F. Marks, G. Furstenberger (Eds.)

Prostaglandins , Leukotrienes and Other Eicosanoids From Biogenesis to Clinical Application

@3 w I LEY-VCH Weinheim - New York Chichester Brisbane Singapore Toronto

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F. Marks, G. Furstenberger (Eds.)

Prostaglandins, Leukotrienes and Other Eicosanoids

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Prof. Dr. Friedrich Marks Dr. Gerhard Furstenberger Abteilung Biochemie der gewebsspezischen Regulation (BOSOO) Deutsches Krebsforschungszentrum Im Neuenheimer Feld 280 D-69120 Heidelberg

This book was carefully produced. Nevertheless, editors, authors and publisher do not warrant the information contained therein to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertentlv be inaccurate.

Library of Congress Card No.: applied for

A catalogue record for this book is available from the British Library.

Deutsche Bibliothek Cataloguing-in-Publication Data: Prostaglandins, leukotrienes and other eicosanoids: from biogenesis to clinical application I Friedrich Marks; Gerhard Fiirstenberger (ed.). - Weinheim; New York; Chichester; Brisbane; Singapore; Toronto: Wiley-VCH, 1999

ISBN 3-527-29360-4

0 WILEY-VCH Verlag GmbH, D-69469 Weinheim (Federal Republic of Germany). 1999 Printed on acid-free and chlorine-free paper. All rights reserved (including those of translation in other languages). No part of this book may be reproduced in any form - by photoprinting, microfilm, or any other means - nor transmitted or translated into machine language without written per-mission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law. Composition: DATA SOURCE SYSTEMS, Timisoara 1900, Romania. Printing: betz druck gmbh, D-64291 Darmstadt. Bookbinding: J. Schaffer GmbH&Co. V.G., D-67269 Griinstadt. Printed in the Federal Republic of Germany.

F. Marks, G. Furstenberger (Eds.)

Pros tag landins, Leukotrienes and Other Eicosanoids From Biogenesis to Clinical Application

Weinheim - New York - Chichester Brisbane Singapore - Toronto

Preface

Although discovered more than 65 and identified more than 30 year ago, eicosanoids were considered for a long time to represent biocompounds of minor importance, for instance , when compared with hormones and cytokines. As a consequence eicosa- noid research was regarded as a subject for narrow specialist rather than being a ma- jor field in biomedical science. This situation has changed completely, since mainly due to the impact of modern analytical and molecular-biological technology, eicosa- noids have become recognized as a physiologically and pathologically very important family of cellular signal transducers; this also underlined by two Nobel prizes.

Eicosanoids and related fatty acid derivatives are produces throughout the eukary- otic kingdom by probably every organism and every cell type. Most of these com- pounds fulfill the functions of local mediators, also called autocoids or tissue hor- mones. As such they modulate the effects of all kinds of hormonal, immunological, and nervous signals as well as of environmental influences. in fact, the eicosanoid system may be understood as a device of biological signal transduction and signal processing which is placed between the environment and the systemic network of signaling on the one side and the intracellular machinery of signal processing on the other side. At the level of the molecular mechanism of prostanoid action there are indeed fluid transitions between extra- and intracellular signaling. The enormous versatility of eicosanoids is a result of the extraordinary multiplicity of poly- unsaturated fatty acid metabolism that leads to innumerable bioactive compounds, which are still far from being known as a whole. This metabolic complex certainly represent one of the most fascinating examples of biochemical evolution in nature. It appears as if eicosanoids - together with other local mediators - form a highly sophis- ticated network of locally restricted inter- and intracellular communication which is not fixed but becomes organized 'on demand. The high degree of multiplicity and feedback interactions indicate that this machinery operates in a non-linear fashion which may help to produces 'order in chaos', that is, to carry out a characteristic per- formance of living matter.

Considering the central role eicosanoids and related compounds play in cellular (patho)-physiology it is everything but surprising that both basic research and phar- maceutical industry undertake tremendous efforts in the development of drugs which interact with this metabolic complex. As a major milestone in this field the identifica- tion of nonsteroidal antiinflammatory drugs, such as aspirin, as inhibitors of prosta- noid biosynthesis has to be mentioned. Today most investigators and clinicians agree that an in-depth elucidation of eicosanoid metabolism is a prerequisite not only for an understanding but also for an advanced treatment or prevention of some of the most serious diseases, such as atherosclerosis, cancel, Alzheimer's dementia, allergic asthma, and others.

This opinion is reflected by the chapters of this book. Following an introductory

VI Prejace

overview (Chapter I ) , Chapters 2-6 deal with the major enzymatic routes of eicosa- noid biosynthesis, while Chapter 7-12 focuss on clinical aspects. The selection of topics may appear to be rather arbitrary. However, considering the enormous and rapidly enlarging extent of the field, any attempt to reach completeness would have been doomed to failure from the very beginning. Thus, the selection of the topics had to aim necessarily at exemplarity, and the goal of this book is to provide an introduc- tion into, rather than a comprehensive review, of the field. As a compensation the authors have done their best in providing up-dated reference lists which may help the reader to follow-up special aspects in more detail and to the roots. Notwithstanding these joint intentions, every contribution reflects the authors' personal approach to handle the subject as far as style, arrangement, content, in-depth treatment, and the emphasis on special aspects are concerned. Since every chapter should stand by itself some overlapping and redundancy was inavoidable.

As a whole the book offers a snapshot of one of the most fascinating subjects of current biological research and its clinical applications. The editors are aware of the fact that in such a rapidly developing fields ideas, hypotheses, conclusions, and the latest information are highly perishable goods. Nevertheless, they hope that the book contains a substantial wealth of hard facts and, thus, will serve for a reasonable period of time as a stimulation and guidance for both students and experts.

Heidelberg, June 1999 Friedrich Marks Gerhard Fiirstenberger

Con tents

1 Arachidonic acid and companions: an abundant source of biological signals ................................................ 1 Friedrich Murks

1.1 1.2 1.3 1.3.1 1.3.2 1.3.3 1.3.4 1.3.5 1.3.6 1.3.7 1.3.8 1.3.9 1.4 1.5 1.6 1.6.1 1.6.2

1.6.3 1.7 1.8

The world of PUFAs .................................................................................. 1 The discovery of prostaglandins and related eicosanoids .......................... 3 Mammalian eicosanoids ............................................................................. 6 Free arachidonic acid: a signaling compound? .......................................... 7

HPETEs, HETEs and leukotrienes ........................................................... 11 Lipoxins ................................................................................................... 15

