulating Autoreceptor S. Z. LangerandD. Graham 249

31 Serotoninergic Function in Neuropsychiatric Disorders D. L. Murphy, E. A. Mueller, C. S. Aulakh, G. Bagdyand N. A. Garrick 257

32 Pharmacology and Function of Melatonin Receptors in the Mammalian Central Nervous System M. L. Dubocovich 265

33 Pathophysiology of 5-Hydroxytryptamine: An Overview J. A. Angus and!. M. VanNueten 274

Author Index 281

Subject Index 283

Preface

This volume contains the proceedings of an international symposium on serotonin which took place on 4th-6th September 1987, on Heron Island, Queensland, Australia. The symposium was a satellite meeting of the IUPHAR lOth International Congress of Pharmacology in Sydney. This was the first time that an international symposium on serotonin had been organized as an IUPHAR Congress satellite meeting. The Serotonin Club, an international group of biomedical scientists, has committed itself to sponsorship of satellite meetings on serotonin in conjunction with future IUPHAR Congresses, and plans are now well in hand for the Second IUPHAR Satellite Meeting on Serotonin in July 1990, in Basel.

The first impetus to the organization of the Heron Island meeting came in 1984; it stemmed from an invitation by the Scientific Programme Committee for the lOth IUPHAR Congress to submit a proposal for a symposium on serotonin at the 1987 Congress in Sydney. Ivan de Ia Lande enlisted the aid of Jim Angus and Ewan Mylecharane to form an ad hoc committee to respond to this invitation. This soon became an international Organizing Committee whose members were J. A. Angus (Australia), I. S. de Ia Lande (Australia), J. R. Fozard (France), P. R. C. Howe (Australia), P. P. A. Humphrey (UK), J. A. Kennedy (Australia), E. J. Mylecharane (Australia), R. E. Purdy (USA), B. P. Richardson (Switzerland), P. R. Saxena (The Netherlands), P.M. Vanhoutte (USA) and J. M. Van Nueten (Belgium).It was immediately agreed that serotonin deserved more than half a day. The Organizing Committee accepted the task of arranging both a short symposium at the IUPHAR Congress itself (comprising 'state-of-the-art' overviews by leading serotonin researchers), and a three-day international satellite meeting aimed at providing an opportunity for specialists in this area to discuss and explore in detail the burgeoning and exciting research on serotonin.

At the same time, Paul Vanhoutte was mobilizing serotonin researchers worldwide. The result of his untiring efforts was the Serotonin Club, which had its first official Dinner Meeting in April 1986 (at the F.A.S.E.B. Meeting in St Louis), and was formally constituted in August 1987 at a Dinner Meeting in Sydney. It was therefore a natural progression for the Serotonin Club to sponsor the Heron Island satellite meeting, with the Organizing Committee acting on behalf of the Club. Ewan Mylecharane in Sydney was entrusted with the responsibility of convening both the IUPHAR symposium and the satellite meeting. Heron Island, a National

X Preface

Marine Park in Australia's Great Barrier Reef, was chosen as the venue for the satellite meeting.

The final programme for the meeting was decided following a call for proposed contributions from intending participants. The response allowed the Organizing Committee an opportunity to assemble a comprehensive programme of very high standard. The programme was based on platform presentations by leading investigators, on the themes of neuronal actions, behavioural actions, cardiovascular and other smooth muscle actions, classification of 5-HT receptors and binding sites, and pathophysiology. The presenters were asked to summarize their own investigations, together with the appropriate background information and work of other investigators on the particular topic of their contribution. These presentations form the substance of this volume. In each instance, the first-named author was the presenting contributor.

The programme also included poster presentations, grouped under the appropriate programme themes, and detailed discussions of their content. Time was also set aside for general discussion of the platform presentations within each of the five programme themes, as well as workshops on 5-HT receptor classification and on the pathophysiological roles of serotonin. Poster abstracts and discussion transcripts have not been included in this proceedings volume. Instead, chairmen for each of the programme themes prepared overviews which have attempted to summarize the findings and identify the areas of consensus and contention, based on the platform and poster presentations, the discussions and the workshops, as appropriate. Each of these overviews has been included in this volume at the end of the section dealing with that particular programme theme. In addition, two invited lectures on the development of 5-HT2 and 5-HT3 receptor antagonists were presented by J. M. Van Nueten and J. R. Fozard, respectively, in recognition of their outstanding work in this area; these contributions are included at the beginning of the book.

To assist the reader, details of the abbreviations and drug code names used have been summarized. Both 'serotonin' and '5-hydroxytryptamine' have been used interchangeably in this book. Consistent forms of abbreviation of chemical, drug and enzyme names, and other non-standard abbreviations, have been used throughout. An index of key words and phrases, referenced to the commencing page numbers of the appropriate contributions, has been provided, together with an author index of all contributing authors and co-authors.

All who participated agreed that the Heron Island meeting was a most successful and enjoyable conference both scientifically and socially, in one of the most beautiful marine parks in the world. Despite the distractions of the island paradise outside, attendance at the scientific sessions was always very high. The meeting itself was conducted in an informal and friendly atmosphere, but the science was high-powered, and the exchanges between

Preface xi

participants were insightful and frank. There were, however, legitimate opportunities for snorkelling and scuba diving, fishing and exploring, relaxing on the beach, and eating, drinking and talking. Of course, not all went completely smoothly. Just when the convenor thought he could relax aboard one of the three boats returning from Heron Island to the mainland at the end of the meeting (a voyage of70 km), its engine failed. Speed-boats had to be called out from Heron Island Resort and the Marine Parks Authority to ferry 30 worried scientists (henceforth known as the 'M. V. Odessa Chapter') and a boat-load of baggage (not necessarily belonging to those on the boat) back to Heron Island, then via helicopter shuttle to the mainland to connect with planned bus and air links to the outside world. Miraculously, all but three participants spent the night where they had intended, and everyone ended up with his own baggage!

The meeting owed its success to the dedicated efforts of many people. In particular, the members of the Organizing Committee provided the convenor with invaluable ideas, advice and assistance, and willingly accepted the demanding tasks of chairing programme theme sessions, preparing overviews and editing this proceedings volume. The Organizing Committee is also especially grateful to Dr S. Z. Langer, who chaired the behavioural actions session and prepared an overview on this theme, and to Dr G. Engel, Dr M. Hamon and Dr D. E. Clarke, who undertook the onerous role of poster discussion chairmen. The untiring assistance provided by Ms Ann Cincotta in the planning and conducting of the meeting was particularly appreciated by the convenor and the Organizing Committee. The vision and energy of Paul Vanhoutte in fostering serotonin research also deserve special mention. At the last moment, Paul was unable to attend the Heron Island meeting, to our (and his!) considerable disappointment.

The Serotonin Club and the Organizing Committee also wish to acknowledge with deep gratitude the generous financial assistance provided by Lipha (West Germany) and Servier (France), and the support for the Heron Island satellite meeting from Duphar (The Netherlands), Glaxo (Australia), Glaxo (UK), Janssen Pharmaceutica (Belgium), Merck Sharp and Dohme (UK), Sandoz (Switzerland), Synthelabo (France) and The Wellcome Foundation (UK). The assistance provided by Australian Airlines, Heron Island Resort, Queensland Government Travel Centre and the Department of Pharmacology of the University of Sydney, is also gratefully acknowledged, as is the editorial advice and assistance given by Mr D. Grist and Mr R. M. Powell of The Macmillan Press, Basingstoke, UK.

The Organizing Committee felt that the meeting deepened the understanding by the participants of each other's points of view regarding the multiple sites and mechanisms of action of serotonin, and the various (all worthy) approaches to their study. It is hoped that the IUPHAR

xii Preface

Nomenclature Committee will heed advice from the Serotonin Club and provide insight into the way ahead for 5-HT receptor classification. In the interim, the moratorium on further subclassification of 5-HT receptors that was suggested at Heron Island should be supported by referees and authors alike, until a universally acceptable scheme is ratified.

We trust that this volume will become a valued record of what was undoubtedly a challenging, memorable and important meeting on the fascinating subject of serotonin.

1989 E.J. M. J. A. A.

I. S. de laL. P.P.A.H.

Abbreviations and Drug Code Names

SI units and symbols are used throughout. Other abbreviations which have been used (without definition) are in accordance with the current recommendations of the British Journal of Pharmacology. Abbreviations of chemical, drug and enzyme names, and other non-standard abbreviations, have been defined in each contribution when first used; these abbreviations are listed below.

ACh AHP ANS A VAs 5-Cf CG CSF CYP cyclic AMP cyclic GMP DA DAG DMPP DOM DOPAC DP-5-Cf DR EDRF ENS EPSP GIT 5-HIAA (3H]-5-Me0-DPAC 5-HT HVA I~ [1 1]-BH-8-MeO-N-PAT

LSD MAO m-CPP MDMA 8-Me0-2'-chloro-PAT

8-MeOClEPAT

acetylcholine after-hyperpolarization autonomic nervous system arteriovenous anastomoses 5-carboxamidotryptamine coeliac ganglion cerebrospinal fluid cyanopindolol adenosine 3' ,5' -monophosphate guanosine 3' ,5' -monophosphate dopamine diacylglycerol 1, 1-dimethyl-4-phenylpiperazinium dimethoxymethylphenylaminopropane dihydroxyphenylacetic acid dipropyl-5-carboxamidotryptamine dorsal raphe endothelium-derived relaxing factor enteric nervous system excitatory post-synaptic potential gastrointestinal tract 5-hydroxyindoleacetic acid (3H]-5-methoxy-3-( di-n-propylamino )chroman 5-hydroxytryptamine (serotonin) homovanillic acid inositol-1 ,4,5-triphosphate [1251]-Bolton-Hunter-8-methoxy-2-( N-propyl-N-

propylamino )tetralin lysergic acid diethylamide monoamine oxidase m-chlorophenylpiperazine 3,4-methylenedioxymethamphetamine 8-methoxy-2-(N-2'-chloropropyl,N-

propyl)aminotetralin 8-methoxy-2-(N-2-chloroethyl-N-n

propyl)aminotetralin

xiv Abbreviations and Drug Code Names

5-MeODMT 8-Me0-3' -NAP-amino-

5-methoxy-N,N-dimethyltryptamine 8-methoxy-2-(N-n-propyl,N-3-[2-nitro-4-

PAT MPTP NA NEM p-CPA 8-0H-DPAT 5-0 HIP 6-0HIP PAPP

PIP2 SCG SDS-PAGE

SH TFMPP TTX

azidophenyl]aminopropyl)aminotetralin N-methyl-4-phenyl-1 ,2,3,6-tetrahydropyridine noradrenaline N-ethylmaleiimide p-chlorophenylalanine 8-hydroxy-2-( di-n-propylamino )tetralin 5-hydroxyindalpine 6-hydroxyindalpine 1-(2-[ 4-aminophenyl]ethyl)-4-(3-

trifluoromethylphenyl)piperazine phosphatidylinositol-4,5-biphosphate superior cervical ganglion sodium dodecyl sulphate-polyacrylamide gel elec-

trophoresis spontaneously hypertensive trifluoromethylphenylpiperazine tetrodotoxin

Some drug code names have been used without further qualification throughout. Chemical names are as follows:

AH 25086 BEA 1654 BRL 24924

BRL 43694

CGS 12066B

DiMe-C7 GR 38032F

GR 43175

GR 65630

ICI 118,551 ICS 205-930 LY 53857

MDL 72222 MDL 72832

MDL 73005

MK212 RU 24969 WB 4101

3-(2-aminoethyl)-N-methyl-1H-indole-5-acetamide N-(3-acetylaminophenyl)piperazine ( ± )-endo-4-amino-5-chloro-2-methoxy-N-( 1-azabicyclo[3 ,3, 1 ]non-

4-yl)benzamide endo-N-(9-methyl-9-azabicyclo[3,3, 1 ]non-3-yl)-1-methylindazole-3-

carboxamide 7-trifluoromethyl-4-( 4-methyl-1-piperazinyl)-pyrolo[1 ,2-

a ]quinoxaline [pGlu5, MePhe8, Sar9_1-substance Ps.-n 1 ,2,3 ,9-tetrahydro-9-methyl-3([2-methyl-1H-imidazol-1-yl]methyl)-

4H-carbazol-4-one 3-(2-dimethylamino )ethyl-N-methyl-1H-indole-5-methane sulpho

namide 3-(5-methyl-1H-imidazol-4-yl)-1-(1-methyl-1H-indol-3-yl)-1-

propanone erythro-oL-1(7-methylindan-4-yloxy)-3-isopropylaminobutan-2-ol (3a-tropanyl)-1H-indole-3-carboxylic acid ester 4-isopropyl-7 -methyl-9-(2-hydroxy-1-methylpropoxycarbonyl)-

4,6,6A,7,8,9,10,10A-octahydroindolo[4,3-FG]quinoline maleate

1aH,3a,5aH-tropan-3-yl-3,5-dichlorobenzoate 8-( 4-[1 ,4-benzodioxan-2-ylmethylamino ]butyl)-8-

azaspiro[4,5]decane-7 ,9-dione 8-(2-[2,3-dihydro-1 ,4-benzodioxin-2-ylmethylamino ]ethyl)-8-

azaspiro[ 4,5]decane-7 ,9-dione 6-chloro-2-( 1-piperazinyl)pyrazine HCl 5-methoxy-3( 1 ,2,3 ,6-tetrahydro-4-pyridinyl)-1H -indole 2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1 ,4-benzodioxane

WY 26392

WY 48723

Abbreviations and Drug Code Names XV

N-(1,3,4,6,7,11b-a-hexahydro-2H-benzo-[a]-quinolizin-2-~-yl),N-methyl-n-propylsulphonamide

decahydro-3-( 4[ 4-(2-pyrimidinyl)-1-piperazinyl]butyl)-1 ,5-methano-6, 7 ,9-metheno-2H-pentaleno[1 ,2-d]azepine-2,4(3H)-dione 2HQ

PART I INVITED LECTURES

1

The Development of 5-HT 2 Receptor Antagonists*

J. M. Van Nueten, 1 P. A. J. Janssen, 1 W. J. Jansse~ and P. M. Vanhoutitl

1 International Research Council and 2Department of Cardiovascular Pharmacology, Janssen Research

Foundation, B-2340 Beerse, Belgium 3Department of Physiology and Biophysics, Mayo Clinic and Mayo

Foundation, Rochester, MN 55905, USA

INTRODUCTION

Binding studies indicate that both serotonin and dopamine receptors are involved in the mechanism of action of neuroleptic drugs (Leysen et al., 1978), and have distinguished between two 5-HT binding sites, labelled with high affinity either by eHJ-serotonin (5-HT1) or by (3H]-spiperone in the frontal cortex (5-HT2) in various brain tissues (Peroutka and Snyder, 1979). Structural modification of these molecules with neuroleptic properties led to the synthesis of a number of quinazolinedione derivatives. These substances prevented formation of gastric ulcers in rats challenged with compound 48/80 (which induces release of both histamine and serotonin from mast cells); these findings indicated inhibition of serotonin activity, since these rats were protected against histamine-induced effects by treatment with antihistaminic drugs (Awouters et al., 1982). The ability of this group of molecules and of a number of known serotonin antagonists to block serotonin-induced contractions was determined on the isolated caudal artery of the rat (Van Nueten et al., 1981). Their activities correlated significantly with their affinities for 5-HT2 receptors, as measured in radioligand binding studies (Leysen et al., 1982). Of these 5-HT2 receptor antagonists, ketanserin (R 41 468; 3-(2-(4-(p-fluorobenzoyl)piperidino]ethyl]-2,4(1H,3H)-quinazolinedione) emerged as being the most selective for 5-HT2 receptor sites (Leysen et al., 1981; Van Nueten and Vanhoutte, 1981).1t inhibited serotonin-induced contraction of isolated blood vessels (Van Nueten et al., 1981).

*Presented by J. M. Van Nueten.

4

A % CONTRAC.TION 100

80

60

40

20

0

10•

B

t t ES

ES p T

Serotonin

1o• SEROTONIN

-;:::- 0 II c:

:::;: 20 w ui I

....... 40 + ., c: 0

" 60 5 c: ~ 80 :.0 :.c: .£ ~ 100

o CONTROL i A KETANSERIN 4. 0 x 10-10 M e KETANSERIN 1.6 x 10 • M • KETANSERIN 6.3 x 10• M • KETANSERIN 2. 5 x 10 • M • KETANSERIN 1. 0 x 10 -7 M

10"" 10-4 M

~

M control 2.5x1o-8 1.0x1o-7 4.0x1o-7

concentration of ketonserin M

Figure 1.1 (A) Increasing concentrations of serotonin cause dose-dependent contractions in rabbit pulmonary arteries. These contractions are competitively antagonized by ketanserin, indicating that 5-HT2 receptors are involved. (B) (left) Typical experiment showing that rat platelets (P: 2 x 109/l) stimulated with thrombin (T: 100 N .I. H. units/1) potentiate the response to electric stimulation of the adrenergic nerves (ES: 9 V, 0.5 ms pulse duration, 10 s, 16Hz) in the rabbit femoral artery. (B) (right) This amplifying effect is inhibited by increasing concentrations of ketanserin, indicating that it is due to interaction of the released

serotonin with 5-HT2 receptors

Development of 5-HT2 Receptor Antagonists 5

VASCULAR TISSUES

Effects and Sources of Serotonin

The effects of serotonin on the cardiovascular system are complex, as it can cause both vasoconstriction and vasodilatation (Page, 1954). The main source of serotonin available to the blood vessel wall is from platelets, which release enough serotonin to cause vasoconstriction, particularly when they aggregate at sites of endothelial damage (DeClerck and Van Nueten, 1982; Vanhoutte, 1985; Vanhoutte and Houston, 1985).

Direct Vasoconstrictor Effects of Serotonin

Contractions induced in most large arteries and veins by serotonin are due to a direct action of the monoamine on vascular smooth muscle; most of these contractile responses to serotonin can be inhibited by ketanserin (Figure 1.1(A)), spiperone, methysergide, and a number of other serotonin antagonists with high affinity for 5-HT2 receptors. Ketanserin also inhibits vascular effects (e.g. vasoconstriction, vascular congestion, necrosis) caused by serotonin in vivo in rats or dogs (Van Reempts et al., 1981; Awouters et al., 1985; Van Nueten et al., 1985). Some 5-HT2 receptor antagonists (but not ketanserin) are partial agonists (Van Nueten et al., 1982).

Indirect Vasconstrictor Effects of Serotonin (Amplification)

In addition to its direct vasoconstrictor effects, serotonin augments ('amplifies') the vasoconstrictor effects of other neurohumoral mediators such as noradrenaline, angiotensin II, histamine, prostaglandin Fza and thromboxane A2 . This was observed in a variety of isolated vascular tissues and beds, including human blood-vessels (de Ia Lande et al., 1966; Van Nueten et al., 1981, 1982).1t also has been reported in vivo in various species including the human (Scroop and Walsh, 1968; Myers et al., 1985). Amplification has been demonstrated with endogenous serotonin and noradrenaline released from aggregating platelets and adrenergic nerve endings, respectively. It can be inhibited by serotonin antagonists such as ketanserin (Figure 1.1(B)), indicating that amplification is mediated by 5-HT2 receptors (Van Nueten et al., 1981, 1982; Vanhoutte, 1987).

Vasodilatation

Serotonin can cause relaxations in various blood vessels (Cocks and Angus,

6 Serotonin

1983; Cohen et al., 1983). These relaxations can be due to the release by endothelial cells of a labile vasodilator substance (endothelium-derived relaxing factor) upon exposure to serotonin, as has been demonstrated with other agonists (Cocks and Angus, 1983; Cohen et al., 1983; Furchgott, 1983). Another important mechanism to explain the vasodilator effects of serotonin is pre-synaptic inhibition of noradrenaline release (McGrath, 1977; Van Nueten et al., 1981). The receptors mediating these relaxations may belong to the 5-HTrlike group (Cohen etal., 1983; Engel et al., 1983; Cohen, 1985; Houston et al., 1985). This explains why, in the intact vascular bed, the 5-HT2 receptor antagonist ketanserin not only prevents vasoconstriction, but can unmask, or potentiate, vasodilator responses to serotonin (Van Nueten et al., 1981; Cocks and Angus, 1983; Vanhoutte, 1987).

PLATELETS

Serotonin interacts with platelets to induce shape change and aggregation (De Clerck et al., 1982). Moreover, it strongly amplifies aggregation of platelets induced by low concentrations of other aggregating agents. Both these direct and amplifying effects are prevented or inhibited by 5-HT 2

receptor antagonists, indicating involvement of 5-HT 2 receptors (Figure 1.2;

70

80

90

100

A

1 min 1-----< c. Solvent

K • Ketanserin

B

5-HT 2 x 10-6 M and/or Collagen 0.3 JIQ/ml

1 sot-----\--------

# &Ot----4---------

+coli

Figure 1.2 (A) Serotonin (5-HT) induces platelet aggregation in cat platelet-rich plasma under control (C) conditions. This aggregation is dose-dependently inhibited by ketanserin (K) administered prior to serotonin (arrows). (B) A low concentration of collagen causes a weak aggregation in human platelet-rich plasma which is amplified by simultaneous administration of serotonin at a concentration not causing aggregation by itself. This amplification is inhibited by administration of ketanserin.

(Modified from De Clerck et al., 1982, 1984)


De Clerck et al., 1982; De Clerck and Herman, 1983). The aggregating effects of serotonin may contribute to thrombus formation, and together with its vasoconstrictor activity may lead to obstruction of blood-vessels. This conclusion is supported by the findings that post-thrombotic peripheral collateral circulation in cats is restored and coronary cyclic flow reductions in dogs are prevented by ketanserin (Nevelsteen et al., 1984; Ashton et al., 1986).

CARDIOVASCULAR PAmOLOGY

Although serotonin by itself induces a weak reversible aggregation of normal human platelets, patients with cardiovascular diseases may show irreversible platelet aggregation in response to the monoamine. 5-HT2

receptors are involved, since treatment of these patients with ketanserin reverses this hypersensitivity to serotonin (De Cree et al., 1985a; Van Nueten et al., 1987).

The direct and amplifying effects of serotonin will be of consequence, particularly when it is released from aggregating platelets during a concomitant vascular hyperreactivity to serotonin, as obseliVed in a number

mmHg 190

180 17 0

160 150

140

130 120

110

ft

100 ~ ~ 90

80

70 ' -30·15 0 1 2 3 4 5 6 7 8

baseline 13 18 23 28 33

minutes

Figure 1.3 Mean systolic (e) and diastolic (0) blood pressure in hypertensive patients treated with ketanserin (10 mg i.v.) at 0 min. Ketanserin reduces both systolic and diastolic blood pressure; significant decreases are indicated by ** and

***.(Reproduced with permission from De Cree et al., 1981)

8 Serotonin

of acute and chronic pathological conditions (for review, see Vanhoutte and Houston, 1985; Van Nueten and Janssens, 1986). Old age and loss of endothelial function predispose to enhancement of the direct and amplifying vasoconstrictor response to serotonin (De Mey and Vanhoutte, 1981; Cohen et al., 1983; Janssens and Van Nueten, 1986; Vanhoutte, 1987).

Hypersensitivity of the vascular smooth muscle of spontaneously hypertensive rats and of hypertensive patients to serotoninergic activation is well documented (Doyle et al., 1959; Ahlund et al., 1977; Collis and Vanhoutte, 1977). Early investigation demonstrated that ketanserin lowers blood pressure in hypertensive rats, dogs and humans (Figure 1.3; De Cree et al., 1981; Van Nueten et al., 1981, 1987). The beneficial effect of orally administered ketanserin has been confirmed in more than 2000 patients treated acutely or chronically (for review, see Robertson et al., 1986; Vanhoutte et al., 1988).

The blood-pressure-lowering effect of ketanserin is more pronounced in subjects aged more than 60 years (De Cree et al., 1985b), which may be explained by the age-related increase in vascular reactivity to serotonin (De Mey and Vanhoutte, 1981; Janssens and Van Nueten, 1986).

Implication of serotonin in hypertension is suggested by the bloodpressure-lowering effect of ketanserin, although its ability to block a-adrenoceptors also contributes (see Van Nueten et al., 1987).

PATHOLOGY OF THE CENTRAL NERVOUS SYSTEM

Whereas the actions of ketanserin are mainly peripheral, other members of the same chemical series act mainly on the brain. Some of them are potent antagonists of centrally mediated pharmacological effects caused by serotonin or serotonin mimetic drugs (see Janssen, 1985). For instance, ritanserin ( 6-[2-[ 4-[bis( 4-fluorophenyl)methylene )-1-piperidinyl)ethyl)-7-methyi-5H-thiazolo[3,2-a ]pyrimidin-5-one) and risperidone (3-[2-[ 4-( 6-fluoro-1 ,2-benzisoxazol-3-yl)-1-piperidinyl]ethyl)-6, 7 ,8,9-tetrahydro-2-methyi-4H-pyrido[1,2-a)pyrimidin-4-one) are currently being investigated in patients with psychiatric disorders (Reyntjens et al., 1986).

CONCLUSIONS

Serotonin causes vasoconstriction and platelet aggregation by direct effects and amplification of the response to other agonists. The direct and amplifying effects are mediated by 5-HT2 receptors. These effects of serotonin may be enhanced in a number of cardiovascular diseases (e.g. hypertension, particularly in old age). Therefore, 5-HT2 receptor antagonists such as ketanserin can reduce blood pressure, and may have


beneficial effects in cardiovascular disorders (Vanhoutte, 1985). Centrally acting 5-HT 2 receptor antagonists may have beneficial effects in psychiatric disorders.

ACKNOWLEDGEMENTS

The authors are indebted to Dr De Clerck and Dr De Cree for providing the data from their studies. Thanks go to D. Verkuringen and L. Leijssen for their help in preparing the manuscript. Part of this work was supported by a grant from IWONL.

REFERENCES

Ahlund, L., Lundgren, Y., Sjoberg, B. and Weiss, L. (1977). Vascular reactivity to 5-hydroxytryptamine (5-HT) in hindquarter vascular beds, aortic strips and portal veins from spontaneously hypertensive and normotensive rats. Acta Physiol. Scand., 101,489-492

Ashton, J. H., Benedict, C. R., Fitzgerald, C., Raheja, S., Taylor, A., Campbell, W. B., Buja, L. M. and Willerson, J. T. (1986). Serotonin as a mediator of cyclic flow variations in stenosed canine coronary arteries. Circulation, 73, 572-578

Awouters, F., Leysen, J. E., DeClerck, F. and Van Nueten, J. M. (1982). General pharmacological profile of ketanserin (R 41 468), a selective 5-HT2 receptor antagonist. In DeClerck, F. and Vanhoutte, P.M. (Eds), 5-Hydroxytryptamine in Peripheral Reactions, Raven Press, New York, pp. 193-197

Awouters, F., Niemegeers, C. J. E. and Janssen, P. A. J.(1985). A pharmacological analysis of the rat mast cell5-HT gastric ulcer test and the effects of ketanserin. Drug Devel. Res., 5, 303-312

Cocks, T. M. and Angus, J. A. (1983). Endothelium dependent relaxation of coronary arteries by noradrenaline and serotonin. Nature, 305, 627-630

Cohen, R. A. (1985). Serotonergic prejunctional inhibition of canine coronary adrenergic nerves. J. Pharmacal. Exp. Ther., 235, 76-80

Cohen, R. A., Shepherd, J. T. and Vanhoutte, P.M. (1983). 5-Hydroxytryptamine can mediate endothelium-dependent relaxation of coronary arteries. Am. J. Physiol., 245, H1077-H1080

Collis, M. G. and Vanhoutte, P.M. (1977). Vascular reactivity of isolated perfused kidneys from male and female spontaneously hypertensive rats. Circ. Res., 41, 759-767

De Clerck, F. and Herman, A. G. (1983). 5-Hydroxytryptamine and platelet aggregation. Fed. Proc., 42, 228--232

De Clerck, F. and Van Nueten, J. M. (1982). Platelet-mediated vascular contractions: inhibition of the serotonergic component by ketanserin. Thromb. Res., 21, 713-727

De Clerck, F., David, J. L. and Janssen, P. A. J. (1982). Inhibition of 5-hydroxytryptamine-induced and amplified human platelet aggregation by ketanserin (R 41468), a selective 5-HT2 receptor antagonist. Agents Actions, 12, 388--397

De Clerck, F., Xhonneux, B., Leysen, J. and Janssen, P. A. J. (1984). The involvement of 5-HTz-receptor sites in the activation of cat platelets. Thromb. Res., 33, 305-321

10 Serotonin

De Cree, J., Leempoels, J., De Cock, W., Geukens, H. and Verhaegen, H. (1981). The antihypertensive effects of a pure and selective serotonin-receptor blocking agent (R 41 468) in elderly patients. Angiology, 32, 137-144

DeCree,J.,Leempoels,J., Demoen,B., Roels, V. andVerhaegen,H. (1985a). The effect of ketanserin, a 5-HTrreceptor antagonist on 5-hydroxytryptamineinduced irreversible platelet aggregation in patients with cardiovascular diseases. Agents Actions, 16, 313-317

De Cree, J., Hoing, M., De Ryck, M. and Symoens, J. (1985b). The acute antihypertensive effect of ketanserin increases with age. J. Cardiovasc. Pharmacol., 7, S126-S127

de Ia Lande, I. S., Cannell, V. A. and Waterson, J. G. (1966). The interaction of serotonin and noradrenaline on the perfused artery. Br. J. Pharmacol. Chemother., 28, 255-272

De Mey, C. and Vanhoutte, P. M. (1981). Effect of age and spontaneous hypertension on the tachyphylaxis to 5-hydroxytryptamine and angiotensin II in the isolated rat kidney. Hypertension, 3, 718--724

Doyle, A. E., Fraser, J. R. E. and Marshall, R. J. (1959). Reactivity of forearm vessels to vasoconstrictor substances in hypertensive and normotensive subjects. Clin. Sci., 18, 441-454

Engel, G., Gothert, M., Miiller-Schweinitzer, E., Schlicker, E., Sistonen, L. and Stadler, P. A. (1983). Evidence for common pharmacological properties of [3H] 5-hydroxytryptamine binding sites, presynaptic 5-hydroxytryptamine autoreceptors in CNS and inhibitory presynaptic 5-hydroxytryptamine receptors on sympathetic nerves. Naunyn-Schmiedeberg's Arch. Pharmacol., 324, 116-124

Furchgott, R. F. (1983). Role of endothelium in the responses of vascular smooth muscle. Circ. Res., 53, 557-573

Houston, D. S., Shepherd, J. T. and Vanhoutte, P.M. (1985). Adenine nucleotides, serotonin and endothelium-dependent relaxations to platelets. Am. J. Physiol., 248, H389-H395

Janssen, P. A. J. (1985). Pharmacology of potent and selective Srserotonergic antagonists. J. Cardiovasc. Pharmacol., 7, S2-811

Janssens, W. J. and Van Nueten, J. M. (1986). The direct and amplifying effects of serotonin are increased with age in the isolated perfused kidney of Wistar and spontaneously hypertensive rats. Naunyn-Schmiedeberg's Arch. Pharmacol., 334, 327-332

Leysen, J. E., Niemegeers, C. J. E., Tollenaere, J.P. and Laduron, P.M. (1978). Serotonergic component of neuroleptic receptors. Nature, 272, 168--171

Leysen, J. E., Awouters, F., Kennis, L., Laduron, P. M., Vandenberk, J. and Janssen, P. A. J. (1981). Receptor binding profile ofR 41468, a novel antagonist at 5-HT2 receptors. Life Sci., 28, 1015-1022

Leysen, J. E., Niemegeers, C. J. E., Van Nueten, J. M. and Laduron, P.M. (1982). [3H] Ketanserin (R 41468), a selective 3H-ligand for serotonin2 receptor binding sites. Binding properties, brain distribution and functional role. Mol. Pharmacol., 21, 301-314

McGrath, M. A. (1977). 5-Hydroxytryptamine and neurotransmitter release in canine blood vessels. Inhibition by low and augmentation by high concentrations. Circ. Res., 41, 428-435

Myers, J. H., Mecca, T. E. and Webb, R. C. (1985). Direct and sensitizing effects of serotonin agonists and antagonists on vascular smooth muscle. J. Cardiovasc. Pharmacol., 7, S44-s48

Nevelsteen, A., De Oerck, F., Loots, W. and De Gryse, A. (1984). Restoration of post-thrombotic peripheral collateral circulation in the cat by ketanserin, a selective 5-HT2 receptor antagonist. Arch. Int. Pharmacodyn., 270, 268--274


Page, I. H. (1954). Serotonin (5-hydroxytryptamine). Physiol. Rev., 34, 563-588 Peroutka, S. J. and Snyder, S. H. (1979). Multiple serotonin receptors. Differential

binding of [3H]-5-hydroxytryptamine, [3H]-lysergic acid diethylamide and [3H]-spiroperidol. Mol. Pharmacol., 16, 687-699

Reyntjens, A., Gelders, Y. G., Hoppenbrouwers, M. J. A. and VandenBussche, G. (1986). Thymosthenic effects of ritanserin (R 55 667), a centrally acting serotonin-Sz receptor blocker. Drug Devel. Res., 8, 205-212

Robertson, J. I. S., Stott, D. J. and Ball, S. G. (1986). The serotonin antagonist ketanserin in the treatment of clinical hypertension: a short review. J. Hypertension, 4, Suppl. 5, S119-S121

Scroop, G. C. and Walsh, J. A. (1968). Interactions between angiotensin, noradrenaline and serotonin on the peripheral blood vessels in man. Aust. J. Exp. Bioi. Med. Sci., 46, 573-580

Vanhoutte, P. M. (Ed.) (1985). Serotonin and the Cardiovascular System, Raven Press, New York

Vanhoutte, P. M. (1987). Serotonin and the vascular wall. Int. J. Cardiol., 14, 189-203

Vanhoutte, P. M. and Houston, D. S. (1985). Platelets, endothelium, and vasospasm. Circulation, 72, 728-734

Vanhoutte, P., Amery, A., Birkenhager, W., Breckenridge, A., Biihler, F., Distler, A., Dormandy, J., Doyle, A., Frohlich, E., Hansson, L., Hedner, Th., Hollenberg, N., Jensen, H.-E., Lund-Johansen, P., Meyer, P., Opie, L., Robertson, J., Safar, M., Schalekamp, M., Symoens, J., Trap-Jensen, J. and Zanchetti, A. (1988). Focus on the effects of ketanserin. Hypertension, 11, 111-133

Van Nueten, J. M. and Janssens, W. J. (1986). Augmentation of vasoconstrictor responses to serotonin by acute and chronic factors: inhibition by ketanserin. Hypertension, 4, S55-S59

Van Nueten, J. M. and Vanhoutte, P.M. (1981). Selectivity of calcium antagonism and serotonin antagonism with respect to venous and arterial tissues. Angiology, 32, 476-484

Van Nueten, J. M., Janssen, P. A. J., VanBeek, J., Xhonneux, R., Verbeuren, T. J. and Vanhoutte, P. M. (1981). Vascular effects of ketanserin (R 41 468), a novel antagonist of 5-HT2 serotonergic receptors. J. Pharmacol. Exp. Ther., 218, 217-230

Van Nueten, J. M., Janssen, P. A. J., De Ridder, W. and Vanhoutte, P.M. (1982). Interaction between 5-hydroxytryptamine and other vasoconstrictor substances in the isolated femoral artery of the rabbit: Effect of ketanserin (R 41 468). Eur. J. Pharmacol., 77, 281-287

Van Nueten, J. M., Janssens, W. J. and Vanhoutte, P. M. (1985). Serotonin and vascular reactivity. Pharmacol. Res. Commun., 17, 585-608

VanNueten, J. M., Janssen, P. A. J., Symoens,J., Janssens, W. J., Heykants, J., De Clerck, F., Leysen, J. E., Vancauteren, H. and Vanhoutte, P. M. (1987). Ketanserin. In Scriabine, A. (Ed.), New Cardiovascular Drugs, Raven Press, New York, pp. 1-56

Van Reempts, J., Borgers, M., Xhonneux, R., DeClerck, F. and Awouters, F. (1981). The inhibition of ischemic lesions of the rat gastric mucosa by a novel serotonin-antagonist: a light and electron microscopic study. Angiology, 32, 529-542

2 The Development of 5-HT 3 Receptor

Antagonists

J. R. Fozard

Preclinical Research Department, Sandoz Limited, CH-4002 Basel, Switzerland

INTRODUCTION

Despite the all-embracing title, this chapter must perforce deal mainly with the development of the Merrell Dow 5-HT 3 antagonists, and, in particular, MDL 72222. Thus, of the other major contributors to the field (Table 2.1), the route to the development of ICS 205-930 at Sandoz has already been described (Richardson et al., 1985); conversely, little or no information is available in this context for BRL 24924 and BRL 43694 (Beecham) and GR 38032F ( Glaxo). Although the 5-HT 3 terminology has been introduced only recently to denote receptors mediating excitation of peripheral autonomic and sensory neurones (Bradley et al., 1986), it will be used for convenience throughout this review.

5-HYDROXYTRYPTAMINE AND THE ISOLATED RABBIT HEART: AN ATYPICAL 5-HT RECEPTOR

The author's interest in 5-HT 3 receptors and their antagonists has its origins in a long-standing preoccupation with 5-hydroxytryptamine (5-HT) and its cardiovascular effects (Fozard, 1968), but is indisputably linked with a post-doctoral period spent in the laboratory of Professor Erich Muscholl of the University of Mainz. In Mainz, the introduction to the rabbit heart as a particularly useful model for the evaluation of drugs acting presynaptically at the autonomic neuroeffector junction (Fozard and Muscholl, 1972) led directly to a detailed analysis of the indirect sympathomimetic effects of 5-HT on this preparation. This work (which formed the PhD project of Dr Gabriel Mwaluko at the University of Manchester) confirmed the entirely indirect nature of the response to 5-HT, established the terminals of the


Table 2.1 The advent of selective 5-HT3 receptor antagonists Compound Structure Reference Metoclopramide 0 ( Fozard and Mobarok Ali (1978b)

Cl fflNH~,...-N,.,/

(-)-Cocaine

MDL 72222

ICS 205-930

BRL 24924

BRL 43694

GR 38032F

H2N~O I

Wl ~H ~N ... NH ' H

/N-N

Fozard et a/. (1979)

Fozard (1984)

Richardson eta/. (1985)

Sanger (1987)

Fake et a/. (1987)

Brittain et a/. (1987)

sympathetic nerves as the exclusive source of the noradrenaline released, and provided strong evidence for receptor-mediated depolarization being the principal mechanism of transmitter release (Fozard and Mwaluko, 1976). However, even high concentrations of morphine or methysergide proved unable to inhibit selectively the response to 5-HT, and hence the

14 Serotonin

receptor could not be designated 'M' or 'D' according to the accepted reference classification of the day (Gaddum and Picarelli, 1957).

CHARACTERIZATION OF THE 5-HT RECEPI'OR MEDIATING SYMPATHETIC NEURONAL EXCITATION IN THE RABBIT HEART

In 1975, Dr AbuT. M. Mobarok Ali took over (as his PhD project) the task of identifying and defining the atypical neuronal5-HT receptor of the rabbit heart. Since no leads for putative antagonists were available, the initial strategy was to characterize the receptor in terms of the relative potencies of selective agonists. The results of these studies established the total dissimilarity between the neuronal receptor of the rabbit heart and the 'D' receptor of the guinea-pig ileum; moreover, although certain similarities to the 'M' receptor were evident, there were sufficient major discrepancies to conclude with some confidence that the sites were not identical (Fozard and Mobarok Ali, 1978a).

An early strategy in the search for selective antagonists at the 5-HT3 receptor of the rabbit heart was to screen a number of compounds with activities at a variety of 5-HT receptor or acceptor (uptake) sites; no useful lead was forthcoming (Mobarok Ali, 1978). An unexpected development came, however, when (-)-cocaine was tested. (-)-Cocaine not only proved to be selective as an antagonist of 5-HT, as opposed to the nicotinic cholinergic receptor agonist, 1,1-dimethyl-4-phenylpiperazinium (DMPP), but also surmountable, and with a potency (pA2=6.24) incompatible with its acting as a local anaesthetic, for which concentrations of 1 mM or higher were required (Fozard et al., 1979). Limited structure-activity relationship studies, using compounds obtained from commercial sources or as gifts, established the key structural features of a series of tropine or pseudotropine derivatives for potency and selectivity at sympathetic neuronal 5-HT3 receptors (Fozard et al., 1979). Nor-(- )-cocaine proved an especially potent compound, and was capable of discriminating the 5-HT3 receptor of the rabbit heart (pA2=7.79) from the 'M' receptor of the ileum (pA2=6.49) (Fozard, 1983).

From the control experiments carried out to verify that local anaesthesia was indeed playing no role in .the mechanism of action of (-)-cocaine and its derivatives came a second important advance. Certain local anaesthetics (e.g. lignocaine, tetracaine, benzocaine and butacaine) were active only at high concentrations and showed selectivity for DMPP (Fozard et al., 1979); others (e.g. procaine and procainamide) were active at concentrations well below those causing local anaesthesia, and showed selectivity for 5-HT (Mobarok Ali, 1978). These data were presented at an internal seminar at the University of Manchester in 1976, and during discussion the similarity between procainamide and metoclopramide, a substituted benzamide with


dopamine D2 receptor-blocking activity, was pointed out. Subsequent testing of metoclopramide revealed it to be a potent (pA2 =7.20), surmountable and silent antagonist of the sympathetic neuronal 5-HT3 receptor of the rabbit heart (Fozard and Mobarok Ali, 1978b).

THE SYNTHESIS OF MDL 72222

The author joined the Merrell International Research Centre at Strasbourg in July 1977. The emerging pharmacophore based on data obtained with the analogues of cocaine and structures similar to metoclopramide was discussed and chemical synthesis initiated under the direction of Dr Maurice Gittos. The criteria set at the onset were for a compound with a pA2 on the rabbit heart of >9.0 and a selectivity of at least a thousandfold relative to DMPP. The projected clinical utility was the treatment of migraine, based on the hypothesis that 5-HT released locally in the blood-vessels of the head would contribute to the headache by activating 5-HT3 receptors on pain fibres innervating key elements of the cranial vasculature (Fozard, 1982). It was also recognized that the availability of a compound meeting the above criteria would afford a unique opportunity of exploring the physiological (and possibly the pathophysiological) role(s) of 5-HT3 receptors. MDL 72222 was one of several substituted benzoic acid esters of tropine which were synthesized and found to possess high affinity and selectivity for 5-HT 3 receptors (Fozard and Gittos, 1983); the compound more than adequately satisfied the criteria set at the outset (Fozard, 1984), and was chosen for further development.

FROM PHARMACOLOGICAL CURIOSITY TO THERAPEUTIC RELEVANCE

The development of any drug requires the availability of reliable models to quantify its principal activity in vivo. Since 5-HT induces a multitude of reflex effects in vivo, it was logical to seek to exploit this activity in the design of a suitable test for 5-HT3 receptor antagonists. The von Bezold-Jarisch effect of 5-HT provided the basis for a simple and reproducible cardiovascular model (Fozard, 1982; Fozard, 1984). MDL 72222 proved a particularly potent antagonist of the von Bezold-Jarisch effect of 5-HT, responses being suppressed by 0.1--0.3 mg/kg i.v. (Fozard, 1984) and for >4 h following 1.25 mg/kg orally. At the time, it was both surprising and somewhat disappointing to find that complete blockade of 5-HT3 receptors did not result in overt pharmacological activity.

The test developed for the evaluation of 5-HT3 receptor blockade in man was based on the fact that intradermal injections of 5-HT induce a flare

16 Serotonin

response, which is believed to reflect axonal reflex-mediated vasodilatation. The response is dose-related, easily quantified, and allowed confirmation in man that an i.v. dose of0.3 mglkg of MDL 72222 markedly suppresses the consequences of 5-HT3 receptor activation (Orwin and Fozard, 1986). Consistent with the putative role of 5-HT3 receptors in migraine, MDL 72222 at similar doses and by the same route proved an effective treatment for severe migraine (Loisy et al., 1985). The development of MDL 72222 was eventually suspended owing to its toxicity in animals (unconnected to 5-HT3 receptor blockade).

THE WIDER PERSPECTIVE

Recent years have seen the advent of a number of 5-HT3 receptor antagonists (Table 2.1), all characterized by high potency and remarkable selectivity for these sites. The availability of these compounds has allowed definition of the 5-HT3 receptor (Bradley et al., 1986), and revealed the existence of 5-HT 3 receptor subtypes (Fozard, 1983, 1985; Richardson et al., 1985). More significantly, perhaps, 5-HT3 receptor antagonists have been used in imaginative animal experiments to disclose important clinical potential in suppression of cytotoxic drug-induced vomiting (for brief review, see Fozard, 1987a), in a variety of gastrointestinal disorders (Sanger, 1987), and in CNS conditions such as psychoses and anxiety (see Fozard, 1987b; Tyers et al., this volume). It augurs well for the future that 5-HT3 receptor antagonists are particularly well tolerated clinically, and have already shown benefit in migraine (MDL 72222), carcinoid syndrome (ICS 205-930), and cytotoxic drug-induced emesis (ICS 205-930 and GR 38032F) (see Fozard, 1987b ). It bears emphasis that the exciting and diverse therapeutic potential of 5-HT3 receptor antagonists became evident only when such compounds were available for evaluation. Certainly, their clinical significance could not have been foreseen some 15 years ago, when the only stimulus to the development of such compounds was a largely academic interest in the mechanism of action of 5-HT on the rabbit isolated heart.

REFERENCES

Bradley, P. B., Engel, G., Feniuk, W., Fozard, J. R., Humphrey, P. P. A., Middlemiss, D. N., Mylecharane, E. J., Richardson, B. P. and Saxena, P. R. (1986). Proposals for the classification and nomenclature of functional receptors for 5-hydroxytryptamine. Neuropharmacology, 15, 563-575

Brittain, R. T., Butler, A., Coates, I. H., Fortune, D. H., Hagan, R., Hill, J. M., Hunter, D. C., Humphrey, P. P. A., Ireland, S. J., Jack, D., Gordon, C. C., Oxford, A., Straughan, D. W. and Tyers, M. B. (1987). GR 38032F, a novel selective 5-HT3 receptor antagonist. Br. J. Pharmacol., 90, 87P


Fake, C. S., King, F. D. and Sanger, G. J. (1987). BRL 43694: a potent and novel 5-HT3 receptor antagonist. Br. J. Pharmacol., 91, 335P

Fozard, J. R. (1968). Studies on the cardiovascular reactivity to 5-HT (5-hydroxytryptamine) in the rat. Unpublished Ph.D. thesis, University of Bradford

Fozard, J. R. (1982). In Critchley, M., Friedman, A., Gorini, S. and Sicuteri, F. (Eds), Headache: Physiopathological and Clinical Concepts. Advances in Neurology, Vol. 33, Raven Press, New York, pp. 295-307

Fozard, J. R. (1983). Differences between receptors for 5-hydroxytryptamine on autonomic neurones revealed by nor-(-)-cocaine. J. Auton. Pharmacol., 3, 21-26

Fozard, J. R. (1984). MDL 72222: a potent and highly selective antagonist at neuronal5-hydroxytryptamine receptors. Naunyn-Schmiedeberg's Arch. Pharmacol., 326, 36--44

Fozard, J. R. (1985). In Lambert, R. W. (Ed.), Proceedings of the 3rd SCIIRSC Medicinal Chemistry Symposium, The Royal Society of Chemistry, London, pp. 37-56

Fozard, J. R. (1987a). 5-HT3 receptors and cytotoxic drug-induced vomiting. Trends in Pharmacol. Sci., 8, 44-45

Fozard, J. R. (1987b). 5-HT: The enigma variations. Trends in Pharmacol Sci., 8, 501-506

Fozard, J. R. and Gittos, M. W. (1983). Selective blockade of 5-HT neuronal receptors by benzoic acid esters of tropine. Br. J. Pharmacol., 80, 511P

Fozard, J. R. and Mobarok Ali, A. T. M. (1978a). Receptors for 5-hydroxytryptamine on the sympathetic nerves of the rabbit heart. NaunynSchmiedeberg's Arch. Pharmacol., 301, 223--235

Fozard, J. R. and Mobarok Ali, A. T. M. (1978b ). Blockade of neuronal tryptamine receptors by metoclopramide. Eur. J. Pharmacol., 49, 109-112

Fozard, J. R. and Muscholl, E. (1972). Effects of several muscarinic agonists on cardiac performance and the release of noradrenaline from sympathetic nerves of the perfused rabbit heart. Br. J. Pharmacol., 45, 616--629

Fozard, J. R. and Mwaluko, G. M. P. (1976). Mechanism of the indirect sympathomimetic effect of 5-hydroxytryptamine on the isolated heart of the rabbit. Br. J. Pharmacol., 51, 115-125

Fozard, J. R., Mobarok Ali, A. T. M. and Newgrosh, G. (1979). Blockade of serotonin receptors on autonomic neurones by (-)-cocaine and some related compounds. Eur. J. Pharmacol., 59, 195-210

Gaddum, J. H. and Picarelli, Z. P. (1957). Two kinds of tryptamine receptor. Br. J. Pharmacol., 12, 323--328

Loisy, C., Beorchia, S., Centonze, V., Fozard, J. R., Schechter, P. and Tell, G. P. (1985). Effects on migraine headache of MDL 72222, an antagonist at neuronal 5-HT receptors. Double-blind, placebo-controlled study. Cephalalgia, 5, 79-82

Mobarok Ali, A. T. M. (1978). Pharmacological characterization ofthe receptors for 5-hydroxytryptamine (5-HT) on the sympathetic nerves of the rabbit heart. Unpublished Ph.D. thesis, University of Manchester

Orwin, J. M. and Fozard, J. R. (1986). Blockade of the flare response to intradermal 5-hydroxytryptamine in man by MDL 72222, a selective antagonist at neuronal 5-hydroxytrypt\lmine receptors. Eur. J. Clin. Pharmacol., 30, 209-212

Richardson, B. P., Engel, G., Donatsch, P. and Stadler, P. A. (1985). Identification of 5-hydroxytryptamine M-receptor subtypes and their specific blockade by a new class of drugs. Nature, 316, 126-131

Sanger, G. J. (1987). Increased gut cholinergic activity and antagonism of 5-hydroxytryptamine M-receptors by BRL 24924: potential clinical importance of BRL 24924. Br. J. Pharmacol., 91, 77-87

PART II NEURONAL ACTIONS

3 5-HT Receptors on Afferent Neurones

B. P. Richardson, G. Engel, P. DonatschandK.-H. Buchheit


INTRODUCTION

Receptors for 5-hydroxytryptamine (5-HT) are widely distributed over the mammalian peripheral nervous system (Wallis, 1981; Fozard, 1984). Those located on afferent neurones generally mediate excitatory actions of 5-HT, with neuronal inhibition being only rarely encountered (Paintal, 1973; Wallis, 1981; Roberts, 1984; Higashi and Nishi, 1982). Until recently it was unclear which receptor subtype mediates the stimulant action of 5-HT on primary afferent neurones, because such responses are refractory to blockade by the older, classical 5-HT receptor antagonists such as methysergide or cyproheptadine (Fozard, 1984). However, the recent discovery of new agonist and antagonist drugs with a high degree of selectivity for the different 5-HT receptor subtypes (Bradley et al., 1986; Richardson and Engel, 1986; Saxena et al., 1986) has stimulated renewed interest in this topic.

The purpose of the current presentation is to review the very recent work which has conclusively demonstrated that the excitatory action of 5-HT on primary afferent neurones is invariably mediated through the 5-HT3 receptor subtype. The criteria for arriving at this conclusion are pharmacological ones, as defined by Bradley et al. (1986): the responses can be mimicked by the selective 5-HT3 receptor agonist 2-methyl-5-HT with a potency similar to that of 5-HT itself; they are potently antagonized by 5-HT3 receptor antagonists such as MDL 72222 or ICS 205-930; but are refractory to inhibition by ketanserin, which selectively blocks 5-HT2 receptors, or methysergide and methiothepin, which block both 5-HT1-like and 5-HT2 receptors. For the reader's convenience, the quantitative data obtained with these discriminatory compounds on the various systems discussed in this review have been summarized in Table 3.1.

~

Tab

le 3

.1

Pote

ncie

s of

var

ious

com

poun

ds fo

r 5-

HT 3

rece

ptor

s on

aff

eren

t neu

rone

s

Com

poun

d Re

lativ

e Va

gus

(Rb)

N

odos

e vo

n B

ezol

d-C

ardi

ogen

ic

Car

otid

Lu

ng (R

b)

Blis

ter

Skin

re

cept

or

gang

lion

(Rb)

Ja

risch

ref

lex

hype

rten

sive

bo

dy (

C, R

) pa

in (

H)

flare

(H

) se

lect

ivity

(R

) re

flex

(D)

Agon

ists

pD

2 E

D50

(nm

o1)

ED

5o(Jt

gfkg

i.v.

) pD

2 pD

2

5-H

T

Non

e 6.

0 17

20

...

25

5.8

1-4

6.0

>5.0

~

2-M

ethy

l-5-H

T 5-

HT 3

5.

7 n.

t. 13

n.

t. n.

t. n.

t. 5.

7 n.

t. ~

a-M

ethy

l-5-

HT

5-H

T 2

Inac

tive

n.t.

Inac

tive

n.t.

n.t.

n.t.

3.7

n.t.

c 5-

Cf

5-H

Tt

Inac

tive

n.t.

Inac

tive

n.t.

n.t.

n.t.

Inac

tive

n.t.

;:s

PBG

5-

HT3

4.

9 -2

00

19

n.t.

n.t.

10-8

0 A

ctiv

e n.

t. s·

Anta

goni

sts

pA2

pA2

1050

(Jtg

fkg

i.v.)

pA2

ID50

(J.tg

/kg

i.v.)

ICS

205-

930

5-H

T3

10.2

10

.2

0.35

...

1 <1

0 n.

t. 11

.2

n.t.

MD

L 7

2222

5-

HT3

7.

9 7.

7 39

n.

t. 10

-100

-8

0

n.t.

<280

M

ethy

serg

ide

5-H

T 1 &

5-H

T 21n

activ

e In

activ

e In

activ

e In

activ

e In

activ

e n.

i:.

Inac

tive

Inac

tive

n.t.

= no

t tes

ted;

Rb

= ra

bbit;

R =

rat;

D =

dog;

C =

cat;

H =

hum

an.

5-C

T =

5-ca

rbox

amid

otry

ptam

ine;

PB

G =

phen

ylbi

guan

ide.

Fo

r ref

eren

ces

see

text

.

Afferent Neuronal 5-HT Receptors 23

IN-VITRO STUDIES

Nodose Ganglion

The nodose ganglion houses the cell bodies of vagal primary afferent neurones. Earlier studies had shown that 5-HT depolarizes a majority of 'C' type neurones in this preparation (Higashi and Nishi, 1982; Wallis et al., 1982) and that this cannot be blocked by the 5-HTrlike and 5-HT2 receptor antagonist methysergide (Higashi and Nishi, 1982). Formal proof of the involvement of 5-HT3 receptors has recently been obtained using the sucrose-gap technique. Depolarizing responses to 5-HT in the isolated rabbit nodose ganglion can be blocked by MDL 72222 or ICS 205-930 in an apparently competitive manner, yielding pA2 values of 7. 7 and 10.2, respectively (Azami etal., 1985; Round and Wallis, 1985, 1986). Although no systematic studies have been performed with selective agonists, previous studies have shown that phenylbiguanide, which is an agonist at 5-HT3 receptors in the rat vagus nerve (Ireland and Tyers, 1987), also has a potent agonist action in the rabbit nodose ganglion (Wallis et al., 1982).

Vagus Nerve

5-HT causes a concentration-dependent depolarization of the rabbit or rat isolated cervical vagus nerve (Neto, 1978; Ireland et al., 1982). Neither cyproheptadine nor methysergide blocks this action of 5-HT, indicating that 5-HTrlike and 5-HT2 receptors are not involved. Recent studies with agonist and antagonist drugs selective for 5-HT 3 receptors show that it is this receptor subtype that mediates the excitatory action of 5-HT in this preparation (Donatsch et al., 1984a; Richardson et al., 1985). Thus the selective 5-HT3 receptor agonist 2-methyl-5-HT is almost equipotent with 5-HT in reducing the amplitude of the compound action potential carried in the non-myelinated 'C' fibres of the rabbit isolated vagus nerve, while the selective 5-HT2 receptor agonist S( + )-a-methyl-5-HT is some 770 times less potent and the selective 5-HTrlike receptor agonist 5-carboxamidotryptamine (5-CT) is totally inactive. The effect of 5-HT in this preparation can be blocked by ICS 205-930 or MDL 72222, with pA2 values of 10.2 and 7.9, respectively, values which are in close agreement with those obtained in the nodose ganglion (Round and Wallis, 1985), suggesting that the 5-HT3 receptors found on the axons and cell bodies of vagal 'C' fibres may be identical. Recent studies employing the rat isolated vagus nerve have yielded essentially similar results (Ireland and Tyers, 1987). In addition, they have shown that phenylbiguanide acts as a 5-HT3 receptor agonist in this preparation, with a potency about one-half that of 5-HT. An agonist action at 5-HT3 receptors also explains why phenylbiguanide induces a von

24 Serotonin

Bezold-Jarisch effect in the cat and pain when applied to a blister on the human forearm (Pastier et al., 1959; and see below).

IN-VIVO STUDIES

The von Bezold-Jarisch Reflex

The von Bezold-Jarisch reflex is a vagally mediated reflex bradycardia and associated fall in systemic blood pressure which occurs when afferent nerve endings in the right ventricle are depolarized by a variety of stimuli (Salmoiraghi et al., 1956; Krayer, 1961; Paintal, 1973). In rats, bolus injection of 5-HT into the jugular vein is one way of eliciting this reflex (Salmoiraghi et al., 1956) and this effect of 5-HT is refractory to pre-treatment of animals with 5-HT1-like or 5-HT2 receptor antagonists (Fozard, 1984). Fozard and Gittos (1983) were the first to demonstrate conclusively the involvement of 5-HT3 receptors in mediating this action of 5-HT, when they showed that the administration of low doses of MDL 72222 and its analogues prior to injection of 5-HT could block this response in an apparently competitive fashion. Subsequently, it was shown that the 5-HT 3 receptor agonist 2-methyl-5-HT is equipotent with 5-HT in inducing the von Bezold-Jarisch reflex, while the 5-HT2 receptor agonist S( + )-a-methyl-5-HT and the 5-HT1-like receptor agonist 5-CT are completely inactive (Richardson et al., 1985; Richardson and Engel, 1986). As mentioned above, the 5-HT3 receptor agonist phenylbiguanide can also produce a von Bezold-Jarisch reflex in cats (Pastier et al., 1959) and rats (Richardson et al., unpublished observations). More recent studies in rats with other 5-HT 3 receptor antagonists such as ICS 205-930, GR 38032F and BRL 43694 have also clearly confirmed the involvement of this receptor subtype.

Figure 3.1 shows the excellent correlation between the pA2 values obtained for a large series of competitive 5-HT 3 receptor antagonists on the rabbit isolated vagus nerve and their in-vivo potency as inhibitors of the 5-HT-induced von Bezold-Jarisch reflex in rats. This again suggests that 5-HT3 receptors mediate both actions of 5-HT.

Cardiogenic Hypertensive Reflex

Bolus injections of 5-HT either into the left atrium or into the small branches of the proximal, but not the distal, left coronary artery of anaesthetized dogs leads to a complex of reflex cardiovascular events: systemic and pulmonary hypertension, changes in heart rate and contractile force, and disturbances in cardiac impulse conduction (James et al., 1975). The afferent limb of this reflex is carried in the vagus nerve and the efferent limb by both


13 • 12

"' ::: "' 11 2

• "' ::I

"" .. 10 > -..CI ..CI .. 9 a: • c • 0 • "' "' 8 ::I c;; > N

• r = -0.829 slope= 1.68 •

<t 7 c. p < 0.001 •

6

4 3 2 0

log Ki (pmoles/kg i.v.) von Bezold-Jarisch Effect

Figure 3.1 Correlation between the pA2 values obtained for a series of competitive 5-HT3 receptor antagonists on the rabbit vagus nerve and their in-vivo potencies as inhibitors of the 5-HT-induced von Bezold-Jarisch effect in rats. For methods, see

Richardson et al. (1985)

parasympathetic and sympathetic nerve fibres. In Figure 3.2 it can be seen that a dose of 5-HT as low as 10 t-tg/kg given directly into the left atrium is sufficient to evoke this reflex, and that the prior administration of 20 t-tg/kg ICS 205-930 almost completely abolishes it. Higher doses of 5-HT are able to overcome the blockade produced by this dose of ICS 205-930, suggesting competitive inhibition. These results indicate that the chemoreceptors for 5-HT present in tissue surrounding the proximal left coronary artery, which, when stimulated, produce the cardiogenic hypertensive reflex, are 5-HT3

receptors.

Pulmonary Depressor and Respiratory Cbemoreflexes

When given intravenously to rabbits, 5-HT and the 5-HT3 receptor agonist phenylbiguanide (see above) both cause dose-related decreases in systemic arterial blood pressure, heart rate and tidal volume, and increases in respiration rate, all of which can be prevented by vagotomy. These reflex events are virtually abolished by the prior administration of low doses of MDL 72222, although reflex respiratory responses to other pulmonary 'C' fibres agonists such as sodium dithionate and veratrine remain unaffected

26 Serotonin

400oi !g

-r~ ------------------------------------------.,r~ 0 "'0

O "'Q fl}~' \JVVJ~'VVVV'JV''VV'VV'.AAA/VVV''-"AJVVVVV\.IV' ~ f 20 polk.i l.•triM ICS 205-030

.OOOI!g

•or ~ 0 "'0

ZOOI~ ~~---------------------------0 "'0

'i ~~)Jio.,.'~A..~~ ~ r;pglk.9lelrlaiSKT

Figure 3.2 Induction of the cardiogenic hypertensive reflex by 5-HT in anaesthetized dogs. ECG = lead II electrocardiogram; BP = phasic systemic blood pressure; MAP= mean arterial blood pressure; PAP= phasic pulmonary arterial blood pressure. Panel A: 10 JA.g/kg 5-HT injected into the left atrium produces changes in cardiac rhythm including sinus bradycardia and ventricular tachycardia. There are also substantial increases in systemic and pulmonary arterial pressure. These effects typically last 3-4 min. Panel B: 5 min later, 20 JA.g/kg ICS 205-930 injected into the left atrium produces no cardiovascular effects. Panel C: 3 min later, 10 JA.g/kg 5-HT injected into the left atrium produces a slight bradycardia and increase in blood pressure. Higher doses of 5-HT overcome blockade by ICS 205-930 and again produce a cardiogenic hypertensive reflex. Experiments in three further

dogs produced similar results

(Armstrong and Kay, 1985). This demonstrates that 5-HT and phenylbiguanide evoke their reflex effects on respiration through stimulation of 5-HT3 receptors located on pulmonary 'C' fibres. Induction of pulmonary embolism in rabbits by the injection of sephadex beads into the right atrium also induces reflex tachypnoea, and again this is inhibited by pre-treatment of animals with low doses of MDL 72222 (Armstrong et al., 1986).


Presumably, 5-HT is released endogenously subsequent to pulmonary embolism and stimulates the 5-HT3 receptors located on pulmonary 'C' fibres, leading to reflex tachypnoea.

Carotid Sinus Nerve Activity

Injection of a small dose of 5-HT into the carotid artery of cats produces a complex pattern of changes in chemoreceptor discharge as recorded by impulse frequency in the carotid sinus nerve. Usually there is a brief period of excitation followed by a longer-lasting depression, which in turn is succeeded by a delayed increase in the neuronal firing rate (Black et al., 1972; Nishi, 1975). Studies with the 5-HT3 receptor antagonist MDL 72222 show that low doses virtually abolish the initial chemoexcitation and increase the dose of 5-HT required to produce subsequent chemodepression (Kirby and McQueen, 1984). This suggests that the latter may occur secondary to neuronal excitation, i.e. that it is due to depolarization blockade. In contrast to the inhibition obtained with MDL 72222 in these studies, previous investigations had shown that the early changes in chemoreceptor discharge are not inhibited by the 5-HTrlike and 5-HT2

receptor antagonist methysergide (Nishi, 1975). Together, these results show that activation of 5-HT3 receptors located in the carotid body is responsible for the abrupt transient increase in chemoreceptor discharge following intracarotid injection of 5-HT, and for the subsequently reduced activity. The delayed increase in sinus nerve activity remains unaffected by MDL 72222, but can be inhibited by low doses of the 5-HT2 receptor antagonist ketanserin (Kirby and McQueen, 1984). Neither MDL 72222 nor ketanserin had any effect on the responses of carotid chemoreceptors to hypoxia or to the chemodepressant action of dopamine, indicating their specificity of action for 5-HT receptors under these circumstances.

Results very similar to those with MDL 72222 in cats have very recently been obtained in rats using ICS 205-930 (Yoshoika et al., 1987). Again, the brief, rapid increase in carotid nerve activity was blocked by this 5-HT3

receptor antagonist, but the 5-HTrlike and 5-HT2 receptor antagonist methysergide was ineffective.

Blister Base Pain

When applied to a blister base on the human forearm, 5-HT causes pain and markedly potentiates the vascular pain caused by bradykinin (Keele and Armstrong, 1964; Sicuteri et al., 1965). More recent studies have shown that the painful effects of 5-HT in the human blister base model are not inhibited by the 5-HTrlike and 5-HT2 receptor antagonist methysergide, but can be

28 Serotonin

potently and competitively blocked by the 5-HT3 receptor antagonist ICS 205-930. The potentiation of bradykinin pain by 5-HT in these studies was also blocked by ICS 205-930 (Donatsch eta/., 1984b; Richardson et al., 1985). These investigations also showed that the 5-HT3 receptor agonist 2-methyl-5-HT was approximately equipotent with 5-HT at producing pain, whereas the 5-HT2 receptor agonist S( + )-a-methyl-5-HT was some 100 times less potent. Further studies have shown that the 5-HT3 receptor agonist phenylbiguanide also produces pain in this model (Fastier et al., 1959), whereas the selective 5-HT1-like receptor agonist 5-CT is inactive (Richardson and Engel, 1986).

Cutaneous Wheal and Flare Responses

Intradermal injection of 5-HT in man produces a wheal and flare reaction (Demis et al., 1960; Greaves and Shuster, 1967), which is believed to result from activation of a cutaneous axonal reflex involving the antidromic release of substance P (Greaves and Shuster, 1967; Lembeck and Holzer, 1979). During the study of the effects of 5-HT on the blister base in man (see above), it had previously been noticed that a wheal and flare reaction occurred concomitantly with the pain response and that, like the pain, it could be inhibited by ICS 205-930 (Donatsch et al., 1984b, Richardson et al., 1984). A more detailed, recent study on the flare component ofthis reaction shows that it can be prevented by giving volunteers a small i.v. dose of the 5-HT3 receptor antagonist MDL 72222 prior to the intradermal injection of 5-HT (Orwin and Fozard, 1986). If higher concentrations of 5-HT are injected, the antagonism by MDL 72222 can be overcome, indicating a competitive type of blockade. These data show that activation of 5-HT3

receptors on sensory nerve endings in human skin produces pain and a cutaneous axonal reflex, the latter leading to the release of vasoactive substances which produce the wheal and flare response.

CONCLUSIONS

5-HT activates afferent nerves in many different species and in a variety of anatomical locations. In all systems where selective agonist and antagonist drugs have so far been employed, it could be clearly shown that 5-HT3

receptors rather than 5-HTrlike or 5-HT2 receptors mediate this action of 5-HT. This suggests the potential therapeutic use of 5-HT3 receptor antagonists in a variety of clinical conditions such as pulmonary embolism, cardiac arrhythmias and migraine, where activation of 5-HT3 receptors on primary afferent nerves may initiate a complex of undesirable reflex events.


ACKNOWLEDGEMENTS

We thank Dr H. Siegl for providing the von Bezold-Jarisch data presented in Figure 3.1, and Messrs W. Bielser, P. Gugger and F. Luh for their expert technical assistance.

REFERENCES

Armstrong, D. J. and Kay, I. S. (1985). MDL 72222 (a5-HTantagonist) antagonizes the pulmonary depressor and respiratory chemoreflexes evoked by phenylbiguanide in anaesthetized rabbits. J. Physiol., 365, 104P

Armstrong, D. J., Kay, I. S. and Russell, N.J. W. (1986). MDL 72222 antagonizes the reflex tachypnoeic response to miliary pulmonary embolism in anaesthetized rabbits. J. Physiol., 381, 13P

Azami, J., Fozard, J. R., Round, A. A. and Wallis, D. I (1985). The depolarizing action of 5-hydroxytryptamine on rabbit vagal primary afferent and sympathetic neurones and its selective blockade by MDL 72222. Naunyn-Schmiedeberg's Arch. Pharmacal., 328, 423-429

Black, A.M. S., Comroe, J. H. and Jacobs, L. (1972). Species difference in carotid body response of cat and dog to dopamine and serotonin. Am. J. Physiol., 233, 1097-1102

Bradley, P. B., Engel, G., Feniuk, W., Fozard, J. R., Humphrey, P. P. A., Middlemiss, D. N., Mylecharane, E. J., Richardson, B. P. and Saxena, P. R. (1986). Proposals for the classification and nomenclature of functional receptors for 5-hydroxytryptamine. Neuropharmacology, 25, 563--576

Demis, J., Davis, M. J. and Lawler, J. C. (1960). A study of the cutaneous effects of serotonin. J. Invest. Dermatol., 34, 34--49

Donatsch, P., Engel, G., Richardson, B. P. and Stadler, P. A. (1984a). Subtypes of neuronal 5-hydroxytryptamine (5-HT) receptors as identified by competitive antagonists. Br. J. Pharmacol., 81, 33P

Donatsch, P., Engel, G., Richardson, B. P. and Stadler, P. A. (1984b). The inhibitory effect of neuronal5-hydroxytryptamine (5-HT) receptor antagonists on experimental pain in humans. Br. J. Pharmacol., 81, 35P

Fastier, F. N., McDowall, M.A. and Waal, H. (1959). Pharmacological properties of phenylbiguanide and other amidine derivatives in relation to those of 5-hydroxytryptamine. Br. J. Pharmacal. Chemother., 14, 527-535

Fozard, J. R. (1984). Neuronal5-HTreceptors in the periphery. Neuropharmacology, 23, 1473--1486

Fozard, J. R. and Gittos, M. W. (1983). Selective blockade of 5-hydroxytryptamine neuronal receptors by benzoic acid esters of tropine. Br. J. Pharmacal., 80, 511P

Greaves, M. and Shuster, S. (1967). Responses of skin blood vessels to bradykinin, histamine and 5-hydroxytryptamine. J. Physiol., 193, 255-267

Higashi, H. and Nishi, S. (1982). 5-Hydroxytryptamine receptors on visceral primary afferent neurones on rabbit nodose ganglion. J. Physiol., 323, 543--567

Ireland, S. J. and Tyers, M. B. (1987). Pharmacological characterization of 5-hydroxytryptamine-induced depolarization of the rat isolated vagus nerve. Br. J. Pharmacol., 90, 229-238

Ireland, S. 1., Straughan, D. W. and Tyers-, M. B. (1982). Antagonism by metoclopramide and quipazine of 5-hydroxytryptamine-induced depolarizations of rat isolated vagus nerve. Br. J. Pharmacol., 15, 16P

30 Serotonin

James, T. N., Isobe, J. H. and Urthaler, F. (1975). Analysis of components in a canliogenic hypertensive chemoreflex. Circulation, 52, 179-192

Keele, C. A. and Armstrong, D. (1964). In Substances Producing Pain and Itch, Arnold, London, 30--66

Kirby, G. C. and McQueen, D. S. (1984). Effects ofthe antagonists MDL 72222 and ketanserin on responses of cat carotid body chemoreceptors to 5-hydroxytryptamine. Br. J. Pharmacal., 83, 259-269

Krayer, 0. (1961). The history of the Bezold-Jarisch effect. Naunyn-Schmiedeberg's Arch. Pharmacal., 240, 361-368.

Lembeck. P. and Holzer, P. (1979). Substance P as neurogenic mediator of antidromic vasodilation and neurogenic plasma extravasation. NaunynSchmideberg's Arch. Pharmacal., 310, 175-182

Neto, F. R. (1978). The depolarizing action of 5-HTon mammalian non-myelinated nerve fibres, Eur. J. Pharmacal., 49, 351-356

Nishi, K. (1975). The action of 5-hydroxytryptamine on chemoreceptor discharge of the eat's carotid body. Br. J. Pharmacal., 55, 27-40

Orwin, J. M. and Fozard, J. R. (1986). Blockade of the flare response to intradermal 5-hydroxytryptamine in man by MDL 72222, a selective antagonist at neuronal 5-hydroxytryptamine receptors. Eur. J. Clin. Pharmacal., 30, 209-212

Pain tal, A. S. (1973). Vagal sensory receptors and their reflex effects. Physiol. Rev., 53, 159-227

Richardson, B. P. and Engel, G. (1986). The pharmacology and function of 5-HT3 receptors. Trends in Neurosci., 9, 424-428

Richardson, B. P., Engel, G., Donatsch, P. and Stadler, P. A. (1985). Identification of 5-hydroxytryptamine M-receptor subtypes and their specific blockade by a new class of drugs. Nature, 316, 126-131

Roberts, M. H. T. (1984). 5-hydroxytryptamine and antinociception. Neuropharmacology, 23, 1529-1536

Round, A. A. and Wallis, D. I. (1985). Selective blockade by ICS 205-930 of 5-hydroxytryptamine (5-HT) depolarizations of rabbit vagal afferent and sympathetic ganglion cells. Br. J. Pharmacal., 86, 734P

Round, A. A. and Wallis, D. I. (1986). The depolarizing action of 5-hydroxytryptamine on rabbit vagal afferent and sympathetic neurones in vitro and its selective blockade by ICS 205-930. Br. J. Pharmacal., 88, 485-494

Salmoiraghi, G. C., Page, I. H. and McCubbin, J. W. (1956). Cardiovascular and respiratory response to intravenous serotonin in rats. J. Pharmacal. Exp. Ther., ll8, 447--481

Saxena, P. R., Richardson, B. P., Mylecharane, E. J., Middlemiss, D. N., Humphrey, P. P. A., Fozard, J. R., Feniuk, W., Engel, G. and Bradley, P. B. (1986). Functional receptors for 5-hydroxytryptamine. Trends in Pharmacal. Sci., 94 (centre fold)

Sicuteri, F., Fanciullacci, M., Franchi, G. and Del Biancho, P. L. (1965). Serotonin-bradykinin potentiation on the pain receptors in man. Life Sci., 4, 303-316

Wallis, D. (1981). Neuronal 5-hydroxytryptamine receptors outside the central nervous system. Life Sci., 29, 2345-2355

Wallis, D. 1., Stansfeld, C. E. and Nash, H. L. (1982). Depolarizing responses recorded from nodose ganglion cells of the rabbit evoked by 5-hydroxytryptamine and other substances. Neuropharmacology, 21, 31--40

Yoshoika, M., Matsumoto, M., Togashi, H., Abe, M., Tochihara, M. and Saito, H. (1987). The 5-hydroxytryptamine-induced increase in chemoreceptor afferent nerve discharge and its blockade by ICS 205-930 in the rat. Res. Commun. Psycho/. Psychiat. Behav., 12, 215-220

4 Electro physiological Investigation of the

Actions of 5-Hydroxytryptamine on Sympathetic Ganglionic Neurones

D. I. Wallis1 andN. J. Dun2

1 Department of Physiology, University of Wales College of Cardiff, PO Box 902, CardiffCFllSS, UK

2Department of Pharmacology, Loyola University Stritch School of Medicine,MaywoodiL60153, USA

INTRODUCTION

Two aspects of the biology of 5-hydroxytryptamine (5-HT) in relation to sympathetic neurones which have been investigated electrophysiologically are the mechanisms by which 5-HT brings about its effect, i.e. the ionic basis of each action, and the identification of the 5-Hf receptors involved. The latter requires the development of quantitative pharmacological techniques. This article outlines some of the approaches that have proved useful, and touches on the relationship between information from extracellular recording and from intracellular measurements of 5-HT actions on single neurones. Reference is made to some recent experiments on guinea-pig ganglia conducted at Loyola University.

Results from a variety of techniques make it clear that 5-HT has a range of actions on sympathetic neurones at a variety of sites (Figure 4.l(A) ). These actions include changes in membrane potential and effects on transmitter release, detected electrophysiologically by analysing excitatory postsynaptic potential (EPSP) amplitude in the post-synaptic cell.

ACTIONS ON PRE-GANGLIONIC NEURONES

5-HT depolarizes (or excites) spinal sympathetic motoneurones. A promising approach is the use of cord slices from neonate rats (Ma and Dun, 1986), which allows intracellular recording of the effects of 5-HT applied by

32

A r - - - - - - - - - Depolarization

~I I \

II

Serotonin

~ -Depolarization - - - -, \

ACh release Hyperpolarization

Release of Noradrenaline

Nor A release

8 i ~~ ~. . 5ms 10 15

15ms 6s

ii 20s

10ms 20 50 jo.snA

Figure 4.1 (A) Schematic representation of pre-ganglionic and post-ganglionic sympathetic neurones, showing the identified sites of 5-HT action and the nature of the 5-HT receptor involved. 1 = 5-HT1-like receptor; 3 = 5-HT3 receptor; ni =not identified. (B) Responses of two guinea-pig SCG cells to 5-HT applied by pressure ejection. Chart records: triangles show times of ejection of 5-HT onto cell. Downward deflections of traces are electrotonic potentials in response to current pulses. (i) Fast responses to 5-HT accompanied by cell discharge. Ejections of longer duration evoked a more sustained depolarization (top line). At slower chart speeds (middle line), a 15 ms ejection evoked an after-hyperpolarization following the sustained depolarization. The latter response was much enhanced when 5-HT was ejected onto the cell for 6 s. (ii) Another cell responded with slow depolarizations to 5-HT whose amplitude was related to the duration ofthe ejection. In the right-hand record, the membrane was manually voltage-clamped for a period during the peak of the response. Under these conditions, there was a small increase in input resistance as indicated by an increase in electrotonic potential amplitude. The lower trace is a

record of the current

superfusion. Responses to 5-HT were accompanied by a fall in membrane conductance. Although responses were antagonized by methysergide and cyproheptadine, the receptor has yet to be fully characterized.

Actions of 5-HT on Sympathetic Ganglionic Neurones 33

Extracellular recording from the rabbit cervical sympathetic nerve, superfused in a perspex chamber in which the nerve passes through a sealed partition (Brown and Marsh, 1978), has revealed a depolarizing response in pre-ganglionic axons (Elliott and Wallis, unpublished observations). With carefully desheathed preparations, a series of cumulative concentrationresponse curves can be constructed, providing an adequate interval (about 1 h) is allowed between curves. This response appears to be mediated by a 5-HT3 receptor, since responses were antagonized in a surmountable manner by MDL 72222 and ICS 205-930, and 2-methyl-5-HT acted as an agonist.

Extracellular recording is facilitated by a diminution in the extracellular shunt of current between two recording points. In the method just cited, the seal at the partition reduces shunting, while in the sucrose-gap method (Wallis et al., 1975) a sucrose solution of high specific resistance replaces the electrolytes of extracellular fluid. This greatly reduces short-circuiting while bathing part of the preparation in a solution of appropriate osmolality. Ganglia are suspended in a series of perspex chambers and superfused. Sucrose-gap records from the proximal pole of the rabbit superior cervical ganglion (SCG) suggest that 5-HT may depolarize pre-ganglionic terminals within the ganglion (Wallis and Woodward, 1975). This requires confirmation, as does the suggestion (Figure 4.1(A) ) that a 5-HT3 receptor mediates this action.

Part of the depression of ganglionic transmission on superfusing 5-HT may result from depolarization block of spike propagation through the branching pre-ganglionic axons. In some preparations of rabbit SCG, depression of transmission by 5-HT was alleviated, at least partially, by 5-HT3 antagonists (Elliott and Wallis, unpublished observations). Alternatively, 5-HT3 receptors on nerve terminals might be associated with the facilitation of transmission which can be induced by 5-HT (Haefely, 1974; Hirai and Koketsu, 1980). Other evidence points to a 5-HTrlike receptor reducing acetylcholine release. Extracellular methods are, in the main, insufficiently incisive to distinguish a pre-synaptic from a post-synaptic modulation of transmission. The use of intracellular recording in conjunction with quanta! analysis of the EPSP has shown that activation of a pre-synaptic 5-HT receptor reduces evoked acetylcholine release (Dun and Karczmar, 1981; Wallis and Dun, 1989). The site is not fully characterized, although both methysergide and 5-carboxamidotryptamine have agonist actions and so a 5-HT3 receptor is unlikely to be involved. Pre-synaptic actions of 5-HT in autonomic ganglia have recently been reviewed (Wallis and Dun, 1989). Unfortunately, intracellular studies to quantify a pre-synaptic 5-HT action are made difficult because (a) some inputs appear insensitive to the amine, and (b) quanta! analysis can properly be applied only to monophasic EPSPs arising from one synaptic location, whereas convergent pathways onto ganglion cells are the general rule.

34 Serotonin

ACTIONS ON GANGLIONIC NEURONES

5-HT depolarizations of somata of rabbit SCG or their axons can be recorded using the sucrose-gap technique, which allows quantitative assessment of 5-HT3 receptor-mediated effects in ganglionic neurones (Azami et al., 1985; Round and Wallis, 1987). The method records selectively from the population of neurones at the interface between saline and sucrose solutions. With appropriate intervals between tests, it is possible to construct repeatable, full dose-response curves for 5-HT. 1,1-Dimethyl-4-phenylpiperazinium (DMPP), which activates nicotinic receptors, can be used as a control agonist. Measurements of antagonist potency gave apparent pA2 values for ICS 205-930, MDL 72222, quipazine and metoclopramide at this receptor of 10.4, 7 .8, 7.6 and 7 .2, respectively. The relative activities as agonists of a number of 5-HT analogues, including a-methyl-5-HT, N-methyl-5-HT, N,N-dimethyl-5-HT, N-methyltryptamine and N,N-dimethyltryptamine, were tested by Wallis and Nash (1981).

