Interaction Of Some Antihypertensive Drugs
I I With
Central Adrenergic & Dopaminergic Receptors
DISSERTATION SUBiMITTED i TO THE v:^
Aligarh Muslim University, Aligarh FOR THE DEGREE OF
Master of Philosophy IN THE
Faculty of Life Sciences
BY
M. Sc.
DIVISION OF PHARMACOLOGY
CENTRAL DRUG RESEARCH INSTITUTE LUCKNOW
1987
1 ' ds logs M &i ^fW
DS1085
C E R T I F I C A T E
TkU, t6 to c.2Ati^y that the. mAk embodizd in tkii di^^zAtation
hcUi bO-Zn acuiAizd out by M^4 Ghaz(tta Hcu o.'Cn, U.Sa. {Zoology], uyidoA
ouA upfiV'Uion, Shz hcu {^u^ltied the. Aequiteme.yvti, ^OA dzgAee. o^
McuteJc 0^ Pkilo^ophy o^ AllqaAh MiuZim UniveJi^ity, AZigoAk, Ae.gaAding
the mXuJie. and pAe^cAibed period OjJ inve^tigouLLonaZ mAk, The MOAk,
inaiadzd in tkii> di^^eAtation, hcu> not be.e.n submitted {^oA any otheA
degAee and, unle^^ othoAwi^z stated, -66 alt oAiginai.
mt PROF. ATMER H.SWVm
Vh.V,[klig.],Ph.V.[PuAduz], ChaiAman, VzpoAtment o^ Zoology AligoAh Miulim UniveA^iity AligoAh 202 001.
PR. R.C. SRIMAL M,V., MAMS
deputy ViAzctoA S Head, division oi VhoAmaaology
CentAol VAug Re^zaAck In^tiXxitz Lucknow 226001
VIA/^AA
PR AWIL t M.P.
Sciznti4t Division oi PhoAmacology
CzntAal VAug Rz4zaAch In^titutz Lucknouo 226001
Vatz:
ACKNOWLEVGEMENT
It 16 my plza^uKU to zxpfiz^^ my gratitude, to Vfi. B.N. Vhawan,
Scie.nti'tt In DiA-Zcto^'^i G^adt, CtntKai V^ug Rz6zaAch Imtitutt, Lucknow
{,0^ introducing me to thi'i, nzw ^izid o{, 6ciznce.. To, V^i. R.C. SnJ.mal, Hzad,
Vivi'tiion oi PhoAmacotogy, C.V.R.I, ^oi ki^ advitz and 6uggz6tion6 itndzizd
to me {jfiom timz to timz. Jo, Vn.. Anil Gutati, I am indzptzd {^on. hi6 cii-
cam6pzct guidancz and timziy Support.
1 am tkankiut to Vr. A.H. Siddiqi, Chairman, VzpoAtmznt o^ Zoology,
AtigoAh Mu/ittm UnivzrMty, A^goAh ^on. hi6 advice and zncoiAagzmznt.
To, Vn.. M.M. Vha/L, Viizcton., CD.R.I. Ion. providing thz nzcz^^oAy •{,acititiz6.
1 apprzoiatz thz hzip givzn to mz by thz mzmbzi^ o^ thz Division o{, Phar-
macology.
Thz financial Support providzd by thz Indian Council oi Mzdical
Rz^zoAch, Nzu) Vzlhi i6 al6o acknowlzdgzd.
Ghazala Hu/^^ain
INTRODUCTION
The primary factors Involved in the regulation of blood pressure
are the cardiac output, peripheral resistance and the circulating blood
volume. The systolic pressure is chiefly dependent upon the cardiac output,
whereas the diastolic blood pressure mainly reflects the peripheral r e s i s
tance in the ar ter ies (Mellander, 1960). Both cardiac output and peripheral
resistance are under the control of the autonomic nervous system.
The central nervous system (CNS) is known to influence and domi
nate the autonomic nervous system. It i s , therefore, not surprising that
the CNS is also involved in the regulation of blood pressure . Certain r e
gions in the medulla oblongata and the hypothalamus, which upon stimulation
produce changes in blood pressure, are known to be involved since a long
time (Schmitt and Schmitt 1969).
Claude Bernard (1878-1879) first observed that a section of the
spinal cord in the lower cervical region caused a fall in blood pressure
indicating the importance of CNS. Cynon and Ludwig (18 66) found that
stimulation of the central end of the cut nerve lying parallel but separate
from the cervical vagus caused bradycardia and hypotension. In 1870,
Dittmar showed the importance of a structure in the medulla oblongata
in the maintenance of normal blood pressure.
The control of the activity of the cardiovascular system by the
CNS is highly complex. It is apparent that various neuronal populations
containing different transmitter substances interact intr icately to maintain
a homeostatic balance of the cardiovascular system. Various authors have
suggested that central mechanisms are involved in certain forms of hyper
tension. Drugs have, therefore, been developed which lower blood pressure
via their influence on certain parts of the central nervous system.
The lowering of raised blood pressure is a pharmacological effect
of numerous compounds, some of which are suitable for the treatment of
hypertension. The onset, degree and duration of the blood pressure lowering
effect, the mode of action, the occurrence of s ide effects and adverse,
reactions are only some features that eventually determine whether a hypo
tensive agent can become an antihypertensive drug. A detailed knowledge
of the areas in the CNS and the study of the involved receptors is vital
in the understanding of the mechanism of action of antihypertensive drugs.
Studies indicate that various adrenergic, cholinergic, dopaminergic,
serotonergic and GABAergic receptors are directly or indirect ly involved
in blood pressure regulation. The o(-adrenoceptors, mainly located post
synaptical ly, in the region of the NTS are involved in the lowering of
blood pressure (ilejong, 1974).
The cholinergic neurons are widely distributed in the CNS and
the cholinergic agents can enter CNS freely. Certain areas in the brain ,
l ike the hypothalamus and area postrem of medulla are involved in central
and peripheral cholinergic mechanisms (Bhargava, 1973).
Dopaminergic neurons in the midbrain and dorsal hypothalamus
are also said to be responsible in blood pressure control. Central dopa
minergic system plays an important role in hypertension (Bhargava, 1983)
Dopamine itself induces a fall in blood pressure mainly after perfusion
in the anterior part of the third ventricle. Serotonergic neurons are present
in the brain stem, hypothalamus and spinal cord. There are many interac
tions between the central serotonergic and sympathetic systems in relation
with the cardiovascular regulation^ \ ? - .> GABAergic
mechanism is also involved in the central blood pressure regulation since
such neurons are present throughout the brain with high concentrations
in the hypothalamus and substantia nigra. Exogenous administration of GABA
or muscimol leads to hypertension or hypotension possibly by an inhibitory
influence on pressor pathways in the brain. Since anti-hypertensive therapy
consists of treatment with drugs which may have primary action in CNS
or outside. We have taken four antihypertensive drugs for our study. The
proposed mechanism of action of each of these drugs is different but the
effect of their long term administration on the brain receptors is not known
but .is of vital importance.
Clonidine ^[Catapres, Catapsesan) was synthesised by Stable in
1962 and originally tested preclinically as a niisal decongestant because
of i t s vasoconstrictor proper t ies . Later experiments by Graubner and Wolf
(1966) indicated that it possessed other pharmacological propert ies like
reducing heart r a t e , lowering blood pressure and causing sedation. Today
clonidine is accepted as a potent antihypertensive agent with a predojninantly
central mode of action. Considering its chemical relationship to various
sympathomimetic agents it is not surprising that clonidine influences adre
nergic receptors both in the periphery and in the central nervous system
(Anden £t_ al_. 1970), Chronic clonidine therapy produces a persistent reduc
tion in arterial pressure.