Monoox ygenase-derived eicosanoids ...................................................... 20 Isoprostanes .............................................................................................. 21 Anandam& ............................................................................................. 24 Eicosanoids in invertebrates ..................................................................... 27 Eicosanoid-related signaling compounds in plants .................................. 30 The cellular functions of eicosanoids in mammals .................................. 34 Eicosanoids as local mediators ................................................................. 34

effects of eicosanoids ............................................................................... 35 Nuclear eicosanoid receptors: a new frontier in research ......................... 38 Addendum: Methods of eicosanoid research ........................................... 40 References ................................................................................................ 40

Prostanoids ................................................................................................. 8

15-Epi-lipoxins ......................................................................................... 17 Hepoxilins ................................................................................................ 18

Specific membrane receptors mediate many biological

2 The generation of free arachidonic acid ............................................... 47 Peter Dieter

Introduction .............................................................................................. 47 (Re)Incorporartion of arachidonic acid into phospholipids ..................... 48

2.3 Phospholipases A ..................................................................................... 49

2.3.2 Phospholipases A2 .................................................................................... 49 Secretory phospholipases A2 .................................................................... 51 Cytosolic phospholipase A2 ..................................................................... 52 Calciurn-independent phospholipases A2 ................................................. 56 DAG lipase and PLC or PLDPA phosphohydrolase ............................... 56

2.1 2.2

2.3.1 Phospholipase A, ..................................................................................... 49

2.3.2.1 2.3.2.2 2.3.2.3 2.4

VIII Contents

2.5 2.5.1 2.5.2 2.6 2.7

3

3.1 3.2 3.3 3.4 3.5 3.5.1 3.5.2 3.5.3 3.5.4 3.6 3.7 3.8 3.9 3.10 3.11

4

4.1 4.2. 4.3 4.4 4.5. 4.6 4.7 4.8 4.9 4.10

5

5.1.

Cellular models ........................................................................................ 57 P388D1 macrophages ............................................................................... 57 Rat liver macrophages .............................................................................. 57 Conclusions .............................................................................................. 58 References. ............................................................................................... 59

C yclooxygenases ..................................................................................... 65 Karin Miiller-Decker

Introduction .............................................................................................. 65 Cloning of cyclooxygenase isoforms ....................................................... 66 Cyclooxygenase gene structures .............................................................. 67 Regulation of cyclooxygenase isoenzyme expression ............................. 69 Cyclooxygenase proteins ......................................................................... 73 Sequence comparisons ............................................................................. 73 Post-translational modification ................................................................ 74 X-ray analysis of crystal structure ............................................................ 74 Subcellular localization ............................................................................ 75 Coupling of COX isoenzymes with phospholipases A2 ........................... 76 Substrate specificities ............................................................................... 77 Mechanism of enzyme catalysis ............................................................... 78 Biological functions of COX isoforms ..................................................... 79 Isoenzyme-specific inhibitors .................................................................. 81 References ................................................................................................ 83

Prostanoid synthases .............................................................................. 89 Christian Martin and Volker Ullrich

Introduction .............................................................................................. 89

Prostaglandin D syntase ........................................................................... 97 Prostaglandin E synthase .......................................................................... 99 Prostaglandin F synthase ........................................................................ 100 Glutathione S-transferases ..................................................................... 101

Summary and outlook ............................................................................ 104 References .............................................................................................. 104

Thromboxane A2 synthase ....................................................................... 91 Prostacyclin synthase ............................................................................... 93

Detection of prostaglandin synthases in various tissues ........................ 102

Lipoxygenases ....................................................................................... 109 Hartmut Kiihn

lntroduction ............................................................................................ 109

Contents IX

5.2. 5.3. 5.4. 5.5 5.5.1. 5.5.2 5.6 5.6.1 5.6.2 5.6.3 5.6.4 5.6.5 5.7 5.7.1 5.7.2 5.7.3 5.7.4 5.8 5.8.1 5.8.2 5.8.3 5.8.4 5.8.4.1

5.8.4.2 5.8.4.3 5.9

Lipoxygenase reaction ........................................................................... 110 Common properties of lipoxygenases .................................................... 111 Classification of lipoxygenases .............................................................. 113 Structural aspects of lipoxygenases ....................................................... 115 X-ray crystallography ............................................................................. 115 Substrate alignment and determinants of positional specificity ............. 118 5-Lipoxygenases .................................................................................... 120 Enzymatic properties., ............................................................................ 120 5-Lipoxygenase activating protein ........................................................ 122 Molecular biology of 5-lipoxygenases ................................................... 123

Biological functions of 5-lipoxygenases ................................................ 124 12-Lipoxygenases .................................................................................. 125 Subclassification and enzymatic properties ........................................... 125 Molecular biology of 12-lipoxygenases ................................................. 126 Tissue distribution and regulation of 12-LOX expression ..................... 127 Biological functions of 12-lipoxygenases .............................................. 127 Mammalian 15 -1ipoxygenases .............................................................. 129 Subclassification and enzyme properties ............................................... 129

Tissue distribution and regulation of 15-LOX expression ..................... 131 Biological functions of 15-lipoxygenases .............................................. 132

Modulation of intracellular lipid signal transducers .............................. 133 Formation of bioactive oxygenated fatty acid derivatives ..................... 134 References .............................................................................................. 134

Tissue distribution and regulation of 5-LOX expression ....................... 123

Molecular biology of the reticulocyte-type 15-lipoxygenases ............... 130

Structural modification of lipid-protein assemblies . Implication in cell maturation and atherogenesis ................................... 132

6 Oxygenation of arachidonic Acid by cytochromes P-450 ................. 143 Ernst H . Oliw and Johanna Ericsson

6.1 6.2 6.3 6.3.1 6.3.2 6.3.3

6.4 6.4.1 6.4.2 6.5. 6.5.1 6.5.2 6.5.3

Introduction ............................................................................................ 143 Early work on oxidation of fatty acids by cytochromes P-450 .............. 144 Oxygenation of arachidonic acid by cytochromes P-450 ....................... 146 Hydroxylation of o side chain ............................................................... 146 Epoxidation ............................................................................................ 147

migration ................................................................................................ 149 Metabolism of epoxides ......................................................................... 152 Epoxide hydrolases ................................................................................ 152 Incorporation into phospholipids ........................................................... 153 Analysis of arachidonic acid metabolites ............................................... 153 Radioimmunoassay ................................................................................ 153 GC-MS and LC-MS analyses ................................................................. 154 Steric analysis of hydroxy fatty acids .................................................... 154

Bisallylic hydroxylation and hydroxylation with double bond

X Contents

6.5.4 6.6 6.6.1 6.6.2 6.6.3 6.6.4 6.6.5 6.7 6.8

7

7.1 7.1.1 7.1.2 7.1.3 7.1.4 7.1.4.1 7.1.4.2 7.1.4.3 7.1.4.4 7.1.4.4.1 7.1.4.4.2 7.1.4.4.3 7.1.5 7.1.6