Surface recordings for cat SCG in situ revealed a complicated, triphasic potential change evoked by i.a. injection of 5-HT (de Groat and Volle, 1966; Machova and Boska, 1969). An initial depolarization was followed by hyperpolarization and a late depolarization. Recently, intracellular studies in our laboratory have clarified the nature of the potential changes by applying 5-HT to the vicinity of the cell by pressure ejection. Studies of guinea-pig coeliac ganglion (CG) cells have shown that three phases of potential change occur, but not in every cell. Over 90 per cent of neurones responded to 5-HT: among responding cells, 43 per cent displayed a fast depolarization, and 26 per cent a slow depolarization alone. A number of cells showed a hyperpolarization following the fast depolarization. The fast phasic depolarization appeared to result from increased Na+ and K+ permeability, and was accompanied by a large drop in membrane input resistance. The ionic basis of the slow depolarization has still to be determined, but responses are associated with little or no change in input resistance. The fast response to 5-HT was blocked by ICS 205-930 (1 !.I.M), MDL 72222 (1-S!J.M), quipazine (1 J.I.M), metoclopramide (10 J.I.M) and tubocurarine (50 !J.M), but not by methysergide (1 J.I.M). Responses showed pronounced tachyphylaxis. Thus the receptor mediating the fast response is a 5-HT3 receptor, while that underlying the slow depolarization, although methysergide-sensitive (0.5--1 !J.M), has yet to be identified.

Interestingly, when 5-HT is applied by superfusion to this ganglion, only depolarizations of the slow type are observed (Dun et al., 1984). When pressure ejection of 5-HT was used, both fast and slow depolarizations were observed, not only in guinea-pig CG, but also in guinea-pig inferior mesenteric ganglion and SCG cells. Figure 4.1(B) shows the responses of two guinea-pig SCG cells, one of which showed a fast depolarization, a hyperpolarization and a slow depolarization. It seems likely that the sucrose-gap method, where responses are generated by bolus injections of

Actions of 5-HT on Sympathetic Ganglionic Neurones 35

5-HT into the superfusion stream to the ganglion, may be rather selective for examining the fast 5-HT 3-mediated response from a restricted population of ganglion cells.

Electrogenesis of hyperpolarizing responses to 5-HT in sympathetic neurones is complex, since in some cases these are after-hyperpolarization& dependent upon the preceding depolarization. However, at least in rat SCG cells, a hyperpolarization directly mediated by a 5-HTrlike receptor has been identified (Ireland, 1987), using an extracellular recording technique similar to that of Brown and Marsh (1978).

Finally, modulatory effects of 5-HT on nicotinic responses have also been reported (Akasu et al., 1981). In bullfrog ganglion cells, 5-HT modulates nicotinic receptor affinity to acetycholine, acting as an antagonist.

At the neuroeffector junction itself, 5-HT acts to cause release of noradrenaline from rabbit cardiac sympathetic nerve endings, and may act in a similar fashion at other sympathetic post-ganglionic neuroeffector junctions (Fozard, 1984). A 5-HT3 receptor is involved, but this excitatory action of 5-HT is not manifested at all sympathetic terminals. More commonly, reduction of noradrenaline release is seen, mediated via 5-HTrlike receptors (Bradley et al., 1986).

CONCLUSIONS

The number and variety of 5-HT effects on sympathetic neurones, demonstrated pharmacologically, suggest that 5-HT has the potential to exert a powerful and complex modulatory action on the sympathetic nervous system. At present, it is unclear whether any or all of these effects have physiological significance. However, the presence of 5-HT in ganglionic neural elements has recently been demonstrated (Dun et al., 1984). 5-HT immunoreactivity was identified in nerve fibres in close proximity to principal ganglion cells, and accompanying the splanchnic nerves, in the guinea-pig coeliac superior mesenteric plexus. Evidence was presented to suggest that 5-HT liberated from these axons might be responsible for slow EPSPs generated in a proportion of neurones.

An intriguing aspect of the recent findings is evocation by 5-HT of fast and slow depolarizing responses, reminiscent of nicotinic and muscarinic actions of acetylcholine on sympathetic neurones. These actions may constitute forms of post-synaptic modulation of transmission, while the effects of 5-HT on transmitter release at various points suggest that this kind of modulation may be deployed selectively on particular inputs to ganglionic and effector cells.

ACKNOWLEDGEMENTS

Supported by the Wellcome Trust and NS 18710.

36 Serotonin

REFERENCES

Akasu, T., Hirai, K. and Koketsu, K. (1981). 5-hydroxytryptamine controls ACh-receptor sensitivity of bullfrog sympathetic ganglion cells. Brain Res., 211, 217-220

Azami, J., Fozard, J. R., Round, A. A. and Wallis, D. I. (1985). The depolarizing action of 5-hydroxytryptamine on rabbit vagal primary afferent and sympathetic neurones and its selective blockade by MDL 72222. Naunyn-Schmiedeberg's Arch. Pharmacol., 328, 423-429


Brown, D. A. and Marsh, S. (1978). Axonal GADA-receptors in mammalian peripheral nerve trunks. Brain Res., 156, 187-191

de Groat, W. C. and Volle, R. L. (1966). The actions of the catecholamines on transmission in the superior cervical ganglion of the cat. J. Pharmacol. Exp. Ther., 154, 1-13

Dun, N. J. and Karczmar, A. G. (1981 ). Evidence for a presynaptic inhibitory action of 5-hydroxytryptamine in a mammalian sympathetic ganglion. J. Pharmacol. Exp. Ther., 217, 714-718

Dun, N. J., Kiraly, M. and Ma, R. C. (1984). Evidence for a serotonin mediated slow excitatory potential in the guinea-pig coeliac ganglia. J. Physiol., 351, 61-76

Fozard, J. R. (1984). Neuronal 5-HT receptors in the periphery. Neuropharmacotogy,23, 1473-1486

Haefely, W. (1974). The effects of 5-hydroxytryptamine and some related compounds on the cat superior cervical ganglion in situ. Naunyn-Schmiedeberg's Arch. Pharmacol., 281, 145-165

Hirai, K. and Koketsu, K. (1980). Presynaptic regulation of the release of acetylcholine by 5-hydroxytryptamine. Br. J. Pharmacol., 70, 499-500

Ireland, S. J. (1987). Origin of 5-hydroxytryptamine-induced hyperpolarization of the rat superior cervical ganglion and vagus nerve. Br. J. Pharmacol., 92, 407-416

Ma, R. C. and Dun, N.J. (1986). Excitation oflateral hom neurons of the neonatal rat spinal cord by 5-hydroxytryptamine. Devel. Brain. Res., 24, 89-98

Machova, J. and Boska, D. (1969). The effects of 5-hydroxytryptamine, dimethylphenylpiperazinium and acetylcholine on transmission and surface potential in the cat sympathetic ganglion. Eur. J. Pharmacol., 1, 152-158

Round, A. and Wallis, D. I. (1987). Further studies on the blockade of 5-HT depolarizations of rabbit vagal afferent and sympathetic ganglion cells by MDL 72222 and other antagonists. Neuropharmacology, 26, 39-48

Wallis, D. I. and Dun, N.J. (1989). Presynaptic action of 5-hydroxytryptamine on autonomic ganglia. In Feigenbaum, J. J. and Hanani, M. (Eds), Presynaptic Regulation of Neurotransmitter Release: a Handbook, Freund, London and Tel Aviv (in press)

Wallis, D. I. and Nash, H. L. (1981). Relative activities of substances related to 5-hydroxytryptamine as depolarizing agents of superior cervical ganglion cells. Eur. J. Pharmacol., 70, 381-392

Wallis, D. I. and Woodward, B. (1975). Membrane potential changes induced by 5-hydroxytryptamine in the superior cervical ganglion of the rabbit. Br. J. Pharmacol., 55, 199-212

Wallis, D. 1., Lees, G. M. and Kosterlitz, H. W. (1975). Recording resting and action potentials by the sucrose-gap method. Comp. Biochem. Physiol., SOC, 199-216

5

Multiple 5-HT Receptors in the Enteric Nervous System

M.D. Gershon, G. M. Maweand T. A. Branchek

Department of Anatomy and Cell Biology, Columbia University, College of Physicians and Surgeons, 630 W 168th Street, New York NY 10032, USA

At the time when peripheralS-hydroxytryptamine (5-HT) receptors were first classified, by Gaddum and Picarelli (1957), the complex nature of the enteric nervous system (ENS) was not appreciated, although many of the essential and distinguishing characteristics of that division of the autonomic nervous system (ANS) had already been published. The pioneering experiments of Bayliss and Starling (1899), in vivo, and Trendelenburg (1917), in vitro, established that all of the components of the peristaltic reflex arc, including pressure receptors, primary sensory neurones, interneurones and excitatory and inhibitory motor neurones, are present in the gut as intrinsic elements of the ENS. Because of this enteric autonomy, and because of the matching anatomical discrepancy between a very large number of neurones in the enteric ganglia and a very small number of pre-ganglionic fibres in the diaphragmatic vagus nerves, Langley (1921) classified the ENS as the third division of the ANS. Nevertheless, despite all the evidence showing that most enteric neurones receive no direct input from the CNS, many texts in 1957 continued to treat enteric ganglia simply as parasympathetic relays.

In their investigation of 5-HT receptors, Gaddum and Picarelli (1957) used the parasympathetic relay model of the ENS; therefore, the possibility that their data might be complicated by confounding effects of interneurones, or that there might be multiple types of 5-HT receptor in the ENS, were not discussed. Gaddum and Picarelli (1957) used the mechanical activity of the enteric musculature to assess the effects of experimental compounds added to segmt;nts of guinea-pig ileum isolated in an organ bath to define what they called 'M' and 'D' 5-HT receptors. 'M' receptors were those that mediated responses to 5-HT which were inhibited by morphine, while 'D' receptors were those that mediated responses inhibited by phenoxybenzamine (dibenzyline). Although this terminology was subse-

38 Serotonin

quently criticized (Drakontides and Gershon, 1968; Humphrey, 1983; Bradley et al., 1986) because neither morphine nor phenoxybenzamine are specific 5-HT receptor antagonists, the distinction between these two types of receptor has been useful because it corresponds to a real difference in receptor location. 'M' receptors are neural and cause contractile responses of the gut; thus, when activated by 5-HT, they directly or indirectly lead to the release of acetylcholine (which is inhibited by morphine) from common excitatory motor neurones (see Gershon, 1981). 'M' receptor-mediated responses to 5-HT, therefore, have now come to mean those effects of 5-HT that can be blocked by neural paralysis with an agent such as tetrodotoxin (Gershon, 1967; Drakontides and Gershon, 1968; Fozard and Mobarok Ali, 1978; Richardson et al., 1985). The tetrodotoxin-resistant 'D' receptormediated responses to 5-HT, in contrast, are due to a direct activation of receptors located on the intestinal smooth muscle itself (Gershon, 1967). Since many of the drugs that block 'D' receptor-mediated responses to 5-HT are 5-HT2 receptor antagonists (Cohen et al., 1983; Peroutka, 1984), it is probable that'D' receptors can be included in the 5-HT2 receptor set (Engel eta/., 1984; Richardson eta/., 1985; Bradley eta/., 1986). The definition of 'M' receptor-mediated responses has recently been updated through the development of antagonists that appear to be specific for 5-HT receptors, and this type of receptor has been renamed 5-HT3 (Bradley et al., 1986; Richardson and Engel, 1986); however, even this recent reclassification has retained the simple model of the ENS and has not entertained the possibility that there may be more than one type of enteric neural receptor for 5-HT.

5-HT has many sites of action within the bowel, affecting epithelium (see Cooke, 1987), as well as muscle and nerve (Gershon, 1982; Furness and Costa, 1987). Neural actions of 5-HT are also of many different types, and include excitation of afferent nerve fibres (BUlbring and Crema, 1959; Paintal, 1964; Lew and Longhurst, 1986), cholinergic ganglion cells (to cause contraction of all enteric muscle layers; Costa and Furness, 1979; Gershon, 1982; Kamikawa and Shimo, 1983), intrinsic inhibitory nonadrenergic non-cholinergic ganglion cells (to relax the smooth muscle; Costa and Furness, 1979; Gershon, 1982), and pre-synaptic inhibition of the release of acetylcholine (North eta/., 1980; Sanger, 1985). As a result of its plethora of effects, many physiological responses of the gastrointestinal tract have been attributed to 5-HT. These include vagal relaxation of the stomach (Biilbring and Gershon, 1967) and lower oesophageal sphincter (Rattan and Goyal, 1978), post-train synaptic excitation in the myenteric plexus (Dingeldine and Goldstein, 1976), ascending peristaltic excitation in the colon (Furness and Costa, 1973; Costa and Furness, 1976), descending inhibition of vagal excitation (Jule, 1980), cholera toxin-induced intestinal secretion (Cassuto eta/., 1982), and regulation of the migrating myoelectric complex (Ormsbee eta/., 1984; Davidson and Pilot, 1986).

An accurate resolution of the neural actions of 5-HT can be obtained by

Enteric Neuronal 5-HT Receptors 39

studying its effects on single neurones with intracellular microelectrodes. By means of intracellular recording techniques, neurones of the myenteric plexus have been classified by different investigators as types liS; III AH, or NS (see Takaki et al., 1985a; Wood, 1987). Application of 5-HT to single myenteric neurones induces a long-lasting depolarization in type III AH neurones associated with an increase in input resistance (Takaki et al., 1985a; Wood, 1987). During this 'slow response', the afterhyperpolarization that characterizes type III AH neurones is inhibited so that the neurones become hyperexcitable. This effect of 5-HT is not blocked by hexamethonium and is a direct response of the type III AH cells themselves. 5-HT may also evoke a short-lived depolarization of types liS and III AH cells associated with a fall instead of a rise in input resistance. This 'fast response' to 5-HT is substantially shorter in duration than the slow response, enabling a single application of 5-HT to elicit sequentially opposite effects on the input resistance in a single cell. Like the slow response, the fast response to 5-HT is a direct neural action of the amine; moreover, both the fast and slow responses to 5-HT are blocked by receptor desensitization using prolonged exposures to high concentrations of 5-HT itself. Both responses to 5-HT, therefore, are probably mediated by subtypes of a neural5-HT receptor. A hyperpolarization, associated with a decrease in input resistance, is also evoked by application of 5-HT, but is rare and has not yet been fully characterized.

The observation that 5-HT has different actions on single enteric neurones suggests that it is likely to be an oversimplification to assume that there is only a single type of enteric neural 'M' or '5-HT 3' receptor for 5-HT. Moreover, since the ENS contains many different types of neurones (Furness and Costa, 1987), which release a variety of neurotransmitters and affect one another, the introduction of a compound, which like 5-HT is neurally active, can be expected to activate a number of neural pathways in the ENS. The effects of this activation cannot be expected to be fully apparent if one looks only at the final mechanical activity of the smooth muscle, which may be the net effect of a complicated series of actions on the intemeurones that comprise enteric microcircuits. As a result, it is very difficult to interpret data from pharmacological experiments in which 5-HT or its antagonists are applied in an organ bath where they can reach all elements of the ENS simultaneously. It may be no easier to infer the effects of drugs on receptors in the ENS from this type of study than similarly to apply drugs to the brain and infer the effects on CNS receptors by monitoring the output of anterior hom cells. To characterize enteric neural receptors for 5-HT, therefore, it is preferable to study each of the actions of the amine on individual enteric neurones and to correlate electrophysiological results with those derived from radioligand binding assays.

Recently, two dipeptides, N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP) and N-hexanoyl-5-hydroxytryptophyl-5-

40 Serotonin

hydroxytryptophan amide, have been demonstrated to antagonize the slow response of myenteric Type III AH neurones to 5-HT (Takaki et al., 1985a ). 5-HTP-DP is a specific 5-HT antagonist and does not inhibit responses of myenteric Type Ill AH neurones to substance P or muscarinic respones to acetylcholine. 5-HTP-DP also fails to affect fast excitatory post-synaptic potentials (EPSPs), nicotinic responses to acetylcholine, or fast responses to 5-HT (Takaki et al., 1985a). Because it blocked a neural response to 5-HT, 5-HTP-DP was originally considered to be an 'M' receptor antagonist (Gershon et al., 1985; Takaki et al., 1985a); however, benzoyltropine analogues (Fozard, 1984) and indoletropanyl esters (Richardson et al., 1985), which have effects quite different from those of 5-HTP-DP, have also been reported to be 'M' receptor antagonists. These latter compounds block atropine- and tetrodotoxin-sensitive contractile responses of the bowel to 5-HT. While it is thus clear that the site of action of benzoyltropine and indoletropanyl ester antagonists is within the ENS, effects on the contraction of the gut do not reveal whether the particular 5-HT receptor at which these compounds act is the same as or different from that acted upon by 5-HTP-DP. The conclusion that indoletropanyl esters, such as ICS 205-930, identify a single peripheral neural 5-HT receptor, now called '5-HT3' instead of 'M' (Bradley et al., 1986), cannot be adequately established from studies of the contractile effects of 5-HT alone.

It is possible to analyse enteric neural 5-HT receptors by means of radioligand binding techniques. [3H]-5-HT has been employed as a ligand in studies of the ENS (Branchek et al., 1984a; Gershon et al., 1985; Branchek and Gershon, 1987). The binding of [3H]-5-HT to membranes derived from the myenteric plexus is saturable and dissociable with a K0 between 1.4 and 3.7 nM. The dissociation rate constant of the [3H]-5-HT-receptor complex (K-1) is about 0.11/min. The association rate constant (K+1) is about 7.5 X

107/mol per min. Kinetic estimates of the K0 for the binding of [3H]-5-HT, from the ratio K_ 1/K+t. 1.5 DM, is close to that obtained from analysis of saturation isotherms, and the Hill coefficient approximates unity. These data indicate that there is only a single high-affinity [3H]-5-HT binding site in enteric neural membranes without positive or negative co-operativity. The binding of [3H]-5-HT to enteric neural membranes is specific and is not inhibited by agonists and/or antagonists of muscarinic, nicotinic, 0 1 and 0 2 dopamine, a- and 13-adrenergic, H1 and H2 histamine, opiate, and y-aminobutyric acid receptors (Table 5.1). Moreover, many agonists and antagonists at other types of 5-HT receptor, including '5-HT 3' receptors, are unable to antagonize the binding of [3H]-5-HT to enteric neural membranes (Table 5.1). On the other hand, the binding of [3H]-5-HT to enteric neural membranes is antagonized by 5-HTP-DP and by hydroxylated indalpines (Takaki et al., 1985a; Mawe et al., 1986; Branchek and Gershon, 1987; Table 5.1). The high-affinity binding site for [3H]-5-HT in enteric neural membranes, therefore, is different from 5-HT binding sites that have


Table 5.1 Binding of [3H]-5-HT to enteric membranes and its inhibition by potential antagonists Compound [3H]-5-HT 5-0 HIP 6-0HIP 2-Methyl-5-HT 5-HTP-DP ICS 205-930 MDL 72222 lndalpine 5-Carboxamidotryptamine

K0 or Ki (nM) 2.7±0.2 2.1±1.7

10.6±1.7 34.0±10.2

450.0±20 >34 000 >34 000 >34 000 >34 000

Potency (relative to 5-HT) 1.0 0.8 3.9

12.6 166.7

00

00

00

00

The following compounds were inactive: atropine, hexamethonium, metoclopramide, spiperone, domperidone, butaclamol, phentolamine, propranolol, cyproheptadine, diphenhydramine, cimetidine, mianserin, naloxone, codeine, muscimol, cinanserin, (+)-lysergic acid diethylamide, metergoline, 5-methoxytryptamine, methysergide, 6-nitroquipazine, trazodone, zimelidine, ketanserin, pirenperone, 8-hydroxy-2-(di-n-propylamino)tetralin, and RU 24969.

5-0HIP = 5-hydroxyindalpine; 6-0HIP = 6-hydroxyindalpine. previously been described in the CNS and is not the '5-HT3' receptor responsible for actions of 5-HT that can be antagonized by benzoyltropine analogues (Fozard, 1984) and indoletropanyl esters (Richardson et al., 1985). On the other hand, it may well be the receptor responsible for those actions of 5-HT that are antagonized by 5-HTP-DP.

The structure-activity relationship of requirements for [3H]-5-HT binding is not dissimilar to that previously reported for agonists at 5-HT 'M' receptors. For example, hydroxylated tryptamines are known to evoke neurally mediated enteric contractions, while tryptamine itself neither mimics nor antagonizes neurally mediated responses to 5-HT (Gyermek, 1966; Drakontides and Gershon, 1968; Fozard and Mobarak Ali, 1978; Costa and Furness, 1979). Similarly, the binding of [3H]-5-HT to enteric neural membranes is antagonized only by hydroxylated tryptamines and not by analogues in which the ring hydroxyl is masked, or by tryptamine itself (Branchek et al., 1984a; Gershon et al., 1985; Mawe et al., 1986; Branchek and Gershon, 1987). The failure of 'classical' 5-HT antagonists to block [3H]-5-HT binding to enteric neural membranes also correlates with the failure of these same compounds to antagonize neurally mediated responses of the bowel (or other peripheral organs) to 5-HT (Gyermek, 1966; Drakontides and Gershon, 1968; Fozard and Mobarok Ali, 1978; Costa and Furness, 1979; Humphrey et al., 1983). 5-Methoxytryptamine, which has been reported to be a 5-HT 'M' receptor antagonist (Fozard and Mobarok Ali, 1978; but see also Gyermek, 1966), does fail to block the binding of [3H]-5-HT to enteric neural membranes (Branchek et al., 1984a); however, the neural action of 5-methoxytryptamine is a long-lasting depolarization of enteric neurones with a conductance change (increase) the opposite of that

42 Serotonin

evoked in the same cells by 5-HT (Takaki et al., 1985a). 5-Methoxytryptamine thus does not appear to act on enteric neural 5-HT receptors.

Tryptamine has an unusual effect on enteric serotoninergic neurones, which illustrates the need for electrophysiological investigation of drug action. Tryptamine releases endogenous 5-HT from enteric serotoninergic neurones, which activates and then desensitizes neural 5-HT receptors (Takaki et al., 1985b). This release of 5-HT is Ca2+-independent. Neural actions of tryptamine, therefore, are not seen in tissue from which 5-HT has been depleted, and tryptamine can be used to deplete enteric neural stores of 5-HT. The initial effect of tryptamine, to release endogenous 5-HT, illustrates another confounding variable that can complicate analyses of 5-HT receptors if not taken into account. Relatively few studies of pharmacological agents introduced into organ baths measure the release of neurotransmitters by the agents under investigation.

Binding sites for eH]-5-HT have been located in peripheral tissues by dry-mount radioautography (Branchek et al., 1984a). The properties of [3H]-5-HT binding assessed by radioautography are identical to those found by rapid filtration of isolated membranes (Branchek et al., 1984a; Gershon et al., 1985; Mawe et al., 1986; Branchek and Gershon, 1987). The same receptor, therefore, is probably demonstrated by each of these methods. Using radioautography, [3H]-5-HT binding sites have been found in both enteric plexuses and in the lamina propria (Branchek and Gershon, 1987). The [3H]-5-HT binding sites of the lamina propria lie just beneath, but not within, the mucosal epithelium and overlie nerve fibres. The resolution of the radioautographic method does not permit [3H]-5-HT binding sites to be definitely identified; however, there is almost no binding of[3H]-5-HT in the lamina propria of the congenitally aganglionic terminal colon of Is/Is mice (Branchekl et al., 1984b). The aganglionic zone of the Is/Is gut is deficient mainly in intrinsic neurites and contains the enteric projections of neurones the cell bodies of which lie outside the bowel (Payette et al., 1987). These observations suggest that [3H]-5-HT binding sites are present on intrinsic nerve fibres in the lamina propria. A possible role for 5-HT receptors on mucosal axons is to fvnction in the initiation of the peristaltic reflex (Biilbring and Crema, 1959). When pressure is applied to the mucosal surface of the gut, 5-HT is released to the lamina propria from enteroendocrine cells and activates the peristaltic reflex. The location of mucosal [3H]-5-HT binding sites is thus consistent with the hypothesis that they are the physiological5-HT receptors upon which mucosal5-HT acts. The virtual absence of these receptors from the aganglionic region of the bowel of Is/Is mice, in which peristalsis is deficient (Branchek et al., 1984b), further supports this hypothesis.

The structural requirements for antagonism of the binding of [3H]-5-HT correlate well with those needed for compounds to mimic or block the slow

Enteric Neurona/5-HT Receptors 43

response of Type III AH neurones to 5-HT (Takaki et al., 1985a; Mawe et al., 1986). This correlation suggests that displacement of [3H]-5-HT from its binding sites is a good predictor of the activity of potential agonists or antagonists at those 5-HT receptors that mediate the slow response. For example, 5-HTP-DP and hydroxylated indalpines inhibit the binding of [3H]-5-HT, and are respectively antagonists and mimics of the slow response to 5-HT (Takaki eta/., 1985a; Mawe eta/., 1986). On the other hand, ICS 205-930 fails to interfere with the binding of eH]-5-HT and fails to antagonize the slow response to 5-HT, although it is an antagonist of the fast response. 2-Methyl-5-HT at low concentrations mimics the fast response to 5-HT, but at higher concentrations 2-methyl-5-HT is an agonist at the receptor mediating the slow response and an antagonist of the binding of [3H]-5-HT.

These observations suggest that further reclassification of peripheral neural5-HT receptors is indicated, since there is not just one type of 'M' = '5-HT3' receptor. A classification scheme that continues the convention that has been developed for cataloguing 5-HT receptors in the CNS is to name enteric neural 5-HT receptors, 5-HT1p and 5-HT2p. Those CNS 5-HT receptors that can be labelled by [3H]-5-HT are subsets of the 5-HT1 receptor (Pedigo eta/., 1981; Pazos eta/., 1985; Pazos and Palacios, 1985; Yagaloff and Hartig, 1985; Heuring and Peroutka, 1987). Those CNS 5-HT receptors that cannot be labelled by [3H]-5-HT are subsets of the 5-HT2 receptor (Pazos eta/., 1985). The 5-HT2 receptor can instead be labelled by antagonists, such as [3H]-ketanserin (Leysen et a/., 1982). One enteric neural 5-HT ree<:eptor (which mediates the slow response of Type lilAH neurones to 5-HT) can be labelled by [3H]-5-HT; therefore, it may be a subtype of the 5-HT1 receptor and can be called 1P for peripheral. In contrast, the receptor that mediates fast responses to 5-HT cannot be labelled by [3H]-5-HT and so may be a subset of the 5-HT2 receptor (5-HT2p). This receptor can be defined physiologically (with ICS 205-930), although there is not yet a radioligand binding assay for its detection. The 5-HT2p receptor would appear to be identical to what has elsewhere been called the '5-HT3' receptor type (Bradley et al., 1986).

In classifying enteric neural5-HT receptors, it is important to bear in mind that 5-HT is a neurotransmitter in the ENS, and that there are enteric serotoninergic neurones (Gershon, 1982; Furness and Costa, 1987). The only physiological response that has been shown to be mediated by 5-HT in the ENS is a slow EPSP in myenteric Type lilAH neurones (Wood and Mayer, 1979; Erde eta/., 1985; Takaki eta/., 1985a, b). Since the slow EPSP is blocked by 5-HTP-DP and not by ICS 205-930, it is mediated by 5-HT1p

and not 5-HT2p ('5-HT3') receptors (Mawe et al., 1986). At the moment, therefore, no action mediated by 5-HT2p ('5-HT3') receptors has been linked to a normal physiological process in the ENS. The role, if any, of fast responses to 5-HT in the physiology of the bowel has yet to be ascertained. A

44 Serotonin

classification scheme for peripheral neural 5-HT receptors should thus include the subtype (here called 5-HT1p) that can be related to the function of the ENS as well as the multiplicity of receptor subtypes.

ACKNOWLEDGEMENTS

Work presented in this review was supported in part by grants NS 12969, NS 15547, NS 22637 and NS 07062 from the National Institutes of Health. Additional support was provided by the Council for Tobacco Research and the Pharmaceutical Manufacturers' Association Foundation.

REFERENCES

Bayliss, W. M and Starling, E. H. (1899). The movements and innervation of the small intestine. J. Physiol., 24, 99-143

Bradley, P. B., Engel, G., Feniuk, W., Fozard, J. R., Humphrey, P. P. A., Middlemiss, D. N., Mylecharane, E. J., Richardson, B. P. and Saxena, P. R. (1986). Proposals for the classification and nomenclature of functional receptors for 5-hydroxytryptantine. Neuropharmacology, 25, 563-576

Branchek, T. A. and Gershon, M. D. (1987). Development of neural receptors for serotonin in the murine bowel. J. Comp. Neurol., 258, 597-610

Branchek, T., Kates, M. and Gershon, M. D. (1984a). Enteric receptors for 5-hydroxytryptamine. Brain Res., 324, 107-118

Branchek, T., Rothman, T. and Gershon, M.D. (1984b ). Serotonin receptors on the processes of intrinsic enteric neurons: Reduction in the aganglionic bowel of the Is/Is mouse. Soc. Neurosci. Abst., 10, 1097

Biilbring, E. and Crema, A. (1959). The release of 5-hydroxytryptamine in relation to pressure exerted on the intestinal mucosa. J. Physiol., 146, 381--407

Biilbring, E. and Gershon, M.D. (1967). 5-Hydroxytryptamine participation in the vagal inhibitory innervation of the stomach. J. Physiol., 192, 823-846

Cassuto, J., Jodal, M., Tuttle, R. and Lundgren, 0. (1982). 5-Hydroxytryptamine and cholera secretion. Scand. J. Gastroenterol., 17, 695--703

Cohen, M. L., Mason, N., Wiley, K. S. and Fuller, R. W. (1983). Further evidence that vascular serotonin receptors are of the 5-HT2 type. Biochem. Pharmacol., 28, 565--571

Cooke, H. J. (1987). Neural and humoral regulation of small intestinal electrolyte transport. In Johnson, L. R. (Ed.), Physiology of the Gastrointestinal Tract, Vol. 2, Raven Press, New York, 1307-1350

Costa, M. and Furness, J. B. (1976). The peristaltic reflex: an analysis of the nerve pathways and their pharmacology. Naunyn-Schmiederberg's Arch. Pharmacol., 294,47-60

Costa, M. and Furness, J. B. (1979). The sites of action of 5-HT in nerve muscle preparations from guinea-pig small intestine and colon. Br. J. Pharmacol., 65, 237-248

Davidson, H. I. and Pilot, M. A. (1986). Does endogenous neuronal 5-hydroxytryptamine influence canine intestinal motility? J. Physiol., 376, 49P

Enteric Neurona/5-HT Receptors 45

Dingledine, R. and Goldstein, A. (1976). Effect of synaptic transmission blockade on morphine action in the guinea pig myenteric plexus. J. Pharmacal. Exp. Ther., 196, 97-106

Drakontides, A. B. and Gershon, M.D. (1968). 5-Hydroxytryptamine receptors in the house duodenum. Br. J. Pharmacal., 33, 480-492.

Engel, G., Hoyer, D., Kalkman, H. 0. and Wick, M. B. (1984). Identification of 5-HT2-receptors on longitudinal muscle of the guinea-pig ileum. J. Recept. Res., 4, 113-126

Erde, S., Sherman, D. and Gershon, M. D. (1985). Morphology of the serotonergic innervation of physiologically identified cells of the guinea pig myenteric plexus. J. Neurosci., 5, 617-633

Fozard, J. R. (1984). Neuronal 5-HT receptors in the periphery. Neuropharmacology, 23, 1473-1486

Fozard, J. R. and Mobarok Ali, A. T. M. (1978). Receptors for 5-hydroxytryptamine on sympathetic nerves of the rabbit heart. NaunynSchmiedeberg's Arch. Pharmacal., 301, 224-235

Furness, J. B. and Costa, M. (1973). The nervous release and the action of substances which affect intestinal muscle through neither adrenoreceptors nor cholinoreceptors. Phil. Trans. R. Soc. Series B, 265, 123-133

Furness, J. B. and Costa, M. (1987). The Enteric Nervous System, Churchill Livingstone, New York, pp. 65-69

Gaddum, J. H. and Picarelli, Z. P. (1957). Two kinds oftryptamine receptor. Br.J. Pharmacal. Chemother., 12, 323-328

Gershon, M. D. (1967). Effects of tetrodotoxin on innervated smooth muscle preparations. Br. J. Pharmacal., 29, 259-279

Gershon, M. D. (1981). The enteric nervous system. Ann. Rev. Neurosci., 4, 227-272

Gershon, M. D. (1982). Enteric serotonergic neurons. In Osborne, N. (Ed.), Biology of Serotonergic Neurotransmission, Wiley, New York, pp. 363-399

Gershon, M. D., Takaki, M., Tamir, H. and Branchek, T. (1985). The enteric neural receptor for 5-hydroxytryptamine. Experientia, 41, 863-868

Gyermek, L. (1966). Drugs which antagonise 5-hydroxytryptamine and release indolealkylamines. In Erspamer, V. (Ed.), Handbook of Experimental Pharmacology, Vol. 19, 5-Hydroxytryptamine and Related Indolealkylamines, Springer, Berlin, Heidelberg, New York, pp. 471-528

Heuring, R. E. and Peroutka, S. J. (1987). Characterization of a novel 3H-5-hydroxytryptamine binding site subtype in bovine brain membranes. J. Neurosci., 7, 894-903

Humphrey, P. P. A. (1983). Pharmacological characterization of cardiovascular 5-hydroxytryptamine receptors. In Bevan, J. A., Fujiwara, M., Maxwell, R. A., Mohri, K., Shibata, S. and Toda, N. (Eds.), Vascular Neuroeffector Mechanisms: 4th International Symposium, Raven Press, New York, pp. 237-242

Humphrey, P. P. A., Feniuk, W. and Watts, A. D. (1983). Prejunctional effects of 5-hydroxytryptamine on noradrenergic nerves in the cardiovascular system. Fed. Proc., 42, 218-222

Jule, Y. (1980). Nerve-mediated descending inhibition in the proximal colon of the rabbit. J. Physiol., 159, 361-368

Kamikawa, Y. and Shimo, Y. (1983). Indirect action of5-hydroxytryptamine on the isolated muscularis mucosa of the guinea pig oesophagus. Br. J. Pharmacal., 78, 103-110

Langley, J. N. (1921). The Autonomic Nervous System. Part I, Heffers, Cambridge Lew, W. Y. W. and Longhurst, J. C. (1986). Substance P, 5-hydroxytryptamine and

46 Serotonin

bradykinin stimulate abdominal visceral afferents. Am. J. Physiol., 250, R465--R473

Leysen, J. E., Awouters, F., Kennis, L., Laduron, P. M., Vandenberk, J. and Janssen, P. A. J. (1982). ReceptorbindingprofileofR41468, anovelanagonistat 5-HT2 receptors. Life Sci., 28, 1015--1022

Mawe, G. M., Branchek, T. and Gershon, M. D. (1986). Peripheral neural serotonin receptors: Identification and characterization with specific agonists and antagonists. Proc. Nat/. Acad. Sci. U.S.A., 83, 9799-9803

North, R. A., Henderson, C., Katayama, Y. and Johnson, S. M. (1980). Electrophysiological evidence of presynaptic inhibition of acetylcholine release by 5-hydroxytryptamine in the enteric nervous system. Neuroscience, 5, 581-586

Ormsbee, H. S., Silver, D. A. and Hardy, F. E. (1984). Effects of 5-hydroxytryptamine on the migrating myoelectric complex in the canine intestine. J. Pharmacal. Exp. Ther., 231, 436-440

Paintal, A. S. (1964). Effects of drugs on vertebrate mechanoreceptors. Pharmacal. Rev., 16, 341-380

Payette, R. F., Tennyson, V. M., Pham, T. D., Mawe, G. M., Pomeranz, H., Rothman, T. P. and Gershon, M.D. (1987). Origin and morphology of nerve fibers in the aganglionic colon ofthe lethal spotted (Is/Is) mutant mouse. J. Comp. Neurol., 257, 237-252

Pazos, A. and Palacios, J. M. (1985). Quantitative autoradiographic mapping of serotonin receptors in the rat brain. I. Serotonin-! receptors. Brain Res., 346, 205--230

Pazos, A., Cortes, R. and Palacios, J. M. (1985). Quantitative autoradiographic mapping of serotonin receptors in the rat brain. II. Serotonin-2 receptors. Brain Res., 346, 231-249

Pedigo, N. W., Yamamura, H. I. and Nelson, D. L. (1981). Discrimination of multiple [3H)5-hydroxytryptamine binding sites by the neuroleptic spiperone in rat brain. J. Neurochem., 36, 220-226

Pemutka, S. (1984). 5-HTt. receptor sites and functional correlates. Neuropharmacology, 23, 1487-1492

Rattan, S. and Goyal, R. K. (1978). Evidence of 5-HT participation in vagal inhibitory pathway to opossum LES. Am. J. Physiol., 234, E273-E276


Richardson, B. P. and Engel, G., Donatsch, P. and Stadler, P. A. (1985). Identification of serotonin M-receptor subtypes and their specific blockade by a new class of drugs. Nature, 316, 126-131

Sanger, G. J. (1985). Three different ways in which 5-hydroxytryptamine can affect choline activity in guinea-pig isolated ileum. J. Pharm. Pharmacal., 37, 584-586

Takaki, M., Branchek, T., Tamir, H. and Gershon, M. D. (1985a). Specific antagonism of enteric neural serotonin receptors by dipeptides of 5-hydroxytryptophan: Evidence that serotonin is a mediator of slow synaptic excitation in the myenteric plexus. J. Neurosci., 5, 1769-1780

Takaki, M., Mawe, G. M., Barasch, J. and Gershon, M.D. (1985b). Physiological responses of guinea-pig myenteric neurons secondary to the release of endogenous serotonin by tryptamine. Neuroscience, 16, 223-240

Trendelenburg, P. (1917). Physiolische und pharmakolische versuche uber die diinndarmperistaltik. Naunyn-Schmiedeberg's Arch. Exp. Pathol. Pharmakol., 81, 55--129

Wood, J. D. (1987). Physiology of enteric neurons. In Johnson, L. R. (Ed.), Physiology of the Gastrointestinal Tract, Vol. 1, 2nd edn, Raven Press, New York, pp. 1-41


Wood, J. D. and Mayer, C. J. (1979). Serotonergic activation of tonic-type enteric neurons in guinea pig small bowel. J. Neurophysiol., 422, 582-593

Yagaloff, K. A. and Hartig, P.R. (1985). 1251-Lysergic acid diethylamide binds to a novel serotonergic site of rat choroid plexus epithelial cells. J. Neurosci., 5, 3178-3183

6 Pre-synaptic 5-HT Receptors Mediating Inhibition of Transmitter Release from

Peripheral Cholinergic and Noradrenergic Nerves

D. E. Clarke, R. A. Bond, K. G. Charlton and D. R. Blue

Department of Pharmacology, University of Houston, Houston, TX 77004, USA

INTRODUCTION

5-Hydroxytryptamine (5-HT) can inhibit acetylcholine {ACh) and noradrenaline (NA) release from certain peripheral cholinergic and noradrenergic nerves, but the receptor site or sites involved have not been defined fully.

CHOLINERGIC NERVES

Inhibition of cholinergic neurotransmission by 5-HT has been studied on a quantitative basis almost exclusively in the gastrointestinal tract. Electrophysiological, biochemical and functional studies support the idea that 5-HT can activate pre-synaptic inhibitory receptors located on the enteric cholinergic nerves to inhibit the action potential-induced release of ACh {for review, see Fozard, 1984).

Evidence for a pre-synaptic inhibitory action of 5-HT on ACh release from guinea-pig myenteric neurones has been provided by North et al. {1980) in electrophysiological studies and by Kilbinger and PfeufferFriederich {1985) in biochemical studies. The latter workers labelled cholinergic nerves with [3H)-choline and measured [3H)-ACh release following electrical stimulation. 5-HT inhibited [3H)-ACh release in a concentration- and frequency-dependent manner. This inhibitory effect of 5-HT did not exhibit desensitization (over a 30 min period) and was resistant to a-adrenoceptor blockade but was antagonized by methiothepin and

Peripheral Pre-synaptic Inhibitory 5-HT Receptors 49

methysergide with apparent pAz values of 7.6 and 7.0, respectively. (+)-Lysergic acid diethylamide (LSD) was found to be about 10 times more potent than 5-HT at inhibiting [3H)-ACh release and was also antagonized by methiothepin with a comparable apparent pAz value of 8.0 (PfuefferFriederich and Kilbinger, 1985). However, there was no clear correlation between inhibition of [3H)-ACh release by 5-HT and (+)-LSD, and inhibition of the functional response to electrical stimulation of enteric cholinergic neurones (the cholinergically mediated 'twitch' response). Earlier workers (Gintzler and Musacchio, 1974; Sanger, 1985) demonstrated inhibition of the 'twitch' response with 5-HT but a concentration dependency for 5-HT has been difficult to establish. One reason for this difficulty is that the inhibitory action of 5-HT and (+)-LSD on ACh release, although potent, is likely to be obscured and/or confounded functionally by the neuroexcitatory and smooth-muscle actions of the agonists ( PfeufferFrederich and Kilbinger, 1985; Sanger, 1985; Richardson and Engel, 1986; Gunning and Humphrey, 1987).

Fozard and Kilbinger (1985) avoided this problem by using the selective 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-0H-DPAT). Figure 6.1(A) illustrates the inhibitory effect of 8-0H-DPAT on the 'twitch' response. The first phase ofthe curve, up to 1 J.tM, but not the second phase, is attributed to 5-HT1A agonism by 8-0H-DPAT, as it is antagonized by metergoline (apparent pAz = 7.71), methiothepin (7.58), (- )-pindolol (7.20), spiperone (7.58), buspirone (7.39), (-)-MDL 72832 (9.90) and urapidil (7.30), but not ketanserin (1 J.tM), MDL 72222 (1 J.tM), ( + )-pindolol (1 J.tM), prazosin (0.01 J.tM) and idazoxan (0.5 J.tM) (Fozard and Kilbinger, 1985; Fozard et al., 1987; Fozard and Mir, 1987). A pre-synaptic site of action for 8-0H-DPAT is indicated because 8-0H-DPAT inhibited [ 3H]ACh output but failed to inhibit contractile responses to carbachol and histamine (Fozard and Kilbinger, 1985).

The conclusion drawn from the above data is that a 5-HT1A receptor is involved (Fozard and Kilbinger, 1985). However, the functional classification of 5-HT receptors, without using 5-HT itself, or an indole-based analogue, is not totally convincing. Some indole-based agonists have been studied by Hagenbach et al. (1986), but the potential for interference from other 5-HT receptors in the ileum was not taken into account. Furthermore, electrical field stimulation may evoke the release of 5-HT (Costall et al., 1986). Therefore, investigations were carried out in our laboratory using 5-HT and the potent 5-HTrlike receptor agonist 5-carboxamidotryptamine (5-CT), in proximal ileum preparations from reserpine-treated Hartley guinea-pigs (325-425 g; 30-40 days old). In order to limit agonism to the putative 5-HT lA receptor, pre-synaptic o.-adrenoceptors were blocked with phentolamine (3 J.tM) and 5-HT3 receptors were inhibited with ICS 205-930 (3 J.tM; Richardson and Engel, 1986). 5-HTz, 5-HT1c (if present), and smooth-muscle 'relaxant' receptors for 5-HT (Feniuk et al., 1983; Kalkman

50 Serotonin

-100 A 8 w ft) z -90 0 II. ft) -80 w It

~ -70 CJ

i -80 t-iii!: -so -20 w CJ z -40 c :z: CJ w -30 CJ c .... -20 z w CJ -10 It w II.

0 0 9 8 7 8 5 4 10

+10 CONCENTRATION (-log M)

Figure 6.1 Effect of agonists on the 'twitch' response in isolated segments of proximal ileum from guinea-pigs. (A) 8-0H-DPAT in Tyrode solution (n = 85), Fozard (personal communication) (0-0); 8-0H-DPAT in Krebs solution (n = 6), this study ( ........ ). (B) Comparative experiments in Krebs solution containing phentolamine (3 f.I.M), ICS 205-930 (3 f.I.M), mesulergine (3 f.I.M), cocaine (30 f.I.M) and ascorbic acid (100 f.I.M), using proximal ileum (30-40 em from the ileo-caecal junction) from reserpine-treated (5 mglkg, i.p. for 18 h) guinea-pigs: WY48 723 (0-0), 5-CT (._.); 8-0H-DPAT (0-0); buspirone (..._.); 5-HT (.6.-.6.). Cumulative concentration-effect curves were made with indoles, and noncumulative curves (single drug additions) with non-indoles. Single drug additions were made every 30 min, followed by washes at 10 min intervals to offset desensitization. Each point is the mean value of 3-6 experiments ± s.e. mean. The

numbers in parentheses give relative IC10 values (WY48 723 = 1)

eta/., 1986) were inhibited with mesulergine {3 !JM; Kalkman eta/., 1986; Richardson and Engel, 1986). The results are shown in Figure 6.1(B), as are concentration-effect curves for 8-0H-DPAT, buspirone, and the new buspirone analogue WY48 723. The relative IC10 values are: WY48 723 {1), 5-CT (3), 8-0H-DPAT (7), buspirone {70) and 5-HT {840). None of the compounds (up to 1 f.IM) inhibited contractile responses to histamine. Responses to buspirone showed considerable fade and maximum inhibitions are shown. WY48 723, 5-CT and 8-0H-DPAT were antagonized in a competitive manner by buspirone {0.5 f.IM) to give apparent pA2 values of 7.25, 7.03 and 7.23, respectively, indicating a common site of action. Quantitative analysis of the interaction between 5-HT and buspirone was


precluded because of the low relative potency of 5-HT and its apparent dual action on the 'twitch' response (see Figure 6.1(B)). Finally, a selectivity of action for buspirone (0.5 !J.M) was demonstrated because it failed to antagonize the agonistic action of UK14, 304-18 at inhibitory pre-synaptic a 2-adrenoceptors (these experiments were performed in the absence of phentolamine).

Employing current criteria and nomenclature (Bradley et al., 1986), the overall findings from our own studies and those of others are consistent with a pre-synaptic 5-HT1A receptor mediating inhibition of ACh release from enteric cholinergic nerves of the guinea-pig. Such a receptor site fits certain of the physiological and pathophysiological actions of 5-HT in the intestine (Gershon et al., 1983), and from an experimental viewpoint may serve as a test model for the evaluation of new 5-HT lA agonists and antagonists. WY 48 723 is an interesting example in this regard. However, the low intrinsic activity of agonists, the absence of an apparent singular response to 5-HT, and the observation that non-indoles may exhibit a variable degree of autoinhibition (see Figure 6.1) limits usefulness. Futhermore, responsiveness varies along the length of the ileum (Costa and Furness, 1979).

NORADRENERGIC NERVES

Evidence for the presence of inhibitory pre-synaptic receptors for 5-HT on peripheral noradrenergic nerves has been reviewed (Fozard, 1984; Humphrey, 1984; Bradley et al., 1986), and additional investigations have now been published (Su and Uruno, 1985; Docherty and Warnock, 1986; Gothert et al., 1986a, b; Moritoki et al., 1986; Molderings et al., 1987). These receptor sites for 5-HT, which function to inhibit the action potentialinduced release of NA, are distributed widely, being claimed on the noradrenergic innervation to several organs and tissues: the saphenous vein of dog and man; the rat kidney; the rat vena cava; the heart of dog, guinea-pig and rat; the rat vas deferens; the rat mesenteric blood vessels; and the femoral arterial bed of dogs. Table 6.1 summarizes findings with agonists and antagonists at the inhibitory pre-synaptic receptor site. The greater potency of 5-CT versus 5-HT, the antagonism by methiothepin, metergoline and methysergide (a partial agonist), and the lack of antagonism with ketanserin, cyproheptadine, mesulergine and metoclopramide, indicates a 5-HT1-like receptor. However, many of the compounds listed as inactive in Table 6.1 rule out the involvement of the 5-HT1A,

5-HT18 and 5-HT1c subtypes, as well as a-adrenoceptors, and a possible prostaglandin involvement ( Bradley et al., 1986; Charlton et al., 1986). Therefore, the receptor is defined by exclusion from currently designated 5-HT receptors; no specific agonist or antagonist has been identified. However, it should be noted that Molderings et al. (1987) claim that the

52 Serotonin

pre-synaptic inhibitory receptor to 5-HT corresponds to the 5-HT 1B subtype in rat vena cava, as evidenced by correlations of functional and ligand binding data.

Table 6.1 also indicates agreement between functional data and data derived from the binding study of Heuring and Peroutka (1987). These authors describe a novel binding site ( 5-HT 10) for [3H]-5-HT in bovine brain which is 5-HTrlike but is clearly distinct from the 5-HT1A, 5-HTm and 5-HT 1c binding sites (compare Charlton et al., 1986). Therefore, as with the functional receptor, the 5-HT10 binding site is defined by exclusion from currently designated sites. However, the binding site and the functional receptor do not correspond exactly. The displacement of specific [3H]-5-HT binding from the 5-HT10 site is sensitive to rauwolscine (Ki = 47 nM; Peroutka, personal communication) but rauwolscine (0.1-10 ~tM) is an ineffective antagonist against 5-HT-induced inhibition of NA release in the human saphenous vein (Gothert et al., 1986b ), the rat vena cava (Gothert et al., 1986a), the rat vas deferens (Docherty and Warnock, 1986) and in the isolated perfused rat kidney (Clarke eta/., unpublished observations). Both rauwolscine and yohimbine (Ki = 59 nM at the 5-HT10 binding site) are potent antagonists towards 5-HT in the rat stomach fundus (Clineschmidt et al., 1985), but the stomach fundus differs from the 5-HT 10 binding site with respect to some other compounds (e.g. its high affinity for mchlorophenylpiperazine).

Table 6.1 Pharmacological profile at the putative inhibitory 5-HT 1-like receptor on peripheral noradrenergic nerves0

1. Compounds claimed as agonists: 5-CT, b 5-HT,RU 24969, 5-methoxytryptamine, N,N-dimethyltryptamine, tryptamine, 5-aminotryptamine, 5-hydroxykynuramine, c,d methysergide

2. Compounds claimed as antagonists: Methiothepin, metergoline, methysergide, quipazine, MK 212

3. Compounds inactive at 111M (functional studies) or with a Ki > 111M (binding studies): 8-0H-DPAT, ipsapirone, c m-chlorophenylpiperazine, c kynuramine, c,d trazodone,c buspirone,C cyproheptadine, ketanserin, mesulergine, (-)-propranolol, ( ±)-pindolol, ( ± )-cyanopindolol, metoclopramide, spiroperidol, phentolamine, rauwolscine, c indomethacin

"For references, see text. b5-CT is more potent than 5-HT. clsolated perfused rat kidney (unpublished observations). dPeroutka, personal communication.

Italic type indicates agreement between data from functional studies and the ligand binding studies of Heuring and Peroutka ( 1987).


CONCLUSIONS

The pharmacological evidence suggests that inhibitory pre-synaptic receptors for 5-Hf differ on peripheral cholinergic nerves from those on peripheral noradrenergic nerves. The former receptors appear to be of the 5-Hf tA subtype, whereas the latter receptors, also of the 5-Hf 1-like group, have not been named specifically. This may be wise, as the method of classifying receptors on the basis of antagonist equilibrium dissociation constants and agonist potency ratios undergoes re-evaluation (Leff eta/., 1986, 1987). The multiplicity of 5-Hf receptor subtypes has grown in proportion to the production of synthetic agonists and antagonists. The molecular size of these compounds is invariably larger than that of 5-Hf and tryptamine (the endogenous ligands), and as a consequence the synthetic molecules may recognize not only the receptor itself but binding sites located within the domain of the receptor (accessory sites). Such sites may vary depending upon cell type, thereby creating an apparent multiplicity of receptors. In addition, allosteric effectors, and the age of the 5-Hf receptor in its membrane life cycle, may influence affinity or efficacy or both. Consideration of these factors, and the measurement of the equilibrium dissociation constants of 5-Hf, tryptamine and other small indole-based analogues for receptor classification (Leff et al., 1986, 1987) may go a long way towards clarifying the field of 5-Hf receptors.

ACKNOWLEDGEMENTS

This work was supported by NIH Grant NS 24871. Dr Gary Stack of Wyeth Laboratories Inc. synthesized and supplied WY48 723.

REFERENCES


Charlton, K. G., Bond, R. A. and Clarke, D. E. (1986). An inhibitory pre junctional 5-IIT 1-like receptor in the isolated perfused rat kidney: Apparent distinction from the 5-IIT1A, 5-IIT18 and 5-IIT1c subtypes. Naunyn-Schmiedeberg's Arch. Pharmacal., 332, 8-15

Clineschmidt, B. V., Reiss, D. R., Pettibone, D. J. and Robinson, J. L. (1985). Characterization of 5-hydroxytryptamine receptors in rat stomach fundus. J. Pharmacal. Exp. Ther., 235, 696-708

Costa, M. and Furness, J. B. (1979). The sites of action of 5-hydroxytryptamine in nerve-musele preparations from the guinea-pig small intestine and colon. Br. J. Pharmacal., 65, 237-248

54 Serotonin

Costall, B., Gunning, S. J., Naylor, R. J. and Tattersall, F. D. (1986). Modification of electrical field stimulation-induced contractions in the guinea-pig ileum by metoclopramide and ICS 205-930 depends on the integrity of the mucosa. J. Pharm. Pharmacol., 38, 811-814

Docherty, J. R. and Warnock, P. (1986). Prejunctional inhibitory 5-hydroxytryptamine (5-HT) receptors in rat heart and vas deferens. Br. J. Pharmacol., 89, 843P

Feniuk, W., Humphrey, P. P. A. and Watts, A. D. (1983). 5-Hydroxytryptamineinduced relaxation of isolated mammalian smooth muscle. Eur. J. Pharmacol., 96, 71-78

Fozard, J. R. (1984). Neuronal 5-HT receptors in the periphery. Neuropharmacology, 23, 1473-1486

Fozard, J. R. and Kilbinger, H. (1985). 8-0H-DPAT inhibits transmitter release from guinea-pig enteric cholinergic neurons by activating 5-HT lA receptors. Br. J. Pharmacol., 86, 601P

Fozard, J. R. and Mir, A. K. (1987). Are 5-HT receptors involved in the antihypertensive effects of urapidil? Br. J. Pharmacol., 90, 24P

Fozard, J. R., Hibert, M., Kidd, E. J., Middlemiss, D. N., Mir, A. K. and Tricklebank, M. D. (1987). MDL 72832: A potent, selective and stereospecific ligand for 5-HT1A receptors. Br. J. Pharmacol., 90, 273P

Gershon, M.D., Sherman, D., Erde, S.M. and Rothman, T. P. (1983). In Chey, W. Y. (Ed.), Functional Disorders of the Digestive Tract, Raven Press, New York, pp. 59-77

Gintzler, A. Rand Musacchio, J. M. (1974). Interaction between serotonin and morphine in the guinea-pig ileum. J. Pharmacol. Exp. Ther., 189, 484-492

Gothert, M., Schlicker, E. and Kollecker, P. (1986a). Receptor mediated effects of serotonin and 5-methoxytryptamine on noradrenaline release in the rat vena cava and in the heart of the pithed rat. Naunyn-Schmiedeberg's Arch. Pharmacol., 332, 124-130

Gothert, M., Kollecker, P., Rohm, N. and Zerkowski, H. R. (1986b). Inhibitory presynaptic 5-hydroxytryptamine ( 5-HT) on the sympathetic nerves of the human spahenous vein. Naunyn-Schmiedeberg's Arch. Pharmacol., 332, 317-323

Gunning, S. J. and Humphrey, P. P. A. (1987). Evidence for a 5-HTrreceptor mediated release of an inhibitory transmitter in guinea-pig isolated ileum. Br. J. Pharmacol., 91, 359P

Hagenbach, A., Hoyer, D., Kalkman, H. 0. and Seiler, M. P. (1986). N,N-Dipropyl-5-carboxamidotryptamine (DP-5CT), an extremely potent and selective 5-HT1A agonist. Br. J. Pharmacol., 87, 136P


Humphrey, P. P. A. (1984). Peripheral 5-hydroxytryptamine receptors and their classification. Neuropharmacology, 23, 1503-1510

Kalkman, H. 0., Engel, G. and Hoyer, D. (1986). Inhibition of 5-carboxamidotryptamine-induced relaxation of guinea-pig ileum correlates with P25I] LSD binding. Eur. J. Pharmacol., 129, 139-145

Kilbinger, H. and Pfeuffer-Friederich, I. (1985). Two types of receptors for 5-hydroxytryptamine on the cholinergic nerves of the guinea-pig myenteric plexus. Br. J. Pharmacol., 85, 529-539

Left, P., Martin, G. R. and Morse, J. M. (1986). The classification of peripheral 5-HT2-like receptors using tryptamine agonist and antagonist analogues. Br. J. Pharmacol., 89, 493-499


Leff, P., Martin, G. R. and Morse, J. M. (1987). Differential classification of vascular smooth muscle and endothelial cell 5-Hf receptors by use of tryptamine analogues. Br. J. Pharmacal., 91, 321-332

Molderings, G. J., Fink, K., Schlicker, E. and Gothert, M. (1987). Inhibition of noradrenaline release in rat vena cava via presynaptic 5-Hf18 receptors. Naunyn-Schmiedeberg's Arch. Pharmacal., 336, 245-250

Moritoki, H., Iwamoto, T., Kanaya, J., Ishida, Y. and Fukuda, H. (1986). Age-related change in serotonin-mediated prejunctional inhibition of rat vas deferens. Eur. J. Pharmacal., 131, 39-46

North, R. A., Henderson, G., Katayama, Y. and Johnson, S. M. (1980). Electrophysiological evidence for presynaptic inhibition of acetylcholine release by 5-hydroxytryptamine in the entric nervous system. Neuroscience, 5, 581-586

Pfueffer-Friederich, I. and Kilbinger, H. (1985). The effects of LSD in the guinea-pig ileum. Inhibition of acetylcholine release and stimulation of smooth muscle. Naunyn-Schmiedeberg's Arch. Pharmacal., 331, 311-315


Sanger, G. J. (1985). Three different ways in which 5-hydroxytryptamine can affect cholinergic activity in the guinea-pig isolated ileum. J. Pharm. Pharmacal., 37, 584-586

Su, C. and Uruno, T. (1985). Excitatory and inhibitory effect of 5-hydroxytryptamine in mesenteric arteries of spontaneously hypertensive rats. Eur. J. Pharmacal., 106, 283-290

7 5-HT Receptors Mediating Pre-synaptic

Autoinhibition in Central Serotoninergic Nerve Terminals

M. Gothert

Institute of Pharmacology and Toxicology, Rheinische Friedrich-Wilhelms Universitiit Bonn, ReuterstraBe 2b, D-5300 Bonn, Federal Republic of

Germany

INTRODUCTION

Pre-synaptic 5-HT autoreceptors are receptors which are located on (or at least close to) the axon terminals of serotoninergic neurones. Accordingly, they can be activated by serotonin (5-hydroxytryptamine; 5-HT) released from those terminals on which they are located. It is compatible with this location that 5-HT autoreceptors probably play a physiological role in fine regulation of 5-HT release (and of 5-HT synthesis: Hamon et al., 1973; Sawada and Nagatsu, 1986).

Most of the studies designed to provide evidence for the existence of such autoreceptors in the CNS and to determine their function were carried out on isolated preparations of the brain (mainly cortex, hippocampus, hypothalamus, cerebellum) or spinal cord, in which stimulation-evoked overflow of 5-HT (or, in most cases, radiolabelled compounds after incubation with [3H)-5-HT) into the superfusion or incubation fluid was determined. 5-HT receptor-mediated effects found in such preparations suggest a pre-synaptic site of action, provided that (a) the preparation exclusively contains 5-HT axon terminals but not their cell bodies, and (b) indirect trans-synaptic modulation (comprising glia or non-5-HT -containing nerve terminals or cell bodies) and modulation via short interneurones are ruled out (e.g., by experiments on synaptosomes). Virtually all brain regions and the spinal cord are innervated by serotoninergic axon terminals, the cell bodies of which are mainly located in the raphe nuclei of the midbrain; such cell bodies occur to a minor extent in the nucleus reticularis

CNS Pre-synaptic Inhibitory 5-HT Autoreceptors 57

paragigantocellularis and nucleus pontis oralis of the brain-stem, from which the axons innervating the rat cerebellum originate.

In the present article, the literature on the identification, function and pharmacological characterization of pre-synaptic 5-HT autoreceptors will be briefly and very selectively reviewed (for previous reviews, see Gothert, 1982; Moret, 1985). Furthermore, the physiological and potential therapeutic implications of autoreceptor-mediated modulation of 5-HT release will be briefly touched upon. This report will be based to a certain extent on the contributions of my laboratory to the present topic; some new unpublished results will be incorporated.

PRE-SYNAPTIC S-HT AUTORECEPTORS IN THE RAT

Identification, Function and Location

Basic evidence for the existence of inhibitory pre-synaptic 5-HT autoreceptors in the CNS was mainly obtained in slices and synaptosomes prepared from various regions of the rat brain and spinal cord. Thus, it was shown in cortical slices that 5-HT (and other non-selective 5-HT receptor agonists) inhibit the electrically evoked release of [3H]-5-HT; this effect was competitively blocked by the non-selective 5-HT receptor antagonist methiothepin, suggesting that the agonist(s) and the antagonist act at the same sites, namely pre-synaptic 5-HT autoreceptors. In agreement with this interpretation, methiothepin given alone disinhibited, i.e. increased, [3H]-5-HT release by preventing endogenous 5-HT from activating the autoreceptors (Gothert and Weinheimer, 1979; Gothert, 1980; Baumann and Waldmeier, 1981; Gothert and Schlicker, 1983).

Evidence for the assumption that the receptors involved are located pre-synaptically in an anatomical sense was derived from experiments in cortical slices superfused with solution containing tetrodotoxin, i.e. under a condition in which no impulse flow occurs along nerve axons of intemeurones, for example. Since the above-mentioned compounds still produced their typical effects on the [3H]-5-HT release evoked by reintroduction of Ca2+ after superfusion with K+ -rich, Ca2+ -free solution, location of the release-modulating 5-HT receptors on intemeurones is ruled out (Gothert and Weinheimer, 1979). Experiments on cortical synaptosomes (i.e. resealed pinched-off nerve terminals) provide more direct evidence for the location of the 5-HT receptors on the serotoninergic nerve terminals themselves (Raiteri et al., 1984); the fact per se that K+ -evoked [3H]-5-HT release was inhibited by 5-HT receptor agonists in a manner sensitive to blockade by methiothepin almost excludes any other interpretation.

By means of these techniques, inhibitory pre-synaptic 5-HT autorecep-

58 Serotonin

tors have been identified not only in the cortex but also in various other regions of the rat CNS. such as the hippocampus (Maura eta/., 1986), striatum (Middlemiss. 1984a). hypothalamus (Cerrito and Raiteri, 1979; Langer and Moret. 1982). cerebellum (Bonanno et a/., 1986), medulla oblongata (area of the nucleus tractus solitarii; Schlicker eta/., 1988), and the spinal cord (Monroe and Smith, 1985).

Pharmacological Characterization

In order to determine the receptor type and subtype to which the pre-synaptic 5-HT autoreceptors belong, a large number of 5-HT receptor agonists and antagonists have been investigated. The 5-HTrlike character of these receptors could be established by the finding that not only methiothepin but also metergoline, another antagonist with affinity for both 5-HT1-like and 5-HT2 receptors, blocked the inhibitory effect of 5-HT on [3H)-5-HT release in rat cortical slices (Engel eta/., 1983).

Since only a few of the classical, relatively non-selective 5-HT receptor antagonists are capable of blocking the pre-synaptic 5-HT autoreceptors in the rat brain cortex and hypothalamus (Martin and Sanders-Bush, 1982; Engel et al., 1983), the observation that the latter receptors are stereoselectively blocked by the ~-adrenoceptor antagonist propranolol (Middlemiss, 1984b) was important. This property was shared by various other ~-adrenoceptor blockers, such as pindolol and cyanopindolol, but not by atenolol and ICI 118,551. Cyanopindolol is the most potent 5-HT autoreceptor antagonist yet described (Schlicker eta/., 1985a); according to recent data, it may possess partial agonist activity (Maura et al., 1987). The potencies of 5-HT receptor agonists in inhibiting eHJ-5-HT release and the potencies of 5-HT receptor antagonists (including the above-mentioned f3-adrenoceptor blockers) in competitively antagonizing the inhibitory effect of 5-HT were compared with their affinities for 5-HT1A, 5-HT18, 5-HT1c and 5-HT2 binding sites (Engel et al., 1986): correlation was best with 5-HT18 sites. Considerable progress in the classification of the 5-HT autoreceptor was brought about by drugs acting relatively selectively at certain 5-HT receptor types and subtypes. In support of the proposed 5-HT 18 character of the pre-synaptic 5-HT autoreceptors, it was found that 8-hydroxy-2-(di-n-propylamino )tetralin (8-0H-DPAT; Middlemiss, 1984a) and ipsapirone, both of which are preferential 5-HT lA receptor agonists, and spiperone, an antagonist at 5-HT1A but not 5-HT18 or 5-HT1c receptors, are ineffective, whereas the preferential5-HT18 receptor agonist CGS 12066B (Neale et al., 1987) inhibited [3H)-5-HT release. In this context, it is of interest to note that the somadendritic 5-HT autoreceptors in the rat midbrain raphe nuclei appear to belong to the 5-HT1A receptor subtype (Sprouse and Aghajanian, 1986).


Mechanism of Action

Little is known about the potential mechanism underlying 5-HT autoreceptor-mediated modulation of 5-HT release. Modification of Ca2+ availability for stimulus-release coupling in depolarized varicosities appears to play an important role in this event (Gothert, 1980; Langer and Moret, 1982). Alteration of Ca2+ influx via individual voltage-dependent Ca2+ channels rather than a change in the number of activated Ca2 + channels or varicosities seems to be involved. However, modification of the intraneuronal effects of Ca2+ or of its inactivation cannot be ruled out (Gothert, 1980). The decrease in availability of Ca2+ ions may also explain the feedback inhibition of 5-HT synthesis; the calcium-calmodulindependent mechanism activating tryptophan hydroxylase is probably inhibited in this situation (Sawada and Nagatsu, 1986).

The events between autoreceptor activation and the changes in Ca2+ availability are unknown. These receptors seem not to be coupled to the adenylate cyclase system of the serotoninergic nerve terminals, since the release-modulating effects of 5-HT and methiothepin in cortical slices were not modified in the presence of forskolin plus an inhibitor of phosphodiesterase (Schlicker et al., 1987).

Autoret:eptor Function In Vivo

Evidence has been obtained by different techniques that pre-synaptic 5-HT autoreceptors are operative in the rat brain in vivo. In an electrophysiological study, the effectiveness of stimulation of the ascending 5-HT pathway on the firing activity of post-synaptic hippocampal pyramidal neurones was measured. Facilitation of serotoninergic synaptic transmission, probably due to pre-synaptic 5-HT autoreceptor blockade, was observed: the responsiveness (i.e. suppression of firing activity) of the pyramidal neurones to electrical stimulation of afferent 5-HT fibres was increased by methiothepin but not by direct application of 5-HT to the pyramidal neurones (Chaput et al., 1986). In another approach, differential pulse voltammetry was applied (Brazell et al., 1985). The interpretation of such experiments was based on the assumption that the brain concentration of 5-hydroxyindoleacetic acid (5-HIAA) reflects neuronal5-HT release. The 5-HIAA signal in the frontal cortex and suprachiasmatic nucleus was decreased by the 5-HTrlike receptor agonist RU 24969, in a manner sensitive to blockade by metergoline and methiothepin but not ketanserin. However, it should be noted that a non-pre-synaptic site of action was not completely excluded by these experiments.

60 Serotonin

PRE-SYNAPTIC 5-HT AUTORECEPTORS IN OTHER SPECIES

Pre-synaptic 5-HT autoreceptor function has been demonstrated in the cat caudate nucleus in situ by means of the push-pull cannula technique: K+ -evoked [3H)-5-HT release was found to be inhibited by lysergic acid diethylamide (Hery et al., 1979). Such autoreceptors have also been identified by in vitro experiments in slices and synaptosomes of other brain areas of several species: the mouse cerebellum (Figueroa et al., 1985); the rabbit cortex (Umberger et al., 1986), hippocampus (Feuerstein et al., 1987) and hypothalamus (Verbeuren et al., 1984); and the human cortex (Schlicker et al., 1985b ). Since no 5-HT 18 binding sites were found in human cortical membranes, the possibility that the pre-synaptic 5-HT autoreceptors in the CNS of man may belong to another 5-HT receptor (sub )type has to be considered. Therefore, the question of an appropriate model of the human CNS with respect to its pre-synaptic 5-HT autoreceptors arises. Since the pig brain also appears to be devoid of 5-HT18 binding sites, it was of interest to investigate whether pre-synaptic 5-HT 1 autoreceptors can also be identified in the CNS of this species. In fact, it was recently found in my laboratory that in both slices and synaptosomes of the pig brain cortex, 5-HT inhibits electrically and K+ -evoked [3H)-5-HT release, respectively, an effect which is antagonized by methiothepin but not ketanserin. In slices, methiothepin given alone facilitates [3H)-5-HT release (Fink et al., unpublished observations). Work is in progress at present to determine the 5-HT 1 receptor subtype involved in this autoreceptor-mediated modulation of 5-HT release.

PHYSIOLOGICAL AND POTENTIAL THERAPEUTIC IMPLICATIONS

Pre-synaptic 5-HT autoreceptors probably play a role in local fine regulation of 5-HT release via a short negative-feedback loop. In this way, local adaptation to changes of 5-HT concentration in the vicinity of a given nerve terminal is made possible. Such changes may be caused, for example, by local modification of the inactivation of 5-HT previously released into the synaptic cleft. Owing to their role in fine regulation of 5-HT release, these autoreceptors may be assumed to be capable of modulating any CNS function influenced by serotoninergic neurones; Accordingly, they may be involved in the control of appetite, prolactin release, body temperature, blood pressure, perception of pain, and emotional behaviour, for example. Furthermore, it is evident from these considerations that 5-HT autoreceptors may play a role in the pathogenesis of diseases in which these functions are disturbed, such as depression (see Gothert, 1986) or hormonal dysfunctions, and that they may become targets of newly developed drugs for the treatment of such diseases.


As an example, it is conceivable that depression may be related to a decreased serotoninergic neurotransmission, which, in turn, may be due to an increased number and/or affinity of pre-synaptic 5-HT autoreceptors. Newly developed, highly potent and selective autoreceptor antagonists may be expected to facilitate 5-HT release, and this may lead to considerable progress in antidepressant therapy (Gothert, 1986).

5-HT autoreceptor agonists may be developed as a new class of antihypertensive drugs. This concept is based on the observation that an increase in the activity of serotoninergic neurones produces a rise in blood pressure, probably by increasing 5-HT release in the relevant brain regions. Accordingly, 5-HT autoreceptor activation may be assumed to decrease blood pressure by inhibiting 5-HT release ( Gothert, 1985). In this context, it should be noted that modulation via pre-synaptic 5-HT 1 autoreceptors in the brain of spontaneously hypertensive rats is not different from that in normotensive rats (Schlicker et al., 1988).

ACKNOWLEDGEMENTS

The author's own work on pre-synaptic 5-HT autoreceptors was supported by a grant from the Deutsche Forschungsgemeinschaft.

REFERENCES

Baumann, P. A. and Waldmeier, P. C. (1981). Further evidence for negative feedback control of serotonin release in the central nervous system. NaunynSchmiedeberg's Arch. Pharmaco/., 317, 36-43

Bonanno, G., Maura, G. and Raiteri, M. (1986). Pharmacological characterization of release-regulating serotonin autoreceptors in rat cerebellum. Eur. J. Pharmaco/., 126, 317-321

Brazell, M.P., Marsden, C. C., Nisbet, A. P. and Routledge, C. (1985). The 5-HT1 receptor agonist RU-24%9 decreases 5-hydroxytryptamine (5-HT) release and metabolism in the rat frontal cortex in vitro and in vivo. Br. J. Pharmaco/ .• 86. 209-216

Cerrito, F. and Raiteri, M. (1979). Serotonin release is modulated by presynaptic autoreceptors. Eur. J. Pharmacol., 57, 427-430

Chaput, Y., Blier, P. and De Montigny, C. (1986). In vivo electrophysiological evidence for the regulatory role of autoreceptors on serotonergic terminals. J. Neurosci., 6, 2796-2801

Engel, G., Gothert, M., Miiller-Schweinitzer, E., Schlicker, E .• Sistonen, L. and Stadler, P. A. (1983). Evidence for common pharmacological properties of [lH]5-hydroxytryptamine binding sites, presynaptic 5-hydroxytryptamine autoreceptors in CNS and inhibitory presynaptic 5-hydroxytryptamine receptors on sympathetic nerves. Naunyn-Schmiedeberg's Arch. Pharmacol., 324, 11&-124

62 Serotonin

Engel, G., Gothert, M., Hoyer, D., Schlicker, E. and Hillenbrand, K. (1986). Identity of inhibitory presynaptic 5-hydroxytryptamine (5-HT) autoreceptors in the rat brain cortex with 5-HT1s binding sites. Naunyn-Schmiedeberg's Arch. Pharmacol., 332, 1-7

Feuerstein, T. J., Lupp, A. and Hertting, G. (1987). The serotonin (5-HT) autoreceptor in the hippocampus of the rabbit: role of 5-HT biophase concentration. Neuropharmacology, 26, 1077-1080.

Figueroa, H. R., Yiirgens, P. B., Newton, D. K and Hall, T. R. (1985). Evidence for negative feedback control of the release of [3H] serotonin from superfused mouse cerebellum slices induced by electrical field stimulation. Gen. Pharmacol., 16, 10~108

Gothert, M. (1980). Serotonin-receptor-mediated modulation of Ca2+ -dependent 5-hydroxytryptamine release from neurones of the rat brain cortex. NaunynSchmiedeberg's Arch. Pharmacol., 314, 223-230

Gothert, M. (1982). Modulation of serotonin release in the brain via presynaptic receptors. Trends in Pharmacol. Sci., 3, 437-440

Gothert, M. (1985). Role of autoreceptors in the function of the peripheral and central nervous system. Arzneimittelforschung, 35, 1909-1916

Gothert, M. (1986). Presynaptic 5-HT autoreceptors and the modulation of the release of 5-HT. Clin. Neuropharmacol., 9, Suppl. 4, 30~310

Gothert, M. and Schlicker, E. (1983). Autoreceptor-mediated inhibition of 3H-5-hydroxytryptamine release from rat brain cortex slices by analogues of 5-hydroxytryptamine. Life Sci., 32, 118~1191

Gothert, M. and Weinheimer, G. (1979). Extracellular 5-hydroxytryptamine inhibits 5·hydroxytryptamine release from rat brain cortex slices. NaunynSchmiedeberg's Arch. Pharmacol., 310, 9~96

Hamon, M., Bourgoin, S. and Glowinski, J. (1973). Feedback regulation of 5-HT synthesis in rat striatal slices. J. Neurochem., 20, 1727-1745

Hery, F., Simonnet, G., Bourgoin, S., Soubrie, P., Artaud, F., Hamon, M. and Glowinski, J. (1979). Effect of nerve activity on the in vivo release of [3H] serotonin continuously formed from L-[3H] tryptophan in the caudate nucleus of the cat. Brain Res., 169, 317-334

Langer, S. Z. and Moret, C. (1982). Citalopram antagonizes the stimulation by lysergic acid diethylamide of presynaptic inhibitory serotonin autoreceptors in the rat hypothalamus. J. Pharmacol. Exp. Ther., 222, 220-226

Limberger, N., Bonanno, G., Spath, L. and Starke, K. (1986). Autoreceptors and aradrenoceptors at the serotonergic axons of rabbit brain cortex. NaunynSchmiedeberg's Arch. Pharmacol., 332, 324-331

Martin, L. L. and Sanders-Bush, E. (1982). Comparison of the pharmacological characteristics of 5-HT1 and 5-HT2 binding sites with those of serotonin autoreceptors which modulate serotonin release. Naunyn-Schmiedeberg's Arch. Pharmacol., 321, 165-170

Maura, G., Roccatagliata, E. and Raiteri, M. (1986). Serotonin autoreceptor in rat hippocampus: Pharmacological characterization as a subtype of the 5-HT1 receptor. Naunyn-Schmiedeberg's Arch. Pharmacol., 334, 32~326

Maura, G., Ulivi, M. and Raiteri, M. (1987). (-)-Propranolol and (±)cyanopindolol are mixed agonists--antagonists at serotonin autoreceptors in the hippocampus of the rat brain. Neuropharmacology, 26, 71~717

Middlemiss, D. N. (1984a). 8-Hydroxy-2-(di-n-propylamino)tetralin is devoid of activity at the 5-hydroxytryptamine autoreceptor in rat brain. Implications for the proposed link between the autoreceptor and the [3H]5-HT recognition site. Naunyn-Schmiedeberger's Arch. Pharmacol., 327, 1~22


Middlemiss, D. N. (1984b). Stereoselective blockade at (3H]5-HT binding sites and at the 5-HT autoreceptor by propranolol. Eur. J. Pharmacal., 101, 289-293

Monroe, P. J. and Smith, D. J. (1985). Demonstration of an autoreceptor modulating the release of (3H]5-hydroxytryptamine from a synaptosomal-rich spinal cord tissue preparation. J. Neurochem., 45, 1886-1894

Moret, C. (1985). Pharmacology of the serotonin autoreceptor. In Green, A. R. (Ed.), Neuropharmacology of Serotonin, Oxford University Press, Oxford, pp. 21-49

Neale, R. F.,Fallon, S. L.,Boyar, W. C., Wasley,J. W. F.,Martin,L. L., Stone, G. A., Glaeser, B. S., Sinton, C. M. and Williams, M. (1987). Biochemical and pharmacological characterization of CGS 12066B, a selective serotonin-lB agonist. Eur. J. Pharmacal., 136, 1-9

Raiteri, M., Bonnano, G., Marchi, M. and Maura, G. (1984). Is there a functional linkage between neurotransmitter uptake mechanisms and presynaptic receptors? J. Pharmacal. Exp. Ther., 231, 671-677

Sawada, M. and Nagatsu, T. (1986). Stimulation of the serotonin autoreceptor prevents the calcium-calmodulin-dependent increase of serotonin biosynthesis in rat raphe slices. J. Neurochem., 46, 963-967

Schlicker, E., Gothert, M. and Hillenbrand, K. (1985a). Cyanopindolol is a highly potent and selective antagonist at the presynaptic serotonin autoreceptor in the rat brain cortex. Naunyn-Schmiedeberg's Arch. Pharmaco/.,,331, 398-401

Schlicker, E., Brandt, F., Classen, K. and Gothert, M. (1985b ). Serotonin release in human cerebral cortex and its modulation via serotonin receptors. Brain Res., 331, 337-341

Schlicker, E., Fink, K., Classen, K. and Gothert, M. (1987). Facilitation of serotonin (5-HT) release in the rat brain cortex by cAMP and probable inhibition of adenylate cyclase in 5-HT nerve terminals by presynaptic a2-adrenoceptors. Naunyn-Schmiedeberg's Arch. Pharmacal., 336, 251-256

Schlicker, E., Classen, K. and Gothert, M. (1988). Presynaptic serotonin receptors and a 2-adrenoceptors on central serotoninergic and noradrenergic neurones of normotensive and spontaneously hypertensive rats. J. Cardiovasc. Pharmacal., 11, 51~528

Sprouse, J. S. and Aghajanian, G. K. (1986). (-)-Propranolol blocks the inhibition of serotonergic dorsal raphe cell firing by 5-HT lA selective agonists. Eur. J. Pharmacal., 128, 295-298

Verbeuren, T. J., Coen, E. P., Schoups. A., van de Velde, R., Baeyens, R. and de Potter, W. P. (1984). Presynaptic serotonin receptors regulate the release of (3H]-serotonin in hypothalamic slices of the rabbit. Naunyn-Schmiedeberg's Arch. Pharmacal., 327, 102-106

8 Need the Autoregulation of Raphe Neurones

Involve 5-Hydroxytryptamine?

J. S. Kelly, N.J. Penington and D. G. Rainnie

Department of Pharmacology, University of Edinburgh, 1 George Square, Edinburgh EH8 9JZ, UK

Early electrophysiological experiments on the dorsal raphe (DR) nucleus using extracellular unit recording showed a slow continuous firing rate of 0.5-3 Hz, which could be inhibited by systemic administration of the potent hallucinogen lysergic acid diethylamide (LSD; Aghajanian et al., 1968). The effects of LSD were assumed to be mediated via 5-hydroxytryptamine (5-HT) receptors. To determine the role of tonic 5-HT release on DR neuronal excitability, animals were pre-treated with the specific 5-HT synthesis inhibitor p-chlorophenylalanine (p-CPA). However, when given alone,p-CPA did not appreciably alter DR neuronal firing rate (Aghajanian et al., 1970). On the other hand, monoamine oxidase inhibitors caused a marked decrease in base-line firing rates, suggesting that negative feedback was due to an extracellular accumulation of 5-HT; pre-treatment with p-CP A almost totally prevented the inhibitory effect of monamine oxidase inhibitors, but it had no effect on ihe LSD-mediated reduction in firing. This confirmed the earlier suggestion that LSD must act at a point distal to 5-HT release.

Although pre-treatment with the 5-HT precursor L-tryptophan also caused a reduction in base-line firing rates, this could not be prevented by p-CP A pre-treatment. This paradox was eventually reconciled by the finding that p-CP A appears to act preferentially on nerve terminals, causing a marked reduction in histofluorescence in forebrain terminals but not in the perikarya, possibly owing to continued synthesis of new tryptophan hydroxylase in cell bodies (Aghajanian et al., 1973).

A role for LSD as a specific 5-HT uptake inhibitor has been excluded. Sheard et al. (1972) showed that depletion of 5-HT stores with p-CPA inhibited the reduction of base-line firing rate normally seen following treatment with tertiary amine 5-HT uptake blockers such as chlorimipramine, but not that produced by LSD.