Rmwolila 6eApenUna is the plant from which reserpine (Bein,
1956) was isolated and used in Indian medicine for the treatment of various
disorders for centuries. It was brought to the notice of Western medicine
only in 1949 by Vakil. Reserpine is widely used in the therapy of hyper
tension, particulariy in combination with other drugs. Reserpine lowers
arter ial pressure in hypertensive and normotensive animals and man. The
depletion of catecholamine stores has been recognized to be an important
mechanism in the antihypertensive action of reserpine and related compounds.
Since noradernaline is the most important neurotransmitter in the peripheral
adrenergic system, the depletion of this neurohumour will probably have
a negative effect upon the activity of the sympathetic nervous system.
The depletion of noradrenaline stores caused by reserpine is not limited
to the peripheral adrenergic system but also extends to the CNS where
it is present in high concentrations. The hypotensive action of reserpine
can therefore, be both the peripheral and central action.
Hydralazine was discovered by Druey and Coworkers in 1945. Al
though hydralazine was originally thougiit to reduce blood pressure by
a central action, it is now accepted that these effects are only minor of
and that the major site of action resides at the l eve l /vascu la r smooth
muscle (Baum e1 al . 1972; David et al_. 1975).
Centhaquin - was synthesized in C D . R.I. in 1971, It was found
to lower blood pressure in several species of animals in low doses. The
main site of action was found to reside in the CNS (Srimal et a l . 1978).
However, the exact mechanism of i ts action is not yet known. It was also
found to lower the blood pressure of hypertensive pat ients .
METHODOLOGY AND TECHNIQUE
Albino rats of the Charles Foster s t ra in , bred in the animal house
of the Central Drug Research Institute were used. Rats weighing 100-150
gE". were taken and kept in standard laboratory conditions of temperature,
humidity and dark light cycle.
Antihypertensive drugs, clonidine (0.1 mg/kg) was dissolved in
water, centhaquin (0.1 mg/kg) synthesised in the Central Drug Research
Institute and was dissolved in /a lcohol . Dihydralazine (0.125 mg/kg) made
by Ciba Geigy. of India Ltd. was diluted in water. Reserpine (Serpasil)
(12.5 mg/kg) made by Ciba Geigy of India Ltd. was diluted in water.
These drugs were orally administered daily for two months to groups of
20 r a t s .
Preparation of Membranes with Receptors
Animals were sacrificed and decapitated at the end of two months.
Specific areas of the brain like the hypothalamus, cortex, caudate and
medulla were dissected out in cold. Each brain area was weighed and
then homogenized in 30 volumes of ice cold 50 nM Tris HCl buffer (pH
7.4 at 20''C) containing 0.1% ascorbic acid, 120 nM pargyline, 5 mM pota
ssium chloride, 2 mM calcium chloride and I mM magnesium chloride. The
neuronal membrane was prepared by centrifugation at 2,000 g for 10 minutes,
pellets were discarded and the supernatant was recentrifuged twice at
50,000 g for 15 minutes each. This time the supernatant was discarded
and the pellets were weighed and suspended in 50 mM Tris buffer (pH
7.4 at 37°C).
The standard assay mixture containing different concentraions of
the hot l igands, fixed concentration of cold ligand and 200 ug of the neuro
nal membrane and buffer to make up the total volume of I ml was incubated
in tr ipl icate in a shaking water bath at 37°C for 15 minutes. The reaction
was discontinued by dipping the tubes in ice cold water. The contents
of the incubated tubes were rapidly filtered under partial vacuum using
a Millipore Manifold filtration unit and Whatman GF/C glass fibre f i l t e r s .
This was followed by three washes with 5 ml ice cold 50 mM Tris buffer
(pH 7.4). The dried discs were transferred to sci-'ntillation liquid [Compo
sition: 2,5-diphenyloxazole 4.9 M; 1,4-bis (5-Phenylonazolyl) benzene
200 mg; naphthalein 60 g, ; ethylene glycol 20 ml; methanol 100 ml; dio-
xane upto 1 l i t r e ) ] . After an overnight equilibration per iod, the radio
act ivi ty in the sample was counted by LKB Rack beta 1217 liquid sciuntilla-
tion spectrometer.
It is necessary, to differentiate between the two types of binding
that takes place under these circumstances, that i s , specific to the recep
tor vinder investigation and non-specific to the other biopolymers of the
t issue. The number of specific receptor sites are limited whereas the
non-specific sites are unlimited. The binding experiment was, therefore,
repeated in the presence of excess amount of non-radio-labelled ligand.
Under these conditions it is unlikely that any radio labelled ligand would
bind to the specific sites while non-specific binding was unaffected. The
difference in the binding values for these two experiments then gave the
amount of specific binding. Repeating the two experiments for a range
of labelled ligand concentrations helps in plotting down the resul t s . This
clearly shows that the amount of specifically bound ligand reached a maxi
mum despite the addition of increasing amounts of ligand. This situation
would be expected if the number of specific receptors are limited. Such
curves are capable of the mathematical analysis common to similar bioche
mical studies from which dissociation constants and the number of receptor
sites are determined.
An extension of these studies is to investigate the binding process
in the presence of agents thought to act as agonists or antagonists for
that particular receptors which were being studied. Varying amounts of
the compound under investigation were added to the incubation medium
which contained a fixed amount of labelled ligand; the amount of specific
binding is plotted against the concentration of the added compotmd.
Receptor Binding Studies
Adrenergic and dopaminergic receptors were taken for the radio
active receptor binding studies.
The hot ligand used for the oC-adrenergic receptors was 3H-Dihydro-
ergocriptine (Amersham). Different concentrations from 0.25 to 12nM ' were
used. Cold ligand used was phentolamine (1 uM).
For the fi -adrenergic receptors 3H-Dihydroalprenalol (Amersham)
was used as the hot ligand at concentrations ranging from 0.12 nM to 4nM.
Propranolol at a fixed concentration of 1 uM was taken" as the cold ligand.
For dopaminergic receptor binding assays 3H-Spiperone (NEN) at
concentrations of 0.1 nM to 5 nM was used as hot ligand. Haloperidol at
a fixed concentration of 1 uM was taken as the cold ligand.
All the experiments were repeated thrice and the data was analysed
using least square regression analysis and analysis of variance.
REVIEW OF LITERATURE
HYPOTHALAMUS
Brezenoff and Jenden (1969) r e p o r t e d tha t injection of noradrena l ine
into the pos te r io r hypothalamus of r a t s r e su l t ed in a s l i g h t and s h o r t
l i ved r i s e of blood p r e s s u r e . E lec t r ica l s t imulat ion of va r ious nuclei of
the hypothalamus led to an enhanced r e l ea s e of r a d i o - a c t i v e no rad rena l ine
which had been ea r l i e r injected in the l a t e r a l ven t r i c l e ( P h i l i p p u , 1970).
It has been suggested tha t p^ r a t h e r than ^ - ad renocep to r s of the h y p o
thalamus a r e involved in the p r e s s o r response (Ph i l ippu and K i t t e l , 1977).