7.1.7 7.2 7.2.1 7.2.3 7.3 7.3.1 7.3.2 7.4 7.4.1 7.4.2 7.4.3 7.4.4 7.4.5 7.4.6 7.5 7.5.1 7.5.2

Steric analysis of epoxy fatty acids and vicinal diols ............................. 155 Biological effects ................................................................................... 156 Kidney .................................................................................................... 156 Heart ....................................................................................................... 158 Vascular tree .......................................................................................... 158 Central nervous system and the pituitary ............................................... 159 Genital glands and endocrine organs ..................................................... 160

References ............................................................................................. 161 Summary ................................................................................................ 161

Renal eicosanoids ................................................................................. 169 Margarete Goppelt-Striibe and Joachim Fader

Renal prostanoids ................................................................................... 169 Localization of prostanoid biosynthesis in the kidney ........................... 170

Cyclooxygenase expression in renal inflammation ................................ 173 Prostanoid receptors ............................................................................... 173 TXA2 receptor ........................................................................................ 174 PGFza receptor ....................................................................................... 175 Prostacyclin (PG12) receptor .................................................................. 175 PGE? receptors ....................................................................................... 176

EP2/EP4 receptors ................................................................................... 177 EP3 receptors .......................................................................................... 177

clinical implications ............................................................................... 180 Cyclooxygenase inhibitors and the kidney ............................................. 181 Renal leukotrienes .................................................................................. 181 Biosynthesis ........................................................................................... 181

Renal lipoxins ........................................................................................ 183 Biosynthesis of lipoxins ......................................................................... 183

Cytochrome P-450 enzyme-generated arachidonic acid metabolites ..... 185 P-450 arachidonic acid o-hydroxylases ................................................. 186 Renal epoxygenases ............................................................................... 186 Functional properties of 19- and 20-HETE ............................................ 187

Monooxygenase products in animal models of renal hypertension ....... 188 P-450-catalysed arachidonic acid metabolism in man ........................... 189 Isoprostanes ............................................................................................ 190 Generation of isoprostanes in the kidney ............................................... 190

Regulation of prostaglandin synthesis in mesangial cells ...................... 172

EP, receptors .......................................................................................... 176

Vasoactive effects of prostanoids: relation to hypertension ................... 178 Regulation of water and electrolyte transport by prostanoids:

Biological activity of leukotrienes in the kidney .................................... 182

Biological activity of lipoxins in the kidney .......................................... 184

Functional properties of epoxides .......................................................... 187

Functional properties of isoprostanes ..................................................... 191

Contents XI

7.6 Perspective ............................................................................................. 192 7.7 References .............................................................................................. 192

8 The role of eicosanoids in reproduction ............................................. 199 H.P. Zahradnik. B . Wetzka and W.R. Schaler

8.1 8.2 8.2.1 8.2. 1 . 1 8.2.1.2 8.2.2 8.2.3 8.2.4 8.3. 8.3.1 8.3.2 8.3.3 8.3.4 8.3.4.1 8.3.4.2 8.3.4.3 8.4 8.4.1 8.4.2 8.4.3 8.5

Introduction ............................................................................................ 199 Female reproductive system ................................................................... 199 Ovarian function .................................................................................... 199 Follicular phase: ovulation ..................................................................... 200 Luteal phase: luteolysis .......................................................................... 201 The Fallopian tube ................................................................................. 202 Menstruation .......................................................................................... 203 Endometriosis ......................................................................................... 207 Pregnancy ............................................................................................... 208 Implantation ........................................................................................... 208 Placenta .................................................................................................. 211 Pregnancy-induced hypertension and pre-eclampsia ............................. 214 Parturition .............................................................................................. 216 Cervical ripening .................................................................................... 218 Labor ...................................................................................................... 220 Pre-term labor ........................................................................................ 222 Male reproductive system ...................................................................... 223 Acrosome reaction ................................................................................. 224 Immunosuppressive actions of PGE ...................................................... 224 Erectile dysfunction ............................................................................... 224 References .............................................................................................. 225

9 The role of eicosanoids in inflammation and allergy ........................ 233 Eva Wikstrom Jonsson and Sven-Erik Dahlkn

9.1 9.2 9.3

9.3.1 9.3.2 9.3.3 9.3.4 9.4

9.4.1 9.4.2 9.4.3

Introduction ............................................................................................ 233 Formation of eicosanoids in allergic inflammation ................................ 234 Biological activities and receptors with relevance for asthma and allergic inflammation ...................................................................... 238 COX products ........................................................................................ 238

Cysteinyl leukotrienes ............................................................................ 241 Lipoxins ................................................................................................. 243

obstruction and bronchial hyper-responsiveness .................................... 243 Biological activity .................................................................................. 244 Endogenous formation ........................................................................... 244

LTB4 ....................................................................................................... 239

Cysteinyl leukotrienes as mediators of allergen-induced airway

Influence of inhibitors of leukotriene synthesis or CysLT, receptor

XI1 Contents

9.4.4 9.5

9.5.1 9.5.2 9.5.3 9.5.4 9.5.5 9.6 9.7 9.7.1 9.7.2 9.7.3

9.7.4 9.7.5 9.8 9.9

antagonists on allergen-induced airway obstruction .............................. 245 Allergen-induced bronchial hyper-responsiveness ................................ 247

other factors ........................................................................................... 249

Adenosine .............................................................................................. 250 Sulfur dioxide ......................................................................................... 250 PAF ........................................................................................................ 250 Intolerance to aspirin and other NSAIDs ............................................... 25 1 Treatment of asthma with anti-leukotrienes ........................................... 253 COX products as modulators of asthma and allergic inflammation ....... 254 Effects of prostaglandins and thromboxane ........................................... 254 Release of COX products ....................................................................... 255

bronchoconstriction ................................................................................ 256 COX products in airway hyper-responsiveness ..................................... 256 Anti-inflammatory and anti-asthmatic effects of PGE2 ......................... 257 Conclusions ............................................................................................ 258 References .............................................................................................. 259

Cysteinyl leukotrienes as mediators of airway obstruction induced by

Exercise .................................................................................................. 2S0

Influence of inhibition of COX products on allergen-induced

10 Prostanoids in the cardiovascular system .......................................... 273 Lukasz Partyka. Arsineh Arakil Aghajanian and Helmut Sinzinger

10.1 10.1.1 10.2 10.3 10.3.1

10.3.2 10.4 10.4.1 10.4.2 10.4.3 10.5 10.6 10.6.1 10.6.2 10.6.3 10.6.4

10.7 10.7.1 10.7.2 10.7.3

Introduction ............................................................................................ 273 Defects in the eicosanoid system related to vascular disease ................. 274 Unsaturated fatty acids, vascular system and atherosclerosis ................ 276 Endothelium, vascular wall. platelet aggregation and eicosanoids ........ 278

‘Resistance to aspirin’ ............................................................................ 280 Eicosanoids and restenosis ..................................................................... 281 Function of eicosanoids in the heart and kidney .................................... 283 Heart muscle activity, contractility, ejection fraction ............................ 283 Myocardial infarction ............................................................................. 283 Kidney, eicosanoids and cardiovascular regulation ............................... 283 Isoprostanes-novel indicators of cardiovascular disease ..................... 285 Prostaglandins as drugs in the therapy of cardiovascular disorders ....... 286 Mechanisms of action ............................................................................ 286 Side-effects ............................................................................................. 290 Route of administration and dosage ....................................................... 291

disorders ................................................................................................. 291

Sample collection and preparation ......................................................... 292 Bioassays ................................................................................................ 293 Physicochemical methods ...................................................................... 293

Aspirin interaction with COX-1 . Role in cardiovascular prevention .