Autoregulation of Raphe Neurones 65

Following pre-treatment with R04-4602, an aromatic amino acid decarboxylase inhibitor, Gallagher and Aghajanian (1976) showed that a reduction occurred in the inhibition of DR neuronal firing caused by L-tryptophan treatment. Furthermore, R04-4602 significantly reduced the amount of 5-HT in the DR nucleus, even in the presence of L-tryptophan. Thus it was proposed that a local increase in 5-HT in the vicinity of the perikarya after tryptophan loading is sufficient to produce inhibition of DR neuronal firing. Consistent with this hypothesis is the finding that pre-treatment with R04-4602leaves unaltered the decrease in DR neuronal firing rate caused by the microiontophoretic application of both LSD and 5-HT.

Among the negative-feedback loops available for the inhibition of DR neurones by tonic 5-HT release, the most direct would be via 5-HT collaterals. Antidromic activation of DR neurones continues to produce a period of post-stimulus inhibition even after the major frontal afferent pathways, including the habenula and the pre-optic hypothalamus, are lesioned (Wang and Aghajanian, 1977). The antidromic inhibition of DR cells, however, is lost after the destruction of 5-HT pathways by 5,7-dihydroxytryptamine but not by the destruction of other pathways, e.g. the noradrenergic pathways by 6-hydroxydopamine. Although yaminobutyric acid can also inhibit DR cells, this effect is preferentially blocked by picrotoxin, leaving the action of iontophoretic 5-HT and antidromic stimulation unaltered (Wang and Aghajanian, 1977).

Recently, with improved immunohistochemical and ultrastructural techniques, serotoninergic dendritic contacts as well as 5-HT-containing axon terminals have been observed in the cat DR nucleus (Chazal and Ralston, 1987). These findings raise three possible ways in which 5-HT may mediate inhibition in the DR nucleus: (a) direct local interaction between serotoninergic neurones; (b) recurrent serotoninergic collaterals; and (c) a serotoninergic input from other raphe nuclei. However, the recent introduction of brain slice preparations (Mosko and Jacobs, 1976) raises the possibility that the post-spike inhibitory period is part of the inherent oscillatory activity of DR neurones (Mosko and Jacobs, 1977).

Initial intracellular records from single DR neurones in vivo showed them to be spontaneously active, and to undergo a pronounced post-spike hyperpolarization followed by a gradual depolarization leading to the onset of the next spike (Aghajanian and VanderMaelen, 1982a). At that time, it was proposed that an excitatory input, possibly noradrenergic, was required to keep this system going. However, the hyperpolarization-depolarization cycle was termed the 'pacemaker potential' and was later assumed to account for the automacity of these cells. In a follow-up study using a double labelling technique, Aghajanian and VanderMaelen (1982b) demonstrated that DR neurones showing 'pacemaker potentials' also displayed 5-HTderived fluorescence.

66 Serotonin

In a preliminary report on the passive membrane properties of DR neurones of the rat mesencephalon in vitro, Crunelli et al. (1983) showed DR neurones to be characterized by a high mean membrane input resistance of 190 MQ, and a long mean time constant of 26 ms. The DR neurones showed slow, spontaneous, rhythmic firing (0.2-4 Hz), consisting of single action potentials followed by marked after-hyperpolarizations (AHP). The AHP was resistant to tetrodotoxin, reduced by the presence of Ca2+ ions, and reversed in polarity between -75 and -90 mV. It was concluded that this potential represented at least in part the activation of a Ca-dependent K+ conductance. The AHP is one of at least two outward K+ currents which may contribute to intrinsic pacemaker activity. Another is a large transient voltage- and 4-aminopyridine-sensitive current, termed /A (Aghajanian, 1985). In our laboratory, experiments employing a dual pulse voltage-clamp protocol have confirmed the voltage dependency of I A and its sensitivity to 4-aminopyridine (Penington et al., 1987a).

Paradoxically, we have now shown this current, /A, to be attenuated by the extracellular Ca2+ concentration (Rainnie et al., 1987b). Replacing Ca2+ by Mgl+ causes a 50 per cent increase in amplitude of lA for a given hyperpolarizing pre-pulse. This finding supports our observation that /A is opposed by at least two voltage-sensitive inward Ca2+ currents most clearly seen in es+ -loaded cells (Penington et al., 1987a, b). The first ofthese was shown to be associated with a low-voltage, low-threshold, long-latency, slow depolarizing potential, and the second a higher-threshold, much larger, faster-rising potential, which often appears to ride on the first. The low-threshold potential is reduced and the high-threshold potential abolished by Cd2 + ions. Cd2 + also caused a small depolarization, which might indicate that at rest the Ca-dependent K + conductance may contribute to the resting membrane potential. Thus at rest the most likely single event leading to pacemaker activity is the transient de-inactivation of a low-threshold inward Ca2 + current by the post-spike AHP. Following the decay of the AHP, the low-threshold Ca2+ potential initiates a tetrodotoxinsensitive, Na +-mediated, propagated action potential. This in turn activates the high-threshold Ca2+ current, which enhances the AHP and ensures the onset of the next pacemaker cycle and regulates the length of the repetitive rate. Presumably, activation of lA by the repolarization of the previous AHP increases the amplitude and duration of the AHP, and hence delays the onset of the next action potential.

The possible modulation of these active currents by neurotransmitters that modify pacemaker activity, such as 5-HT, is also under investigation (Rainnie et al., 1987a; Penington et al., unpublished observations). Intracellular recordings from the in-vitro brain slice preparation show that the majority of neurones are hyperpolarized by up to 20m V by a superfusion of the DR nucleus with micromolar concentrations of 5-HT ( 50-200 !.IM); the hyperpolarization is accompanied by a relatively voltage-independent


decrease in input resistance of20-50 per cent. These effects are unaltered by the presence of tetrodotoxin. The hyperpolarizing action of 5-HT persists when 3 M KCl electrodes are used; when on the same cells, the increase in conductance evoked by y-aminobutyric acid was accompanied by a depolarization. This suggests that an increase in Cl- conductance is not involved in the action of 5-HT. The 5-HT1A ligand 8-hydroxy-2-(di-npropylamino )tetralin (8-0H-DPAT; Middlemiss and Fozard, 1983) at rather lower micromolar concentrations (1 JA.M) had a similar action to 5-HT, except that recovery took about 30 min longer. The estimated reversal potential for both 8-0H-DP AT and 5-HT was approximately -90 m V. Both these values are close to that derived from the Nernst equation for an event mediated by an increase inK+ conductance. Hence it may be that 5-HT released tonically from DR neurone cell bodies acts via 5-HT lA receptors to cause an increase in K+ conductance, and so regulates the spontaneous activity of the DR neurone from which it arises, or that of adjacent neurones.

The high input resistance and long time constant of DR neurones could be of great importance to the phasic and tonic synaptic inputs to these neurones. The high membrane resistance will enhance even the smallest synaptic input, and the long time constant will increase the effectiveness of these events during temporal summation. Clearly, under such circumstances inhibitory post -synaptic potentials may activate h and thus decrease cell firing rate. Focal electrical stimulation of the DR nucleus elicits slow inhibitory post-synaptic potentials (Yoshimura and Higashi, 1985), which are thought to be 5-HT-mediated, because the response could be reduced by methysergide, a 5-HT2 receptor antagonist, and enhanced by imipramine.

Recent reports suggest that a number of compounds may be of use in further elucidating the nature of the 5-HT receptor(s) on DR neurones, and thus the role of 5-HT in autoregulation. (-)-Propranolol reduces the effect of 5-HT recorded extracellularly from DR neurones in vivo (Sprouse and Aghajanian, 1986), and spiperone has been shown to prevent the hyperpolarizing action of 5-HT on hippocampal CA1 neurones (Andrade et al., 1986). The selective 5-HT2 receptor antagonist ketanserin has been shown to neither attenuate nor potentiate the effects of 5-HT on DR neurones when recorded extracellularly in vivo (Lakoski and Aghajanian, 1985). Thus the DR somatic 5-HT receptor is unlikely to be of the 5-HT2

type. Although one study (Colino and Halliwell, 1986) has shown low doses of

8-0H-DPAT to be a potent antagonist of 5-HT actions in the rat, most intracellular studies show selective 5-HT lA receptor compounds to be similar in their modes of action to 5-HT itself. In a recent report, Sprouse and Aghajanian (1987) used intracellular recording techniques to compare the potency of putative selective 5-HT lA and 5-HT 18 receptor agonists, and concluded that DR neurones appear highly responsive to 5-HT1A but not to

68 Serotonin

5-HT 18 compounds. Indeed, there may be reason to believe the suggestion of Martin and Sanders-Bush (1982) that the autoreceptors of the DR neurones are different from those on the rostral nerve terminals thought to regulate 5-HT release, and that the classes of 5-HT receptors at the somatic and terminal sites are restricted (see Verge et al., 1985; Maura et al., 1986).

Thus experiments to exclude 5-HT autoreceptors from a physiological role in the regulation of DR basal firing frequency await the development of a selective and potent antagonist.

ACKNOWLEDGEMENTS

J. S. K. and N. J. P. are grateful to the Scottish Home and Health Department and the Medical Research Council for support. D. G. is a SERC CASE student in collaboration with the Lilly Research Centre.

REFERENCES

Aghajanian, G. K. (1985). Modulation of a transient outward current in serotonergic neurones by a 1-adrenoceptors. Nature, 315, 501-503

Aghajanian, G. K. and VanderMaelen, C. P. (1982a). Intracellular recordings from serotoninergic dorsal raphe neurons: pacemaker potentials and the effect of LSD. Brain Res., 238, 463-469

Aghajanian, G. K. and VanderMaelen, C. P. (l982b). Intracellular identification of central noradrenergic and serotonergic neurones by a new double labelling procedure. J. Neurosci., 2, 1786-1792.

Aghajanian, G. K., Foote, W. E. and Sheard, M. H. (1968). Lysergic acid diethylamide: sensitive neuronal units in the midbrain raphe. Science, 161, 706-708

Aghajanian, G. K., Graham, A. W. and Sheard, M. H. (1970). Serotonincontaining neurons in brain: depression of firing by monoamine oxidase inhibitors. Science, 189, 1100--1102

Aghajanian, G. K., Kuhar, M. J. and Roth, R. H. (1973). Serotonin-containing neuronal perikarya and terminals: differential effects of p-chlorophenylalanine. Brain Res., 54, 85-101

Andrade, R., Malenka, R. C. and Nicoll, R. A. (1986). A G protein couples serotonin and GABAB receptors to the same channels in hippocampus. Science, 234, 1261-1265

Chazal, G. and Ralston, III, H. J. (1987). Serotonin-containing structures in the nucleus raphe dorsalis of the cat: an ultrastructural analysis of dendrites, presynaptic dendrites, and axon terminals. J. Comp. Neurol., 259, 317-329

Colino, A. and Halliwell, J. V. (1986). 8-0H-DPAT is a strong antagonist of 5-HT action in rat hippocampus. Eur. J. Pharmacol., 130, 151-152

Crunelli, V., Forda, S., Brooks, P. A., Wilson, K. C. P., Wise,J. C. M. andKelly,J. S. (1983). Passive membrane properties of neurones in the dorsal raphe and periaqueductal grey recorded in vitro. Neurosci. Len., 40, 263-268

Gallagher, D. W. and Aghajanian, G. K. (1976). Inhibition of firing of raphe neurones by tryptophan and 5-hydroxytryptophan: blockade by inhibiting serotonin synthesis with R0-4-4602. Neuropharmacology, 15, 149-156


Lakoski, J. M. and Aghajanian, G. K. (1985). Effects of ketanserin on neuronal responses to serotonin in the prefrontal cortex, lateral geniculate and dorsal raphe nucleus. Neuropharmacology, 24, 265-273

Martin, L. L. and Sanders-Bush, E. (1982). Comparison of the pharmacological characteristics of 5-Hf1 and 5-Hf2 binding sites with those of serotonin autoreceptors which modulate serotonin release. Naunyn-Schmiedeberg's Arch. Pharmacol., 321, 165-170

Maura, G., Roccatagliata, E. and Raiteri, M. (1986). Serotonin autoreceptor in rat hippocampus: pharmacological characterization as a subtype of the 5-Hf1 receptor. Naunyn-Schmiedeberg's Arch. Pharmacol., 334, 323-326

Middlemiss, D. N. and Fozard, J. R. (1983). 8-hydroxy-2-(di-npropylamino)tetralin discriminates between subtypes of the 5-HT1 recognition site. Eur. J. Pharmacol., 90, 151-153

Mosko, S. S. and Jacobs, B. L. (1976). Recording of dorsal raphe unit activity in vitro. Neurosci. Lett., 2, 195-200

Mosko, S. S. and Jacobs, B. L. (1977). Electrophysiological evidence against negative neuronal feedback from the forebrain controlling midbrain raphe unit activity. Brain Res., 119, 291-303 ·

Penington, N.J., Rainnie, D. G. and Kelly, J. S. (1987a). Intracellular studiesofrat dorsal raphe neurones in vitro. Neurosci. Lett., Suppl. 29, S17

Penington, N.J., Rainnie, D. G. and Kelly, J. S. (1987b). Studies on the pacemaker potentials of rat dorsal raphe neurones in vitro. Proc. Inti. Union Pharmacol., X, P260

Rainnie, D. G., Penington, N.J. and Kelly, J. S. (1987a). On the hyperpolarising action of 5-hydroxytryptamine on rat dorsal raphe neurones in vitro. Neurosci. Lett., Suppl. 29, S72

Rainnie, D. G., Penington, N.J. and Kelly, J. S. (1987b). Activationof'A' currents in dorsal raphe neurones in vitro. Neuroscience, 22, Suppl., S696, Abst. 2078P

Sheard, M. H., Zolovick, A. and Aghajanian, G. K. (1972). Raphe neurons: effect of tricylic antidepressant drugs. Brain Res., 43, 690-694

Sprouse, J. S. and Aghajanian, G. K. (1986). (-)-Propranolol blocks the inhibition of serotonergic dorsal raphe cell firing by 5-Hf1A selective agonists. Eur. J. Pharmacol., 128, 295-298

Sprouse, J. S. and Aghajanian, G. K. (1987). Electrophysiological responses of serotoninergic dorsal raphe neurons to 5-Hf1A and 5-Hf1B agonists. Synapse, 1, 3-9

Verge, D., Daval, G., Patey, A., Gozlan, H., El Mestikawy, S. and Hamon, M. (1985). Presynaptic 5-Hf autoreceptors on serotonergic cell bodies and/or dendrites but not terminals are of the 5-Hf1A subtype. Eur. J. Pharmacol., 113, 463-464

Wang, R. Y. and Aghajanian, G. K. (1977). Antidromically identified serotonergic neurons in the rat midbrain raphe: evidence for collateral inhibition. Brain Res., 132, 186-193

Yoshimura, M. and Higashi, H. (1985). 5-hydroxytryptamine mediates inhibitory postsynaptic potentials in rat dorsal raphe neurones. Neurosci. Lett., 53, 69-74

9

In vivo Electrophysiology of Receptors Mediating the Central Nervous System Actions

of 5-Hydroxytryptamine

M. H. T. Roberts and M. Davies

Department of Physiology, University of Wales College of Cardiff, PO Box 902, CardiffCF11SS, UK

INTRODUCTION

The existence of classifiable groupings of the 5-hydroxytryptamine (5-HT) receptor have been clearly demonstrated by the use of novel and selective agonists and antagonists on peripheral tissues (Bradley et al., 1986). To date, however, little is known about the nature of functional receptors on central neurones, although one of the earliest reports on the central effects of 5-HT reported the heterogeneity of neuronal responses to 5-HT (Roberts and Straughan, 1967).

Heterogeneity of 5-HT binding in the CNS has been reported by Peroutka and Snyder (1979). They provided evidence for two distinct 5-HT binding sites, which they called 5-HT 1 and 5-HT 2 • The centralS-HT 1 binding site has since been further subdivided into 5-HT1A, 5-HT18, 5-HT1c and 5-HT10 sites (see Pedigo et al., 1981; Pazos et al., 1985; Heuring and Peroutka, 1987). Following closely behind these binding studies were attempts to relate functional responses to activation of these binding sites. Greatest progress has been made using functional responses to 5-HT and its selective agonists and antagonists in studies on isolated peripheral neurone and vascular preparations. These and other functional studies have been summarized by Bradley et al. (1986), and a classification of functional receptors has been proposed, using terminology which is clearly derived from, but importantly not identical to, the classification of binding sites. Three groupings were suggested: 5-HT1-like, 5-HT2 and 5-HT3• The 5-HT2 receptor closely resembles the 5-HT2 binding site and may be defined by the high antagonistic potency of ketanserin and methysergide. The 5-HT3

receptor is defined by the potent and selective antagonists MDL 72222 and

CNS Receptors for 5-HT 71

ICS 205-930. The 5-HTrlike receptor is not defined adequately by a potent and selective antagonist, although methysergide is effective but with a lower potency than at 5-HT2 receptors. The agonist 5-carboxamidotryptamine (5-CT) is at least as potent as 5-HT at the 5-HTrlike receptor. Another agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-0H-DPAT) is potent on some 5-HT 1-like receptors, but Bradley et al. ( 1986) did not suggest at the time that this difference was sufficient to justify subdivision of the 5-HTrlike receptor into 1A and 1B subtypes.

It has long been known that microiontophoretically applied 5-HT has both excitatory and depressant effects upon the firing rate of central neurones (Roberts and Straughan, 1967). The relationship between the receptors mediating these central neuronal actions of 5-HT and the 5-HTrlike, 5-HT2 and 5-HT3 receptor types outlined by Bradley et al. (1986) is uncertain. The effects of some of the new selective compounds have therefore been examined on (a) the excitatory responses of brain-stem neurones to 5-HT, (b) the depressant responses of brain-stem neurones to 5-HT, and (c) the excitatory responses of spinal motoneurones to 5-HT. All the experiments were conducted in vivo on rats anaesthetized with halothane. Some of these data are described in detail elsewhere (Davies et al., 1988a, b; Roberts et al., 1988).

THE EXCITATORY RESPONSES OF BRAIN-STEM NEURONES TO 5-HT

The majority of mid-line brain-stem neurones at the level of raphe obscurus and magnus were excited by iontophoretically applied 5-HT. These excitatory effects of 5-HT were selectively and reversibly attenuated by iontophoretic application of the 5-HT2 receptor antagonists ketanserin and methysergide. The effects of ketanserin are illustrated in Figure 9.1. Both antagonists were also tested systemically. Ketansarin (0.3 mg/kg i.v.) and methysergide (1 mg/kg i.v.) reversibly antagonized the excitatory responses to 5-HT, without reducing excitatory responses to noradrenaline or glutamate.

This response to 5-HT was not selectively reduced by iontophoretic or systemic administration of the 5-HT3 receptor antagonist MDL 72222 (1 mg/kg i.v.) or the u-adrenoceptor antagonist prazosin (0.8 mg/kg i.v.).

The 5-HT rlike receptor agonists 5-CT and 8-0H-D PAT very rarely, and then only weakly, mimicked the excitatory effects of 5-HT. This was not due to inadequate release of these compounds from the microelectrode, because in-vitro testing did not result in electrophoretic transport numbers significantly different from that for 5-HT. It is concluded that the excitatory receptor for 5-HT on brain-stem neurones is a functional 5-HT2 receptor.

72 Serotonin

a) CONTROL

4] 5HT G 50 50

b) KETANSERIN 5nA

§ 40( ~ 0 a.. en

5HT G

c) RECOVERY

4] c • -5HT G ...............

1min > + + + ~L

5ms

a b c Figure 9.1 The effect of ketanserin on excitatory responses to iontophoretically applied 5-HT and glutamate (G) on a single brain-stem neurone. (a) Control responses to the agonists, both of which were applied with a current of 50 nA. (b) Responses to the agonists during the continuous application of ketanserin. Ketanserin was applied with a current of 5 nA and had been applied continuously for 28 min at the start of trace (b). Ketanserin blocked the response to 5-HT without reducing the response to glutamate. (c) Recovery of the responses to 5-HT 42 min after the application of ketanserin had been terminated. Below the ratemeter records are samples of extracellularly recorded action potentials taken at those times indicated by the arrows. It can be seen that antagonism of the 5-HT response was not accompanied by any change in spike amplitude. (Reproduced with permission from

Davies eta/., 1988a)

CNS Receptors for 5-HT

THE DEPRESSANT RESPONSES OF BRAIN-STEM NEURONES TOS-HT

73

A minority of mid-line brain-stem neurones are depressed by 5-HT. These responses were very resistant to antagonism by ketanserin applied iontophoretically with high currents for long periods or administered systemically with doses up to 2 mglkg i. v. Similarly, MDL 72222 also failed to antagonize depressant responses to 5-HT. Thus it is likely that the depressant response is mediated by either 5-HT2 or 5-HT3 receptors. Attempts were made to determine the ability of methysergide to block these depressant responses to 5-HT. At systemic doses of 1 mg/kg i.v. (the dose which potently reduced excitatory responses), methysergide did not affect depressant responses. At much higher doses (30 mglkg i. v. ), the depressant response was severely attenuated. At such high doses, non-specific actions of methysergide may be anticipated, but the responses of the cells to y-aminobutyric acid were unaffected, and the antagonism was not accompanied by any change in spike amplitude or duration. Selective antagonism by methysergide at high doses is compatible with an action at a 5-HT1-like receptor.

The 5-HT1-like receptor agonist 5-CT had marked depressant effects on neurones depressed by 5-HT (Figure 9.2). As transport number measurements showed no difference in the release of 5-HT and 5-CT from electrodes, it was possible to make an approximate estimate of the comparative potency of the two compounds. 5-CT evoked much larger and more prolonged depressant responses than 5-HT. This is compatible with the depressant actions of 5-HT being due to an action at 5-HT1-like receptors. Studies were also made of the effects of 8-0H-DPAT on these neurones; it was seen to have very similar effects to those of 5-CT. It is concluded therefore that the depressant effects of 5-HT on brain-stem neurones may be due to its action on an 8-0H-D PAT -sensitive, 5-HT ~-like receptor.

fo[ 0 0 ~~~--~~~~~~--~~~~~~~

5CT ,_...., (nA) 0 5 10 20 2 min

Figure 9.2 The depressant effects of 5-Cf on the spontaneous activity of a single brain-stem neurone. The horizontal bars below the trace refer to iontophoretic applications of 5-Cf and the numbers refer to the intensity of the ejecting current in

nA. (Reproduced with permission from Davies et al., 1988b)

74 Serotonin

THE EXCITATORY RESPONSES OF SPINAL MOTONEURONES TO 5-HT

Spinal motoneurones do not discharge spontaneously in the fluothaneanaesthetized rat. Therefore, the amplitude of the antidromically evoked population spike was used as an index of motoneurone excitability (Barasi and Roberts , 1974; Lipski, 1981). Iontophoretically applied 5-HT frequently increased the excitability of motoneurones and never had the opposite effect. These effects of 5-HT could be mimicked by electrical stimulation (20 Hz, 3 V) of nucleus raphe obscurus. Intracellular recordings showed that the stimulation of this nucleus resulted in depolarization of motoneurones. Responses of motoneurones to 5-HT were resistant to antagonism by ketanserin, suggesting that 5-HT2 receptors were not involved. The increased excitability of motoneurones by 5-HT was, however, selectively and reversibly attenuated by iontophoretic application of methysergide with modest currents (see Figure 9.3) .

The 5-HT1-like receptor agonist 5-Cf mimicked the effects of 5-HT on spinal motoneurones with a potency similar to , or slightly greater than , 5-HT. Interestingly, 8-0H-DPAT had no effect on the excitability of spinal motoneurones, even when applied with large iontophoretic currents.

The involvement of a 5-HT.-like receptor in these responses of motoneurones to 5-HT is suggested by the potent agonistic effects of 5-CT and the effectiveness of methysergide but not ketanserin. This receptor, however, would appear to be distinct from the 5-HT.-like receptor which mediates the depressant effects of 5-HT on brain-stem neurones, because of the greater sensitivity of motoneurones to methysergide , the lack of effect of 8-0H-DPAT on these cells, and the failure of cyanopindolol, an antagonist with high affinity for 5-HT1A and 5-HTm binding sites (Engel et al., 1986), to block the spinal motoneurone effects of 5-HT, even when cyanopindolol is applied with currents sufficient to abolish the responses to noradrenaline.

Methysergide 20 5HT NA 100 100

1!!!1 (J c:J 0 c 0 1!!!1 0 0 0 CJ 0 CD

r 5mV 10 20 30 40 50 eo ro 00 eo •·m!IUTES

Figure 9.3 The effects of methysergide on the responses of spinal motoneurones to iontophoretically applied 5-HT and noradrenaline (NA). Each upward deflection represents the amplitude of the antidromically evoked spinal motoneurone population spike. Both 5-HT and noradrenaline were applied with a current of 100 nA, and each caused an increase in the amplitude of the population spike. The response to 5-HT but not that to noradrenaline was abolished by iontophoretic application of methysergide with a current of 20 nA. Partial recovery of the response

to 5-HT may also be seen

CNS Receptors for 5-HT 75

CONCLUSIONS

It seems likely that the central effects of 5-HT may be mediated by at least three types of 5-HT receptor: two 5-HT 1-like receptor subtypes and a 5-HT 2

receptor. As 8-0H-DPAT is a potent agonist at the brain-stem depressant 5-HT 1-like receptor but not at the other, it is tempting to speculate that the former resembles the 5-HT tA binding sites. Our findings with cyanopindolol suggest that the receptor mediating the spinal motoneurone excitatory effect of 5-HT is different from the 5-HT1A and 5-HTlB sites.

ACKNOWLEDGEMENTS

The technical assistance of Tim Gould is gratefully acknowledged. M. D. is a SERC CASE scholar with Glaxo Group Research Ltd.

REFERENCES

Barasi, S. and Roberts, M. H. T. (1974). The modification oflumbar motoneurone excitability by stimulation of a putative 5-hydroxytryptamine pathway. Br. J. Pharmacal., 52, 339-348


Davies, M., Wilkinson, L. S. and Roberts, M. H. T. (1988a). Evidence for excitatory 5-HT2 receptors on rat brainstem neurones. Br. J. Pharmacal., 94, 483-491

Davies, M., Wilkinson, L. S. and Roberts, M. H. T. (1988b). Evidence for depressant 5-HT 1-like receptors on rat brainstem neurones. Br. J. Pharmacal., 94, 492-499

Engel, G., Gothert, M;, Hoyer, D., Schlicker, E. and Hillenbrand, K. (1986). Identity of inhibitory presynaptic 5-hydroxytryptamine (5-HT) autoreceptors in the rat brain cortex with 5-HT18 binding sites. Naunyn-Schmiedeberg's Arch. Pharmacal., 332, 1-7


Lipski, J. ( 1981). Antidromatic activation of neurones as an analytic tool in the study of the central nervous system. J. Neurosci. Methods, 4, 1-32

Pazos, A., Hoyer, D. and Palacios, J. M. (1985). The binding of serotonergic ligands to the porcine choroid plexus: characterization of a new type of serotonin recognition site. Eur. J. Pharmacal., 106, 539-546

Pedigo, N. W., Yamamura, H. I. and Nelson, D. L. (1981). Discrimination of multiple 3H-5-hydroxytryptamine binding sites in rat brain by the neuroleptic spiperone in rat brain. J. Neurochem., 36, 220-226

Peroutka, S. J. and Snyder, S. H. (1979). Multiple serotonin receptors: differential binding of PH]5-hydroxytryptamine, [3H]lysergic acid diethylamide and PH]spiroperidol. Mol. Pharmacal., 16, 687-699

76 Serotonin

Roberts, M. H. T. and Straughan, D. W. (1967). Excitation and depression of cortical neurones by 5-hydroxytryptamine. J. Physiol., 193, 269-294

Roberts, M. H. T., Davies, M., Girdlestone, D. and Foster, G. (1988). The effects of 5-hydroxytryptamine agonists and antagonists on the responses of rat spinal motoneurones to raphe obscurus stimulation. Br. J. Pharmacol., 95, 437-448

10 Neuronal Actions of 5-Hydroxytryptamine:

An Overview

I. S. de la Landel and E. J. Mylecharant?

1 Department of Clinical and Experimental Pharmacology, University of Adelaide, Box 498, GPO, Adelaide, SA 5001, Australia

2Departrnent of Pharmacology, University of Sydney, Sydney NSW 2006, Australia

INTRODUCTION

The diversity of the effects of 5-hydroxytryptarnine ( 5-HT) is clearly evident when its neuronal actions are considered. The data presented in this session described excitatory and inhibitory actions, in the periphery and the CNS; all three of the recognized 5-HT receptor types may be involved, as is at least one other type. Particular aspects of the contributions in this session attracted much comment, as did some of the data presented in the poster discussion session.

5-HT3 RECEPTOR-MEDIATED NEUROEXCITATION

Evidence for the mediation of neuroexcitation by 5-HT3 receptors in a variety of afferent neuronal systems was described by Brian Richardson and his colleagues. In accordance with the criteria suggested by Bradley et al. (1986), studies with selective antagonists such as MDL 72222 and ICS 205-930, and the selective agonist 2-methyl-5-HT, confirmed the involvement of 5-HT 3 receptors in the depolarizing effects of 5-HT in vitro in rabbit nodose ganglion and in rat and rabbit vagus nerve preparations, as well as their involvement in the von Bezold-Jarisch reflex in rats in vivo. The agonist effects of phenylbiguanide at these 5-HT3 receptors were mentioned. Fred Fastier commented on the old observations made by himself and his colleagues, and by Gyerrnek, that arnidines including phenylbiguanide might have given a lead to compounds that could have blocked these effects of 5-HT. No pure antagonists ever eventuated,

78 Serotonin

however, partly because of the difficulties in discriminating between stimulant and depressant effects of 5-HT in some of the electrophysiological models then in use, and partly because of the limitations imposed by the highly toxic nature of some of the intermediates used to develop putative antagonists.

Michael Tyers commented on some of the problems involved in assessment of the 5-HT3 receptor antagonists in vitro. Often, apparent competitive antagonism can be obtained, but the antagonists may not be at equilibrium; with equilibration times in excess of 30 min, non-surmountable blockade became apparent in the rat vagus nerve. These sorts of problems were relevant to the general consensus in the subsesquent 5-HT receptor workshop session that the antagonists available were not able to discriminate definitively between possible 5-HT3 receptor subtypes (see Humphrey and Richardson, this volume).

The 5-HT 3-receptor-mediated von Bezold-Jarisch reflex was subsequently described in more detail by Pramod Saxena (this volume). However, Richardson summarized the evidence for involvement of 5-HT3-receptormediated neuroexcitation in a variety of other afferent pathways in vivo: cardiogenic hypertensive reflexes elicited by chemoreceptor stimulation in the coronary circulation; pulmonary chemoreflexes resulting in decreased blood pressure, heart rate and tidal volume, and increased respiratory rate; complex effects on carotid sinus nerve activity, which may involve an initial excitation followed by a depolarization blockade; pain pathways, as exemplified by the human forearm blister base model; and activation of cutaneous axonal reflexes leading to wheal and flare responses as a consequence of the antidromic release of substance P.

David Wallis described a series of excitatory effects of 5-HT in sympathetic neuronal tissue preparations; again, these appeared to fulfil the Bradley et al. (1986) criteria for mediation by 5-HT 3 receptors. These effects comprised depolarization of rabbit superior cervical pre-ganglionic axons, pre-ganglionic terminals, post-ganglionic cell bodies, and post-ganglionic axons. In guinea-pig coeliac ganglion cells, 5-HT-induced fast but not slow depolarization is also mediated by 5-HT3 receptors. Both fast and slow depolarization& can also be produced by pressure ejection of 5-HT in guinea-pig inferior mesenteric ganglion and superior cervical ganglion cells, although the receptors involved have not yet been established. Wallis also mentioned the 5-HT 3 receptor-mediated neuroexcitatory effects of 5-HT on rabbit cardiac sympathetic nerve terminals and at some other postganglionic neuroeffector junctions; these were described in more detail by John Fozard (this volume).

5-HT clearly has complex effects on the enteric nervous system. Among the actions described by Michael Gershon and colleagues was a fast response to 5-HT (a short-lived depolarization) in types liS and IIlAH cells in guinea-pig myenteric neurones, mediated by a receptor that appears to be

Overview: Neuronal Actions 79

identical to the 5-HT 3 receptor. This action, however, has not been linked to any normal physiological process in the enteric nervous system. Gershon also alluded to neuroexcitatory effects of 5-HT on myenteric neurones which activated inhibitory pathways culminating in smooth-muscle relaxation. Patrick Humphrey provided evidence in a poster presentation that 5-HT3 receptors mediated a fast relaxant phase in guinea-pig ileum preparations pre-contracted by histamine. A neuronal mechanism was responsible, because it was susceptible to tetrodotoxin. The possibility of a cholinergic interneurone in this relaxant pathway was suggested by the fact that atropine could inhibit the relaxant effect of 5-HT. Ewan Mylecharane commented that he and Sutherland had reported a neuronally mediated relaxant effect of 5-HT in guinea-pig ileum in 1983. At that time, potent selective 5-HT3 receptor antagonists were not available, but the effect was blocked by tetrodotoxin, and could not be obtained in K+ -depolarized tissues; however, it was readily elicited in the presence of atropine. Gershon commented that similar neuronally mediated inhibitory effects could be obtained with 5-HT in rabbit gut, mouse gut and guinea-pig stomach, mediated perhaps by vasoactive intestinal peptide or purinergic mechanisms. Data derived from recordings of net mechanical smooth-muscle activity in gastrointestinal preparations are therefore difficult to interpret in terms of the myenteric neuronal actions of 5-HT. The use of other agonists may also complicate the analysis; Gershon pointed out that tryptamine was able to release enteric neuronal 5-HT which can both activate and desensitize neuronal 5-HT receptors. Release from enterochromaffin cells was not involved, because the preparations used (longitudinal muscle plus myenteric plexus only) contained none of these cells. The potential problems posed by the blockade of 5-HT uptake in the gastrointestinal tract with higher concentrations of ICS 205-930 were also mentioned.

5-HT1 RECEPTOR-MEDIATED NEUROEXCITATION

Malcolm Roberts described neuroexcitatory effects in the CNS which were mediated by 5-HT2 receptors. Brain-stem raphe neuronal excitatory effects of 5-HT in rats were blocked by ketanserin and methysergide, and were unaffected by MDL 72222. This action of 5-HT was occasionally but only weakly mimicked by 5-carboxamidotryptamine ( 5-CT) and 8-hydroxy-2-( din-propylamino)tetralin (8-0H-DPAT); thus a 5-HT2 receptor is clearly responsible for these effects. Stephen Peroutka, in a poster presentation, also described a central neuroexcitatory effect mediated by 5-HT 2 receptors, in guinea-pig cortical pyramidal neurones.

80 Serotonin

5-HT .-LIKE RECEPTOR-MEDIATED NEUROEXCITATION

Roberts also described a neuroexcitatory effect of 5-HT in rat spinal motoneurones, which was unaffected by ketanserin but blocked by methysergide and mimicked by 5-CT; 8-0H-DPAT was inactive. It was concluded that a 5-HTrlike receptor was involved, but that it was different from the 5-HTlA binding site subtype. This effect of 5-HT was able to be mimicked by electrical stimulation of the nucleus raphe obscurus. Wallis noted that 5-HT could elicit depolarization responses in spinal cord slices from neonate rats. Whether this involved a 5-HTrlike or a 5-HT2 receptor was not yet clear, since the response was blocked by methysergide and cyproheptadine.

5-HT.-LIKE RECEPTOR-MEDIATED NEUROINHIBITION

Peripheral neuroinhibitory effects of 5-HT on cholinergic neurones in the gastrointestinal tract and on sympathetic adrenergic terminals in a variety of vascular and other preparations were discussed by David Clarke and his colleagues. Inhibition of acetylcholine release from enteric neurones in guinea-pig ileum is clearly produced by a 5-HT rlike receptor; the inhibitory effect of 5-HT is blocked by methiothepin and (-)-pindolol but not by ketanserin or MDL 72222, and is mimicked by 5-CT and 8-0H-DPAT. Clarke concluded that a 5-HT1A receptor subtype was responsible, on the basis of results from his own studies and those of others using selective agonists such as buspirone and WY 48 723. The pre-synaptic inhibitory effect of 5-HT on noradrenaline release from sympathetic nerve terminals is also clearly 5-HT 1-like, but the receptor subtype involved is more difficult to establish. While that in rat vena cava may correspond to the 5-HT lB binding site, those in other vascular and non-vascular tissues seem to be distinct from 5-HT1A, 5-HTm and 5-HT1c binding sites. Although there is considerable similarity to the 5-HT 10 binding site (see Peroutka, this volume), the lack of activity of rauwolscine at the functional pre-synaptic sympathetic inhibitory receptor indicates a lack of exact correlation.

Wallis noted that 5-HT-induced inhibition of sympathetic ganglionic transmission could be due in part to a desensitization block following 5-HT3

receptor-mediated depolarizing effects, but pointed out that there was also evidence that activation of a pre-synaptic 5-HT receptor reduced acetylcholine release from pre-ganglionic nerve terminals. This may be a 5-HTrlike receptor, because both 5-CT and methysergide had agonistic effects. Also, a 5-HTrlike receptor mediating hyperpolarization has recently been identified in rat superior cervical ganglionic cells.

In the CNS, the extensive evidence for the existence of pre-synaptic 5-HT


autoreceptors on the terminals of serotoninergic axons was reviewed by Manfred Gothert. Most work on the pre-synaptic inhibition of [3H)-5-HT release has been done using slices and synaptosomes prepared from rat brain and spinal cord, and shows clearly that a 5-HTrlike receptor is involved. The use of selective agonists and antagonists indicates that these autoreceptors in the rat correlate most closely with 5-HT 18 binding sites, but the 5-HT1 binding site subtype correlate for pig and human cortex autoreceptors has not yet been identified. Gothert also discussed the potential therapeutic implications of autoreceptor agonists and antagonists in the control of a variety of physiological functions and behaviours, including the development of novel antihypertensive and antidepressant agents.

The apparent absence of 5-HT 18 binding sites in human brain was commented on. Salomon Langer mentioned data suggesting that human brain may have functional 5-HTIB autoreceptors, and that the failure to detect 5-HT 18 binding sites might reflect factors such as low density, fragility (and the associated problems of freshness of material, post-mortem delays, freezing, thawing and storage), inappropriate conditions, and inappropriate ligands and binding inhibitors. Gothert confirmed that functional human autoreceptors were sensitive to methiothepin but not ketanserin, and that 8-0H-DPAT was inactive; thus a 5-HT1A receptor subtype was not involved. Fozard argued that it was difficult to accept that a functional 5-HTIB receptor was present until evidence of susceptibility to fiadrenoceptor blockers such as propranolol or cyanopindolol was forthcoming.

John Kelly and his colleagues outlined the inhibitory effects of 5-HT on dorsal raphe neuronal cell bodies, and delineated the evidence available on physiological or autoregulatory control of raphe neuronal activity by 5-HT. Serotoninergic dendritic contacts and 5-HT-containing axon terminals have recently been demonstrated in the dorsal raphe, but an equally important factor appears to be the inherent oscillatory function or automaticity in these cells as revealed by electrophysiological investigation. It was suggested that tonic release of 5-HT from dorsal raphe cell bodies might regulate spontaneous activity in the neurone from which it arises, or in adjacent neurones. Functional evidence available thus far suggests that a 5-HT1A

receptor subtype might be involved. Consistent with this are recent autoradiographic studies, which have shown high densities of 5-HT1A

binding sites in the rat dorsal raphe nucleus (see Hamon et al., this volume). Roberts also described 5-HT -induced depressant responses in rat mid-line

brain-stem neurones. The effect was resistant to antagonism by ketanserin and MDL 72222, and was mimicked by 5-CT and 8-0H-D-PAT, suggesting the involvement of a 5-HT 1-like receptor similar to the 5-HT lA binding site. Although very high doses of methysergide were required to block the effect, the antagonism did not appear to be a non-specific action.

82 Serotonin

OTHER NEURONAL EFFECTS

Gershon and colleagues reviewed in detail their evidence for an enteric neuronal excitatory receptor for 5-HT which cannot be equated with any of the 5-HT receptor types proposed by Bradley et al. (1986). 5-HT produces a long-lasting depolarization, termed a 'slow response', in many Type IIlAH enteric neurones. The slow response can be blocked by 5-hydroxytryptophan dipeptides, and mimicked by 5- and 6-hydroxyindalpine, but it is unaffected by ICS 205-930. Radioligand binding studies with [3H)-5-HT in myenteric plexus membrane preparations have identified a high-affinity binding site which correlates with the functional receptor (termed 5-HT1p), and which is clearly a specific but novel 5-HT receptor. Binding is unaffected by ICS 205-930, MDL 72222 and a wide range of agents with affinity for 5-HTrlike, 5-HT2 , cholinergic, adrenergic, dopaminergic, histaminergic, opiate and y-aminobutyric acid receptors. Although 2-methyl-5-HT inhibited [3H)-5-HT binding, it was 13-fold less potent than 5-HT itself, and it acted as an agonist of the 5-HT-induced slow response only at concentrations considerably higher than those which produced the 5-HT3 receptor-mediated fast depolarization response.

Theresa Branchek described additional autoradiographic data on the location of the novel 5-HT1p binding site. Using both [3H)-5-HT and [3H)-5-hydroxyindalpine, binding in mouse colon was localized in the myenteric and submucosal plexuses and in the lamina propria (i.e., in the same locations as in guinea-pig ileum). Binding was also demonstrated in discrete locations in guinea-pig heart (in the pericardium, often perivascular, and in the subendocardial region; Ag staining revealed dense innervation of these areas), and in skin (in both the dermis and hypodermis, in association with areas of dense innervation around hair follicles and sebaceous glands). Autoradiographic displacement studies in these tissues showed their similarity to the 5-HT lP sites in the gastrointestinal tract. Indalpine itself and other 5-HT uptake inhibitors have no effect on this binding. Gershon and Branchek also discussed the possibility that the 5-HT1p binding sites in the lamina propria may be on intrinsic axonal nerve fibres which are involved in the initiation of the peristaltic reflex.

Another neuronal effect of 5-HT was briefly mentioned by Gothert; the pre-synaptic adrenergic inhibitory effect of 5-HT in pig coronary artery preparations, which also seems to be mediated by a specific but novel5-HT receptor. This inhibitory effect of 5-HT cannot be mimicked or blocked by any of the recognized 5-HT receptor agonists or antagonists, respectively.

These novel neuronal actions of 5-HT were further discussed in the 5-HT receptor classification workshop (see Humphrey and Richardson, this volume).


REFERENCE


PART III BEHAVIOURAL

ACTIONS

11

Behavioural Correlates of the Activation of 5-HT Receptors

M. D. Trickle bank

Neuroscience Research Centre, Merck Sharp and Dohme Research Laboratories, Terlings Park, Eastwick Road, Harlow, Essex CM20 2QR,

UK

INTRODUCTION

The precise role of neurones containing 5-hydroxytryptamine (5-HT) in behavioural control is unclear. A contributing factor to our lack of understanding has undoubtedly been the use of pharmacological tools devoid of selectivity for the three major populations of 5-HT receptor, 5-HTrlike, 5-HT2 and 5-HT3 (and their subtypes), thought to exist in the CNS. A number of novel drugs with appreciable degrees of affinity and selectivity for these sites have recently become available and are proving to be useful tools for the re-examination of the functional roles of 5-HT. In this review of the behavioural pharmacology of 5-HT, attention is concentrated on some of the behavioural responses (largely in rodents) that have been proposed to reflect activation of specific 5-HT receptor subtypes. Unfortunately, little is known about some of the more complex behavioural paradigms that may help to predict the therapeutic potential of selectively manipulating 5-HT receptor subtypes in man.

MOTOR RESPONSES

Forepaw Treading

There is strong evidence that at least one component of the behavioural syndrome induced by the 5-HT receptor agonists 8-hydroxy-2-( di-npropylamino )tetralin (8-0H-DPAT) and 5-methoxy-N,N-dimethyltryptamine, namely, reciprocal forepaw treading, reflects activation of the 5-HT lA receptor subtype (Lucki et al., 1984; Trickle bank et al., 1984, 1985).

88 Serotonin

Reciprocal forepaw treading is also seen following administration of the selective5-HT1Aligand(±)-MDL 12832(Miretal., 1988). (-)-MDL 72832 is more active in this respect than either the racemic mixture or the ( + )-enantiomer, which is consistent with the stereoselectivity of the compound for the 5-HT1A recognition site (Mir et al., 1988).

A number of other compounds with high affinity and selectivity for the 5-HT1A site have recently been identified. These include 1-(2-[4-aminophenyl]ethyl)-4-(3-trifluoromethylphenyl)piperazine (PAPP; L Y 165163, Ransom eta/., 1986), flesinoxan (DU29373; Bevan eta/., 1986),and the putative anxiolytic compounds buspirone and ipsapirone (Peroutka, 1985). While buspirone induces forepaw treading (Hjorth and Carlsson, 1982; Tricklebank, unpublished observations), it is absent or only weakly present after administration of PAPP (Donohoe et al., 1987), flesinoxan (Bevan et al., 1986) and ipsapirone (Tricklebank, unpublished observations). Buspirone induces the behaviour more prominently in reserpinized than in intact animals (Hjorth and Carlsson, 1982; Tricklebank, unpublished observations), perhaps reflecting its weak neuroleptic properties (see also Donohoe et al., 1987), but it is not known whether reserpinization would also reveal forepaw treading with P APP and flesinoxan. Other evidence suggests that some of these compounds have partial agonist properties at 5-HT1A receptors (Bockaert et al., 1987). Thus their efficacy may be insufficient to induce forepaw treading, a response that is rarely seen in non-drug-treated animals, and that may require full occupation of 5-HT lA receptors by high-efficacy agonists for expression.

Head Shakes

The ability of 5-HT receptor antagonists to inhibit the head shake response to 5-HT receptor agonists correlates significantly with their affinity for the 5-HT 2 recognition site (Amt et al., 1984). Furthermore, the selective 5-HT 2 receptor antagonist ketanserin antagonizes the behaviour at doses well below those required to block forepaw treading (Lucki et al., 1984). The behaviour seems likely, therefore, to be a specific response to the activation of the 5-HT2 receptor.

Dopamine-mediated Behaviours

The 5-HT 3 receptor antagonists GR 38032F and ICS 205-930 antagonize the hyperactivity induced by infusion of dopamine or amphetamine into the nucleus accumbens ( Costall et al., 1987). Together with the enhancement of amphetamine-induced hyperactivity by the 5-HT3 receptor agonist 2-methyl-5-HT (Costall et al., 1987), these results are consistent with

Behavioural Correlates of 5-HT Receptors 89

mediation of the resultant motor stimulation by the 5-HT 3 receptor. Motor stimulation associated with increased dopaminergic activity induced by the injection into the ventral tegmentum of the neurokinin receptor agonist DiMe-C7 can be similarly blocked by GR 38032F (Hagan et al., 1987).

SENSORY SYSTEMS

Startle Reftex

How serotoninergic drugs might alter stimulus reactivity and reflex excitability is a basic question that can be answered by monitoring the acoustic startle reflex in the rat. Depletion of 5-HT, either by lesions or by synthesis inhibition, increases the amplitude of the startle response. On the other hand, 5-HT receptor agonists can also increase the reflex, an apparent paradox that seems to revolve around different sites of action. Thus when ascending 5-HT systems are activated, the startle reflex is depressed; when 5-HT is applied to the spinal cord, startle is enhanced (Davis, 1980). Interestingly, the selective 5-HT1A agonist 8-0H-DPAT, given systemically, increases the startle amplitude, while the putative 5-HT 18 receptor agonist m-chlorophenylpiperazine (m-CPP) has a depressant action (Davis et al., 1986). Intrathecal administration of 8-0H-DPAT again reveals an excitatory action, but the compound is without effect when given intracerebroventricularly. For m-CPP, the reverse holds: a depressant effect is seen after intracerebroventricular injection, while intrathecal administration is without effect (Davis et al., 1986). Antagonist studies of these behavioural responses have not yet been completed.

Nociception

The decrease in responsiveness to painful stimuli induced by increasing serotoninergic tone, and the reversal of these effects by administration of 5-HT synthesis inhibitors or neurotoxins, argue strongly for an inhibitory influence of 5-HT systems on nociceptive processes (Messing et al., 1976). However, little is known of the 5-HT receptors involved. Consistent with many of its other properties, 8-0H-DPAT diminishes rather than enhances the analgesia induced by morphine (Fozard and Tricklebank, 1983), perhaps reflecting an agonist action at 5-HT cell body autoreceptors (Dourish et al., 1986a). Identification of the post-synaptic 5-HT receptor involved in nociceptive pathways might reveal uovel therapeutic agents for the management of pain.

90 Serotonin

Interoceptive Stimuli and Drug Discrimination

Rats can be trained to discriminate from saline the interoceptive stimulus induced by a number of psychotropic drugs. For 8-0H-DPAT, the resultant discriminative stimulus, or cue, is mimicked by the 5-HT1A-selective ligands buspirone and ipsapirone (Cunningham et al., 1987; Tricklebank et al., 1987). In addition, stereoselective generalization is seen with MDL 72832 (Mir et al., 1988), while the cue is antagonized, also stereoselectively, by pindolol and alprenolol (Tricklebank et al., 1987), which is consistent with mediation of the 8-0H-DPAT discriminative stimulus by the 5-HT1A receptor.

Animals can also be trained to discriminate the putative 5-HT18 receptor agonist trifluoromethylphenylpiperazine (TFMPP) from saline. This cue is mimicked by m-CPP and RU 24969, but not by 8-0H-DPAT or the 5-HT2-selective agonist dimethoxymethylphenlyaminopropane (DOM; McKenney and Glennon, 1986). Antagonists of this cue have not been reported.

Perhaps most well-defined is the discriminative stimulus associated with activation of 5-HT2 receptors: lysergic acid diethylamide, quipazine and DOM all induce cross-generalizing discriminative stimuli that can be dose-dependently antagonized by 5-HT2 receptor antagonists (see, for example, Glennon et al., 1983).

5-HT 3 receptor antagonists have an anxiolytic profile in some behavioural paradigms (Tyers et al., 1987) and might, therefore, induce interoceptive stimuli. Preliminary experiments with ICS 205-930 (0.05--0.1 mg/kg, i. p.) suggest that this may be the case, but the stimulus is nevertheless weak, only 10 per cent of rats achieving 9 out of 10 correct discriminations after 60 training sessions (Singh and Tricklebank, unpublished observations).

FOOD INTAKE

Compounds inhibiting 5-HT reuptake (e.g. fluoxetine), enhancing 5-HT release (e.g. fenfluramine), or acting as direct agonists at post-synaptic 5-HT receptors (e.g. RU 24969, TFMPP and m-CPP), decrease food intake in rodents. The pharmacological profile of the effects of R U 24969, TFMPP and m-CPP suggests an action at 5-HT18 recognition sites located post-synaptically (Kennett et al., 1987). On the other hand, the 5-HT1A

ligands 8-0H-DPAT, buspirone, gepirone, ipsapirone (Dourish et al., 1986b) and PAPP (Hutson et al., 1987) all increase food intake. While this i~ more consistent with an antagonist rather than agonist action, two lines of evidence suggest that 8-0H-DPAT-induced hyperphagia is mediated by an agonist action at 5-HT lA receptors located not post -synaptically to 5-HT neurones, but on 5-HT-containing neuronal cell bodies.


First, the selective 5-HT neurotoxin 5,7-dihydroxytryptamine and the 5-HT synthesis inhibitor p-chlorophenylalanine, whilst hyperphagic in their own right, block the hyperphagic effect of 8-0H-DPAT (Bendotti and Samanin, 1986; Dourish et al., 1986b), suggesting that 5-HT must be available for 8-0H-DPAT to exert its action. Second, 8-0H-DPAT injected directly into the dorsal or medial raphe, areas rich in 5-HT neuronal cell bodies, elicits feeding (Bendotti and Samanin, 1986; Dourish et al., 1986b ).

The non-selective 5-HT1/5-HT2 receptor antagonists methiothepin, metergoline, methysergide and mesulergine also increase food intake. The lack of effect of selective 5-HT2 and 5-HT3 antagonists again suggests that post-synaptic 5-Hr1-like receptors play an important role in the control of feeding in the rat (Dourish et al., 1989).

SLEEP

Very early work from Jouvet's laboratory first implicated 5-HT in the control of sleep: 5-HT synthesis inhibition induced insomnia in cats, an effect reversed by 5-hydroxytryptophan (Delorme et al., 1966). However, 5-HT receptor agonists also disrupt sleep. Thus, 5-carboxamidotryptamine, RU 24969 and 8-0H-DPAT decrease sleep time (Dzoljic et al., 1986), although such effects might be secondary to their activation of motor systems. On the other hand, the 5-HT2 receptor antagonist ritanserin increases slow-wave sleep in humans (Idzikowski et al., 1986) and antagonizes the disruption of slow-wave sleep induced in the rat by the 5-HT2 receptor agonist DOM (Dugovic and Wauquier, 1987). These findings will undoubtedly rekindle interest in the role(s) of 5-HT receptor subtypes in sleep mechanisms.

ANXIETY

Disinhibition of punished responding, suggestive of anxiolysis, is seen following depletion of brain 5-HT, and following treatment with a number of non-selective 5-HT receptor antagonists (Iversen, 1984). It is perhaps not too surprising, therefore, that compounds acting selectively at each of the three major populations of 5-HT receptor show anxiolytic-like profiles in some anxiety-provoking paradigms.

Thus there is a strong possibility that central5-HT1A receptors mediate the anxiolytic effects of the selective 5-HT1A ligands buspirone and ipsapirone, possibly via an agonist action on 5-HT cell body autoreceptors (Dourish et al., 1986a). However, caution must be exercised, since the major metabolite of buspirone, 1-(2-pyrimidyl)piperazine, is active in the Vogel conflict test, yet is devoid of significant affinity for the 5-HT1A recognition

92 Serotonin

site, and, unlike many other 5-HT1A-like agonist effects, the increase in punished drinking in the Vogel test is not blocked by pindolol (Gower and Tricklebank, 1988; Gower and Tricklebank, unpublished observations). More antagonist studies using different anxiolytic paradigms are required.

Interestingly, MDL 73005, a 5-HT1A ligand that does not contain a 1-(2-pyrimidyl)piperazine moiety, is also active in the Vogel test, an effect thatisblockedby8-0H-DPAT(Hiberteta/., 1988;Moseretal., 1988). This suggests that an antagonism of 5-HT1A receptors (possibly post-synaptic) is responsible for anxiolysis, and might explain why pindolol does not block the anxiolytic effects of buspirone.

Few studies have rigorously examined 5-HT2 receptor antagonists in rodent anxiolytic paradigms, although there is evidence that some beneficial effects may be obtained in the clinic (Arriaga et al., 1984). On the other hand, much of the evidence for the actual existence of 5-HT 3 receptors in the CNS is derived from the effects of selective 5-HT 3 receptor antagonists in rodent anxiolytic paradigms (light/dark box exploration in the mouse, social interaction in the rat; Tyers et al., 1987). Curiously, however, GR 38032F is inactive in the Vogel conflict test (Jones et al., 1987). Such a profile raises the questions of whether each of the varied anxiolytic tests employed is equally predictive of anxiolytic activity in man: the answer must await the outcome of clinical trials. Nevertheless, studies with selective 5-HT1A, 5-HT2 and 5-HT 3 ligands strongly support a role for 5-HT in anxiety, even though it is not clear which, if any, receptor is of dominating importance.

CONCLUSIONS

A wealth of behavioural evidence indicates the importance of 5-HT receptors in normal and abnormal brain function. However, in some areas, little progress has been made. Thus while there is little doubt about the effectiveness of 5-HT reuptake inhibitors in depression, little is known of the relative importance of 5-HT receptors that are subsequently stimulated, or down-regulated. Similarly, a plethora of animal experiments suggests a link between 5-HT and memory, but there is little in the way of hard evidence. With the continuing development of selective 5-HT receptor agonists and antagonists, perhaps the answers to these questions are not too far away.

REFERENCES

Amt, J., Hyttel, J. and Larsen, J. J. (1984). The citalopram/5-HfP-induced head shake syndrome is correlated to 5-HT2 receptor affinity and also influenced by other transmitters. Acta Pharmacal. Toxicol., 55, 363-372

Arriaga, F., Leitao, J., Mills, F. J., Padma, J., Ruiz, 1., Tropa, J. and Sousa, M.P. (1984). R55667, an effective non-benzodiazepine anxiolytic. Proc. 14th CINP Congress, 126


Bendotti, C. and Samanin, R. (1986). 8-Hydroxy-2-{di-n-propylamino)-tetralin {8-0H-DPAT) elicits eating in free-feeding rats by acting on central serotonin neurons. Eur. J. Pharmacol., 121, 147-150

Bevan,P., Tulp,M. T.M.andWouters, W. (1986).Are5-HT1Abindingsitesrelevant for the antihypertensive effects of D U 29373? Br. J. Phartnllcol., 89, 637P

Bockaert, J., Dumuis, A., Bouhelal, R., Sebben, M. and Cory, R. N. (1987). Piperazine derivatives including the putative anxiolytic drugs, buspirone and ipsapirone, are agonists at 5-HT1A receptors negatively coupled with adenylate cyclase in hippocampal neurons. Naunyn-Schmiedeberg's Arch. Pharmacol., 335, 588-592

Costall, B., Domeney, A.M., Naylor, R. J. and Tyers, M. B. (1987). Effects of the 5-HT3 receptor antagonist GR 38072F, on raised dopaminergic activity in the meso limbic system of the rat and marmoset brain. Br. J. Pharmacol., 92, 881-894

Cunningham, K. A., Callahan, P. M. and Appel, J. B. (1987). Discriminative stimulus properties of 8-hydroxy-2-(di-n-propylamino)tetralin {8-0H-DPAT): implications for understanding the actions of novel anxiolytics. Eur. J. Pharmacol., 138, 29-36

Davis, M. (1980). Neurochemical modulation of sensory-motor reactivity: acoustic and tactile startle reflexes. Neurosci. Biobehav. Rev., 4, 241-263

Davis, M., Cassella, J. V., Wrean, W. H. and Kehoe, J. H. (1986). Serotonin receptor subtype agonists: Differential effects on sensorimotor reactivity measured with acoustic startle. Psychopharmacol. Bull., 22, 837-843

Delorme, F., Froment, J. L. and Jouvet, M. {1966). Suppression du sommeil par Ia p-chloromethamphetamine et Ia pCP A. Comp. Rend. Soc. Bioi., 160, 2347-2349

Donohoe, T. P., Hutson, P. H. and Curzon, G. {1987). Blockade of dopamine receptors explains the lack of 5-HT stereotypy on treatment with the putative 5-HT1A agonist LY 165163. Psychopharmacology, 93, 82-86

Dourish, C. T., Hutson, P. H. and Curzon, G. {1986a). Putative anxiolytics 8-0H-DP AT, buspirone and TVX Q 7821 are agonists at 5-HT lA autoreceptors in the raphe nuclei. Trends in Pharmacol. Sci., 1, 212-214

Dourish, C. T., Hutson, P. H., Kennett, G. A. and Curzon, G. {1986b). 8-0H-DPAT-induced hyperphagia: the neural basis and possible therapeutic relevance. Appetite, 1, Suppl., 127-140

Dourish, C. T., Clark, M. L., Fletcher, A. and Iversen, S.D. {1989). Evidence that blockade of 5-HT 1 receptors elicits feeding in satiated rats. Psychopharmacology, 97, 54-58

Dugovic, C. and Wauquier, A. (1987). 5-HT2 receptors could be primarily involved in the regulation of slow-wave sleep in the rat. Eur. J. Pharmacol., 137, 145-146

Dzoljic, M. R., Saxena, P. R. and Ukponmwan, 0. E. {1986). Activation of '5-HT1-like' receptors stimulates wakefulness. Br. J. Pharmacal., 89, 522P

Fozard, J. R. and Tricklebank, M.D. {1983). Differential effects of putative 5-HT1 receptor agonists on responses to noxious stimuli. Naunyn-Schmiedeberg's Arch. Pharmacal., 324, Suppl., R20

Glennon, R. A., Young, R., Jacyno, J. M., Slusher, R. M. and Rosecrans, J. A. (1983). DOM-stimulus generalization to LSD and other hallucinogenic indolealkylamines. Eur. J. Pharmacol., 86, 453-459

Gower, A. J. and Tricklebank, M. D. (1988). Alpha 2-adrenoceptor antagonist activity may account for the effects of buspirone in an anticonflict test in the rat. Eur. J. Phartnllcol., 155, 129-137

Hagan, R. M., Butler, A., Hill, J. M., Jordan, C. C., Ireland, S. J. and Tyers, M. B. (1987). Effect of the 5-HT3 receptor antagonist, GR 38032F, on responses to injection of a neurokinin agonist into the ventral tegmental area of the rat brain. Eur. J. Pharmacol., 138, 303-305

94 Serotonin

Hi bert, M., Mir, A. K., Maghioros, G., Moser, P., Middlemiss, D. N., Tricklebank, M. D. and Fozard, J. R. (1988). The pharmacological properties of MDL 73005EF: A potent and selective ligand at 5-HT1A receptors. Br. J. Pharmacol., 93, 2P

Hjorth, S. and Carlsson, A. (1982). Buspirone: effects on central monoamine transmission - possible relevance to animal experimental and clinical findings. Eur. J. Pharmacol., 83, 299--303

Hutson, P. H., Donohoe, T. P. and Curzon, G. (1987). Neurochemical and behavioural evidence for an agonist action of 1-[2-(4-aminophenyl)ethyl]-4-(3-trifluoromethylphenyl)piperazine (L Y 165163) at central5-HT receptors. Eur. J. Pharmacol., 138, 215-223

ldzikowski, C., Mills, F. J. and Glennard, R. (1986). 5-Hydroxytryptamine-2 antagonist increases human slow wave sleep. Brain Res., 378, 164-168

Iversen, S. D. (1984). 5-HT and anxiety. Neuropharmacology, 23, 1553-1560 Jones, B. J., Oakley, N. R. and Tyers, M.D. (1987). The anxiolytic activity of GR

38032F, a 5-HT3 receptor antagonist, in the rat and Cynomolgus monkey. Br. J. Pharmacol., 90, 88P

Kennett, G. A., Dourish, C. T. and Curzon, G. (1987). 5-HT1B agonists induce anorexia at a postsynaptic site. Eur. J. Pharmacol., 141, 429-435

Lucki, 1., Nobler, M. S. and Frazer, A. (1984). Differential actions of serotonin antagonists on two behavioural models of serotonin receptor activation in the rat. J. Pharmacol. Exp. Ther., 228, 133-139

McKenney, J.D. and Glennon, R. A. (1986). TFMPP may produce its stimulus effects via a 5-HT1B mechanism. Pharmacol. Biochem. Behav., 24, 43-47

Messing, R. B., Fisher, L.A., Phebus, L. and Lytle, L. D. (1976). Interaction of diet and drugs in the regulation of brain 5-hydroxyindoles and the response to painful electric shock. Life Sci., 18, 707-714

Mir, A. K., Hibert, M., Tricklebank, M.D., Middlemiss, D. N., Kidd, E. J. and Fozard, J. R. (1988). MDL 72832: A potent, selective and stereospecific ligand with mixed agonist, antagonist properties at both central and periphera15-HT1A receptors. Eur. J. Pharmacol., 149, 107-120

Moser, P., Hibert, M., Middlemiss, D. N., Mir, A. K., Tricklebank, M. D. and Fozard J. R. (1988). Effects of MDL 73005EF in animal models predictive of anxiolytic activity. Br. J. Pharmacol., 93, 3P

Peroutka, S. J. (1985). Selective interaction of novel anxiolytics with 5-hydroxytryptamine1A receptors. Bioi. Psychiat., 20, 971-979

Ransom, R. W., Asarch, K. B. and Shih, J. C. (1986). [3H]1-[2-(4-Aminophenyl)ethyl]-4-(3-trifluoromethylphenyl)piperazine: a selective radioligand for 5-HT1A receptors in rat brain. J. Neurochem., 46, 68-75

Tricklebank, M. D., Farler, C. and Fozard, J. R. (1984). The involvement of subtypes of the 5-HT1 receptor and of catecholaminergic systems in the behavioural response to 8-hydroxy-2-( di-n-propylamino )tetralin in the rat. Eur. J. Pharmacol., 106, 271-282

Tricklebank, M. D., Farler, C., Middlemiss, D. N. and Fozard, J. R. (1985). Subtypes of the 5-HT receptor mediating the behavioural responses to 5-methoxy-N,N-dimethyltryptamine in the rat. Eur. J. Pharmacol., 117, 15-24

Tricklebank,M. D., Neill,J., Kidd,E. J. andFozard,J. R. (1987). Mediation of the discriminative stimulus properties of 8-hydroxy-2-( di-n-propylamine )tetralin by the putative 5-HT1A receptor. Eur. J. Pharmacol., 133, 47-56

Tyers, M. B., Costall, B., Domeney, A., Jones, B. J., Kelly, M. E., Naylor, R. J. and Oakley, N. R. (1987). The anxiolytic activities of 5HT3 antagonists in laboratory animals. Neurosci. Lett., Suppl. 29, S68.

12 5-HT3 Receptors in the Central Nervous System

M. B. Tyers, 1 B. Costalf and R. J. Naylor

1Neuropharmacology Department, Glaxo Group Research Limited, Ware, Hertfordshire SG 12 ODP, UK

2Postgraduate School of Studies in Pharmacology, University of Bradford, Bradford, WestYorkshireBD71DP, UK

INTRODUCTION

The presence of 5-HT3 receptor-mediated responses in peripheral tissues is well established. Their presence in the CNS is implied by the powerful behavioural effects produced by 5-HT3 receptor antagonists in laboratory animals (Costall et al., 1987a, b; Jones et al., 1987, 1988; Tyers et al., 1987). However, despite the high selectivity of action of these compounds for the 5-HT 3 receptor, there is no direct evidence that 5-HT 3 receptors are present in the brain. Three series of experiments have been conducted to address this issue: functional studies on central 5-HT pathways; effects on mesolimbic dopamine metabolism; and binding studies with a novel [3H]-Iabelled 5-HT3 receptor antagonist.

DISCRETE INJECTION STUDIES

The effects of parenterally administered 5-HT3 receptor antagonists in models of meso limbic hyperactivity and anxiety have been described (see above, and Hagan et al., 1987). Bilateral injections of amphetamine into the nucleus accumbens of rats causes a hyperactivity response through the release of dopamine. Co-administration of the 5-HT3 receptor agonist 2-methyl-5-HT (10 ~-tg) into the nucleus accumbens markedly exacerbates the hyperactivity response to amphetamine. Furthermore, administration of the selective 5-HT3 receptor antagonist GR 38032F (Butler eta/., 1988), together with 2-methyl-5-HT and amphetamine, into the nucleus accumbens not only antagonizes the effect of 2-methyl-5-HT but reduces the hyperactivity to levels below the amphetamine-alone response (Cos tall et a/., 1987b ). The intracerebral dose of GR 38032F used in this study (0.01-10

96 Serotonin

ng) is considerably below the parenteral dose (10 f..tg/kg i.p.) which is necessary to inhibit the amphetamine hyperactivity, indicating that the effect is through an action in the nucleus accumbens. The observation that 2-methyl-5-HT and GR 38032F produce opposite and mutually antagonistic effects strongly suggests that this action is mediated through 5-HT3 receptors.

In the mouse light-dark discrimination test for anxiolytic activity (Costall et al., 1987c), 2-methyl-5-HT (0.1-10 ng) injected into the dorsal raphe nucleus or the central amygdala (Figure 12.1) reduced behavioural activity in the light compartment, with corresponding increases in the dark compartment. Such effects are similar to those produced by anxiogenic substances such as 13-ethylcarboline. Higher doses of 2-methyl-5-HT given into the medial raphe nucleus have a similar effect, but there are no effects on behaviour in this test when injected into the nucleus accumbens or striatum. In contrast, the 5-HT3 receptor antagonists GR 38032F and ICS

80 • <{ w 60 a: <{

~ 40 a: c ~ .E 20

.... 0 ~ 80

60

40

20

0

DAN ACE DAN ACE DAN ACE DAN ACE 2 Methyi-5HT GA38032F IC$205-930 Diazepam

Figure 12.1 Anxiolytic activities of 2-methyl-5-HT, GR 38032F, ICS 205-930 and diazepam injected into the dorsal raphe nucleus (DRN) or central amygdala (ACE) in conscious mice. Activities are determined using the light-dark discrimination test (see text). The histograms show the mean number of rears for 6 mice per group in the light and dark areas. Hatched columns are the control values for each pair of data. The first pairs show data following injection of 1 ng of each drug as shown into the dorsal raphe nucleus, while the second pairs show data following injection into the central amygdala. Values of s.e. means are < 14 per cent of means (6 mice per

group). * Shows significant differences from respective controls (p < 0.001)

5-HT3 Receptors in the CNS 97

205-930 (Richardson et al., 1985), at doses of 0.01-1 ng, like diazepam, increase responding in the light compartment with corresponding decreases in the dark. As for 2-methyl-5-HT, these effects of GR 38032F and ICS 205-930 were induced by injection given into the dorsal raphe nucleus and the central amygdala (Figure 12.1), and (to a lesser extent) the medial raphe nucleus, with no effects being produced following administration into the nucleus accumbens or striatum. The doses used in each of these studies also suggest a selective action on 5-HT 3 receptors in the brain.

EFFECTS ON MESOLIMBIC DOPAMINE METABOLISM

The second series of experiments extends those reported by Hagan et al. (1987), who investigated the effects of GR 38032F on dopamine turnover in the nucleus accumbens. In these experiments, the stable neurokinin receptor agonist DiMe-C7 was injected discretely into the ventral tegmental area as described by Elliot eta/. (1986). The resulting hyperactivity, as well as the increased turnover of dopamine in the nucleus accumbens, were

Table 12.1 Effect of pre-treatment with GR 38032F (100 JJ.g/kg s.c.) on DiMe-C7-induced changes in dopamine metabolism in the nucleus accumbens of the rat

Treatment Metabolite (nucleusaccumbens)

s.c Ventral Ratio HVA tegmental area DOPACIDA (nglmg protein)

Vehicle Vehicle 0.129 4.19 (0.111-0.159) (2.47-7.08)

Vehicle DiMe-C7 0.227°[ + 76%] 7.62°[+82%] (0.188-0.274) (5.51-10.54)

GR 38032F Vehicle 0.137 5.02 (0.118-0.158) (3.69--6.82)

GR 38032F DiMe-C7 0.173b, c[+26%] 5.85 [+16%] (0.148-0.202) (3.05-11.24)

The rats were killed 50 min after injection into the ventral tegmental area of vehicle or DiMe-C7. Levels of dopamine (DA), dihydroxyphenylacetic acid (DOPA C) and homovanillic acid (HVA) are expressed as ng/mg tissue protein. Values in the table are geometric means with 95 per cent confidence limits for 5 or 6 animals. Percentage increases with respect to appropriate controls are also shown. Control levels (ng/mg tissue protein) were as follows: dopamine 85.19 (73.68--98.40); DOPAC, 10.99 (9.33--12.93).

op < 0.05 with respect to vehicle/vehicle. bp < 0.05 with respect to GR 38032F/vehicle. cp < 0.05 with respect to vehicle/DiMe-C7.

98 Serotonin

inhibited by GR 38032F (Table 12.1). In further experiments (Hagan, personal communication), ICS 205-930, MDL 72222 (Fozard, 1984) and the novel selective 5-HT3 receptor antagonist GR 65630 (see below), all mimicked the effects reported for GR 38032F. Another antagonist, BRL 43694 (Fake et al., 1987), also caused some inhibition of the hyperactivity response, but the effects were not dose-related and at higher doses the response was unaffected or even increased. This latter observation may well have been due to a direct locomotor stimulant effect observed for BRL 43694 in other experiments (Costall, unpublished observations).

These experiments not only demonstrate a potent effect of 5-HT 3 receptor antagonists on dopamine turnover in mesolimbic terminal areas, but also provide important information on the mechanisms involved in mesolimbic hyperactivity initiated in the ventral tegmental area in the rat. Since 5-HT3

antagonists have no overt effects on normal behaviour (except for BRL 43694; see above), it would seem that a 5-HT pathway is recruited during mesolimbic excitation. The anatomical location of this pathway remains to be established.

5-HT3 BINDING SITE

5-HT1 and 5-HT2 binding sites have been identified in the brain receptors (Peroutka and Snyder, 1979; Peroutka et al., 1981). Recent studies from Kilpatrick et al. (1987) have now demonstrated the presence of 5-HT3

binding sites in the rat brain. These studies, using the novel5-HT3 receptor antagonist [3H]-GR 65630 (85 Ci/mmol) as ligand, have identified a saturable, reversible, high-affinity binding site which appears to be present predominantly in cortical and limbic terminal areas, particularly the entorhinal cortex, amygdala and nucleus accumbens. Competition studies with 5-HT3 receptor antagonists show a high correlation between the affinities of these compounds for the [3H]-GR 65630 binding site and for antagonism of 2-methyl-5-HT-induced depolarization of the rat isolated vagus nerve (Figure 12.2). The agonists 5-HT and 2-methyl-5-HT also compete for [3H]-GR 65630 binding sites, but the Hill slopes of these compounds are significantly greater than 1. This deviation from unity may indicate positive co-operativity. The pattern of distribution of the 5-HT3

binding sites is particularly striking when the behavioural effects seen with 5-HT3 receptor antagonists are considered. For instance, the effects in putative models of anxiety may be mediated in limbic areas, such as the entorhinal cortex, amygdala and hippocampus. Effects in models of mesolimbic hyperactivity may be mediated through dopamine-containing areas such as the amygdala, frontal cortex and nucleus accumbens/olfactory tubercle. These areas contain the highest densities of 5-HT3 receptors.

5-HT3 Receptors in the CNS 99

0 11

10

(!l z iS 9 z • iii 0 eiCS205·930

~ 8 lr t • I!... eMDL72222 ~ 7 Q.

• 6

ecoCAINE

5

5 6 7 8 9 10 11

pA, (vs. 5-HT) RAT VAGUS NERVE

Figure 12.2 Correlation of pK; values of compounds for the [3H)-GR 65630 binding site and pA2 values for antagonism of S-liT-induced depolarization of the rat isolated vagus nerve preparation. Slope = 1.01; r = 0.91 (P < 0.001). mCPP = m-chlorophenylpiperazine. Unlabelled points represent data obtained for novel 5-IIT 3 receptor antagonists synthesized at Glaxo Group Research Ltd. The structure of GR 65630 is shown (top left). Vagus nerve data are from Ireland and Tyers (1987)

CONCLUSIONS

The experimental studies described above show that 5-HT3 receptors are present in the CNS, and that their distribution complements the potent effects reported for 5-HT3 receptor agonists and antagonists on animal behaviour.

REFERENCES

Butler, A., Hill, J. M., Ireland, S. J. and Tyers, M. B. (1988). Pharmacological properties of GR 38032F, a novel antagonist at 5-HT 3 receptors. Br. J. Pharmacol., 94, 397-412.

Costall, B., Domeney, A.M., Kelly, M. E., Naylor, R. J. and Tyers, M. B. (1987a). The antipsychotic potential of GR 38032F, a selective antagonist of 5-HT3 receptors in the central nervous system. Br. J. Pharmacol., 90, 89P

Costall, B., Domeney, A.M., Naylor, R. J. and Tyers, M. B. (1987b). Effectsofthe 5-IIT3 receptor antagonist, GR 38032F, on raised dopaminergic activity in the mesolimbic system of the rat and marmoset brain. Br. J. Pharmacol., 92, 881-894

100 Serotonin

Costall, B., Hendrie, C. A., Kelly, M. E. and Naylor, R. J. (1987c). Actions of sulpiride and tiapride in a simple model of anxiety in mice. Neuropharmacology, 26, 195--200

Elliott, P., Alpert, J. E., Bannon, M. J. and Iversen, S. D. (1986). Selective activation of mesolimbic and mesocortical dopamine metabolism in rat brain by infusion of a stable substance P analogue into the ventral tegmental area. Brain Res., 363, 145--147

Fake, C. S., King, F. D. and Sanger, G. J. (1987). BRL 43694: a potent and novel 5-HT3 receptor antagonist. Br. J. Pharmacol., 91, 335P

Fozard, J. R. (1984). MDL 72222: a potent and highly selective antagonist at neuronal5-hydroxytryptamine receptors. Naunyn-Schmiedeberg's Arch. Pharmacol., 326, 36-44

Hagan, R. M., Butler, A., Hill, J. M., Jordan, C. C., Ireland, S. J. and Tyers, M. B. (1987). Effect of the 5-HT3 receptor antagonist GR 38032F, on responses to injection of a neurokinin agonist into the ventral tegmental area of the rat brain. Eur. J. Pharmacol., 138, 303-305

Ireland, S. J. and Tyers, M. B. (1987). Pharmacological characterisation of 5-hydroxytryptamine-induced depolarisation of the rat isolated vagus nerve. Br. J. Pharmacol., 90, 229-238

Jones, B. J., Oakley, N. R. and Tyers, M. B. (1987). The anxiolytic activity of GR 38032F, a 5-HT3 receptor antagonist, in the rat and Cynomolgus monkey. Br. J. Pharmacol., 90, 88P

Jones, B. J., Costall, B., Domeney, A.M., Kelly, M. E., Naylor, R. J., Oakley, N. R. and Tyers, M. B. (1988). The potential anxiolytic activity of GR 38032F, a 5-HT3 receptor antagonist. Br. J. Pharmacol., 93, 985--993

Kilpatrick, G. J., Jones, B. J. and Tyers, M. B. (1987). Identification and distribution of 5-HT3 receptors in rat brain using radio ligand binding. Nature, 330, 746-748

Peroutka, S. J. and Snyder, S. H. {1979). Multiple serotonin receptors: differential binding of [3H]-5-hydroxytryptamine, [3H]-lysergic acid diethylamide and [3H)-spiroperidol. Mol. Pharmacol., 16, 687--699

Peroutka, S. J., Lebovitz, R. M. and Snyder, S. H. (1981). Two distinct central serotonin receptors with different physiological functions. Science, 212, 827-829

Richardson, B. P., Engel, G., Donatsch, P. and Stadler, P. A. (1985). Identification of serotonin M-receptor subtypes and their specific blockade by a new class of drugs. Nature, 316, 126-131

Tyers, M. B., Costall, B., Domeney, A.M., Jones, B. J., Kelly, M. E., Naylor, R. J. and Oakley, N. R. (1987). The anxiolytic activities of 5-HT3 antagonists in laboratory animals. Neurosci. Lett., Suppl. 29, S68

13 Serotoninergic Function and Aggression in

Animals

P. Bevan, B. Olivier, J. Schipper and!. Mos

Department of Pharmacology, Duphar BV, PO Box 2, 1380 AA Weesp, The Netherlands

INTRODUCTION

Serotonin (5-hydroxytryptamine; 5-HT) has for some time been implicated in the control of aggression. Early work on 5-HT and aggression indicated that general 5-HT activation decreased aggression, whereas an overall inactivation of 5-HT by various means enhanced it (Valzelli, 1981).

The anatomical distribution and localization of cell groups and pathways of 5-HT in the CNS and their differential projections (Peroutka et al., 1986) are, however, strong arguments against a simple general inhibitory role of 5-HT in any given aggression paradigm. Moreover, the recent differentiation of 5-HT binding site subtypes (Peroutka et al., 1986) and their putative functional roles are suggestive of functional differentiations in the 5-HT systems in the CNS with regard to different kinds of aggressive behaviour. This review therefore focuses on the role that different types of serotoninergic receptors may play in aggression.

Apart from a general modulatory role, it may well be possible that the role of 5-HT in aggression depends upon the type of paradigm used. Therefore several aggression paradigms were used in our ethological (animal behavioural) studies (Olivier et al., 1987), reflecting diverse aspects of agonistic behaviour including offensive and defensive types, and also predatory aggression. Moreover, different species and genders have been used. By using psyehoactive drugs with several different serotoninergic mechanisms of action in these various agonistic behaviour paradigms, it was hoped that it would prove possible to give a more precise description of the mode of action of 5-HT in agonistic behaviour, and to delineate whether 5-HT is involved in a more general way in aggression or if its role depends on the paradigm used.

In the following discussion, the effects of several drugs with diverse

102 Serotonin

serotoninergic neurochemical and functional profiles will be exemplified in the various agonistic paradigms used. This is prefaced by a short description of the neurochemical profile of these drugs.

NEUROCHEMICAL CHARACTERIZATION

Table 13.1 shows the affinities of the drugs used in the aggression studies for 5-HTrlike and 5-HT2 receptors, as determined in our laboratory. Although some uncertainties remain over the functional relevance of the 5-HT1

binding site subtypes (see Bradley et al., 1986), it is convenient in this discussion to distinguish between drugs showing affinity for the 5-HT lA

receptor (defined by rat cortex binding of 8-hydroxy-2-( di-npropylamino)tetralin; 8-0H-DPAT), the 5-HT18 receptor (defined by rat

Table 13.1 Affinities for 5-HT1 and 5-HTz receptors of the compounds used in the study

5-HT TFMPP Eltoprazine Fluprazine RU 24969 5-MeODMT Quipazine 8-0H-DPAT Buspirone Ipsapirone Fluvoxamine Methysergide Ritanserin Fenfluramine GR 38032F MDL 72222

8.4 6.7 7.4 6.4 8.1 8.2 5.6 8.6 7.8 8.3

<5.2 7.7 6.1 5.7

<5.2 <5.2

7.7 1.0 7.7 0.7 7.6 0.4

<5.5 0 8.0 0.8 5.5 0.9 5.5 0

<5.0 0 <6.0 0 <6.0 0

<6.0 0 <6.0 0

5.7 0 <6.0 0 <6.0 0

6.5 1.0 6.4 0.6 5.6 0

<6.0 0 6.1 0.3 5.4 0.3

<6.6 0 <5.0 0 <6.6 0 <6.0 0 <5.6 0

7.4 0 <6.0 0 <6.0 0 <6.0 0

5-HT/

pK;

5.9 6.1 5.8 5.8 5.8 5.6 6.0

<5.2 6.0 5.6 5.2 7.8 8.5

<5.2 <5.3 <5.3

a5-HTtA affinity (as pKi values), determined from binding experiments using 8-0H-DPAT. For details, see Gozlan et al. (1983).

b5-HT1B affinity (pDz) and intrinsic activity (a), determined from measurement of K+-evoked [-'H)-5-HT release from rat cortex slices. Neurona15-HT uptake was blocked during superfusion by fluvoxamine (10 J.lM). For details of methods, see Engel et a/. (1986). Where a = 0, the value in the pDz column is the pAz.

c5-HT1c affinity (pDz) and intrinsic activity (a), determined from measurement of stimulated inositol phosphate turnover in slices of pig choroid plexus. For details of methods, see Conn et al. (1986). Where a= 0, the value in the pDz column is the pAz.

d5-HTz affinity (as pKi values), determined from binding experiments using [3H)-spiperone. For details of methods, see Creese and Snyder (1978).