Superfusion of hypothalamus with procaine at a concentrat ion which was
equ ianaes the t i c to tha t of propranolol ( P h i l i p p u , 1970', Ph i l i ppu and K i t t e l ,
1977) was ineffective in prevent ing noradrenal ine effect . Microinjection
of clonidine into the pos te r ior hypothalamus of r a t led to a fall in blood
p r e s s u r e and hea r t r a t e (Struyker Boudier et^ al^., 1972). It was presumed
tha t the hypotens ive act ion of c lonidine was p a r t l y due to i t s ac t ion on
the n o r - a d r e n e r g i c system of the pos te r io r hypothalamus (S t ruyker Boudier, ,
et a l . . 1972). Phi l ippu et^ al_. , (1973a) p rov ided ev idence for the ac t ion
of c lonidine on both p r e - and pos t - synap t i c r ecep to r s of pos t e r io r h y p o t h a
lamus in ca t . Injection of clonidine into the l a t e ra l ven t r i c l e of ca t s i n h i b i
ted the p re s so r response to e l ec t r i ca l s t imulat ion of the hypo tha lamus
(Dhawan et_ al_., 1975).
Bhargava (1984) , suggested tha t cyclo (Leucyl-glycine) i n t e r ac t s wi th b r a i n
dopamine r ecep to r s and tha t b ra in dopamine r e c e p t o r s may be invo lved
in the et iology of hype r t ens ion . He also found a number of (3H) s p i r o p e r i
dol binding s i t e s in the hypothalamus of spontaneously h y p e r t e n s i v e r a t s
(SHR). In e a r l i e r s tud ies Bhargava , (1983 ) has ind ica ted t h a t chronic
t reatment of SHR with CLG - inhibi tes the enhanced hypo the rmic r e sponse
to apomorphine .
The pa raven t r i cu la r nucleus of the hypothalamus (PVNH) has been
suggested to p a r t i c i p a t e in the control of the autonomic nervous s y s t e m ,
p a r t i c u l a r l y the ca rd iovascu la r functions (Kannan and Yamash i ta , 1985).
This nucleus has been shown to r ece ive a v a r i e t y of ca rd iovascu la r af ferent
information (Calaresu and C i r i e l l o , 1980). The s t imulat ion and les ion of
the PVNH area have been r e p o r t e d to modify ca rd iovascu la r functions (Zhang
and C i r i e l l o , 1982).
Neuroanatomical s tud ies demonstrate tha t the PVNH neurons send
axons to the lower brains tem and the spinal cord including the nucleus
t r ac tus so l i t a r ius (NTS) and the in termedio la te ra l cel l column of the sp ina l
c o r d , which may u l t imate ly control ca rd iovascu la r function (Mnltra et_ a l . t
19W). Some neurons which project to the dorsomedial medulla r ece ive
afferent information from ca ro t id ba ro recep to r and i t suggests t h a t PVNH
may p lay a ro le in the neural control of the ba ro recep to r re f lex a t the
level of the NTS region (Kannan and Yamashita , 1985).
Cir iel lo ,^ Caver son (1986) suggested tha t e levat ion of a r t e r i a l p r e
s su re obse rved following lesion of the PVNH was not due to des t ruc t ion
of i t s neurons , but i t was a non-specif ic effect of the les ion of the b ra in
t i s s u e . Elec t r ica l s t imulat ion of PVNH e l i c i t s increase in the a r t e r i a l p r e
ssure and the hear t r a t e .
NUCLEUS TRACTUS SOLITARIUS (NTS)
The nucleus t r ac tu s so l i t a r ius (NTS) i s t h e s i t e of terminat ion
of afferent f ib res from a r t e r i a l and cardiopulmonary mechanoreceptors and
chemoreceptors (Muira and Reis , 1969). It thus mediates and in tegra tes
the barorecep tor re f lex (Muira and Reis , 1972).
The NTS is thought to be the s i t e of action of noradrena l ine con
taining neurons which have been found in t h i s nucleus (Dahlstrom and Fuxe ,
1965). Injection of catecholamines including alpha'methyl noradrena l ine into
the NTS causes decrease of blood p r e s s u r e and hea r t r a t e (ctsjong, 1974).
This may be the r e s u l t of d i r ec t st imulation of post synap t i c a l p h a - a d r e n o -
ceptor . ' The most sens i t ive region for t h i s action
i s A (Dahlstrons and Fuxe , 1964). Alpha, type of ad renocep to r s a r e thought
to be involved in b r a d y c a r d i a (dejong, 1974).
Infusion of c lonidine into t h i s nucleus has been r e p o r t e d to reduce
b l o o d - p r e s s u r e and h e a r t r a t e (Sinha et^ al_. , 1975). However, consenses
i s t ha t the NTS is not the only or the main s i t e of t h e hypo t ens ive action
of clonidine in cats and dogs .
Laguzzi et^ al_., (1984) have suggested tha t a l though serotonergic
mechanisms in the NTS may be involved in the modulation of e l e c t r o - c o r
t ica l and card iovascu la r a c t i v i t y , t h e y a r e not i n t e g r a r to the ba ro recep to r
re f l ex a r c . Sved (1985) ind ica ted tha t hyper tens ion by b i l a t e r a l NTS d e s
t ruc t ion in r a t s is p roduced by complex in terac t ions among t h e sympa tho-
adrenal systems vasopressin and the renin-angiotensin system.
MEDULLA
The ventral surface of the medulla oblongata (VSMO) is the focus
of attention as an important brain region in the central regulation of the
cardiovascular system. The basis for this interest lies largely in functional
studies carried out in cats and rabbi ts . Bousquet and Guertzenstein (1973)
claimed that the site of action of clonidine is located on a small area
of the ventral surface of medulla oblongata called Schlafke zone. Clonidine
applied in small concentration on this area induced a marked fall in blood
pressure and caused bradycardia. This was confirmed by Dhawan et_ al. ,
(1975), Bousquest ei^ al., (1975 ) further reported that bilateral destruction
of this area abolished the depressor effect of clonidine. Reis and Coworkers
(1984) have provided evidence that adrenaline containing neurons of C^
area may represent the tonic vasomotor neurons of the rostral ventrolateral
medulla. They have hypothesized that the decrease in blood pressure
provoked by clonidine is mediated via a decrease in adrenaline turnover
in the A^/C area. Electrical stimulation, glutamate microinjection or kainic
acid application to the ventral medulla raises ar ter ial blood pressure
(McAllen et_ ah., 1982). Bilateral destruction with electrolytic lesions, cold
block, or neurotoxic doses of kainic acid lower blood pressure (McAllen
et a l . . 1982). This area is highly chemosensitive. Three specific areas
( ros t ra l , intermediate and caudal) in this region of medulla have been
defined according to the cardiovascular and ventilatory effects elicited
by topical application of various agents . (Schlaefke,
1981). However, the intermediate zone is the area from which the most
profound cardiovascular effects have been evoked (McAllen et al_., 1982;
Keeler e^ al^., 1984).
When topically applied to cat VSMO, GABA and i ts potent agonist,
musimol, produced depressor and bradycardiac responses. These responses
were reversed by similar application of the specific GABA receptor anta
gonist bicuculline (Yamada et al., 1982). These studies suggested that GABA-
ergic system' in the VSMO in the cat is inhibitory to sympathetic out flow
(Keeler et al_., 1984).