Gene therapy-experiments with prostaglandins in cardiovascular

Methods of clinical prostaglandin research ............................................ 292

Contents XI11

10.7.4 Immunochemical methods ..................................................................... 294 10.8 References .............................................................................................. 295

11 Eicosanoids and cancer ........................................................................ 303 Friedrich Murks and Gerhard Fiirstenberger

11.1. 11.2 11.3

11.4.

11.5 11.5.1 11.5.2 11 S.3

11.5.4 1 1 S.5 11.6 11.7 11.8

Carcinogenesis: a multistage process ..................................................... 303 Immunosuppression by prostaglandins .................................................. 307 Metabolic activation of carcinogens in the course of prostaglandin synthesis ................................................................................................. 308 The generation of agressive products in the course of eicosanoid formation ................................................................................................ 308 The role of eicosanoids in tumor promotion .......................................... 309 COX inhibitors suppress tumor promotion in animal models ................ 309

phenomenon? ......................................................................................... 315 Mechanistic aspects ................................................................................ 316 Lipoxygenase-catalyzed arachidonic acid metabolism .......................... 321 Effects of eicosanoids on tumor cell metastasis ..................................... 322 Cyclopentenone prostaglandins: a new class of anti-cancer drugs? ....... 324 References .............................................................................................. 324

Prevention of colorectal cancer in man by COX inhibitors ................... 312 Promotion of tumor development by prostaglandins: a widespread

12 Synthetic eicosanoids: development and clinical applications ......... 331 Bemd Buchmann. Ulrich Klar. Hartmut Rehwinkel and Werner Skuballa

Introduction ............................................................................................ 12.1 331 12.2 Prostaglandins ........................................................................................ 331 12.3 Prostacyclins .......................................................................................... 336

Thromboxane receptor antagonists ........................................................ 343 12.5 Cystenilleukotriene antagonists ............................................................. 350

12.7 References .............................................................................................. 360

12.4

12.6 LTB4 antagonists .................................................................................... 354

Index ...................................................................................................... 375

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List of Contributors

Arisineh Arakil Aghajanian Institut fur Nuklearmedizin Universitat Wien Wahringer Giirtel 18-20 1009 Wien Austria

Bernd Buchmann Schering AG Tnstitut fur Arzneimittelchemie MullerstraBe 170- 178 13342 Berlin Germany

Sven-Erik DahlCn Institute of Environmental Medicine Karolinska Institute xx 17177 Stockholm Sweden

Peter Dieter Institut fur Physiologische Chemie Technische Universitat Dresden Karl-Marx-StraBe 3 0 1 109 Dresden Germany

Joachim Fauler Universitatsklinikum Carl Gustav Carus Institut fur Klin. Phhmakologie Techn. Universitat Dresden FriedlerstraBe 27 0 1307 Dresden Germany

Gerhard Furstenberger Abteilung des Biochemie

gewebsspezifischen Regulation (BOSOO) Deutsches Krebsforschungszentrum Im Neuenheimer Feld 280 69 1 20 Heidelberg Germany

Margarete Goppelt-Striibe Medizinische Klinik IV Nephrologische Forschungslaboratorien Universitat Erlangen-Nurnberg LoschgestraBe 8 9 1054 Erlangen Germany

Ulrich Klar Schering AG Institut fur Arzneimittelchemie MiillerstraBe 170- 178 I3342 Berlin Germany

Hartmut Kiihn Institut fur Biochemie/Charitt Humboldt Universitat SchumannstraBe 20-2 1 101 17 Berlin

Friedrich Marks Abteilung Biochemie der gewebsspezifischen Regulation (BOSOO) Deutsches Krebsforschungszentrum Im Neuenheimer Feld 280 69 120 Heidelberg Germany

Christian Martin Fakultiit fur Biologie Universitat Konstanz UniversitatsstraBe 10

XVJ List of Contributors

78464 Konstanz Germany

Karin Muller-Decker Abteilung Biochemie der gewebsspezifischen Regulation (BOSOO) Deutsches Krebsforschungszentrm Im Neuenheimer Feld 280 69 120 Heidelberg Germany

Ernst H. Oliw Institute for Pharmacology Uppsala University Box 59 1, Biomedicum 75124 Uppsala Sweden

Lukasz Partyka Department of Clinical Biochemistry Collegium Medicum Jagiellonian University 15a Kopernika Str. 31-501 Krak6w Poland

Hartmut Rehwinkel Schering AG Institut fur Arzneimittelchemie MullerstraBe 170- 178 13342 Berlin Germany

Wolfgang Schafer Universitats-Frauenklinik Hugstetter Stral3e 55 79106 Freiburg Germany

Helmut Sinzinger Institut fur Nuklearmedizin Universitat Wien Wlihringer Giirtel 18-20 1009 Wien Austria

Werner Skuballa Schering AG Institut fur Arzneimittelchemie MullerstraBe 170-178 13342 Berlin Germany

Volker Ullrich Fakuitat fur Biologie Universitat Konstanz Universitatsstrafie 10 78464 Konstanz Germany

Birgit Wetzka Universitats-Frauenklinik Hugstetter StraBe 55 79 106 Freiburg Germany

Eva Wikstrom Jonsson Institute of Environmental Medicine Karolinska Institute xx 17177 Stockholm Sweden

Hans-Peter Zahradnik Universitats-Frauenklinik Hugstetter StraBe 55 79 106 Freiburg Germany

Abbreviations

ADH AEA AMP AOD AOS APC ASA ATL bFGF CaLB CDNB CHD CHO CLASP cox cPLA~ CRH CSF CSF CYP DAG DART DGLA DHEA DHET(E) DMSO DRE Dsh DTT EDHF EDRF ECP EDTA EET EFA EGF EGFR EPR EPR