Serotoninergic Function and Aggression 103

cortex 5-HT release modulation; Engel et al., 1986), and the 5-HT1c receptor (defined by stimulation of inositol phosphate turnover in rat choroid plexus; Conn et al., 1986), respectively. The serotoninergic properties of the drugs used in the study are based on the profile as presented in Table 13.1.

Thus, agonists having specificity for both the 5-HT tA site (8-0H-DPAT) and the 5-HT18 site (trifluoromethylphenylpiperazine; TFMPP) have been included. Apart from TFMPP and RU 24969, which appear to be weak partial agonists at the 5-HT1c receptor, no potent 5-HT1c agonists have been tested. Quipazine has been included as an example of an agonist with affinity for 5-HT2 sites, although it is not very potent or specific.

The effects of some 5-HT receptor antagonists have also been studied, although ethological methodology is unfortunately not suitable for meaningful studies of interactions. The 5-HT1 receptor antagonist methysergide and the 5-HT2 receptor antagonist ritanserin are included, as well as buspirone, a compound often claimed to be an agonist, but with clear 5-HT1A antagonistic properties in some systems (Fozard and Kilbinger, 1985). The 5-HT3 receptor antagonists MDL 72222 and GR 38032F are included for completeness.

Two drugs which modulate 5-HT neurotransmission other than through direct receptor interaction have also been investigated: fluvoxamine, which is a specific 5-HT uptake blocker, and fenfluramine, which releases 5-HT from storage granules.

The behavioural profiles of the above types of drugs on agonistic behaviour have also been compared with those of the serenics fluprazine (D U 27716) and eltoprazine (DU 28853), drugs thought to exert their effects via 5-HT1-like receptors.

ISOLATION-INDUCED AGGRESSION AND INTER-MALE AGGRESSION IN MICE

When a male mouse is isolated for some time and then confronted with a male intruder, heavy fighting may occur, including threat, chasing and biting (Miczek, 1987). The behaviour of aggressive isolated males has been considered as offensive; effects of drugs on this behaviour can be studied using ethological methodology (Olivier et al., 1987). A simple first approach is determination of an ED50 for lowering the number of aggressive interactions. A substantial number of drugs inhibit aggression (Table 13.2), but it is unclear from this paradigm which behavioural inhibitory mechanisms are involved. This was further investigated by means of extensive ethological observations (Olivier et al., 1987). By dividing the behaviour into several categories, namely exploration, social interest, aggression, defence, avoidance and inactivity, a general view of the main effects of the drugs can be obtained. Such an analysis shows that several

104 Serotonin

Table 13.2 Effects of several serotoninergic drugs with different mechanisms of action in various animal paradigms in rats and mice

IIA IMA RI EBS MA MK FID DB Putative serotoninergic Drug mouse mouse rat rat rat rat mouse rat mechanism of action TFMPP J, (l) (l) (l) (l) (l) (l) 0 ~-HT1B11c agonist (partial) Eltoprazine J, (l) (l) (l) (l) (l) 0 0 5-HT IAIIB agonist (partial) Fluprazine J, (l) (l) (l) (l) (l) (l) 0 Weak 5-HT1AJ1BJ2 agonist RU 24969 J, (l) (l) (l) J, - 5-JITIA/IB/IC agonist 5-MeODMT J, J, J, J, - 5-HT IA agonist Quipazine 0 0 J, J, J, J, J, - Weak 5-HT 112 agonist 8-0H-DPAT J, J, J, 0 J, 0 0 - 5-HT1A agonist Buspirone 0 0 J, J, J, - 5-HT1A agonist lpsapirone 0 0 J, 0 - 5-HT1A agonist/antagonist Fluvoxamine J, J, J, J, (l) J, - 5-HT reuptake blocker Methysergide 0 0 0 0 0 - 5-HT 112 antagonist Ritanserin 0 0 0 0 - 5-HT2 antagonist Fenfluramine J, J, J, J, - 5-HT releaser GR 38032F 0 0 0 0 - 5-HT3 antagonist MDL 72222 0 0 0 - 5-HT3 antagonist (l) = specific decrease; J, = non-specific decrease; o = no effect;- = not tested. IIA = isolation-induced aggression; IMA = inter-male aggression; RI = resident-intruder; EBS = electrical stimulation of the brain; MA = maternal aggression; MK = mouse killing; FID = footshock-induced defence; DB = defensive behaviour.

drugs exert a non-specific anti-aggressive action, because the inhibitory effects on aggression are accompanied by a simultaneous decrease in, for example, social interest, as well as increases in inactivity which can be indicative of a non-specific reduction in aggression, e.g. via sedation, muscle relaxation or motor disturbances (Olivier et al., 1984, 1987; Olivier and Mos, 1986). Serenics (fluprazine, eltoprazine), TFMPP and RU 24969, however, exert a quite specific behavioural profile: reduction in aggression, without any concomitant inactivity, or reductions in exploration and social interest (which is even enhanced). GR 38032F, up to a dose of 10 mglkg, was not active.

RESIDENT-INTRUDER AGGRESSION IN RATS

Introduction of a strange male rat into the territory of another male rat (resident) evokes a rich natural agonistic repertoire, which can be used in an ethopharmacological approach (Miczek, 1987; Olivier eta/., 1987). Analysis shows (Table 13.2) that quite a number of drugs exert non-specific anti-aggressive actions caused by sedation: 5-methoxy-N,Ndimethyltryptamine (5-MeODMT), quipazine, buspirone and fenfluramine. This non-specific anti-aggressive activity is evident as increases in inactivity, concomitant with decreases in social interest and exploration. Detailed ethological analysis (Olivier et al., 1984) also reveals in this test the specific nature of the anti-aggressive action of serenics (fluprazine,


eltoprazine) and TFMPP, because there is no decrease in social interest, a slight increase in exploration, and no increase in inactivity.

BRAIN-STIMULATION-INDUCED AGGRESSION IN RATS

Electrical stimulation of parts of the hypothalamus of rats may evoke aggression; locomotion, teeth chattering and switch-off behaviour can also be evoked via the same electrodes (Kruk et al., 1987). Measurement of drug effects on these parameters indicates the specificity of the behavioural effects on aggression. By estimating threshold current intensities for the different behaviours, specific anti-aggressive effects of drugs can be determined (i.e. enhancing aggression and teeth-chattering thresholds) simultaneously with effects on other behaviours, primarily locomotion thresholds (Olivier and Mos, 1986).

Serenics and TFMPP have a specific profile in this paradigm (Table 13.2). These agents enhance aggression thresholds without enhancing locomotion or switch-off. 8-0H-DPAT has no effect, up to doses which almost completely incapacitate animals. Quipazine and fluvoxamine have a non-specific anti-aggressive influence (indicated by a concomitant enhancement of locomotion thresholds).

MATERNAL AGGRESSION IN RATS

Lactating female rats attack intruders intensively with very short attack latencies (Olivier et al., 1987). The lactation period, between 3 and 12 days post-partum, is a relatively stable period for measuring aggression, using females as their own controls. The behaviour performed by lactating females against intruders is quite different from that in male aggression paradigms, as, apart from aggression, pup-care also occurs. Therefore drug effects were evaluated after extensive ethological analysis of the behavioural 'make-up' of this paradigm (Olivier et al., 1987). Table 13.2 summarizes the findings, showing that this test clearly differentiates between drugs: sedation (increased inactivity, disruption of pup-care) or inattentiveness for the pups occurred in a number of cases (e.g. quipazine, 8-0H-DPAT, ipsapirone, fenfluramine), whereas serenics, TFMPP and RU 24969 exert specific behavioural effects. GR 38032F and MDL 72222, up to doses of 10 mg/kg, had no behavioural effects.

MOUSE KILLING BY RATS

Although mouse killing reflects aspects of aggression other than

106 Serotonin

intraspecific agonistic behaviour (predation), it is of value in determining psychoactive properties of drugs. By measuring sedation concomitantly, a simple rating of the specificity of the inhibitory effect of a drug is obtained (Olivier et al., 1987). A number of drugs (8-0H-DPAT, GR 38032F, ipsapirone, methysergide and ritanserin) do not inhibit mouse killing (except at very high doses which completely debilitate the animals), whereas all other drugs do, although sometimes in a non-specific way (quipazine, buspirone, fluvoxamine), as indicated in Table 13.2.

DEFENSIVE BEHAVIOUR

When animals are attacked, they normally defend themselves quite adequately, or if necessary flee. This aspect of agonistic behaviour is quite important for species survival, and anti-aggressive drugs preferably should not interfere with such defence/flight capacities of animals. Two paradigms to test defence/flight effects of drugs have been used: footshock-induced defence, in which pairs of mice are shocked electrically via their paws and consequently perform defensive behaviours; and a more natural intruder paradigm (Olivier and Mos, 1986) in rats.

Although only a limited number of drugs have been tested (Table 13.2), the available data suggest that serenics do not affect the defensive capabilities of animals. The decrease observed in footshock-induced fighting after TFMPP and fluprazine presumably can be ascribed to the analgesic properties of these drugs, which is absent in eltoprazine.

CONCLUSIONS

The findings presented show that a variety of drugs which can modulate 5-HT neurotransmission also share in common the ability to reduce agonistic behaviour in mice and rats. The anti-aggressive property seems to be a feature of agonists of the 5-HT 1-like receptor, although good examples of 5-HT2 and 5-HT3 receptor agonists have not been available for testing.

Anti-aggressive activity is present in drugs with affinity for 5-HT1A

receptors. Marked differences in the behavioural profiles are apparent, however, if for example the profiles of 5-MeODMT and 8-0H-DPAT are compared. Drugs with intrinsic activity at the 5-HT 18 receptor seem to show a much more consistent pattern of activity. TFMPP, for example, is a potent and specific inhibitor of agonistic behaviour.

Anti-aggressive activity is often a consequence of the sedative properties of a drug. However, some drugs, notably the serenics, specifically suppress agonistic behaviour without concomitant inhibitory effects on locomotion, for example. All serenics share with TFMPP an intrinsic activity at the


5-HT18 subtype. Although TFMPP is also a weak partial agonist at the 5-HT 1c site, this is not a property shared by either fluprazine or eltoprazine.

The available evidence, then, points to a specific modulatory role of the 5-HT18 receptor site in aggression. Relatively specific 5-HT18 agonists (TFMPP) or mixed 5-HT tAJB agonists (fluprazine, eltoprazine, RU 24969) exert specific anti-aggressive effects in several aggression paradigms, whereas drugs with other mechanisms of action either have no, or non-specific, anti-aggressive effects.

There are claims that the 5-HT 18 receptor may be peculiar to rodents and may not be present in other species such as pigs and primates (Hoyer et al., 1985). Nevertheless, specific anti-aggressive properties of the serenic eltoprazine have been demonstrated in our laboratory in both pigs (Olivier et al., 1987), and monkeys (Bevan et al., unpublished observations). Although it is too early to exclude categorically the involvement of other 5-HT sites in aggression, a 5-HT1-like receptor (defined as 5-HT18 in rodents) seems an attractive candidate for the substrate of agonistic behaviour. The available evidence suggests that the differential role of the various 5-HT receptor mechanisms holds for most, if not all, aggression paradigms used. This may point to a general role of a 5-HT 1-like receptor subtype in aggression.

REFERENCES


Conn, P. J., Sanders-Bush, E., Hoffman, B. J. and Hartig, P.R. (1986). A unique serotonin receptor in choroid plexus is linked to phosphoinositide hydrolysis. Proc. Nat[. Acad. Sci. U.S.A., 83, 4086-4088

Creese, I. and Snyder, S. H. (1978). ([3H])Spiroperidollabels serotonin receptors in rat cerebral cortex and hippocampus. Eur. J. Pharmacol., 49, 201-202

Engel, G., Gothert, M., Hoyer, E., Schlicker, E. and Hillenbrand, K. (1986). Identity of inhibitory presynaptic 5-hydroxytryptamine (5-HT) autoreceptors in the rat brain cortex with 5-HTlB binding sites. Naunyn-Schmiedeberg's Arch. Pharmacol., 332, 1-12

Fozard, J. R. and Kilbinger, H. (1985). 8-0H-DPAT inhibits transmitter release from guinea-pig enteric cholinergic neurons by activating 5-HT tA receptors. Br. J. Pharmacol., 86, 601P

Gozlan, H., El Mestikawy, S., Pichat, L., Glowinsky, J. and Hamon, M. (1983). Identification of presynaptic serotonin autoreceptors using a new ligand: [3H)-PAT. Nature, 305, 140-142

Hoyer, D., Engel, G. and Kalkman, H. 0. (1985). Molecular pharmacology of 5-HT1 and 5-HT2 recognition sites in rat and pig brain membranes: radioligand binding studies with [3H]5-HT, [3H]8-0H-DPAT, P25I) iodocyanopindolol, [3H)mesulergine and [3H)ketanserin. Eur. J. Pharmacol., 118, 13-23

108 Serotonin

Kruk, M. R., VanderPoel, A.M., Lammers, J. H. C. M., Hagg, T., de Hey, A.M. D. M. and Oostwegel, S. (1987). Ethopharmacology of hypothalamic aggression in the rat. In Olivier, B., Mos, J. and Brain, P. F. (Eds), Ethopharmacology of Agonistic Behaviour in Humans and Animals, Martinus Nijhoff, Dordrecht, pp. 33-45

Miczek, K. A. (1987). The psychopharmacology of aggression. In Iversen, L. L., Iversen, S.D. and Snyder, S. H. (Eds), Handbook of Psychopharmacology, Vol. 19, Behavioural Pharmacology, Plenum Press, New York, pp. 183-328

Olivier, B. and Mos, J. (1986). Serenics and aggression, Stress Medicine, 2, 197-209 Olivier, B., VanAken, H., Jaarsma, I., van Oorschot, R., Zethof, T. and Bradford,

L. D. (1984). Behavioural effects of psychoactive drugs on agonistic behaviour of male territorial rats (resident-intruder paradigm). In Miczek, K. A., Kruk, M. R. and Olivier, B. (Eds), EthopharmacologicalAggression Research, Alan R. Liss, New York, pp. 137-156

Olivier, B., Mos, J., Schipper, J., Tulp, M., Beekelmans, B. and Bevan, P. (1987). Serotonergic modulation of agonistic behaviour. In Olivier, B., Mos, J. and Brain, P. F. (Eds), Ethopharmacology of Agonistic Behaviour in Humans and Animals, Martinus Nijhoff, Dordrecht, pp. 162-186

Peroutka, S. J., Heuring, R. E., Mauk, M.D. and Kocsis, J.D. (1986). Serotonin receptor subtypes: biochemical, physiological, behavioral, and clinical implications. Psychopharmacol. Bull., 22, 813-817

Valzelli, L. (1981). Psychopharmacology of aggression: an overview. Int. Pharmacopsychiat., 16, 39-48

14 Behavioural Actions of 5-Hydroxytryptamine:

An Overview

S. Z. Langer

Department of Biology, Synthelabo Recherche (LERS), 58, rue de Ia Glaciere, 75013 Paris, France

INTRODUCTION

Many open questions persist concerning the precise role of serotoninergic mechanisms in behavioural control. One of the major reasons for incomplete understanding has been the use of pharmacological tools devoid of selectivity for the three populations of 5-HT receptors described so far, namely 5-HT1-like, 5-HT2 and 5-HT3 , and the various subtypes of 5-HT1 binding sites thought to exist in the CNS. With the availability of novel drugs showing appreciable degrees of affinity and selectivity for these sites, it is now possible to re-examine the functional roles of 5-hydroxytryptamine (5-HT). A general survey of the behavioural actions of 5-HT has been provided by Mark Tricklebank; detailed data on specific aspects were presented by Michael Tyers and by Paul Bevan, and their colleagues. The following paradigms are of interest in relation to several 5-HT receptor subtypes.

MOTOR RESPONSES

There is strong evidence that one component of the 5-HT behavioural syndrome, reciprocal forepaw treading in the rat, reflects activation of the 5-HT1A receptor. Drugs such as 8-hydroxy-2-(di-n-propylamino)tetralin (8-0H-DPAT), 5-methoxy-N,N-dimethyltryptamine, MDL 72832, buspirone and gepirone all induce forepaw treading. The lack of effect of some putative 5-HT1A agonists, such as 1-(2-[4-aminophenyl)ethyl)-4-(3-trifluoromethylphenyl)piperazine (P APP), flesinoxan or ipsapirone, on this behaviour may be due to the partial agonist properties of these compounds.

It is well-established that the ability of 5-HT receptor antagonists to

110 Serotonin

inhibit the head shake response to 5-HT receptor agonists correlates significantly with their affinity for the 5-HT2 receptor. This behaviour is therefore specific for the activation of this 5-HT receptor type.

The hyperactivity induced by injection of dopamine or amphetamine into the nucleus accumbens of rodents is antagonized by selective 5-HT 3 receptor antagonists ( GR 38032F and ICS 205-930), while the 5-HT 3 receptor agonist 2-methyl-5-HT potentiates amphetamine-induced hyperactivity. These results, described in detail by Tyers, support the view that a 5-HT pathway with 5-HT3 . receptors is recruited during mesolimbic excitation. The anatomical location of this pathway, however, remains to be established.

SENSORY SYSTEMS

The involvement of 5-HT in altering stimulus reactivity and reflex excitability can be studied by monitoring the acoustic startle reflex in the rat. The selective 5-HT1A receptor agonist 8-0H-DPAT increases the startle amplitude, while the putative 5-HT 18 receptor agonist mchlorophenylpiperazine (m-CPP) has a depressant action. The availability of selective ligands for 5-HT 1 receptor subtypes may help to clarify this question.

It is thought that 5-HT systems exert an inhibitory influence on nociceptive processes, and it is well documented that increasing serotoninergic tone reduces the responsiveness to painful stimuli. However, little is known of the receptor subtypes involved. Identification of the post-synaptic 5-HT receptor involved might be useful in the development of novel therapeutic agents for the management of pain.

Drug discrimination has been observed for the 5-HT1A, 5-HT18 and 5-HT2 receptors. This is an indication of potential involvement of these receptors on interoceptive stimuli (used to select psychoactive drugs).

FOOD INTAKE

There is good evidence for the involvement of 5-HT in decreasing food intake (based on the effects of inhibitors of 5-HT uptake, or 5-HT -releasing agents such as fenfluramine). In addition, it was reported that activation of post-synaptic 5-HT 18 receptors decreases food intake in rodents. On the other hand, the 5-HT 1A receptor agonist-mediated increase in food intake is probably associated with an effect on the autoreceptors of 5-HT -containing neuronal cell bodies.

The lack of effect of selective 5-HT2 and 5-HT3 receptor antagonists on food intake also supports the view that post-synaptic 5-HT1-like receptors play an important role in the control of feeding in the rat.

Overview: Behavioural Actions 111

SLEEP

While it is now well established that 5-HT is implicated in the control of sleep, the receptor subtypes have not yet been fully characterized. It has been reported that the 5-HT2 receptor antagonist ritanserin increases slow-wave sleep in humans, and antagonizes the disruption of slow-wave sleep induced in the rat by the 5-HT2 receptor agonist dimethoxymethylphenylaminopropane (DOM). However, additional work is necessary to examine the role of other 5-HT receptors.

ANXIETY

There is increasing evidence for the view that central 5-HT1A receptors mediate the anxiolytic effects of drugs such as buspirone and ipsapirone, and the possibility of an agonist action on 5-HT cell body autoreceptors has been proposed. However, some recent observations suggest that an antagonism at the level of post-synaptic 5-HT1A receptors may be responsible for anxiolysis.

Recent studies support the view that 5-HT 2 or 5-HT 3 receptor antagonists could be active in rodent anxiolytic paradigms, in spite of some inconsistencies reported among the different tests. Details of studies with 5-HT3 receptor antagonists were presented by Tyers. The potential role of 5-HT2 and 5-HT3 receptors in anxiety remains to be determined.

DEPRESSION

The clinical effectiveness of several inhibitors of 5-HT uptake in the treatment of depression is well established. It is, however, an open question as to whether specific post-synaptic 5-HT receptors are directly involved in the antidepressant action obtained through the chronic inhibition of neuronal uptake of 5-HT (which is the main mechanism for the inactivation of released transmitter). It has been reported that 8-0H-DPAT, buspirone and ipsapirone are active in the restraint stress animal model, which may suggest antidepressant activity. This action is probably mediated by 5-HT IA

receptors.

AGGRESSION

A variety of drugs which can modulate 5-HT neurotransmission also share in common the ability to reduce aggressive behaviour in several paradigms in mice and rats, as described in detail by Bevan. The anti-aggressive property

112 Serotonin

seems to be a feature of agonists of the 5-HT1-Iike receptor, although selective 5-HT2 and 5-HT3 receptor agonists have not been extensively tested.

Relatively selective 5-HT 18 receptor agonists exert specific antiaggressive effects in several behavioural models of aggression, whereas drugs acting on other 5-HT receptors are either inactive or possess non-specific, anti-aggressive effects (e.g. due to sedation). One important question is that the 5-HT 18 receptor may be peculiar to rodents and may not be present in other species such as pigs and primates. It is therefore possible that the 5-HT 1-like receptor (defined as 5-HT 18 in rodents) could be a target for agonist drugs with anti-aggressive action. Confirmation that this activity can be obtained in man, however, awaits clinical data. with the appropriate selective compounds.

S-HT3 RECEPTORS AND BEHAVIOUR

The presence in the CNS of specific recognition sites for the 5-HT3 receptor antagonist [3H]-GR 65630, and the predominance of these sites in cortical and limbic terminal areas, as described by Tyers, is of particular interest in connection with behavioural effects in putative models of anxiety and of meso limbic hyperactivity. The demonstration of the apparent presence of functional5-HT3 receptors in the CNS should further encourage studies on animal behaviour with selective agonists and antagonists at this 5-HT receptor.

LEARNING AND MEMORY

The possible role of serotoninergic systems in learning and memory is at present rather speculative. A preliminary study reported that 8-0H-DPAT impairs performance in the water maze task, suggesting an effect on spatial learning.

CONCLUSIONS

A wealth of behavioural evidence points towards the importance of 5-HT receptors in normal and abnormal brain functions. With the continuing development of selective agonists and antagonists for the different 5-HT receptors, answers to the current questions in these fields are likely to become available. At the same time, drug discovery is likely to lead to novel therapeutic applications.

PART IV CARDIAC, VASCULAR AND OTHER SMOOTH

MUSCLE ACTIONS

15 Cardiac Actions of 5-Hydroxytryptamine

P.R. Saxena

Department of Pharmacology, Faculty of Medicine, Erasmus University Rotterdam, PO Box 1738, 3000 DR Rotterdam, The Netherlands

INTRODUCTION

Most animals respond to the i. v. administration of 5-hydroxytryptamine (5-HT) initially with a decrease in heart rate, generally via similar mechanisms and receptors. In isolated heart preparations or in whole animals without vagal influences, 5-HT usually increases heart rate and, sometimes, cardiac contractility, but a variety of mechanisms and receptors appear to be involved in different species. Although the cardiac effects of 5-HT have been studied extensively, the precise nature of the receptors involved is emerging only now (Saxena, 1986). This is particularly due to the availability of selective drug tools and the characterization of 5-HT receptor subtypes into 5-HT1-like, 5-HT2 and 5-HT3 receptors (see Bradley et al., 1986). This present article reviews the mechanisms and the nature of the 5-HT receptors mediating the cardiac effects of 5-HT (Table 15.1).

BRADYCARDIAC EFFECTS OF 5-HT

The bradycardia caused by 5-HT lasts for a few seconds and is due to stimulation of chemoreceptors mainly in the left ventricular myocardium. Both the afferent and efferent limbs of the ensuing von Bezold-Jarisch reflex are located in the vagus. MDL 72222 (Fozard, 1984a; Saxena et al., 1985b) and ICS 205-930 (Richardson et al., 1985) are selective antagonists; the mediation of the responses is by 5-HT3 receptors present on afferent cadiac nerves.

5-HT can elicit bradycardia in rabbit isolated heart preparations depleted of catecholamines. This response is due to acetylcholine release and is also mediated by 5-HT3 receptors (Fozard, 1984b). These receptors may not be present on post-ganglionic cholinergic fibres, as suggested by Fozard (1984b), but are probably located on intramural cardiac parasympathetic

116 Serotonin

Table 15.1 Mechanisms involved in the heart rate responses to 5-HT in different species Species Condition Receptor LoCIJtion Remarks Bradycardia Rat Intact 5-Hf3 Aff.N., heart von Bezold-Jarisch reflex Cat Intact 5-HT3 Aff.N., heart von Bezold-Jarisch reflex Rabbit Isolated, Res. 5-Hf3 Heart Parasympathetic ganglia? Cardiostimulation Molluscs Isolated not known Heart 5-Hf 1-like receptor? Rat Ganglion-block 5-Hfa Heart

Pithed Heart 'Tyramine-like' action* Guinea-pig Isolated fl-adr Heart Indirect action?

Spinal Heart? 'Tyramine-like' action Hamster Isolated n.e. Heart Direct + CA release Rabbit Isolated 5-Hf3 Sym.N., heart CA release Cat Isolated 5-Hf1-like Heart Atria

Isolated ? Heart Papillary muscle Spinal 5-Hf1-like Heart Subtype not known

Dog Isolated Heart 'Tyramine-like' action* Ganglion-block 5-Hf2 Adrenal medulla CA release

Pig Intact ? Heart New receptor type? Man Intact n.e. Heart

Aff.N., afferent nerves; ~adr, ~adrenoceptor; CA, catecholamine; n.e., not established; Res., reserpinized; Sym.N., sympathetic nerve; *,high concentration of 5-HT is needed.

ganglia, since 5-HT is known to excite parasympathetic ganglia by an action on 5-HT3 receptors (Saxena et al., 1985a).

CARDIOSTIMULATORY EFFECTS OF 5-HT

Invertebrate Species

The hearts of certain lamellibranch and gastropod species (Mercenaria mercenaria, Tapes watlingi, Patella vulgata, Helix, Aplysia) are extremely sensitive to 5-HT (threshold concentration 0.1 nM; for references, see Walker, 1985). In the Mercenaria heart, 5-HT increases the rate and amplitude of contraction. Analogues of 5-HT have the following order of activity (equipotent molar ratio; 5-HT = 1): N, N-dimethyl-5-HT (0.028), N-methyltryptamine (3.7), a-methyl-5-HT (6.0), N,N-diethyltryptamine (7.9), a-methyltryptamine (8.6), N-ethyltryptamine (9.1), tryptamine (9.9), N,N-dimethyltryptamine (10.7), 2-methyl-5-HT (31.4). The effect of 5-HT is antagonized by methysergide and 2-bromolysergide; however, lysergic acid diethylamide (LSD) mimics 5-HT with an incredible potency (threshold concentration 1 fM). There is evidence that 5-HT may be the excitatory transmitter in the Mercenaria heart; the effects of stimulation of the cardioaccelerator nerve are reduced by methysergide, 2-bromolysergide

Cardiac Actions of 5-HT 117

and reserpinization, and the perfusate shows the presence of 5-HT upon nerve stimulation.

The exact nature of the 5-HT receptors in the mollusc heart is uncertain, but the high sensitivity for 5-HT and LSD, and the blockade of their effects by methysergide and 2-bromolysergide, suggest that 5-HT1-like receptors are involved.

Rat

The tachycardiac response to 5-HT is blocked by cyproheptadine and ketanserin, and thus appears to be mediated by 5-HT2 receptors present in the heart (Saxena and Lawang, 1985). In addition, high doses of 5-HT may release catecholamines by a 'tyramine-like' action (Gothert et al., 1986).

Guinea-pig

The mechanism of the cardiostimulatory action of 5-HT in the guinea-pig is somewhat contentious. Using guinea-pig isolated atria, Trendelenburg (1960) suggested the involvement of both direct (LSD-sensitive) and indirect (morphine-, cocaine- and dichloroisoprenaline-sensitive but reserpine-insensitive) mechanisms in the positive inotropic action of 5-HT, while recently, Eglen et al. (1985) claimed that 5-HT increases heart rate by acting directly on ~1-adrenoceptors. In spinal guinea-pigs, 5-HT-induced tachycardia remains unaffected by methiothepin, ketanserin or MDL 72222, but is antagonized by propranolol and atenolol. Moreover, in reserpinetreated animals the responses to 5-HT are not dose-dependent and show quickly developing tachyphylaxis (Dhasmana et al., 1988). These results suggest that catecholamines released by a 'tyramine-like' action are mainly responsible for the responses to 5-HT in the guinea-pig.

Hamster

5-HT increases heart rate in isolated atrial preparations, probably by combination of a direct effect on the myocardium and an indirect effect via liberation of cardiac catecholamines (Gonzalez Alvarez and Garcia Rodriguez, 1977). The nature of the receptor involved is unknown.

Rabbit

Rabbit isolated atria respond to 5-HT with tachycardia due to release of

118 Serotonin

catecholamines from cardiac sympathetic nerves (Trendelenburg, 1960). This effect, antagonized by MDL 72222 (Fozard, 1984a) and ICS 205-930 (Richardson et al., 1985), is mediated by pre-synaptic excitatory 5-HT3 receptors.

Cat

Trendelenburg (1960) reported that 5-HT-induced cardiostimulation in cat isolated atria is not affected by reserpine, dichloroisoprenaline or cocaine, but is susceptible to LSD. Recently, it was shown that the 5-HT-induced tachycardia is little affected by bilateral adrenalectomy, guanethidine, propranolol, burimamide, MDL 72222 or by a number of 5-HT2 receptor antagonists (cyproheptadine, ketanserin, ritanserin, mianserin and pizotifen), but methysergide and methiothepin are potent antagonists, and 5-carboxamidotryptamine (5-CT) mimics 5-HT (Figure 15.1; Saxena et al., 1985b; Connor et al., 1986). In isolated atria also, the two agonists cause tachycardia, and methysergide but not ketanserin behaves as an antagonist (Kaumann, 1986). Therefore, the tachycardiac response to 5-HT in the cat is mediated by 5-HT1-like receptors present on the sinoauricular node. However, these receptors are probably not related to specific 5-HT 1 binding site subtypes; only high doses of the selective 5-HT tA ligands BEA 1654 and 8-hydroxy-2-(di-n-propylamino)tetralin (8-0H-DPAT), but not the nonselective agent RU 24969, increase heart rate (Figure 15.1), and mesulergine, which is selective for 5-HT 1c binding sites, is not a particularly effective antagonist (Saxena, 1988).

5-HT increases the force of contraction of isolated papillary muscles

-1 Beats min

w 1-<

80

a: 60 1-a:: ~ :r:: lfO ~ w Ill ~ 20 a: u ~

0 0.0001 0.0003 0.001 0.003

5-HT

0.01 0.03 0.1 0.3 1.0 3.0

DOSE (mg kg-11

Figure 15.1 Heart rate responses (mean± s.e. mean) to 5-carboxamidotryptamine (5-Cf), 5-HT, BEA 1654, 8-0H-DPAT and RU 24969 in spinal cats (n = ~).

(Reproduced with permission from Saxena, 1988)


(Kaumann, 1986). The receptors mediating this response, unlike those mediating 5-HT-induced tachycardia, are neither stimulated by 5-CT nor blocked by methysergide (Kaumann, 1986). The exact nature of these ventricular receptors is not yet established.

Dog

In anaesthetized dogs, treated with mecamylamine or subjected to stellate ganglionectomy, 5-HT causes cardioacceleration which is antagonized by cyproheptadine, methysergide, bilateral adrenalectomy, syrosingopine and the f}-adrenoceptor antagonist bufetolol (Feniuk et al., 1981; Kimura and Satoh, 1983). Therefore, this effect is mainly mediated by adrenomedullary release of catecholamines via 5-HT2 receptors.

In isolated blood-perfused heart preparations, high concentrations of 5-HT have a stimulatory effect which is due to a 'tyramine-like' action (Chiba, 1977).

Pig

The mechanism of the tachycardiac response to 5-HT in the pig has recently been studied (Born et al., 1988). A number of 5-HT receptor antagonists, namely phenoxybenzamine, methiothepin, metergoline, methysergide and mesulergine (active at 5-HT1-like and 5-HT2 receptors), ketanserin, cyproheptadine, pizotifen and mianserin (5-HT2 receptors), and MDL 72222 and ICS 205-930 (5-HT3 receptors), do not attenuate chronotropic responses to 5-HT (Figure 15.2). The same is the case with antagonists at aand f}-adrenoceptors, cholinoceptors, histamine H1 and H2 receptors, dopamine receptors, Ca2+ channels or 5-HT uptake sites; drugs which are active at 5-HT uptake sites (indalpine and fluvoxamine) themselves increase heart rate and potentiate 5-HT-induced tachycardia. Moreover, several putative selective agonists at 5-HTrlike receptors or their subtypes (5-CT, 8-0H-DPAT, BEA 1654 and RU 24969) or at 5-HT3 receptors (2-methyl-5-HT) elicit no or only a weak tachycardiac response. It can therefore be concluded that the tachycardia produced by 5-HT does not involve receptors for common neurotransmitters, and is possibly mediated by a new 5-HT receptor type that is clearly different from 5-HT 1-like, 5-HT 2 or 5-HT3 receptors.

This new type of receptor may resemble the 5-HT receptor mediating either the positive inotropic effect in the cat papillary muscle (see above) or the slow depolarization of myenteric Type III AH neurones in the guinea-pig small intestine (Mawe et al., 1986; Gershon et al., this volume). This intestinal 5-HT receptor is insensitive to ICS 205-930 and LSD, but is

120 Serotonin

150 CONTROL

lOOt _r--....__ ~ 50 • •

' AFTER MDL 72222 (0.3 mg kg- 1) c: 150t ·elOO~~ ..

'CO 50 • • cu al

•

•

~ AFTER CYPROHEPTADINE (0.5 mg kg-l) ~ 150[ r-----__ 1- 100[ ~ _r---_ _; ---~ 50 • • • w J:

AFTER METHYSERG I DE ( 0. 5 mg kg -1)

150[ 100[~ 50 •

5-HT -1 3 llg kg

• 5-HT

-1 10 llg kg

• 5-HT

-1 30 llg kg

Figure 15.2 The effects of 5-HT (3, 10 and 30 j.tg/kg) on heart rate in an anaesthetized pig before and after successive administration of MDL 72222 (0.3 mglkg), cyproheptadine (0.5 mglkg) and methysergide (0.5 mglkg). Note that 5-HT causes a dose-dependent tachycardia which is not antagonized by these drugs.

(Reproduced with permission from Born et at., 1988)

antagonized by dipeptides of 5-hydroxytryptophan and excited by hydroxylated indalpines (Mawe et al., 1986; Gershon et al., this volume). However, the possibility that 5-HT (whether acting by a receptor mechanism or not) may be releasing an unidentified neurotransmitter must still be kept in mind.

Man

Slow i.v. infusions of 5-HT elicit tachycardia in man before any changes in arterial blood pressure occur (LeMessurier et al., 1959). This may indicate a myocardial site of action for 5-HT, although adrenomedullary catecholamine release and other mechanisms cannot be excluded.

ACKNOWLEDGEMENTS

I am grateful to my colleagues who collaborated in the studies mentioned in


this review, and to the editors of Drug Development Research, and the British Journal of Pharmacology, for permission to reproduce Figures 15.1 and 15.2, respectively.

REFERENCES

Born, A. H., Duncker, D. J., Saxena, P. R. and Verdouw, P. D. (1988). 5-Hydroxytryptamine-induced tachycardia in the pig: possible involvement of a new type of 5-hydroxytryptamine receptor. Br. J. Pharmacol., 93, 663--671


Chiba, S. (1977). Pharmacologic analysis of chronotropic and inotropic responses to 5-hydroxytryptamine in the dog heart. lap. J. Pharmacol., 27, 727-734

Connor, H. E., Feniuk, W., Humphrey, P. P. A. and Perren, M. J. (1986). 5-Carboxamidotryptamine is a selective agonist at 5-HT receptors mediating vasodilatation and tachycardia in anaesthetized cats. Br. J. Pharmacol., 87, 417-426

Dhasmana, K. M., de Boer, H. J., Banerjee, A. K. and Saxena, P.R. (1988). Analysis of the tachycardiac response to 5-hydroxytryptamine in the spinal guinea-pig. Eur. J. Pharmacol., 145, 67-73

Egfen, R. M., Park, K. C. and Whiting, R. L. (1985). Analysis of the positive chronotropic action of 5•HT on the isolated guinea-pig atria. Br. J. Pharmacol., 86,658P

Feniuk, W., Humphrey, P. P. A. and Watts, A. D. (1981). An analysis of the mechanism of 5-hydroxytryptamine-induced vasopressor responses in ganglionblocked anaesthetized dogs. J. Pharm. Pharmacol., 33, 155-160

Fozard, J. R. (1984a). MDL 72222: a potent and highly selective antagonist at neuronal5-hydroxytryptamine receptors. Naunyn-Schmiedeberg's Arch. Pharmacol., 326, 36-44

Fozard, J. R. (1984b ). Neuronal5-HT receptors in the periphery. Neuropharmacology, 23, 1473-1486

Gonzalez Alvarez, R. and Garcia Rodriguez, M. (1977). Sobre el mecanismo de accion de Ia serotonina en Ia auricula aislada de hamster.!. Serotonina: amina de accion mixta. Acta Physiol. Lat. Am., 27, 239-247

Gothert, M., Schlicker, E. and Kollecker, P. (1986). Receptor-mediated effects of serotonin and 5-methoxytryptamine on noradrenaline release in the rat vena cava and in the heart ofthe pithed rat. Naunyn-Schmiedeberg's Arch. Pharmacol., 332, 124-130

Kaumann, A. J. (1986). Further differences between 5-HT receptors of atrium and ventricle in cat heart. Br. J. Pharmacol., 89, 546P

Kimura, T. and Satoh, S. (1983). Presynaptic inhibition by serotonin of cardiac sympathetic transmission in dogs. Clin. Exp. Pharmacol. Physiol., 10, 535-542

LeMessurier, D. H., Schwartz, C. J. and Whelan, R. F. (1959). Cardiovascular effects of intravenous infusions of 5-hydroxytryptamine in man. Br. J. Pharmacol., 14, 24Cr250

Mawe, G. M, Branchek, T. A. and Gershon, M. D. (1986). Peripheral neural serotonin receptors: Identification and characterization with specific antagonists and agonists. Proc. Nat/ Acad. Sci. U.S.A., 83, 9799-9803

122 Serotonin

Richardson, B. P., Engel, G., Donatsch, P. and Stadler, P. (1985). Identification of serotonin M-receptor subtypes and their specific blockade by a new class of drugs. Nature, 316, 126-131

Saxena, P. R. (1986). Nature of the 5-hydroxytryptamine receptors in mammalian heart. Prog. Pharmacol., 6, 173-185

Saxena, P. R. (1988). Further characterization of 5-hydroxytryptamine.-like receptors mediating tachycardia in the cat: no apparent relationship to known subtypes of the 5-hydroxytryptamine1 binding site. Drug Devel. Res., 13, 245-258

Saxena, P. R. and Lawang, A. (1985). A comparison of cardiovascular and smooth muscle effects of 5-hydroxytryptamine and 5-carboxamidotryptamine, a selective agonist of 5-Hf1 receptors. Arch. Int. Pharmacodyn., 277, 235-252

Saxena, P. R., Heiligers, J., Mylecharane, E. J. and Tio, R. (1985a). Excitatory 5-hydroxytryptamine receptors in the cat urinary bladder are of the M- and 5-Hf 2 type. J. Auton. Pharmacol., 5, 101-107

Saxena, P.R., Mylecharane, E. J. and Heiligers, J. (1985b). Analysis of the heart rate effects of5-hydroxytryptamine in the cat; mediation by 5-Hf1-like receptors. Naunyn-Schmiedeberg's Arch. Pharmacol., 330, 121-129

Trendelenburg, U. (1960). The action of histamine and 5-hydroxytryptamine on isolated mammalian atria. J. Pharmacol. Exp. Ther., 130, 450-460

Walker, R. J. (1985). Thepharmacologyofserotoninreceptorsininvertebrates. In Green, A. R. (Ed.), Neuropharmacology of Serotonin, Oxford University Press, Oxford, pp. 366-408

16 Amplifying Action of 5-Hydroxytryptamine in

the Rabbit Ear Artery

I. S. de Ia Lande, J. A. Kennedy and B. J. Stanton

Department of Clinical and Experimental Pharmacology, University of Adelaide, Box 498, GPO, Adelaide, SA 5001, Australia

INTRODUCTION

An ability of 5-hydroxytryptamine (5-HT) in a low concentration to potentiate the responses to a second contractile agent (termed variously its sensitizing, synergistic, or amplifying action) has been documented in virtually all in-vitro artery preparations in which the interaction has been studied (Table 16.1). In some arteries (Table 16.2), both the contractile and the synergistic actions of 5-HT are inhibited by ketanserin in concentrations at which it is a selective antagonist of the 5-HT 2 receptor (Table 16.2). When both actions of 5-HT are mediated by a common receptor, it is reasonable to propose that the synergism reflects excitability changes which lead to or are associated with the contractile responses to the interacting agonists. However, there are some examples where constriction but not synergism is inhibited by ketanserin (Table 16.2), and in one artery (the perfused central artery of the rabbit ear) the reverse applies in that synergism appears to be mediated by a 5-HT-specific receptor (de Ia Lande eta/., 1966) which is probably of the 5-HT2 subtype (Meehan et al., 1986), whereas the contractile response appears to be mediated by the a-adrenoceptor (Apperley et al., 1976; Fozard, 1976). These exceptions draw attention to the possibility that the synergism in some vessels may reflect a true sensitizing action on the part of 5-HT, i.e. an action which is separate from its contractile action. In the case of the perfused rabbit ear artery, the sensitizing action may reflect failure of the maximal stimulus generated by occupation of the 5-HT2 receptor to reach a contractile threshold. Alternatively, as suggested by Manzini et al. (1986) on the basis of an analysis of the interaction between 5-HT and noradrenaline (NA), a tryptaminergic mechanism may impair the relaxation phase of the extracellular Ca2+ -dependent contractile response to NA.

124 Serotonin

Table 16.1 5-HT synergistic interactions Species Artery Interacting Agonist References Human Digital NA, thromboxane Arlike 1, 2

Temporal NA 3

Dog Basilar Prostaglandin F2a 4

Rat Caudal }NA Mesenteric 5, 6, 7, 8

Hind limb Aorta Acetylcholine 9

Rabbit Aorta NA, methoxamine 10 Femoral NA, histamine, prostaglandin F2a 11 Coronary Acetylcholine 12 Ear NA, adrenaline, histamine, 1, 3, 13, 14 (perfused) angiotensin II Reserpinized NA, methoxamine, histamine, ear (strips 2-pyridylethylamine 15, 16 and rin~s)

References: 1 de Ia Lande et al. {1966); 2 Young et al. (1986);

9 Asano and Hidaka {1980); 10 Stupecky et al. {1986);

3 Carroll et al. {1974); 4 Van Nueten {1985); 5 Van Nueten et al. {1981);

11 Van Nueten et al. {1982); 12 de Ia Lande et al. (1974); 13 Manzini et al. (1986); 14 Meehan et al. (1986); 6 Seabrook and Nolan (1983);

7 Su and Uruno {1985); 8 Wilton and McCalden {1977);

15 de Ia Lande and Kennedy (1985); 16 this study.

Table 16.2 Receptors mediating contractile and synergistic actions of 5-HT Artery Synergistic

receptor References Contractile

receptor

Rabbit femoral 5-HT2 10 5-HT2

Rat caudal 5-HT2 5 5-HT2 Rat mesenteric 5-HT2 7 5-HT2 Human digital Not 5-HT2 2 5-HT2

Rabbit ear 5-HT2 13 a-adreno-(perfused) ceptor Rabbit ear Not 5-HT2 15 5-HT2 and (reserpinized a-adreno-strips) ceptor

References

10 5 7

17 18, 19

15

References: see Table 16.1, also: 17 Ameklo-Nobin and Owman (1985); 18 Apperly et al. (1976); 19 Fozard. (1976).

Amplification of Vasoconstrictor Responses by 5-HT 125

The present report summarizes an analysis of the interaction between 5-HT, and agonists of either a-adrenoceptors or histamine H 1 and Hz receptors, in ear artery strips and rings from reserpine-treated rabbits. The results suggest that the receptor mechanisms involved in synergism may be more complex than is suggested by the earlier results obtained on perfused ear arteries.

METHODS

Ear artery strips and rings were from rabbits pre-treated with reserpine 2.5 mglkg 24 h previously to eliminate effects which may have been due to release of endogenous NA. The strips and rings were mounted under 1 g and 2 g tension, respectively, in Krebs solution at 37°C, and when the tension had achieved a stable base-line, full concentration-response curves for an agonist were elicited. The curves elicited in the absence and presence of 5-HT in low concentrations (0.01 or 0.1 !JM) were compared, and the leftward shift produced by 5-HT at the EC30 level of response to the interacting agonist was used to quantify the synergism. The shift is termed the sensitivity ratio. The response was measured from the base-line at the time of commencing the cumulative application of the agonist; frequently, although not always, the base-line had been elevated by 5-HT, but the elevation was usually small and less than 10 per cent of the maximal response to the agonist.

RESULTS

Interaction with a-Adrenoceptor Agonists

Descriptions of the interaction between 5-HT and the a-adrenoceptor agonists NA and methoxamine are summarized in de Ia Lande and Kennedy (1985). A feature was the relatively minor (and non-significant) inhibitory effect of ketanserin on the 5-HT (0.01-0.1 !J.M)-NA interaction in a concentration (0.05 J.tM) which was about 50-fold greater than the equivalent of the reported pAz for blockade of the 5-HTz receptor, namely 0.001 ~-tm (rat caudal artery; Van Nueten etal., 1981). In contrast, the interaction was strongly inhibited by ketanserin at a concentration (0.5 J.tM) which produced significant a-adrenoceptor blockade. The latter effect was reproduced by prazosin (0.08 !J.M). Although these results suggested that the synergism was mediated by the action of 5-HT on the a-adrenoceptor, there was no evidence of an inhibitory effect of prazosin on the synergism between 5-HT (0.1 !J.M) and methoxamine. Nevertheless, when 5-HT was present at the lower concentration (0.01 !J.M), its interaction with methoxamine was depressed by ketanserin (0.5 !J.M).

126 Serotonin

Interaction with Histamine H1 and H2 Receptor Agonists

A surprising finding was an ability of ketanserin 0.5 JAM to increase the synergistic interaction between 5-HT (0.1 JAM) and histamine. Ketanserin at this concentration shifted the concentration-response curve for histamine to the right by about an order of magnitude, in accord with evidence that the drug is a histamine H1 receptor antagonist (Van Nueten et al., 1982).

To assess whether histamine H1 receptor antagonism influenced the synergistic action of 5-HT, the effect of the H1 receptor antagonist mepyramine on the 5-HT-histamine interaction was studied. Synergism with histamine was increased by mepyramine at concentrations (0.05 and 0.2 JAM) which produced about 30- and 140-fold shifts of the histamine concentration-response curve to the right in strips and rings, respectively (Figure 16.1 (A, B)). Since the effect of mepyramine on synergism was abolished by cimetidine 100 JAM (Figure 16.1 (C)), it seemed likely that the effect was related to an increased stimulation of histamine Hz receptors resulting from the high concentrations of histamine required to produce contraction in the presence of H1 receptor blockade. Further evidence in support of this suggestion was provided by the finding that the specific Hz receptor agonist impromidine (0.01 JAM) increased the synergistic interaction between 5-HT (0.1 JA.M) and 2-pyridylethylamine, a specific agonist at H1

receptors (Figure 16.1 (D)). Interestingly, the results in Figure 16.1 (D) can equally well be interpreted as evidence that the vasodilator action of impromidine is prevented by 5-HT.

In view of the above findings, it was decided to assess the effects of ketanserin and of prazosin on the 5-HT (0.01 JA.M)-histarnine interaction under conditions where histamine Hz receptor stimulation was prevented by cimetidine (100 JA.M}. The interaction was unaffected by prazosin (0.08 JA.M) and by ketanserin (0.05 JAM); although ketanserin (0.5 JAM) tended to depress the interaction, the effect was not significant (paired t-test, n = 6).

DISCUSSION

In the introduction, it was pointed out that the constrictor and synergistic actions of 5-HT in non-reserpinized perfused preparations of rabbit ear artery segments appear to be mediated by different receptors, the former action by the a-adrenoceptor, and the latter by the 5-HTz receptor. However, there is evidence to suggest that, under different experimental conditions, a 5-HT receptor as well as the a-adrenoceptor can contribute to the contractile responses of ear artery strips and rings to 5-HT. 2-Bromolysergide partly antagonized responses of the chemically denervated artery ring to 5-HT (Purdy et al., 1981}, while ketanserin, at a 5-HTz receptor-specific blocking concentration (0.008 JAM}, antagonized responses

120

100

~ 80 ~ 60 1&1

!2 40 2 f3 II:

120 100

>< 80 < :s !! 60 1&1

!2 2 ~

A

c

8


B

f --+-- -; v/, /'

/l / ,t' )/

.. - ,v • +y 3 8 7 6 54 3

-LOG CONCN. HISTAMINE <Ml

D

7 6 54 38 7 6 54 3 -LOG CONCN. HISTAMINE (Ml -LOG CONCN. 2·PYRIDYLETHYLAMINE <Ml

Figure 16.1 Cumulative contractile responses to histamine or 2-pyridylethylamine in the absence(--) or presence(---) of5-HT(O.l f.tM), in rabbit ear artery strips or rings. In each preparation, responses were expressed as percentages of the maximal response in the concentration-response curve in the absence of 5-HT which immediately preceded the corresponding curve in the presence of 5-HT. The arrows show the magnitude of the contractile response to 5-HT, and represent the base-lines at the time of applying histamine or 2-pyridylethylamine. In (A) (strips) and (B) (rings), responses to histamine were obtained in the absence (e) or presence (0) of mepyramine (A, 0.05 f.tM, n = 6; B, 0.2 f.tM, n = 5). InC (strips), responses were obtained as in A, except that cimetidine (100 JAM, n = 6) was present throughout. In D (rings), responses to 2-pyridylethylamine were obtained in the absence (e) or

presence (0) of impromidine (0.01 f.tM, n = 5)

of reserpinized strips to low concentrations of 5-HT (de Ia Lande and Kennedy, 1985),

Despite the presence of both receptors (5-HT2 and a-adrenoceptors), the present analysis of synergism in the reserpinized preparations has indicated only a role for the a-adrenoceptor, and then only when NA is the interacting agonist. Although ketanserin inhibited synergism when either methoxamine or NA were the interacting agonists, the effect was significant only at a high concentration (0.5 JA-M) where the drug exerted significant blockade of a-adrenoceptors and histamine H 1 receptors. For this reason, the inhibitory effect of ketanserin does not constitute evidence that the synergistic interaction between 5-HT and a-adrenoceptor agonists is mediated by the 5-HT2 receptor. The use of more selective 5-HT2 antagonists, e.g. ritanserin, may help to resolve the question, and, in the process, shed further

128 Serotonin

light on the possible relationship between the synergistic and contractile actions of 5-HT.

That the relationship is likely to prove a complex one is indicated by the failure of prazosin to affect 5-HT-methoxamine synergism, which suggests that the mechanisms of synergism may differ even when, as is the case for NA and methoxamine, the agonists interacting with 5-HT are believed to exert their contractile effects via the same receptor (the a 1-adrenoceptor). The complexity is further highlighted by the present evidence that concomitant stimulation of a receptor mediating vasodilatation (histamine H2) results in a marked increase in the synergistic interaction between 5-HT and H 1 receptor agonists. Whether the increase is a consequence of vasodilatation per se, and whether vasodilator stimuli influence synergy between 5-HT and a-adrenoceptor agonists, are questions which remain to be answered.

ACKNOWLEDGEMENTS

This work was supported by a grant from the National Heart Foundation of Australia.

REFERENCES

Apperley, A., Humphrey, P. P. A. and Levy, G. P. (1976). Receptors for 5-hydroxytryptamine and noradrenaline in rabbit isolated ear artery and aorta. Br. J. Pharmacol., 58, 211-221

Ameklo-Nobin, B. and Owman, Ch. (1985). Adrenergic and serotoninergic mechanisms in human hand arteries and veins studies by fluorescence histochemistry and in vitro pharmacology. Blood Vessels, 22, 1-12

Asano, M. and Hidaka, H. (1980). Potentiation of the contractile response to acetylcholine in aorta strips by low concentrations of vascular contractile agonists. Br. J. Pharmacol., 69, 639--646

Carroll, P. R., Ebeling, P. W. and Glover, W. E. (1974). The responses of the human temporal and rabbit ear artery to 5-hydroxytryptamine and some of its antagonists. Aust. J. Exp. Bioi. Med. Sci., 52, 813-823

de Ia Lande, I. S. and Kennedy, J. A. (1985). Receptors mediating the vasoconstrictor and amplifying actions of serotonin on the rabbit ear artery. Clin. Exp. Pharmacol. Physiol., Suppl. 9, fr7

de la Lande, I. S., Cannell, V. A. and Waterson, J. G. (1966). The interaction of serotonin and noradrenaline on the perfused artery. Br. J. Pharmacol. Chemother., 28, 255-272

de IaLande, I. S.,Harvey,J. A. and Holt, S. (1974). Responseoftherabbitcoronary arteries to autonomic agents. Blood Vessels, 11, 319-337

Fozard, J. R. (1976). Comparative effects of four migraine prophylactic drugs on an extracranial artery. Eur. J. Pharmacol., 36, 127-139


Manzini, S., Maggi, C. A. and Meli, A. (1986). 5-Hydroxytryptamine delays relaxation time of norepinephrine-induced vasoconstriction. Am. J. Physiol., 250, H121-H130

Meehan, A. G., Rand, M. J. and Medgett, I. C. (1986). Effects of serotonin on sympathetic noradrenergic transmission in rabbit isolated ear artery. J. Cardiovasc. Pharmacol., 8, 1144-1153

Purdy, R. E., Hurlbut, D. E. and Rains, L. A. (1981). Receptors for 5-hydroxytryptamine in rabbit isolated ear artery and aorta. Blood Vessels, 18, 16-27

Seabrook, J. M. and Nolan, P. L. (1983). The vascular interaction of noradrenaline and 5-hydroxytryptamine. Eur. J. Pharmacol., 89, 131-135

Stupecky, G. L., Murray, D. L. and Purdy, R. E. (1986). Vasoconstrictor threshold synergism and potentiation in the rabbit isolated thoracic aorta. J. Pharmacol. Exp. Ther., 238, 802-808

Su, C. and Uruno, T. (1985). Excitatory and inhibitory effects of 5-hydroxytryptamine in mesenteric arteries of spontaneously hypertensive rats. Eur. J. Pharmacol., 106, 283-290

Van Nueten, J. M. (1985). Calcium entry blockers and vascular smooth muscle reactivity. In Godfraind, T. (Ed.), Calcium Entry Blockers and Tissue Protection, Raven Press, New York, pp. 69-79

VanNueten,J. M.,Janssen, P. A. J., VanBeek,J.,Xhonneux, R., Verbeuren, T. J. and Vanhoutte, P.M. (1981). Vascular effects ofketanserin (R 41468), a novel antagonist of 5-HT2 serotonergic receptors. J. Pharmacol. Exp. Ther., 218, 217-230

Van Nueten, J. M., Janssen, P. A. J., De Ridder, W. and Vanhoutte, P.M. (1982). Interaction between 5-hydroxytryptamine and other vasoconstrictor substances in the isolated femoral artery of the rabbit; effect of ketanserin (R 41 468). Eur. J. Pharmacol., 77, 281-287

Wilton, P. B. and McCalden, T. A. (1977). Interaction between noradrenaline and 5-hydroxytryptamine in the isolated perfused rat hindlimb. Eur. J. Pharmacol., 46, 213-219

Young, M. S., Iwanov, V. and Moulds, R. F. W. (1986). Interaction between platelet-released serotonin and thromboxane A2 on human digital arteries. Clin. Exp. Pharmacol. Physiol., 13, 143-152

17 Serotonin-induced Vasoconstriction and

Contractile Synergism with Noradrenaline: Role of a-Adrenoceptors

R. E. Purdy and D. L. Murray

Department of Pharmacology and Cardiovascular Biology Group, University of California, Irvine CA 92717, USA

Serotonin acts primarily on serotonin receptors in most vascular and non-vascular smooth muscle. However, this important endogenous substance has two additional actions: it elicits contraction by stimulating a-adrenoceptors in a number of tissues, and it amplifies the vasoconstrictor response to a-adrenoceptor agonists. In the present review, studies of these latter two actions of serotonin are outlined; it is proposed that these actions may be interrelated.

Fozard (1976) was among the first to suggest that serotonin activates a-adrenoceptors in the rabbit ear artery. However, the study by Apperley et al. (1976) firmly established this action of serotonin in a comparison of the rabbit ear artery and thoracic aorta. These authors also demonstrated that serotonin acts exclusively on serotonin receptors in the latter vessel. Black et al. (1981) extended these observations to other distributing arteries of the rabbit, and found that serotonin-induced vasoconstriction was mediated by a-adrenoceptors in the external carotid but not the common carotid or femoral arteries. In our laboratory, it was found that serotonin acted primarily on a-adrenoceptors in rabbit ear artery (Purdy et al., 1981), confirming the findings of Apperley et al. (1976). However, we also demonstrated the presence of a small population of serotonin receptors.

Further studies in our laboratory have been aimed at re-examining the rabbit thoracic aorta and extending those investigations to other rabbit blood-vessels, including the abdominal aorta, and femoral, saphenous, mesenteric and radial arteries (Purdy et al., 1985; present results). Activation of a-adrenoceptors was detected by pre-treatment of the blood-vessel rings with benextramine, a selective, irreversible aadrenoceptor antagonist (Purdy et al., 1986). In the thoracic aorta,

a-Adrenoceptor Agonism and Synergism with Serotonin 131

benextramine pre-treatment had no effect on the serotonin concentrationresponse curve. However, there was a progressively greater rightward shift and depression of the serotonin maximal response in abdominal aorta, femoral artery and mesenteric artery, respectively (see Figure 17.1). The radial and saphenous arteries closely resembled the femoral and mesenteric in terms of the degree of contribution of a-adrenoceptors to the serotonin-induced vasoconstrictor response. The selective serotonin antagonist 2-bromolysergide was used to demonstrate the presence of serotonin receptors. Excluding the ear artery, this antagonist caused between 100- and 1000-fold rightward shifts of the serotonin concentrationresponse curves in all blood vessels studied. Thus, it is concluded that serotonin acts on both a-adrenoceptors and serotonin receptors to cause vasoconstriction in these blood vessels (except the thoracic aorta; see below).

The results described above raise the following question. Why does serotonin activate a-adrenoceptors in some but not all blood vessels? It is possible that certain vessels possess novel 'serotonin-sensitive' aadrenoceptors. If this is true, then the a-adrenoceptors of the rabbit thoracic aorta must be insensitive to serotonin and cannot be activated by this agonist under any circumstances. We tested this possibility by assessing the effect of

120 --- CONTROL

100 -o- ABDOMINAL AORTA z 0 _.,.. 1- 80 () <( a: 60 1-z 0 40 () ~ 0 20

0 -10 -9 -8 -7 -6 -5 -4 -3

[5-HT ] (LOG M)

Figure 17.1 Effect of benextramine pre-treatment on serotonin concentrationresponse curves in rabbit abdominal aorta and femoral artery. The control curve is normalized to represent both blood vessels without exposure to benextramine; contractions to serotonin are expressed as percentages of the control maximal response. The two remaining curves illustrate the differential effects of benextramine pre-treatment (10 !lM) on abdominal aorta and femoral artery. Control experiments showed that the exposure to benextramine abolished the responses of

these vessels to noradrenaline

132 Serotonin

a-adrenoceptor antagonism in both the presence and absence of serotonin receptor blockade (Purdy eta/., 1986). Neither benextramine pre-treatment nor exposure to 0.1 f.tM prazosin had any effect in control thoracic aortas. However, after shifting the serotonin curve far to the right with 2-bromolysergide, both benextramine and prazosin caused a further displacement of the serotonin concentration-response curve. This argues against the existence of a 'serotonin-sensitive' a-adrenoceptor. Our proposed explanation is that serotonin is a partial agonist at aadrenoceptors, and that differences in a-adrenoceptor activation between vessels are a result of different serotonin and a-adrenoceptor reserves. It is likely that serotonin fails to activate a-adrenoceptors in control thoracic aortas because, in relative terms, serotonin receptor reserve is high, and a-adrenoceptor reserve is low (Purdy and Stupecky, 1984). Thus, in this vessel, the maximal response to serotonin, acting on serotonin receptors, is achieved at concentrations below that necessary for serotonin to activate a-adrenoceptors. In contrast, the other vessels studied, such as the abdominal aorta and the femoral and mesenteric arteries, possess a relatively higher a-adrenoceptor reserve and a relatively lower serotonin receptor reserve. In keeping with this argument, the rabbit ear artery possesses a very high a-adrenoceptor reserve (Purdy and Stupecky, 1984) and almost no serotonin receptors (Apperley eta/., 1976; Purdy et al., 1981).

Among the earliest studies demonstrating contractile synergism between serotonin and noradrenaline was the landmark work by de Ia Lande eta/. (1966). These authors found that sub-pressor and threshold pressor concentrations of serotonin markedly enhanced the response of the isolated perfused rabbit ear artery to noradrenaline. Fozard (1976) described the reverse situation in which the a-adrenoceptor agonist clonidine markedly amplified the vasoconstrictor responses to serotonin. Van Nueten et al. (1982) demonstrated that serotonin, acting on 5-HT2 receptors, amplifies the vasoconstrictor responses to the endogenous agonists angiotensin II, histamine and prostaglandin F2a in the rabbit femoral artery.

The contractile synergism between serotonin and a-adrenoceptor agonists including noradrenaline and methoxamine has also been studied in our laboratory (Stupecky et a/., 1986; Murray, 1986). When threshold concentrations (i.e. concentrations eliciting threshold contractions) of serotonin and noradrenaline were added to the medium bathing the rabbit thoracic aorta ring, the combination of these agonists caused a far greater than additive contraction, and changing the order of agonist addition had no effect. In addition, a full concentration-response curve for one of the agonists was shifted to the left in the presence of a threshold concentration of another. However, in this case the order of agonist administration changed the degree of shift: in the thoracic aorta, methoxamine caused a slightly greater leftward shift of the serotonin curve than did serotonin of the methoxamine concentration-response curve. This difference was much


120 --- CONTROL -o- THORACIC AORTA

z 100 -A- FEMORAL ARTERY 0 ..... EAR ARTERY 1- 80 0 <( a: 60 1-z 0 40 0 ~ 0 20

0 .01 1 100 10,000

[5-HT] Figure 17.2 Effect of a threshold concentration of methoxamine on serotonin concentration-response curves in rabbit thoracic aorta, femoral artery and ear artery. The control curve is normalized to represent all three blood vessels; contractions to serotonin are expressed as percentages of the control maximal response. The remaining curves illustrate the different magnitudes of amplification which resulted from exposure to a threshold concentration of methoxamine prior to

obtaining the serotonin concentration-response curves

more marked in the rabbit femoral artery, in which methoxamine caused a 100-fold leftward shift of the serotonin concentration-response curve, while serotonin caused only an 8-fold leftward shift of the methoxamine curve.

The degree of amplification of the serotonin concentration-response curve by threshold concentrations of methoxamine varied between blood vessels. As shown in Figure 17 .2, methoxamine shifted the serotonin concentration-response curves to the left by approximately 10-, 100-, and 1000-fold in the rabbit thoracic aorta, femoral artery and ear artery, respectively. The order of these differences in the magnitude of amplification corresponded to the same order of a-adrenoceptor activation by serotonin in the respective blood-vessels. For example, in the rabbit aorta benextramine exerted a modest inhibition of the serotonin response only after serotonin receptor blockade. In contrast, benextramine reduced the maximal response of the femoral artery by 60 per cent, indicating greater a-adrenoceptor activation. In the ear artery, all (Apperley et al., 1976) or nearly all (Purdy et al., 1981) of the response to serotonin is mediated by a-adrenoceptors. The correlation between a-adrenoceptor activation and vasoconstrictor synergism is illustrated in Figure 17.3. A causal relationship

134

1000

z 0 ~ ~100 ::::i a.. :::iii <C

Serotonin

• EAR ARTER'i

• FEMORAL ARTERY

10 • AORTA

<10% 60% 100%

BHC BLOCKADE OF 5-HT

Figure 17.3 The relationship between amplification of the serotonin concentration-response curve by a threshold concentration of methoxamine (as leftward shifts in the serotonin concentration-response curves) and the activation of a-adrenoceptors by serotonin, in rabbit thoracic and abdominal aorta, femoral artery and ear artery. The magnitude of blockade of the serotonin responses (average per cent decrease in serotonin maximal response) by benextramine (BHC)

was taken as a measure of serotonin a-adrenoceptor activation

between these parameters is clearly not established. Nevertheless, as a basis for future experiments, we propose that a-adrenoceptor activation and serotonin-noradrenaline vasoconstrictor synergism are correlated. The present results suggest that when serotonin receptor reserve is high and a-adrenoceptor reserve is low, both a-adrenoceptor activation and amplification by serotonin are modest. However, both actions increase as a-adrenoceptor reserve increases and, possibly, as serotonin receptor reserve declines.

ACKNOWLEDGEMENTS

This work was supported by NIH Grants HL-27049 and HL-31637.


REFERENCES

Apperley, E., Humphrey, P. P. A. and Levy, G. P. (1976). Receptors for 5-hydroxytryptamine and noradrenaline in rabbit isolated ear artery and aorta. Br. J. Pharmacal., 58, 211-221

Black,J. L., French, R. J. andMylecharane, E. J. (1981). Receptor mechanisms for 5-hydroxytryptamine in rabbit arteries. Br. J. Pharmacal., 74, 619-626

de Ia Lande, I. S., Cannell, V. A. and Waterson, J. G. (1966). The interaction of serotonin and noradrenaline on the perfused artery. Br. J. Pharmacal. Chemother., 28, 255-272

Fozard, J. R. (1976). Comparative effects offour migraine prophylactic drugs on an isolated extracranial artery. Eur. J. Pharmacal., 36, 127-139

Murray, D. L. (1986). Receptor interactions in peripheral blood vessels. Unpublished doctoral dissertation, University of California, Irvine

Purdy, R. E. and Stupecky, G. L. (1984). Characterization of the alpha adrenergic receptor properties of rabbit ear artery and thoracic aorta. J. Pharmacal. Exp. Ther., 229, 459-468

Purdy, R. E., Hurlbut, D. E. and Rains, L. A. (1981). Receptors for 5-hydroxytryptamine in rabbit isolated ear artery and aorta. Blood Vessels, 18, 16-27

Purdy, R. E., Murray, D. L. and Stupecky, G. L. (1985). 5-Hydroxytryptamine is an alpha agonist in rabbit blood vessels. Proc. West. Pharmacal. Soc., 28, 123-125

Purdy, R. E., Murray, D. L. and Stupecky, G. L. (1986). Receptors for 5-hydroxytryptamine in rabbit blood vessels: activation of alpha adrenoceptors in rabbit thoracic aorta. J. Pharmacal. Exp. Ther., 240, 535-541

Stupecky, G. L., Murray, D. L. and Purdy, R. E. (1986). Vasoconstrictor threshold synergism and potentiation in the rabbit isolated thoracic aorta. J. Pharmacal. Exp. Ther., 238, 802-808

Van Nueten, J. M., Janssen, P. A. J., De Ridder, W. and Vanhoutte, P.M. (1982). Interaction between 5-hydroxytryptamine and other vasconstrictor substances in the isolated femoral artery of the rabbit; effect of ketanserin (R 41 468). Eur. J. Pharmacal., 77, 281-287

18 Pre-synaptic Sympathetic Inhibition and

5-Hydroxytryptamine-induced Vasodilatation

E. J. Mylecharane and C. A. Phillips

Department of Pharmacology, University of Sydney, Sydney NSW 2006, Australia

INTRODUCTION

Within 5 years of the isolation of the serum vasoconstrictor factor, 5-hydroxytryptarnine (5-HT), its vasodilating properties had been recognized; Haddy (1953) described 5-HT-induced vasodilation in the forelimb in anaesthetized dogs. A real understanding of the mechanisms responsible for the vasodilator effects of 5-HT, however, has taken a much longer time to emerge.

Vasodilator responses to 5-HT in vivo have now been described in many other circulations and species, including the dog coronary, hind limb, renal, mesenteric and hepatic circulations, the human forearm, various vessels in rats, the baboon external carotid vasculature, and the pig and cat common carotid vascular beds (see Garattini and Valzelli, 1965; Saxena and Verdouw, 1982; Phillips et al., 1985b). In-vitro evidence for the underlying mechanisms which could contribute to 5-HT-induced vasodilatation in vivo dates from reports of a direct vascular smooth muscle relaxation (Eyre, 1975), the release of an endothelium-derived relaxing factor (Cocks and Angus, 1983; Cohen eta/., 1983), and pre-synaptic inhibition of sympathetic adrenergic nerve terminals (McGrath, 1977). These mechanisms are discussed briefly below, followed by a more detailed assessment of the contribution made by pre-synaptic sympathetic inhibition to the vasodilator effect of 5-HT in vivo, and the receptor mechanism responsible for this effect.

MECHANISMS OF S-OT-INDUCED VASODILATATION

Direct Vascular Smooth-muscle Relaxation

The early in-vivo studies on the vascular actions of 5-HT were all interpreted

Pre-synaptic Sympathetic Inhibition 137

in terms of contractile and relaxant effects on vascular smooth muscle, despite the absence of any firm in-vitro evidence fot the latter. Haddy et al. (1959) developed the so-called 'neurogenic tone' hypothesis: 5-HT causes a net vasodilatation when a vascular bed is constricted (for example, by sympathetic adrenergic tone), and a net vasoconstriction when it is dilated, because of the contractile and relaxant effects of 5-HT on vascular smooth muscle in different segments of the same vascular bed; the 'neurogenic tone' hypothesis was really a 'vascular tone' hypothesis.

McCubbin et al. (1962), who studied the in-vivo effects of 5-HT on different segments of the dog hind-limb, renal and mesenteric circulations, recognized that 5-HT-induced vasodilatation occurred only during sympathetic nerve stimulation or adrenaline infusion, and concluded that 5-HT produced an indirect sensitization of vascular smooth-niuscle adrenergic vasodilator receptors. Blackshear et al. (1985), using more sophisticated techniques, confirmed the differential effects of 5-HT in large and small arterial vessels in the dog hind-limb, but excluded an involvement of 13-adrenoceptors, and also ruled out eicosanoid mechanisms. McCubbin et al. (1962) had used the 13-adrenoceptor antagonist dichloroisoprenaline, which may have simply masked the vasodilator effect of 5-HT because of its marked partial agonist activity; Blackshear et al. ( 1985) used propranolol. In the dog cranial circulation, there have been conflicting reports of both vasoconstrictor and vasodilator responses to 5-HT (Swank and Hissen, 1964; Saxena, 1972; Vidrio and Hong, 1976). Mena and Vidrio (1979) found that the vasodilator action of 5-HT depended upon the cervical sympathetic nerve trunks remaining intact.

Involvement of a direct relaxant effect in 5-HT-induced vasodilatation in vivo has been reinforced by in-vitro findings of relaxant responses to 5-HT in a variety of vascular preparations (Eyre, 1975; Edvinsson et al., 1978; Chand, 1981; Feniuk et al., 1983; Trevethick et al., 1984; Martinet al., 1987). 'the use of various antagonists and inhibitors in these studies has excluded the involvement of muscarinic cholinergic receptors, 13-adrenoceptors, histamine H 1 and H2 receptors, eicosanoid mediators, or neuronally derived inhibitory mediators.

Release of Endothelium-derived Relaxing Factor

An endothelium-dependent component in 5-HT-induced relaxant responses in vitro has been recognized (Cocks and Angus, 1983; Cohen et al., 1983). Of the in-vitro studies cited above as demonstrating a direct vascular smooth-muscle relaxant action of 5-HT, the tissue preparation techniques used by Edvinsson et al. (1978) and Trevethick et al. (1984) would be expected to have preserved the endothelial cells, which could have contributed to the responses obtained. Most of the investigations into this

138 Serotonin

action of 5-HT have of necessity been conducted in vitro, but some recent in-vivo studies have shown that endothelium-derived relaxing factor is involved in the overall vascular response to 5-HT, at least in large vessels such as the dog common carotid artery (Angus et al., 1987).

Pre-synaptic Inhibition of Sympathetic Adrenergic Nerve Terminals

In-vitro studies have shown that 5-HT inhibits the electrically evoked release of [3H)-noradrenaline from several vascular preparations, such as the dog saphenous vein (McGrath, 1977; Miiller-Schweinitzer, 1981; Watts et al., 1981; Engel et al., 1983; Fishburn etal., 1987), the rat mesenteric artery (Su and Uruno, 1985), the rat renal vasculature (Charlton et al., 1986), the rat vena cava (Gothert et al., 1986b; Molderings et al., 1987) and the human saphenous vein (Gothert et al., 1986a). In the dog saphenous vein, contractile responses to electrical stimulation were also inhibited by 5-HT, but responses to exogenous noradrenaline were unaffected or enhanced (McGrath, 1977; Feniuk et al., 1979; Miiller-Schweinitzer, 1981; Van Nueten et al., 1981; Watts et al., 1981); these experiments provide indirect evidence for pre-synaptic sympathetic inhibition, as do similar observations in the rabbit basilar artery (Bevan et al., 1975), despite the complicating factors of the direct contractile effects of 5-HT and its amplifying effect on responses to other vasoconstrictors. Evidence from some of these indirect studies has also excluded the apparent involvement of muscarinic cholinergic receptors, histamine H 1 and H2 receptors, dopamine receptors, and eicosanoid mediators, in the pre-synaptic inhibitory effect of 5-HT. The influence of pre-synaptic a- and f3-adrenoceptors is discussed subsequently.

CONTRIBUTION OF PRE-SYNAPTIC SYMPATHETIC INHIBITION TO 5-HT-INDUCED VASODILATATION IN VIVO

A series of in-vivo experiments with 5-HT was undertaken in our laboratory, using anaesthetized dogs, to assess the relative contribution to 5-HTinduced vasodilatation made by pre-synaptic inhibition of sympathetic adrenergic nerve terminals. Studies were conducted in the femoral (Phillips et al., 1985b) and common carotid (Markus et al., 1989b) arterial circulations; vascular resistances and conductances were calculated from local perfusion pressures across the circulation and blood flow data measured using electromagnetic flow probes. It was reasoned that any direct vascular smooth muscle relaxant effect of 5-HT should be in evidence as a vasodilator response regardless of whether vascular tone was maintained by sympathetic or non-sympathetic means.

The i.a. infusion of 5-HT (0.1-50 f.lg/kg per min) elicited reproducible


vasodilator responses in both circulations, the maximal effect being approximately 75 per cent of that produced by i.a. infusion of acetylcholine. The response to 5-HT was abolished following removal of sympathetic vascular tone (by means of the ganglion-blocking agent mecamylamine in the femoral experiments, and bilateral cervical vagosympathectomy in the common carotid studies). In some experiments, a small vasoconstrictor response was unmasked. Removal of sympathetic vascular tone could have simply masked any vasodilator response to 5-HT regardless of its site of action. However, the vasodilator effect of acetylcholine was still in evidence after removal of sympathetic tone, suggesting that it is unlikely that a simple masking of vasodilator effects alone is responsible. When vascular tone in these circulations was restored by the i.a. infusion of omipressin, the vasodilator response to 5-HT remained abolished, but that to acetylcholine was retained.

If the vasodilator effect of 5-HT depended only on vascular tone, as was implied by Haddy et al. (1959), then 5-HT should have produced vasodilatation following restoration of vascular tone with omipressin. Any direct relaxant effect of 5-HT would be expected to be manifested as a physiological or functional antagonism irrespective of the means by which vascular tone was maintained, as indicated by the fact that acetylcholine, which acts on the vascular endothelium to initiate vascular smooth muscle relaxation (Furchgott and Zawadzki, 1980), retained its vasodilator effect in these circumstances. The results obtained therefore suggest that a direct relaxant effect of 5-HT makes little contribution to 5-HT-induced vasodilatation in the dog femoral and common carotid arterial circulations. The effect of 5-HT appears to be directly related to sympathetic adrenergic function. Its site of action must be pre-synaptic; in our experiments, 5-HT had no effect on the dog common carotid vasoconstrictor responses to noradrenaline (before or after cervical sympathectomy), in accordance with findings that vasoconstrictor responses to noradrenaline in the dog forelimb or hind limb are either unaffected or enhanced by 5-HT (Haddy et al., 1959; McCubbin et al., 1962; Feniuk et al., 1981). Thus in these circulations, the vasodilator effect of 5-HT appears to depend predominantly on a pre-synaptic inhibition of sympathetic vascular tone, most probably at the adrenergic nerve terminals.

Evidence of a more direct nature for a pre-synaptic sympathetic inhibitory effect of 5-HT on vascular tone in vivo comes from studies in the femoral arterial circulation of anaesthetized dogs by Feniuk et al. (1981), who showed that 5-HT inhibited the vasoconstrictor response to lumbar sympathetic chain stimulation, but did not reduce that to i.a. noradrenaline; it was noted that 5-HT itself tended to constrict the femoral vascular bed. Studies in our laboratory have demonstrated that 5-HT (0.2-51J.g/kg per min i.a.) reproducibly inhibits the vasoconstrictor responses to cervical sympathetic nerve stimulation (0.25, 0.5. 1 and 2.5 Hz) in the dog common

140 Serotonin

carotid circulation (Markus et al., 1989a), without affecting those to exogenous noradrenaline (Markus et al., 1989b); the degree of inhibition tended to be a little more marked at the lower frequencies of stimulation. As expected, 5-HT itself occasionally produced a small vasoconstrictor response, because the cervical sympathetic nerve trunks were sectioned at the outset. While this could possibly complicate the interpretation of the data, control experiments in which a similar degree of vasoconstriction was produced by i.a. ornipressin showed that the responses to cervical sympathetic nerve stimulation were unchanged, indicating that the effect of 5-HT was due to a specific pre-synaptic inhibitory action.

5-HT normally elicits an external carotid vasoconstrictor response in anaesthetized monkeys (Spira et al., 1978; Mylecharane et al., 1978). This could be due to the apparent absence of resting sympathetic tone to this vasculature under these experimental conditions; sectioning of the cervical sympathetic nerve trunk does not alter base-line vascular tone (Mylecharane et al., 1980). Preliminary findings suggest, however, that 5-HT also has a pre-synaptic sympathetic inhibitory effect in this circulation (Phillips and Mylecharane, 1987), because i.a. 5-HT, but not ornipressin at a dose producing equivalent vasoconstriction, inhibited the vasoconstrictor responses to nerv~ stimulation.

The predominant contribution of pre-synaptic sympathetic inhibition to the in-vivo vasodilator action of 5-HT in some specific circulations should not detract from the possibility that direct vascular smooth muscle relaxation or release of endothelium-derived relaxing factor may be important in others. Saxena and Verdouw (1982) have shown that vasodilator responses to 5-HT in the arteriolar-capillary (nutrient) fractions of pig and cat common carotid circulations do not appear to depend on pre-synaptic sympathetic adrenergic inhibitory effects. Likewise, Kalkman et al. (1983) demonstrated a direct vasodepressor response to 5-HT in pithed rats following restoration of vascular tone with vasopressin. It is also likely that direct relaxation and release of endothelium-derived relaxing factor plays at least some part in 5-HT-induced vasodilation, even in those circulations where pre-synaptic inhibition seems to be the dominant mechanism. In our own experiments, the vasodilator responses to 5-HT in dogs with intact sympathetic innervation exceeded the level to which vascular tone fell when sympathetic activity was interrupted, although this could also have been at least partly due to sympathetic tone not being completely abolished.

THE 5-HT RECEPTORS MEDIATING PRE-SYNAPTIC SYMPATHETIC INHIBITION

The effects of several antagonists on 5-HT-induced vasodilatation in dogs


have been studied in our laboratory. Ketanserin (0.1-4 mglkg) and pizotifen (0.1-0.4 mglkg) were studied in the dog femoral (Phillips et al., 1985b) and common carotid (Markus eta/., 1989b) arterial circulations; methysergide (0.02-0.4 mglkg) and pirenperone (0.001-0.01 mglkg) were tested only in the dog common carotid circulation. All are potent 5-HT2 receptor antagonists, but methysergide also possesses appreciable affinity for 5-HT1-like receptors (Bradley et al., 1986). The vasodilator responses to 5-HT were inhibited by all the antagonists, but the degree of blockade was relatively modest, and the doses required were higher than those normally used to block 5-HT2 receptors. Methysergide was the only one of these antagonists which had no direct effects on resting vascular tone. Ketanserin, and to a lesser extent pirenperone, produced appreciable vasodilatation; pizotifen markedly dilated the common carotid circulation, but did not produce consistent significant femoral vasodilatation. The apparent blockade of 5-HT -induced vasodilatation produced by the latter antagonists may therefore have been due to a simple masking resulting from their intrinsic vasodilating effects. None of these 5-HT antagonists affected the vasoconstrictor responses to noradrenaline in these experiments, except for ketanserin at higher doses (1-4 mglkg). In anaesthetized dogs, ketanserin at doses of 0.1-0.4 mglkg produces a centrally mediated inhibition of sympathetic vascular tone (Phillips et al., 1985a). The mechanisms of the vasodilator actions of pizotifen and pirenperone are not known.