Sved et al^., (1985) have indicated that vasopressin release and
arterial pressure responds differently to treatments of the caudal ventro
lateral medulla, suggesting that different cells in the caudal ventrolateral
10
medulla are involved in the regulation of vasopressin release and arter ial
pressure .
The cardiovascular effects associated with the micro-injections
of carbachol, phytostigmine and atropine, into the pressor area of ventro
lateral medulla were studied by Willete et al_. , (1984). They found that
the hypertensive responses evoked by muscarinic-receptor stimulation in
the ventrolateral medulla were mediated by increased sympathetic ac t iv i ty .
Ciriello and Caverson (1986) suggested that neurons in the ventro
lateral medulla may be a medullary feed back reflex loop though which
afferent information from cardiovascular receptors exer ts an influence on
NTS neurons involved in the control of the circulation.
Smialowska et_ a2_. » (1985), by histological examination and scanning
electron microscopy of the medulla oblongata, found that there is a wide
dense area of catecholamine terminals in the external layer of the ventral
medulla oblongata. 5-Hydroxytryptamine-containing terminals and nerve
cell bodies on and near the surface were also found. They suggested that
due to their superficial location these monoamine nerve terminals may in
fluence the content of cerebrospinal fluid and in this way have effects
on cardiovascular and other physiological functions.
CAUDATE NUCLEUS
The corpus striatum comprises of caudate nucleus, putamen and
globus pallidus. The size of the caudate nucleus depends upon the size
of the cerebral cortex. It has a large head which tapers to a body and
then' t a i l . The large bulbous head forms the lateral wall of the posterior
horn of the lateral ventricle. Caudate nucleus receives afferent input from
the thalamus, subthalamus, cerebral cortex, brainstem nuclei and substantia
nigra.
The main afferent projection from caudate is to the globus pal l idus ,
thalamus, subthalamus, brain stem nuclei, olivary nuclei and substantia
nigra.
.Neurons which contain dopamine are found with cell bodies in
substantia nigra and terminals in the caudate nucleus (Anden et_ al_., 1970).
It has been shown that there is a close relationship between the two nigro-
str iatal dopaminergic pathways in cat and rat (Hull et a l . , 1974).
11
Elec t r ica l s t imulat ion of caudate nucleus r e l ea ses dopamine in the
ven t r i cu la r sys tem (Goldstein et a l . . 1969).
CLONIDINE
History
Clonidine was syn thes i sed in 1962 by Stable as a d e r i v a t i v e of
the imidazol ine nuc leus , the vasoconst r ic tor p r o p e r t i e s of which were well
known, in the hope tha t i t would be a nasal decongestant . It caused cons i
de rab l e consternat ion, when after the nasal ins ta l l a t ion of a few d r o p s ,
the f i r s t two volunteers to rece ive c lonidine became ve ry seda ted and
hypo tens ive (Graubner and Wolf, 1966). This chance finding t r igge red a
large number of inves t iga t ions (Nayler et_ ah., 1968', McRaven et^ al_., 1971)
which have shown tha t clonidine is an effect ive cen t r a l ly act ing a n t i h y p e r
tens ive agent .
Mechanism of Action
Clonidine produces an in i t ia l increase in blood p r e s s u r e followed
by a f a l l . The in i t i a l increase in blood p r e s s u r e was found to be due
to s t imulat ion of p e r i p h e r a l ad renocep to r s . The h y p e r t e n s i v e effect was
eas i e r to inves t iga te after suppress ion of the sympathe t i c tone by s p i n a l i -
zation (Constantine and McShane, 1968), p i th ing (Autret et^ al_., 1971), admi
n i s t r a t ion of ganglion blocking agents and (Boisser et^ al_. 1968; Nayler
et a l . , 1968), resei :pine (Boissier et^ a\_., 1968) guanethidine (Robson and
Kaplan, 1969) or b re ty l ium (Nayler et_ al_., 1966).
Phenoxybenzamine has been r e p o r t e d to be a poor antagonist of
c lonidine induced hyper tens ion in cat (Nayler et^ aj_., 1966) but o t h e r s have
r e p o r t e d i t to be effect ive in dogs and r a b b i t s (Nayler et^ ah., 1968) as
well as ca t (Schmitt and Schmitt 1970).
I t has been demonstrated tha t c lonidine ac t s as a sympathomimetic
agent on J^-adrenoceptors . L ipski et^ al_., (1976a) found tha t b i l a t e r a l d e s
t ruct ion of the nucleus t r ac tus so l i t a r i i induced in u re than ized r a t s a r i s e
in blood p r e s s u r e and. reduced the hypotens ive effect of c lon id ine .
The effects of clonidine on ^ - a d r e n o c e p t o r s a p p e a r to be v e r y
weak or a b s e n t . In the i so la ted aur ic le of guinea p i g s , c lonidine in high
concent ra t ion , i nc reased the cont rac t i le force but d id not change the r a t e
(Hoefke and Kobinger, 1966). On the h e a r t lung p r e p a r a t i o n of the dog
(Hoefke and Kobinger 1966) or in the i so la t ed h e a r t of the cat (Constantine
12
and McShane, 1968) or the rabbit (Werner et_ aj_., 1972) clonidine reduced
the contractile force only at high concentrations. All these investigations
demonstrate that clonidine is an oC-sympathomimetic agent and is probably
not taken up by the sympathetic nerve terminals.
Clonidine appears to be a partial agonist on ^ - a d r e n o c e p t o r s ;
in fact the maximum effect was less than that of noradrenaline on rabbit
pulmonary ar tery (Starke ejt a2_.. 1974).
Mechanism of Hypotension
Low doses of clonidine caused hypotension and bradycardia in
dog, cat and rat (Boissier et_ al_., 1968). This potential of hypotension
and bradycardia can be explained in part by loss of the reflex inhibition
of the sympathetic tone after clonidine, thus the vascular smooth muscle
is unopposed in i ts response to the pressor agents. However, since noradre
naline is also potentiated after clonidine in pithed rats (Boissier et_ al . ,
1968) some peripheral component must also be involved. In man, no signi
ficant interaction of clonidine with catecholamines was observed (Onesti
et_ al_., 1971).
In high doses, clonidine reduced the effect of noradrenaline and
sometimes reversed the hypertensive effects of adrenaline (Boissier et
a l . > 1968). Coupar and Kirby (1972) suggested that clonidine may cause
venodilation by blocking endogenously released noradrenaline, an effect
which may contribute to the hypotensive action of the drug.
The concept that a pre-synaptic of-adrenoceptor stimulation could
inhibit noradrenaline release is generally accepted (Starke et al_., 1975).