Anti-diuretic hormone Arachidonyl-ethanolamide Adenosine monophosphate Arterial occlusive disease Allene oxide synthase Adenomatous polyposis coli Acetyl-salicylic acid Aspirin-triggered lipoxin Basic fibroblast growth factor Calcium-dependent lipid binding 1 -Chloro-2,4-dini trobenzene Coronary heart disease Chinese hamster ovary Collaborative Low-dose Aspirin Study in Pregnancy C ycloox ygenase Cytoplasmic phospholipase A2 Corticotropin-releasing hormone Cerebrospinal fluid Colony-stimulating factor Cytochrome P-450 Diac ylglycerol Diet and Reinfarction Trial Dihomo-y-linoleic acid Deh ydroepiandrostendione Dihydroxyeicosatrienoic acid Dimeth ylsulfoxide Dioxin-responsive element Dishevelled Dithiothreitol Endothelium-derived hyperpolarizing factor Endothelium-derived relaxing factor Eosinophil cationic protein Ethylenediamine tetraacetate Epoxyeicosatrienoic acid Essential unsaturated fatty acids Epidermal growth factor Epidermal growth factor receptor Electron paramagnetic resonance Endoplasmic reticulum

XVIII A bbrc4rrtiotz.s

EXAFS FABP FAP FEN 1

FLAP FMRF FPR FS H Fz GAP

GLA

GnRH GRE GSH GSK GST GT HAT/HIT hCG HHT HETE HNPCC HODE HOT HOTE HPETE HPLC HPODE HX IFN Ig IL IP I p 3 IRE isoP IUGR KETE LASS

LDL LEF LH LIF

GC-MS

GM-CSF

LC-MS

Extended X-ray absorption fine structure Fatty acid-binding protein Familial adenomatous polyposis Forced expiratory volume in 1 s 5-Lipoxygenase-activating protein Phenylalan yl-methionyl-arginy 1-phenylalanin PGF receptor Follicle-stimulating hormone Frizzled GTPase-activating protein Gas chromatography mass spectrometry y-Linoleic acid Granulocyte/monocyte colony-stimulating factor Gonadotropin-releasing hormone Glucocorticoid-responsive element Glutathione Glycogen synthase kinase Glutathione S-transferase Gonadotropin Heparin-associated thrornboc ytopenia Human chorion gonadotropin Hydroxy-heptadecatrienoic acid Hydrox yeicosatetraenoic acid Hereditary non-polyposis colorectal carcinoma Hydroxyoctadecadienoic acid Hydroxyoctadecatrienoic acid Hydroxyoctadecatetraenoic acid Hydroperoxyeicosatetraenoic acid High-performance liquid chromatography Hydroperoxyoctadecadienoic acid Hepoxilin Interferon Immunoglobulin Interleukin Isoelectric point Inositor - 1,4,5 - triphosphate Insulin-responsive element Isoprostanes Intrauterine growth retardation Ketoeicosatetraenoic acid Labile (platelet) aggregation-stimulating substance Liquid chromatography mass spectrometry Low density lipoprotein Leukocyte-enhancing factor Luteinizing hormone Leukemia inhibitory factor

Abbreviations XIX

Lipopol y saccharide Leukotriene Luteotropic hormone Lipoxin Mitogen-activated protein kinase Monocyte chernotactic protein MAP kinase/Erk kinase Microsornal glutathione S-transferase Multiple intestinal neoplasia Mass spechornetry Nicotinarnide adenine dinucleotide (reduced form) Nicotinarnide adenine dinucleotide phosphate (reduced form) N-Ethyl-maleirnide Nerve growth factor Negative-ion chemical ionization Natural kdler cell N-Methyl-D-aspartic acid Nitric oxide Nitric oxide synthase Non-steroidal anti-inflammatory drug Phosphatidic acid Platelet-activating factor Polyacrylamide gel electrophoresis Peripheral arterial occlusive disease Phosphatidylcholine Phytodienoic acid Platelet-derived growth factor Prostaglandin dehydrogenase Phosphatidylethanolarnine Peak expiratory flow rate Prostaglandin Prostaglandin H synthase Phospholipid hydroperoxide glutathione peroxidase p-H ydrox ymercuribenzoate Phosphatidylinositol Pregnanc y-induced hypertension Protein kinase C Phospholipase Phospholipase-activating protein Phospholipase C Polyrnorphonuclear leukocyte Peroxisome proliferator-activated receptor Phosphatidylserine Percutaneous translurninal coronary angioplasty Polyunsaturated fatty acid Peripheral vascular direase

LPS LT LTH LX MAPkinase MCP MEK MGST Min MS NAD(H) NADP(H) NEM NGF NICI NKcell NMDA NO NOS NSAID PA PAF PAGE PAOD PC PDA PDGF PGDH PE PEFR PG PGHS PHGPx pHMB PI PIH PKC PL PLAP PLC PMNL PPAR PS PTCA PUFA PVD

XX Abbreviutioris

RBP RCS

SDS SH SHR SMC

SRS

TGF TLC TMD TNF TX UTR VLDL

RT-PCR

sPLA~

SRS-A

Retinol-binding protein Rabbit aorta-contracting substance Reverse transcriptase polymerase chain reaction Sodium dodecyl sulfate Sulfhydryl Spontaneously hypertensive rat Smooth muscle cell Secretory phospholipase A2 Slow-reacting substance Slow-reacting substance of anaphylaxis Transforming growth factor Thin layer chromatography Transmembrane domain Tumor necrosis factor Thromboxane Untranslated region Very low density lipoprotein

1 Arachidonic acid and companions: an abundant source of biological signals

Friedrich Marks

1.1 The world of PUFAs

Polyunsaturated fatty acids (PUFAs), such as linoleic acid, a-linolenic acid, dihomo- y-linolenic acid, arachidonic acid, eicosapentaenoic acid, etc., are found throughout the eucaryotic kingdom. For men and most animals these fatty acids--or at least the precursors linoleic and a-linolenic acid-are ‘essential’, i.e. they cannot be synthe- sized de novo but have to be taken up in a vegetable diet (Fig. 1-1). While the major function attributed to these compounds in earlier textbooks of biochemistry was the control of membrane viscosity, it is now known that they play a key role in intercel- lular communication as well serving as precursors of a very large and extremely ver- satile family of signaling compounds formed primarily along oxidative pathways.

A characteristic structural feature of the PUFAs found in living matter is that the double bonds are cis-configurated and not conjugated, i.e. are each separated by a CH2 group (1.4-cis, cis-pentadiene structure). Upon contact with air, such structures become rapidly autoxidized resulting in a bewildering variety of peroxy, hydroxy and epoxy compounds which easily undergo further reactions. Biological evolution has made use of this chemical versatility by forcing the individual reactions step by step under the control of enzymatic catalysis. As a result, cells then dispose of a very large collection of oxygenated fatty acid derivatives called ‘oxylipins’ [3] which they mainly use for signaling purposes. At least in animals, the C20 fatty acid-derived eico- sanoids [4] constitute the most abundant subfamily of oxylipins with arachidonic acid being the major precursor.