Identification of the in-vivo pre-synaptic vascular sympathetic inhibitory receptor for 5-HT was therefore pursued by investigating 5-HT-induced inhibition of the dog common carotid vasoconstrictor responses to cervical sympathetic nerve stimulation (Mylecharane and Phillips, 1987; Markus et al., 1989a). In control experiments, alteration of resting vascular tone with i.a. acetylcholine or omipressin had no effect on the responses to nerve stimulation; thus any intrinsic actions of antagonists on vascular tone will not interfere with assessment of their effects on the pre-synaptic inhibitory receptor-mediated response. Ketanserin (0.4 mglkg), pizotifen (0.1-0.4 mglkg), and the selective 5-HT3 receptor antagonists MDL 72222 (0.5 mglkg) and ICS 205-930 (0.1 mglkg), had no effect on the pre-synaptic inhibitory response to 5-HT, nor did they alter the vasoconstrictor responses to noradrenaline or nerve stimulation alone; thus neither 5-HT2 nor 5-HT3

receptors are involved. Methysergide (0.2 and 0.4 mglkg) acted as a partial agonist; the vasoconstrictor responses to noradrenaline were not inhibited, but those to nerve stimulation alone were, and the pre-synaptic inhibitory effect of 5-HT was enhanced. Methysergide increased resting common carotid vascular tone in these experiments, in contrast to its lack of effect in dogs with intact cervival sympathetic nerve trunks (Markus et al., 1989b), suggesting that a post-synaptic vasoconstrictor component is normally balanced by its pre-synaptic inhibitory effect. The selective 5-HT1-like receptor agonist 5-carboxamidotryptamine (5-CT) mimicked the inhibitory

142 Serotonin

action of 5-HT on the vasoconstrictor responses to nerve stimulation, but did not affect noradrenaline-induced vasoconstriction, and also mimicked the vasodilator response to 5-HT in dogs with intact cervical sympathetic nerve trunks, its potency (on a molecular basis) being approximately 16 and 22 times greater, respectively, than that of 5-HT. These observations, together with the previously mentioned finding that methysergide at similar dose levels inhibited 5-HT-induced common carotid vasodilatation, indicate that a 5-HTrlike receptor mediates the pre-synaptic sympathetic inhibitory effect of 5-HT in vivo.

In accordance with this conclusion, methiothepin (0.1-0.5 mglkg) partly reversed 5-HT- and 5-Cf-induced pre-synaptic inhibition of dog common carotid vasoconstrictor responses to nerve stimulation. Methiothepin is a 5-HT2 receptor antagonist with appreciable 5-HT1-like receptor blocking activity. However, the vasoconstrictor response to noradrenaline was inhibited, while that to nerve stimulation alone was unaltered, suggesting that the post-synaptic a-adrenoceptor blocking action of methiothepin may have been balanced by an augmentation of nerve stimulation-induced noradrenaline release via a pre-synaptic a-adrenoceptor blocking action. It is therefore possible that the reversal by methiothepin of 5-HT- and 5-Cf -induced pre-synaptic inhibition could involve physiological antagonism (via enhancement of noradrenaline release) as well as blockade of 5-HT1-like receptors. An unexpected finding in these experiments was a partial reversal of 5-HT -induced pre-synaptic inhibition following the higher doses of pirenperone (0.005 and 0.01 mg/kg); vasoconstrictor responses to noradrenaline or nerve stimulation alone were not affected, and thus pirenperone may have some affinity for 5-HT 1-like receptors. Partial agonist actions of methysergide in the dog femoral circulation (Feniuk et al., 1981) and the monkey external carotid vasculature (Phillips and Mylecharane, 1987) suggest that a 5-HTrlike receptor mediates 5-HT-induced presynaptic inhibition of vasoconstrictor responses to nerve stimulation in these circulations as well.

The conclusion that a 5-HT1-like receptor mediates pre-synaptic inhibition of sympathetic vascular tone in vivo is therefore based on the actions of 5-Cf and methysergide, and the lack of effect of selective 5-HT2

and 5-HT 3 antagonists. This conclusion is in agreement with in-vitro findings in vascular and non-vascular preparations (see Clarke eta/., this volume). Included among the antagonists which have been used in attempts to identify the 5-HT1-like receptor subtypes responsible for pre-synaptic sympathetic inhibition in vitro are several agents which also act at a- and ~-adrenoceptors (e.g. methiothepin, phentolamine, propranolol, pindolol and cyanopindolol). Effects at pre-synaptic a- and ~-adrenoceptors may in tum influence noradrenaline release, which thereby complicates the interpretation of functional studies of this type. Clearly, the availability of more specific 5-HT1-like receptor antagonists would be helpful in defining the receptor


mediating pre-synaptic inhibition of sympathetic vascular tone, which makes an important contribution to the in-vivo vasodilator action of 5-HT.

ACKNOWLEDGEMENTS

Investigations conducted in the authors' laboratory were supported in part by the National Health and Medical Research Council of Australia, and Janssen Pharmaceutica (Australia).

REFERENCES

Angus, J. A., Cocks, T. M. and Wright, C. E. (1987). Mechanismsof5-HTinduced vasospasm and vasodilatation in large and small arteries. Clin. Exp. Pharmacol. Physiol., Suppl. 11, 20

Bevan, J. A., Duckles, S. P. and Lee, T. J.-F. (1975). Histamine potentiation of nerve- and drug-induced responses of a rabbit cerebral artery. Circ. Res., 36, 647--653

Blackshear, J. L., Orlandi, C., Gamic, J. D. and Hollenberg, N. K. (1985}. Differential large and small vessel responses to serotonin in the dog hindlimb in vivo: role of the 5-HT2 receptor. J. Cardiovasc. Pharmacol., 1, 42-49


Chand, N. (1981). 5-Hydroxytryptamine induces relaxation of goat pulmonary veins: evidence for the noninvolvement of M and D-tryptamine receptors. Br. J. Pharmacol., 72, 233-237

Charlton, K. G., Bond, R. A. and Clarke, D. E. (1986). Aninhibitoryprejunctional 5-HT1-like receptor in the isolated perfused rat kidney: apparent distinction from the 5-HT1A, 5-HT18 and 5-HT1c subtypes. Naunyn-Schmiedeberg's Arch. Pharmacol., 332, 8-15

Cocks, T. M. and Angus, J. A. (1983). Endothelium-dependent relaxation of coronary arteries by noradrenaline and serotonin. Nature, 305, 627--630


Edvinsson, L., Hardebo, J. E. and Owman, Ch. (1978). Pharmacological analysis of 5-hydroxytryptamine receptors in isolated intracranial and extracranial vessels of cat and man. Circ. Res., 42, 143-151

Engel, G., Gothert, M., Miiller-Schweinitzer, E., Schlicker, E., Sistonen, L. and Stadler, P. A. (1983). Evidence for common pharmacological properties of [3H]-5-hydroxytryptamine binding sites, presynaptic 5-llydroxytryptamine autoreceptors in CNS and inhibitory presynaptic 5-hydroxytryptamine receptors on sympathetic nerves. Naunyn-Schmiedeberg's Arch. Pharmacol., 324, 116-124

Eyre, P. (1975). Atypical tryptamine receptors in sheep pulmonary vein. Br. J. Pharmacol., 55, 329-333

144 Serotonin

Feniuk, W., Humphrey, P. P. A. and Watts, A. D. (1979). Presynaptic inhibitory action of 5-hydroxytryptamine in dog isolated saphenous vein. Br. J. Pharmacol., 67, 247-254

Feniuk, W., Humphrey, P. P. A. and Watts, A. D. (1981). Modification of the vasomotor actions of methysergide in the femoral arterial bed of the anaesthetized dog by changes in sympathetic nerve activity. J. Auton. Pharmacol., l, 127-132


Fishburn, P. A., Phillips, C. A. and Mylecharane, E. J. (1987). 5-Hydroxytryptamine-induced inhibition of [3H]-noradrenaline overflow responses in transmurally stimulated dog saphenous vein preparations. Clin. Exp. Pharmacol. Physiol., Suppl. 10, 72

Furchgott, R. F. and Zawadzki, J. V. (1980). The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature, 288,373--376

Garattini, S. and Valzelli, L. (1965). Serotonin, Elsevier, Amsterdam, pp. 169-198 Gothert, M., Kollecker, P., Rohm, N. and Zerkowski, H.-R. (1986a). Inhibitory

presynaptic 5-hydroxytryptamine (5-HT) receptors on the sympathetic nerves of the human saphenous vein. Naunyn-Schmiedeberg's Arch. Pharmacol., 332, 317-323

Gothert, M., Schlicker, E. and Kollecker, P. (1986b ). Receptor-mediated effects of serotonin and 5-methoxytryptamine on noradrenaline release in the rat vena cava and in the heart of the pithed rat. Naunyn-Schmiedeberg's Arch. Pharmacol., 332, 124-130

Haddy, F. J. (1953). Physiological studies of the venous system. Unpublished Ph.D. thesis, University of Minnesota

Haddy, F. J., Gordon, P. and Emanuel, D. A. (1959). The influence of tone upon responses of small and large vessels to serotonin. Circ. Res., 7, 123--130

Kalkman,H. O.,Boddeke,H. W. G. M.,Doods,H. N., Timmermans,P. B. M. W. M. and van Zwieten, P. A. (1983). Hypotensive activity of serotonin receptor agonists in rats is related to their affinity for 5-HT 1 receptors. Eur. J. Pharmacol., 91, 155--156

McCubbin, J. W., Kaneko, Y. and Page, I. H. (1962). Inhibition of neurogenic vasoconstriction by serotonin. Circ. Res., 11, 74-83

McGrath, M. A. (1977). 5-Hydroxytryptamine and neurotransmitter release in canine blood vessels: inhibition by low and augmentation by high concentrations. Circ. Res., 41, 428-435

Markus, J. K., Phillips, C. A. and Mylecharane, E. J. (1989a). Receptor mechanisms involved in the pre-synaptic sympathetic inhibitory action of 5-hydroxytryptamine in the dog common carotid arterial circulation. Eur. J. Pharmacol. (submitted for publication)

Markus, J. K., Phillips, C. A., Mylecharane, E. J. and Shaw, J. (1989b). The mechanism of the vasodilator action of 5-hydroxytryptamine in the dog common carotid arterial circulation in vivo. Eur. J. Pharmacol. (submitted for publication)

Martin, G. R., Leff, P., Cambridge, D. and Barrett, V. J. (1987). Comparative analysis of two types of 5-hydroxytryptamine receptor mediating vasorelaxation: differential classification using tryptamines. Naunyn-Schmiedeberg's Arch. Pharmacol., 336, 365--373

Mena, M. A. and Vidrio, H. (1979). Reversal of serotonin vasodilatation in the dog external carotid bed by sympathetic denervation. J. Cardiovasc. Pharmacol., 1, 149-154

Molderings, G. J., Fink, K., Schlicker, E. and Gothert, M. (1987). Inhibition of


noradrenaline release via presynaptic 5-HT18 receptors of the rat vena cava. Naunyn-Schmiedeberg's Arch. Pharmacal., 336, 245-250

Miiller-Schweinitzer, E. (1981). Agonist potencies of tryptamine derivatives at preand postjunctional receptors in canine saphenous vein. Postgrad. Med. 1., 57, Suppl. 1, 36-44

Mylecharane, E. J. and Phillips, C. A. (1987). 5-HT1-like receptor-mediated pre-synaptic sympathetic inhibition in the dog common carotid arterial circulation. Clin. Exp. Pharmacal. Physiol., Suppl. 11, 30

Mylecharane, E. J., Spira, P. J., Misbach, J., Duckworth, J. W. and Lance, J. W. (1978). Effects of methysergide, pizotifen and ergotamine in the monkey cranial circulation. Eur. J. Pharmacal., 48, 1-9

Mylecharane, E. J., Duckworth, J. W., Lord, G. D. A. and Lance, J. W. (1980). Effects of low doses of clonidine in the monkey cranial circulation. Eur. J. Pharmacal., 68, 163-173

Phillips, C. A. and Mylecharane, E. J. (1987). The actions of 5-hydroxytryptamine in the external carotid circulation of the monkey. Clin. Exp. Pharmacal. Physiol., Suppl. 11, 188

Phillips, C. A., Mylecharane, E. J., Markus, J. K. and Shaw, J. (1985a). Hypotensive actions of ketanserin in dogs: involvement of a centrally mediated inhibition of sympathetic vascular tone. Eur. J. Pharmacal., Ill, 319-327

Phillips, C. A., Mylecharane, E. J. and Shaw, J. (1985b). Mechanisms involved in the vasodilator action of 5-hydroxytryptamine in the dog femoral arterial circulation in vivo. Eur. J. Pharmacal., 113, 325-334

Saxena, P. R. (1972). The effects of antimigraine drugs on the vascular responses by 5-hydroxytryptamine and related biogenic substances on the external carotid bed of dogs: possible pharmacological implications to their antimigraine action. Headache, 12, 44-54

Saxena, P.R. and Verdouw, P. D. (1982). Redistribution by 5-hydroxytryptamine of carotid arterial blood at the expense of arteriovenous anastomotic blood flow. J. Physiol., 332, 501-520

Spira, P. J., Mylecharane, E. J., Misbach, J., Duckworth, J. W. and Lance, J. W. ( 1978). Internal and external carotid vascular responses to vasoactive agents in the monkey. Neurology, 28, 162-173

Su, C. and Uruno, T. (1985). Excitatory and inhibitory effects of 5-hydroxytryptamine in mesenteric arteries of spontaneously hypertensive rats. Eur. J. Pharmacal., 106, 283-290

Swank, R. L. and Hissen, W. (1964). Influence of serotonin on cerebral circulation. Arch. Neurol., 10, 468-472

Trevethick, M. A., Feniuk, W. and Humphrey, P. P. A. (1984). 5-Hydroxytryptamine-induced relaxation of neonatal porcine vena cava in vitro. Life Sci., 35, 477-486

VanNueten,J. M.,Janssen,P.A.J., VanBeek,J.,Xhonneux,R., Verbeuren, T.J. and Vanhoutte, P.M. (1981). Vascular effects of ketanserin (R 41468), a novel antagonist of 5-HT2 serotonergic receptors. J. Pharmacal. Exp. Ther., 218, 217-230

Vidrio, H. and Hong, E. (1976). Vascular tone and reactivity to serotonin in the internal and external carotid vascular beds of the dog. J. Pharmacal. Exp. Ther., 197, 49-56

Watts, A. D.,Feniuk, W. andHumphrey,P. P. A. (1981). A pre-junctional action of 5-hydroxytryptamine and methysergide on noradrenergic nerves in dog isolated saphenous vein. J. Pharm. Pharmacal., 33, 515-520

19 5-HT tA Receptors and Cardiovascular Control

J. R. Fozard, 1 A. K. Mir and A. G. Ramage

1Preclinical Research Department, Sandoz Limited, CH-4002 Basel, Switzerland

2 Academic Department of Pharmacology, Royal Free Hospital Medical School, Hampstead, London NW3 2PF, UK

INTRODUCTION

Substantial evidence implicates 5-hydroxytryptamine (5-HT) neurones in the control of cardiovascular function. The precise nature of that role remains, however, undefined, a reflection in part of the plurality of 5-HT receptors and, in particular, the absence until recently of truly selective agonists and antagonists for these sites (Bradley et al., 1986). However, a number of compounds are now recognized to have both high affinity and a certain selectivity for the 5-HT lA subtype of high-affinity [3H]-5-HT binding sites, including 8-hydroxy-2-( di-n-propylamino )tetralin (8-0H-DPAT), dipropyl-5-carboxamidotryptamine (DP-5-CT), MDL 72832, ipsapirone and flesinoxan (Middlemiss and Fozard, 1983; Dompert et al., 1985; Hagenbach et al., 1986; Bevan eta/., 1986; Fozard eta/., 1987a). Described below are the results from experiments in rats and cats in which these compounds have been used to explore the role of the putative 5-HT1A

receptor in cardiovascular control.

EXPERIMENTS IN THE RAT

The prototype selective 5-HT1A receptor ligand 8-0H-DPAT lowers both blood pressure and heart rate in conscious, spontaneously hypertensive (SH) and normotensive anaesthetized rats ( Gradin et al., 1985; Fozard et al., 1987b), predominantly, if not exclusively, by an action in the CNS (see Fozard et al., 1987b; Mir and Fozard, 1987). A detailed pharmacological analysis (see Table 19.1) allows the involvement of aradrenoceptors and dopamine, 5-HT2 and 5-HT3 receptors to be ruled out, and strongly implicates 5-HT1A receptors as the primary site of action (Fozard et al.,

5-HT1A Receptors and Cardiovascular Control 147

1987b; Mir and Fozard, 1987). Such a conclusion receives further support from the results obtained with the highly potent, selective and stereospecific 5-HT1A receptor ligand (±)-MDL 72832 (Fozard et al., 1987a). (±)-MDL 72832 dose-dependently and selectively inhibited the cardiovascular effects of 8-0H-DPAT in anaesthetized rats, as opposed to an equieffective dose of clonidine; (-)-MDL 72832 was some 10-20 times more potent than (+)-MDL 72832 as an antagonist of 8-0H-DPAT, consisteat with its higher affinity for the 5-HT1A recognition site (Fozard et al., 1987a).

In the rat, but not in the cat (see below), an indirect catecholaminergic component to the mechanism of action of 8-0H-DPAT is suggested by susceptibility to blockade by «radrenoceptor antagonists (Table 19.1) and the fact that responses are absent in animals in which central monoamine stores are depleted following DL-a-mono-fluoromethyldopa, yet unchanged following pre-treatment with p-chlorophenylalanine, which causes a greater than 90 per cent decrease in central 5-HT concentrations (Fozard et al., 1987b).

In contrast to the results obtained with 8-0H-DPAT, a second highly potent and selective ligand at 5-HT1A receptors, DP-5-Cf (4-128 !J.g/kg i. v. ), causes hypotension associated with reflex tachycardia in conscious SH rats(Miretal., 1987). Moreover, unlike8-0H-DPAT, whichdoesnotlower blood pressure in pithed normotensive or SH rats, even when blood pressure is raised to normal with angiotensin II (Fozard et al., 1987b) or vasopressin (Gradin et al., 1985), DP-5-Cf caused dose-related falls in blood pressure with no significant effect on heart rate in pithed SH rats (Mir et al., 1987).

Table 19.1 Pharmacological dissection of the cardiovascular effects of 8-0HDPAT in the normotensive anaesthetized rat

Response to Compound Site 8-0H-DPAT Clonidine Metergoline 5-HT1-like, 5-HT2 block Methiothepin 5-HT1-like, 5-HT2 block (-)-Pindolol 5-HT1A, 5-HT18 block (±)-Cyanopindolol 5-HT1A, 5-HT18 block Spiperone 5-HT1A, 5-HT2 toxic Buspirone 5-HT1A block 8-MeO-ClEPAT' 5-HT1A block Ketanserin 5-HT2 no block MDL 72222 5-HT3 no block Yohimbine aradrenoceptor, 5-HT10 block WY 26392 aradrenoceptor block Idazoxan aradrenoceptor block Prazosin aradrenoceptor no block Cis-flupenthixol dopamine no block

no block no block block block toxic block no block

block block block no block

88-MeO-CIEPAT = 8-methoxy-2-(N-2-chloroethyl-N-n-propyl)aminotetralin. Full quantitative data are given in Fozard eta/. (1987b).

148 Serotonin

Clearly, the cardiovascular effects of DP-5-Cf are quite different from those of 8-0H-DPAT, a similarly selective 5-HT1A receptor agonist. It seems likely that the difference between DP-5-Cf and 8-0H-DPAT reflects poor penetration of the former into the brain, and thus an inability to activate the centra/5-HT tA receptors (Fozard eta/., 1987b ). The mechanism of the peripheral vasodilator action of DP-5-Cf remains to be established. However, it must involve a site quite different from that activated by 8-0H-DPAT, since (±)-MDL 72832, at a dose (100 tJ.g/kg s.c.) which completely and selectively blocks the cardiovascular response to 8-0HDPAT (Fozard eta/., 1987a), failed to inhibit the vasodepressor effects of submaximal hypotensive doses of DP-5-Cf in anaesthetized rats (Mir and Fozard, unpublished observations). It is interesting to note that the close structural analogue 5-carboxamidotryptamine (5-Cf), which has high affinity not only for 5-HT1A recognition sites but also for 5-HTm and 5-HT10 sites (Heuring and Peroutka, 1987; Hoyer, 1989), also results in a lowered blood pressure associated with marked reflex tachycardia in conscious SH rats (Dalton et al., 1986). Since DP-5-Cf has moderate and 8-0H-DPAT negligible affinity for 5-HT10 sites (Hoyer, 1989), it is possible that DP-5-Cf and 5-Cf lower blood pressure by activating a 5-HT10 site in the peripheral vasculature.

EXPERIMENTS IN THE CAT

The effects of drugs that show selectivity for the 5-HT1A recognition site have also been examined in anaesthetized cats in which central sympathetic tone and blood flow in at least one vascular bed (femoral) are monitored in addition to blood pressure and heart rate (Ramage, 1984). This model has been used to investigate the cardiovascular effects of 8-0H-DPAT, ipsapirone (Ramage and Fozard, 1987) and flesinoxan (Bevan eta/., 1986).

Cumulative i. v. doses of 8-0H-DP AT (0.5-128 tJ.g/kg), ipsapirone (2-512 tJ.g/kg) and flesinoxan (3-300 tJ.g/kg) cause dose-related falls in blood pressure which are associated with moderate thoracic sympathoinhibition. At higher doses, all three drugs cause profound bradycardia which can be substantially inhibited by an injection of atropine methonitrate or by bilateral vagotomy. A comparison of the effect of a single bolus injection of 8-0H-DPAT on the above parameters in an intact and a bilaterally vagotomized cat is shown in Figure 19.1. These observations indicate that a primary property of these compounds is to cause a bradycardia by increasing vagal tone. Figure 19.1 also shows that a bolus injection of these compounds causes an increase in femoral arterial conductance, therefore indicating some degree of femoral vasodilatation along with the fall in blood pressure.


(a) VAGI INTACT (b) VAGI CUT

8-0H-DPAT 8-0H-DPAT

mlmmHg.1 min'~ ~ ~ FAC 01l ~32~- l32fJg kg"

0 soo ~oo-

PSNA 300 ,J"lr'~ 100~ ~~ spikes 10s"" _ \{ - - ~

o o-

FAF ~1 ~ mlminrlJ~ 1 -~

HR :::J~ --.._m_'"-v----------beatsmiri' -~

100

~~ ........ _. .... Figure 19.1 Traces comparing the effects of bolus i.v. injection of 8-0H-DPAT (32 11g/kg) in (a) an intact and (b) a bilaterally vagotomized anaesthetized cat, on femoral arterial conductance (FAC), pre-ganglionic sympathetic nerve activity (PSNA), femoral arterial flow (FAF), heart rate (HR) and blood pressure (BP)

It is somewhat surprising, considering that these compounds cause such large falls in blood pressure, that the degree of femoral arterial vasodilatation was minimal. In this context, it is interesting that injections of both 8-0H-DPAT and ipsapirone (although not flesinoxan) are associated with transient femoral arterial vasoconstriction (Ramage and Fozard, 1987). The results indicate that vascular beds other than the femoral are playing a more important role in the reduction of total peripheral resistance; clearly, these drugs may be having more marked sympathoinhibitory actions at levels of sympathetic outflow other than the thoracic.

The characteristic haemodynamic profile of putative 5-HT tA receptor agonists in the cat differs markedly from that observed for the aradrenoceptor agonist clonidine, in that the fall in blood pressure caused by clonidine was associated with a parallel reduction in thoracic sympathetic tone and the associated bradycardia was due exclusively to withdrawal of sympathetic tone to the heart (Ramage and Fozard, 1987). This suggests that unlike in the rat, activation of 5-HT tA sites in the cat does not result in an indirect activation of aradrenoceptors. Like the putative 5-HT1A receptor agonists, clonidine failed to cause vasodilatation in the femoral bed, which may reflect the well-known peripheral vasoconstrictor action of this agent, thereby masking the effects of withdrawal of sympathetic drive.

150 Serotonin

CONCLUSIONS

There is growing evidence for a key role for central 5-HT1A receptors in cardiovascular control in the rat and the cat. Thus, compounds like 8-0H-DPAT, flesinoxan and ipsapirone, which have good affinity and a certain selectivity for central 5-HT1A recognition sites, show hypotensive activity which is associated with an increase in vagal tone and sympathoinhibition. Further, a detailed pharmacological analysis of the response to 8-0H-DP AT in the rat has provided strong evidence that central 5-HT1A receptors mediate the effects. There is evidence of an indirect involvement of az-adrenoceptors in the mediation of the cardiovascular effects of 8-0H-DPAT in the rat, but not in the cat. Activation of central 5-HT1A receptors is not involved in the vasodepressor effects of DP-5-CT, presumably a reflection of its poor penetration to the brain. A peripheral site different from the 5-HT1A receptor is responsible for the peripheral vasodilator effects of DP-5-CT.

The question as to whether the capacity to modulate central sympathetic activity and/or vagal tone via activation of central 5-HT1A receptors is of therapeutic importance must await the outcome of clinical experience with compounds having a high selectivity for these sites, such as flesinoxan. In the meantime, however, the validity of such an antihypertensive principle is supported by the finding that urapidil, a marketed antihypertensive drug with a 1-adrenoceptor antagonist activity, has appreciable potency and mixed agonist/antagonist effects at 5-HT1A receptors (Fozard and Mir, 1987), and hypotensive activity accompanied by inhibitory effects on sympathetic nerve activity and vagal tone in animal experimental models (Sanders and Juma, 1985).

REFERENCES

Bevan, P., Ramage, A. G. and Wouters, W. (1986). Investigation of the effects of DU 29373 on the cardiovascular system of the anaesthetized cat. Br. J. Pharmacol., 89, 506P

Bradley, P. B., Engel, G., Feniuk, W., Fozard, J. R., Humphrey, P. P. A., Middlemiss, D. N., Mylecharane, E. J., Richardson, B. P. and Saxena, P. R. (1986). Proposals for the classification and nomenclature of functional receptors for 5-hydroxytryptamine. Neuropharmacology, 25, 56>-576

Dalton, D. W., Feniuk, W. and Humphrey, P. P. A. (1986). An investigation into the mechanisms of the cardiovascular effects of 5-hydroxytryptamine in conscious normotensive and Doca-Salt hypertensive rats. J. Auton. Pharmacol., 6, 219--229

Dompert, D. U., Glaser, T. and Traber, J. (1985). [3H)-TVX Q 7821: identification of 5-HT1 binding sites as targets for a novel putative anxiolytic. NaunynSchmiedeberg's Arch. Pharmacol., 328, 467-470

Fozard, J. R. and Mir, A. K. (1987). Are 5-HT1A receptors involved in the antihypertensive effects of urapidil? Br. J. Pharmacol., 90, 24P


Fozard, J. R., Hibert, M., Kidd, E. J., Middlemiss, D. N., Mir, A. K. and Tricklebank, M. D. (1987a). MDL 72832: a potent, selective and stereospecific ligand for 5-HT1A receptors. Br. J. Pharmacol., 90, 273P

Fozard, J. R., Mir, A. K. and Middlemiss, D. N. (1987b). The cardiovascular response to 8-hydroxy-2-( di-n-propylamino) tetralin (8-0H-DP AT) in the rat: site of action and pharmacological analysis. J. Cardiovasc. Pharmacol., 9, 328-347

Gradin, K., Petterson, A., Hedner, T. and Persson, B. (1985). Acute administration of 8-hydroxy-2-(di-n-propylamino) tetralin (8-0H-DPAT), a selective 5-HT receptor agonist, causes a biphasic blood pressure response and a bradycardia in the normotensive Sprague-Dawley rat and the spontaneously hypertensive rat. J. Neural Transm., 62, 305-319

Hagenbach, A., Hoyer, D., Kalkman, H. 0. and Seiler, M. P. (1986). N,N-Dipropyl-5-carboxamidotryptamine (DP-5Cf), an extremely potent and selective 5-HT1A agonist. Br. J. Pharmacol., 87, 136P

Heuring, R. E. and Peroutka, S. J. (1987). Characterization of a novel (3H]-5-hydroxytryptamine binding site subtype in bovine brain membranes. J. Neurosci., 7, 894-903

Hoyer, D. (1989). In Fozard, J. R. (Ed.), The Peripheral Actions of 5-Hydroxytryptamine, Oxford University Press, Oxford, pp. 72-99

Middlemiss, D. N. and Fozard, J. R. (1983). 8-Hydroxy-2-(di-n-propylamino) tetralin discriminates between subtypes of the 5-HT recognition site. Eur. J. Pharmacol., 90, 150-153

Mir, A. K. and Fozard, J. R. (1987). Cardiovascular effects of 8-hydroxy-2-(di-npropylamino) tetralin (8-0H-DPAT). In Dourish, C. T., Ahlenius, S. and Hutson, P. H. (Eds), Brain 5-HT1A Receptors, Ellis Horwood, Chichester, pp. 120-134

Mir, A. K., Hibert, M. and Fozard, J. R. (1987). In Nobin, A., Owman, Ch. and Ameklo-Nobin, B. (Eds), Neuronal Messengers in Vascular Function, Elsevier, Amsterdam, pp. 21-29

Ramage, A. G. (1984). The effect of propranolol and other antihypertensive drugs on preganglionic sympathetic nerve activity. Neuropharmacology, 13, 43-48

Ramage, A. G. and Fozard, J. R. (1987). Evidence that the putative 5-HT1A

receptor agonists, 8-0H-DP AT and ipsapirone have a central hypotensive action that differs from that of clonidine in anaesthetised cats. Eur. J. Pharmacol., 138, 179-191

Sanders, K. H. and Juma, I. (1985). Effects of urapidil, clonidine, prazosin and propranolol on autonomic nerve activity, blood pressure and heart rate in anaesthetized rats and cats. Eur. J. Pharmacol., liD, 181-190

20 Cardiac, Vascular and other Smooth-muscle

Actions of 5-Bydroxytryptamine: An Overview

R. E. Purdyl and P. R. Saxentl

1Department of Pharmacology and Cardiovascular Biology Group, University of California, Irvine CA 92717, USA

2Department of Pharmacology, Faculty of Medicine, Erasmus University Rotterdam, PO Box 1738, 3000 DR Rotterdam, The Netherlands

The session on the effects of 5-hydroxytryptamine (5-HT) on cardiac, vascular and other smooth muscle systems revealed a multiplicity of actions mediated through a variety of receptors.

The cardiac actions of 5-HT were reviewed by Pramod Saxena. Most animals with intact vagi respond to 5-HT primarily with a reflex bradycardia mediated by 5-HT3 receptors. After elimination of reflexes, 5-HT generally elicits cardiostimulation. Studies using cultured cells, isolated tissues and whole organisms from a variety of animal species were outlined; it was concluded that the mechanism and the nature of 5-HT receptors involved in the tachycardiac responses to 5-HT vary widely. 5-HT acting directly on the myocardium increases heart rate via either 5-HT1-like receptors in the cat, 5-HT2 receptors in the rat, or a new type of putative 5-HT receptor in the pig. The 5-HT1-like receptors mediating tachycardia in the cat do not seem to correlate with any of the 5-HT 1 binding site subtypes described so far. The pig heart 5-HT receptor is unaffected by any of the recognized agonists or antagonists which act at 5-HT1-like, 5-HT2 or 5-HT3 receptors. Manfred Gothert commented on his findings regarding the 5-HT receptor responsible for pre-synaptic sympathetic inhibition in the pig coronary artery, and suggested that this receptor was similar to that mediating tachycardia in the pig. 5-HT also increases heart rate indirectly by releasing endogenous catecholamine; such an effect may involve either 5-HT2 receptors on the adrenal medulla in the dog, 5-HT3 receptors present on the cardiac sympathetic nerve fibres in the rabbit, or even a tyramine-like action in the guinea-pig.

The amplifying action of 5-HT was the subject of an interesting presentation by Ivan de Ia Lande and his colleagues. In many arteries, both

Overview: Cardiovascular Actions 153

the synergistic and contractile actions of 5-HT are mediated by the 5-HT2

receptor. However, an apparent exception is the perfused rabbit ear artery, where synergism with noradrenaline is mediated by the 5-HT 2 receptor, and 5-HT-induced constriction (almost paradoxically) by the a-adrenoceptor. More recent data outlined by de la Lande demonstrated an interesting difference between ear artery strips and rings obtained from reserpine pre-treated rabbits, compared with perfused artery segments from non-reserpinized rabbits. In the arterial strips and rings, the 5-HT2 receptor contributes to constriction, but the synergistic interaction is less pronounced than in the perfused segments and does not involve the 5-HT2 receptor. It was suggested that these differences may reflect a high threshold in the 5-HT2 receptor-mediated co-vasoconstrictor response in the perfused artery. Alternatively, it is possible that the reserpine pre-treatment of the artery strips and rings caused an important qualitative change in the receptor mediating constriction. It was also noted that the synergistic action of 5-HT is influenced by the nature of the interacting agonist. Thus, prazosin (0.08 !J.M) attenuates the interaction between 5-HT and noradrenaline, but is without effect on the interaction between 5-HT and methoxamine. The interaction between 5-HT and histamine is unaffected by prazosin, but is markedly increased when sensitivity to histamine is depressed by the histamine H1 receptor antagonist mepyramine (0.1 !J.M) or by ketanserin (0.5 !J.M). The increase in sensitivity may be related to stimulation of histamine H2 receptors, since it is prevented by the H2 receptor antagonist cimetidine, and the specific H2 receptor agonist impromidine increases the synergistic interaction between 5-HT and a specific H1 receptor agonist, 2-pyridylethylamine.

The role of a-adrenoceptors in both 5-HT-induced vasoconstriction and contractile synergism with noradrenaline was reviewed by Ralph Purdy and his colleagues. Data from the literature indicate that 5-HT acts almost exclusively on a-adrenoceptors of the rabbit ear artery and on 5-HT2 receptors in rabbit thoracic aorta, but on both 5-HT2 receptors and a-adrenoceptors in other rabbit arteries (abdominal aorta, femoral, saphenous, mesenteric and radial). Under conditions of 5-HT receptor blockade or tachyphylaxis, 5-HT was found also to stimulate aadrenoceptors of the rabbit thoracic aorta. It was proposed that 5-HT is an agonist at the a-adrenoceptors on all blood vessels. Whether or not 5-HT stimulates a-adrenoceptors in a given vessel is dependent on the relative densities of 5-HT and a-adrenoceptors in that vessel. A synergistic interaction between 5-HT and the a-adrenoceptor agonist methoxamine was also observed in several rabbit blood vessels. When added to the bathing medium at threshold concentrations, the combination of these agonists caused a far greater than additive contraction, and changing the order of addition had no effect. A full concentration-response curve for one of these agonists is shifted to the left in the presence of a threshold concentration of

154 Serotonin

the other. However, order of administration changes the degree of shift. Using a threshold concentration of methoxamine caused a 100-fold shift of the 5-HT concentration-response curve in the rabbit femoral artery, while 5-HT caused an 8-fold shift of the curve for methoxamine.

The two preceding studies had drawn attention to some anomalous aspects of the interactions between a-adrenoceptor and 5-HT2 receptor agonists in the rabbit ear artery. Coincidentally, evidence was presented (as a poster) by Rosemary Bevan which suggested that the activity of the membrane pump influenced the relative importance of the adrenoceptor Na+/K+ and a 5-HT receptor in mediating contractile effects of 5-HT in the rabbit ear artery. The evidence was based on the appearance of a prazosin-insensitive response to 5-HT under conditions (chronic sympathetic denervation or oubain treatment) which are known to decrease the activity of the electrogenic component of the pump. Discussion between the 'rabbit ear' workers centred around the possibility that the anomalous aspects of synergy may relate to an altered level of pump activity imposed by pre-treatment procedures or by the agonist interacting with 5-HT.

The role of 5-HT and pre-synaptic sympathetic inhibition in 5-HTinduced vasodilatation in vivo was explored by Ewan Mylecharane and his colleagues. It was pointed out that vasodilatation is a prominent action of 5-HT in vivo. Studies utilizing in-vitro vascular preparations have revealed three mechanisms by which 5-HT could produce vasodilation: (a) a direct relaxant effect on vascular smooth muscle; (b) release of endotheliumderived relaxing factor; and (c) pre-synaptic inhibition of the release of noradrenaline from sympathetic nerve terminals in the vascular wall. The vasodilator responses to 5-HT in the hind limb and common carotid circulations of anaesthetized dogs were characterized. These responses were abolished after ganglion blockade or acute cervical sympathectomy, even when tone was restored with ornipressin. 5-HT did not alter the constrictor response to exogenous noradrenaline. These results support the possibility that 5-HT produced a pre-synaptic inhibition of sympathetic vascular tone, making a major contribution to 5-HT-induced vasodilatation in vivo. This pre-synaptic inhibitory effect of 5-HT could also be demonstrated in vivo as an inhibition of the dog common carotid constrictor response to cervical sympathetic nerve stimulation; a similar inhibition could be shown in the monkey external carotid circulation. Studies in dogs (in vivo) with ketanserin, pizotifen, pirenperone, MDL 72222 and ICS 205-930 ruled out mediation by 5-HT2 and 5-HT3 receptors. In contrast, experiments using methysergide, methiothepin and 5-carboxamidotryptamine indicated that a 5-HT 1-like receptor may be responsible for the pre-synaptic inhibitory effect of 5-HT on sympathetic vascular tone in vivo.

A role for 5-HT1A receptors in cardiovascular control was described by John Fozard and his colleagues, who studied the effects of 8-hydroxy-2-( din-propylamino)tetralin (8-0H-DPAT), a selective 5-HT1A agonist, in

Overview: Cardiovascular Actions 155

anaesthetized cats. This and two other 5-HT1A receptor agonists, flesinoxan and ipsapirone, caused moderate central sympathoinhibition and a pronounced vagally mediated bradycardia. In contrast, the az-adrenoceptor agonist clonidine caused profound sympathoinhibition and only a minimal vagally mediated bradycardia. 8-0H-DPAT caused both hypotension and bradycardia in anaesthetized normotensive rats, and in conscious spontaneously hypertensive rats. These effects of 8-0H-DPAT were mediated by a direct central effect on 5-HT1A receptors, and an indirect catecholaminergic link. Dipropyl-5-carboxyamidotryptamine (D P-5-CT), a potent and selective ligand at 5-HT1A binding sites, was also studied. However, this agent produced unexpected haemodynamic effects, some of which were not blocked by selective 5-HT1A antagonists. On the basis of these findings, a key role was suggested for central 5-HT1A receptors in cardiovascular control; it was concluded that DP-5-CT may be of limited usefulness in the definition of functional 5-HT1A receptors in the cardiovascular system.

As well as being involved in the investigations of 5-HT lA receptors and cardiovascular control, Andrew Ramage has also utilized the anaesthetized cat model to investigate the effects of 5-HT2 receptor antagonists on cardiovascular control. At dose levels specific for 5-HT2 receptor blockade (i.e. lower than those producing vascular a-adrenoceptor blocking activity), ketanserin reduces blood pressure and heart rate, in parallel with central sympathoinhibition. Mylecharane and colleagues have also described evidence for a central sympathoinhibitory effect of ketanserin in anaesthetized dogs at similar dose levels. In addition Ramage outlined the variable cardiovascular effects of some other 5-HT2 receptor antagonists. Ritanserin (which is more selective than ketanserin for 5-HT2 receptors as opposed to a-adrenoceptors) appears to have a direct vasodilator effect and modest central sympathoinhibitory activity, because it produced femoral vasodilatation and a slight hypotensive and bradycardiac effect, but sympathetic outflow was unchanged, perhaps reflecting a balance between central inhibition and a reflex increase following vasodilatation. In line with this interpretation, L Y 53857 (which is devoid of a-adrenoceptor-blocking activity) may have no central sympathoinhibitory activity, because it produces femoral vasodilatation, no change in blood pressure or heart rate, and an increase in sympathetic outflow. However, the effects of cinanserin on femoral blood flow, blood pressure and heart rate were similar to those of L Y 53857, but sympathetic activity was unaltered. Evidence suggestive of variable peripheral vascular and sympathoinhibitory activity with pizotifen and pirenperone in anaesthetized dogs was mentioned by Mylecharane. Ramage suggested that central a 1-adrenoceptor blocking effects with the 5-HT2 receptor antagonists might be related to their sympathoinhibitory activity.

Changes in serotoninergic function related to age were described briefly

156 Serotonin

by Ian Creese and Noburu Toda. Creese outlined data suggesting that rat frontal cortex 5-HI' 2 receptors were down-regulated with increasing age; functional correlates are not known at present. In contrast, Toda described an enhancement of 5-HI'-induced contractile responses in dog coronary artery preparations with increasing age; the change is both 5-HI'-specific (other constrictors such as angiotensin II and noradrenaline do not exhibit age-related enhanced responses) and vessel-specific (dog cerebral and mesenteric arteries do not exhibit age-related changes). The dog coronary 5-HI' contractile responses were blocked by ketanserin and cinanserin. These observations may be of relevance in assessing serotoninergic cardiovascular control mechanisms.

The difficulties of assessing antagonist activity against 5-HI'-induced smooth muscle contraction in rat stomach fundus and guinea-pig ileum preparations were alluded to briefly, the main problems being the various complicating effects of 5-HI' at other sites in guinea-pig ileum, and the fact that many antagonists produce non-surmountable blockade in rat stomach fundus. These aspects were further explored in the receptor classification workshop (see Humphrey and Richardson, this volume).

PARTV CLASSIFICATION OF

5-HT RECEPTORS AND BINDING SITES

21 The Subclassification ofFunctiona15-HT 1-like

Receptors

P. P. A. Humphrey and W. Feniuk

Pharmacology Division, Glaxo Group Research Limited, Ware, Hertfordshire SG 12 ODP, UK

INTRODUCTION

Since the identification of 'D' and 'M' receptors by Gaddum and Picarelli (1957), 5-hydroxytryptamine (5-HT) receptor types have been reclassified into 5-HT1-like, 5-HT2 and 5-HT3 (Bradley et al., 1986). With good receptor-blocking drugs now available for 5-HT2 and 5-HT3 receptors, their distribution and classification is relatively well studied. However, numerous examples of 5-HT receptor types have been described which are of neither the 5-HT 2 nor the 5-HT 3 type, and the majority of these can be classified as 5-HT1-like according to the criteria proposed by Bradley et al. (1986; see below). Unfortunately, these receptors cannot be fully characterized and subclassified, owing to the absence of good selective drug tools. Nevertheless, the term 5-HTrlike conveys the view that these receptors have characteristics which are the same or similar to one of the 5-HT 1

binding sites in the brain, of which at least four subtypes are currently known. However, we have consistently argued that any pharmacological receptor classification should be based on functional studies, and that many 5-HT1-like receptors, notably but not exclusively those found in isolated smooth-muscle preparations, are not the same as the known 5-HT 1 binding sites (Humphrey, 1983, 1984; Bradley et al., 1986; Humphrey and Feniuk, 1987a, b). This is now discussed further in the light of recent new data from studies in functional preparations using ligands for 5-HT1 binding sites, and a novel tryptamine analogue AH 25086, which appears to be a potent, selective agonist at a subtype of 5-HTrlike receptor (Humphrey et al., 1987).

160 Serotonin

SUBCLASSIFICATION OF 5-HT1 BINDING SITES AND THEIR FUNCTIONAL RELEVANCE

5-HT1 binding sites in rat brain were first identified by Peroutka and Snyder (1979); these sites have subsequently been subdivided into 5-HT lA, 5-HT 18,

5-HT1c and 5-HT10 (Table 21.1). All of these 5-HT1 sites are characterized by the high affinity of 5-HT. Each 5-HT1 subtype has a different profile of affinities for different ligands, many of which have greater affinity for other non-5-HT receptor types. Within the limitations of such drug tools, evidence is starting to accumulate that specific functions are mediated via some of these sites.

The 5-HT lA site appears to be involved in inhibition of transmitter release and hyperpolarization of central neurones (Table 21.1). However, much of the evidence is based on studies with the agonist 8-hydroxy-2-( di-npropylamino)tetralin (8-0H-DPAT), which has many actions including antagonism at a-adrenoceptors as well as the ability to interfere with neuronal uptake of 5-HT (Langer and Schoemaker, 1986; Crist and Suprenant, 1987). Nevertheless, there is recent evidence to suggest that 8-0H-DPAT and 5-HT stimulate a common 5-HT1A receptor which mediates activation of adenylate cyclase in guinea-pig hippocampus (Shenker et al., 1987).

The 5-HT18 site has been definitively correlated with the rat 5-HT autoreceptor on cortical serotoninergic nerves, which inhibits 5-HT release (Table 21.1). The same receptor appears to be involved in the potentiation of neuronally mediated contraction of mouse bladder (Holt et al., 1986). However, enigmatically, the 5-HT 18 receptor site appears to be restricted to the mouse and rat (Hoyer et al., 1986).

The 5-HT1c binding site has a very discrete localization, largely in the choroid plexus, where its functional importance, if any, is not clear. It is characterized by a high affinity for mesulergine and methysergide but not spiperone, all of which have high affinity for the 5-HT2 binding site (Table 21.1). Despite the close similarities between the two sites, evidence has been provided to show that in the choroid plexus phosphatidylinositol turnover by 5-HT is mediated via a receptor more like the 5-HT1c than the 5-HT2 site (Sanders-Bush and Conn, 1986).

The newly identified 5-HT10 binding site in the basal ganglia of bovine brain has no known functional correlate, althougb it has been speculated that it is like the 5-HT receptor which mediates inhibition of noradrenaline release from sympathetic nerves in rat kidney (Heuring and Peroutka, 1987).

SUBCLASSIFICATION OF 5-HT1-LIKE RECEPTORS IDENTIFIED FROM FUNCTIONAL STUDIES

5-HT1-like receptors can be characterized by (a) the high potency of

Functional 5-HTrlike Receptor Subtypes 161

5-carboxamidotryptamine (5-CT), which is greater than that of 5-HT, (b) specific antagonism by methiothepin, and (c) lack of blockade by 5-HT2 and 5-HT3 receptor antagonists. These criteria were proposed to encompass not only the 5-HT1 binding sites within this classification but also those receptors, identified from functional studies independently of ligand binding studies (Apperley et al., 1977; Feniuk et al., 1979), which are obviously similar but not the same as the binding sites (Bradley et al., 1986). Interestingly, 5-CT was identified by us as a selective agonist for these receptors in such functional studies before its high affinity for the various 5-HT1 binding sites (except 5-HT1c; Table 21.1) had been determined (see Humphrey and Feniuk, 1987a).

Following the identification of 5-CT, it was proposed that 5-HT1-like receptors identified from functional studies can be subdivided into two types, based on the relative potencies of 5-CT and some other tryptamine agonists (Humphrey, 1984). One receptor type is typified by that which mediates contraction of dog saphenous vein, and the other receptor type by that which mediates relaxation of various isolated veins (Table 21.1). Discussed below are new data with an agonist, AH 25086, which is exclusively selective for the 5-HT1-like receptor in dog saphenous vein and provides further evidence for the subdivision of peripheral 5-HT rlike receptors (Humphrey et al., 1987; Feniuk and Humphrey, 1989).

AH 25086 (0.05-5 J.tM) produces contraction in dog isolated saphenous vein with a concentration-effect curve which is parallel to that for 5-HT (0.01-5 J.tM), the maximal response (92 ± 8 per cent of the 5-HT maximal; mean ± s.e. mean of 4 determinations) not being significantly different from that for 5-HT. The EC50 value for AH 25086 (concentration to produce 50 per cent of maximal response) is about 0.3 J.tM, compared with that of about 0.08 J.I.M for 5-HT. The contractile action of AH 25086 is specifically antagonized by methiothepin, to a similar degree·as for 5-HT, indicating that AH 25086 acts via the same receptor as 5-HT (Apperley and Humphrey, 1986; Feniuk and Humphrey, 1989). This, together with the lack of antagonism by ketanserin and MDL 72222, suggests that the receptor involved is not of the 5-HT2 or 5-HT3 type, and implicates a 5-HTrlike receptor (Figure 21.1).

Although AH 25086 is only about 4-fold less potent than 5-HT as a contractile agent in the dog saphenous vein, it is virtually devoid of activity as an agonist or antagonist (10 J.tM) at 5-HT2 receptors mediating vascular contraction or at 5-HT 3 receptors mediating neuronal depolarization (Table 21.2). AH 25086 is also inactive at 5-HT rlike receptors mediating relaxation in the cat saphenous vein and porcine vena cava, but is potent in inhibiting neuronally mediated contractions in the dog saphenous vein, being only about 6-fold weaker than 5-HT in this respect (Table 21.2). This is consistent with our previous claim that the pre-and post-junctional 5-HTrlike receptors in the dog saphenous vein are the same, and different from those

Tab

le 2

1.1

Cha

ract

eris

tics

of 5

-HT 1

bin

ding

site

sub

type

s

5-H

TtA

5-

HTl

B Ra

diol

abel

led

by

[3H

]-5-

HT

[3 H]-

5-H

T [3

H]-

8-0 H

-DP A

T

P25I]

-Cya

nopi

ndol

ol

Hig

h-de

nsity

re

gion

s

Dru

g po

tenc

ies*

<lO

nM

10-1

()()()

OM

Pote

ntia

l fu

nctio

nal

corr

elat

es

Rap

he n

ucle

i H

ippo

cam

pus

5-C

T 8-

0H-D

PAT

Met

ergo

line

Spip

eron

e

Rap

he a

nd

hipp

ocam

pal

inhi

bitio

n,

and

inhi

bitio

n/

activ

atio

n of

bra

in

aden

ylat

e cy

clas

e *K

i(nM

); LS

D =

lyse

rgic

aci

d di

ethy

lam

ide.

M

odifi

ed fr

om H

euri

ng a

nd P

erou

tka

(198

7).

(rat

and

m

ouse

onl

y)

Subs

tant

ia n

igra

RU

249

69

5-C

T

Met

ergo

line

Syna

ptos

omal

'a

utor

ecep

tor'

5-H

Ttc

[3H

]-5-

HT

[3H

]-M

esul

ergi

ne

Cho

roid

ple

xus

Mes

uler

gine

M

eter

golin

e

Mia

nser

in

5-C

T

Phos

phat

idyl

in

ositi

de

turn

over

5-H

Tm

[3

H]-

5-H

T

Bas

al g

angl

ia

5-C

T M

eter

golin

e

Met

hyse

rgid

e M

ians

erin

8-

0H-D

PAT

(+

)-LS

D

RU

249

69

? (see

text

)

...... ~

~ a 8' :II

s·

Tab

le 2

1.2

Rel

ativ

e ag

onis

t pot

enci

es o

f var

ious

tryp

tam

ine

agon

ists

at 5

-HT

rece

ptor

s Eq

uipo

tent

mol

ar c

once

ntra

tion

ratio

s (5

-HT=

1)

Dog

sap

heno

us v

ein

Dog

sap

heno

us v

ein

Cat

sap

heno

us v

ein

Porc

ine

vena

cav

a Ra

bbit

aort

a Ra

t vag

us n

erve

(c

ontr

actio

n:

(neu

rona

l inh

ibiti

on:

(rel

axat

ion:

(r

elax

atio

n:

(con

trac

tion:

(d

epol

ariz

atio

n:

Agon

ist

5-H

Trli

ke)

5-H

Trli

ke)

5-H

Trli

ke)

5-H

Trli

ke)

5-H

T 2)

5-H

T 3)

5-H

T 1

1 1

1 1

1

5-C

f 0.

4 0.

3 0.

2 0.

04

26

>100

0 (0

.1-0

.9)

(0.2

-0.5

) (0

.006

-0.0

65)

(0.0

02-0

.12)

(1

4-49

)

a-M

ethy

l-5-

HT

13

25

571

>100

2.

2 >1

000

(5-3

2)

(13-

46)

(128

-256

2)

(1.1

-4.1

)

2-M

ethy

l-5-H

T 24

2 n.

d.

>100

52

55

3.

9 (2

14-2

70)

(25-

79)

(6-1

04)

(3.0

-4.9

)

AH

250

86

4.0

6 >1

00

>500

>1

92

>100

0 (1

.5-1

0.5)

(4

-11)

V

alue

s ar

e eq

uipo

tent

mol

ar c

once

ntra

tion

ratio

s (5

-HT

= 1)

, and

are

geo

met

ric m

eans

(95

% c

onfid

ence

lim

its)

of a

t le

ast f

our

dete

rmin

atio

ns.

n.d.

=

not

dete

rmin

ed.

Dat

a fr

om H

umph

rey

(198

4), F

eniu

k et

a/.

(198

5), S

umne

r eta

/. (

1987

), Fe

niuk

and

Hum

phre

y (1

989)

and

Hum

phre

y an

d Fe

niuk

(u

npub

lishe

d ob

serv

atio

ns).

~

;:: ~ o· ;:: ~ - v, ~ ,:l

I - ~ ~ ~ ~ c ~ ~

<::l" ~

~

~

~

164

120

" E so .. ~ c -~

8 40 "#

Serotonin

METHIOTHEPIN (0.1 pM) KETANSERIN (I JJM) MDL 72222 (I JJM)

Cone. AH25086 Hog M)

Figure 21.1 Effects of methiothepin (0.1 IJ.M), ketanserin (l!J.M) and MDL 72222 (l!J.M) on contractile effects of AH 25086 in dog isolated saphenous vein. Concentration-effect curves in presence of antagonist (e); time-matched control

responses <•>· All values are means ± s.e. mean from at least 4 experiments

mediating smooth-muscle relaxation (Watts.etal., 1981; Humphrey, 1984). The pharmacological profile of AH 25086 contrasts with that of 5-Cf, which is a potent agonist at all of these 5-HT1-like receptors (Table 21.2).

The 5-HT 1-like receptor mediating contraction of dog saphenous vein can be differentiated from 5-HT1-like receptors mediating relaxation on the basis of antagonist, as well as agonist, studies. Thus the contractile action of 5-HT or 5-Cf in dog saphenous vein is not antagonized by spiperone (10 J.tM), (±)-cyanopindol (1 J.tM), mesulergine (1 J.tM) or metergoline (0.5 !J.M) (Feniuk et al., 1985; Humphrey and Feniuk, unpublished observations). However, 5-Cf -mediated relaxation in porcine vena cava is antagonized by spiperone with a pA2 (slope) of 7.5 (0.9) but not by cyanopindolol at a concentration as high as 1 !JM (Sumner et al., 1987). These data also indicate that neither of these 5-HT 1-like receptors identified from functional studies corresponds with any of the four 5-HT1 binding sites (see Table 21.3). Nevertheless, these two distinct and reasonably well-characterized peripheral 5-HT1-like receptors appear widespread and functionally important in vivo as well as in vitro in a number of different animal species (see Humphrey and Feniuk, 1987a; Feniuk and Humphrey, 1989).

However, some functional receptors tentatively classified as 5-Hrrlike such as those of the rat stomach fundus and vascular endothelium, have characteristics different from the receptors described above. Thus the rat stomach fundus strip contains a 5-HT receptor which mediates contraction and can barely be called 5-HT rlike (because 5-Cf is less potent than 5-HT; Feniuk, unpublished observations), but it has been suggested recently that it is like the 5-HT1c binding site where 5-Cf has only modest affinity (Richardson and Engel, 1986). However, the action of 5-HT on intestinal

Tab

le 2

1.3

Rel

ativ

e af

finiti

es o

f ant

agon

ists

at 5

-HT 1

-like

rec

epto

rs a

nd 5

-HT 1

bin

ding

site

s•

Rece

ptor

/bin

ding

site

M

ethi

othe

pin

Met

ergo

line

(±)-

Cya

nopi

ndol

ol

Spip

eron

e

5-H

T 1 A

,,:::

:::::'::::

:':::!

R:~ii:~41

5-H

Tts

5-H

Ttc

5-H

TID

Dog

sap

heno

us v

ein

Porc

ine

vena

cav

a

£:%%

:::~

:!

!::,::

:::lli

t)

H::<

ttd -

1!'?4'

:::::1 - - D -

- D

n.a.

D

K; o

r an

tilog

pA

2: -<

lO

nM; ,

,,,,,:,:

,~,,~,!

10-1

00 n

M;

D

> 10

00 n

M;

n.a.

not

ava

ilabl

e.

E.i1

E.

D

D

D

@l::

:::~

::!

Mes

uler

gine

me

D

D

D

p:::::

!::~ii

l

•Act

ual v

alue

s ha

ve n

ot b

een

give

n, a

s th

e an

tago

nist

s do

not

alw

ays

appe

ar to

beh

ave

as s

impl

e co

mpe

titiv

e lig

ands

com

petin

g at

a

sing

le s

ite. T

he d

epic

ted

affin

ity e

stim

ates

hav

e be

en b

ased

on

data

from

var

ious

pub

licat

ions

cite

d in

the

text

. See

als

o H

oyer

et a

l. (1

985)

and

Eng

el e

t al.

(198

6).

~

~ [ f.J) :l: ~ I ::::-: rt ~ ~ s .... ~ """ ~ ~ ..... ~

166 Serotonin

preparations involves complex responses resulting from activation of various 'inhibitory' and 'excitatory' neurones as well as direct smoothmuscle effects, which may complicate true measures of potencies of both agonists and antagonists (e.g. see Drakontides and Gershon, 1968; Feniuk, 1984; Gunning and Humphrey, 1987). Certainly, more specific antagonists are needed to determine the importance of the 5-HT1c site. Interestingly, the endothelium of rabbit jugular vein appears to contain a 5-HTrlike receptor which has unique characteristics and where, like the rat stomach fundus receptor, 5-CT is less potent than 5-HT itself (Leff et al., 1987). It remains to be seen whether these receptors really represent other 5-HT 1-like receptor subtype(s) and whether similar receptors are functionally important elsewhere.

CONCLUSIONS

There is good evidence for the functional involvement of four subtypes of the 5-HTrlike receptor in the mediation of responses to 5-HT (see Humphrey and Feniuk, 1987b ). Two of these correspond to the 5-HT lA and 5-HT18 binding sites in brain (Table 21.1), although the latter site appears restricted to mouse and rat. The full significance ofthe 5-HT1c and 5-HT10

binding sites remains to be determined. The other two important receptor subtypes have been identified in functional studies on peripheral tissues (Table 21.3) and have still to be named.

The 5-HTrlike receptor which mediates contraction and inhibition of noradrenergic neurotransmission in dog saphenous vein may also be functionally important elsewhere on peripheral neurones and in the brain (Charlton et al., 1986; Lim berger et al., 1986; Raiteri et al., 1986). The recent identification of a selective agonist, AH 25086, for this receptor should be invaluable in determining its distribution and functional importance. Indeed, the clinical efficacy of AH 25086 in migraine suggests that this receptor is functionally important in man (Brand et al., 1987; Humphrey et al., 1987).

The other 5-HTrlike receptor, which mediates smooth-muscle relaxation, as in the porcine vena cava, is, like the 5-HT1A receptor, linked to adenylate cyclase (Trevethick et al., 1986; Shenker et al., 1987). Both these receptors are similar too in being blocked by spiperone to similar degrees, but they can be distinguished by cyanopindolol, which has high affinity for the 5-HT1A receptor but not for the other (Table 21.3).

It remains to be seen how many 5-HTrlike receptor subtypes can be shown to be important functionally in vivo. Clearly, this will only become evident when more specific and selective drugs have been identified as receptor probes.

Functional 5-HTrlike Receptor Subtypes 167

REFERENCES

Apperley, E. and Humphrey, P. P. A. (1986). The interaction of 5-hydroxytryptamine and methysergide with methiothepin at '5-HT1-like' receptors in dog saphenous vein. Br. J. Pharmacol., 87, 131P

Apperley, E., Humphrey, P. P. A. and Levy, G. P. (1977). Two types of excitatory receptor for 5-hydroxytryptamine in dog vasculature? Br. J. Pharmacol., 61, 465P


Brand, J., Hadoke, M. and Perrin, V. (1987). Placebo controlled study of a selective 5-HTrlike agonist, AH25086B, in relief of acute migraine. Cephalalgia, 1, 402--403

Charlton, K. G., Bond, R. A. and Clarke, D. E. (1986). Aninhibitoryprejunctional 5-HT 1-like receptor in the isolated perfused rat kidney. Apparent distinction from the 5-HT1A, 5-HTIB and 5-HT1c subtypes. Naunyn-Schmiedeberg's Arch. Pharmacol., 332, S--15

Crist, J. and Surprenant, A. (1987). Evidence that 8-hydroxy-2-(ndipropylamino)tetralin (8-0H-DPAT) is a selective aradrenoceptor antagonist on guinea-pig submucous neurones. Br. J. Pharmacol., 92, 341-347

Drakontides, A. B. and Gershon, M. D. (1968). 5-Hydroxytryptamine receptors in the mouse duodenum. Br. J. Pharmacol. Chemother., 33, 480-492

Engel, G., Gothert, M., Hoyer, D., Schlicker, E. and Hillenbrand, K. (1986). Identity of inhibitory presynaptic 5-hydroxytryptamine (5-HT) autoreceptors in the rat brain cortex with 5-HT18 binding sites. Naunyn-Schmiedeberg's Arch. Pharmacol., 332, 1-7

Feniuk, W. (1984). An analysis of 5-hydroxytryptamine receptors mediating contraction of isolated smooth muscle. Neuropharmacology, 23, 1467-1472

Feniuk, W. and Humphrey, P. P. A. (1989). Mechanisms of 5-hydroxytryptamineinduced vasoconstriction. In Fozard, J. R. (Ed.), The Peripheral Actions of 5-Hydroxytryptamine, Oxford University Press, Oxford, pp. 100-122

Feniuk, W., Humphrey, P. P. A. and Watts, A. D. (1979). Presynaptic inhibitory action of 5-hydroxytryptamine in dog isolated saphenous vein. Br. J. Pharmacol., 67, 247-254

Feniuk, W., Humphrey, P. P. A., Perren, M. J. and Watts, A. D. (1985). A comparison of 5-hydroxytryptamine receptors mediating contraction in rabbit aorta and dog saphenous vein: evidence for different receptor types obtained by use of selective agonists and antagonists. Br. J. Pharmacol., 86, 697-703

Gaddum, J. H. and Picarelli, Z. P. (1957). Two kinds oftryptamine receptor. Br. J. Pharmacol. Chemother., 12, 323--328

Gunning, S. J. and Humphrey, P. P. A. (1987). Evidence for 5-HT3-receptor mediated release of an inhibitory transmitter in guinea-pig isolated ileum. Br. J. Pharmacol., 91, 359P

Heuring, R. E. and Peroutka, S. J. (1987). Characterization of a novel PH]-5-hydroxypyptamine binding site subtype in bovine brain membranes. J. Neurosci., 7, 894-903

Holt, S. E., Cooper, M. and Wyllie, J. H. (1986). On the nature of the receptor mediating the action of 5-hydroxytryptamine in potentiating responses of the mouse urinary bladder strip to electrical stimulation. Naunyn-Schmiedeberg's Arch. Pharmacol., 334, 333--340

Hoyer, D., Engel, G. and Kalkman, H. 0. (1985). Molecular pharmacology of 5-HT1 and 5-HT2 recof!ition sites in rat and pig brain membranes: radioligand binding studies with [ H]5-HT, [3H]8-0H-DPAT, (-}P25I]iodocyanopindolol, PH]mesulergine and [3H]ketanserin. Eur. J. Pharmacol., 118, 13--23

168 Serotonin

Hoyer, D., Pazos, A., Probst, A. and Palacios, J. M. (1986). Serotonin receptors in the human brain. I. Characterization and autoradiographic localization of 5-HT tA

recognition sites. Apparent absence of 5-HTlB recognition sites. Brain Res., 376, 85-96

Humphrey, P. P. A. (1983). Pharmacological characterization of cardiovascular 5-hydroxytryptamine receptors. In Bevan, J. A., Fujiwara, M., Maxwell, R. A., Mohri, K., Shibata, S. and Toda, N. (Eds), Vascular Neuroeffector Mechanisms: 4th International Symposium, Raven Press, New York, pp. 237-242

Humphrey, P. P. A. (1984). Peripheral 5-hydroxytryptamine receptors and their classification. Neuropharmacology, 23, 1503-1510

Humphrey, P. P. A. and Feniuk, W. (1987a). Pharmacological characterization of functional neuronal receptors for 5-hydroxytryptamine. In Nobin, A., Owman, Ch. and Arneklo-Nobin, B. (Eds), Neuronal Messengers in Vascular Function, Fernstrom Foundation Series, Vol. 10, Elsevier, Amsterdam, pp. 3-19

Humphrey, P. P. A. and Feniuk, W. (1987b). Classification of functional 5-hydroxytryptamine receptors. In Rand, M. J. and Raper, C. (Eds), Pharmacology, Proceedings of the Xth International Congress of Pharmacology, Sydney, Elsevier, Amsterdam, pp. 277-280

Humphrey, P. P. A., Feniuk, W., Perren, M. J., Oxford, A. W., Brittain, R. T. and Jack, D. (1987). The pharmacology of selective 5-HT1-like receptor agonists for the acute treatment of migraine. Cephalalgia, 7, 400-401

Langer, S. Z. and Schoemaker, H. ( 1986). High affinity inhibition by ergometrine of [3H]-8-0H-DPAT binding to hippocampal5-HT1A receptors. Br. J. Pharmacol., 89, 524P

Leff, P., Martin, G. R. and Morse, J. M. (1987). Differential classification of vascular smooth muscle and endothelial cell5-HT receptors by use of tryptamine analogues. Br. J. Pharmacol., 91, 321-331

Limberger, N., Bonanno, G., Spath, L. and Starke, K. (1986). Autoreceptors and alpharadrenoceptors at the serotonergic axons of rabbit brain cortex. NaunynSchmiedeberg's Arch. Pharmacol., 332, 324-331

Peroutka, S. J. and Snyder, S. H. (1979). Multiple serotonin receptors: differential binding of [3H]-5-hydroxytryptamine, [3H]-lysergic acid diethylamide and [3H]-spiroperidol. Mol. Pharmacol., 16, 687-699

Raiteri, M., Maura, G., Bonnano, G. and Pittaluga, A. (1986). Differential pharmacology and function of two 5-HT 1 receptors modulating transmitter release in rat cerebellum. J. Pharmacol. Exp. Ther., 237, 644-648


Sanders-Bush, E. and Conn, P. J. (1986). Effector systems coupled to serotonin receptors in brain: serotonin stimulated phosphoinositide hydrolysis. Psychopharmacol. Bull., 22, 829-836

Shenker, A., Maayani, S., Weinstein, H. and Green, J.P. (1987). Pharmacological characterization of two 5-hydroxytryptamine receptors coupled to adenylate cyclase in guinea pig hippocampal membranes. Mol. Pharmacol., 31, 357-367

Sumner, M. J., Humphrey, P. P. A. and Feniuk, W. (1987). Characterisation ofthe 5-HT rlike receptor mediating relaxation of porcine vena cava. Br. J. Pharmacol., 92, 574P

Trevethick, M. A., Feniuk, W. and Humphrey, P. P. A. (1986). 5-Carboxamidotryptamine: a potent agonist mediating relaxation and elevation of cyclic AMP in the isolated neonatal porcine vena cava. Life Sci., 38, 1521-1528

Watts, A. D.,Feniuk, W. and Humphrey, P.P. A. (1981). A pre-junctional action of 5-hydroxytryptamine and methysergide on noradrenergic nerves in dog isolated saphenous vein. J. Pharm. Pharmacol., 33, 515-520

22 New Pharmacological Tools for Studies of

Central5-HT tA Binding Sites

M. Hamon1, M. B. Emeril, M. Ponchanr, J. M. Cosse,Y, S. ElMestikawyl, D. Vergtf, G. Davaf and H. Gozlan1

1 INSERM U. 288, Neurobiologie Cellulaire et Fonctionelle, Faculte de Medecine Pitie-Salpetriere, 91, Boulevard de l'Hopital,

75634 Paris cedex 13, France 2Service des Molecules Marquees, Departement de Biologie,

CEA-CEN Saclay, 91191 Gif-sur-Yvette, France 3Departement de Cytologie, Institut des Neurosciences, CNRS-U A 4199,

Universite Pierre et Marie Curie, 7, Quai Saint-Bernard, 75005 Paris, France

INTRODUCTION

The present knowledge of central serotonin (5-hydroxytryptamine; 5-HT) receptors largely derives from binding studies with appropriate radioligands. Thus it is commonly accepted that two main classes of 5-HT receptors, 5-HT1 and 5-HT2, exist in the CNS (Peroutka and Snyder, 1979). Apparently the 5-HT 2 class is homogeneous and corresponds to binding sites with low (micromolar) affinity for 5-HT and most agonists, and high (nanomolar) affinity for selective antagonists. In contrast, the 5-HT1 class, with high (nanomolar) affinity for 5-HT, is heterogeneous, and four binding site subtypes, 5-HT 1A, 5-HT 18, 5-HT 1c and 5-HT 10, have been identified so far. Correspondence of these binding sites with authentic receptors has been demonstrated: for example, the 5-HT1A site which is negatively coupled to adenylate cyclase (De Vivo and Maayani, 1986), and the 5-HT1c site, which is positively coupled to phospholipase C in the choroid plexus (Conn et al., 1986). Finally, another class, 5-HT3 receptors, has been identified in the periphery (Richardson and Engel, 1986), and their existence within the CNS has recently been reported (see Tyers et al., this volume).

Progress in the pharmacological and biochemical characterization of central neurotransmitter receptors largely depends on the development of

170 Serotonin

new selective (radio)ligands, and during the last 3 years efforts have been made in our laboratory to synthesize such molecules for studies on 5-HT1A

receptors. Two reversible radioligands, an alkylating agent, and a photosensitive irreversible radioligand were developed and successfully used for a better characterization of central 5-HT1A binding sites. The present review is a summary of the main findings recently obtained with these new molecules.

SYNTHESIS AND APPLICATIONS OF NEW REVERSmLE RADIOLIGANDS OF CENTRAL S-HT tA RECEPTORS

[lfi]-S-Methoxy-3-(di-n-propylamino)chroman ([3H]-S-Me0-DPAC)

Originally, the real demonstration of the existence of 5-HT lA binding sites in rat brain membranes derived from studies of the displacement of specifically bound [3H]-5-HT by the neuroleptic spiperone (Pedigo et al., 1981) and by the po.tent 5-HT agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-0HDPAT; Arvidsson eta/., 1981). Indeed, inhibition of [3H]-5-HT binding by the latter drug is clearly biphasic in cortical and hippocampal membranes (Middlemiss and Fozard, 1983; Hamon et al., 1984), as expected from the existence of two distinct classes of 5-HT 1 binding sites in both preparations, namely the 5-HT1A sites with nanomolar affinity for 5-HT, 8-0H-DPAT and spiperone, and the 5-HT18 sites with nanomolar affinity for 5-HT, but much lower affinity for the other two drugs.

Owing to the synthesis of a [3H] derivative of 8-0H-DPAT, direct labelling of 5-HT1A sites became possible in brain membranes and on brain sections (Gozlan et al., 1983; Marcinkiewcz et al., 1984). Such studies revealed notably that 5-HT1A sites are abundant within limbic regions (hippocampus, septum) but virtually absent in extrapyramidal areas. Nevertheless, specific binding of [3H]-8-0H-DPAT could be detected in striatal membranes, with pharmacological properties markedly different from those of 5-HT1A receptors (Gozlan eta/., 1983). Indeed, subcellular fractionation and lesion studies led to the demonstration of the location of striatal [3H]-5-0H-DPAT binding sites on pre-synaptic serotoninergic terminals, in contrast to 5-HT1A sites, mainly located on post-synaptic targets of serotoninergic projections (Gozlan et al., 1983; Hallet a/., 1985, 1986). These data were recently confirmed by Schoemaker and Langer (1986), who proposed that the pre-synaptic binding sites, termed 5-HTPre• may correspond to the membrane carrier responsible for 5-HT reuptake into serotoninergic terminals.

Obviously, the availability of [3H]-8-0H-DPAT has largely contributed to the present knowledge of central5-HT lA receptors, but the recognition of 5-HT Pre sites by this radio ligand also led to some confusion. As the best

Reversible and /"eversible 5-HT1A Binding Site Ligands 171

radioligand would be a molecule with absolute selectivity for 5.-HT1A sites, i.e. one which does not recognize 5-HTPre sites, a program was tentatively set up for the possible development of this ideal molecule.

First, a large series of 8-0H-DPAT derivatives were synthesized and tested in appropriate binding assays to determine the structure-activity relationships which order the recognition of 5-HT tA and/or 5-HT Pre sites by these compounds. It was found that the OH group on C-8 can be changed to a methoxy group with no marked alteration in the pharmacological properties of the derivatives, and that one of the two propyl chains is required for the optimal recognition of 5-HT1A sites. Replacement of the C-4 atom by an 0 in the saturated cycle gave a chroman derivative with interesting properties. Also, 5-0H- and 5-methoxy-3-{di-npropylamino )chroman were still recognized in the nanomolar range by 5-HT1A sites but exhibited very low affinity (IC50 > 1 mM) for 5-HTPre sites. These observations led to the synthesis of [3H)-5-methoxy-3-(di-n-propylamino )chroman ([3H)-5-Me0-DPAC) for possible selective labelling of 5-HT1A sites in the rat brain {Cossery et al., 1987). As expected from findings with the cold compound, eHJ-5-MeO-DPAC bound to only one class of specific sites in any brain area examined, and the pharmacological properties of these sites were identical to those of 5-HT tA sites in hippocampal membranes. Furthermore, the total number of specific sites labelled by [3H)-5-Me0-DPAC was equal to that of [3H)-8-0H-DPAT binding sites in the rat hippocampus, and no significant specific binding of [3H)-5-Me0-DPAC was detected in the rat striatum {Cossery et al., 1987).

All these data indicated that [3H)-5-Me0-DPAC is in fact the best radioligand presently available for the selective labelling of 5-HT1A sites in the CNS. Like [3H)-8-0H-DPAT, [3H)-5-Me0-DPAC is quite stable in spite of a high specific radioactivity (usually higher than 100 Ci/mmol), and exhibits a low degree of non-specific binding. Fortunately, unlike [3H)-8-0H-DPAT, eHJ-5-MeO-DPAC does not bind to 5-HT Pre sites in the rat striatum.

[ 1151]-Bolton-Hunter-8-methoxy-2-(N-propyl-N-propylamino)tetralin ([1151]-BH-8-MeO-N-PAT)

Quantitative autoradiography has proved to be extremely helpful for investigations of central neurotransmitter receptors, particularly in discrete brain regions for which binding studies using membranes are impossible. For instance, densitometric analysis of [3H)-8-0H-DPAT binding to brain sections from 5,7-dihydroxytryptramine-lesioned rats revealed that 5-HT1A

sites within the dorsal raphe nucleus are located on serotoninergic cell bodies and/or dendrites, and may correspond to 5-HT autoreceptors {Weissman-Nanopoulos et a/.,1985; Verge et a/.,1986). Another example

172 Serotonin

concerns the developmental changes in 5-Hf 1A binding sites during the early post-natal period, when the small sizes of brain structures make studies of ligand binding to membranes particularly difficult. Using the autoradiographic technique, Daval et al. (1987) discovered transient [3H]-8-0H-DPAT binding sites within deep nuclei and cortex of the cerebellum in new-born rats.

However, with [3H] ligands such as [3H]-8-0H-DPAT and eH]-5-MeODPAC, the procedure is time-consuming, since labelled sections must be exposed to [3H]-sensitive film for 2-4 months in order to obtain satisfactory autoradiographs. In contrast, 2-3 days are enough to achieve similar data with [1251]-labelled ligands. Therefore, a P25I] derivative of 8-0H-DPAT was of great potential interest for autoradiographic investigations of 5-HT 1A

sites, and a program was initiated aimed at the synthesis of such a probe. Given the structural requirements for the binding of 8-0H-DPAT

derivatives of 5-Hf1A sites, it was theoretically possible to keep intact one propyl chain and add the P251] reagent, N-succinimidyl-3-( 4-hydroxy-3-P25I]-iodophenyl)propionate {P251]-Bolton-Hunter reagent) to the other chain in order to make a P251]-labelled 5-Hf1A ligand. The resulting compound, [1251]-Bolton-Hunter-8-methoxy-2-(N-propyl-N-propylamino)-tetralin (P23I])BH-8-Me0-N-PAT), is clearly a selective 5-HT1A

ligand as shown by the following observations: (a) it binds to specific sites which exhibit nanomolar affinity for 5-Hf, 8-0H-DP AT, and other 5-Hf 1A

ligands such as ipsapirone, buspirone and gepirone (see Hamon et al., 1986), and the same Bmax as that of 5-HT1A sites labelled by [3H]-8-0H-DPAT or [3H]-5-Me0-DPAC in hippocampal membranes; (b) at least micromolar concentrations of drugs acting at other receptor types (5-Hf18, 5-Hf1c, 5-Hf2 , dopamine, adrenergic) are required to inhibit the specific binding of [1251]-BH-8-MeO-N-PAT to hippocampal membranes; and (c) the regional distribution of specific sites labelled by P251]-BH-8-Me0-N-PAT in 9 different structures dissected from the adult rat brain is significantly correlated (r = 0.984) with that of 5-HT1A sites labelled by [3H]-8-0HDPAT (Gozlan et.al., 1988).

The initial goal for which the [1251] probe was synthesized, i.e. the autoradiographic visualization of 5-HT tA sites, has been reached, since similar autoradiographs were obtained after a 3-day exposure of brain sections incubated with P25I]-BH-8-Me0-N-PAT or a 3-month exposure of those incubated with [3H]-8-0H-DP AT ( Gozlan et al., 1988). As previously reported with the [3H] ligand (Verge et al., 1986), intense labelling is found in the hippocampus, septum, entorhinal cortex and dorsal raphe nucleus of a horizontal brain section incubated with P25I]-BH-8-Me0-N-PAT (Figure 22.1). Thus P25I]-BH-8-Me0-N-PAT is the first P251] probe for studies of 5-Hf1A binding sites; it may soon become a commonly used ligand for quantitative autoradiography of these sites, not only in animals but also in human brain.

Reversible and Irreversible 5-HT1A Binding Site Ligands 173

cereb. ex

Sept

Figure 22.1 Autoradiogra£hic localization of P25I]-BH-8-Me0-N-PAT binding sites in the rat brain. Total [1 51]-BH-8-MeO-N-PAT binding is increasing from white to grey with highest levels in dark regions. In this horizontal section: DRN =dorsal raphe nucleus; hippo= hippocampus; Sept= septal area; Str =striatum; cere b. ex=

cerebral cortex; ent. ex = entorhinal cortex

SYNTHESIS AND APPLICATIONS OF IRREVERSIBLE LIGANDS OF CENTRAL 5-HT tA RECEPTORS

8-Methoxy-2-(N-2' -chloropropyi,N-propyl)aminotetralin (8-Me0-2' -chloroPAT)

Synthesis of 8-methoxy-2-(N-2'-hydroxypropyl,N-propyl)aminotetralin from 8-methoxy-tetralone, and subsequent reaction with thionyl chloride, gave 8-methoxy-2-(N-2'-chloropropyl,N-propyl)aminotetralin (8-Me0-2'-

174 Serotonin

chloro-PAT), which was revealed to be an irreversible alkylating ligand of 5-HT tA sites in the rat brain (Emerit et al., 1985). Probably via its conversion to an aziridinium ion, 8-Me0-2'-chloro-PAT reacts with various nucleophilic groups and achieves irreversible occupancy of 5-HT tA sites in brain membranes and sections. However, further investigations using a radioactive derivative of 8-Me0-2'-chloro-PAT were not attempted, since subsequent studies (Radja et al., 1989) demonstrated that the unlabelled compound interacts not only with 5-HT1A sites but also with 5-HT1c sites in the choroid plexus. Nevertheless, under conditions resulting in the irreversible blockade of half the 5-HT1A sites in brain membranes, neither adrenergic, dopamine, y-aminobutyric acid nor benzodiazepine receptor binding sites are affected (Emerit et al., 1985; Radja et al., 1989).

The relative selectivity of 8-Me0-2' -chloro-PAT for 5-HT tA binding sites led to attempts to achieve their irreversible blockade in vivo in rats. Although the peripheral (i.p. or i.v.) administration of 8-Me0-2'-chloroPAT (up to 5 mglkg) was ineffective, the intracerebroventricular and particularly the intracerebral injection of the alkylating agent produced a long-lasting blockade of 5-HT1A sites in selected brain regions. Thus, the stereotaxic microinjection of 1 tJ.g 8-Me0-2' -chloro-PAT into the ventral hippocampus of chloral hydrate-anaesthetized rats resulted in a local40-50 per cent loss of 5-HT1A sites, as assessed by the decrease in Bmax of [3H]-8-0H-DP AT binding sites in hippocampal membranes (Hamon et al., 1988). This effect was noted 24-48 h after the treatment, and disappeared progressively thereafter, with full recovery of the control density of 5-HT tA

sites on the ninth day following 8-Me0-2'-chloro-PAT microinjection. When larger doses were used (5-20 tJ.g 8-Me0-2'-chloro-PAT), recovery was only partial even at longer times after the treatment, because the highly reactive alkylating agent produced local lesions of the brain parenchyma (Hamon et al., unpublished observations). However, under appropriate conditions, i.e. using low doses of 8-Me0-2'-chloro-PAT (0.5-1 tJ.g), an estimate of the half-life of hippocampal 5-HT tA sites could be made on the basis of the time-dependent recovery of [3H]-8-0H-DPAT binding sites. The value obtained, 2.3 days (Hamon et al., 1988), is in the same range as half-lives of other membrane-bound proteins, including neurotransmitter receptors (Steinman et al., 1983).

Thus 8-Me0-2' -chloro-PAT is a relatively selective irreversible ligand of 5-HT tA sites in the rat brain; it can be used for the determination of the turnover rate of these sites in vivo, and for the functional (i.e. behavioural, metabolic) consequences of irreversible in vivo blockade of 5-HT1A sites.

8-Methoxy-2-(N-n-propyl,N-3-[2-nitro-4-azidophenyl]aminopropyl)aminotetralin (8-Me0-3' -NAP-amino-PAT)

Starting with 1,7-dihydroxynaphthalene, it was possible to synthesize


'Hcpm A

' H-8-methoxy-3'-NAP-aminO-~T

HIPPOCAMPUS

100

0 5 'W) 15 20 25 30 35 40 45

Gel slice I"K),

'Hcpm 8

f 'H-8-methoxy-3:NAP-amno.~T 8-0H-DPAT- ('W) jiM)

200

HIPPOCAMPUS

Pr

100

0 5 'W) 15202530354045

Gel slice na.