Phentolamine increases noradrenaline release and in addition antagonises
the effect of clonidine (Starke et a2^.. 1974). Since the f i rs t synapses of
baroreceptor fibres are located in the nucleus tractus sol i tar i i (NTS) ,
it is thought that clonidine night mimic the effects of baroreceptor stimula
tion. Greenberg and Wilborn (1982) suggested that clonidine
may act directly on the veins, or indirectly by inhibiting the release
or synthesis of a trophic noradrenergic factor, to reverse the functional
and structural changes in the veins. Lorez et_ al^.. (1983) proposed that
the effect of clonidine on noradrenaline turnover is most l ikely the result
of a local feed back inhibition through presynaptic 0(-adrenoceptors. Medgett
and Rand (1983) suggested that since the NTS and intermediolateral cell
13
column of the spinal cord are the prime centers for the site of the cardio
vascular action of clonidine and since the cardiovascular effects of clonidine
can be elicited in the virtual absence of neuronal noradrenaline, the de
crease in central noradrenaline turn over and the cardiovascular effects
of clonidine are not interrelated phenomena. Bentley e t _ ^ . , (1986) suggested
that clonidine causes a selective reduction in sympathetic tone to the veins
that is mediated at least partly by a central action as well as an expan
sion of the collateral venous routes. This together with the selective im
pairment of venoconstrictor responses to both noradrenaline and adrenaline,
may account for the decrease in cardiac output that is most often reported
following clonidine,
Clonidine in hypertensive patients reduced the excretion of urinary
catecholamines (Hokfelt et_ al_., 1975). This effect is believed to be due
to the decreased sympathetic tone. Clonidine does not change the level
of catecholamines in the brain (Hoefke and Kobinger 1966; Persson and
Waldeck, 1970; Bralet £t_ ail ., 1973). A slight increase in noradrenaline
or dopamine levels has been reported after high doses of clonidine (Persson
and Waldeck, 1970). The increased dopamine levels in the corpus striatum
could account for the stereotypes seen with high doses of the drug (Persson
and Waldeck, 1970).
The elevation of cerebrospinal fluid and noradrenaline act ivi ty
may be increased in patients with mild to moderate essential hypertension
and that can be reduced by treatment with clonidine (Cubeddu et_ a]_., 1984).
Stimulation of peripheral prejunctional pC ^-adrenoceptors in anesthetized
rats may contribute to the fall in the plasma catecholamine level produced
by i . v . clonidine and further suggests that the hypotensive effect may
be centrally mediated (Brown and Harland, 1984).
The centrally mediated fall in blood pressure induced by clonidine
has been ascribed to a reduced sympathetic nerve act iv i ty . This was shown
to be dose-dependent after intravenous injection (Waite, 1975) as well
as after it was injected into the 3rd Ventricle of the brain (Waite, 1975).
Franz and Madsen (1982) suggested that although the - bulbo
spinal reflex pathway was- more sensitive to depression by clonidine than
i ts efferent descending intraspinal pathway, analysis of the relat ive depre
ssion of transmission at spinal and at brainstem level indicated that the
spinal site is more sensitive to clonidine than it has been considered
14
Prolonged treatment of spontaneously hypertensive rats with cloni-
dine decreased the mean arter ial pressure and heart rate (Pegram et a l . ,
1982). On stopping chronic clonidine treatment, an abrupt rebound of the
blood pressure to values higher than those recorded before treatment has
been reported by several authors.
Adrenergic Link
Studies have shown that centrally acting agents l ike clonidine
are usually effective in low doses and that increase in dosage tend to
exaggerate the possibil i ty of side effects without providing much additional
antihypertensive efficacy (Schultz et al_. , 1981). Titeler and Seeman (1982)
have suggested that the antihypertensive action of clonidine is more likely
due to interactions with oC^-adrenergic receptors than oC^-receptors.
Pharmacological evidence shows that central ©(--adrenoceptor acti
vation is associated with sedation and reduction in blood pressure (Bousquet
et a l . . 1983). It has been further suggested that the bradycardiac effect
of clonidine results from activation of both central and peripheral o i -adre
nergic receptors of the •'^.-subtype (Park et al_. , 1983). Neurotransmitter
concentration in the synaptic cleft may be responsible for the transsynaptic
modulation of o( -adrenoceptors postsynaptic population. The fiC -adrenoceptor
which are presynaptically located on the serotonergic terminals but are
post-synaptic in relation to the noradrenergic neurons also show increased
sensit ivi ty after chronic clonidine treatment (Cerrito et_ al_. , 1984). Chronic
clonidine administration can induce down-regulation of both of_-presynaptic
autoreceptors located on noradrenaline terminals and of -presynaptic hetro
receptors located on serotonin terminals (Maura et aj_. , 1985). The increased
pressor resonsiveness of both spontaneously hypertensive ra ts and Wistar-
Kyoto rats to arginine vasopressin following clonidine is an unexpected
finding and may be related to peripheral interaction between oc-adrenergic
agonist and arginine vasopressin (Dat-a-r et al_. , 1986).
Cholinergic
Srimal et al_., (1977) suggested that intact cholinergic link in
the brain stem is essential for the hypotensive action of clonidine. Criscione
et a l . , (1983) have proposed that cholinergic mechanism in the NTS tends
tonically to lower ar ter ial pressure after clonidine administration and may
modulate the baroreceptor reflex without being an integral par t of the
reflex a r c .
15
Opioid
Naloxone completely blocked the c lonidine induced hypotens ion
in spontaneously hype r t ens ive r a t s and c a t s , and p a r t i a l l y b locked i t in
Wistar Kyoto r a t s , suggesting the pa r t i c ipa t ion of opioid l ink in the d e v e
lopment of c lonidine evoked b radyca rd i a and hypotension both in h y p e r t e n
s ive and normotensive an imals . Experiments conducted on anes the t i zed ca t s
(Rogers and Cubeddu, 1983) show tha t c lonidine a p p a r e n t l y reduces blood
p r e s s u r e , hea r t r a t e and efferent sympathe t ic nerve f i r i ng , by a mechanism
independent of an opia te receptor ac t iva t ion wi th in the CNS. Contradic t ing
t h i s , Er iksson and Tuomisto, (1983) suggested tha t opioid mechanism p o
s s i b l y p a r t i c i p a t e s in the hypotens ive action of c lon id ine , but l e s s in
normotensive than in spontaneously h y p e r t e n s i v e an imals . Farsang et_ al_. ,
(1984) i s of the view tha t the haemodynamic differences between the cent
r a l l y act ing oC -adrenoceptor agonist a n t i h y p e r t e n s i v e drugs may at l eas t
in p a r t r e s u l t from the involvement of opioid mechanism only in the act ion
of c lon id ine .
RESERPINE
History
The use of Rauwoiiiahy Indian worke r s was r eco rded in 1918,
but i t s usefulness in hyper tens ion was pointed out by Vakil in 1949 and
l a t e r confirmed by Wilkins and Judson (1953). In the e a r l y 19^0, Rauwo^^a
6Q.n.pZntina p r e p a r a t i o n s and r e s e r p i n e , the main a l k a l o i d , were in t roduced
for the t rea tment of l ess seve re form of hype r t ens ion .
Rauwot^a p r epa ra t ions were a lso in t roduced in p s y c h i a t r y for the
t rea tment of Sch izophren ia . However, g radua l ly r e s e r p i n e passed out of
p s y c h i a t r i c use and was rep laced by more efficacious syn the t i c drugs (Luby ,
1968). Reserpine continued to be used as a n t i h y p e r t e n s i v e drug .
MODE OF ACTION
Depletion of Catecholamines
Treatment of animals with r e s e r p i n e r e su l t ed in deple t ion of c a t e
cholamines from the sympathe t ic ganglia , blood v e s s e l s , s p l e e n , i r i s and
o ther t i s sues wi th an adrenerg ic innervat ion (Berkowitz et^ al_., 1971).