In cells PUFAs are normally found to be sequestered in membranes, i.e. esterified in phospholipids. They are released from this store by phospholipases which are un- der the control of environmental or cellular signals. Such signal-activated phospholi- pases [5 ] also regulate the formation of other signaling molecules, such as diacylglyc- erols, inositol-phosphates, lyso-phosphatidic acid, ceramides, etc. Most of them are released into the cytoplasm where they act as intracellular signal transducers, whereas others, such as lysophosphatidic acid, operate as mediators of intercellular communi- cation [6]. In contrast, fatty acid-derived signaling molecules can be released into the cell’s interior as well as into the extracellular space, acting both as second mes- senger-like intracellular and hormone- and even pheromone-like intercellular sig- nals. Moreover, at the receptor level they share the properties of hydrophilic hor- mones, i.e. they activate cellular signaling cascades by interaction with receptors at

2 1 Ar~ichidonic acid und companions: an abundunt source c.f biological s ip ids

the cell surface, with the ability of lipophilic hormones, i.e. they penetrate the plasma membrane and modulate the activity of intracellular receptor proteins such as tran- scription factors. It appears, therefore, that fatty acid-derived oxylipins provide the most versatile signaling molecules in living nature.

ESSENTIAL FATTY ACIDS

9

oleic acid linoleic acid a - linolenic acid

I t

6.9-octadacadienoic acid

i

8.1 I-eicosadienoic acid

mead acid

t

(22zYooH 7.10.1 3dowsatrienoic acid

1

y-linolenic acid stearidonic acid

i i ELONGASE 1

I 1 14 11

dihomo-y-linolenic acid 8.1 1,14,17-eicosatetraenoic acid

AS-DESATL‘RASE i i I 1 I 4 eez? 11 11 17

5,8,11,14,174cosapentaenoic acid

e2x7 arachidonic acid

ELONGASE i t

m C O O H m C O O H

- - 13 16 19

adrenic acid 7,10,13,16,19-docosapentaenoic acid

Figure 1-1. Biosynthetic pathways for the formation of PUFAs. Linoleic and a-linolenic acids are synthesized from oleic acid in plants only (upper line) and, therefore, are essential for animals. In animal cells, these plant fatty acids undergo chain elongation with acetyl CoA at the carboxylic end (broken arrows) and desaturation at A 5 and 6. Beyond carbon atom 9 no double bonds can be introduced by animal cells, strictly limiting the interconversibility between linoleic acid- and a-linolenic acid-derived compounds. The major oxilipin precursors in vertebrates are arachidonic acid, dihomo-y-linolenic acid, eicosapentaenoic acid and linoleic acid. In invertebrates and plants, oxilipins are also synthesized from a- linolenic acid and stearidonic acid. The role of other PUFAs in oxilipin biosynthesis is unknown, although some of them (i.e. mead acid and adrenic acid) have been shown to be substrates of oxilipin-forming oxygenases. As far as the nomenclature of unsaturated fatty acids is concerned, the positions of the double bonds are given as prefix numbers (beginning with the terminal carboxylic group as number 1 ) . In another nomenclature system the position of the double bond nearest to the terminal methyl group (carrying the so-called w C-atom) is designated in order to set out the biosynthetic interrelationships of fatty acid families (for instance, the a-linolenic acid-derived w3-fatty acids and the a-linoleic acid-derived o6-fatty acids). Fish oil is particularly rich in 03-fatty acids. For more details see Willis [ I ] and Brenner [Z].

As far as evolutionary aspects are concerned, lipid mediators such as oxylipins confirm the hypothesis that the formation of signaling molecules may have developed as a ‘by-product’ of ancient metabolic pathways which were initially required for the biosynthesis and metabolism of basic biomolecules such as lipids, amino acids, pep- tides, nucleotides and carbohydrates.

1.2 The discovery of prostuglandins and related eicosunoids 3

1.2 The discovery of prostaglandins and related eicosanoids

For a detailed treatment of this subject the reader is referred to Willis [ 11 and von Euler and Eliasson [7]. Historically, prostaglandins are the leading compounds of the eicosanoid subfamily of oxylipins. In 1930 Kurzrock and Lieb [8] discovered that seminal fluid exerted pronounced pharmacological effects on uterus preparations. Depending on whether the tissue was obtained from formerly pregnant or from sterile women it responded either by relaxation or by contraction. Analogous results ob- tained with extracts from the sheep vesicular gland or human seminal fluid were pub- lished a few years later by von Euler [9] and Goldblatt [ 101. Von Euler characterized the active principle as an unsaturated acidic lipid thus ruling out a hypothesis put forward by others, i.e. that acetylcholine had caused the observed effects. Since the active lipid was also initially found in extracts from the prostate von Euler [ 1 I ] pro- posed the name ‘prostaglandin’. Only later was it realized that the prostaglandin in prostate extracts came mostly from the vesicular glands [7]. However, by that time it was already too late to change the name which had become established in the litera- ture.

Because of the inadequacy of the analytical methods available at that time it still took another 20 years until prostaglandins became identified and the whole field of research could leave the gallery of curios.

In cooperation with the Bayer Company (formerly part of the IG Farben Company) in Elberfeld (Germany), von Euler and his group in Stockholm. prepared prostaglandin as barium salt from sheep vesicular glands in amounts large enough for further biological and chemical characterization. In the course of this work it soon became clear that prostaglandin represented a whole family of related factors rather than being a single compound. This finding and the chemical characterization as well as partial synthesis of prostaglandin Fza from arachidonic acid were primarily the merit of Bergstrom and Sjovall [12]. In 1957 these authors published the isolation of the first prostaglandin, i.e. PGF2a, in a crystalline form [12] and, in 1960, PGE2 was purified to homogeneity [ 131. The structures of both these prostaglandins were elucidated by Bergstrom et al. [ 141 and the absolute configuration was determined by them, in collaboration with van Doorp’s [15] group at the Unilever Research Laboratories in Vlaardingen, The Netherlands, in the early 1960s. Moreover, the biosynthesis of prostaglandin from radioactively labeled Cl0 PUFAs, such as dihomo- y-linoleic acid, arachidonic acid and 5,8,11 , 14,17-eicosapentaenoic acid, was investigated by both groups using homogenates from sheep vesicular glands as enzymatic preparations [ 16,171. The involvement of molecular oxygen and the formation of a cyclic peroxide intermediate was demonstrated in these studies. This breakthrough in Prostaglandin research depended critically on the introduction of novel preparative and analytical methods such as thin-layer and gas chromatography and nuclear magnetic resonance and mass spectrometry. It left the way clear for the start of a steep increase in the scientific career of the prostaglandins and other eicosanoids, i.e. the number of corresponding publications began to increase exponentially El]. Nevertheless, it appears as if eicosanoid research has still not

4 I Arachidonic acid and companions: an abundunt tsource ojbiological signals

become fully integrated into the mainstream of today’s biomedical science, probably because it requires sophisticated analytical equipment which only a very limited number of laboratories possess and is not easily accessible for current methods of molecular biology.