Figure 22.2 SDS-PAGE pattern of irreversible labelling of hippocampal microsomal membranes by rH]-8-Me0-3'-NAP-amino-PAT. Membranes were incubated and UV-irradiated with 20 nM [3H]-8-Me0-3'-NAP-amino-PAT in the absence (A) or presence (B) of 10 JA.M8-0H-DPAT, solubilized, and then processed for SDS-PAGE analysis. Bars correspond to the radioactivity (cpm: counts per min) in 2 mm slices cut all along the gel. P1 = peak of radioactivity due to the 63 kdalton

binding subunit of the 5-HT1A receptor

176 Serotonin

8-methoxy-2-(N-n-propyl,N-3-[2-nitro-4-azidophenyl]amino-propyl)aminotetralin (8-Me0-3'-NAP-amino-PAT), with a nitro-azidophenyl group added to one of the propyl chains (Emerit et al., 1986). In the dark, 8-Me0-3' -NAP-amino-PAT interacts selectively with 5-HT lA sites in the nanomolar range (IC50 = 6.6 nM). Ultraviolet irradiation of hippocampal membranes incubated with 8-Me0-3'-NAP-amino-PAT resulted in a marked reduction in the Bmax of 5-HT lA sites, and this effect persisted even after extensive washing. However, prior occupancy of 5-HT1A sites by a reversible ligand (such as 5-HT or 8-0H-DPAT) prevented any subsequent blockade by 8-Me0-3'-NAP-amino-PAT under UV light (Emerit et al., 1986).

Because ofthe high selectivity of 8-Me0-3' -NAP-amino-PAT for 5-HT lA

sites (for instance, neither 5-HT18, 5-HT2 nor dopamine receptor binding sites were affected by the photosensitive probe), a [3H] derivative was synthesized for the possible irreversible labelling of the corresponding membrane protein(s). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of the material solubilized from hippocampal membranes and UV-irradiated in the presence of [3H]-8-Me0-3'-NAPamino-PAT revealed a (3H] band of 63 kdaltons (Figure 22.2). This band was not detected when membranes were pre-incubated with various 5-HT lA

ligands (8-0H-DPAT, ipsapirone, gepirone) to prevent the irreversible occupancy of 5-HT1A sites by [3H]-8-Me0-3'-NAP-amino-PAT. Furthermore, the regional and subcellular distributions of the 63 kdalton protein were identical to those of specific 5-HT1A binding sites: both were particularly abundant in the microsomal fraction from hippocampus and absent in any fraction from cerebellum (Emerit et al., 1987; Gozlan et al., 1987).

These data indicated that the binding subunit of the 5-HT1A receptor is a 63 kdalton protein, which can be visualized directly in the SDS-PAGE gels with the photoaffinity probe [3H]-8-Me0-3'-NAP-amino-PAT. Evidence has been recently published in support of these findings: first, on the basis of a radiation inactivation procedure applied to intact tissues, Gozlan et al. (1986) estimated a molecular weight of =60 kdaltons for the 5-HT1A

receptor binding site; and second, Ransom et al. (1986) calculated a molecular weight of =55 kdaltons for a membrane protein irreversibly labelled by eH]-1-(2-[ 4-azidophenyl]ethyl)-4-(3-trifluoromethylphenyl) piperazine, another photoaffinity probe for 5-HT1A sites in the rat hippocampus. Finally, partial purification of the 5-HT1A receptor binding subunit by affinity chromatography on an agarose column coupled to an 8-0H-DPAT derivative gave a main band of 63 kdaltons in SDS-PAGE (El Mestikawy et al., unpublished observations).

CONCLUSIONS Since the introduction of 8-0H-DPAT as a selective 5-HT agonist by


Arvidsson et al. (1981), considerable progress has been made in our knowledge of central 5-HT1 receptors. Thus concentration-dependent inhibition by 8-0H-DPAT of [3H)-5-HT binding to cortical and hippocampal membranes (Middlemiss and Fozard, 1983; Hamon eta/., 1984) has revealed biphasic curves, in support of the heterogeneity of 5-HT 1 binding sites initially proposed by Pedigo et al. (1981). The synthesis of [3H)-8-0H-DPAT has allowed direct studies of the 5-HT1A binding site subtype (and also 5-HTPre sites in the membranes of serotoninergic terminals; see Gozlan et al., 1983; Hall et al., 1985, 1986). Finally, as summarized in this review, 8-0H-DPAT was also a lead compound for the synthesis of new reversible and irreversible ligands of great interest for in-vivo and in-vitro studies of 5-HT1A receptors. Among these compounds, the chroman derivative [3H)-5-Me0-DPAC appears to be even better than [3H)-8-0H-DPAT for binding assays aimed at studying 5-HT1A sites in membranes, and the iodinated probe P25I)-BH-8-Me0-N-PAT is particularly adapted to autoradiographic studies using brain sections. The irreversible alkylating agent 8-Me0-2'-chloro-PAT has allowed an estimate of the turnover rate of 5-HT1A sites in the rat brain in vivo, and the photoaffinity probe [3H)-8-Me0-3'-NAP-amino-PAT has led to the identification of the 5-HT tA receptor binding subunit.

Numerous problems are still pending about 5-HT tA receptors, such as the development of a selective antagonist, and the mechanism of their coupling with adenylate cyclase or other effector systems; it is likely that 8-0H-DP AT and derivatives will be of great help at least for their study, and possibly their answer.

ACKNOWLEDGEMENTS

This research has been supported by grants from INSERM, Bayer-Pharma, CEA and la Fondation pour la Recherche Medicate.

REFERENCES

Arvidsson, L. E., Hacksell, U., Nilsson, J. L. G., Hjorth, S., Carlsson, A., Lindberg, P., Sanchez, D. and Wikstrom, H. (1981). 8-Hydroxy-2-(di-npropylamino )tetralin, a new centrally acting 5-hydroxytryptamine receptor agonist. J. Med. Chem., 24, 921-923

Conn P. J., Sanders-Bush, E., Hoffman, B. J. and Hartig, P. R. (1986). A unique serotonin receptor in choroid plexus is linked to phosphatidylinositol turnover. Proc. Natl. Acad. Sci. U.S.A., 83, 4086-4088

Cossery, J. M., Gozlan, H., Spampinato, U., Perdicakis, C., Guillaumet, A., Pichat, L. and Hamon, M. (1987). The selective labelling of central 5-HT1A

receptor binding sites by [3H]-5-methoxy-3-(di-n-propylamino)chroman. Eur. J. Pharmacol., 140, 143-155

178 Serotonin

Daval, G., Verge, D., Becerril, A., Gozlan, H., Spampinato, U. and Hamon, M. (1987). Transient expression of 5-HT lA receptor binding sites in some areas of the rat CNS during postnatal development. Int. J. Devel. Neurosci., 5, 171-180

De Vivo, M. and Maayani, S. (1986). Characterization of the 5-hydroxytryptarnine1A receptor-mediated inhibition of forskolin-stimulated adenylate cyclase activity in guinea-pig and rat hippocampal membranes. J. Pharmacol. Exp. Ther., 238, 248-253

Emerit, M. B., Gozlan, H., Hall, M.D., Hamon, M. and Marquet, A. (1985). Irreversible blockade of central5-HT binding sites by 8-methoxy-2'-chloro-PAT. Biochem. Pharmacol., 34, 88H92

Emerit, M. B., Gozlan, H., Marquet, A. and Hamon, M. (1986). Irreversible blockade of central 5-HT1A receptor binding sites by the photoaffinity probe 8-methoxy-3'-NAP-amino-PAT. Eur. J. Pharmacol., 127, 67-81

Emerit, M. B., El Mestikawy, S., Gozlan, H., Cossery, J. M., Besselievre, R., Marquet, A. and Hamon, M. (1987). Identification of the 5-HT1A receptor binding subunit in rat brain membranes using the photoaffinity probe [3H]8-methoxy-2-[N-n-propyl,N-3-(2-nitro-4-azidophenyl)aminopropyl]aminotetralin. J. Neurochem., 49, 373-380

Gozlan, H., El Mestikawy, S., Pichat, L., Glowinski, J. and Hamon, M. (1983). Identification of presynaptic serotonin autoreceptors using a new ligand: [3H]PAT. Nature, 305, 140-142

Gozlan, H., Emerit, M. B., Hall, M.D., Nielsen, M. and Hamon, M. (1986). In situ molecular sizes of the various types of 5-HT binding sites in the rat brain. Biochem. Pharmacol., 35, 1891-1897

Gozlan, H., Emerit, M. B., El Mestikawy, S., Cossery, J. M., Marquet, A., Besselievre, R. and Hamon, M. (1987). Photoaffinity labelling and solubilization of the central 5-HT1A receptor binding site. J. Recept. Res., 1, 195-221

Gozlan, H., Ponchant, M., Daval, G., Verye, D., Menard, F., Vanhove, A., Beaucourt, J. P. and Hamon, M. &1988). [ 25I]Bolton-Hunter-8-methoxy-2-(Npropyl-N-propylamino)tetralin W2 I]BH-8-MeO-N-PAT) as a new selective radioligand of 5-HT1A sites in the rat brain. In vitro binding and autoradiographic studies. J. Pharmacol. Exp. Ther., 244, 751-759

Hall, M.D., El Mestikawy, S., Emerit, M. B., Pichat, L., Hamon, M. and Gozlan, H. (1985). [3H]-8-Hydroxy-2-(di-n-propylamino)tetralin binding to pre- and post-synaptic 5-hydroxytryptamine sites in various regions of the rat brain. J. Neurochem., 44, 1685-1696

Hall, M.D., Gozlan, H., Emerit, M. B., El Mestikawy, S., Pichat, L. and Hamon, M. (1986). Differentiation of pre- and post-synaptic high affinity serotonin receptor binding sites using physico-chemical parameters and modifying agents. Neurochem. Res., 11, 891-912

Hamon, M., Bourgoin, S., Gozlan, H., Hall, M.D., Goetz, C., Artaud, F. and Horn, S. (1984). Biochemical evidence for the 5-HT agonist properties of PAT [8-hydroxy-2-(di-n-propylamino)tetralin] in the rat brain. Eur. J. Pharmacol., 100, 263-276

Hamon, M., Cossery, J. M., Spampinato, U. and Gozlan, H. (1986). Are there selective ligands for 5-HT1A and 5-HTlB receptor binding sites in brain? Trends in Pharmacol. Sci., 7, 336-338

Hamon, M., Gozlan, H., El Mestikawy, S., Emerit, M. B., Cossery, J. M. and Lutz, 0. (1988). Biochemical properties of central serotonin receptors. In Osborne, N. N. and Hamon, M. (Eds),NeuronalSerotonin, Wiley, Chichester, pp. 393-422

Marcinkiewicz, M., Verge, D., Gozlan, H., Pichat, L. and Hamon, M. (1984). Autoradiographic evidence for the heterogeneity of 5-HT1 sites in the rat brain. Brain Res., 291, 159-163

Reversible and Irreversible 5-HTIA Binding Site Ligands 179

Middlemiss, D. N. and Fozard, J. R. (1983). 8-Hydroxy-2-(di-npropylamino)tetralin discriminates between subtypes of 5-Hf1 recognition sites. Eur. J. Pharmacol., 90, 151-153

Pedigo, N. W., Yamamura, H. I. and Nelson, D. L. (1981). Discrimination of multiple [3H]5-hydroxytryptamine binding sites by the neuroleptic spiperone in rat brain. J. Neurochetn., 36, 220-226

Peroutka, S. J. and Snyder, S. H. (1979). Multiple serotonin receptors: differential binding of [3H]5-hydroxytryptamine, [3H]lysergic acid diethylamide and [3H]spiroperidol. Mol. Pharmacol., 16, 687--{)99

Radja, F., Daval, G., Emerit, M. B., Gallissot, M. C., Hamon, M. and Verge, D. (1989). Selective irreversible blockade of 5-HT1A and 5-Hf1c receptor binding sites in the rat brain by 8-Me0-2'-chloro-PAT: a quantitative autoradiographic study. Neuroscience (in press)

Ransom, R. W., Asarch, K. B. and Shih, J. C. (1986). Photoaffinity labelling ofthe 5-hydroxytryptamine1A receptor in rat hippocampus. J. Neurochem., 47, 1066-1072


Schoemaker, H. and Langer, S. Z. (1986). [3H]8-0H-DPAT labels the serotonin transporter in the rat striatum. Eur. J. Pharmacol., 124, 371-373

Steinman, R. M., Mellman, I. S., Muller, W. A. and Cohn, Z. A., (1983). Endocytosis and the recycling of plasma membrane. J. Cell. Bioi., 96, 1-27

Verge, D., Daval, G., Marcinkiewicz, M., Patey, A., El Mestikawy, S., Gozlan, H. and Hamon, M. (1986). Quantitative autoradiography of multiple 5-Hf 1 receptor subtypes in the brain of control and 5,7-dihydroxytryptamine-treated rats. J. Neurosci., 6, 3474-3482

Weissman-Nanopoulos, D., Mach, E., Magre, J., Demassey, Y. and Pujol, J. F. (1985). Evidence for the localization of 5-Hf1A binding sites on serotonin containing neurons in the raphe dorsalis and raphe centralis nuclei of the rat brain. Neurochem. Int., 1, 1061-1072

23 Serotonin 5-HT 1c Receptors: What Do They

Do?

P. R. Hartigt

Environmental Neurobiology Group, Department of Environmental Health Sciences, Johns Hopkins Medical Institutions, 615 N. Wolfe St,

BaltimoreMD21205, USA

INTRODUCTION

Few, if any, receptor systems have moved from initial discovery to detailed molecular characterization as quickly as the serotonin 5-HT 1c receptor. This rapid progress was triggered by the timely coincidence of a series of discoveries and developments, some taken from quite different fields of science, culminating in the recent molecular cloning of this receptor. These findings will be recounted in roughly historical order in this short review, with an emphasis on functional responses of the 5-HT1c receptor.

DISCOVERY

As has been the case in many neurotransmitter systems, the initial discovery and characterization of the serotonin 5-HT1c receptor arose from radio ligand binding experiments. Studies in the author's laboratory, u&ing P25I)-lysergic acid diethylamide (Yagaloff and Hartig, 1985), and in Palacios's laboratory using [3H)-mesulergine (Pazos et al., 1985), pointed out the presence of an unusually high density of binding sites in the brain ventricles on a richly vascularized secretory tissue known as the choroid plexus. These sites displayed a unique binding profile that did not match any previously characterized serotonin binding site. In particular, they displayed high affinity for serotonin (6.5-30 nM), as is characteristic of a 5-HT1

binding site, but they also displayed selective high affinity for certain serotoninergic antagonists such as mianserin and metergoline, and low

tPresent address: Neurogenetic Corporation, 215 College Road, Paramus NJ 07652, USA.

Serotonin 5-HT1c Receptors 181

affinity for others such as spiperone. The choroid plexus site was named the serotonin 5-HT1c binding site (Pazos et al., 1985), since two other binding sites with high affinity for serotonin (5-HT1A and 5-HT18) had already been described (Pedigo et al., 1981).

SECOND MESSENGER SYSTEM

These early studies on the 5-HT 1c binding site clearly established that it was a novel site, but did not settle the question of whether it was a functional receptor. This question can be raised on two levels: are there biochemical responses triggered by occupancy of the site, and what are the physiological consequences of this activation? The first question was answered by the discovery that the choroid plexus 5-HT1c site is linked to the phosphatidylinositol second messenger system (Conn et al., 1986), whose consequences normally include release of intracellular Ca2+ and activation of protein kinase C. Recent work in pig choroid plexus indicates that the 5-HT1c receptor can produce one of the largest elevations of intracellular inositol phosphate (in the presence of Li+) of any phosphatidylinositol-coupled receptor (Hoffman et al., 1986). The other common receptor-activated second messenger system, the adenylate cyclase system, does not appear to be linked to the 5-HT1c receptor in either an excitatory or an inhibitory manner (Palacios et al., 1986).

TISSUE LOCALIZATION

Progress in elucidating the physiological role of 5-HT 1c receptor activation has been much slower. Localization studies have, however, helped to focus our search. High-resolution autoradiographs of choroid plexus have suggested that this receptor is localized on the secretory epithelial cell layer of the choroid plexus (Yagaloff and Hartig, 1985). This assignment has been confirmed by isolating highly purified epithelial cell fractions in our laboratory. It was found that 95 per cent of the 5-HT 1c sites are on epithelial cells and cell fragments, while only 5 per cent could be found on the tissue remnant containing mostly blood vessels and connective tissue (Pihl and Hartig, unpublished observations). This study clearly rules out a vascular site for this receptor and focuses attention on its possible involvement in secretory and transport functions of the choroid plexus epithelium.

These selective transport roles of the choroid plexus are especially important because this tissue forms a unique element of the blood-brain barrier. The outermost cell layer of this tissue contains circumferential tight junctions which separate the apical face of epithelial cells, bathed in cerebrospinal fluid (CSF), from the basal and lateral faces, bathed in blood.

182 Serotonin

Further localization of the 5-HT 1c receptor to the CSF or blood side of the epithelium would obviously help focus our search for its physiological functions. This question was approached by attempting to label the choroid plexus 5-HT1c receptor in vivo by injection of a water-soluble, membraneimpermeable ligand, [3H)-serotonin, either into the circulation via i.v. injection, or into the CSF via intracerebroventricular injection. lntracerebroventricular injections produced labelling of a choroid plexus binding site whose site density and pharmacological binding characteristics closely resemble in-vitro assays of 5-HT1c binding, whereas i.v. injection produces no evidence of labelling of the 5-HT1c site (Giordano and Hartig, unpublished observations). Thus, the 5-HT 1c receptor is responsive to CSFbut not to blood-borne serotonin, and it appears to be localized on the apical face of the epithelial cell layer, in direct contact with ventricular CSF.

The choroid plexus receives little direct innervation by serotoninergic fibres, and those few indoleaminergic fibres which are present appear to terminate primarily on blood elements rather than epithelial cells (Napoleone et al., 1982). Thus, CSF-borne serotonin would appear to be the sole source for activation of the choroid plexus 5-HT1c receptor. Further support for this idea comes from recent denervation studies in which intracerebroventricular injection of 5,7-dihydroxytryptamine, to deplete serotoninergic innervation, caused supersensitivity of the 5-HT 1c phosphatidylinositol response in choroid plexus (Conn et al., 1987), and from the fact that recent estimates of the CSF concentration of serotonin suggest values near 50 nM (Volicer et al., 1985), which is very close to the midpoint for activation of the receptor in both binding (Yagaloff and Hartig, 1985) and functional assays (Conn et al., 1986). The most likely direct source of CSF serotonin for activation of the choroid plexus 5-HT1c receptor lies in supra-ependymal serotoninergic fibres which line the ventricles. These fibres do not form synapses, have no serotonin receptors in their near vicinity, and appear to release serotonin directly into the CSF (Richards and Guggenheim, 1982). It is not yet known which physiological process is regulated by this system. Several groups are studying the possible regulation of CSF secretion, but it is possible that a more subtle process, such as a specific ion or substrate transport, is controlled by the choroid plexus 5-HT1c receptor.

Although most work up to now has focused on the choroid plexus, it is important to note that 5-HT1c binding sites have been observed in layers CA1 and CA3 of the human hippocampus, and in the frontal cortex (Hoyer et al., 1986; Peroutka, 1986). The presence of cortical5-HT1c receptors has also been demonstrated in oocyte injection experiments described below. A recent preliminary report suggests the presence of 5-HT1c receptors in rat stomach fundus, where they appear to regulate smooth-muscle contraction (Buchheit et al., 1986). Progress in studying these other 5-HT1c receptor sites has been slowed by the very low density of these sites, by the lack of


selective agonists or antagonists for the 5-H1"tc site, and by the fact that close similarity of antagonist profiles may have led to misassignment of 5-HT1c actions to the well-studied 5-HT2 receptor. Until more selective ligands become available, the high (nanomolar) potencies of serotonin, metergoline and mianserin and the micromolar potency of spiperone serve best to differentiate the 5-HT 1c site from other serotonin binding sites.

MOLECULAR CLONING

Two independent lines of research from the field of molecular genetics have recently established a major new direction in 5-HT1c receptor research. Brinster et al. (1984) reported the injection ofSV40viral DNA into fertilized mouse embryos. In a low percentage of the injections, the foreign DNA was stably incorporated into the resulting mouse pup, and a line of trans-genic mice was produced by selective breedings. This foreign DNA produced no obvious effects until the mice reached approximately 5 months of age, at which time essentially all the mice developed large tumours of the choroid plexus, which often reached several hundred milligrams in weight (compared with 0.5 mg for the native plexus). These papillomas were identified as epithelial cell in origin. When these tumours were examined for serotonin binding sites, it was found (Yagaloff et al., 1986) that very high densities of 5-HT1c receptors were present (6.6 pmoVmg protein), making this the richest known source of 5-HT1c sites (a 25-fold richer source than any other serotonin receptor-containing tissue).

In a separate line of research, Gundersen et al. (1983) observed that injection of rat brain poly-A+ RNA into Xenopus oocytes led to the translation of the foreign mRNA and the appearance of a serotoninactivated cr current. Dascal et al. (1986) and Liibbert et al. (1987a) extensively characterized this response and determined that it arose from a serotonin 5-HT 1c receptor. In support of this assignment, they showed that the signal was mediated by a G protein, that it involved release of intracellular Ca2+ via the inositol phosphate pathway, that it was preferentially present in choroid plexus mRNA (although weaker 5-HT1c signals could be obtained from the cortex and substantia nigra), and that it exhibited the appropriate pharmacology for a 5-HT1c receptor.

Based on these discoveries, a cloning strategy (Figure 23.1) was devised. The strategy employed electrophysiological screening of mRNA-injected oocytes in order to identify a 5-HT1c receptor clone from a eDNA library. Poly-A+ RNA was prepared from choroid plexus tumours and sizefractionated to produce a 4.8-5.2 kilobase fraction, which is highly enriched in the 5-HT 1c message. A eDNA library was prepared from this fraction by a directional cloning approach utilizing a plasmid vector which facilitates isolation of single-stranded DNA. The library was screened for the 5-HT 1c

184 Serotonin

eDNA by isolating DNA from groups of 20 clones, and using this DNA to deplete 5-HT1c mRNA from RNA obtained from the tumours. The depletion was achieved by separation of DNA-RNA hybrids on a density gradient, and this hybrid-depleted RNA was injected into oocytes. If the injected RNA failed to produce the serotonin receptor response, then the DNA isolated from those particular 20 clones may contain the sequence coding for the 5-HT1c receptor. This strategy was successful in pulling out a partial clone (1.9 kilobase out of 5 kilobase) for the serotonin 5-HT1c

Cloning Strategy

Isolate mRNA from choroid plexus tumors

Size fractloMte mRNA by gel electrophoresis

~repare a eDNA library In a plasmi d vector contai ning lhe M 13 lntergenlc region, allowing production or single stranded DNA

Isolate single stranded DNA from groups of 20 clones; linearize the plasmids

Hybrid depletion: Hybridize linearized pl asmlds to tumor RNA end separate DNA- RNA hybrt ds from RNA one CsCl - guenidine HCl dens1ty gradient

Isolate the unhybrldized RNA end Inj ect Into Xenopus oocytes

Record Cl- curr ents following appl ication of serotonin Select clones that cause loss of the 5-HT IC receptor

mediated signal

··~ ...... .._.._ .......... ,..~........ (,.,. tumor mRNR

-~~ ... "'-... .......... . • J l

jl .. - .... .....--· 4.8 to 5.2 kb .., J' • ., fraction .r---1-

l (0 plumids

with cDNII Inserts

linearize ·-·-·~~ t /'""' \~,__ hybridize

mltroinjett

Figure 23.1 Strategy for cloning the 3-HT1c receptor (Liibbert eta/., 1987b)


receptor after screening 1200 bacterial colonies (Liibbert et a/. , 1987b). Subsequently, Julius (1988) isolated a eDNA clone for the 5-HT1c receptor which contained the entire protein coding region.

CONCLUSIONS

At present, our understanding of 5-HT1c receptor function in the most studied tissue, mammalian choroid plexus, is summarized in Figure 23.2. The 5-HT1c receptor appears to be localized to the apical face of choroid plexus epithelial cells, where it interacts with serotonin in the CSF, perhaps arising from the ventricular plexus of supra-ependymal serotoninergic fibres. Activation of this receptor triggers hydrolysis of

. .. ... .. . . . . . . . . . . . . . . . . . . . . . . 0 • •••••• . . . . . . . . . . . . . . . . . . . . . . • • • • • • 0 • . . . . . . . . . . . . . . . . . • • • • 0 ••• . . . . . . . 0 •• ' ••• • . . . . . . . . . . . . . . . . .. . ... . . . . . . . . . . . . . . . . . . . . . . . . . ' . . . .

' .... ' ..

. . . ... .. . .. .. .... . .. . ........................ . . ·.· .=· .·.·.·.·.·.·.·.·.·.·.·.·.·.·. ·.·. ·.· .·. ·.·.·.·.·. ·.·.·.·.·.·.· ...... ' . .. .

1 supraependymal fibers

5-HT Cerebrospinal fluid

1

Epithelial Cell Layer

Figure 23.2 Summary of 5-Hf1c receptor actions in the choroid plexus. PIP2 = phosphatidylinositol-4,5-biphosphate; IP3 = inositol-1,4,5-triphosphate; DAG =

diacylglycerol

186 Serotonin

phosphatidylinositol-4,5-bisphosphate to produce inositol-1 ,4,5-triphosphate and diacylglycerol, which in turn would release intracellular Ca2+ and activate protein kinase C. These responses are likely to open specific ion channels in the cell membrane (just as they open Cl- channels in the oocyte) by a combined effect of elevated intracellular Ca2 + and phosphorylation of specific target proteins.

In addressing the title line of this article, it can be said that much has been learned about the coupling of the 5-HT1c receptor to second messenger systems and to ion channels, but that the rapid pace of molecular studies on this receptor has somewhat eclipsed our understanding of its physiological roles. Progress has been made in localizing this site within brain tissues and in determining its activation pathways, and these provide important clues regarding its possible physiological roles and cellular regulation. Since the 5-HT 1c receptor is the first serotonin receptor to be cloned from any source, it protnises to provide many novel insights and new directions for the study of serotoninergic processes on the molecular and cellular level.

ACKNOWLEDGEMENTS

This work was supported by NIH grants NS23048 and NS24628 and by a grant from the Office of Naval Research.

REFERENCES

Brinster, R. L., Chen, H. Y., Messing, A., van Dyke, T., Levine, A. J. and Palmiter, R. D. (1984). Transgenic mice harboring SV40 T-antigen genes develop characteristic brain tumors. Cell, 37, 367-379

Buchheit, K. H.,Engel, G., Hagenbach,A.,Hoyer,D., Kalkman, H. 0. and Seiler, M. P. ( 1986). The ratisolated stomach fundus strip, a modelfor 5-HT 1c receptors. Br. J. Pharmacol., 88, 367P

Conn, P. J., Sanders-Bush, E., Hoffman, B. J. and Hartig, P.R. (1986). A unique serotonin receptor in choroid plexus is linked to phosphatidylinositol turnover. Proc. Nat/. Acad. Sci. U.S.A, 83, 4086-4088

Conn, P. J., Janowsky, A. and Sanders-Bush, E. (1987). Denervation supersensitivity of 5-HT1c receptors in rat choroid plexus. Brain Res., 400, 396-398

Dascal, N., Ifune, C., Hopkins, R., Snutch, T. P., Liibbert, H., Davidson, N., Simon, M. I. and Lester, H. A. (1986). Involvement of a GTP-binding protein in mediation of serotonin and acetylcholine responses in Xenopus oocytes injected with rat brain messenger RNA. Mol. Brain Res., 1, 201-209

Gundersen, C. B., Miledi, R. and Parker, I. (1983). Serotonin receptors induced by exogenous messenger RNA in Xenopus oocytes. Proc. R. Soc. B., 219, 103-109

Hoffman, B. J., Hartig, P.R., Conn, P. J. and Sanders-Bush, E. (1986). Thirtyfold stimulation of phosphatidylinositol hydrolysis by serotonin 5-HT1c receptors in pig choroid plexus. Soc. Neurosci. Abst., 12, 576


Hoyer, D., Pazos, A., Probst, A. and Palacios, J. M. (1986). Serotonin receptors in the human brain. II. Characterization and autoradiographic localization of 5-Hf1c and 5-Hf2 recognition sites. Brain Res., 376, 97-107

Julius, D., MacDermott, A. B., Axel, R. and Jessen, T. M. (1988). Molecular characterization of a functional eDNA encoding the serotonin 5-Hf1c receptor. Science, 241, 55&-564

Liibbert,H., Snutch, T. P.,Dascal,N.,Lester,H. A. andDavidson,N. (1987a). Rat brain 5-HF1c receptors are encoded by a 5-6 kbase mRNA size class and are functionally expressed in injected Xenopus oocytes. J. Neurosci., 1, 1159-1165

Liibbert, H., Hoffman, B. J., Snutch, T. P., van Dyke, T., Levine, A. J., Hartig, P. R., Lester, H. A. and Davidson, N. (1987b ). eDNA cloning of a serotonin 5-Hf 1c receptor by using electrophysiological assays of mRNA-injected Xenopus oocytes. Proc. Natl Acad. Sci. U.S.A., 84, 4332-4336

Napoleone, P., Sancesario, G. and Amenta, F. (1982). Indoleaminergic innervation of rat choroid plexus: a fluorescence histochemical study. Neurosci. Len., 34, 143-147

Palacios, J. M., Markstein, R. and Pazos, A. (1986). Serotonin-1C sites in the choroid plexus are not linked in a stimulatory or inhibitory way to adenylate cyclase. Brain Res., 380, 151-154

Pazos, A., Hoyer, D. and Palacios, J. M. (1985). The bindingofserotonergicligands to the porcine choroid plexus: characterization of a new type of serotonin recognition site. Eur. J. Pharmacol., 106, 539-546

Pedigo, N. W., Yamamura, H. I. and Nelson, D. L. (1981). Discrimination of multiple (3H]5-hydroxytryptamine binding sites by the neuroleptic spiperone in rat brain. J. Neurochem., 36, 220-226

Peroutka, S. J. (1986). Pharmacological differentiation and characterization of 5-Hf lA• 5-Hf 18, and 5-Hf 1c binding sites in rat frontal cortex. J. Neurochem., 47, 529-540

Richards, J. G. and Guggenheim, R. (1982). Serotonergic axons in the brain: A bird's eye view. Trends in Neurosci., 5, 4-5

Volicer, L., Direnfeld, L. K., Freedman, M., Albert, M. L., Langlais, P. J. and Bird, E. D. (1985). Serotonin and 5-hydroxyindoleacetic acid in CSF. Arch. Neurol., 42, 127-129

Yagaloff, K. A. and Hartig, P.R. (1985). 1251-Lysergic acid diethylamide binds to a novel serotonergic site on rat choroid plexus epithelial cells. J. Neurosci., 5, 317&-3183

Yagaloff, K. A., Lozano, G., van Dyke, T., Levine, A. J. and Hartig, P.R. (1986). Serotonin 5-Hf1c receptors are expressed at high density on choroid plexus tumors from transgenic mice. Brain Res., 385, 389-394

24

Characterization of 5-HT 1 Binding Site Subtypes Labelled by

[38]-5-Hydroxytryptamine

S. J. Peroutka

Departments of Neurology and Pharmacology, Stanford University School of Medicine, Stanford CA 94305, USA

The first radioligand used in an attempt to label receptors for 5-hydroxytryptamine (5-HT) receptors in the CNS was [3H]-5-HT (Marchbanks, 1966). It was not until1976, however, that a specific population of high-affinity, saturable binding sites in brain membranes was identified using this radioligand by Bennett and Snyder (1976). In 1979, it was determined that the binding sites labelled by [3H]-5-HT were distinct from a second class of 5-HT binding sites labelled by [3H]-spiperone (Peroutka and Snyder, 1979; Peroutka et al., 1981). The sites labelled by [3H]-5-HT were designated 5-HT1 binding sites, and initially were believed to be homogeneous.

However, largely on the basis of the work of Nelson and colleagues, it soon became clear that [3H]-5-HT binding was heterogeneous. Slopes of spiperone competition curves for [3H]-5-HT-labelled sites were shallow, with Hill coefficients significantly less than unity. Nelson and colleagues suggested that [3H]-5-HT labelled at least two distinct sub-populations of 5-HT1 sites, and designated them 5-HT1A and 5-HT18 binding site subtypes (Pedigo et al., 1981; Schnellmann et al., 1984).

Over the past few years, it has become obvious that [3H]-5-HT labels more than just two subtypes of the 5-HT1 binding site in the CNS (Blurton and Wood, 1986; Hoyer et al., 1986; Peroutka, 1986, 1988). For example, (+)-lysergic acid diethylamide (LSD), methysergide, spiperone and mianserin display Hill coefficients ranging from 0.56-0.78 at [3H]-5-HTlabelled sites (Peroutka, 1986). These values are significantly less than unity. Computer-assisted iterative curve fitting analysis of these drug competition studies in rat frontal cortex suggests that at least two distinct sub-populations of 5-HT1 sites exist. In addition, the Hill slope of the

5-HT1 Binding Site Subtypes 189

competition curve for RU 24969 in rat frontal cortex is only 0.51±0.04 (mean±s.e. mean). Computer-assisted iterative curve fitting analysis of RU 24969 interactions with eHJ-5-HT-labelled sites indicates that the competition data are best fitted by a three-site model of (3H]-5-HT binding in this brain region.

The 5-HT1A binding site has been the most extensively characterized 5-HT1 binding site subtype, owing to the large number of potent and selective agents which interact with this site. Besides (3H]-5-HT, radio ligands such as (3H]-8-hydroxy-2-( di-n-propylamino )tetralin ((3H]-8-0H-DPAT), (3H]-ipsapirone and (3H]-WB 4101 can also be used to label 5-HT1A sites. 5-Carboxamidotryptamine (5-CT), 8-0H-DPAT, 5-HT, RU 24969 and (+)-LSD display less than 10 nM affinity for 5-HT lA sites. Autoradiographic studies have confirmed that the raphe nuclei and the hippocampus contain extremely high densities of this 5-HT 1 binding site subtype (Marcinkiewicz et al., 1984; Pazos and Palacios, 1985; Hoyer et al., 1986). A number of functional correlates have been proposed for this receptor. For example, modulation of adenylate cyclase activity (De Vivo and Maayani, 1986; Markstein et al., 1986), inhibition of raphe cell firing (Trulson and Aresteh, 1986; VanderMaelen et al., 1986; Sprouse and Aghajanian, 1987), canine basilar artery contraction (Peroutka et al., 1986; Taylor et al., 1986), hypotension (Doods et al., 1985), thermoregulation (Gudelsky et al., 1986; Trickle bank et al., 1986), and certain aspects of the 5-HT behavioural syndrome (Tricklebank et al., 1984, 1985; Kwong et al., 1986; Smith and Peroutka, 1986) are believed to be mediated by the 5-HT1A

receptor. 8-0H-DPAT displays more than three orders of magnitude selectivity for

5-HT lA versus non-5-HT lA sites in rat frontal cortex. The competition curve for 8-0H-DPATversus (3H]-5-HT is biphasic, with approximately one-third of the sites labelled by (3H]-5-HT displaced by nanomolar concentrations of 8-0H-DPAT (Ki=3 nM). These sites represent 5-HT1A sites. A second component of the competition curves is observed with micromolar concentrations of 8-0H-DPAT.

Therefore, in the presence of 100 nM 8-0H-DPAT, (3H]-5-HT binding is restricted to non-5-HT1A sites. Drug competition studies under this condition reveal extremely shallow Hill slopes for a number of drugs. For example, RU 24969 begins to compete for specific (3H]-5-HT binding in the presence of 100 nM 8-0H-DPAT at a concentration of approximately 0.1 nM. Competition continues over five orders of magnitude, with complete displacement not observed until a concentration of 10 1-tM RU 24969 is present. Computer-assisted iterative curve fitting analysis reveals that the data are best explained by at least two sub-populations of non-5-HT1A sites.

In the author's laboratory, 5-HT18 binding has been defined as specific [ 3H]-5-HT binding in the presence of 100 nM 8-0H-DPAT plus 3 1-tM

190 Serotonin

mianserin. Under this condition, RU 24969 is an extremely potent agent with a Ki value of approximately 0.7 nM. Moreover, the Hill slope of the competition curve for RU 24969 versus [3H)-5-HT -labelled sites approaches unity. The absolute potencies of a series of pharmacological agents correlate quite well with 5-HT 18 binding sites labelled by [125I)-cyanopindolol {Hoyer et al., 1985).

Interestingly, the 5-HT 18 site as defined above has only been identified in rat and mouse brain. Studies of human, dog, cow, chicken, turtle and frog brain membranes have not documented any evidence for 5-HT18 binding sites {Heuring et al., 1986; Hoyer et al., 1986). In functional studies, it has become apparent that the synaptosomal release of 5-HT is modulated by 5-HT18 receptors in rat brain (Engel et al., 1986; Raiteri et al., 1986). Although 5-HT modulates synaptosomal release of 5-HT in other species (including human), the 5-HT receptor mediating this effect remains unknown.

Table 24.1 Characteristics of 5-HT1 binding site subtypes

Radio/abelled by [3H)-5-HT [3H]-5-HT [3H)-5-HT [3H)-5-HT [ 3H)-8-0H-DPAT [1251)-CYP [3H)-Mesulergine [ 3H)-Ipsapirone (Rat and [1251)-LSD [3H]-WB 4101 mouse only) [3H)-Buspirone [ 3H)-PAPP [3H)-spiroxatrine

High-density Raphe nuclei Substantia Choroid Basal regions nigra plexus ganglia

Hippocampus Globus pallid us

Drug potencies (K.., nM) <10DM 5-CT RU 24969 Mesulergine 5-CT

8-0H-DPAT 5-CT Metergoline 5-HT 5-HT 5-HT Methysergide Metergoline RU 24969 (+)-LSD

10-1000 DM Metergoline Metergoline Mianserin Methysergide Methysergide Methysergide 5-HT Mianserin Spiperone (+)-LSD RU 24969 8-0H-DPAT Mesulergine 5-CT (+)-LSD

(+)-LSD RU 24969 >1000 DM Mianserin Mianserin Spiperone Mesulergine

Spiperone 8-0H-DPAT Spiperone Mesulergine 8-0H-DPAT

CYP=cyanopindolol; P APP= 1-(2-[4-aminophenyl]ethyl)-4-(3-trifluoromethylphenyl) piperazine.

Data given are derived from Peroutka and Snyder (1979), Hoyer eta/ (1986), Peroutka (1986), Heuring and Peroutka (1987), and Peroutka (1987).


The 5-HT1c binding site was first identified in the choroid plexus in autoradiographic studies (Pazos et al., 1985; Yagaloff and Hartig, 1985). In the author's laboratory, 5-HT 1c binding was defined initially as specific [3H]-5-HT binding in the presence of 100 nM 8-0H-DPAT and 10 nM RU 24969 (Peroutka, 1986). Under this condition, the rank order of drugs is again shifted as compared with their effects on total [3H]-5-HT binding. Drugs such as mianserin and methysergide become more potent and drugs such as RU 24969 become weaker as compared to their affinities for 5-HT 1A

and 5-HTlB sites. Functionally, the 5-HT1c receptor seems to mediate phosphatidylinositol turnover in choroid plexus membranes (Conn et al., 1986).

Most recently, we have identified a 5-HT1 binding site subtype in bovine brain which does not coincide with previously described 5-HT 1A, 5-HT 18, or 5-HT1c binding site subtypes (Heuring and Peroutka, 1987). Drugs such as 8-0H-DP AT and mesulergine are extremely weak at this site (with Ki values in the micromolar range). 5-CT is the most potent agent, with an apparent Ki value of 0.75±0.1 nM (mean±s.e. mean), and 5-HT also displays a nanomolar affinity for this site. It has been designated the 5-HT 10 subtype of the 5-HT 1 binding site, and is most dense in the basal ganglia, although it appears to be present in all bovine brain regions. To date, no direct correlation between drug affinities for this site and any biochemical or neurophysiological effects of 5-HT has been documented. However, the pharmacological characteristics of the 5-HT10 site appear to be similar to those of the receptor mediating the effects of 5-HT and related agents in the rat stomach fundus (Clineschmidt et al., 1985; Cohen and Wittenauer, 1986) and the isolated perfused rat kidney preparation (Charlton et al., 1986).

In summary, [3H]-5-HT can be shown to label at least four distinct 5-HT1 binding site subtypes in brain membranes. Each of the four sites has a distinct pharmacological profile and each displays a unique anatomical localization (summarized in Table 24.1). The recent development of potent subtype-selective agents has facilitated the correlation of these sites with receptor-mediated effects of 5-HT in various biochemical, neurophysiological and behavioural systems. Future studies are needed to determine if even more than four 5-HT 1 binding site subtypes exist in the CNS. Moreover, the complete pharmacological characterization of all binding sites labelled by [3H]-5-HT must be determined in order to identify their functional relev.ance. This type of information should greatly elucidate the role of 5-HT in the CNS.

ACKNOWLEDGEMENTS

This work was supported in part by the John A. and George L. Hartford Foundation, the McKnight Foundation, the Alfred P. Sloan Foundation and National Institutes of Health grants NS 12151-13 and NS 23560-01.

192 Serotonin

REFERENCES

Bennett, J. P., Jr and Snyder, S. H. (1976). Serotonin and lysergic acid diethylamide binding in rat brain membranes: Relationship to postsynaptic serotonin receptors. Mol. Pharmacol, 12, 373-389

Blurton, P. A. and Wood, M.D. (1986). Identification of multiple binding sites for [3H]5-hydroxytryptamine in the rat CNS. J. Neurochem., 46, 1392-1398

Charlton, K. G., Bond, R. A. and Clarke, D. E. (1986). Aninhibitoryprejunctional 5-HT1-like receptor in the isolated perfused rat kidney. Naunyn-Schmiedeberg's Arch. Pharmacol, 332, 8--15

Cline:;chmidt, B. V., Reiss, D. R., Pettibone, J. and Robinson, J. L. (1985). Characterization of 5-hydroxytryptamine receptors in rat stomach fundus. J. Pharmacol. Exp. Ther., 235, 696--708

Cohen, M. L. and Wittenauer, L. A. (1986). Further evidence that the serotonin receptor in the rat stomach fundus is not 5-HT1A or 5-HTIB. Life Sci., 38, 1-5

Conn, P. J., Sanders-Bush, E., Hoffman, B. J. and Hartig, P.R. (1986). A unique serotonin receptor in choroid plexus is linked to phosphatidylinositol turnover. Proc. Nat/. Acad. Sci. U.S.A, 83, 4086-4088

De Vivo, M. and Maayani, S. (1986). Characterization of the 5-hydroxytryptamine1A receptor-mediated inhibition offorskolin-stimulated adenylate cyclase activity in guinea pig and rat hippocampal membranes. J. Pharmacol. Exp. Ther., 238, 248--253

Doods, H. N., Kalkman, H. 0., De Jonge, A., Thoolen, M., Wilffert, B., Timmermans, P. and Van Zwieten, P. A. (1985). Differential selectivities of RU 24969 and 8-0H-DPAT for the purported 5-HT1A and 5-HT1B binding sites. Correlation between 5-HT1A affinity and hypotensive activity. Eur. J. Pharmacol., 112, 363-370

Engel, G., Gothert, M., Hoyer, D., Schlicker, E. and Hillenbrand, K. (1986). Identity of inhibitory presynaptic 5-hydroxytryptamine (5-HT) autoreceptors in the rat brain cortex with 5-HT1B binding sites. Naunyn-Schmiedeberg's Arch. Pharmacol, 351, 1-7

Gudelsky, G. A., Koenig, J. I. and Meltzer, H. Y. (1986). Thermoregulatory responses to serotonin (5-HT) receptor stimulation in the rat. Neuropharmacology, 25, 1307-1313


Heuring, R. E., Schlegel, J. R. and Peroutka, S. J. (1986). Species variations in 5-HT tB and 5-HT 1c binding sites defined by R U 24969 competition studies. Eur. J. Pharmacol, 122, 279-282

Hoyer, D., Engel, G. and Kalkman, H. 0. (1985). Characterization ofthe 5-HTIB recognition site in rat brain: Binding studies with (-)P25I] iodocyanopindolol. Eur. J. Pharmacol., 118, 1-12

Hoyer, D., Pazos, A., Probst, A. and Palacios, J. M. (1986). Serotonin receptors in the human brain: I. Characterization and autoradiographic localization of 5-HT tA recognition sites. Apparent absence of 5-HT IB recognition sites. Brain Res., 376, 85-96

Kwong, L. L., Smith, E. R., Davidson, J. M. and Peroutka, S. J. (1986). Differential interactions of 'prosexual' drugs with 5-hydroxytryptamine1A and alphar adrenergic receptors. Behav. Neurosci., 100, 664-668

Marchbanks, R. M. (1966). Serotonin binding to nerve ending particles and other preparations from rat brain. J. Neurochem., 13, 1481-1493


Marcinkiewicz, M., Verge, D., Gozlan, H., Pichat, L. and Hamon, M. (1984). Autoradiographic evidence for the heterogeneity of 5-HT1 sites in the rat brain. Brain Res., 291, 159-163

Markstein, R., Hoyer, D. and Engel, G. (1986). 5-HT1A receptors mediate stimulation of adenylate cyclase in rat hippocampus. Naunyn-Schmiedeberg's Arch. Pharmacal, 333, 335---341

Pazos, A. and Palacios, J. M. (1985). Quantitative autoradiographic mapping of serotonin receptors in the rat brain. I. Serotonin-1 receptors. Brain Res., 346, 205---230

Pazos, A., Hoyer, D. and Palacios, J. M. ( 1985). The binding of serotonergicligands to the porcine choroid plexus: Characterization of a new type of serotonin recognition site. Eur. J. Pharmacal., 106, 539-546

Pedigo, N. W., Yamamura, H. I. and Nelson, D. L. (1981). Discrimination of multiple [3H]5-hydroxytryptamine binding sites by the neuroleptic spiperone in rat brain. J. Neurochem., 36, 220-226

Peroutka, S. J. (1986). Pharmacological differentiation and characterization of 5-HT1A, 5-HT18 and 5-HT1c binding sites in rat frontal cortex. J. Neurochem., 41, 529-540

Peroutka, S. J. (1988). 5-Hydroxytryptamine receptor subtypes. Ann. Rev. Neurosci., 11, 45---60

Peroutka, S. J. and Snyder, S. H. (1979). Multiple serotonin receptors: differential binding of [3H]5-hydroxytryptamine, [3H]lysergic acid diethylamide and [3H]spiroperidol. Mol. Pharmacal., 16, 687-699

Peroutka, S. J., Lebovitz, R. M. and Snyder, S. H. (1981). Two distinct central serotonin receptors with different physiological functions. Science, 212, 827-829

Peroutka, S. J., Huang, S. and Allen, G. S. (1986). Canine basilar artery contractions mediated by 5-hydroxytryptamine1A receptors. J. Pharmacal. Exp. Ther., 237, 901-906

Raiteri, M., Maura, G., Bonanno, G. and Pittaluga, A. (1986). Differential pharmacology and function of two 5-HT 1 receptors modulating transmitter release in cerebellum. J. Pharmacal. Exp. Ther., 237, 644-648

Schnellmann, R. G., Waters, S. J. and Nelson, D. L. (1984). [3H]5-Hydroxytryptamine binding sites: Species and tissue variation. J. Neurochem., 42, 65---70

Smith, L. M. and Peroutka, S. J. (1986). Differential effects of 5-hydroxytryptamine1A selective drugs on the 5-HT _behavioral syndrome. Pharmacal. Biochem. Behav., 24, 1513-1519

Sprouse, J. S. and Aghajanian, G. K. (1987). Electrophysiological responses of serotonergic dorsal raphe neurons to 5-HT tA and 5-HT lB agonists. Synapse, 1, 3-9

Taylor, E. W., Duckles, S. P. and Nelson, D. L. (1986). Dissociation constants of serotonin agonists in the canine basilar artery correlate to Ki values at the 5-HT1A binding site. J. Pharmacal. Exp. Ther., 236, 11~125

Tricklebank, M. D., Forler, C. and Fozard, J. R. (1984). The involvement of subtypes of the 5-HT1 receptor and of catecholaminergic systems in the behavioural response to 8-hydroxy-2-( di-n-propylamino )tetralin in the rat. Eur. J. Pharmacal., 106, 271-282

Tricklebank, M. D., Forler, C., Middlemiss, D. N. and Fozard, J. R. (1985). Subtypes of the 5-HT receptor mediating the behavioural responses to 5-methoxy-N,N-dimethyltryptamine in the rat. Eur. J. Pharmacal., 117, 15---24

Tricklebank, M. D., Middlemiss, D. N. and Neill, J. (1986). Pharmacological analysis of the behavioral and thermoregulatory effects of the putative 5-HT1 receptor agonist, RU 24969, in the rat. Neuropharmacology, 25, 877-886

Trulson, M. E. and Arasteh, K. (1986). Buspirone decreases the activity of

194 Serotonin

5-hydroxytryptamine-containing dorsal raphe neurons in-vitro. J. Pharm. Pharmacol., 38, 380-382

VanderMaelen, C. P., Matheson, G. K., Wilderman, R. C. and Patterson, L.A. (1986). Inhibition of serotonergic dorsal raphe neurons by systemic and iontophoretic administration of buspirone, a non-benzodiazepine anxiolytic drug. Eur. J. Pharmacol., 129, 123-130

Yagaloff, K. A. and Hartig, P.R. (1985). 1251-LSD binds to a novel serotonergicsite on rat choroid plexus epithelial cells. J. Neurosci., 5, 317~3183

25 The Classification of 5-HT Receptors Using

Tryptamine Analogues P. Lefft and G. R. Martin

Analytical Pharmacology Group, Department of Pharmacology (I), Wellcome Research Laboratories, Beckenham, Kent, BR3 3BS, UK

INTRODUCTION

The object of this article is to promote a rigorous approach to the classification of 5-HT receptors. Rigour in receptor classification means: (a) the provision of quantitative information about the interactions between agonists, antagonists and receptors; that is, estimates of dissociation constants, and, in the case of agonists, efficacies; and (b) attention to practical conditions and analytical criteria which allow valid estimation of these quantities. This article will briefly examine the extent to which 5-HT receptor classification, as currently practised, fulfills these demands and how some of the problems which emerge may be approached using tryptamine analogues.

CONDITIONS AND CRITERIA

A number of practical conditions have been established which serve to maximize the possibility that only a single receptor type is studied at a time, and which ensure, as far as possible, that the interactions studied are at equilibrium when measurements are made. For example, the presence of multiple receptor types and agonist removal mechanisms in the assay tissue are factors to be considered, as are inadequate equilibration times and multiple, post-receptor actions by antagonists (Furchgott, 1968}.

Then there are analytical criteria which serve to check whether the practical conditions have been effective and to test the quality of the affinity and efficacy estimates made. Among them are graphical tests for concentration-effect curve parallelism and unit Schild plot slopes in the analysis of antagonism (Black et al., 1983), and curve shape considerations in the analysis of agonism (Leff, 1987). These criteria derive from the

tPresent address: Fisons PLC, Pharmaceuticals Division, Bakewell Road, Loughborough, Leicestershire LEU ORN, UK.

196 Serotonin

occupancy theory of receptor interactions. When fulfilled, they provide support that simple bimolecular interactions between agonist, antagonist and receptor molecules are taking place. The resulting estimates of, for example, dissociation constants can be accepted with some confidence under these conditions. However, when the criteria are not fulfilled, the operation of simple mechanisms cannot be assumed and quantities such as dissociation constants cannot be estimated. It may be possible to describe the observed interaction by an empirical parameter, but clearly such a quantity does not carry the same weight in receptor classification as a dissociation constant.

Even when the analytical criteria are fulfilled, the estimated dissociation constants must be reproducible to be useful in classification. For example, a pKa estimate for an antagonist should be independent of the agonist-tissue combination assuming the same receptor operates. Only under such conditions can the receptor be positively identified and anomalies exposed.

QUANTITATIVE PROBLEMS IN 5-HT RECEPTOR CLASSIFICATION

Non-surmountable Antagonism

Non-surmountable antagonist action is frequently observed in studies of 5-HT receptor interactions. This phenomenon is not restricted to a particular receptor type. For example: 5-HT 1-like receptor-mediated effects on post-ganglionic sympathetic neurones (Charlton et al., 1986) and on vascular endothelium (Van Nueten et al., 1984) are antagonized in a non-surmountable fashion by methiothepin; 5-HT2 receptor-mediated agonist effects in bovine coronary arteries (Kaumann and Frenken, 1985), in rat jugular vein and in rat caudal artery (Leff and Martin, 1986) are antagonized non-surmountably by methysergide, and ketanserin demonstrates this action in the rat caudal artery (Leff and Martin, 1986), in rat renal vasculature (Charlton eta/., 1984) and in guinea-pig trachea (Lemoine and Kaumann, 1986); 5-HT3 receptor-mediated effects in the rat vagus nerve (Ireland and Tyers, 1987), in rabbit superior cervical ganglion (Fozard eta/., 1985) and in rabbit nodose ganglion (Fozard eta/., 1985) are antagonized non-surmountably by MDL 72222, and ICS 205-930 produces similar effects in these tissues (Round and Wallis, 1986; Ireland and Tyers, 1987).

The failure of these compounds to meet the criteria for competitive antagonism in these systems rules out accurate estimation of dissociation constants. However, knowledge of the mechanism responsible for the non-surmountable profile may conceivably assist. If, for example, it was due to a post-receptor property superimposed on competitive antagonism, then the latter component may be resolvable from the resultant as in the case of verapamil which exhibits 5-HT2 receptor antagonism as well as Ca

-2 .. c 8 1-l: I

10 .... 0 c .2 t;

l 4D e .2 .5 I • ! u .E

Classification of 5-HT Receptors Using Tryptamines

1.5 A

1.0

0.5

0

1.5 B

1.0

0.5

0

1.5 c 1.0

0.5

0 8

[Methysergide J 0.01pM 0.03pM 0.1 pM 0.3 pM 1.0 pM

[Methysergide I 0.001pM 0.01 pM 0.10 pM

-0.001pM ,....y"...--- 0.01 pM

~-=----..... 0.10 pM

7 s 5 4 3 [5-HT] (-log10M)

197

Figure 25.1 Antagonist effects of methysergide in (A) rabbit aorta, (B) rat jugular vein and (C) rat caudal artery. (Reproduced with permission from Left and Martin,

1986)

198 Serotonin

antagonism (Leff and Morse, 1987). On the other hand, if other explanations apply, such as slow dissociation kinetics or allosteric mechanisms, it is unclear how these may be quantified. The problem is compounded by variations in the expression of non-surmountable antagonism by particular compounds. Methysergide, for example, behaves as an apparently competitive antagonist at 5-HT2 receptors in the rabbit aorta but is a non-surmountable antagonist in the rat jugular vein and rat caudal artery (See Figure 25.1) (Leff and Martin, 1986). Furthermore, the concentration of methysergide required to produce a given amount of curve depression varies between the latter two tissues. Therefore, even empirical measures of antagonist activity (such as pOi values) are variable in such circumstances, meaning that they are not useful for classification purposes.

Variable Antagonist Aff'mity

Variable antagonist activity is not only associated with the expression of non-surmountable action. Even when the analytical criteria are fulfilled, variation in antagonist affinity is evident. For example, estimates of dissociation constants for ketanserin vary by about two orders of magnitude between different tissues ostensibly containing the same 5-HT 2 receptor type (Leff et al., 1986, and literature cited therein). Some of this variation could be accounted for by differences in methods of analysis by different authors, but even when steps are taken to ensure equivalence of experimental conditions in different assays, differences in affinity estimates for ketanserin and other antagonists are still exposed (Leff and Martin, 1986). Such variation may be another expression of the mechanisms which produce non-surmountable effects. Alternatively, it may be a manifestation of receptor heterogeneity among the 5-HT2 class. Regardless of the explanation, the absence of a stable estimate of affinity undermines rigour in classification, because under these circumstances positive identification of receptors is not possible and anomalous behaviour is difficult to detect.

USE OF TRYPTAMINE ANALOGUES IN 5-HT RECEPTOR CLASSIFICATION

Quantitative Evaluation Using Agonists

In the light of the problems discussed above and the association of these problems with compounds which are structurally unrelated to 5-HT, we have been prompted to investigate whether quantitative information of a more robust kind can be obtained using tryptamine analogues. By its nature, this exercise has involved the use of agonist as well as antagonist analogues,

Classification of 5-HT Receptors Using Tryptamines 199

and therefore efficacy as well as affinity information has been generated. The essence of this approach is to produce a 'fingerprint' for each receptor

6 a

4

2

-0)

- 0

• (,) ··············································································· .. 0 LL.

1.5

1.0

0.5

0

b

-a -7 -6 -5 -4

[Agonist] Figure 25.2 Agonist effects of 5-HT (e), 5-cyanotryptamine (0), N,Ndimethyltryptamine <•). and N-benzyl-5-methoxytryptamine (t,.), in (a) rabbit aorta, and (b) rat jugular vein. Lines drawn through the data were produced by operational model-fitting (Black et al., 1985) which provided affinity and efficacy

estimates. (Reproduced with permission from Leff et al., 1986)

200 Serotonin

consisting of affinity and efficacy information which can be used for positive receptor identification or for discriminatory purposes, as exemplified below.

Confll"mation of Receptor Similarity

Figure 25.2 illustrates the constrictor effects produced by a series of three agonist analogues of 5-HT, and 5-HT itself, in isolated preparations of the rabbit aorta and the rat jugular vein. Analysis of these data using methods described elsewhere (Black et al., 1985) showed that the estimates of affinity and relative efficacy for each agonist are identical in the two assays (Leff et al., 1986). Furthermore, a tryptamine antagonist, «,a-dimethyltryptamine, fulfilled the criteria for simple competition and exhibited the same affinity in the two tissues (the pK8 values were 5.67 in the aorta and 5.53 in the jugular vein).

These data positively and quantitatively identified the receptor in the two tissues as the same. Moreover, they did so in a situation where non-tryptamine antagonists exhibited discrepant behaviour between the two tissues. Methysergide, for example, behaved as an apparently competitive antagonist in the aorta but non-surmountably in the jugular vein; ketanserin, spiperone and trazodone, although they behave competitively in both tissues, exhibited different affinities, of at least 0.4 log10 unit differences in pK8 (Leff and Martin, 1986).

Differential Receptor Classification

Figure 25.3 illustrates constrictor and relaxant effects produced by another series of agonist analogues in three vascular preparations. The object of this analysis was to determine whether endothelium-dependent and endothelium-independent 5-HT-mediated vasorelaxant effects were mediated by the same or different receptors, and simultaneously to discriminate them from 5-HT2 receptor-mediated constrictor effects. In this case, the four agonists exhibited different affinities and relative efficacies between the three assays, indicating that distinct classes of 5-HT receptors subserve these actions (Martinet al., 1987).

This example illustrates how the tryptamine 'fingerprint' approach substantiates and extends the currently proposed classification of 5-HT receptors. First, adopting the recommended nomenclature proposed by Bradley et al. (1986), the classification of 5-HT receptors subserving vasorelaxation as 5-HT1-like, as distinct from 5-HT2, is confirmed; second, quantitative evidence for subclasses of functional 5-HT rlike receptors is provided. Although evidence for such subclassification is provided by the butyrophenone spiperone (Martinet al., 1987), in the light of the variable pharmacology of such non-tryptamines, the availability of the quantitatively reliable information supplied by tryptamines is important.


8 ·············································

--• u

1.2

0.8

.. 0.4 0 IL 0.0

a

0.8 ··········································· c

0.6

0.4

0.2

0.0 -10 -9 -8 -7 -6 -5 -4

[Agonist] log 10M Figure 25.3 Agonist effects of 5-HT (e), a-methyl-5-HT (0), 5-methyltryptamine (A), and 5-carboxamidotryptamine (D.), in (a) rabbit aorta, (b) rabbit jugular vein with intact endothelium, and (c) rabbit jugular vein with denuded endothelium. Responses in (a) are constrictor (increases in force); responses in (b) and (c) are relaxant (decreases in force, following pre-contraction with U-46619). The lines drawn through the data were obtained by operational model-fitting (Black et al.,

1985)

202 Serotonin

REFERENCES

Black, J. W., Gerskowitch, V. P. and Leff, P. (1983}. Reflections on the classification of histamine receptors. In Parsons, M. E. and Ganellin, C. R. (Eds.), Pharmacology of Histamine Receptors, Wright, Bristol, London, Boston, pp. 1-9

Black, J. W., Leff, P., Shankley, N. P. and Wood, J. (1985). An operational model of agonism: the effect of EJ[ A] curve shape on agonist dissociation constant estimation. Br. J. Pharmacol., 84, 561-571


Charlton, K. G., Johnston, T. D. and Clarke, D. E. (1984). Vasoconstrictor and norepinephrine potentiating action of 5-hydroxykynuramine in the isolated perfused rat kidney: involvement of serotonin receptors and aradrenoceptors. Naunyn-Schmiedeberg's Arch. Pharmacol., 328, 154-159

Charlton, K. G., Bond, R. A. and Clarke, D. E. (1986). An inhibitory pre junctional 5-HT 1-like receptor in the isolated perfused rat kidney. Apparent distinction from the 5-HT1A, 5-HT18 and 5-HT1c subtypes. Naunyn-Schmiedeberg's Arch. Pharmacol, 332, 8-15

Fozard, J. R., Humphreys, A., Round, A. and Wallis, D.l. (1985). Further studies on the actions of MDL 72222 on responses to 5-HT in rabbit nodose (NG) and superior cervical (SCG) ganglia. Br. J. Pharmacol., 85, 309P

Furchgott, R. F. (1968). A critical appraisal of the use of isolated organ systems for the assessment of drug action at the receptor level. In Tedeschi, D. H. and Tedeschi, R. E. (eds), Importance of Fundamental Principles of Drug Evaluation, Raven Press, New York, pp. 277-297

Ireland, S. J. and Tyers, M. B. (1987). Pharmacological characterisation of 5-hydroxytryptamine-induced depolarisation of the rat isolated vagus nerve. Br. J. Pharmacol., 90, 229-238

Kaumann, A. J. and Frenken, M. (1985). A paradox: the 5-HT-receptor antagonist ketanserin restores the 5-HT-induced contraction depressed by methysergide in large coronary arteries of calf. Allosteric regulation of 5-HT_receptors. Naunyn-Schmiedeberg's Arch. Pharmacol., 328, 295-300

Leff, P. (1987). Can operational models of agonism provide a framework for classifying hormone receptors? In Black, J. W., Jenkinson, D. H. and Gerskowitch, V. P. (Eds), Perspectives on Receptor Classification, Receptor Biochemistry and Methodology, Vol. 6, Alan R. Liss, New York, pp. 157-167

Leff, P. and Martin, G. R. (1986). Peripheral 5-HT2-like receptors. Can they be classified with the available antagonists? Br. J. Pharmacol., 88, 585-593

Leff, P. and Morse, J. M. (1987). Resultant pharmacological actions ofverapamil: quantification of competitive 5-hydroxytryptamine antagonism in combination with calcium antagonism. J. Pharmacol. Exp. Ther., 240, 284-287

Leff, P., Martin, G. R. and Morse, J. M. (1986). The classification of peripheral 5-HTz-like receptors using tryptamine agonist and antagonist analogues. Br. J. Pharmacol., 89, 493-499

Lemoine, H. and Kaumann, A. J. ( 1986). Allosteric properties of 5-HT 2-receptors in tracheal smooth muscle. Naunyn-Schmiedeberg's Arch. Pharmacol., 333, 91-97

Martin, G. R., Leff, P., Cambridge, D. and Barrett, V. J. (1987). Comparative analysis of two types of 5-hydroxytryptamine receptor mediating vasorelaxation: differential classification using tryptamines. Naunyn-Schmiedeberg's Arch. Pharmacol., 336, 365-373


Round, A. and Wallis, D.l. (1986). The depolarising action of 5-hydroxytryptamine on rabbit vagal afferent and sympathetic neurones in vitro and its selective blockade by ICS 205-930. Br. J. Pharmacol., 88, 485-494

Van Nueten, J. M., Leysen, J. E., de Clerck, F. and Vanhoutte, P. M. (1984). Serotonergic receptor subtypes and vascular reactivity. J. Cardiovasc. Pharmacol., 6, S564-S574

26

Classification of 5-HT Receptors and Binding Sites: An Overviewt

P. P. A. Humphrey1 and B. P. Richardson2

1Pharmacology Division, Glaxo Group Research Limited, Ware, Hertfordshire SG 12 ODP, UK

2Preclinical Research Department, Sandoz Limited, CH-4002 Basel, Switzerland

INTRODUCTION

The history of the development of our overall understanding of the classification of receptors for 5-hydroxytryptamine (5-HT) was extensively discussed by the participants at the workshop on 5-HT receptor classification. The general consensus was that, despite major recent advances, a clear picture has yet to emerge because specific drug tools are not available to characterize definitively all the known/putative 5-HT receptor types. Furthermore, although all the workshop participants expressed a general desire to collate data from functional studies with data from ligand binding studies, this is not always as easy as it has been with the 5-HT2 receptor. Nevertheless, despite these shortcomings, it was agreed that the attempted all-embracing 5-HT receptor classification of Bradley et al. (1986) still provided a framework of understanding on which to build. Indeed, the majority of new data presented reinforced the classification, although some observations were difficult to encompass within it.

The classification of 5-HT receptors can be updated on the basis ofcurrent knowledge, and this is discussed in detail below. Although more data is now available on putative subtypes of the 5-HTrlike receptor, and our understanding of 5-HT2 receptors is relatively uncomplicated, the subclassification of 5-HT3 receptors remains controversial and complicated by the identification of neuronal 'M' receptors which are not blocked by

tThis overview incorporates material presented during the classification of 5-HT receptors and binding sites session, and material discussed in a workshop chaired by the authors; an attempt has been made to reflect all the views expressed and not just those of the authors (see acknowledgements).

Overview: Classification of 5-HT Receptors 205

5-HT3 receptor antagonists. It was therefore decided to impose a moratorium on the further subclassification of 5-HT receptors and on naming new types of putative 5-HT receptors. Thus, for example, it was agreed that the subclassification of 5-HT 3 receptors into 5-HT 33 , 5-HT Jb and 5-HT Jc may not be particularly useful at this stage because of the lack of ideal pharmacological tools and the complexity of measuring a neuronal effect by measuring the end-organ response, as in the rabbit heart and guinea-pig ileum.

Currently, only the three broad classes of 5-HT receptor are recognized, as indicated in Table 26.1, but a more definitive characterization and classification of the various receptor subtypes may be possible later on. This will be regularly reviewed by the Serotonin Club Receptor Nomenclature Committee (see Footnote, p. 219) formed in Sydney on 28 August 1987. The view was expressed that continual liaison with the recently appointed IUPHAR Receptor Nomenclature Committee was imperative in order to achieve a consistent overall scheme for 5-HT receptors within the general classification of neurotransmitter receptors.

PRINCIPLES OF RECEPI'OR CLASSIFICATION

Much of the workshop discussion was devoted to the philosophy of receptor characterization, and a number of diverse views were expressed. Thus there were strong pleas for rigour in attention to experimental protocols and analytical criteria. There was a strong feeling too that a receptor should be named only when it had been fully characterized according to universally acceptable techniques and standards.

Definition of a Receptor

Several participants expressed concern about the ever-increasing number of different 5-HT receptor subtypes reported in the literature, and wondered if there was any limit to the number which might eventually be identified. David Clarke suggested that this may be the inevitable outcome of a pharmacological approach to receptor classification that depends heavily on the use of synthetic compounds which are of appreciably greater molecular size than the natural ligand, 5-HT. Such synthetic agonists and antagonists possess the potential to bind to accessory sites within or outside the receptor domain which are not used by the smaller natural agonist. Both he and Paul Leff thought that there is a real danger that almost as many different 'receptors' will be recognized as the drugs used to identify them, if such an approach is adopted. This could be avoided if the key compounds used to

Tabl

e 26

.1

Cur

rent

pro

posa

ls fo

r cl

assi

ficat

ion

and

nom

encl

atur

e of

func

tiona

l5-H

T re

cept

orsa

P

ropo

sed

rece

ptor

no

men

clat

ure

5-H

T 1-li

ke

5-H

T2

5-H

T3

Typi

cal r

espo

nses

Pre j

unct

iona

l inh

ibiti

on o

f ne

uron

al tr

ansm

itter

re

leas

e, c

ontr

actio

n of

som

e va

scul

ar sm

ooth

mus

cle,

sm

ooth

-mus

cle r

elax

atio

n,

tach

ycar

dia i

n th

e cat

G

astr

oint

estin

al an

d va

scul

ar sm

ooth

-mus

cle

cont

ract

ion,

pla

tele

t ag

greg

atio

n, n

euro

nal

depo

lari

zatio

n D

epol

ariz

atio

n of

peri

pher

al (a

nd ce

ntra

l?)

neur

ones

Sele

ctiv

e Se

lect

ive

agon

ists

an

tago

nist

s

5-C

arbo

xam

idot

rypt

amin

e ( 5-

Cf)

M

ethi

othe

pinb

(m

ore s

elec

tive a

goni

sts w

ill m

imic

so

me b

ut n

ot al

l the

actio

ns o

f 5-C

f,

see t

ext)

a-M

ethy

l-5-h

ydro

xytry

ptam

inec

K

etan

serin

M

ethi

othe

pin

2-M

ethy

l-5-h

ydro

xytry

ptam

ine

GR

3803

2F

ICS2

05-9

30

MD

L722

22

Equi

vale

nt br

ain

bind

ing s

ite

5-H

T 1 su

bsite

s are

in

clud

ed w

here

an

alog

ous

char

acte

ristic

s ap

ply

5-H

T2

5-H

T3d

This

upd

ated

clas

sific

atio

n en

com

pass

es m

uch

of w

hat is

kno

wn

abou

t 5-H

T, b

ut a

few

exa

mpl

es o

f put

ativ

e 5-H

T re

cept

ors o

r sub

type

s ca

nnot

at p

rese

nt o

bvio

usly

be

incl

uded

in th

is s

chem

e (s

ee te

xt).

~odified f

rom

Bra

dley

et a

l. (1

986)

. ~ss p

oten

t ant

agon

ist t

han

at 5

-HT 2

rece

ptor

s bu

t ina

ctiv

e at

5-H

T 3 re

cept

ors.

cs

ee R

icha

rdso

n et

al.

(198

5) a

nd H

umph

rey

and

Feni

uk (t

his

volu

me)

. dS

ee T

yers

et a

l. (t

his

volu

me)

.

~ ~ a ~ ;: s·


classify 5-HT receptor subtypes were limited to small synthetic indole analogues, although the question of when such compounds become relevantly larger than 5-HT may be difficult to decide.

These deliberations led to the central question of what is a receptor and whether we should be characterizing 'drug receptors' or 'hormone receptors'. Leff recalled Stephenson's definition of a receptor as 'that spatial arrangement of atoms to which a substance endogenous to the organism attaches itself as an essential step in modifying cellular function' (see Stephenson, 1975). Leff pointed out the inherent danger in a definition,

HJ

N H

5-HYDROXYTRYPTAMINE 0

N H

5-CARBOXAMIDOTR.YPTAMINE (ALL 5-Hr 1-LIKE RECEPTORS)

N H

I CH3

" CH3

GR43175 (SOME 5-Hr1-UKE RBCEPTORS)

HO

Figure 26.1 Selective agonists at 5-Hf receptors

208 Serotonin

based on sites where drugs act, which does not distinguish between drug effects on hormone action sites and those on other cell constituents, such as enzymes, nucleic acids and ion channels. This leads to conceptual and terminological confusion. Clearly Stephenson's definition is a physiological one, and makes unlikely the situation in which a new 'drug' implies a new 'receptor' because greater weight is attached to the action of close structural analogues of the natural transmitter, in our case 5-HT (see Figure 26.1). Larger molecules may mimic or block the action of the endogenous agonist in one tissue and not in another, not because the receptors in the two tissues are structurally different, but because accessory binding sites are available in the former but not in the latter (see Figure 26.2). This may reflect differences in the membrane environment in which the receptor protein is located, so that tissue selectivity for the drug is confused with receptor heterogeneity. It was generally agreed that the question of whether receptor classification

~o"'"@ UNJ H

ICS 205-930 (SIIT3 RECEPTORS)

N~ IN CH3

N

\CH3

GR 380321'(51IT3 RECEPTORS)

H

o~Ny

~ UN~CN

H

CYANOPINDOLOL (51ITIA AND SliT to RECEPTORS)

0 F~N0-1

SPIPERONE (5HT1A AND 5HT2 RECEPTORS)

Figure 26.2 Selective antagonists at 5-HT receptors


should be a physiological or pharmacological one was fundamental, and must be addressed by the IUPHAR Receptor Nomenclature Committee with some urgency. In addition, it was stressed by many that pharmacologists should attempt to reach a classification scheme for all neurotransmitter and hormone receptors, which approaches the conciseness and clarity of that which has been achieved by biochemists in classifying enzymes.

Techniques for Characterization

The relative merits of the different techniques currently used for receptor classification were discussed at length. Paul Hartig and Stephen Peroutka pointed out that receptors will ultimately be characterized through gene-cloning techniques, which will permit determination of their amino acid sequences and tertiary structures. In-situ hybridization will also indicate in which cells the various receptor types are expressed in the body. At the moment we have not yet reached that point, but the cloning of the 5-HT1-like receptor found in the choroid plexus, and of several types of muscarinic receptor, has already occurred, suggesting that the amino acid sequences of most receptors will probably be known within the next few years, possibly even before the next IUPHAR meeting! Today we are in an interim situation, where information regarding the molecular structure of receptors is indirect and has to be inferred from data obtained using a variety of different biological techniques. The pharmacological model systems employed are usually not understood at the molecular or even cellular level, and often depend on recording complex biological responses in tissues which express several different 5-HT receptor subtypes. Nevertheless, defining receptor subtypes according to physiological responses has the advantage of functional relevance, which is not always apparent with approaches based on radio ligand binding studies. The need for increased rigour in the choice of biological system and in the way in which experiments are performed and analysed was stressed by several participants. Often the drugs used are simply not specific enough to provide reliable information regarding the subtype of receptor involved in mediating the particular response measured.

Leff mentioned that in the past, receptor classification has relied almost entirely on traditional bioassay procedures, although data of similar quality can, at least in principle, now be obtained from biochemical, electrophysiological and ligand binding studies. However, he stressed that scientists using these latter techniques should erect criteria by which the quality of their information can be judged, just as pharmacologists have done in the past. This would then provide a more integrative approach to receptor classification, and provide additional information to help dispel some of the uncertainties in the interpretation of data obtained from measuring complex functional end-organ responses. In addition, the defining of the second

210 Serotonin

messenger systems or ion channels involved in bringing about different end-organ responses should help to define the various subtypes of receptor involved, since implicit in Stephenson's original definition is the assumption that, if two receptors process chemical information imparted to a target cell by the natural agonist differently, then the receptors involved must also differ.

Nomenclature

The view of the meeting was that before attempting to name further 5-HT receptors, we should strive in collaboration with the IUPHAR Nomenclature Committee for a consensus on the criteria which should be fulfilled before a receptor binding and response system, or a pharmacological preparation, can usefully serve as a model system for defining a particular receptor subtype. Hartig suggested that these criteria should include the requirement that such systems be readily studied and easily reproduced, and that the response measured be shown to result from activation of a single homogeneous population of receptors. The response may be biochemical, physiological or electrophysiological, but it should be kept in mind that biologists have made most rapid advances when restricting themselves to simple, well-defined systems. He and Leff urged that receptor nomenclature should incorporate information derived from both functional and ligand binding studies to provide a single uniform and consistent scheme.

Referring to the classification of 5-HT receptors proposed by Bradley et al. (1986), several participants criticized the appellation of '5-HTrlike' receptor, and suggested that '5-HT1' receptor would be less cumbersome. However, there was no consensus on this point. Patrick Humphrey adamantly reminded everyone that the term '5-HT1-like' receptor had originally been chosen because a number of such receptors had been shown to be functionally important, and yet even today cannot be equated with any of the known 5-HT1 binding subsites in brain tissue. Although an all-embracing term has been used, the 5-HT1-like receptor group has clear commonality in that these receptors: (a) are resistant to blockade by 5-HT2 or 5-HT3 receptor antagonists; (b) can be activated potently and selectively by 5-carboxamidotryptamine (5-CT); and (c) are blocked potently by methiothepin. This group does not therefore merely represent a dumping ground for miscellaneous uncharacterizable receptors. However, Michael Gershon supported the concept expressed earlier by Leff that 5-HT receptors can essentially be defined on the basis of their affinity for 5-HT. He referred to the original binding studies of Peroutka and Snyder (1979), where two broad categories of 5-HT binding sites in rat brain had been identified, based on affinity. Thus 5-HT1 sites have high nanomolar affinity for [3H)-5-HT, whereas 5-HT2 sites have lower micromolar affinity.


Gershon indicated that these cortical5-HT 1 binding sites represent just one of several different varieties of 5-HT 1 receptor, and that subsites within this category can now be distinguished by the ability of a variety of specific compounds such as 8-hydroxy-2-( di-n-propylamino )tetralin (8-0H-DPAT) to displace [3H)-5-HT. Clearly, these agonists and antagonists can be used in functional studies to identify receptors which may be equivalent to each particular binding site. Gershon pointed out that using this criterion, the receptor identified by his group, first in the gut and later in heart, skin and lymphoid organs, which binds [3H]-5-HT with high affinity, should continue to be called a 5-HT1p receptor (subscript 1 = high affinity for 5-HTI> subscript P = peripheral). This view does not seem generally acceptable within our current understanding, and certainly the receptor cannot be called 5-HTrlike on the basis of the Bradley et al. (1986) criteria until it can be shown that it is potently stimulated by 5-CT and blocked by methiothepin. One major barrier to accepting the concept of the high affinity of 5-HT as the main criterion for classification of 5-HT 1 receptors is that the affinity of 5-HT for a number of 5-HTrlike receptors identified from functional studies remains to be determined. Furthermore, reliable measures of affinity of agonists are more difficult to obtain in functional studies compared with radioligand binding studies. In addition, the affinity of [3H]-5-HT at 5-HT3 binding sites appears intermediate between that at 5-HT 1 and 5-HT 2 binding sites (see Tyers et al., this volume; Kilpatrick et al., 1987). It remains to be seen whether the affinity of the natural ligand for the different receptor types really does fall into three distinct categories.

Future Approaches

It was agreed that the fundamental considerations on how receptors should be classified will, in the future, be decided by the main IUPHAR Nomenclature Committee, who will ultimately make recommendations, obviously with input from those of us who wish to contribute to the debate. Thus, a receptor might only be fully characterized by a complete understanding of: (a) the gene responsible for its synthesis; (b) its protein structure and sequence; and (c) its intracellular biochemical coupling, in addition to the more conventional measurement of (d) its binding characteristics determined from radioligand binding studies; and (e) its classical pharmacological characteristics from functional studies. It was felt that the Serotonin Club, through its Receptor Nomenclature Committee, and regular workshops such as this one, could provide the 'building blocks' for use in any overall scheme eventually to be recommended internationally by the IUPHAR Nomenclature Committee, which would probably involve all the above criteria.

212 Serotonin

UPDATE OF THE CURRENT CLASSIFICATION

A number of major new observations consistent with the Bradley eta/. (1986) classification of 5-HT receptors were described at the meeting and focused on in the discussion. The main points will be outlined here in relation to the three broad groups of 5-HT receptor (see Table 26.1).

5-HT .-like Receptors

There appear to be at the very least six subtypes of 5-HTrlike receptor, although it has been argued by one of us that on the basis of the evidence to date only four subtypes seem functionally important (Humphrey and Feniuk, 1987a). Despite the debate aboutthe term '5-HTrlike' (see above), it would still be preferred as the all-embracing term, at least for the time being, as two of the important subtypes, which occur in smooth muscle, do not have the characteristics of any of the 5-HT1 binding sites currently known (see Humphrey and Feniuk, 1987a). These two different 5-HTrlike receptors can now be subdivided more convincingly with the recent identification of AH 25086 and GR 43175. Thus, AH 25086 and GR 43175 are very selective agonists for the 5-HTrlike receptor which mediates contraction of smooth muscle in a restricted number of blood vessels, notably in the cerebral circulation (Humphrey et a/., 1987a, b, 1988; Humphrey and Feniuk, this volume). The same receptor also appears to mediate inhibition of transmitter from some peripheral nerves (Humphrey et al., 1988). All the published evidence shows that this receptor cannot be blocked by even high concentrations of cyanopindolol, mesulergine or metergoline (Charlton eta/., 1986; Humphrey eta/., 1988; Humphrey and Feniuk, this volume). It remains to be seen whether this receptor exists in the brain, despite the lack of evidence for an equivalent binding site. The exciting discovery of the selective agonists will allow this question to be explored.

The other 5-HTrlike receptor, which on the basis of current knowledge is not equatable with a 5-HT1 binding site, is the receptor which mediates vascular smooth muscle relaxation in a number of animal species (Feniuk et a/., 1983; Connor eta/., 1986; Dalton eta/., 1986). This receptor also appears to stimulate adenylate cyclase; evidence has recently been presented to show that, like the smooth-muscle relaxant response, the increase in adenosine 3' ,5'-monophosphate (cyclic AMP) can be potently effected by 5-CT, and this is antagonized potently and specifically by methiothepin and methysergide, but not by cyanopindolol, at the same concentrations which antagonize the concomitant relaxation (Sumner eta/., 1987).

There was general agreement that considerable data now confirm that the 5-HT1A binding site is a functional receptor. Ligand binding studies have


shown its widespread distribution in the brains of different species, including man (Hoyer et al., 1986a). Functionally, it mediates many of the hyperpolarizing actions of 5-HT on brain neurones (Martin and Mason, 1987), and also the stimulating (or inhibitory) actions of 5-HT and 5-Cf on adenylate cyclase (Shenker et al., 1985; Markstein et al., 1986). The 5-HT tA

receptor can be distinguished from the 5-HT1-like receptor stimulating adenylate cyclase in smooth muscle (described above) by the fact that at the former but not the latter, cyanopindolol is a potent antagonist, while spiperone is an antagonist at both (Humphrey and Feniuk, 1987a; Sumner et al., 1987).