It a l so dep l e t ed the b r a i n of i t s content of noradrena l ine (Holzbauer and
Vogt, 1956) and serotonin (Karki and Paasonen, 1959). Reserpine a l so d e p
le t ed t h e r a b b i t and sheep b ra in of dopamine, which occur red more r a p i d l y
16
than that of noradrenaline (Bertler et al_. , 1956). In pigeons, reserpine
caused depletion of the serotonin of the brain, more rapid ly than dopamine
and finally noradrenaline (Juorio and Vogt, 1967).
Kopin and Gordon (1962) showed that the reserpine - induced
depletion of catecholamines is accompanied by the release of inactive dea-
minated metabolites. Thus the appearance of active catecholamines at the
site of action is minimal.
The interaction of reserpine with indirectly acting sympathomimetic
amines have been reviewed by Muscholl (1966).
Reduction in Transmission
The administration of reserpine results in reduction of responses
of effector organs to stimulation of post ganglionic adrenergic nerves , even
though the effector organ remains responsive to noradrenaline (Gaffney
et a l . , 1963). The loss of peripheral adrenergic transmission has a latency
and occurs sometime after the appearance of the usual signs of the reser
pine syndrome such as sedation, bradycardia, decrease in blood pressure
and relaxation of the nictitating membrane. The loss of adrenergic t rans
mission after reserpine treatment is not complete. Thus, adrenergic nerve
stimulation sti l l produces positive inotropic responses in guinea pig atria
(Barnett and Benforado 1966) and chicks heart (Bolton, 1967). But the
response is abolished in rabbit (Hukovic, 1959).
Cholinergic Component
A cholinergic component is reported in the response of the nictitat
ing membrane of reserpinized cat (Mirkin and Cervoni, 1962). It is sugges
ted that there are cholinergic fibres running with the adrenergic fibres
in the sympathetic nerve trunk or a cholinergic mechanism is present in
the adrenergic nerves (Burn and Rand 1965).
Effect in Hypertensive Animals
Reserpine gradually lowers blood pressure without causing an initial
pressor response in most animals and in man (Bein, 1956). However, a
primary hypertensive response to reserpine has been observed in spinal
animals (Schmitt and Schmitt, 1969) and in animals pretreated with ganglion
blocking drugs (Bech ^ aj^., 1957). Both these procedures result in consi
derable enhancement of pressor responses to noradrenaline and adrenaline.
17
Reserpine treated r a t s , subjected to procedures for producing renal hyper
tension failed to become hypertensive. The isolated perfused hind quarters
of these rats exhibit a lesser vasoconstrictor action of noradrenaline than
do those of untreated hypertensive r a t s . The hypotensive
action of reserpine is greater in hypertensive subjects than in normotensive
ones, although the incidence of side effects does not differ between the
two groups (Fluckiger, 1969). In spontaneously hypotensive r a t s , reserpine
has a more pronounced antihypertensive activi ty than in renal hypertensive
rats (Ebinhera and Martz, 1970). A single dose of 0.05 mg/kg po lowered
the ar ter ial pressure and reduced the heart rate of dogs with neurogenic
hypertension, whereas inconsistent effects were obtained with the same
dose of reserpine in renal hypertensive or normotensive animals (Ehrre ich,
1978).
Peripheral Mechanism
The antihypertensive action of reserpine is often at t r ibuted to
the loss of vasomotor and cardiac sympathetic tone consequent to the deple
tion of noradrenaline from the peripheral adrenergic nerves subserving
cardiovascular control (Green, 1962).
Central Mechanism
The depletion of noradrenaline from cardiovascular control centres
in the hypothalamus, medulla, and from neurons in the lateral horns of
the spinal cord which make synaptic connection with thoracolumbar sympa
thetic preganglionic neurones, could also be responsible for the fall in
blood pressure (Green, 1962).
Greenberg and Weiss (1979) gave repeated doses of reserpine to
3 month old rats which produced dose related increase in (3H) dihydro
alprenolol (DHA) bincjing in pineal gland, cerebral cortex and cerebellum.
Reserpine increased DHA binding by increasing the density of ^ - a d r e n e r g i c
receptors , according to these authors.
Reserpine-induced supersensitivity to isoprenaline does not appear
to involve a change in the affinity for ^-adrenoceptor or in receptor num-3
ber as determined by ( H) DHA binding (Hawthorn and Broadley, 1982).
The evidence against the central site of action for the antihyper
tensive action has been provided by several authors. Thus after treatment
18
with reserpine, no diminution in electrical discharge in the preganglionic
sympathetic nerves was observed which is expected following depression
of sympathetic centres (Kobinger and Pichler, 1976).
The initial enthusiasm for the clinical use of reserpine was dampen
ed by i ts side effects, in particular severe depression resulting in some
cases committing suicide. It is now used in combination with other anti
hypertensives. However, reserpine is a useful pharmacological tool.
HYDRALAZINE
History
The hypotensive effect of hydralazine, which is characterized
by its gradual onset, limited degree and long duration, was observed in
the laboratory as early as 1945. But only four years later the first paper
on this new group of blood pressure lowering agent was published (Gross
et a l . , 1950). One of the reasons for the delay was unpleasant, acute side
effects such as headache, nausea, vomitting, flushing, tachycardia and
angina.
Mechaitlsm of Action
The onset of hypotensive action is gradual, the maximum response
is reached after 10 to 20 min, the degree of blood pressure reduction
is limited and the duration of action is long. Higher doses do not induce
a marked fall in blood pressure, but a prolongation of the effect lasting
for several hours is observed (Gross et al_. , 1950; Bein et stJL'» 1953).
In the spinal cat , in which the basal blood pressure is reduced
to about 50 to 60 mmHg hydralazine does not induce a further fall of blood
pressure . From this it was erronously concluded that the drug may have
a central site of action. Although on the basis of the f i rs t pharmacological
studies of hydralazine i t was supposed that the drug acted directly on
the arteriolar smooth muscle, some data suggested only a central site of
action (Gross e^ al_. , 1950; Bein et a]_., 1953).
In further studies hydralazine was administered into a fourth cere
bral ventricle; , the carotid arteries of cats and the hypotensive response
obtained was taken as an indication that the drug acted centrally (Lim
et a l . , 1955). However, other investigators reported contradictory resul ts .
When hydralazine was injected into the cisterna magna or the th i rd ventricle
19
of ca t s , only a slight fall in the blood pressure was observed accompanied
by a reduced pressor response to i . v . injected adrenaline (Schmitt and
Gicguel, 1956). Furthermore injections of hydralazine into the vertebral
ar tery of the cat caused a similar hypotensive response to intravenous
injection (van Zwieten, 1968). Increase of the hydralazine dose from 1
to 2 mg/kg, which resulted in a greater fall in blood pressure , produced
no significant reduction of spontaneous sympathetic outflow (Baum et_ al_. ,
1972).
The reduction in the peripheral vascular resistance is the conse
quence of a general vasodilation of the precapil lary resistant vessels ( Is-
rai l i and Dayton, 1977). Simultaneously with the fall in the blood pressure ,
an increase in the heart rate and cardiac output occurs as a result of
enhanced peripheral sympathetic act iv i ty . The increase in heart rate is
more pronounced in the conscious animals than in the anesthetized one
and is of shorter duration than the fall in blood pressure (Brunner ei_
a l . , 1967). Since hydralazine has only a weak action on the capacitance
vesse ls , venous return is not diminished and cardiac output increased
simultaneously with the r ise in heart rate (Zacest et al_. , 1972). To a
certain degree the increase in myocardial contractility was repor ted, which
could be inhibited by <?f-adrenergic blockers (Barrett et_ al_. , 1965). In
spontaneously hypertensive rats hydralazine lowers blood pressure (Nagaoka
et_ a l . , 1969).