An important step forward in the development of eicosanoid research was the dis- covery of the prostaglandin endoperoxides PGG2 and PGH2 [ 18,191. In the beginning, these intermediates of enzymatic prostaglandin synthesis were thought to be identical with so-called rabbit aorta-contracting substance (RCS) [20] and labile (platelet) ag- gregation-stimulating substance (LASS) [21]. Both were found in pharmacological experiments as short-lived activities which were released, for instance, from allergen- challenged guinea-pig lung. Later on, thromboxane A2 was identified as the major component of RCS and LASS [22]. At about the same time, the Vane and his col- legues. [23] postulated the existence of an endogenous thromboxane A2 antagonist, ‘PGX’, which was then characterized as PGI2 or prostacyclin. Vane[24] and another group [25] made the most important and remarkable discovery that cyclooxygenase, i.e. the enzyme controlling the initial step of prostaglandin, thromboxane and prosta- cyclin biosynthesis, is specifically inhibitied by non-steroidal antiinflammatory drugs such as aspirin.

In 1974, while studying thromboxane and prostacyclin formation in platelets, Hamberg and Samuelsson [26] as well as Nugteren [27] observed the appearance of non-cyclic hydroxylated arachidonic acid derivatives. This finding opened a new avenue of eicosanoid research which led to the discovery of the lipoxygenase enzyme family [28] and the leukotrienes as powerful pro-inflammatory and pro-anaphylactic mediators. However, the fact that the first lipoxygenase (then called ‘lipoxidase’) had already been discovered in plants in the early 1930s should not be overlooked [29]. Eventually, lipoxygenase-catalyzed fatty acid metabolism in both animal and plant cells will move to the center of interest since it is the source of a large array of bio- logically hghly active substances with putatively important functions in the healthy organism and in numerous diseases. This may also hold true for eicosanoids derived from cytochrome P-450-catalyzed monooxygenation of arachidonic acid.

The latter reaction, together with the cylooxygenase and lipoxygenase pathways, have been thought to represent the three major routes of eicosanoid biosynthesis. Recently, however, additional pathways, such as the formation of arachidonyl-A- ethanolamide (anandamide, see Section 1.3.9), a putative neuromodulator or neuro- transmitter, and the non-enzymatic biosynthesis of isoprostanes (see Section 1.3.8) have been discovered (Table 1-1) . This may not be the end of the story, however. Thus, the possibility of a.large group of compounds synthesized by an interaction of the major pathways is just beginning to emerge. Moreover, there is an increasing body of evidence that, besides arachidonic acid, other unsaturated fatty acids seques- tered in membrane phospholipids may give rise to a wide variety of signaling com- pounds which rival the eicosanoids in functional importance. Presently, the linoleic acid-derived octadecanoids are gaining increasing attention, whereas the correspond- ing linolenic acid derivatives are still almost entirely neglected as far as animal cells are concerned, although for plants their fundamental role in cellular signaling is fully recognized (see Sectionl.5).

For the regulation of eicosanoid formation in cells, it is of central importance that,

1.2 The discovery of prostaglandins and related eicosanoids 5

in 'resting' cells, the concentrations of the free polyunsaturated precursor fatty acids are far below the KM values of the enzymes involved in the biosynthesis of eicosa- noids. Instead, these fatty acids are sequestered in membrane phospholipids and are released from these stores only upon demand, i.e. through endogenous or environ- mental signaling (Fig. 1-3). Since eicosanoid precursor fatty acids are predominantly esterified in the sn-2 position of glycerol phospholipids, signal-activated phospholi- pases of the A2 type play a critical role in controlling the rate-limiting step of eicosa- noid biosynthesis, although alternative pathways of fatty acid release have been iden- tified (see Chapter 2).

Table 1-1. Eicosanoid families

Name Biosynthetic pathway Major function

Prostaglandins

Thromboxanes

Prostacyclins

HPETEs and HETEs~

Leukotrienes

Lipoxins 15-Epi-lipoxins Hepoxilins

Trioxilins Epoxyeicosatrienoic acids

Isoprostanes

Anandamide

Cycloox ygenaselprostaglandin synthases

C ycloox ygenase/Thromboxane s ynthases CyclooxygenaseProstacyclin synthases Lipoxygenases and cytochrome P-450-dependent monooxygenases Lipoxy genase/Leukotriene synthases Lipoxygenase Acetylated cyclooxygenase-2 Lipox ygenase

Lipoxygenase Cytochrome P-450-dependent monooxygenases Non-enzymatic lipid peroxidation Unclear

Control of smooth muscle activity, secretory processes, immunosuppression, luteinization, etc. Platelet aggregation and aorta constriction Thromboxane antagonists

Control of blood pressure, renal function, synaptic transmission and inflammation Bronchoconstriction and leukotaxis Anti-inflammatory (?) Anti-inflammatory (?) Intracellutar Ca2+ release neuromodulation (?) Unknown (hepoxilin metabolites) Vasodilatation and control of renal function Signaling of oxidative stress (?)

Inhibitory neurotransmitter (?), endogenous cannabinoid receptor ligand

"HPETEs, mono-hydroperoxy eicosatetraenoic acids; HETEs, mono-hydroxy eicosatetraenoic acids.

Once released, the precursor fatty acid immediately becomes transformed into eicosanoids (the type of which depends on the pattern of biosynthetic enzymes that are expressed by a given cell) or is re-esterified. Eicosanoids are short-lived, i.e. they either hydrolyze spontaneously (such as thromboxane A2 and prostacyclin) or un- dergo rapid metabolic inactivation by enzymatic dehydrogenation, o-hydroxylation and fatty acid &oxidation (Fig. 1-2). As a consequence eicosanoids are not stored in cells. An exception to this rule is provided by certain invertebrates which are able to store large amounts of prostaglandins as esters or lactones (see Section 1.4).

6 I Aruchidonic mid and compunions: an abundunt source of biological signals

k O O H

HO OH

15-hydroxy prostaglandin debydrogenase NAD+ 1 (PDGH)

-OH

HO 0

1 5-ketoprostaglandin-AI3-reduetase i 0

fatty acid P-oxidation (2x)

. t C O O H

HO 0

o-hydroxylation and oxidation

- \GOOH

0

Figure 1-2. A major pathway of prostaglandin metabolism. E- and F-prostaglandins are rapidly inacti- vated by oxidation of the hydroxyl group at C15. This reaction is catalyzed by a ubiquitous cytosolic NAD+-dependent prostaglandin dehydrogenase (PDGH), the key enzyme of prostaglandin inactivation. The subsequent steps of prostaglandin degradation involve reduction of the A13 double bond (by a cytoso- lic 15-ketoprostaglandin A'3-reductase plus NADH), w-hydroxylation and oxidation and shortening of the side chains by fatty acid B-oxidation.