The functional relevance of the other 5-HT 1 binding sites is still not clear. There is general agreement that the 5-HT18 site does equate with a functional receptor, and yet it seems to be restricted to rats and mice (Hoyer et al., 1986a; Holt et al., 1986). Interestingly, this receptor, like the GR43175-sensitive receptor in dog saphenous vein, mediates inhibition of transmitter release (Engel et al., 1983). It is tempting to speculate that the latter receptor is the 'non-rodent' equivalent of the 5-HT18 receptor (Humphrey et al., 1988).

The 5-HT1c binding site is found in the choroid plexus of various species, including man (Hoyer et al., 1986b), and recent studies have linked it to 5-HT activation of phosphatidylinositol turnover (Conn et al., 1986). Furthermore, recent elegant studies have led to the cloning of the receptor protein, with its subsequent incorporation into oocyte membranes, where it is linked to a Cl- channel (Hartig, this volume). Nevertheless, no gross functional response to 5-HT 1c activation has yet been identified, and thus it remains to be seen whether it is physiologically or pharmacologically relevant in vivo. Furthermore, it is not well characterized, as the drugs used lack selectivity, with most being potent ligands for 5-HT2 receptors.

The 5-HT10 binding site, first identified in bovine brain, is also poorly defined, but, as at all 5-HT1-like sites (except 5-HT1c and rat stomach fundus), 5-Cf has high affinity (or in the case of functional studies, is a potent agonist). The antagonist methiothepin also has high affinity for the 5-HT10 site (Heuring and Peroutka, 1987). However, there is no obvious functional correlation with the 5-HT10 site. Nevertheless, it has been shown that this site is widespread in the brain of various species, including man (Hoyer and Schoeffler, 1988). A recent report suggests that the 5-HT 10 site is negatively linked to adenylate cyclase in bovine brain (Hoyer and Schoeffler, 1988).1t remains to be seen of what importance, if any, this site is in the whole animal, although some investigations suggest that it may resemble the putative 5-HT 1-like receptors which mediate contraction in the rat stomach fundus and pre-synaptic sympathetic inhibition in the rat kidney (see Clarke eta/. and Peroutka, this volume).

A putative 5-HT 1-like receptor subtype of functional relevance is that on vascular endothelium. This receptor has been described by Cocks and

214 Serotonin

Angus (1983), and has been recently more fully pharmacologically characterized by Leff et al. (1987). This receptor mediates the release of endothelium-derived relaxing factor (EDRF) by 5-HT, and is not blocked by 5-HT2 and 5-HT3 receptor antagonists but is blocked by methiothepin. Nevertheless, this receptor, like the 5-HT receptor found in rat stomach fundus, cannot be convincingly called a 5-HTrlike receptor because of the relatively low potency of 5-Cf.

5-HT2 Receptors

It is clear that the 5-HT 2 receptor is perhaps the best characterized of all 5-HT receptor types, with broad agreement between data from ligand binding and functional pharmacological studies. Thus the receptor seems ubiquitous throughout the body, and its characteristics from functional studies in the periphery bear remarkable similarity to the characteristics of the brain ligand binding site (see Humphrey and Feniuk, 1987b for discussion on this topic). It was generally accepted at the workshop that Gaddum's 'D' receptor, which he first described in the guinea-pig ileum, is equatable with the 5-HT 2 receptor, allowing an easily recognized link with the current nomenclature and Gaddum's important but now outdated classification (see Humphrey and Feniuk, 1987b for references).

There was some discussion on possible subtypes of 5-HT2 receptor, although most were rather sceptical about the evidence considering the poor specificity of the antagonists used. There was a heated debate about the wide variation in pA2 values published for ketanserin from studies using a range of vascular smooth muscle preparations. Whether this represents poor experimentation in some cases, lack of attention to possible complicating factors in others, or, as strongly advocated by Leff (see Leff and Martin, 1986), that the antagonists are inherently unsuitable for classification of 5-HT receptors, remains to be established. The latter point gives way to the much wider argument, already discussed above, as to what we are attempting to characterize, namely, drug receptors or the actual receptor for the particular hormone or transmitter under study (see Leff et al., 1987). All agreed that unless we know the answer to this crucial question, further discussion about subtypes of the 5-HT2 receptor was semantic. Nevertheless, on the basis of pragmatism, one can observe that ketanserin is a very potent and relatively specific antagonist at 5-HT receptors which mediate a wide variety of actions, including smooth muscle contraction, platelet aggregation and neuronal depolarization. Ketanserin will not antagonize responses to 5-HT mediated via 5-HT 1-like or 5-HT 3 receptors, even at very high concentrations. Clearly, these observations lead to the general conclusion that 5-HT 2 receptors, which have broadly similar pharmacological characteristics and are identifiable with ketanserin, are widespread throughout the mammalian body.


5-HT3 Receptors

The 5-HT 3 receptor is now widely accepted as a neuronal receptor mediating afferent stimulation or efferent release of neurotransmitter. On isolated neuronal preparations, such as the rat or rabbit vagus, the selective 5-HT3 receptor agonist 2-methyl-5-HT produces neuronal depolarization. These responses can be easily quantified and shown to be antagonized by a variety of 5-HT3 receptor antagonists now available, including MDL 72222, ICS 205-930 and GR 38032F. However, until recently there was great scepticism about whether the 5-HT 3 receptor existed centrally, even though there were behavioural observations in both rats and primates for which there was no other obvious explanation (e.g. Costall et al., 1987). However, a major breakthrough was reported for the first time at this meeting concerning a novel [3H)-labelled 5-HT3 receptor antagonist GR 65630, which is suitable for use as a radioligand in binding studies. In such studies, clear evidence was provided that 5-HT3 binding sites exist in cortical and limbic terminal areas of rat brain (Kilpatrick et al., 1987). The distribution of these receptors appears consistent with the putative anxiolytic effects of 5-HT3 receptor antagonists (Tyers et al., this volume). On the basis of data obtained with a wide range of 5-HT 3 receptor antagonists of varying chemical structure, the brain 5-HT3 binding site appears remarkably similar to the 5-HT3 receptor mediating depolarization in the rat vagus nerve (Kilpatrick et al., 1987). The 5-HT3 receptor must now be seen as a very important entity which is relatively well defined pharmacologically, and which appears to mediate a whole variety of neuronal excitatory actions of 5-HT peripherally, and probably centrally. However, an electrophysiological response has yet to be ascribed to a 5-HT3 receptor-mediated response in the brain. Obviously, a lot more work is needed to complete a full understanding of the nature and biological significance of 5-HT3 receptors.

The question of subtypes of 5-HT 3 receptor was discussed in some detail. In the original 5-HT receptor classification of Bradley et al. (1986), it was proposed (probably incorrectly; see below) that the 'M' receptor of Gaddum in the guinea-pig ileum should be termed a 5-HT3 receptor on the basis that cocaine was an antagonist at this receptor as well as at other 5-HT 3 receptor sites, such as those on the sympathetic nerves innervating the rabbit heart. However, more potent and selective 5-HT3 receptor antagonists were subsequently showq to be relatively much weaker in antagonizing responses to 5-HT in guinea-pig ileum than in rabbit heart or the vagus nerve, and indeed MDL 72222 cannot be shown to antagonize specifically the neuronal responses to 5-HT in guinea-pig ileum at all (Fozard, 1984; Richardson et al., 1985). Attempts to determine pA2 values for the various antagonists have led to the claim that there are three subtypes of the 5-HT3 receptor (Richardson and Engel, 1986). However, there was general agreement that such claims cannot be substantiated on the basis of current evidence. Thus, in the rabbit heart and guinea-pig ileum, the response measured is the

216 Serotonin

end-organ response to neuronal release of transmitter(s) by 5-HT. In the guinea-pig ileum, the response to 5-HT is particularly complicated, because of the apparent presence of numerous other 5-HT receptors, mediating relaxation as well as contraction, with some on the smooth muscle and some ontheneurones(Feniuketa/., 1983;Engeleta/., 1984;Buchheiteta/., 1985; Gunning and Humphrey, 1987). In discussion, Gunter Engel presented data with several indole 5-HT3 receptor antagonists with different potencies on the various 5-HT3 receptor-containing preparations. However, it was agreed that antagonists had not yet been identified with the required specificity for one or other of the putative 5-HT3 receptor subtypes. Nevertheless, Engel felt that 5-HT3 receptor heterogeneity should be conceded but, at the same time, acknowledged that a satisfactory means of discriminating the sites is not yet available.

With regard to the 5-HT3 receptor populations in the guinea-pig ileum, they appear to be distributed on different types of neurone. Buchheit eta/. (1985) have suggested that they exist both on the cholinergic nerves and on substance P-containing nerves, and that 5-HT3 receptor activation causes release of both acetylcholine and substance P. The situation is further complicated, because substance P will itself activate cholinergic nerve terminals, and, in the contracted guinea-pig ileum, 5-HT will cause relaxation via a putative 5-HT3 receptor mechanism yet to be fully elucidated (Gunning and Humphrey, 1987). Enigmatically, the situation is even more complex because of the presence of 'M' receptors on cholinergic nerves, which, unlike the 5-HI'3 receptors also present, cannot be blocked by 5-HT3 antagonists such as ICS 205-930 (Buchheit et al., 1985). With the surfeit of 5-HT receptors in the guinea-pig ileum, many of which occur on neurones, it may be unrealistic to think of Gaddum's 'M' receptor, which was only characterized using morphine in whole ileum, as a single homogeneous pharmacological entity (but see below). What is clear, however, is that 5-HT 3 receptors, as defined by Bradley eta/. (1986), appear to occur on various neurone types throughout the body, but that at least one neurona15-HT receptor identified in guinea-pig ileum is not of the 5-HT3 type (on the basis of the lack of blockade by ICS 205-930) and cannot -be characterized in terms of the Bradley et al. (1986) classification as it stands at present.

OBSERVATIONS INCONSISTENT WITH THE PRESENT CLASSIFICATION

Several 5-HT receptors which cannot be obviously incorporated into the Bradley eta/. (1986) classification were discussed in some detail. It is clear that Gershon and Branchek's work, on the 5-HT receptor that mediates slow excitatory post-synaptic potentials in Type ll/AH neurones in the guinea-pig ileum (Gershon eta/., this volume), needs to be understood in relation to the


current classification. It was appreciated that this receptor has been classified on the basis of radioligand binding and electrophysiological studies, but its presence cannot be demonstrated in studies of contraction in the whole ileum (see below). However, Gershon pointed out that well over 95 per cent of the neurones in the bowel do not 'talk to' the muscle, but to one another, so that when the contraction of a whole intestinal preparation in an organ bath is recorded, it is the resultant of a very complex response. Clearly, the definitive characterization of 5-HT receptors in gastrointestinal preparations such as the guinea-pig ileum and rat stomach fundus using simple pharmacological techniques may be overambitious.

Another 5-HT receptor type which defies classification is a putative 5-HT receptor which appears to mediate the tachycardia induced by 5-HT in the anaesthetized pig. In a very comprehensive study, Pramod Saxena has been unable to antagonize the response to 5-HT with a whole variety of antagonists, thereby excluding the possible involvement of 5-HT .-like, 5-HT2 or 5-HT3 receptors (Saxena, this volume). Although 5-CTcan mimic the effect of 5-HT, it is not very potent or effective. It was suggested that this receptor is similar or identical to the high-affinity 5-HT receptor in the guinea-pig ileum described by Gershon above, and that the effects of the 5-hydroxytryptophan dipeptide antagonist and the agonist 6-hydroxyindalpine should be examined.

John Fozard explained why the original 'M' receptor described by Gaddum and Picarelli in the guinea-pig ileum which mediates acetylcholine release does not fit into the Bradley et al. (1986) classification scheme. He pointed out that Gaddum's work on the 'M' receptor provided evidence that it mediated release of acetylcholine from enteric cholinergic neurones at concentrations of 5-HT up to 0.2 J.I.M. In contrast, Buchheit et al. (1985) showed that it is only when one uses concentrations greater than 0.2 J.I.M that a second-phase contraction occurs. The latter is also a neuronal stimulant effect, but seems to be mediated by a transmitter other than acetylcholine, perhaps substance P (which by definition cannot be Gaddum's 'M' receptor). Unlike the second-phase contraction, which involves a 5-HT3 receptor, the 'M' receptor-mediated response is not antagonized by ICS 205-930 (Buchheit et al., 1985). Furthermore, the 'M' receptor is not blocked by ketanserin, methysergide or methiothepin, and the agonists S( +)-amethyl-5-HT, 2-methyl-5-HT and 5-CT are all extremely weakly active at this receptor. Fozard went on to say that it was unlikely that the 'M' receptor is the same as the high-affinity 5-HT receptor (5-HT1p) described by Gershon in the guinea-pig ileum, since some benzamides such as cisapride, metoclopramide and BRL 24924 act as agonists at the 'M' receptor, but are inactive on Type IIlAH neurones. Moreover, 6-hydroxyindalpine does not produce cholinergic-dependent contractions in the guinea-pig ileum; nor does 5-hydroxytryptophan dipeptide block this response to 5-HT (Buchheit, unpublished observations).

David Wallis wondered whether the Bradley et al. (1986) classification

218 Serotonin

was comprehensive enough to incorporate receptors characterized on the basis of electrophysiological data. Thus he referred not only to the data of Gershon and Branchek but also to some of his own unpublished data in which 5-HT-mediated depolarization of an isolated hemisected spinal cord preparation could not be antagonized by the antagonists he had tested. Manfred Gothert also mentioned his studies in pig coronary arteries, where he had studied the 5-HT receptor mediating inhibition of transmitter release from the sympathetic nerves. He found 5-HT to be a very potent agonist in the nanomolar range, and yet none of the antagonists tested, including methiothepin, ketanserin or yohimbine, was effective. Clearly, efforts need to be made to characterize these receptors further, and, if novel, to see if evidence can be provided for their presence elsewhere.

Nevertheless, despite the exceptions described here, the vast majority of responses mediated by 5-HT can still be assigned to one of the three main receptor types defined by Bradley et al. (1986}. The strength of the classification is that it is supported by data from studies with both agonists and antagonists. Unquestionably, the scheme has proven practical value, and although synoptic revision willclearly be necessary in time, this cannot occur until better pharmacological tools become available. Thus, for example, the present lack of specific antagonists for the putative subtypes of ·s~HT1-like' and 5-HT3 receptors prevents their definitive characterization and hence classification. It was agreed that in such circumstances, no attempt should be made to provide names for putative novelS-HT receptors or their subtypes.

GENERAL CONCLUSION

While it was generally agreed that neurotransmitter receptors may well ultimately be classified according to their amino acid sequences, improved fingerprints of these molecules can already be constructed by integrating information provided by reliable pharmacological, biochemical, electrophysiological and ligand binding studies. However, in all cases it is essential to obtain quantitative data with selective agonists and antagonists under established, reproducible conditions using generally accepted criteria, in systems for which the response measured can be shown to result from the interaction of 5-HT with a single homogeneous population of receptors. If such information cannot be provided, the naming and classification of a particular receptor would be invalid. In time, this list of requirements will undoubtedly be extended to include details of the molecular structure of the receptor. This implies that additions and modifications to the scheme proposed by Bradley et al. (1986} are required, but there was no strong advocate for an entirely new approach at the present time.


ACKNOWLEDGEMENTS

We would like to thank all the participants who contributed to this most stimulating workshop, and in particular Professor David Clarke, Dr Paul Leff, Dr Paul Hartig and Professor Michael Gershon, who provided their written comments to assist in the preparation of this manuscript.

FOOTNOTE

The following members of the Serotonin Club have agreed to serve on the Club's Receptor Nomenclature Committee: Dr G. K. Aghajanian, Professor P. B. Bradley (Liaison with IUPHAR), Dr M. L. Cohen, Dr J. R. Fozard, Professor J. P. Green (Vice-chairman), Dr P. P. A. Humphrey (Chairman), Dr J. E. Leysen, Dr E. J. Mylecharane, DrS. J. Peroutka, Dr B. P. Richardson and Professor P. R. Saxena.

REFERENCES


Buchheit, K. H., Engel, G., Mutschler, E. and Richardson, B. P. (1985). Study of the contractile effect of 5-hydroxytryptamine (5-HT) in the isolated longitudinal muscle strip from guinea-pig ileum. Evidence for two distinct release mechanisms. Naunyn-Schmiedeberg's Arch. Pharmacol., 329, 36-41

Charlton, K. G., Bond, R. A. and Clarke, D. E. (1986). Aninhibitoryprejunctional 5-HT 1-like receptor in the isolated perfused rat kidney. Apparent distinction from the 5-HT1A, 5-HTIB and 5-HT1c subtypes. Naunyn-Schmiedeberg's Arch. Pharmacol., 332, 8-15

Cocks, T. M. and Angus, J. A (1983). Endothelium-dependent relaxation of coronary arteries by noradrenaline and serotonin. Nature, 305, 627-630

Conn, P. J., Sanders-Bush, E., Hoffmann, B. J. and Hartig, P.R. (1986). A unique serotonin receptor in choroid plexus is linked to phosphatidylinositol turnover. Proc. Nat/. Acad. Sci. U.S.A., 83, 4086-4088

Connor, H. E., Feniuk, W., Humphrey, P. P. A. and Perren, M. J. (1986). 5-Carboxamidotryptamine is a selective agonist at 5-hydroxytryptamine receptors mediating vasodilatation and tachycardia in anaesthetized cats. Br. J. Pharmacol., 87, 417-426

Costall, B., Domeney, A.M., Naylor, R. J. and Tyers, M. B. (1987). Effects of the 5-HT3 receptor antagonist, GR 38032F, on raised dopaminergic activity in the meso limbic system of the rat and marmoset brain. Br. J. Pharmacol., 92, 881-894

Dalton, D. W., Feniuk, W. and Humphrey, P. P. A. (1986). An investigation into the mechanisms of the cardiovascular effects of 5-hydroxytryptamine in conscious normotensive and doca-salt hypertensive rats. J. Auton. Pharmacol., 6, 219-228

220 Serotonin

Engel, G., Gothert, M., Muller-Schweinitzer, E., Schlicker, E., Sistonen, L. and Stadler, P. A. (1983). Evidence for common pharmacological properties of [3H)5-hydroxytryptamine binding sites, presynaptic 5-hydroxytryptamine autoreceptors in CNS and inhibitory presynaptic 5-hydroxytryptamine receptors on sympathetic nerves. Naunyn-Schmiedeberg's Arch. Pharmacol., 324, 116-124

Engel, G., Hoyer, D., Kalkman, H. 0. and Wick, M. B. (1984). Identification of 5-IIT2 receptors on longitudinal muscle ofthe guinea pig ileum. J. Recep. Res., 4, 113-126


Fozard, J. R. (1984). MDL 72222, a potent and highly selective antagonist at neuronal5-hydroxytryptamine receptors. Naunyn-Schmiedeberg's Arch. Pharmacol., 326, 35-44

Gunning, S. J. and Humphrey, P. P. A. (1987). Evidence for 5-IIT3-receptor mediated release of an inhibitory transmitter in guinea-pig isolated ileum. Br. J. Pharmacol., 91, 359P

Heuring, R. E. and Peroutka, S. J.(1987). Characterization of a novel Ju-S-hydroxytryptamine binding site in bovine brain membranes. J. Neurosci., 7, 894-903

Holt, S. E., Cooper, M. and Wyllie, J. H. (1986). On the nature of the receptor mediating the action of 5-hydroxytryptamine in potentiating responses of the mouse urinary bladder strip to electrical stimulation. Naunyn-Schmiedeberg's Arch. Pharmacol., 334, 333-340

Hoyer, D. and Schoeffter, P. (1988). 5-IIT10 receptor-mediated inhibition of forskolin-stimulated adenylate cyclase activity in calf substantia nigra. Eur. J. Pharmacol., 147, 145-147

Hoyer, D., Pazos, A., Probst, A. and Palacios, J. M. (1986a). Serotonin receptors in the human brain. I. Characterization and autoradiographic localization of 5-IIT IA recognition sites. Apparent absence of 5-IIT 18 recognition sites. Brain Res., 376, 85-96

Hoyer, D., Pazos, A., Probst, A. and Palacios, J. M. (1986b ). Serotonin receptors in the human brain. II. Characte~ation and autoradiographic localization of 5-IIT1c and 5-HT2 recognition sites. Brain Res., 376, 97-107

Humphrey, P. P. A. and Feniuk, W. (1987a). Classification of functional 5-hydroxytryptamine receptors. In Rand, M. J. and Raper, C. (Eds), Pharmacology, Proceedings of the Xth International Congress of Pharmacology, Sydney, Elsevier, Amsterdam, pp. 277-280

Humphrey, P. P.·A. and Feniuk, W. (1987b). Pharmacological characterisation of functional neuronal receptors for 5-hydroxytryptamine. In Nobin, A., Owman, Ch. and Arneklo-Nobin, B. (Eds), Neuronal Messengers in Vascular Function, Fernstrom Foundation Series, Vol. 10, Elsevier, Amsterdam, pp. 3-19

Humphrey, P. P. A., Feniuk, W., Perren, M. J., Oxford, A. W., Brittain, R. T. and Jack, D. (1987a). The pharmacology of selective 5-IITrlike receptor agonists for the acute treatment of migraine. Cephalalgia, 7, 400--401

Humphrey, P. P. A., Feniuk, W., Perren, M. J., Oxford, A. W., Coates, I. H., Butina, D., Brittain, R. T. and Jack, D. (1987b). GR43175- A selective agonist for functional S-liT 1-like receptors in dog saphenous vein. Br. J. Pharmacol., 92, 616P

Humphrey, P. P. A., Feniuk, W., Perren, M. J., Connor, H., Oxford, A. W., Coates, I. H. and Butina, D. (1988). GR 43175, a selective agonist for the 5-IITrlike receptor in dog isolated saphenous vein. Br. J. Pharmacol., 94, 1123-1132


Kilpatrick, G. J., Jones, B. J. and Tyers, M. B. (1987). Identification and distribution of 5·Hf3 receptors in rat brain using radioligand binding. Nature, 330, 746-748

Leff, P. and Martin, G. R. (1986). Peripheral 5-Hf2-like receptors. Can they be classified with the available antagonists? Br. J. Pharmacol., 88, 585-593

Leff, P., Martin, G.R. and Morse, J. M. (1987). Differential classification of vascular smooth muscle and endothelial cell5-HT receptors by use of tryptamine analogues. Br. J. Pharmacol., 91, 321-331

Markstein, R., Hoyer, D. and Engel, G. (1986). 5-Hf1A receptors mediate stimulation of adenylate cyclase in rat hippocampus. Naunyn-Schmiedeberg's Arch. Pharmacol., 333, 335-341

Martin, K. F. and Mason, R. (1987). Ipsapirone is a partial agonist at 5-hydroxytryptamine 1A (5-HT1A) receptors in the rat hippocampus: electrophysiological evidence. Eur. J. Pharmacol., 141, 479--483

Peroutka, S. J. and Snyder, S. H. (1979). Multiple serotonin receptors: differential binding of [3H)5-hydroxytryptamine, [3H)lysergic acid diethylamide and [3H)spiroperidol. Mol. Pharmacol., 16, 687-699

Richardson, B. P. and Engel, G. (1986). The pharmacology and function of5-HT3 receptors. Trends in Neurosci., 9, 424-428

Richardson, B. P. , Engel, G. , Donatsch, P. and Stadler, P. A. ( 1985). Identification of serotonin M-receptor subtypes and their specific blockade by a new class of drugs. Nature, 316, 126-131

Shenker, A., Maayani, S., Weinstein, H. and Green, J. P. (1985). Two 5-HT receptors linked to adenylate cyclase in guinea-pig hippocampus are discriminated by 5-carboxyamidotryptamine and spiperone. Eur. J. Pharmacol., 109, 427-429

Stephenson, R. P. (1975). Interaction of agonists and antagonists with their receptors. INSERM, 50, 15-28

Sumner, M. J., Humphrey, P. P. A. and Feniuk, W. (1987). Characterisation of the 5-Hf 1-like receptor mediating relaxation of porcine vena cava. Br. J. Pharmacol., 92, 574P

PART VI PATHOPHYSIOLOGY

27 Vascular Actions of Serotonin in Large and

Small Arteries are Amplified by Loss of Endothelium, Atheroma and Hypertension

J. A. Angus, C. E. Wright and T. M. Cocks

Baker Medical Research Institute, PO Box 348, Prahran Vic 3181, Australia

INTRODUCTION

Free serotonin (5-hydroxytryptamine; 5-HT) levels in blood are kept low because platelets avidly trap the autacoid released from neuronal or enterochromaffin cells. If platelets aggregate and release serotonin at sites of atheroma or endothelium denudation, the artery wall will generally constrict. The discovery that endothelial cells can release endotheliumderived relaxing factor (EDRF), a powerful relaxant now known to be NO (Palmer et al., 1987), has stimulated much work in trying to unravel the complex interactions that can occur for serotonin within the artery wall in large and small vessels.

This brief review describes studies in our laboratory aimed at determining what changes may occur to the reactivity to serotonin in the coronary artery (a) when there is an acute loss of endothelium, and (b) if the arteries are in an atheromatous or atherosclerotic-like state. In addition, the effect of hypertension and medial hypertrophy on the reactivity of serotonin in the rabbit hindquarter was tested. Here, paradoxically, serotonin is a vasodilator of this intact vascular bed, presumably at the level of the small resistance vessels.

METHODS

Coronary Arteries

Large coronary arteries were removed from mongrel dog, greyhound dog and pig hearts, and cut into 3 mm long ring segments for isometric force

226 Serotonin

recording in isolated organ baths (Cocks and Angus, 1983). Arteries were stretched passively to 4 g and readjusted again 30 min later. Cumulative concentration-response curves were obtained for serotonin in the presence or absence of endothelium, which was removed by rubbing the intima with a filter paper taper (Cocks and Angus, 1983). Arteries were also pre-contracted with K+ (20-40 mM) or with the thromboxane Az-mimetic U-46619 (1~30 nM).

Rabbits were fed a normal pellet diet (control), or one supplemented with 1 per cent cholesterol for 16-20 weeks prior to experiment, resulting in the production of atheromatous lesions in the coronary vessels. Hearts were removed and 2 mm long segments of large (8~ 1300 1.1.m internal diameter) or small (15~350 fJ.m internal diameter) coronary arteries were suspended on 40 fJ.ffi stainless-steel wires in a Mulvany double myograph for force recording. These vessels were normalized by setting the passive stretch according to a routine method from the length-tension relationship (Mulvany and Halpern, 1976).

Hindquarter Reactivity

Rabbits were instrumented with a chronic indwelling catheter in the lower abdominal aorta just proximal to a pulsed Doppler flow transducer at the iliac bifurcation. The kidneys were left undisturbed (sham) or wrapped in cellophane (wrap) to induce perinephritis, cortical compression and hypertension over the next 5 weeks. On the day of the experiment, the rabbit was given mecamylamine (10 mglkg i.v.) to block autonomic reflexes, before infusing serotonin via the aortic catheter and measuring hindquarter resistance as the ratio: ear artery blood pressure/hindquarter limb flow (Wright et al., 1987).

RESULTS

Acute Loss of Endothelium

In the absence of active tone, dog and pig large coronary arteries contracted rather poorly to serotonin. When endothelium was removed, however, the concentration-response curves were markedly enhanced in range (up to 3-fold), and increased in sensitivity in terms oflower EC50 values (Cocks and Angus, 1983; and see Figure 27.1). An explanation was that serotonin has a dual action, to release EDRF and to contract the smooth muscle. Removal of the endothelium would leave the contractile response unopposed. In these experiments, serotonin was delivered to the bathing fluid, i.e. to both the lumen and adventitial surfaces. With endothelium-intact rings, EDRF

Vascular Pathology and Reactivity to Serotonin 227

release was sufficient to attenuate the smooth-muscle contraction, indicating the important modulating influence of this factor. More direct evidence that EDRF was released by serotonin came from experiments where tone was raised by the thromboxane Armimetic U-46619. With endothelium intact, serotonin only poorly relaxed the rings, but the addition of ketanserin uncovered a powerful relaxation to serotonin that was abolished by endothelium removal (Cocks and Angus, 1983; and see Figure 27.1). Similarly, aggregating platelets induced an endothelium-dependent

endothelium

smooth muscle

tone: low

Q) 0 ~

0 -5-HT

5-HT -like 1

EDRF

5-HT 2

high

5-HT

-E

+E

5-HT Figure 27.1 Schema illustrating the proposed serotonin receptor populations on the dog coronary artery. 5-HT 1-like receptors on endothelium mediate the release of EDRF and relaxation of smooth muscle cells. 5-HT2 receptors on smooth muscle cells mediate contraction. The lower graphs summarize the pattern of concentration-response curves for serotonin ( 5-HT) in the absence (left) or presence (right) of active tone induced by U-46619. +E with, -E without endothelium. To demonstrate relaxation (on the right, +E), ketanserin is often necessary to

antagonize 5-HT2 receptors

228 Serotonin

relaxation, a response that was antagonized by methysergide but not by ketanserin (Cohen et al., 1983). These endothelial 5-HT receptors are clearly distinguished from the 5-HT2 receptors mediating smooth-muscle contraction. Recently, receptors for serotonin on endothelium have been loosely classified as 5-HTrlike on the basis of agonist activities of tryptamine analogues in rabbit jugular vein (Leff et al., 1987). This subtype is considered to be different from the 5-HT1-like receptors reported to mediate contraction of dog saphenous vein or direct relaxation of vascular and intestinal smooth muscle (Feniuk et al., 1983, 1985; Humphi"ey and Feniuk, this volume).

Atheroma and Serotonin

Large coronary artery segments (901 ± 74 f..tm internal diameter, mean± s.e. mean) removed from a group of rabbits (n = 8) on normal diet contracted poorly to cumulative concentrations of serotonin (0.01-3 f..tM), as illustrated in Figure 27.2. The increase in force was less than 16 per cent of the maximum contraction to K+ 50 mM depolarizing solution (5.8 mN/mm segment length). In contrast, coronary segments (1113 ± 84 f..tm internal diameter) removed from rabbits fed on the 1 per cent cholesterolsupplemented diet (n = 10) contracted to 51.4 per cent of the maximum K+ response (3.9 mN/mm segment length), as shown in Figure 27.2. The sensitivity (EC50) to serotonin was not enhanced in atheroma. Enhancement of contraction in atheromatous vessels was not observed for histamine- or K+ -induced contractions. For these latter agents, the range was significantly decreased. Small coronary arteries (250---300 f..tm internal diameter) from rabbits on either diet did not contract to serotonin.

A role for EDRF could explain the responses to serotonin. In normal arteries, acetylcholine and substance P relaxed the vessels, the former having a dual action of relaxation and contraction at higher concentration (Figure 27.2). In atheromatous vessels, where the average lesion-free internal circumference was reduced to only 26 per cent, substance P relaxation responses were initially absent, while acetylcholine only caused further contraction (Figure 27.2). These studies suggest that large coronary arteries of the rabbit contract more forcefully for a given concentration of serotonin in the presence of atheroma; a loss of EDRF activity because of poor penetration through the lipid-filled thickened intima or inactivation by 02 radicals could increase the contraction to serotonin. This hypothesis assumes that rabbit coronary endothelial cells release EDRF in response to serotonin, a point where direct evidence still remains to be found.

Serotonin in Intact Vascular Beds

Serotonin i.v. infusions in conscious rabbits with ganglion blockade caused


diverse responses in the vasculature: dilatation in the hindquarter bed but simultaneously renal artery spasm (Wright and Angus, 1987). Clearly, serotonin has powerful constrictor actions unrelated to the autonomic nervous system in some larger arteries, but relaxes the resistance vessels. By giving infusions to steady-state via a chronic aortic catheter, the sensitivity (EC50) and range of the dose--dilatation curve for serotonin in the rabbit hindquarter were able to be measured. Perinephritic hypertension caused a marked increase in resting resistance from 16.9 units in 7 sham rabbits (mean blood pressure 79 mmHg) to 29.9 units in 5 hypertensive rabbits (mean blood pressure 125 mmHg). After ketanserin (0.5 mglkg i.v.), given to

z E

16

CP 8 ()

0 1&.

0

12

z 8 E II ~ 0

1&. 4

0

Normal diet 878um

ACH

Cholesterol diet 1145um

ACH

A K+30mM

5-HT

.. ~6 8 7

ash

Figure 27.2 Chart records from the myograph of isolated large coronary arteries from rabbits fed a normal diet (top) or a 1 per cent cholesterol-supplemented diet for 16 weeks (bottom). Acetylcholine (ACH) caused relaxation and contraction in normal rings but only contraction in the atheromatous artery. Serotonin ( 5-HT) was a powerful constrictor in the atheromatous vessel. Concentrations are -log M at half-log unit increments. Artery lumen internal diameters: normal diet, 878 J.l.m;

cholesterol diet, 1145 11m

230 Serotonin

antagonize 5-HT2 receptors, serotonin dose-response curves were similar in sensitivity (ED50, log10 (nglkg per min), mean ± s.e. mean: sham 4.06 ± 0.04; hypertensive 3.92 ± 0.08), while the range (d resistance for the full curve, mean± s.e. mean) was 7.0 ± 1.3 and 12.2 ± 2.3 resistance units for the sham and hypertensive rabbits, respectively (Figure 27.3) . Changes in range but similar sensitivity were also found for acetylcholine and adenosine. These results are consistent with the non-agonist specific amplifier action of medial hypertrophy in the resistance arteries of hypertensive rabbits (Wright et al., 1987).

DISCUSSION

Abnormal reactlVlty of blood vessels, especially spasm, captures the imagination of clinical and basic scientists. Serotonin is a potential candidate

?P. CQ CD ... CQ

c CD E .2 CD CD ... u..

Rabbit Coronary Artery

100 o ••nnal

0 50

@ atheroma

....... 0 8 7

5-HT -logM

6

In -·c: = CD (.) c CQ -r/1 ·u; CD a:

Rabbit Hindquarter Vascular Bed

20 •

10 • hypertensive

... R 0

5 - HT i.a.

Figure 27.3 Theoretical patterns of serotonin concentration-response curves for lumen cross-sectional area in a normal artery and in an atheromatous coronary artery (left), and for hindquarter resistance in a hypertensive rabbit with medial hypertrophy compared with a normotensive control (right). Left: at rest, lumen cross-sectional area is severely reduced by the atheroma, and serotonin-induced constriction obliterates the lumen. Right: the hypertensive vascular bed with higher resting (R) resistance appears to be more reactive to the vasodilator action of serotonin (greater fall in resistance per dose of serotonin), but the effects may be fully accounted for by medial hypertrophy in comparison with the normotensive

rabbit. The arrows indicate the similar ED50 values


for inducing vasospasm given its source in platelets and its powerful constrictor action on many arteries in crucial locations. Our studies have explored the ways by which changes to the architecture of the artery wall can alter the integrated response of the smooth muscle to serotonin. EDRF decreases contractility; it appears that atheromatous intimal thickening increases the contractility of the smooth muscle cells in response to serotonin, perhaps because ofthe loss ofEDRF. With atheroma, the intimal thickening has another function: that of luminal encroachment. This reduction in lumen cross-sectional area would not alter measurements of isometric force in the myograph but would be important in the determination of blood flow in vivo. Resistance (R) to blood flow increases dramatically as the lumen radius (r) falls (R ex: Vr4). Thus, given that the muscle cells contract more strongly to serotonin (i.e. shorten further), and in an environment of reduced lumen diameter with atheroma, the possibility of enhancement of serotonin reactivity arises, perhaps leading to spasm and zero lumen diameter (Figure 27.3).

Hypertension is associated with medial hypertrophy, and luminal encroachment again as the wall thickness/lumen radius ratio increases. Thus, at rest, the hindquarter resistance was higher in the hypertensive rabbits. Any vasodilator substance will cause a greater fall in resistance for a given concentration because equivalent increases in artery diameter result in greater changes in resistance for hypertensive vessels compared with control arteries (Figure 27.3). This 'amplification' of the vasodilatation response to serotonin simply follows a structural alteration of the vasculature. There is no need to invoke any receptor-specific changes for serotonin.

In conclusion, this work emphasizes that changes in the reactivity of arteries to vasoactive substances like serotonin may involve the architecture of the artery wall and be relatively specific for serotonin if EDRF release is normally involved. Atheroma and medial hypertrophy may simply represent additional 'amplifiers' that are not specific to particular constrictor or dilator agents.

ACKNOWLEDGEMENTS

This work was supported by an Institute Grant from the National Health and Medical Research Council of Australia. We thank Peter Coles and Clara Chan for assistance with the manuscript preparation.

REFERENCES

Cocks, T. M. and Angus, J. A. (1983). Endothelium-dependent relaxation of coronary arteries by noradrenaline and serotonin. Nature, 305, 627-630

232 Serotonin



Feniuk, W., Humphrey, P. P. A. and Watts, A. D. (1985). A comparison of 5-hydroxytryptamine receptors mediating contraction in rabbit aorta and dog saphenous vein: evidence for different receptor types obtained by use of selective agonists and antagonists. Br. J. Pharmacol., 86, 697-704

Leff, P., Martin, G. R. and Morse, J. M. (1987). Differential classification of vascular smooth muscle and endothelial cell5-HT receptors by use of tryptamine analogues. Br. J. Pharmacol., 91, 321-331

Mulvany, M. J. and Halpern, W. (1976). Mechanical properties of vascular smooth muscle cells in situ. Nature, 260, 617--619

Palmer, R. M. J., Ferrige, A. G. and Moncada, S. (1987). Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature, 327, 524--526

Wright, C. E. and Angus, J. A. (1987). Diverse vascular responses to serotonin in the conscious rabbit: effects of serotonin antagonists on renal artery spasm. J. Cardiovasc. Pharmacol., 10, 415-423

Wright, C. E., Angus, J. A. and Komer, P.l. (1987). Vascular amplifier properties in renovascular hypertension in conscious rabbits. Hindquarter responses to constrictor and dilator stimuli. Hypertension, 9, 122-131

28 Sympathetic Nerves Associated with Brain Vessels Store and Release Serotonin which

Interacts with Noradrenaline in Cerebrovascular Contraction

J. E. Hardebo, J.-Y. Chang and Ch. Ow man

Department of Medical Cell Research, University of Lund, Biskopsgatan 5, S-223 62 Lund, Sweden

INTRODUCTION

Using the formaldehyde histofluorescence method, it was demonstrated some 20 years ago in our laboratory that the blood vessels of the brain are innervated by an extensive system of sympathetic nerves originating in the superior cervical ganglia (for references, see Owman, 1986). With the introduction of immunohistochemical techniques, whereby not only neuropeptides but also transmitter amines such as 5-hydroxytryptamine (5-HT) can be visualized microscopically, it has recently been shown that brain vessels of various species, including man, are supplied with fibres containing this amine (Griffith et al., 1982; Edvinsson et al., 1983; Griffith and Bumstock, 1983). Previously utilized histochemical methods (e.g. the formaldehyde technique) were obviously not sufficiently sensitive for microscopic visualization of the relatively low levels of 5-HT stored in perivascular nerve fibres. This finding has attracted considerable interest because 5-HT is a prominent vasoconstrictor substance in the brain circulation, and has been implicated as one pathophysiological factor in serious clinical disorders such as vasospasm and migraine.

The close similarity of the distribution pattern of the 5-HT-containing nerve fibres to that of the noradrenergic nerve plexus was intriguing; therefore, a series of studies (primarily on rat and guinea-pig) were carried out in our laboratory, to elucidate the possible sympathetic origin of the immunofluorescent perivascular nerve fibres (Chang and Owman, 1986). Attempts have also been made to characterize the post-junctional 5-HT

234 Serotonin

receptors which mediate the action of neuronally released 5-HT, and to determine the functional importance of a putative dual release of noradrenaline and 5-HT.

IMMUNOHISTOCHEMICAL STUDIES ON GUINEA-PIG VESSELS

Our experiments confirmed that the distribution of the 5-HT-containing fibres around guinea-pig pial arteries at the base of the brain corresponded well with that of the well-established noradrenergic plexus. In order to allow for a more detailed analysis of the distribution patterns, dopamine-~hydroxylase (DBH, a specific enzyme in the synthesis of noradrenaline) was used as a more sensitive marker for noradrenergic nerve fibres. In the sequential immunohistochemical reaction, the 5-HT antiserum was first applied and the vessel photographed, followed by elution with acid KMn04 ,

subsequent application of the DBH antiserum, and rephotography. The neural localization of the two antisera was found to correspond in all details (Figure 28.1), and was particularly evident when the distribution of single immunofluorescent varicosities along the nerve terminal was compared (Chang et al., 1988). In order to exclude any cross-reaction between the antisera, they were applied to sympathetically innervated peripheral structures, such as atria and vas deferens. In these tissues, an extensive system of DBH-containing sympathetic nerves could be demonstrated in the absence of any immunoreactivity for 5-HT.

The sympathetic origin of the 5-HT-containing perivascular nerves in the brain was able to be confirmed by the complete disappearance of

Figure 28.1 Immunofluorescence in a guinea-pig pial artery with 5-HT antibodies, washing with H2S04 and KMn04, followed by application of DBH antiserum. The nerve plexuses are identical, which is particularly well seen at the arrows, where

single varicosities visualized with the two antisera appear identical (x250)

Serotonin in Cerebrovascular Sympathetic Nerves 235

immunoreactivity following excision of the superior cervical ganglia (Chang et al., 1988). However, no 5-HT immunoreactive neurones could be demonstrated within the ganglia, which instead harboured groups of small, chromaffin-like cells storing 5-HT. The results indicated that 5-HT was primarily located in the distal portion of the nerves, and was perhaps not even synthesized within the neurones.

IMMUNOHISTOCHEMICAL STUDIES ON RAT VESSELS

In studies on rat pial vessels (Chang et al., 1989), which seem to contain lower levels of 5-HT, it was found that the 5-HT is avidly taken up into perivascular sympathetic fibres (distinguished by DBH immunofluorescence and sympathetic denervation) when incubated in concentrations as low as 1 nM. The ability of the nerves to take up and store 5-HT was further elucidated by administration of selective neurotoxic agents. Thus, intraventricular administration of 5,6-dihydroxytryptamine (200 ~tg), which is sufficient to eliminate intracerebral serotoninergic nerves, had no effect on the 5-HT content of the perivascular sympathetic nerves, whereas 250 11g of 6-hydroxydopamine, which selectively destroys catecholaminergic fibres, completely abolished the 5-HT immunofluorescence. This would indicate that the fibres demonstrable by immunohistochemistry in the large pial vessels are not truly 'serotoninergic', but rather reflect uptake and storage of 5-HT in adrenergic nerve fibres, where it may act as a false co-transmitter. This possibility was tested in release experiments using pial arteries from rats.

Preparations of pial vessels were incubated in a modified Krebs-Ringer buffer solution containing 3 nM [3H)-5-HT, together with nialamide in order to inhibit deamination of 5-HT. In some experiments, the preparations were pre-incubated with cocaine to block axonal uptake, or the vessels were denervated beforehand through surgical ganglionectomy to eliminate the perivascular sympathetic fibres. It was found that not only K+ depolarization but also tyramine, which is taken up into the sympathetic nerves and displaces the stored amine, markedly increased the efflux of radioactivity compared with the basal level of outflow. This effect was eliminated by cocaine pre-treatment or sympathetic denervation, whereby the neuronal uptake of eHJ-5-HT was reduced or eliminated. A similar marked release by more than 100 per cent above base-line levels was obtained by electrical activation of the nerves through field stimulation (Figure 28.2). Also, the electrically induced release was eliminated by cocaine pre-treatment or by previous surgical sympathectomy. The results show that 5-HT, taken up into the cerebrovascular sympathetic nerves by an efficient axonal mechanism (Chang et al., 1989), can be released by pharmacological or electrical activation of the nerve fibres.

236 Serotonin

CHARACTERIZATION OF POST-JUNCTIONAL 5-HT RECEPTORS

The post-junctional receptors mediating contraction of pial arteries in rat (Chang and Owman, 1989) and monkey (Chang et al., 1987) are ofthe 5-HT 2 type, as evidenced by the competitive inhibition produced by ketanserin (Figures 28.3(a) and (b) ). The affinity for the antagonist is high, with pA2 values in the order 9.15-9.40.

Experiments on basilar arteries from guinea-pig (Chang and Owman, 1989) suggested that the contractile 5-HT receptor was different in this species. The sensitivity to 5-HT was higher, but the intrinsic activity lower, in guinea-pig than in rat basilar artery. The contractile potency of the 5-HT 1

agonist, 5-carboxamidotryptamine, was 4 times higher than that of 5-HT in guinea-pig, but 16 times lower in rat vessels. In the guinea-pig, ketanserin at

cpm% 300 -

200

100 -

0

12

7

+d-*

C Co SyX

51

6

J ..1.. 6 ~··~ **

C Co SyX TTX

52

4

r-

7 3 4 +.-1-L

r;-;- * *

C Co SyX TTX

sa

Figure 28.2 Fractional release of [3H] (as per cent counts per min; cpm) from rat pial arteries during three periods of electrical field stimulation (S1-S3) following pre-incubation with 3 nM [3H]-5-HT (together with 30 1-tM nialamide, 100 1-tM ascorbic acid, and 30 ~-tM Na2Ca edetate). Electrical activation markedly increases the [3H] efflux in control preparations (C). No increase is seen when stimulation is carried out in preparations pre-incubated in the presence of 10 1-tM cocaine (Co) or following surgical sympathectomy (SyX), or if stimulation is performed in the presence of0.3 J.I.M tetrodotoxin (TIX). Values are means± s.e. mean, and number of animals (preparations) is indicated. Analysis of variance:* P < 0.05, **0.001 < P

< O.Gl

c .2 a 10

0

~80

c: 0 u6Q

~ E

0

40

c: ~ 2

0 .f

0

5-H

T I k

etan

seri

n (r

at B

A)

1 =u

nblo

cked

2

= 3•

10"9

M k

etan

serin

3

• 1•

1ITB

M

4= 3

•10"

8 M

5:1

x1

0"7

M -"-

. .-

--9

-8

-7

-6

-5

-4

lo

g 5-

HT

con

e. l

Ml

~

b 100

c 0 tlso

:! c: 8 60

~ E

40

0 c ~20

... ~

0

5-H

T I k

etan

serin

(m

onke

y B

A l

1 =u

rblo

cl<l

'd

2 = 1

Q·9

M k

etan

seri

n

3o1Q

-BM

4

·10-

7M

5

·10

-•M

-··

-

T I

I I

-8

-7

-6

-5

-4

-3

log

5-H

T co

nc.

lMl

C

5-H

T lk

eta

nse

rin

!gu

inea

-pig

BA

)

100-

l 1

• unb

lock

ed

2 = 'K

)"9

M k

etan

seri

n

3 •

11T8

M

6 ~ 80

c 8

so 0

I, s1

0""7

M

5•10

-<>M

6

.1o

-5M

·

3 4

//_

__

,5

-9

-8

-7

-6

-5

-4 lo

Q

5-H

T co

nc.

lMl

~ a S' :: s· s· Q

~ ~ a J ~ ~ 1:::

1 s. (\ ~·

Figu

re 2

8.3

Phar

mac

olog

ical

in v

itro

anal

ysis

of b

asila

r ar

terie

s (B

A)

show

ing

dext

ral

shift

with

inc

reas

ing

conc

entra

tions

of

~

keta

nser

in in

rat

and

mon

key,

and

a s

light

inhi

bitio

n at

con

cent

ratio

ns b

elow

1 !1

M, c

onsi

stin

g on

ly o

f dep

ress

ion

of th

e m

axim

al

~ re

spon

se, i

n gu

inea

-pig

. Mea

n va

lues

± s

.e.

mea

n ba

sed

on 5

-10

vess

el p

repa

ratio

ns a

t eac

h le

vel o

f ant

agon

ist c

once

ntra

tion

~ ~

238 Serotonin

concentrations up to 1 JLM had only a slight effect on the contractile response, and was limited to depression of the maximal (Figure 28.3(c)), whereas methiothepin, a potent antagonist at 5-HTrlike receptors, inhibited the contraction in a competitive manner.

AMPLIFYING ACTION OF 5-HT

The post-junctional interaction of 5-HT with noradrenaline-induced contractions was tested in basilar arteries of guinea-pigs. Contractions were recorded before and after priming with a low concentration of 5-HT, which in itself had little or no contractile effect (Chang and Owman, unpublished observations). Under these conditions, 5-HT markedly amplified the subsequent contraction induced by noradrenaline. This potentiating effect was not significantly affected by ketanserin, but was almost entirely abolished by administration of methiothepin. In parallel experiments on monkeys, it had been shown that the vasoconstriction induced by transmural electrical stimulation and which is primarily adrenergic in nature was not substantially influenced by 5-HT (Chang et al., 1987). This could indicate that 5-HT, released together with noradrenaline during electrical stimulation, markedly potentiates the vasoconstriction induced by the primary sympathetic transmitter post-junctionally, which may be one explanation why noradrenaline previously has been considered to be a fairly weak vasoconstrictor in the brain circulation when tested alone. It is not unlikely that pre-junctional effects of neuropeptide Y, also present in the sympathetic fibres and liberated during more intense sympathetic activation, may help to economize the effect of the amine transmitters through its pre-junctional action on the sympathetic terminals.

ACTIONS OF Ca2 + -CHANNEL ANTAGONISTS

In a recent series of experiments on monkeys in our laboratory, it has been shown that the Ca2+ antagonist nimodipine markedly impairs the ability of the cerebral circulation to autoregulate during angiotensin-induced hypertension (Sahlin et al., 1987). Thus i. v. infusion of nimodipine at a rate 0.5 JLg/kg per min increased the autoregulatory index (the amount of flow increase in relation to the degree of elevation of systemic blood pressure) from a normal mean value of 0.06 to 1.58 ml/100 g per min. Although this impairment of autoregulation has not previously been reported, it is predictable in view of the action nimodipine has on the contractile process, whereby the resistance vessels would constrict less efficiently to withstand the increased intraluminal pressure in order to maintain a constant blood flow in the face of progressive hypertension. The effect of Ca2+ -channel blockers on the contraction induced by 5-HT was therefore elucidated.

Serotonin in Cerebrovascular Sympathetic Nerves 239

Experiments were performed on rat isolated basilar artery preparations. The effects of 5-HT- and K+ -induced contractions were compared with those obtained in the rat tail artery (Chang and Owman, 1987). High K+ levels contracted both arteries biphasically; the contraction included a prazosin-sensitive a-adrenoceptor activation in the tail artery. Cyproheptadine, nimodipine, verapamil and diltiazem inhibited the basilar artery 5-HT-induced contraction, in that order of potency. Verapamil was more potent than diltiazem in the tail artery also, but nimodipine inhibited the contraction only by 35 per cent. Also, the tonic contraction induced by a high K+ concentration was attenuated by the test agents, with the same relative potency as in the presence of 5-HT, except for cyproheptadine, which was less efficient than nimodipine. The IC50 values were characteristically lower in the basilar than in the tail artery, irrespective of whether the contraction had been produced by 5-HT or by high K+. It was concluded from these experiments that Ca2 + -channel antagonists, particularly nimodipine, inhibit the contractile response to 5-HT preferentially in cerebral arteries.

CONCLUSIONS

In summary, the cerebral vascular bed is supplied with sympathetic fibres in which 5-HT co-exists with noradrenaline (and also neuropeptide Y), originating from the superior cervical ganglia. There is evidence that 5-HT is taken up locally rather than being synthesized in the nerves. 5-HT is released by electrical nerve stimulation and markedly amplifies the vasoconstrictor effect of noradrenaline. The serotoninergic vasoconstriction was mediated by 5-HTrlike receptors in guinea-pig basilar arteries, and 5-HT2 receptors in monkey and rat pial arteries; the contraction in rat basilar arteries is inhibited by the Ca2+ -channel antagonist nimodipine.

ACKNOWLEDGEMENTS

Grant support from the Swedish Medical Research Council (14X-732).

REFERENCES

Chang, J.-Y. and Owman, Ch. (1986). Immunohistochemical and pharmacological studies of serotonergic nerves and receptors in brain vessels. Acta Physiol. Scand., 552, 49--53

Chang, J .-Y. and Owman, Ch. (1987). Involvement ofspecific receptors and calcium mechanisms in serotonergic contractile response of isolated cerebral and peripheral arteries from rats. J. Pharmacol. Exp. Ther., 242, 329--336

240 Serotonin

Chang, J.-Y. and Owman, Ch. (1989). Cerebrovascular serotonergic receptors mediating vasoconstriction: Further evidence for the existence of 5-liT 2 receptors in rat and 5-HT 1-like receptors in guinea-pig basilar arteries. Acta Physiol. Scand., 136, 59-67

Chang, J.-Y., Hardebo, J. E., Owman, Ch., Sahlin, Ch. and Svendgaard, N.-Aa. (1987). Nerves containing serotonin, its interaction with noradrenaline, and characterization of serotonin receptors in cerebral arteries of monkey. J. Auton. Pharmacol, 7, 317-329

Chang, J.-Y., Owman, Ch. and Steinbusch, H. W. M. (1988). Evidence for co-existence of serotonin and noradrenaline in sympathetic nerves supplying brain vessels of guinea-pig. Brain Res., 438, 237-246

Chang, J.-Y., Ekblad, E., Kannisto, P. and Owman, Ch. (1989). Serotonin can be demonstrated by immunohistochemistry in cerebrovascular sympathetic nerve fibers of rat and is released during electrical stimulation. Brain Res. (in press)

Edvinsson, L., Degueurce, A., Duverger, D. MacKenzie, E. T. and Scatton, B. (1983). Central serotonergic nerves project to the pial vessels ofthe brain. Nature, 306, 55-57

Griffith, S. G. and Bumstock, G. (1983). Immunohistochemical demonstration of serotonin in nerves supplying human cerebral and mesenteric blood-vessels. Some speculations about their involvement in vascular disorders. Lancet, 1, 561-562

Griffith, S. G., Lincoln, J. and Bumstock, G. (1982). Serotonin as a neurotransmitter in cerebral arteries. Brain, 247, 388-392

Owman, Ch. (1986). Neurogenic control of the vascular system: Focus on cerebral circulation. In Mountcastle, V. B., Bloom, F. E. and Geiger, S. R. (Eds), Handbook of Physiology. The Nervous System. Intrinsic Regulatory Systems of the Brain, Sect. 1, Vol. 4, American Physiological Society, Bethesda, pp. 525-580

Sahlin, Ch., Brismar, J., Delgado, T., Owman, Ch., Salford, L. G. and Svendgaard, N.-Aa. (1987). Cerebrovascular and metabolic changes during the delayed vasospasm following experimental subarachnoid hemorrhage in baboons, and treatment with a calcium antagonist. Brain Res., 403, 313-332

29 5-HT 3 Receptors in the Gastrointestinal Tract

G. Engel, K.-H. Buchheit and B. P. Richardson


INTRODUCTION

About 90 per cent of the total body content of 5-hydroxytryptamine (5-HT) is found within the intestine (Erspamer, 1966), almost all of it being stored in the enterochromaffin cells which are scattered throughout the gastrointestinal tract (GIT). Although only a small fraction of the GIT 5-HT is located within neurones of the enteric nervous plexus, this neuronally located 5-HT has attracted much attention in recent research. The actions of 5-HT in the GIT are mediated via a heterogeneous population of receptors. Some are located on smooth muscle cells, mediating contractile and relaxant processes in the GIT. However, the neuronally located 5-HT receptors may be more important; these receptors are also heterogeneous, and can modulate the release of different neurotransmitters, and activate enteric reflex mechanisms. Over the last few years, study of these neuronally located 5-HT receptors has become possible due to the discovery of specific antagonists to one of these receptors, the so-called 5-HT3 receptor antagonists (Richardson et al., 1985).

IN-VITRO STUDIES IN GUINEA-PIG ILEUM

A well-established pharmacological model for the investigation of 5-HT receptors in the GIT is the guinea-pig isolated whole-ileum preparation. On the basis of studies in this preparation, Gaddum and Picarelli (1957) proposed the first 5-HT receptor classification, which distinguished between contractile effects mediated by neuronally located 5-HT 'M' receptors, and the 5-HT 'D' receptors on the smooth-muscle cells. According to a recent receptor classification (Bradley et al., 1986; Richardson and Engel, 1986), the 5-HT 'D' receptors in the GIT are identical to 5-HT2 receptors, and the neuronal 'M' receptors are designated 5-HT3 receptors. In the guinea-pig

242 Serotonin

isolated longitudinal smooth-muscle preparation (with the Auerbach plexus adherent to it), 5-HT elicits a biphasic concentration-response curve for the contractile effect (Figure 29.1), indicating an interaction with two independent receptor systems (Buchheit eta/., 1985b).

Using tetrodotoxin (TIX), it is possible to block all the indirect neurotransmitter-releasing effects of 5-HT (see Figure 29.1). The remaining contraction produced by 5-HT at high concentrations is mediated by 5-HT2

receptors located on smooth-muscle cells. While atropine blocks only the first phase of the concentration-response curve, the second phase (at higher concentrations of 5-HT) is influenced exclusively by agents which interfere with the substance P system, or by the newly developed, highly specific 5-HT3 antagonist ICS 205-930 (Richardson et al., 1985; Buchheit et al., 1985b), as shown in Figure 29.1. Interestingly, this antagonist, which is devoid of substantial affinity for other common neurotransmitter receptors, competitively blocks the neuronal receptors for 5-HT (pA2 = 7.95, slope= 1.1), which, when activated, cause the release of substance P, which in turn acts subsequently on smooth-muscle cells of the GIT longitudinal or circular-muscle layers. ICS 205-930 (at nanomolar concentrations) does not antagonize the first phase of the concentration-response curve, i.e. the atropine-sensitive phase, produced by lower concentrations of 5-HT; a specific agonist or antagonist at these 5-HT receptors remains to be found.

In the guinea-pig isolated whole-ileum preparation, the peristaltic reflex can be elicited by the method described by Trendelenburg (1917), whereby the activity of circular and longitudinal muscle can be investigated simultaneously. 5-HT at concentrations in the nanomolar range induces a sensitization to intraluminal pressure stimuli, and thereby facilitates the

% EFFECT 100

50

0

8 7 6 5 4

-Log [ 5-HT] mol/ I

Figure 29.1 Log concentration-response curves for 5-HT alone (e), and in the presence of ICS 205-930 (0.1 !J.M, *), physostigmine (0.1 I'M, •), and TIX (1 I'M,

T), in the isolated longitudinal smooth-muscle strip from guinea-pig ileum

Gastrointestinal 5-HT3 Receptors 243

peristaltic reflex; in comparison with untreated preparations, the pressure threshold necessary to elicit peristalsis was lower in the 5-HT pre-treated preparations. It was not possible to inhibit the peristaltic reflex per se using ICS 205-930 (0.001-10 !!M); however, the 5-HT-induced sensitization to pressure stimuli (hypermotility) was inhibited with a pA2 value of7.8 (slope 1.2) (Buchheit, unpublished observations). Introduction of 5-HT at concentrations of 0.01-1 !!M to the serosal side of the preparation led to an increase of irregular movements of the ileum, even in the absence of a rise in the intraluminal pressure. Duration, amplitude and frequency of the rhythmic contractions of circular and longitudinal muscle were dependent on the 5-HT concentration applied. These 5-HT-induced irregular movements were also reduced or inhibited by pre-treatment with ICS 205-930.

IN-VIVO STUDIES

Gastric Emptying in the Guinea-pig

The ability of 5-HT3 antagonists to stimulate gastric emptying delayed by fasting in conscious guinea-pigs was shown for the first time by Buchheit et al. (1985a). The passage of special glass spheroids from the stomach to the duodenum was determined by an X -ray device and was taken as a measure of the velocity of gastric emptying. After i. p. administration of 0.01 mg/kg ICS 205-930 or 0.1 mg/kg metoclopramide, a significantly enhanced gastric emptying rate was observed, whereas MDL 72222 was only weakly active. The question of the site of action of these 5-HT receptor antagonists was investigated by Costall et al. (1986b ), by injecting ICS 205-930 directly in the guinea-pig hypothalamus. Gastric emptying was able to be stimulated by this route in the same way as i.p. administration. In addition, in fed animals with a higher rate of gastric emptying, the central administration of the selective 5-HT3 receptor agonist 2-methyl-5-HT inhibited the rate of emptying, and with ICS 205-930 it was possible to reverse the effect of 2-methyl-5-HT. This observation points to a central site of action, where 5-HT3 receptor antagonists stimulate and agonists inhibit gastric emptying. However, more in-vivo and in-vitro work needs to be undertaken in order to clarify the mechanism by which 5-HT3 antagonists modify gastric motility.

Antidiarrhoeal Activity in the Mouse

5-Hydroxytryptophan (5-HTP), the biological precursor of 5-HT, induces diarrhoea if applied in a high dose ( 40 mg/kg i.p.), increasing both intestinal secretion and motility. A second type of experimental diarrhoea (Jacoby

244 Serotonin

and Marshall, 1972) can be provoked in mice by administration of cholera toxin, which stimulates adenylate cyclase and thereby releases large amounts of 5-HT from the enterochromaffin cells (Nilsson et al., 1983). If mice with 5-HTP-induced diarrhoea are pre-treated with ICS 205-930, the intensity of diarrhoea is dose-dependently reduced; the IC50 for ICS 205-930 is 150 f..tg/kgorallyor60 f..tg/kgi.p. (Buchheit, unpublished observations). In comparison, ketanserin, a 5-HT2 receptor antagonist, was without any antidiarrhoeal effect. ICS 205-930 was also effective in cholera-toxininduced diarrhoea, but its maximal reduction was only 60 per cent, with an IC50 of 300 f..tg/kg. Raising the dose of ICS 205-930 did not result in a further reduction of the stimulated secretion. In control experiments, castor oil-induced diarrhoea (as a common model for evaluation of antidiarrhoeal drugs) was tested. ICS 205-930 was completely inactive up to a high dose of 32 mg/kg orally, whereas loperamide, the standard antidiarrhoeal drug, resulted in a dose-dependent inhibition (Shearman, unpublished observations).

Cytotoxic Drug-induced Emesis in the Ferret

Miner and Sanger (1986) reported that MDL 72222 blocked cisplatininduced emesis in ferrets, and in this model it has been established that the highly specific 5-HT 3 receptor antagonists which are devoid of antidopaminergicactivity, e.g. GR 38032F, BRL24924, BRL43694 and ICS205-930, are also potent inhibitors of cytotoxic drug-induced emesis (Table 29.1). Vomiting is also a major side-effect of radiotherapy, and may be of such severity that further treatment is refused. In the ferret, it has been demonstrated that either the administration of BRL 24924 (5 mg/kg s.c.) or abdominal vagotomy markedly reduced the vomiting evoked by X-radiation (Andrews et al., 1987). The innervation of the abdominal viscera plays a major role in cytotoxic drug-induced vomiting; furthermore, it is now evident from the inhibitory action of 5-HT3 receptor antagonists that 5-HT3 receptors are involved.

CLINICAL FINDINGS

Carcinoid Syndrome

The antidiarrhoeal activity of ICS 205-930 found in the mouse suggested its administration to carcinoid patients, whose diarrhoea is thought to stem mainly from an overproduction of 5-HT, as measured by their 5-hydroxyindoleacetic acid (5-HIAA) plasma levels. Table 29.2 (Anderson et al., 1987) summarizes the clinical effects of ICS 205-930 in 3 patients with

Gastrointestinal 5-HT3 Receptors 245

carcinoid syndrome accompanied by high 5-HIAA plasma levels, and in 2 patients with no signs of 5-HT overproduction but with diarrhoea, due to a tumour producing vasoactive intestinal peptide (VIP). As can be seen from Table 29.2, ICS 205-930 improved the symptoms only in those patients with 5-HT overproduction, and was inactive in the patients suffering from diarrhoea due to a YIP-producing tumour. These results indicate the involvement of enteric 5-HT3 receptors in the secretory process.

Cytotoxic Drug-induced Emesis

Chemotherapy in man, especially with the antineoplastic agent cisplatin, causes severe nausea and vomiting which can be antagonized by extremely high doses of metoclopramide (Gralla et al., 1981). Very recently, two examples of 5-HT3 antagonists, ICS 205-930 and GR 38032F, have been studied in man ( Cuningham et al., 1987; Leibundgut and Lancranjan, 1987).

Table 29.1 Effect of 5-HT3 receptor antagonists on cisplatin-induced emesis in the ferret

No. of vomits,

Onset of retches in Dose emesis Emetic 2 hfrom

Compound (mglkg i.v.)(min) episodes onset References Vehicle 45-84 14-16 42

Metoclopramide 2 87 4 25 1 4 none none none

ICS 205-930 0.01 96 51 n.a. 2 0.1 none none none

GR 38032F 0.01 92 4 28 1 0.1 none none none 1.0 none none none

BRL 43694 0.01 183 1.0 n.a. 3 0.1 230 0.5 n.a. 1.0 240 none n.a.

MDL 72222 0.1 94 5.8 n.a. 4 1.0 234 0.8 n.a.

BRL 24924 2.5 >197 2.8 n.a. 5 5.0 >169 2.8 n.a.

Emesis was induced by administration of cisplatin at a dose level of 10 mglkg i.v. n.a. = data not available. References: 1. Costall et al. (1987);

2. Costall et al. (1986a); 3. Boyle eta[. (1987); 4. Miner and Sanger (1986); 5. Miner et al. (1986).

246 Serotonin

Table 29.2 Symptoms and treatment in patients with carcinoid syndrome and with tumours producing vasoactive intestinal peptide (VIP)

Stool weight Stool frequency Patient and (g) (motions/day) diagnosis Before Carcinoid syndrome 1163 Carcinoid syndrome 421 Carcinoid syndrome 471 YIP-producing 2767 tumour YIP-producing tumour

516

During 702 173 294

2107

584

Daily dose of ICS 250-930 ""' 30/30/30 mg. Data are from Anderson et al. (1987).

Before During 12 6.3 7.0 2.0 6.7 1.7 9.3 10.3

3.7 4.7

Urine 5-H/AA (f.U11oV24 h) Before During 1100 1107 793 511

1333 1421 6 34

23 16

ICS 205-930 was investigated in 11 patients, and with a total daily dose of 20-40 mg showed a complete blockade of nausea and vomiting in 31 of 47 courses of treatment with a range of cytotoxic compounds. The 5-HT3 antagonist GR 38032F also had a good efficacy against cytotoxic drug-induced vomiting in 15 patients, using a dose of 4 mg i.v. and 4 mg orally. In accordance with the selective pharmacological profile of these new 5-HT 3 receptor antagonists, dystonic reactions, as observed with high metoclopramide dosage, were not reported.

CONCLUSIONS

There exists a heterogeneous population of neuronal 5-HT receptors in the GIT; among them is a 5-HT3 receptor at which 5-HT is a relatively weak (micromolar) agonist, as observed in guinea-pig isolated ileum. Studies in animals and man indicate that 5-HT3 receptors seem to be involved in diseases in which high amounts of 5-HT are released from intestinal stores. These 5-HT3 receptors might be located on afferent sensory neurones, leading to the stimulation of intrinsic reflexes. As the activation of 5-HT3 receptors seems to be the beginning of a cascade of events, the use of5-HT3 receptor antagonists should have beneficial effects in a variety of GIT disorders.

REFERENCES

Anderson, J. V., Coupe, M. 0., Morris, J. A., Hodgson, H. J. F. and Bloom, S. R. (1987). Remission of symptoms in carcinoid syndrome with a new 5-hydroxytryptamine M receptor antagonist. Br. Med. J., 294, 1129

Gastrointestinal5-HT3 Receptors 247

Andrews, P. L. R., Hawthorn, J. and Sanger, G. J. (1987). The effect of abdominal visceral nerve lesions and a novel5-HT-M receptor antagonist on cytotoxic and radiation induced emesis in the ferret. J. Physiol., 382, 47P

Boyle, E. A., Miner, W. D. and Sanger, G. J. (1987). Different anti-cancer therapies evoke emesis by mechanisms that can be blocked by the 5-HT 3 receptor antagonist BRL 43694. Br. J. Pharmacal., 91, 418P

Bradley, P. B., Engel, G., Feniuk, W., Fozard, J. R., Humphrey, P. P. A., Middlemiss, D. N., Mylecharane, E. J., Richardson, B. P. and Saxena, P. R. (1986). Proposals for the classification and nomenclature of functional receptors for 5-hydroxytryptamine. Neuropharmacology, 25,563-576

Buchheit, K. H., Costall, B., Engel, G., Gunning, S. J., Naylor, R. J. and Richardson, B. P. (1985a). 5-Hydroxytryptamine receptor antagonism by metoclopramide and ICS 205-930 in the guinea-pig leads to enhancement of contractions of stomach muscle strips induced by electrical field stimulation and facilitation of gastric emptying in vivo. J. Pharm. Pharmacal., 37, 664--667

Buchheit, K. H., Engel, G., Mutschler, E. and Richardson, B. P. (1985b). Study of the contractile effect of 5-hydroxytryptamine (5-HT) in the isolated longitudinal muscle strip from guinea-pig ileum. Evidence for two distinct release mechanisms. Naunyn-Schmiedeberg's Arch. Pharmacal., 329, 36-41

Costall, B., Domeney, A. M., Naylor, R. J. and Tattersall, F. D. (1986a). 5-Hydroxytryptamine M-receptor antagonism to prevent cisplatin-induced emesis. Neuropharmacology, 25, 959-961

Costall, B., Kelley, M. E., Naylor, R. J., Tan, C. C. W. and Tattersall, F. D. (1986b). 5-Hydroxytryptamine M-receptor antagonism in the hypothalamus facilitates gastric emptying in the guinea-pig. Neuropharmacology, 25, 1293-1296

Costall, B., Domeney, A.M., Gunning, S. J., Naylor, R. J., Tattersall, F. D. and Tyers, M. B. (1987). GR 38032F: a potent and novel inhibitor of cisplatin-induced emesis in the ferret. Br. J. Pharmacal, 90, 90P

Cunningham, D., Hawthorn, J., Pople, A., Gazet, J.-C., Ford, H. T., Challoner, T. and Coombes, R. C. (1987). Prevention of emesis in patients receiving cytotoxic drugs by GR38032F, a selective 5-HT receptor antagonist. Lancet, I, 1461-1462

Erspamer, V. (1966). Occurrence of indolealkylamines in nature. In Erspamer, V. (Ed.), Handbook of Experimental Pharmacology, Vol. 19, 5-Hydroxytryptamine and Related Indolealkylamines, Springer, Berlin, Heidelberg, New York, pp. 132-181

Gaddum, J. H. and Picarelli, Z. P. (1957). Two kinds of tryptamine receptor. Br. J. Pharmacal. Chemother., 12, 323-328

Gralla, R. J., Hri, L. M., Pisko, S. E., Squillante, A. E., Kelsen, D.P., Braun, D. W., Bardin, L.A., Braun, T. J. and Young, C. W. (1981). Antiemetic efficacy of high dose metoclopramide: randomized trials with placebo and prochlorperazine in patients with chemotherapy-induced nausea and vomiting. New Engl. J. Med., 305, 905-909

Jacoby, H. J. and Marshall, C. H. (1972). Antagonism of cholera enterotoxin by anti-inflammatory agents in the rat. Nature, 235, 163-165

Leibundgut, U. and Lancranjan, I. (1987). First results with ICS 205-930 (5-HT3 receptor antagonist) in prevention of chemotherapy-induced emesis. Lancet, I, 1198

Miner, W. D. and Sanger, G. J. (1986). Inhibition of cisplatin-induced vomiting by selective 5-hydroxytryptamine M-receptor antagonism. Br. J. Pharmacal., 88, 497-499

Miner, W. D., Sanger, G. J. and Turner, D. H. (1986). Comparison of the effect of BRL 24924, metoclopramide and domperidone on cisplatin-induced emesis in the ferret. Br. J. Pharmacal., 88, 374P

248 Serotonin

Nilsson, 0., Cassuto, J., Larsson, P. A., Jodal, M., Lidberg, P., Ahlman, H., Dahlstrom, A. and Lundgren, 0. (1983). 5-Hydroxytryptamine and cholera secretion: a histochemical and physiological study in cats. Gut, 24, 542-548

Richardson, B. P., Engel, G., Donatsch, P. and Stadler, P. A. (1985). Identification of serotonin M-receptor subtypes and their specific blockade by a new class of drugs. Nature, 316, 126-131


Trendelenburg, P. (1917). Physiologische und pharmakologische Versuche iiber die Diinndarmperistaltik. Naunyn-Schmiedeberg's Arch. Exp. Path. Pharmakol., 81, 55-129

30 Different Recognition Sites for Serotonin: The Neuronal Na +-dependent Transporter and the

Release-modulating Autoreceptor

S. Z. Langer and D. Graham

Department of Biology, Synthelabo Recherche (LERS), 58, rue de Ia Glaciere, 75013 Paris, France

INTRODUCTION

A number of pharmacologically distinct recognition sites for serotonin (5-hydroxytryptamine; 5-HT) have been identified at serotoninergic synapses of the CNS. Post -synaptically, these 5-HT binding sites have been associated with 5-HT1-like, 5-HT2 and 5-HT3 receptors. In this short overview, however, attention will be restricted to pre-synaptic recognition sites for 5-HT which are involved in the release and neuronal uptake of 5-HT.

THE NEURONAL Na+ -DEPENDENT 5-HT TRANSPORTER

The plasmolemmal5-HT transporter located on pre-synaptic serotoninergic terminals belongs to a family of Na + symport transporter proteins in which the transport of one solute, the substrate, is dependent upon the co-transport of Na+. The trans-membranal electrochemical gradient for Na +, generated by the enzyme Na + /K+ -adenosine triphosphatase, enables this plasmolemmal Na+/5-HT symporter (or Na+-dependent 5-HT transporter) to serve as an active transport mechanism for the uptake of 5-HT from the synaptic cleft into pre-synaptic serotoninergic nerve terminals (Figure 30.1). As such, this transporter has a key role to play in serotoninergic synaptic transmission in that it functions to inactivate released 5-HT by reducing high synaptic cleft concentrations of this neurotransmitter. By this process, not only is the trans-synaptic signal terminated, but the neurotransmitter molecule itself can be recycled to be

250 Serotonin

stored in synaptic storage vesicles through a pharmacologically distinct intracellular 5-HT transporter, at the level of the membrane of the storage granule.

The uptake of 5-HT by the neuronal Na+-dependent 5-HT transporter has been studied using both brain slice and synaptosomal preparations. This uptake process occurs with relatively high affinity (using synaptosomal preparations, KM values ranging between 50 and 100 nM have been reported; see Ross, 1982). Moreover, because blood platelets are considered to accumulate and store 5-HT in a manner similar to that occurring in serotoninergic nerve terminals (Sneddon, 1973; Stahl, 1977), many studies have employed platelets as a convenient model to investigate the mechanism whereby 5-HT is transported across the plasma membrane. Interestingly, the use of plasma membrane vesicle preparations derived from platelets has provided direct evidence that the platelet Na +-dependent 5-HT transporter can operate only when appropriate Na+/K+ transmembranal gradients are constructed across the membrane wall of these vesicles (Nelson and Rudnick, 1982).

In addition to uptake studies, in-vitro binding assays using radiolabelled forms of a number of potent and selective inhibitors of N a+ -dependent 5-HT

~ 5·HT

Figure 30.1 Schematic representation of a serotoninergic synapse. (A) Exocytotic release of 5-HT from the pre-synaptic terminal; (B) post-synaptic 5-HT receptor binding site; (C) pre-synaptic inhibitory 5-HT autoreceptor; (D) neuronal

Na+-dependent 5-HT transporter. MAO= monoamine oxidase

5-HT Neuronal Transporter and Pre-synaptic Autoreceptor 251

uptake have helped to obtain information at the molecular level on the Na+-dependent 5-HT transporter. In conjunction with 5-HT uptake studies, these radiolabelled in-vitro binding assays have contributed to the elucidation of structure-activity relationships for the binding and uptake of substrate and to the development of novel selective 5-HT uptake inhibitors such as SL 81.0385 (Scatton et al., 1988). Moreover, these binding assays offer an amenable approach to study the neuronal Na+-dependent 5-HT transporter of human post-mortem brain samples obtained from certain pathophysiological states.

Inhibitors of 5-HT uptake which have been used to label the neuronal Na+-dependent 5-HT transporter in in-vitro binding assays have included [3H]-imipramine, [3H]-Ro 11-2465, [3H]-paroxetine, [3H]-indalpine, [3H]norzimelidine and eHJ-citalopram. [3H]-Imipramine, in particular, has been used extensively for this purpose (Graham and Langer, 1988). Nevertheless, [3H]-imipramine as a ligand poses certain problems, such as the need to conduct routine binding assays at 4 oc and the presence of binding site heterogeneity for this ligand in brain membrane preparations. For this reason, the binding of [3H]-paroxetine, a highly selective 5-HT uptake inhibitor (Thomas et al., 1987), to mammalian cerebral cortical membranes was chosen for examination in our laboratory.

Equilibrium saturation analyses of [3H]-paroxetine binding to cerebral cortical membranes from rat, mouse and human brain indicate that [3H]-paroxetine selectively labels a single class of high-affinity binding sites in these tissues. The equilibrium dissociation constants obtained in our laboratory for [3H]-paroxetine binding to cerebral cortical membranes from these different animal species are close to the potency of paroxetine as an inhibitor of Na +-dependent 5-HT uptake into rat whole brain synaptosomes (Ki = 0.26 nM; Hytel, 1982). Moreover, [3H]-paroxetine binding to these cerebral cortical membrane preparations can be potently inhibited by Na+-dependent 5-HT uptake inhibitors (Table 30.1). Drugs which label other receptors gave Ki values > 1 IJ.M for inhibiting [3H]-paroxetine binding (Table 30.1, and Habert et al., 1985). An excellent correlation exists between the potencies of various compounds as inhibitors of [3H]paroxetine binding to rat cerebral cortical membranes and Na+ -dependent 5-HT uptake into rat brain synaptosomes (Habert et al., 1985). Also, lesioning experiments with the neurotoxin 5,7-dihydroxytryptamine indicate that [3H]-paroxetine binding sites are located on serotoninergic neurones (Habert et al., 1985). These results indicate that [3H)-paroxetine is a highly selective ligand for studies of the neuronal Na+-dependent 5-HT transporter using in-vitro binding assays.

Considerable data exist on structure-activity relationships which are important for the binding and transport of substrate by the neuronal Na+ -dependent 5-HT transporter. In contrast, little is known regarding the topography of the transporter and the amino acid groups on this protein

252 Serotonin

which have an important function for its mechanism of action. The effect of functional-group modifying agents on [3H]-paroxetine binding to rat cerebral cortical membranes has therefore been examined in our laboratory in an attempt to identify key amino acid groups located at the paroxetine binding site of the neuronal N a+ -dependent 5-HT transporter.

Covalent modification of cysteine residues by the sulphydryl-alkylating reagent N-ethylmaleiimide (NEM), or of tyrosine residues by the alkylating reagent p-nitrobenzylsulphonyl fluoride, produced large decreases in the extent of [3H]-paroxetine binding to rat cerebral cortical membranes compared to controls. These results suggest that sulphydryl groups and tyrosine hydroxyl groups are located at, or very close to, the paroxetine binding site on the neuronal Na+-dependent 5-HT transporter. In this respect, it is interesting to note that previous studies of [3H]-imipramine binding to both the platelet and neuronal forms of the N a+ -dependent 5-HT transporter have indicated that sulphydryl groups on these proteins are involved in the binding of this tricyclic antidepressant (Davis, 1984; Biassoni and Vaccari, 1985).

Pre-incubation of rat cerebral cortical membranes with saturating concentrations of fluoxetine, imipramine or 5-HT before NEM treatment protected against the inactivation of [3H]-paroxetine binding produced by NEM treatment alone (Table 30.2). This observation could indicate that the non-tricyclics (fluoxetine and paroxetine), the tricyclic (imipramine), and the substrate (5-HT) all bind to a similar, or overlapping, site on the neuronal N a+ -dependent 5-HT transporter. Our earlier report that each of these compounds inhibits [3H]-paroxetine binding to rat cerebral cortical

Table 30.1 Inhibition of [3H)-paroxetine binding to rat cerebral cortical membranes

Compounds

Citalopram Indalpine Chlorimipramine Fluoxetine Desipramine

Verapamil Phentolamine Prazosin Nomifensine Diazepam Spiperone

1.0 1.7 2.0

13.5 834.5

>1000 >1000 >1000 >1000 >1000 >1000

Inhibition of fH]-paroxetine binding to rat cerebral cortical membranes was determined at 22 oc using a radioligand concentration of 0. 2 nM (protocol of Habert et a/. , 1985). The Ki values are the means of at least three experiments.


membranes with Hill coefficients close to unity (Habert et al., 1985) would be in accordance with this interpretation. However, other reports would not be consistent with this concept. For example, chronic drug administrations which lead to alterations in 5-HT concentrations failed to induce changes in the parameters of [3H)-paroxetine binding to rat cerebral cortical membranes (Graham et al., 1987), whereas the same chronic administrations have been reported in some (Raisman et al., 1980; Barbaccia et al., 1983; Racagni et al., 1983), but not all (Lee eta/., 1983; Abel et al., 1985), studies to reduce the Bmax values of [3H)-imipramine binding. Equivocal reports of [3H)-imipramine binding site plasticity upon similar drug administrations nevertheless question the interpretation of past [3H)imipramine binding results when this ligand has been used to label the neuronal N a+ -dependent 5-HT transporter.

In addition to chemical modification studies, the effect of disruption of the lipid or protein environment of rat cerebral cortical membranes on [3H)-paroxetine binding to the neuronal N a+ -dependent 5-HT transporter has also been investigated. Treatment of these membranes with trypsin (100 J.tg/ml) and chymotrypsin (100 J.tg/ml) for 30 min at 37 oc produced reductions in [3H)-paroxetine binding of 41 per cent and 57 per cent, respectively. Also, [3H)-paroxetine binding was decreased by 54 per cent and 90 per cent after treatment of rat cerebral cortical membranes for 30 min at 37 oc in the presence of 1 mM CaCiz with phospholipase C (10 J.tg/ml) and phospholipase A2 (0.1 J.tg/ml), respectively. These enzyme treatments thus suggest that the neuronal Na+ -dependent 5-HT transporter requires an intact protein and lipid environment in order to maintain its ability to bind [3H)-paroxetine.