Extensive studies of Ablad and associates (1963) have shown that
hydralazine and related compounds exert their hypotensive effect by means
of direct action on the vascular smooth muscle. It has no influence on
centers regulating blood pressure.
The mechanism of the hypotensive effect of hydralazine in produc
ing vasodilation both IL v^vo ^^d on contracted arterial s t r ips is uncertain.
The scant evidence that exists suggests an interference with the influx 2
of Ca + into the smooth muscle cell or i ts release from intracellular stores
(Mclean el^ al_. , 1978).
In conscious rabbi t s , the release of catecholamines and renin asso
ciated with hydralazine induced hypotension could be blocked by the p ros
taglandin synthetase inhibitor, indomethacin; the hypotensive responses
was enhanced (Graham et al_., 1979).
20
Shepherd et al_. , (1981) have stated that determining the acetylator
index before giving hydralazine to the hypertensive patient is useful as
the plasma hydralazine levels depend on the acetylator index hydralazine
caused a slight increase in plasma renin activity and urinary excretion
of noradrenaline in patients with essential hypertension (Velasco et_ a]_. ,
1985).
The increased formation of kinins within the kidney could be invol
ved in the vasodilator and blood pressure lowering effects of dihydralazine
as suggested by Boenner et_ al_. , (1982).
21
•^NE 5-HT
MESENCEPHALON
PONS
MEDULLA OBLONGATA
SPINAL CORD
Schematic drawing showing, in highly simplified form, the main monoamine neuron projection systems in the central nervous system (Anden et a l . . 1966).
22
RESULTS
The c r i t e r i a for receptor cha rac te r i za t ion were ful f i l led as the
binding s i t e s were found to be r e v e r s i b l e , sa tu rab le and t empera tu re d e
penden t . The d i s t r ibu t ion of adrenerg ic and dopaminergic r e c e p t o r s in
normal r a t bra in i s shown in Table 1. The affinily (Kd) of «^-adrenergic
r e c e p t o r s ranges from 0.i>6 nM to 1.24 nM indicat ing tha t t he se a r e high
aff ini ty binding s i t e s . The populat ion (Bmax) of these r e c e p t o r s i s f a i r l y
constant in al l the four a reas ranging from 30.21 to 41.49 p Mol/g t i s s u e .
The y9-ad rene rg ic r ecep to r s have high aff ini ty binding s i t e s in
c o r t e x , medul la , hypothalamus and caudate ranging from 0.58 to 1.76 nM.
Thei r populat ion ranges from 24.88 to 38.76 p Mol/g t i s sue in different
a r e a s .
The dopaminergic binding s i t s in caudate show high aff in i ty (0.032
nM) and high densi ty (58.68 p/Mol/g t i s s u e ) . In the o the r t h r e e a r ea s
of the r a t b r a i n , the affinity ranges from 0.31-0.43 nM and dens i ty from
25.23 to 46.63 p Mol/g t i s s u e .
The changes produced in t he se r e c e p t o r s by chronic admin i s t r a t ion
of cen thaqu in , c lonidine , hyd ra l az ine or r e se rp ine a re d e s c r i b e d be low.
CENTHAQUIN
Effect on cC-Adrenergic Receptors
Centhaquin did not produce any change in the cor t i ca l of-adrenergic
r e c e p t o r s . The affinity (Kd) as well a s the populat ion (Bmax) remained
unchanged. In the caudate nucleus a l s o , t h e r e was no change in t h e af f in i ty
and dens i ty of these r e c e p t o r s . In the medulla t h e r e was a dec rease (40.91)
in the aff in i ty but an increase in t h e populat ion. In the hypotha lamus
the drug decreased (38.2%) the aff ini ty but inc reased (52.4%) the dens i ty
of t h e s e r e c e p t o r s s ignif icant ly (Table 2; F ig . I & I I ) .
Effect on ^-Adrenergic Receptors
Centhaquin did not produce any change in the aff ini ty or the den
s i t y of any of the four a reas of the b r a i n v i z . c o r t e x , c a u d a t e , medulla
and hypo tha l amus .
23
Effect on Dopaminergic Receptors
An insignificant increase in the aff ini ty (Kd) and a s ignif icant
decrease in the populat ion (Bmax) of the cor t i ca l dopaminergic r e c e p t o r s
was o b s e r v e d . There was no change in the cauda te , medul lary and h y p o
thalamic dopaminergic r e c e p t o r s (Table 2jf^^-^ ' ^
CLONIDINE
Effects on oC-Adrenergic Receptors
There was no change in the aff ini ty or the dens i ty of the oc-adre-
nergic r ecep to r s in the cor tex and the caudate following clonidine t r ea tmen t .
However, the re was a significant decrease in the aff in i ty and a significant
increase in the dens i ty of the oC-adrenergic r e c e p t o r s in the medulla and
hypothalamus (Table 3^ft'^1-
Effects on -Adrenerg ic Receptors
In none of the four a reas of the b ra in viz c o r t e x , cauda te , medulla «
and hypotha lamus , t h e r e was any change in the aff ini ty or the dens i ty
of >^-adrenergic r e c e p t o r s (Table 3; Fig. I l l & IV) on chronic t reatment
with c lonid ine .
Effects on Dopaminergic Receptors
There was no change in the aff ini ty but a s l i g h t increase in the
dens i ty of the cor t i ca l dopaminergic r e c e p t o r s following chronic t reatment
wi th clonidine was ob ta ined . In the o the r t h r e e a r e a s of the b ra in v i z .
cauda te , medulla and hypothalamus no change in t h e af f in i ty or dens i ty
of the r ecep to r s was o b s e r v e d (Table 3 j A^ • ' ^ ' ^ • ^
HYDRALAZINE
Effects on of-Adrenergic Receptors
Chronic t reatment of r a t s wi th hyd ra l az ine (0.125 m g / k g / p o / d a y )
produced no significant change in the aff ini ty eC-adrenergic r e c e p t o r s in
any of the four b ra in a r e a s s tud ied but i t could s ign i f ican t ly decrease
the dens i ty of r e c e p t o r s in the cor tex and the hypothalamus and a s ign i f i
cant ly increase in caudate and medulla oblongata (Table 4; F ig . I & I I ) .
Effects on -Adrenerg ic Receptors
In the cortex , caudate and hypothalamus, hydralazine treatment
caused an increase in the affinity of beta-adrenergic receptors . There
24
was a lso an increase in the populat ion of ^ - r e c e p t o r s in the cor tex (35.2%),
caudate (15.9%) and the hypothalamus (28.6%). However, t h e r e was no
change in the aff ini ty and a decrease in the receptor popula t ion was noted
in the medulla (Table 4; F ig . I l l & IV) .
Effects on Dopaminergic Receptors
The affinity of the cor t i ca l dopaminergic r e c e p t o r s was not changed
whi le the densi ty decreased (32 .41) . There w a s , howeve r , no change in
the r e c e p t o r s of caudate nucleus following chronic t rea tment with h y d r a l a
z ine . In the medulla and the hypo tha lamus , the dens i ty of r e c e p t o r s d e c
r ea sed by 37.7% and 24.6% r e s p e c t i v e l y whereas the af f in i ty dec reased
in the medulla (24.8%) and increased in the hypothalamus (Table 4; Fig.V
& VI) .