1.3 Mammalian eicosanoids

As a concise and comprehensible introduction into the field Piomelli 1301 is highly recommended.

1.3 Manirnaliari eicosanoids 7

1.3.1 Free arachidonic acid: a signaling compound?

Eicosanoids are thought to play a modulatory role, i.e. they either promote or attenu- ate the reactions of cells to systemic signals, as well as collecting various cell types in an overall tissue response. Prominent examples of such integrated responses are de- fense reactions becoming apparent as tissue inflammation or anaphylatic reactions, wound repair and functional blood supply of organs (functional hyperemia) caused by vasodilatation. Cytoplasmic phospholipase A*, a major enzyme of eicosanoid biosyn- thesis (see Section 2.3.2.2.), is activated by MAPkinase (mitogen-activated protein kinase; see Lin et al. [311) through phosphorylation. In the network of cellular signal processing this lunase is located at a central point where several signaling cascades converge [32]. This clearly shows that arachidonic acid release is controlled by a wide variety of extracellular signals and activated concomitantly with many other cellular responses, let alone the fact that additional mechanisms of arachidonic acid release and probably also of phospholipase A2 activation exist (see Chapter 2 and Smith [33]).

Once released from membranes free arachidonic acid is both metabolized to eico- sanoids and reincorporated into phospholipids. For the latter reaction the formation of arachidonyl-CoA through an ATP- and Mg”-dependent synthase is required [34]. The CoA derivative rapidly delivers the arachidonyl residue to the sn-2 position of lysophospholipids yielding the corresponding arachidonic acid ester. This reaction is catalyzed by a specific arachidonyl-CoA-lysophospholipid transferase. Other meta- bolic reactions of arachidonyl CoA, which are probably of minor importance, include the formation of diacylglycerol esters and cholesterol esters.

A major question concerns the transport of arachidonic acid within cells since the enzymes catalyzing eicosanoid formation are not necessarily found at the sites of arachidonic acid release and, moreover, free arachidonate has a high tendency to stay in a membranous (i.e. lipophilic) environment rather than to diffuse into the cytosol. There is now evidence that so-called fatty acid-binding proteins (FABPs) may control the intracellular distribution of arachidonic acid [35]. Due to their structural features these ubiquitous 14-20 kDa proteins, which are tissue-specifically expressed in vari- ous isoforms, successfully compete with lipid bilayers for free fatty acids [36].

The signal-dependent release of arachidonic acid and its rapid removal from the cytosol has led to the proposal that, besides the eicosanoids, arachidonic acid itself may serve as an intracellular signaling molecule [37]. Considerable effort has been made to test this hypothesis. However, due mainly to the technical problems inherent in lipid analysis in cells, the issue has not yet been settled. Instead, much more infor- mation on the actual concentrations of free arachidonic acid in different compartments of signal-activated cells, on the role of FABPs in arachidonate signaling and on the specific interaction with putative target proteins is required. Progress in this field is hampered in particular by the lack of a simple arachidonate assay (such as an immu- noassay or a chemoluminescense reaction) and the tendency of arachidonic acid (and other lipophilic compouds) to interact non-specifically with a vast number of proteins.

Nevertheless, several proteins have been shown to become functionally modulated upon interaction with free arachidonic acid [30]. The most prominent example is protein kinase C [PKC], a family of at least 1 1 Ser/Thr kinases [38] which occupy a central position in cellular signal processing 1391. A characteristic property of these

8 I Arachidonic acid and companions: un ubundunt source of biologicul signals

enzymes is that their activity is strictly controlled by lipid mediators such as diacylglyc- erol, phospholipids, phosphatidic acid, cholesterol sulfate, sphingolipids and unsatu- rated fatty acids, with diacylglycerol being a bona fide cellular protein kinase C acti- vator, whereas the physiological role of the other activators is still a matter of dispute.

It has been argued that the arachidonate concentrations required for PKC activation in vitro appear to be unphysiologically high. However, the fact that PKC activation in general takes place upon contact with cellular membranes and that in this environ- ment free arachidonic acid may temporarily approximate levels of more than 0.1 mM due to its lipophilicity should not be overlooked [30]. These concentrations are suffi- cient for PKC activation in vitro as well as for functional modulation of other putative target proteins of free arachidonic acid. On the other hand, based on in vitro experi- ments it has been proposed that unsaturated fatty acids may preferentially interact with cytosolic rather than with membrane-bound PKC [38]. Such a mechanism, which would considerably increase the intracellular range of PKC effects, requires a release of free arachidonic acid from membranes into the cytosol as perhaps promoted by FABPs.

Another signaling protein, the function of which is possibly modulated by a direct interaction with free arachidonic acid, is the GTPase-activating protein (GAP) of the small G protein Ras. Activation of GAP terminates the function of Ras. As shown in vitro, arachidonic acid specifically inhibits the RasGAP activity [40,41] which would result in the activation of the signaling cascades controlled by Ras [3 11. The inactiva- tion of RasGAP by arachidonic acid might be mediated by PKC since RasGAP is an in vitro substrate of PKC [42]. On the other hand, RasGAP has been found to directly and specifically associate with arachidonic acid-containing lipid micelles [43].

As a third example of a direct regulatory effect of arachidonic acid the modulation of ion channels may be mentioned [44]. In patch-clamp experiments with amphibian smooth muscle [45] and other tissues 1301 activation of potassium channel conduc- tance by free arachidonate rather than by eicosanoids was found. However, this re- sponse could be evoked by a wide variety of negatively charged lipids ruling out a specific second messenger effect of arachidonic acid. Whether this argument also holds true for the activation of cation channels regulated by the N-Methyl-D-aspartic- type glutamate receptor in rat brain neurons by arachidonate [46] remains an open question. Since arachidonic acid is released upon stimulation of NMDA receptors a positive feedback loop would develop, which has been speculated to participate in long-lasting synaptic potentiation (long-term potentiation, LTP) which is thought to play a role in memory storage [30,47].

1.3.2 Prostanoids

For Gerrnan-speaking readers the earlier literature has been thoroughly reviewed by Schror [48].

Enzymatically generated eicosanoids with ring structures are called prostanoids. The prostanoid family includes prostaglandins and thromboxanes.

The common biosynthetic precursor of each prostanoid series is an endoperoxide, prostaglandin H (PGH). PGH is produced from the corresponding fatty acid in two steps. The first step, a twofold dioxygenation, yields a labile 15-hydroperoxy-