Clearly, although functional-group modifying reagents can help to identify amino acids located at certain 'active centres' of a protein, their

Table 30.2 Protection against NEM-induced inactivation of (3H)-paroxetine binding to rat cerebral cortical membranes

Treatment

Membranes alone Membranes + 10 mM NEM

Membranes+ 0.5 ftM fluoxetine + 10 mM NEM Membranes + 1 ftM imipramine + 10 mM NEM Membranes+ 100 ftM 5-HT + 10 mM NEM

Specific [3H}-paroxetine binding (%) 100 26

60 74 74

Rat cerebral cortical membranes were pre-incubated with or without test compound for 5 min at 25 oc before addition of NEM. After 2 h, the incubations were terminated by centrifugation, followed by resuspension and centrifugation of each pellet three more times to ensure removal of the test compound. Aliquots of the final resuspended membrane pellets were then assayed with 0.5 OM eHJ-paroxetine. The values above represent a typical experiment replicated 3 times.

254 Serotonin

application to elucidate the molecular details by which the N a+ -dependent 5-HT transporter binds and translocates 5-HT across the neuronal plasma membrane is limited. In order to gain a detailed insight of these molecular mechanisms, the application of molecular biological techniques to clone this transporter would prove beneficial. Knowledge of the primary and tertiary structure of this protein and the effects of site-directed mutagenesis on carrier function should ultimately improve our understanding of this transport process. As a first step, therefore, a programme has been undertaken to purify the Na+ -dependent 5-HT transporter of rat cerebral cortex. Using the non-ionic detergent digitonin, this neuronal transporter was successfully solubilized out from its membrane-bound environment with retention of the drug inhibitor and substrate binding site(s) of this macromolecule (Habert eta/., 1986). Subsequently, affinity chromatography utilizing a citalopram derivative coupled to an agarose resin was employed as a purification step. In this procedure, the solubilized 5-HT transporter of rat cerebral cortical membranes was adsorbed to the resin, and then the resin was extensively washed. The purified 5-HT transporter, as defined by eHJ-paroxetine binding activity, was then recovered from the resin by elution with 10 f.!M indalpine. Experiments to characterize the affinity-purified 5-HT transporter are currently in progress.

THE PRE-SYNAPTIC 5-HT AUTORECEPTOR

The autoreceptor for 5-HT located on pre-synaptic serotoninergic nerve terminals is involved in the regulation of 5-HT release through a mechanism of negative feedback inhibition (Cerrito and Raiteri, 1979; Langer and Moret, 1982). As such, activation of this pre-synaptic autoreceptor by the binding of 5-HT leads to a subsequent inhibition of 5-HT release (Figure 30.1). The reduction by lysergic acid diethylamide and 5-methoxytryptamine of the Ca2 + -dependent release of [3H]-5-HT from brain slices has led to a classification of these compounds as 5-HT autoreceptor agonists. Also, methiothepin is considered to be a 5-HT autoreceptor antagonist as it blocks the effect of the agonist, and, on its own, leads to an augmentation of Ca2+ -dependent [3H]-5-HT release.

The pre-synaptic inhibitory 5-HT autoreceptor shows a pharmacological profile of a 5-HTrlike receptor, with the indication in rat brain that it belongs to the 5-HTm subtype classification (Engel et a/., 1986). Nevertheless, the definitive characterization of this terminal autoreceptor in mammalian brain still awaits the development of potent and selective antagonists that act at this 5-HT recognition site.

CONCLUSIONS

The importance of the neuronal N a+ -dependent 5-HT transporter and the


pre-synaptic inhibitory 5-HT receptor in the regulation of serotoninergic neurotransmission render these 5-HT recognition sites possible targets for drug therapy. Selective inhibitors of 5-HT uptake have already been used successfully in the treatment of depression, and there are initial indications that these compounds could be effective anti-obesity drugs. Interestingly, selective 5-HT uptake inhibitors have been reported to produce, by an as yet unknown mechanism, subsensitivity of inhibitory 5-HT autoreceptors (Galzin et al., 1985; Chaput et al. 1986). Thus, the time-dependent development of enhanced serotoninergic transmission as a consequence of 5-HT autoreceptor subsensitivity following the administration of selective 5-HT uptake inhibitors could well account for the latency period noted before clinical improvement during the course of treatment of depressive disorders with this class of drugs.

ACKNOWLEDGEMENTS

The authors wish to thank Mrs G. Darles for technical assistance and Miss Fran~ise Pechoux for secretarial aid during the preparation of this manuscript.

REFERENCES

Abel, M.S., Clody, M. E., Wennogle, L. P. and Meyerson, L. R. (1985). Effect of chronic desmethylimipramine or electroconvulsive shock on selected brain and platelet neurotransmitter recognition sites. Biochem. Pharmacol., 34, 679-683

Barbaccia, M. M., Gandolfi, 0., Chuang, D. M. and Costa, E. (1983). Modulation of neuronal serotonin uptake by a putative endogenous ligand of imipramine recognition sites. Proc. Nat/. Acad. Sci. U.S.A., 80, 5134-5138

Biassoni, R. and Vaccari, A. (1985). Selective effects ofthiol reagent on the binding sites for imipramine and neurotransmitter amine in the brain. Br. J. Pharmacol., 85, 447-456

Cerrito, F. and Raiteri, M. (1979). Serotonin release is modulated by presynaptic autoreceptors. Eur. J. Pharmacol., 57, 427-430

Chaput, Y., de Montigny, C. and Blier, P. (1986). Effects of a selective 5HT reuptake blocker, citalopram, on the sensitivity of 5HT autoreceptors: electrophysiological studies in the rat brain. Naunyn-Schmiedeberg's Arch. Pharmacol., 333, 342-348

Davis, A. (1984). Temperature-sensitive conformational changes in [3H]imipramine binding sites and the involvement of sulphur-containing bonds. Eur. J. Pharmacol., 102, 341-347

Engel, G., Gothert, M., Hoyer, D., Schlicker, E. and Hillenbrand, K. (1986). Identity of inhibitory presynaptic 5-hydroxytryptamine (5-HT) autoreceptors in the rat brain cortex with 5-HTlB binding sites. Naunyn-Schmiederberg's Arch. Pharmacol., 332, 1-7

256 Serotonin

Galzin, A. M., Moret, C., Verzier, B. and Langer, S. Z. (1985). Interactions between tricyclic and nontricyclic 5-hydroxytryptamine uptake inhibitors and the presynaptic 5-hydroxytryptamine inhibitory autoreceptors in the rat hypothalamus. J. Pharmacal. Exp. Ther., 235,200-211

Graham, D. and Langer, S. Z. (1988). The neuronal sodium-dependent serotonin transporter: studies with [3H]imipramine and [3H)paroxetine. In Osborne, N. N. and Hamon, M. (Eds), Neuronal Serotonin, Wiley, Chichester, pp. 367-391

Graham, D., Tahraoui, L. and Langer, S. Z. (1987). Effects of chronic treatments with selective monoamine oxidase inhibitors and specific 5-hydroxytryptamine uptake inhibitors on [3H]paroxetine binding to rat cerebral cortical membranes. Neuropharmacology, 26, 1087-1092

Habert, E., Graham, D., Tahraoui, L., Claustre, Y. and Langer, S. Z. (1985). Characterization of [3H)paroxetine binding to rat cortical membranes. Eur. J. Pharmacal., 118, 107-114

Habert, E., Graham, D. and Langer, S. Z. (1986). Solubilization and characterization of the 5-hydroxytryptamine transporter complex from rat cerebral cortical membranes. Eur. J. Pharmacal., 122, 197-204

Hyttel, J. (1982). Citalopram: pharmacological profile of a specific serotonin uptake inhibitor with antidepressant activity. Prog. Neuropsychopharmacol. Bioi. Psychiat., 6, 277-295

Langer, S. Z. and Moret, C. (1982). Citalopram antagonizes the stimulation by lysergic acid diethylamide of presynaptic inhibitory serotonin autoreceptors in the rat hypothalamus. J. Pharmacal. Exp. Ther., 222, 22G-226

Lee, C. M., Javitch, J. A. and Snyder, S. H. (1983). Recognition sites for norepinephrine uptake; regulation by neurotransmitter. Science, 220, 626-629

Nelson, P. J. and Rudnick, G. (1982). The role of chloride ion in platelet serotonin transport. J. Bioi. Chern., 257, 6151--6155

Racagni, G., Mocchetti, 1., Calderini, G., Battistella, A. and Brunello, N. (1983). Temporal sequence of changes in central noradrenergic system of rat after prolonged antidepressant treatment: receptor densitization and neurotransmitter interactions. Neuropharmacology, 22, 415--424

Raisman, R., Briley, M.S. and Langer, S. Z. (1980). Specific tricyclic antidepressant binding sites in rat brain characterized by high-affinity [3H)imipramine binding. Eur. J. Pharmacal., 61, 373--380

Ross, S. B. (1982). In Osborne, N. N. (Ed.), Biology of Serotonergic Transmission, Wiley, London, pp. 159-195

Scatton, B., Claustre, Y., Graham, D., Dennis, T., Serrano, A., Arbilla, S., Pimoule, C., Schoemaker, H., Bigg, D. and Langer, S. Z. (1988). SL 81.0385, a novel selective and potent serotonin uptake inhibitor. Drug Devel. Res., 12, 29-40

Sneddon, J. M. (1973). Blood platelets as a model for monoamine-containing neurons. Prog. Neurobiol., 1, 153--198

Stahl, S. M. (1977). The human platelet: a diagnostic and research tool for the study of biogenic amines in psychiatric and neurologic disorders. Arch. Gen. Psychiat., 34, 509-516

Thomas, D. R., Nelson, D. R. and Johnson, A.M. (1987). Biochemical effects of the antidepressant paroxetine, a specific 5-hydroxytryptamine uptake inhibitor. Psychopharmacology, 93, 193--200

31 Serotoninergic Function in Neuropsychiatric

Disorders

D. L. Murphy, E. A. Mueller, C. S. Aulakh, G. Bagdy and N. A. Garrick

Laboratory of Clinical Science, National Institute of Mental Health, NIH Clinical Center, 10-3D41,BethesdaMD20892, USA

INTRODUCTION

The problem our group has been approaching is the applied question of how to evaluate the apparent contributions of changes in brain serotoninergic function to neuropsychiatric disorders and to the effects of drugs active in treating these disorders. For reasons detailed elsewhere (Murphy et al. , 1986), our studies have moved from static, single-point measures of the levels of serotonin and its major metabolite 5-hydroxyindoleacetic acid in cerebrospinal fluid, platelets, post-mortem brain samples or urine (for reviews see Murphy et al., 1986; Meltzer and Lowy, 1987), to attempts to evaluate the state of functional responsiveness (and possibly receptor sensitivity) of the brain serotoninergic system using drugs with serotoninselective actions as in-vivo probes of the system.

Obviously, most of our investigations in humans are based on the ground-breaking work exploring the neuroanatomy, physiology and pharmacology of serotonin which has been accomplished in rodents and other species. Our group likewise has turned to rodent and non-human primate models to test the validity of some of our so-called pharmacological challenge studies in humans, particularly when more novel agents or dosage regimens are being studied. Some of this data will be summarized below. However, there are some not-unexpected neuroanatomical differences in brain serotoninergic systems between primates and rodents (Azmitia and Gannon, 1986); similarly, major species differences have become evident in the rapidly developing area of brain serotonin receptors and binding sites (Heuring et al. , 1986). Thus ultimately, serotoninergic function needs to be studied directly in humans, particularly when, as some of the preliminary data indicate, disorder-specific changes in responsiveness to serotoninergic agents occur.

258 Serotonin

The agents used as pharmacological probes to study serotoninergic responsiveness in humans in our studies (as in those of other investigators) comprise the serotonin metabolic precursors L-tryptophan and 5-hydroxytryptophan; reuptake inhibitors such as clomipramine, fluoxetine and fluvoxamine; the releasing agent fenfluramine; and the serotonin receptor agonist m-chlorophenylpiperazine (m-CPP), alone and together with some serotonin antagonists such as metergoline, methysergide and ritanserin. This review provides an overview of the most recent work of our group and that of others using m-CPP. This agent has led to some quite provocative findings in patients with suspected alterations in serotoninergic function, although many of the studies are still in preliminary stages.

NEUROENDOCRINE AND TEMPERATURE EFFECTS OF m-CPP

m-CPP is a metabolite of the antidepressant trazodone which acts post-synaptically in rodents to produce elevations in plasma prolactin (Fuller et al., 1981). Prior studies with L-tryptophan, 5-hydroxytryptophan and fenfluramine had shown differences in prolactin and/or cortisol responses to these agents in depressed patients compared with controls (Meltzer et al., 1983; Heninger et al., 1984; Siever et al., 1984). Our first studies with m-CPP were initiated to evaluate whether this agent might also lead to serotonin-mediated neuroendocrine changes in non-human primates and humans, and thus provide us with end-points to investigate whether the altered responses to serotoninergic agonists in depressed patients represented pre-synaptic or post-synaptic alterations.

A preliminary study in rhesus monkeys demonstrated that m-CPP (0.5-3 mg/kg i.v.) led to increases in plasma prolactin, cortisol, and growth hormone, accompanied by a calming behavioural effect (Aloi et al., 1984). Approval was gained to conduct the first studies of m-CPP in humans in our laboratory; dose-related increases in plasma cortisol and prolactin, as well as a hyperthermic effect at a dose of0.5 mg/kg given orally, were demonstrated (Mueller et al., 1985a, b). Subsequently, increases in plasma adrenocorticotrophin, vasopressin and ~-endorphinllipotrophin after 0.5 mg/kg m-CPP were found, but, unlike the results in rhesus monkeys, plasma growth hormone was not increased (Figure 31.1; Mueller et al., 1986; Murphy et al., unpublished observations). Pre-treatment with the 5-HT1/5-HT2 receptor antagonist metergoline (4 mg, orally) prevented the m-CPP-induced increases in cortisol, prolactin, adrenocorticotrophin and temperature (Mueller et al., 1986), in agreement with studies in rodents and monkeys which had previously demonstrated that metergoline prevented m-CPPinduced behavioural and neuroendocrine changes (Samanin et al., 1979; Fuller et al., 1981; Aloi et al., 1984). A smaller dose of m-CPP (0.1 mg/kg) given i. v. to normal humans has subsequently been shown to lead to

Serotonin in Neuropsychiatric Disorders 259

equivalent increases in plasma prolactin and to produce similar plateau concentrations of m-CPP in plasma to those following the 0.5 mg/kg oral m-CPP dose (Figure 31.2).

20

E ---Cl E. 15 2 f= u :5 10 0 a: a.. >< ~ 5 <I

8

E 0, 6 a. :I: 1-u <( 4 >< <(

~ <I

2

**

PLACEBO m-CPP MTG +

m-CPP

PLACEBO m-CPP MTG +

m-CPP

E 8 ---Cl E. w 2 0 ~ a: 0 :I: :I: ,_ ~ 0 a: (.!) 2

~ ~ <I

PLACEBO m-CPP MTG

PLACEBO m-CPP

+ m-CPP

• p<0.05, **p<0.01 Compared to Placebo tp<0.05, tt p<0.01 Compared to m-CPP

Figure 31.1 Hormonal effects of m-CPP (0.5 mglkg orally) in healthy human subjects before and after pre-treatment with metergoline (MfG; 4 mg orally). Bars represent mean maximum increase in plasma levels (peak minus baseline). Error

bars represent s.e. mean)

260 Serotonin

The neuroendocrine studies conducted in humans and reviewed above have all been done in normal volunteers. As yet, there are no published data regarding neuroendocrine responses to m-CPP in depressed patients, or in depressed patients receiving antidepressant drugs, to help in evaluating the studies with pre-synaptically acting serotonin agonists noted above. Studies in rodents have found altered neuroendocrine and food intake responses during chronic antidepressant drug treatment (Aulakh et al., 1987). Two studies evaluating m-CPP in patients with non-depressive psychiatric disorders (panic anxiety and obsessive-compulsive disorder) have found no differences in neuroendocrine responses to m-CPP between patients and controls (Charney et al., 1987; Zohar et al., 1987).

-...-E

.......... en c -:z -t-(.J <C _J CJ Ck: a...

30

20

10

0

ORAL vs IV m-CPP

60

t::. ORAL m-CPP • IV m-CPP o ORAL PRL e IV PRL

120 180 TIME (min)

60

40

20

3 I

n -u -u -:J (.Q

.......... 3 ....... -

Figure 31.2 A comparison of the effects of m-CPP administered orally (0.5 mg/kg) or i.v. (0.1 mglkg) on plasma prolactin in healthy human volunteers. Plasma prolactin concentrations are indicated by the circles and plasma m-CPP

concentrations by the triangles


BEHAVIOURAL EFFECTS OF m-CPP

In rodents, m-CPP differs from many other serotonin agonists in decreasing locomotor activity, decreasing food intake, and in not producing any components of the so-called serotonin syndrome of behaviours; similarly, in drug discrimination paradigms, the close structural analog of m-CPP, trifluoromethylpiperazine, was not distinguished from m-CPP but was differentially discriminated from 8-hydroxy-2-( di-n-propylamino )tetralin (8-0H-DPAT), quipazine, lysergic acid diethylamide and 5-methoxydimethyltryptamine (McKenney and Glennon, 1986).

Minimal subjective or behavioural changes occurred in normal humans receiving 0.5 mg/kg m-CPP orally (Mueller et al., 1985b ). However, patients with obsessive-compulsive disorder receiving the same m-CPP dose under double-blind, placebo-controlled conditions experienced marked anxiety and other behavioural symptoms including a striking exacerbation of their obsessive-compulsive symptoms (Zohar et al., 1987). Patients with panic anxiety also experienced an exacerbation of symptoms when given m-CPP (Charney et al., 1987), but in this study, which used 0.1 mg/kg m-CPP administered i. v. over a 20 min period, the normal controls also responded with similar anxiety and other symptoms which were as severe as those found in the patients. Studies from our group have documented somewhat greater increases in anxiety, dysphoria, and activation self-ratings following the i. v. administration of 0.1 mg/kg m-CPP compared to the oral administration of 0.5 mg/kg of the drug (Murphy et al., unpublished observations). However, the behavioural changes and side-effects were not as great as those reported by Charney et al. (1987) after the same i.v. dose of m-CPP. One experimental condition which may have contributed to these different results came from our pilot studies, which showed that administering m-CPP at the same total dosage (0.1 mg/kg) over longer periods of time (5-20 min) was associated with more severe side-effects, compared with our finally chosen experimental procedure of administering m-CPP in bolus fashion over a 90 s time period. More studies are clearly needed in animals and humans of the influence of time of administration factors on the effects of m-CPP, because in addition to the bolus vs. slow i. v. injection vs. oral effect differences, anxiolytic rather than anxiogenic affects of m-CPP have been observed when the drug was given chronically over a period of 2 weeks to psychiatric patients (Mellow et al., unpublished observations). Fuller et al. (1981) had speculated that the antidepressant effects of the m-CPP parent molecule trazodone might be attributable to m-CPP, and while our findings have not yet provided any confirmatory data to support this hypothesis, it seems possible that m-CPP, like some other 5-HTrlike receptor agonists such as buspirone and gepirone (Goa and Ward, 1986), may prove to have anxiolytic therapeutic actions when given chronically.

262 Serotonin

CARDIOVASCULAR AND OTHER EFFECTS OF m-CPP

In our initial studies in humans, m-CPP given orally had negligible blood pressure or heart rate effects (Mueller et al., 1985b), but both in rhesus monkeys (Aloi et al., 1984) and in humans in our recent studies, modest elevations in systolic blood pressure ( + 11 ± 1 mmHg mean ± s.e. mean, P < 0.01), diastolic blood pressure and heart rate were observed after i.v. m-CPP. In rats, i.v. m-CPP produced dose-dependent blood pressure elevations which were antagonized by metergoline and ritanserin, but not by a-adrenoceptor blockade using a combination of prasozin and yohimbine (Bagdy et al., 1987). m-CPP was found to have similar effects in pithed adrenal-demedullated rats and in conscious intact rats, indicating that m-CPP was apparently acting directly on the peripheral cardiovascular system (Bagdy et al., 1987). An additional example of the sensitivity of certain patient groups to the effects of m-CPP was an unexpected observation of possible relevance to migraine. The vascular theory of migraine postulates an initial vasoconstrictor phase followed by the development of migraine headaches during a secondary phase of vascular dilatation (Olesen, 1987). In an incomplete study exploring the possible anorexic effects of m-CPP in patients with bulimia given m-CPP orally, the most striking observation initially was that of an unusual frequency of headaches, many of the migrainous type (Brewerton et al., 1988). These headaches did not occur during the time period of the immediate neuroendocrine, temperature, behavioural or cardiovascular responses to m-CPP, but rather 8-12 h after 0.5 mg/kg m-CPP administered orally. Headache occurrence was significantly more frequent in individuals with a personal or family history of migraine headaches than in those witho-ut such a history. Since the discovery of this side-effect, administration of m-CPP to individuals with a migraine history has been avoided. However, the possible usefulness of the cardiovascular stimulatory effect of m-CPP and similar agents may be worthy of further exploration because their mode of action is different from that of conventional sympathomimetic drugs.

One other effect of m-CPP deserves mention. Penile erections were regularly observed following i. v. m-CPP in our studies in rhesus monkeys (Aloi et al., 1984), but not in humans given m-CPP orally (Mueller et al., 1985b). Further studies in monkeys in our laboratory revealed that the erectile effect of m-CPP was also obtained using other piperazine-type serotonin agonists and fenfluramine, but not the 5-HT1A agonists 8-0H-DPAT or buspirone (Szele et al., unpublished observations). Similar observations have recently been made in rodents (Berendsen and Broekkamp, 1987). Trazodone, the parent compound of m-CPP, is unusual among antidepressants in being associated with an uncommon but noteworthy occurrence of priapism, which in some instances has required surgical intervention.


CONCLUSIONS

This review has presented an overview indicating that m-CPP and other agents believed to work through central and/or peripheral serotoninergic mechanisms have behavioural, neuroendocrine, cardiovascular and other physiological effects in humans which, in general, resemble those seen in other species, especially rodents and non-human primates. As reviewed above, limited antagonist studies in humans suggest that many of the central effects of m-CPP are most likely mediated via 5-HTrlike receptors, while evidence from animal studies indicates that m-CPP may possess cardiovascular actions mediated by 5-HT2 receptors (Bagdy et al., 1987) and that m-CPP may also interact at 5-HT3 receptors (Ireland and Tyers, 1987) which are located both peripherally and centrally. Additional pharmacological studies should be of value in gaining a better understanding of drugand disease-related changes in serotoninergic function in humans.

REFERENCES

Aloi,J. A., Insel, T. R., Mueller,E. A. and Murphy, D. L. (1984). Neuroendocrine and behavioural effects of m-chlorophenylpiperazine administration in rhesus monkeys. Life Sci., 34, 1325-1331

Aulakh, C. S., Cohen, R. M., Hill, J. L., Murphy, D. L. and Zohar, J. (1987). Long-term imipramine treatment enhances the locomotor and food intake suppressant effects of m-CPP in rats. Br. J. Pharmacal., 91, 747-752

Azmitia, E. C. and Gannon, P. J. (1986). In Fahn, S., Marsden, C. D. and Van Woert, M. H. (eds), Advances in Neurology, Vol. 43, Myoclonus, Raven Press, New York, pp. 407-468

Bagdy, G., Szemeredi, K., Zukowska-Grojec, Z., Hill, J. and Murphy, D. L. (1987). m-Chlorophenylpiperazine increases blood pressure and heart rate in pithed and conscious rats. Life Sci., 41, 775-782

Berendsen, H. H. G. and Broekkamp, C. L. E. (1987). Drug-induced penile erections in rats: indications of serotonin18 receptor mediation. Eur. J. Pharmacal., 135, 279-287

Brewerton, T. D., Murphy, D. L., Mueller, E. A. and Jimerson, D. C. (1988). Induction of migraine-like headaches by the serotonin agonist, mchlorophenylpiperazine. Clin. Pharmacal. Ther., 43, 605-609

Charney, D. S., Woods, S. W., Goodman, W. K. and Heninger, G. R. (1987). Serotonin function in anxiety. II. Effects of the serotonin agonist MCPP in panic disorder patients and healthy subjects. Psychopharmacology, 92, 14-24

Fuller, R. W., Snoddy, H. D., Mason, N. R. and Owen, J. E. (1981). Disposition and pharmacological effects of m-chlorophenylpiperazine in rats. Neuropharmacology, 20, 155-162

Goa, K. L. and Ward, A. (1986). Buspirone: A preliminary review of its pharmacological properties and therapeutic efficacy as an anxiolytic. Drugs, 32, 114-129

Heninger, G. R., Charney, D. S. and Sternberg, D. E. (1984). Serotonergicfunction in depression: prolactin response to intravenous tryptophan in depressed patients and healthy subjects. Arch. Gen. Psychiat., 41, 398-402

264 Serotonin

Heuring, R. E., Schlegel, J. R. and Peroutka, S. J. (1986). Species variations in RU 24969 interactions with non-5-HT1A binding sites. Eur. J. Pharmacal., 122, 279-282

Ireland, S. J. and Tyers, M. D. (1987). Pharmacological characterization of 5-hydroxytryptamine-induced depolarization of the rat isolated vagus nerve. Br. J. Pharmacal., 90, 229-238

McKenney, J. D. and Glennon, R. A. (1986). TFMPP may produce its stimulus effects via a 5-HT1B mechanism. Pharmacal. Biochem. Behav., 24, 43-47

Meltzer, H. Y. and Lowy, M. T. (1987). The serotonin hypothesis of depression. In Meltzer, H. Y. (Ed.), Psychopharmacology: The Third Generation of Progress, Raven Press, New York, pp. 513-526

Meltzer, H. Y., Uberkoman-Wiita, B., Robertson, A., Tricou, B. J. and Lowy, M. (1983). Enhanced cortisol response to 5-hydroxytryptophan in depression and mania. Life Sci., 33, 2541-2549

Mueller, E. A., Murphy, D. L., Sunderland, T. and Jones, J. (1985a). A new postsynaptic serotonin receptor agonist suitable for studies in humans. Psychopharmacol. Bull., 21, 701-704

Mueller, E. A., Murphy, D. L. and Sunderland, T. (1985b). Neuroendocrine effects of m-chlorophenylpiperazine, a serotonin agonist, in humans. J. Clin. Endocrinol. Metab., 61, 1179-1184

Mueller, E. A., Murphy, D. L. and Sunderland, T. (1986). Further studies of the putative serotonin agonist, m-chlorophenylpiperazine: Evidence for a serotonin receptor mediated mechanism of action in humans. Psychopharmacology, 89, 388-391

Murphy, D. L., Mueller, E. A., Garrick, N. A. and Aulakh, C. S. (1986). Use of serotonergic agents in the clinical assessment of central serotonin function. J. Clin. Psychiat., 41, 9-15

Oleson, J. (1987). The ischemic hypotheses of migraine. Arch. Neural., 44, 321-322 Samanin, R., Mennini, T., Ferraris, A., Bendotti, C., Borsini, F. and Garattini, S.

(1979). m-Chlorophenylpiperazine: A central serotonin agonist causing powerful anorexia in rats. Naunyn-Schmiedeberg's Arch. Pharmacal., 308, 159-163

Siever, L. J., Murphy, D. L., Slater, S., de Ia Vega, E. and Lipper, S. (1984). Plasma prolactin changes following fenfluramine in depressed patients compared to controls: An evaluation of central serotonergic responsivity in depression. Life Sci., 34, 1029-1039

Zohar, J., Mueller, E. A., Insel, T. R., Zohar-Kadouch, R. and Murphy, D. L. (1987). Serotonergic responsivity in obsessive-compulsive disorder: comparison of patients and healthy controls. Arch. Gen. Psychiat., 44, 946-951

32 Pharmacology and Function of Melatonin

Receptors in the Mammalian Central Nervous System

M. L. Dubocovich

Department of Pharmacology, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, IL 60611, USA

INTRODUCTION

The hormone melatonin is synthesized in the pineal gland, retina and Harderian gland of the vertebrates from its precursor serotonin (5-hydroxytryptamine). Serotonin is N-acetylated by a relatively specific N-acetyltransferase to yield N-acetylserotonin, which in tum is methylated by hydroxyindole-o-methyltransferase to melatonin (5-methoxy-Nacetyltryptamine) (Axelrod, 1974).

An increasing body of evidence suggests that the hormone melatonin, whose synthesis and secretion exhibits a circadian rhythm, regulates a number of physiological functions in vertebrates. These include regulation of reproduction in photoperiodic mammals (Darrow and Goldman, 1985), control of circadian rhythms in birds and reptiles (Menaker, 1982), modulation of retinal physiology (Besharse and Dunis, 1983; Dubocovich, 1983), and a possible role in the regulation of chronobiological mood and sleep disorders (Lewy and Sack, 1986; Lewy et al., 1987). These effects of melatonin are thought to occur primarily in the CNS; however, the understanding of the site and mechanism of action of this hormone has been hampered by technical difficulties in receptor characterization, and the lack of a soundly based pharmacology. The recent discovery that melatonin is a potent modulator of dopamine release in retina (Dubocovich, 1983, 1985), and the synthesis of the new radioligand 2-[1251]-iododomelatonin (Vakkuri et al., 1984; Dubocovich et al., 1986; Dubocovich and Takahashi, 1987) have allowed us to begin the pharmacological characterization and localization of melatonin receptor sites in the CNS, and the search for potent and selective melatonin receptor antagonists.

266 Serotonin

This review will focus on the pharmacology and function of melatonin receptors in the CNS, their pharmacological differences from serotonin receptor sites and the role of this hormone in regulating photoperiodicinduced changes in physiological processes.

MELATONIN RECEPTORS MEDIATING FUNCTIONAL RESPONSES IN THE CNS

Classically, melatonin responses have been studied on amphibian dermal melanocytes (Heward and Hadley, 1975). Recently, melatonin has been shown to inhibit the release of [3H]-dopamine from the mammalian CNS (Dubocovich, 1983). In the chicken and rabbit retina, picomolar concentrations of melatonin selectively inhibit the Ca2+ -dependent release of dopamine through activation of a site possessing the pharmacological and functional characteristics of a specific receptor (Figure 32.1; Dubocovich, 1983, 1984, 1985). Melatonin is about 1000 times more potent than D-2 dopamine, opiate and aradrenoceptor agonists in inhibiting the Ca2+dependent release of [3H]-dopamine (Figure 32.1; Dubocovich, 1983, 1985). The site activated by melatonin in retina is pharmacologically distinct from serotonin receptors (Dubocovich, 1983). Serotonin did not affect the Ca2 + -dependent release of [3H]-dopamine, and serotonin antagonists such as spiperone, methysergide and methiothepin did not change the responses to melatonin. The potent inhibitory effect of melatonin on dopamine release was mimicked by other 5-methoxyindoles possessing an acetamidoethyl group in the C-3 position (i.e. 2-iodomelatonin, 6-chloromelatonin, 6, 7 -dichloro-2-methylmelatonin, 6-hydroxymelatonin, 6-methoxymelatonin). Structure-activity relationships suggest that the efficacy of melatonin and related indoles in inhibiting the Ca2+ -dependent release of [3H]-dopamine from rabbit retina is determined by the moiety (methoxy) on C-5 of the indole nucleus, whereas the affinity for the receptor is determined primarily by the moiety (acetamidoethyl) on C-3 (Heward and Hadley, 1975; Dubocovich, 1985). In support of this conclusion, the most potent inhibitors (agonists) of [3H]-dopamine release were 5-methoxy-Nacetyltryptamines such as 2-iodomelatonin, 6-chloromelatonin and 6,7-dichloro-2-methylmelatonin (Dubocovich, 1985; Dubocovich et al., 1986). According to this hypothesis, N-acetyltryptamines lacking a 5-methoxy group will be melatonin receptor antagonists (Heward and Hadley, 1975; Dubocovich, 1985).

N-Acetyltryptamine, an indole with no substitution on the indole nucleus, antagonized the melatonin-induced lightening of the frog skin (Heward and Hadley, 1975). Similarly, in the chicken retina N-acetyltryptamine (10-1000 nM), although not inhibiting the Ca2+ -dependent release of [3H]-dopamine, competitively antagonized the melatonin-induced inhibition of [3H]-

Melatonin Receptors 267

dopamine release (Dubocovich, 1984, 1985). In the rabbit retina, N-acetyltryptamine (10, 30 and 100 nM) shows the pharmacological properties of a partial agonist, inhibiting the Ca2 + -dependent release of dopamine when tested alone, and shifting the concentration-effect curve for melatonin to the right (Dubocovich, 1985).

Luzindole (N-0774) is an N-acetyltryptamine with a novel chemical structure that was recently found to be a competitive melatonin receptor antagonist (Dubocovich, 1988). Luzindole did not modify the spontaneous or stimulation-evoked release of [3H] from rabbit retina labelled in vitro with [3H]-dopamine (Dubocovich, 1988). Figure 32.1 shows that luzindole (1 ~-tM)

3 0 _.J u.. a: UJ > o-.... wen :z' H N 2:E:!! < a.. 0 Cl I

:I: 111

1.0

* 0.5

0 .0 ...___.__.__ c 0.1 nil

MEL

*

0.1 ...

APO

*

1,111

CI..O

*

c 0.1 nil 0.1 ....

MEL APO

LUZINOOLE 1 11M

Figure 32.1 Luzindole antagonizes the melatonin-induced inhibition of [3H)dopamine release from rabbit retina. Retinas from albino rabbits were dissected and labelled with fH]-dopamine as previously described (Dubocovich, 1985). Ordinate: [3H)-Dopamine overflow is the percentage of total tissue radioactivity released by field stimulation (3Hz, 2 min, 20 rnA, 2 ms) above the spontaneous levels of release. Results are expressed as means ± s.e. mean of the ratios obtained between the second (S2) and the first (S1) stimulation periods within the same experiment. The spontaneous outflow of radioactivity during the 4 min preceding the first period of stimulation was 1.1 ± 0.08 per cent (n = 14). The per cent of total tissue radioactivity release above basal levels of release was 2.3 ± 13 per cent in S1 and the ratio Sz/S1 in the absence of any drugs (left column C) was 0.98 ± 0.05 (n = 14). Melatonin (MEL, 0.1 nM), apomorphine (APO, 0.1 JA.M) orclonidine (CLO, 1 JA.M) was added 20 min before the second period of stimulation. Luzindole (1 JlM) was added 40 min before the first period of stimulation (S1) and remained present during S2 • *Significantly different from the corresponding control overflow ratio (C) in the

absence of release inhibitors (P < 0.05)

268 Serotonin

completely antagonized the inhibition of the Ca2 + -dependent release of [3H]-dopamine elicited by melatonin (0.1 nM). Luzindole at concentrations of 0.1, 1 and 10 !.I.M produced parallel shifts to the right of the concentration-effect curves for melatonin. The K8 of lizindole for the retinal melatonin receptor determined by Schild analysis is 20 OM (Dubocovich, 1988). In the rabbit retina, luzindole did not modify the inhibition of [3H]-dopamine release elicited through activation of either D-2 dopamine autoreceptors by apomorphine or az-adrenoceptor by clonidine (Dubocovich, 1983, 1986). In summary, luzindole is a competitive melatonin receptor antagonist with high potency and selectivity, which can be used to understand further the functional role of melatonin in mammals.

PHARMACOLOGICAL PROFILE OF THE MELATONIN RECEPTOR SITES LABELLED BY 2-[1251]-IOOOMELATONIN IN THE RETINA AND

BRAIN

Although [3H]-melatonin has been reported to bind to sites in cytosolic and membrane fractions of several CNS tissues, including the retina of lower vertebrates, these sites have not been associated with biological effects of melatonin and related indoles (Cardinali, 1981; Weichmann et al., 1986). Further, the relatively low specific activity of [3H]-melatonin, compared with that of radioiodinated ligands, may have hindered detection of high-affinity binding sites in tissues with a low density of receptors (Cardinali, 1981). To detect melatonin receptor sites in vertebrate retina, 2-[125I]-iodomelatonin, an iodinated ligand of high specific activity and high potency, has been utilized to inhibit the Ca2+ -dependent release of [3H]-dopamine from chicken and rabbit retina in vitro (Dubocovich et al., 1986; Dubocovich and Takahashi, 1987).

The specific binding of 2-F25I]-iodomelatonin both in chicken retina and hamster brain membranes fulfils all the criteria for binding to a receptor site, being stable, reversible, saturable and of high affinity (Dubocovich et al., 1986; Duncan et al., 1986, 1988; Dubocovich and Takahashi, 1987). The affinity constants for 2-F25I]-iodomelatonin derived from Scatchard analysis in saturation experiments were: K0 = 0.45 ± 0.05 nM (mean ± s.e. mean, n = 5) in chicken retina; and K0 = 3.8 ± 0.2 nM (n = 4) in hamster brain (Duncan et al., 1986, 1988; Dubocovich and Takahashi, 1987). The number of 2-P25I]-iodomelatonin binding sites found in chicken retina (Bmax = 74.0 ± 13.6 fmoVmg of protein) was similar to that in the hamster brain (Duncan et al., 1986). 2-P25I]-Iodomelatonin appears to label a single class of sites in membranes from chicken retina or hamster brain.

Pharmacological characterization of the 2-P25I]-iodomelatonin binding site in the retina shows that melatonin and related indoles inhibited binding with the same order of potency as that found for inhibition of [3H]-dopamine


release from chicken and rabbit retina (Table 32.1; Dubocovich, 1985 and Takahashi, 1987). On the basis of both the 2-P25I]-iodomelatonin binding and the functional responses to indoleamines, it was suggested that a retinal melatonin receptor is characterized by the following pharmacological order of potencies: 2-iodomelatonin > 6-chloromelatonin ~ melatonin ~ 6,7-dichloro-2-methylmelatonin > 6-hydroxymelatonin ~ 6-methoxymelatonin > N-acetyltryptamine > N-acetyl-5-hydroxytryptamine > 5-methoxytryptamine >> 5-hydroxytryptamine (inactive).

In hamster brain membranes, 2-[1251]-iodomelatonin labels a melatonin binding site whose pharmacological characteristics, although similar, are not identical to those found for the binding site on retinal membranes (Table 32.1). The most striking difference is the high potency of N-acetyl-5-hydroxytryptamine and 6-methoxymelatonin in competing for 2-P251]iodomelatonin binding in hamster brain (Duncan et a/., 1986). In brain membranes, N-acetyl-5-hydroxytryptamine is equipotent with melatonin in binding (Table 32.1). Furthermore, in hamster brain membranes the affinity of 2-P25I]-iodomelatonin is lower, and the rates of association and dissociation are higher than in the chicken retina (Duncan eta/., 1986, 1988; Dubocovich and Takahashi, 1987). These results suggest that 2-[1251]iodomelatonin labels two distinct sites in hamster brain and retina

Table 32.1 Competition of 2-[1251]-iodomelatonin and [3H]-melatonin binding by various indoles in retina and brain membranes

Inhibitor

2-Iodomelatonin 6-Chloromelatonin Melatonin 6, 7 -Dichloro-2-methylmelatonin 6-Hydroxymelatonin 6-Methoxymelatonin N-Acetyltryptamine N-Acetyl-5-hydroxytryptamine 5-Methoxytryptophol 5-Methoxytryptamine 5-Hydroxytryptamine 5-Methoxy-N,N-dimethyltryptamine 5-Methoxyindole-3-acetic acid 5-Hydroxytryptophol Tryptamine 5-Hydroxyindoleacetic acid 5-Hydroxytryptophan

aDubocovich and Takahashi (1987).

2-[1251]-Iodomelatonin Ki(nm)

Chickena retina

2.5 4 6.3

10 74

460 1 600 3 000 4 600 4 600

10 000 >1 000 000

>100 000 >30 000

>100 000 >100 000 >100 000

Rabbit retina

1.6 0.8 1.3 2 1.6

63 100 300

>100 000

hDubocovich (unpublished observations). cDuncan et al. (1986, 1988). dCardinali (1981).

[3H]-Melatonin Ki(nm)

Hamster< Bovined brain hypo-

thalamus

5 4

11 10 130 110 5 000

9 1000

8 125 300 40

1000 125 3300 130

1400 >100 000 50

100 55 000 125

>100 000 1000 100 000 1300

270 Serotonin

membranes (Table 32.1). Whether the differences in binding characteristics of 2-[125I]-iodomelatonin reflect differences in species (i.e. hamster versus chicken or rabbit) or tissue (i.e. brain versus retina) remains to be determined.

The selectivity of 2-P25I]-iodomelatonin for melatonin sites is further strengthened by our data showing that drugs which interact with either dopamine, serotonin, a- or (3-adrenergic receptors are less potent than the N-acetyltryptamines in competing for this site (Duncan et al., 1986; Dubocovich and Takahashi, 1987). Moreover, 5-methoxy-N-acetyltryptamines which are potent at the 2-[125I]-iodomelatonin binding site (i.e. 6-chloromelatonin, 2-iodomelatonin, N-acetyl-5-hydroxytryptamine, 6,7-dichloro-2-methylmelatonin) do not compete for the binding of [3H]-5-hydroxytryptamine to 5-HT1 and [3H]-ketanserin to 5-HT2 sites in hamster brain membranes (Krause, personal communication). Taken together, these results indicate that 2-[125I]-iodomelatonin is a selective radioligand for labelling melatonin binding sites, without appreciable affinity for serotonin binding sites.

Other investigators (Zisapel et al., 1987) reported binding of 2-[125I]iodomelatonin to rat brain synaptosomal membranes, with affinities of the radioligand for the binding sites 10 times higher than those reported by us (Duncan et al., 1986, 1988). In addition, N-acetyl-5-hydroxytryptamine and 5-methoxytryptamine did not compete for 2-P25I]-iodomelatonin in rat brain membranes (Zisapel et al., 1987; Table 32.1). A number of differences in the experimental conditions used by the two laboratories (i.e. specific activity of 2-[125I]-iodomelatonin; temperature of incubation; animal species) could account for the discrepancies in the results.

The order of potency of melatonin and related indoles at the [3H]-melatonin binding site of bovine hypothalamus (Cardinali, 1981) is pharmacologically different from the site labelled by 2-[l25I]-iodomelatonin in hamster brain and chicken or rabbit retinal membranes (see Table 32.1). The most distinct difference is that with 6-hydroxymelatonin, which is very potent in inhibiting [3H]-dopamine release and in competing with 2-P25I]-iodomelatonin in retina and hamster brain, but is a poor inhibitor of [3H]-melatonin binding in bovine hypothalamus (Table 32.1). In contrast, indoles such as 5-hydroxytryptamine and 5-hydroxytryptophol, which are relatively poor competitors at the melatonin binding sites in chicken and rabbit retina and hamster brain, are the most potent inhibitors of [3H]-melatonin binding in bovine hypothalamus (Cardinali, 1981; Table 32.1). The results reported by Cardinali (1981) suggest that [3H]-melatonin binds to a site different from the melatonin binding site described here.

Several biochemical studies support the presence of melatonin sites within the brain. Melatonin and related indoles also affect the release of monoamines from the hypothalamus (Cardinali, 1981; Zisapel et al., 1982), and the levels of serotonin in rat mid-brain (Anton-Tay et al., 1968).


Biochemical responses mediated by melatonin in the central nervous system include stimulation of guanosine 3' ,5'-monophosphate (cyclic GMP) synthesis, decreases of prostaglandin ~ and adenosine 3' ,5'monophosphate (cyclic AMP) synthesis (Cardinali, 1981), and inhibition of depolarization-evoked Ca2+ uptake (Vacas et al., 1984).

ROLE OF MELATONIN IN MODULATING PHYSIOLOGICAL FUNCTIONS

Evidence suggests a role for melatonin in regulating reproduction and metabolism in mammals (Cardinali, 1981; Tamarkin et al., 1985; Darrow and Goldman, 1985). Melatonin appears to mediate photoperiodically induced changes in gonadal function and pituitary hormone secretion by acting primarily within the hypothalamus (Steger et al., 1984; Tamarkin et a/., 1985). In support of this view, high levels of 2-[125I]-iodomelatonin binding sites were found in the hypothalamus and pituitary gland of hamsters, a species in which the reproductive system is very sensitive to photoperiod (Duncan et al., 1988).

In the retina, melatonin has been implicated in photoreceptor outer segment disc shedding and phagocytosis, melanosome aggregation in retinal pigment epithelium, cone photoreceptor retinomotor movement (for references see Iuvone, 1985), as well as modulation of dopamine release in vitro and in vivo (Dubocovich, 1983, 1985, 1986). Melatonin is synthesized in a diurnal rhythm, with peak levels during the dark period, suggesting that melatonin secreted by photoreceptors may regulate diurnal events that normally occur in the retina (Besharse and Dunis, 1983; Iuvone, 1985; Dubocovich, 1986).

Disorders of melatonin and rhythmicity in humans have been related to chronobiological psychic and sleep disorders due to shift work, jet lag, depression, schizophrenia and sexual maturation. Administration of melatonin at scheduled times has been found to be effective in shifting circadian rhythms (Redman eta/., 1983), and is being effectively used to treat sleep disturbances due to jet lag (Arendt eta/., 1986). Exposure to bright light is now being used experimentally to treat seasonal mood and sleep disorders, which are characterized by altered circadian patterns of melatonin secretion (Lewy and Sack, 1986; Lewy et al., 1987). Recent experiments from the author's laboratory suggest that the melatonin receptor antagonist luzindole may exert antidepressant activity in the mouse behavioural despair test by blocking the effect of endogenous melatonin in the CNS (Mogilnicka and Dubocovich, 1987). Luzindole decreased the duration of immobility during swimming in C3H/HeN mice; this effect was more pronounced at midnight when the levels of melatonin in the pineal gland are high (Mogilnicka and Dubocovich, 1987). The melatonin receptor

272 Serotonin

antagonist luzindole may mimic the effect of light by blocking the effects of melatonin, and have potential therapeutic value to treat chronobiological mood and sleep disorders involving changes in the pattern of melatonin secretion.

ACKNOWLEDGEMENTS

The author thanks Nelson Research for the generous supply of luzindole (N-0774), Mr Patrick Rita for excellent technical assistance, Dr Diana N. Krause for helpful discussions when conducting this research and Ms Vicky James-Houff for excellent secretarial assistance. Supported by Nelson Research and by USPHS grants RR 05470 and MH 42922.

REFERENCES

Anton-Tay, F., Chou, C., Anton, S. and Wortman, R. J. (1968). Brain serotonin concentration: Elevation following intraperitoneal administration of melatonin. Science, 162, 277-279

Arendt, J., Aldhous, M. and Marks, V. (1986). Alleviation of jet lag by melatonin: Preliminary results of controlled double blind trial. Br. Med. ]., 292, 1170

Axelrod, J. (1974). The pineal gland: a neurochemical transducer. Science, 184, 1341-1348

Besharse, J. C. and Dunis, D. A. (1983). Methoxyindoles and photoreceptor metabolism: activation of rod shedding. Science, 219, 1341-1343

Cardinali, D. P. (1981). Melatonin: A mammalian pineal hormone. Endocrinol. Rev., 2, 327-346

Darrow, J. M. and Goldman, B. D. (1985). Circadian regulation of pineal melatonin and reproduction in the Djungarian hamster. J. Bioi. Rhythms, l, 39--54

Dubocovich, M. L. (1983). Melatonin is a potent modulator of dopamine release in the retina. Nature, 306, 782-784

Dubocovich, M. L. (1984). N-acetyltryptamine antagonizes the melatonin-induced inhibition of (3H]-dopamine release from retina. Eur. J. Pharmacol., lOS, 193--194

Dubocovich, M. L. (1985). Characterization of a retinal melatonin receptor. J. Pharmacol. Exp. Ther., 234, 395-401

Dubocovich, M. L. (1986). Modulation of dopaminergic activity by melatonin in retina. In O'Brien, P. J. and Klein, D. C. (Eds), Pineal and Retinal Relationships, Academic Press, New York, pp. 239--252

Dubocovich, M. L. (1988). Luzindole (N-0774): a novel melatonin receptor antagonist. J. Pharmacol. Exp. Ther., 246, 902-910

Dubocovich, M. L. and Takahashi, J. S. (1987). Use of 2-P25I]-iodomelatonin to characterize melatonin binding sites in chicken retina. Proc. Natl. Acad. Sci. U.S.A., 184, 3916-3920

Dubocovich, M. L., Nikaido, S. S. and Takahashi, J. S. (1986). 2-P25I]Iodomelatonin: A new radioligand for characterization of melatonin receptors. Soc. Neurosci. Abst., 12, 995

Duncan, M. J., Takahashi, J. S. and Dubocovich, M. L. (1986). Characterization of 2-P25I]-iodomelatonin binding sites in hamster brain. Eur. J. Pharmacol., 132, 333--334


Duncan, M. J., Takahashi, J. S. and Dubocovich, M. L. (1988). 2-[1251]Iodomelatonin binding sites in hamster brain membranes: Pharmacological characteristics and regional distribution. Endocrinology, 122, 1825-1833

Heward, C. B. and Hadley, M. E. (1975). Structure-activity relationships of melatonin and related indoleamines. Life Sci., 17, 1167-1178

luvone, P. M. (1985). Neurotransmitters and neuromodulators in the retina: regulation, interactions, and cellular effects. In Adler, R. and Farber, D. (Eds), The Retina: A Model for Cell Biology Studies, Academic Press, New York, pp. 2-72

Lewy, A. J. and Sack, R. L. (1986). Light therapy and psychiatry. Proc. Soc. Exp. Bioi. Med., 183, 11-18

Lewy, A. J., Sack, R. L., Miller, L. S. (1987). Antidepressant and circadian phase-shifting effects of light. Science, 235, 352-354

Menaker, M. (1982). The search for principles of physiological organization in vertebrate circadian systems. In Aschoff, J., Daan, S. and Groos, G. (Eds), Vertebrate Circadian Systems, Springer, Berlin, pp. 1-12

Mogilnicka, E. and Dubocovich, M. L. (1987). Effect of the melatonin receptor antagonist luzindole (N-0774) in the mouse behavioral despair test. Soc. Neurosci. Abst., 13, 1039

Redman, J., Armstrong, S. and Ng, K. T. (1983). Free-running activity rhythms in the rat: Entrainment by melatonin. Science, 219, 1080--1081

Steger, R. W., Bartake, A., Matt, K. S., Soares, M. J. and Talamantes, F. (1984). Neuroendocrine changes in male hamsters following photostimulation. J. Exp. Zoo/., 229, 467-474

Tamarkin, L.,Baird, C. J. and Almeida, 0. F. X. (1985). Melatonin: A coordinating signal for mammalian reproduction. Science, 227, 714-720

Vacas, M. 1., Keller-Sarmiento, M. I. and Cardinali, D. P. (1984). Pineal methoxyindoles depress calcium uptake by rat brain synaptosomes. Brain Res., 294, 166-168

Vakkuri, 0., Leppaluoto, J. and Vuolteenaho, 0. (1984). Development and validation of a melatonin radioimmunoassay using radioiodinated melatonin as tracer. Acta Endocrinol., 106, 152-157

Weichmann, A. F., Bok, D. and Horowitz, J. (1986). Melatonin-binding in the frog retina: Autoradiographic and biochemical analysis. Invest. Ophth. Vis. Sci., 27, 153--163

Zisapel, N., Egozi, Y. and Laudon, M. (1982). Inhibition of dopamine release by melatonin: Regional distribution in the rat brain. Brain Res., 246, 161-163

Zisapel, N., Shaharabani, M. and Laudon, M. (1987). Regulation of melatonin's activity in the female rat brain by estradiol: effects on neurotransmitter release and on iodomelatonin binding sites. Neuroendocrinology, 46, 207-216

33 Pathophysiology of 5-Hydroxytryptamine:

An Overview+

J. A. Angus1 and J. M. Van Nueten2

1Baker Medical Research Institute, PO Box 348, Prahran, Vic. 3181, Australia

2International Research Council, Janssen Research Foundation, B-2340 Beerse, Belgium

INTRODUCTION

In the workshop forum, the possible pathophysiological role of serotonin (5-hydroxytryptamine; 5-HT) was discussed, especially in relation to the vascular system and the CNS. The following is a summary of the main points, which also incorporates material presented in the pathophysiology session.

MIGRAINE

Migraine is characterized as an episodic headache, commonly unilateral, associated with nausea, vomiting and photophobia. There are three phases: premonitory symptoms, prodromal aura associated with constriction of cerebral vessels, and the headache phase; in approximately two-thirds of cases, however, the aura phase is absent.

James Lance and his colleagues have established that the mean blood level of serotonin falls during migraine headache, and also during chronic tension headache (chronic daily headache). Serotonin infusion prevented the headache, while serotonin depletion after reserpine induced a migraine headache. Recent work from his laboratory on monkeys has explored the possibility that the CNS can induce the cerebral cortical blood flow changes associated with migraine, and can affect the endogenous pain control pathway. Stimulation of the monkey locus coeruleus at 5-10Hz caused a fall in blood flow of 20 per cent on the ipsilateral side, while stimulation of the +This overview incorporates material presented during the pathophysiology session, and material discussed in a workshop chaired by the authors.

Overview: Pathophysiology 275

raphe raised the· flow to the cerebral cortex by a similar margin. It was proposed that activation of the brain stem nuclei by emotional, hypothalamic activity or external trigger factors produces the cortical microcirculation changes and hence neurological phenomena of migraine. During a phase of monoamine depletion, the pain pathway (or gating) is opened and the flow of neuronal traffic from the head and neck is disinhibited, thus contributing to the pain of migraine.

To investigate the vascular aspects, Pramod Saxena has used the technique of radioactive microspheres for determining cerebral blood flow distribution, principally in pigs. Microspheres 15 JLm in diameter pass up the carotid artery, and are trapped by the carotid resistance-capillary network or pass through opened arteriovenous anastomoses (A VAs) and are ultimately trapped in the lung vasculature. According to Heyck's theory, these A VAs or shunts open up in migraine attacks, causing a relative ischaemia or steal of blood flow in the cerebral resistance beds. The fact that serotonin infusion relieved migraine suggests the possibility that serotonin may divert blood through the resistance bed by closing the A VAs. Saxena showed that in the pig, serotonin did not substantially alter the total carotid blood flow, but markedly reduced flow in the A VAs while increasing the capillary flow. These actions in closing the A VAs can be mimicked by the 5-HT 1 receptor agonist 5-carboxamidotryptamine ( 5-Cf), by methysergide, or by ergotamine; methiothepin will block these effects. The 5-HT receptor antagonists mianserin, cyproheptadine and ketanserin, and also phentolamine, were ineffective, thus the receptor on the A VAs can be classed as 5-HT1-like.

Recent data from Patrick Humphrey and his group showed that AH 25086 (100 JLg/kg i.v.) in anaesthetized cats has a remarkably selective vasoconstrictor action localized to the carotid A VAs. Preliminary studies in patients with migraine indicate that AH 25086 is an effective treatment, supporting the concept that cranial A VAs may be associated with migraine and therefore a target for antimigraine therapy. These 5-HT1-like receptor agonists (e.g. AH 25086, 5-Cf), as classified from functional studies, contract the dog saphenous vein (see Humphrey and Feniuk, this volume), but some agonists within this broad classification (e.g. 5-Cf but not AH 25086) may also relax vascular tissue via 5-HT 1-like receptor subtypes which mediate either an endothelium-independent direct relaxant action, as in the cat saphenous vein, or an endothelium-dependent mechanism, as in the rabbit jugular vein (see Humphrey and Feniuk, and Leff and Martin, this volume). One important consideration raised by a number of speakers was that constriction of the A VAs could also be produced by ergotamine and, to a lesser extent, methysergide. In addition, ergometrine is a powerful constrictor agent on the dog saphenous vein. Therefore, since ergometrine has been used to induce Prinzmetal's variant angina as a diagnostic tool, would not the 5-HT1-like receptor agonist AH 25086 and related

276 Serotonin

antimigraine drugs run the risk of causing coronary vasospasm? It was pointed out by Humphrey that there is no evidence for this in either the animal or the clinical studies to date. It was also noted that there is no hard evidence that patients with migraine also suffer from variant angina, suggesting that the underlying causes of the two vascular pathologies are probably different.

Turning to the pain and nausea associated with migraine, John Fozard reported that there was some evidence suggesting that the 5-HT 3 receptor antagonist MDL 72222 reduced these symptoms. However, Brian Richardson reported that another 5-HT3 receptor antagonist, ICS 205-930, did not affect the pain if given within 30 min of the headache starting, but reduced the nausea and vomiting. Clearly, the value of blocking 5-HT3 receptors in the treatment of migraine remains to be determined. The discovery of 5-HT 3 binding sites in the CNS, and their possible involvement in some behavioural actions (see Tyers et al., this volume), raises the question of the relative importance of peripheral and central 5-HT3 receptors in migraine.

HYPERTENSION

The potential for serotonin to be released from platelets, or from sympathetic nerve endings, and then to amplify other vasoconstrictor stimuli, is an attractive hypothesis to explain the prime initiating cause or maintenance of hypertension. The advent of ketanserin, a 5-HT2 receptor antagonist that lowers blood pressure, especially in the elderly, sparked the expected reaction from scientists to determine whether serotonin has a role in hypertension. Since ketanserin was found to have both 5-HT2 receptorand a 1-adrenoceptor-blocking activity in vascular preparations at therapeutic antihypertensive plasma concentrations, it was not as useful a tool as was initially thought. Indeed, recent trials have shown that ritanserin, a 5-HT2 receptor antagonist with a higher 5-HTiaradrenergic receptor selectivity ratio, does not lower blood pressure. One interesting point was that ketanserin has been shown to lower blood pressure in patients with autonomic insufficiency whereas phentolamine does not. This finding may distinguish the 5-HT2 receptor-blocking activity of ketanserin from that at the aradrenoceptor. However, phentolamine is a partial agonist at a 2-adrenoceptors, and will raise blood pressure, especially if the autonomic reflexes are blocked (see Angus and Lew, Br. J. Pharmacol., 81,423-425, 1984). Therefore, the key experiment would be to give an aradrenoceptor antagonist prior to ketanserin in these patients to determine whether the 5-HT2 receptor is the locus of the hypotensive action of ketanserin. The hypothesis has been put forward that the mechanism of action of ketanserin in hypertension is a rather complex one, involving a combination of its

Overview: Pathophysiology 2TI

vascular 5-HT 2 receptor-blocking activity, thereby inhibiting both the direct and the amplifying effects of serotonin, and its vascular aradrenoceptor blocking activity which is observed at higher doses. The central sympathoinhibitory effects of ketanserin, and the age-related variations in central and peripheral vascular 5-HT2 receptor function, as described in the overview on cardiac, vascular and other smooth muscle actions of serotonin (Purdy and Saxena, this volume), may also be of significance in attempting to evaluate the pathophysiological role of serotonin in hypertension.

VASCULAR REACTIVITY AFI'ER INTIMAL DAMAGE

Vascular reactivity after intimal damage was studied by James Angus and his colleagues. Endothelial cell damage or removal causes a platelet carpet to line the lumen; intimal thickening, comprising synthetic-state smooth muscle cells, then occurs ovet the next few weeks. In the normal carotid artery studied in instrumented conscious greyhound dogs, serotonin caused contraction of the large vessel (measured by sonomicrometry) but dilatation of the distal bed (Doppler flowmeter). Usually this large artery contraction was not sufficient to be flow-limiting, but when intimal thickening occurred, the contracted artery would often reduce blood flow to zero (spasm). In addition, new endothelial cells may produce less endothelium-derived relaxing factor (EDRF), further offsetting the balance of dilatation/ contraction in favour of contraction. In coronary arteries of dogs and pigs, serotonin causes a release of EDRF that offsets the 5-HT2 receptormediated contraction of the smooth muscle. Any loss of endothelium would thus induce a platelet adhesion-release reaction and focal constriction. In carotid arteries removed from rabbits 4 weeks after intimal damage, there was a small increase in sensitivity (i.e. lower EC50) to serotonin compared with control arteries, but the EDRF response to cholinomimetics was unchanged. These studies suggest that intimal thickening as a result of intimal damage may alter the reactivity of blood vessels through luminal encroachment, while EDRF function appears to be restored after 4 weeks. There still remains the possibility that alteration in the smooth-muscle cells in response to serum mitogens may enhance the constrictor reactivity to serotonin. These constrictor actions of serotonin are antagonized by ketanserin, while the vasodilatation of the resistance bed is unaffected. Saxena suggested that the proportion of 5-HT1-like to 5-HT2 receptors increases from the large carotid towards the smaller resistance vessels.

COLLATERAL REACTIVITY

Hollenberg has reported that collateral vessels of the dog and rabbit hind

278 Serotonin

limb, which are responsible for blood flow after saphenous artery ligation, are very sensitive to the constrictor action of serotonin. Similar findings have been reported in rats. This is an area of research that clearly requires more work to identify the agent specificity of the altered reactivity and the underlying mechanism. Involvement of 5-HT2 receptors is suggested by the observation that this constrictor action of serotonin is reversed by ketanserin. Altered reactivity of collateral vessels could be especially important in the coronary and cerebral circulations.

SEROTONIN AND DEPRESSION

Salomon Langer emphasized in his presentation that antidepressant drugs generally have in common the property of inhibiting serotonin uptake. Experimentally, the Na +-dependent uptake of serotonin in human platelets is significantly decreased in untreated depressed patients. Imipramine and paroxetine bind to the platelet serotonin transporter, and this marker may be a useful biochemical test in depressed patients. Langer showed that the binding-site density values in platelets from control patients were significantly higher than in the platelets from untreated depressed patients, both in males and in females, without any change in binding affinity. Since the original work in 1980, however, not all studies have found this relationship. Clearly, there needs to be caution in using platelet-binding data to predict binding activity in the CNS. However, Langer showed an example where the density of binding of imipramine in the cortex of suicide patients (post mortem) was significantly decreased compared with the binding in appropriately matched control brains. Also in post mortem studies on human brain from patients with a history of endogenous depression, hippocampus and occipital cortex binding densities of imipramine were lower than in matched brains from patients who had no history of depression (see Stanley et al., Science, 216, 1337-1339, 1982). Manfred Gothert raised the possibility that 5-HT autoinhibitory receptors could be antagonized, thereby increasing the concentration of serotonin in the synapse. Langer agreed that both inhibition of neuronal uptake and inhibition of autoinhibitory receptors might be suitable targets for the treatment of depression by increasing serotoninergic transmission.

SEROTONINERGIC NEUROTOXICITY IN THE CNS AND DESIGNER DRUG ABUSE

Stephen Peroutka recalled the tragic evidence that the 'designer drug' N-methyl-4-phenyl-1 ,2,3,6-tetrahydropyridine (MPTP) is a neurotoxic by-product of an illicit heroin substitute which depletes CNS dopamine

Overview: Pathophysiology 279

levels and causes idiopathic Parkinsonism. Studies by James Gibb and his colleagues showed that the 'designer drug' 3,4-methylenedioxymethamphetamine (MDMA; 'ecstasy') which is becoming increasingly popular, may be a serotonin neurotoxin. In rats given MDMA, there were long-term decreases in tryptophan hydroxylase activity in the neostriatum, hippocampus and frontal cortex that recovered to only 65 per cent, 40 per cent and 75 per cent of control levels after 110 days. Peroutka suggested that, as MPTP did for dopamine, the 'designer drugs' based on amphetamine analogues may damage the serotoninergic system, but may shed some light on the role of serotonin in the CNS.

Author Index

Angus, J.A. and Van Nueten, J.M. Pathophysiology of 5-hydroxytryptamrne: an overview 274

Angus, J.A., Wright, C.E. and Cocks, T.M. Vasculaf actions of serotonin in large and small arteries are amp1ified by loss of endothelium, atheroma and h1JT)erlension 225

Aulakh, C.S., see M~hy, D.L. 257 Bagdy, G., see Murphy, D.L. 257 BeVan, P., Olivier, B., Schipper, J.

and Mos, J. Serotoninergrc function and aggression m animals 101

Blue, D.R., see Clarke, D.E. 48 Bond, R.A., see Clarke, D.E. 48 Branchek, T.A., see Gershon, M.D.

37 Buchheit, K.-H., see Engel, G. 241 Buchheit, K.-H., see Richardson, B.P.

21 Chang, J.-Y., see Hardebo, J.E. 233 Charlton, K.G., see Oarke, D.E. 48 Clarke, D.E., Bond, R.A., Charlton,

K.G. and Blue, D.R. Pre-synaptic 5-HT receptors mediating inhibition ~transmitter releilse from perip~al cholinergic and noradrenergrc nerves 48

Cocks, T.M., see Angus, J.A. 225 Cossery, J.M., see Hamon, M. 169 Costalf, B., see Tyers, M.B. 95 Daval, G., see Hamon, M. 169 Davies, M., see Roberts, M.H.T. 70 de la Lande, I.S., Kennedy, J.A. and

Stanton, B.J. Amplifying action of 5-hydroxytryptamine in the rabbit ear artery f23

de la Lande, I.S. and Mylecharane, E.J. Neur01Ull actions of ~xyt;rtamine: an

Donatsch, P., see Richardson, B.P. 21

Dubocovich, M.L. Pharmacology and function ~melatonin receptors in tlie mammalian central neroous system 265

Dun, N.J., see Wallis, D.l. 31 El Mestikawy, S., see Hamon, M.

169 Emerit, M.B., see Hamon, M. 169 Engel, G., Buchheit, K.-H. and

Richardson, B.P. 5-HT3 receptors in the gastrointestinal tract 241

Engel, G., see Richardson, B.P. 21 Feniuk, W., see Humphrey, P.P.A.

159 Fozard, J.R. The deuelopment of

5-HT a receptor antagonists 12 Fozard, J.R., Mir, A.IC and Ramage,

A.G. 5-HT1A receptcJJ'S and cardiovascular control 146

Garrick, N.A., see Murphy, D.L. 257

Gershon, M.D., Mawe, G.M. and Branchek, T.A. Multiple 5-HT receptors in the enteric neroous system 37

GOthert, M. 5-HT receptors mediating pre-synaptic autoinhilJition in central serotoninergic nerve terminals 56

Gozlan, H., see Hamon, M. 169 Graham, D., see Langer, S.Z. 249 Hamon, M., Emerit, M.B., Ponchant,

M., Cossery, J.M., m Mestikawy, S., Verge, D., Daval, G. and Gozlan, H. New~hlmnacological tools for studies central 5-HT lA bindrng sites 1 9

Hardebo, J.E., Chang, J.-Y. and Owman, Ch. Sympathetic nerves associated with liram vessels store and releilse serotonin which interacts with noradrenaline in cerebrovascular contraction 233

Hartig, P.R. Serotonin 5-HT1c receptors: what do thev doT 180

Humphrey, P.P.A. and Feniuk, W. The subclassification of functional 5-HT rlike receptors 159

Humphrey, P.P.A. and Richardson, B.P. Clilssification of 5-HT receptors aiul binding sites: an croerview 204

282 Author Index

Janssen, P.A.J., see Van Nueten, J.M. 3

Janssens, W.J., see Van Nueten, J.M. 3

Kelly, J.S., Penington, N.J. and Rainnie, D.G. Need the autoregulation of raphe neurones involve 5-hydroxytryptamine? 64

Kennedy, J.A., see de la Lande, I.S. 123

Langer, S.Z. Behavioural actions of 5=hydroxytrwtamine: an overview 109

Langer, S.Z. and Graham, D. Different recognition sites for serotonin: the neuronal Na+ -dependent transporter and the releizse-modulating autoreceptor 249

Leff, P. and Martin, G.R The classification of 5-Iff receptors using tryptamine analogues 195

Martin, G.lt, see Leff, P. 195 Mawe, G.M., see Gershon, M.D. 37 Mir, A.K., see Fozard, J.R. 146 Mos, J., see Bevan, P. 101 Mueller, E.A., see M':J~Phy, D.L. 257 Murphy, D.L., Mueller, E.A.,

AUlakh, C.S., Bagdy, G. and Garrick, N.A. Sirotoninergic f!tnction in neuropsychilltric disorders 257

Murray, D.L., see Purdy, RE. 130 Mylecharane, E.J. and Phillips, C.A.

Pre-~naptic sympathetic inhibition and-5-hydroxytryptamine-induc:id VtiSodilatation 136

Mylecharane, E.J., see de la Lande, I.S. 77

Naylor, RJ., see Tyers, M.B. 95 Olivier, B., see Bevan, P. 101 Owman, Ch., see Hardebo, J.E. 233 Penington, N.J., see Kelly, J.S. 64 Peroutka, S.J. Charactenzation of

5-m1 bindTr site subtypes labelled by t Hl-5-hydroxytryptamine 188

PbiflJ's, C.A., see Mylecharane, E.J.

Ponchant, M., see Hamon, M. 169 Purdy, R.E. and Murray, D.L.

Serotonin-induced vasoconstriction and contractile syn~· with noradrenaline: role CHdrenoceptors 130

Purdy, E. and SaXena, P.R. Cardillc, vascular and other smooth muscle actions of 5-hydroxy~ine: an overview 152

Rainnie, D.G., see Kelly, J.S. 64 Rama~, A.G., see Fozard, J.R 146 Richardson, B.P., EnKel, G.,

Donatsch, P. and Buchheit, K.-H. 5-Iff receptors on afferent neurones 21

Richardson, B.P., see Engel, G. 241 Richardson, B.P., see Humphrey,

P.P.A. 204 Roberts, M.H.T. and Davies, M. In

vivo electrophysiology of receptors medlllting-the central nervous system actions of 5-hydroxytryptamine 7b

Saxena, P.R. Ciudillc actions of 5-hydroxytryptlmline 115

Saxena, P.R., see Purdy, RE. 152 Schipper, J., see Bevan, P. 101 Stanton, B.J., see de la Lande, I.S.

123 Tricklebank, M.D. Beluwioural

correlates of the activation of 5-Iff recep!ors 87

Tyers, M.B., Costall, B. and Naylor, RJ. 5-HT3 receptors in the central nervous system 95

Vanhoutte, P.M., see Van Nueten, J.M. 3

Van Nueten, J.M., Janssen, P.A.J., Janssens, W.J. and Vanhoutte, P.M. The development of 5-Iff2 receptor antagonists 3

Van N'ueten, J.M., see Angus, J.A. 274

Verg~, D., see Hamon, M. 169 Wallis, D.I. and Dun, N.J.

Electrophysiological investigation of the actions Of 5-hydro~~ine on sympathetic 81lnglionic neurones 31

Wnght, C.E., see Angus, J.A. 225

Subject Index

(Pa~e numbers cited are the commencing page numbers of the appropriate individual chapters. Abbreviations are used in accordance with the listing on pages xiii-xv)

a-adrenoceptor interactions 123, 130 AH25086 159 atheroma 225

BEA 1654 115 behavioural actions

activation 257 aggression 101 anxiety 87, 95, 257 dopamine-mediated motor

responses 87, 95, 257 dysphona 257 food intake 87, 257 forepaw treading 87 head shakes 87 human 257 interoceptive stimuli and drug

discrimination 87, 257 nociception 87 obsessive-compulsive activity

257 overview 109 sleep 87 startle reflex 87

blister base pain 21 BRL 24924 12,241 BRL 43694 12, 21, 95, 241 2-bromolysergide 115, 130 buspirone 4S, 87, 101, 146, 169, 188

Ca2+ current modulation in dorsal raphe neurones 64

Ca2+ -channel antagonists 233 carcinoid s~drome 12, 241 cardiac inhibition

CNS ~HT1A.receptors 146 overview 152 parasympathetic ganglion

stimulation ana ACh release 115

von Bezold-Jarisch reflex activation 115

cardiac stimulation catecholamine release via 5-HT 2

receptors 115

direct via atypical 5-HT receptors 115

direct via 5-HT 1-like receptors 115

indirect 'tyramine-like' 115 m-CPP '157 overview 152 sympathetic nerve depolarization

and NA release via 5-HT 3 receptors 12, 31, 115

cerebral vasospasm 233 cerebrovascular s~pathetic nerves

co-localization of 5-HT, NA and neuropeptide Y 233

uptake, storage and release of 5-HT 233

CGS 12066B 56 chlorimipramine 249, 257 chronob10logical mood disorders

265 circadian rhythms 265 citalopram 249 classification

5-HT binding sites overview 204 5-HT receptors

analytiCal criteria 195 overview 204 quantitative problems 195 use of tryptamine analogues 48,

195 CNS disorders 3, 56, 249, 257, 265 cocaine 115 (-)-cocaine 12 5-CT 31, 48, 70, 115, 136, 159, 188,

195,233 cyanopindolol 56,146, 159 cyproheptadine 31, 115 cytotoxic drug-induced emesis 12,

241

depression 56, 249, 257, 274 desi~ramine 249 diarrhoea 241 dopamine turnover in CNS 95 DP-5-CT 146

284 Subject Index

eltoprazine 101 enterochromaffin cells 241

feedback inhibition Ca2+ influx modulation 56 CNS serotoninergic nerve

terminal autoreceptors 56, 249

dorsal raphe neuronal cell body autoreceptors 64

fenfluramine 101, 257 flesinoxan 87,146 fluoxetine 87,249,257 fluprazine 101 fluvoxamine 101, 115, 257

gastric emptying inhibition 241 gepirone 87,169 GR 38032F 12, 21, 87, 95, 241 GR65630 95 guinea-pig ileum contraction

direct via smooth muscle 5-Hr 2 receptors 241

myenteric neurone stimulation ACh release 241 substance P release via 5-Hr 3

receptors 241

histamine H1 and H2 receptor interactions 123

[3H]-5-Me0-DPAC 169 [3H]-paroxetine 249 [3H]-spiroxatrine 188 5-Hrloinding sites

au oradiography in CNS preparations 169, 188

CNS membrane preparations 48, 159, 169, 188

functional correlates 188 5-Hr lA subunit purification 169 5Hr lC sites 180 irreversible alkylating ligands

169 photoaffinity probes 169 selective 5-ll'rlA._ligands 169, 188 subtypes 48, 15~, 188

5-Hr1A receptors behavioural actions

anxiety 87 food intake 87 forepaw treading 87 interoceptive stimuli and drug

discrimination 87 nociception 87 startle reflex 87

CNS cardiac inhibition 146 hypotension 146

dorsal raphe neuronal cell body hyperpolarization via autoreceptors 64

myenteric cholinergic nerve inhibition of ACh release 48

5-~lB receptors behavioural actions

aggression 101 interoceptive stimuli and drug

discnmination 87 startle reflex 87

rat CNS serotoniner~c nerve terminal inhib1tion of 5-Hr release via autoreceptors 56,249

5-Hr lC receptors choroid p1exus phosphatidyl

inositol response 180 cloning 180 rat stomach fundus contraction

180 Xen'!P.us oocyte CI- current 180

5-m 1-like receptors agonist structure-activity 48, 195 behavioural actions

aggression 101 anxiety 257 food intake 87 sleep 87

brain-stem neuronal inhibition of firing 70

cardiac stimulation 115 CNS serotoninergic nerve

terminal inhibition of 5-Hr release via autoreceptors 56,249

hyperthermia 257 neuroendocrine actions 257 spinal motoneurone increased

firing 70 subtypes 48,56,70,159,195,225 sympathetic post-ganglionic

nerve 1:\yperpolarization 31 sympathetic post-~anglionic

nerve inhibit10n of NA release 31, 48, 136, 159

sympathetic pre-~anglionic nerve iiiliib1tion of ACh release 31

vasoconstriction 159, 233 vasodilatation 159, 195, 225

5-Hrlp binding sites au oradiography in enteric

preparations 37

Subject Index 285

myenteric plexus membrane preparations 37

5-HT 1p receptors and myenteric neuronal slow depolarization 37

5-HT2 receptors agonist structure-activi~ 195 antagonist structure-activity 3 behavioural actions

anxiety 87 head shakes 87 interoceptive stimuli and drug

discrunination 87 sleep 87

brain-stem neurone increased firing 70

cardiac stimulation via catecholamine release 115

CNS 3 guinea-pig ileum smooth muscle

contraction 241 platelet ag~egation 3 vasoconstriction 3, 123, 130, 195,

225,233 vasoconstrictor amplification 3,

123,130 5-HT 3 binding sites in rat CNS

membrane preparations 95 5-HT3 receptors

antagorust structure-activity 12 behavioural actions

anxiety 87, 95 dopamine-mediated motor

responses 87, 95 CNS 12 CNS dopamine turnover 95 diarrhoea 241 g~tric e~p.tving inhibitio~ 241 gumea-ptg ifeum myentenc

neurone stimulation and substance P release 241

myenteric neuronal fast depolarization 37

nodose ganglion depolarization 21

rabbit cardiac sympathetic nerve depolarization and NA release 12, 31, 115

reflex activation cardiac 21 carotid 21 chemoreflexes 21 cutaneous axonal 12,21 enteric 241 pulmonary 21 von Bezolo-Jarisch 12, 21

sympathetic post-ganglionic neuronal depolarization 31

sympathetic pre-ganglionic neuronal depolarization 31

vagus nerve depolarization 21 5-HTP-DP 37 [3H]-WB 4101 188 hypertension 3, 56, 146, 225, 274 hyperthermia 257 h}'l'Otension

CNS 5-HTlA receptors 146 vasodilatation 146

[1251]-BH-8-MeO-N-PAT 169 ICS 205-930 12, 21, 31, 37, 87, 95, 115,

241 [125~-cyanopindolol 188 2-[1 '1]-iodomelatonin 265 imipramine 249 indalpine 115, 249 ipsaprrone 87, 101, 146, 169, 188

K+ current modulation in dorsal raphe neurones 64

ketanserin 3, 70, 87, 115, 123, 136, 195,225,233

LSD 56, 87, 115, 180, 188 luzindole 265

m-CPP 87, 257 MDL 72222 12, 21, 31, 95, 115, 241 MDL 72832 48, 87, 146 MDL73005 87 MDMA 274 melatonin

actions chronobiological mood 265 circadian rhythms 265 reproduction 265 retina 265 sleep 265

binding sites CNS membrane preparations

265 retina 265 subtypes 265

receptors agonist structure-activio/ 265 an~nist structure-activity

retinal dopamine release 265 8-Me0-2'-chloro-PAT 169 8-MeOCIEPAT 146 8-Me0-3'-NAP-amino-PAT 169 mesulergine 87, 159, 180, 188

286 Subject Index

metergoline 48, 56, 87, 146, 159, 180, 188,257

methiothepin 48, 56, 87, 115, 136, 146,159,233

2-methyl-5-HT 21, 31, 37, 87, 95, 115, 241

methysergide 31, 48, 70, 87, 115, 136, 188,195,225

metoclopramide 12, 31, 241 mianserm 180, 188 migraine 12, 233, 257, 274 MK212 48

neuroendocrine actions adrenocorticotrophin 257 cortisol 257 growth hormone 257 human and non-human primates

257 prolactin 257

neuroexcitation ACh release from guinea-pig

myenteric neurones 241 deJ?Olanzation

fast response in myenteric neurones via 5-HT3 receptors 37

nodose ganglion 21 slow response in myenteric

neurones via 5-HT1p receptors 37

sympathetic post-ganglionic neurones 31

sympathetic pre-ganglionic neurones 31

vagus nerve 21 increased firing

brain-stem neurones 70 spinal motoneurones 70

myenteric plexus 37 NA release from rabbit cardiac

sympathetic nerves 12, 31, 115

overview 77 substance P release from

guinea-pig myenteric neurones 241

neuroinhibition hyperpolarization

dorsal raphe neurones 64 sympathetic post-ganglionic

neurones 31 inhibition of ACh release

myenteric cholinergic nerves 48

sympathetic pre-ganglionic nerves 31

inhibition of firing brain-stem neurones 70 dorsal rarhe neurones 64

inhibition o 5-HT release via CNS serotoninergic nerve terminal autoreceptors 56, 249

inhibition of NA release from sympathetic post-ganldionic nerves 31, 48, 136, 15~

overview 77 neurotoxicity 274 nor-(-)-cocame 12

obesity 249 8-0H-"OPAT 48, 64, 70, 87,101,115,

146,159,169,188 5- and 6-0HIP 37

PAPP 87,188 pathophysiolog}" overview 274 penile erection 257 peripheral vascular disease 3 phen}'lbiguanide 21 pindolol 56 (-)-pindolol 48, 146 piren.r.erone 136 pizotifen 136 platelet aggregation 3, 225 propranolOl 56, 115

quipazine 31,48,87,101

rauwolscine 48 reflex activation

cardiac 21 carotid 21 chemoreflexes 21 cutaneous axonal 12, 21 enteric 241 pulmonary 21 von Bezold-Jarisch 12, 21, 115

release from cerebrovascular ~pathetic nerve terminals

retinal function 265 risperidone 3 ritanserin 3, 257 RU 24969 48, 87, 101, 159, 188

serenics 101 sleep disorders 265 smooth muscle overview 152

Subject Index 287

spiperone 48, 159, 188, 195 storage

cerebrovascular sympathetic nerve terminals 233

CNS serotoninergic nerve terminals 249

platelets 249

TFMPP 87, 101 trazodone 195, 257 tryptamine analogue agonists 48,

87,101,115, 19~,265

uptake cerebrovascular sympathetic

nerve terminalS 233 CNS serotoninergic nerve

terminals 169, 249 inhibition 249 ligand binding assays 249 N"a+-dependent 5-Hr transporter

249 platelets 225, 249

urapidil 146

vascular medial hypertrophy 225

vascular pathophysiology overview 274

vasoconstriction amplification

atheroma 225 endothelium denudation 225 interactions with

vasoconstrictors 3, 123, 130, 233

overview 152 via a-adrenoceptors 123, 130 via 5-HT1-like receptors 159,233 via 5-HT...2 receptors 3, 123, 130,

195, 225, ~' 257 vasodilatation

amplification via medial hypertrophy 225

direct relaxation 136, 159, 195,225 EDRF release 3, 136, 195, 225 inhibition of noradrenaline

release 3, 136, 159 overview 152

wheal and flare 12, 21 WY48723 48

yohimbine 48, 146

Date post:	11-Sep-2021
Category:	Documents
Upload:	others
View:	2 times
Download:	0 times

Serotonin: Actions, Receptors, Pathophysiology

Documents