RESERPINE
Effects on oC-Adrenergic Receptors
No significant change was o b s e r v e d in t h e aff ini ty of the oC-adreno-
c e p t o r s in any of the four a r ea s of the b ra in v i z . c o r t e x , c a u d a t e , medulla
and hypothalamus following chronic r e s e r p i n e treatment (12.5 m g / k g / p o / d a y ) .
The r ecep to r population in the cor tex (27.3%) medulla (36.7%) and h y p o t h a
lamus (28.0%), however , decreased s ignif icant ly whereas i t i nc reased in
the caudate (32.1%) (Table 5; F ig . I & I I ) .
Effects on yo-Adrenergic Receptors
No significant change occured in the affinity of the > o - r e c e p t o r s
of c o r t e x , cauda te , medulla and the hypotha lamus . The d e n s i t y of t he se
r e c e p t o r s , however , inc reased in the cor tex (23.7%), caudate (28.9%) and
hypothalamus (44.1%) and dec reased in the medulla (32.0%) (Table 5; F ig .
I l l & IV) .
Effects on Dopaminergic Receptors
There was a decrease in t h e affinity of dopaminergic r e c e p t o r s
in t h e caudate and an increase in the medulla but no change in t h e cor tex
and hypothalamus following chronic t reatment with r e s e r p i n e . The populat ion
of t h e s e r e c e p t o r s decreased in t h e cor tex (19.8%) and medulla (16.5%),
i nc reased in the hypothalamus (24.4%) whi le i t was not affected in t h e
caudate (Table 5; Fig.IV & V) .
25
Table 1: Dis t r ibu t ion of a lpha and be t a - ad rene rg i c and dopaminergic r e c e p t o r s in var ious a reas of the normal r a t b r a in
Region Receptor Type Affinity Kd (nM)
Population Bmax (pMol/g Tissue)
Cortex oC-adrenergic
^ - a d r e n e r g i c
Dopaminergic
0.81 ± 0.12 30.21 ± 1.38
0.80 ± 0.11 38.76 ± 2.57
0.43 ± 0.18 34.86 ± 1.39
Caudate oC-adrenergic
y3-adrenergic
Dopaminergic
1.24 ± 0.23 41.49 ± 2.19
1.76 ± 0.14 35.77 ± 1.44
0.032±0.016 53.03 ± 1.33
Medulla ^ - ad rene rg i c
^ - a d r e n e r g i c
Dopaminergic
0.66 ± 0.20 31.09 ± 1.30
0.68 ± 0.12 31.77 ± 1.73
0.39 ± 0.11 44.49 ± 1.63
Hypothalamus (Jf-adrenergic
/S-adrenergic
Dopaminergic
0.68 ± 0.11 32.89 ± 2.19
0.58 ± 0.09 24.88 ± 2.16
0.31 ± 0 . 0 9 25.23 ± 0.92
26
Table 2: Alteration in (fi- and ^-adrenergic and dopaminergic receptors following administration of centhaquin to rats for 2 months at a dose of
Clmg/kg^c/day
Region
Cor tex
Caudate
Medulla
Hypothalamus
Receptor type
of-adrenergic
yS-adrenergic
Dopaminergic
oC -adrenergic
^ - a d r e n e r g i c
Dopaminergic
^ - a d r e n e r g i c
yS-adrenergic
Dopaminergic
sx'-adrenergic
^ - a d r e n e r g i c
Dopaminergic
Kd (Percentage) Change
-4 .9
- 7 . 5
-18 .6
+13.7
-12 .5
+6.2
+40.9*
0.0
+10.2
+38.2*
+13.7
-3 .2
Bmax (percen tage)
Change
- 3 . 9
+1.9
-25 .2*
+7.9
- 5 . 2
- 1 . 1
+85.8*
- 0 . 2
- 2 . 5
+52.4*
+1.8
- 1 . 9
P^1D.05 compared to control
27
Table 3: Alteration in c(-- and ^-adrenergic and dopaminergic receptors following chronic administration of clonidine (0. Img/kg/p/day) to r a t s .
Region
Cortex
Caudate
Medulla
Hypothalamus
Receptor Type
oC-adrenergic
j 3 - ad rene rg i c
Dopaminergic
o(-adrenergic
^ - a d r e n e r g i c
Dopaminergic
of-adrenergic
yS-adrenergic
Dopaminergic
oC-adrenergic
/^ -adrenergic
Dopaminergic
Kd (Percentage) Change
+ 2.4
-10.0
+11.6
+15.3
+ 7.3
- 3.1
+31.8*
- 2.9
+10.2
+27.9*
+10.3
+ 3.2
Bmax {Percentage)
Change
+ 1.0
- 1.0
+17.1*
+ 9.0
- 1.5
- 1.0
+58.8*
+ 1.5
- 1.4
+31.7*
- 3.4
+ 5.7
* P ^0.05 compared to control.
28
Table 4: Alterat ions in of- a n d ^ ' a d r e n e r g i c and dopaminergic r e c e p t o r s following chronic administration" of hyd ra l az ine (8>Jmg/kg/p/day) to r a t s .
ans
Region
Cortex
Caudate
Medulla
Hypothalamus
Receptor Type
o( -adrenergic
jP -adrenerg ic
Dopaminergic
oC adrenergic
^ - a d r e n e r g i c
Dopaminergic
o( -adrenerg ic
^ - a d r e n e r g i c
Dopaminergic
©(-adrenergic
>^-adrenergic
Dopaminergic
Kd (Percentage) Change
- 6.1
-20 .0*
+ 3.0
- l / . l
-15 .9*
+ 6.2
- 1.3
+ 0.5
+24.8*
- 4.8
-20 .0*
+ 0.6
Bmax (Percentage)
Change
-23 .2*
+35.2*
-32 .4*
+25.9*
+15.9*
+ 0.8
+24.4*
-23 .0*
-37 .7*
- 2 8 . 0 1 *
+28.6*
-24 .6*
• < * P< 0.05 compared to cont ro l .
29
Table 5: Alteration in <*'- and^-adrenerg ic and dopaminergic receptors following chronic administration of Reserpine (lE5nig/kg/p/day) for
2 months to r a t s .
Region
Cortex
Caudate
Medulla
Hypothalamus
Receptor Type
of-adrenergic
^ - a d r e n e r g i c
Dopaminergic
o( -adrenergic
y^-adrenergic
Dopaminergic
of-adrenergic
^ - a d r e n e r g i c
Dopaminergic
cC-adrenergic
^ - a d r e n e r g i c
Dopaminergic
Kd (Percentage) Change
- 1.2
-15 .0
0 . 0
-13 .2
- 9.7
+28.1*
+17.9
-12 .6
-23 .0*
-17 .2
- 4.8
+ 4.5
Bmax {Percentage)
Change
- 2 7 . 3 *
+23.7*
-19 .85*
+32.1*
+28.9*
- 3.8
-36 .7*
-32 .0*
- 1 6 . 5 *
-37 .2*
+44.1*
+24.4*
* P^O.65 compared to control.
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DISCUSSION
Till recently most investigations have been confined to study
the process of neurotransmitter synthesis , frequency of nerve impulse,
the process of release of neurotransmitter and its uptake mechanism or
enzymatic degradation. Not much attention has been paid to the status
of the receptors in the brain of the hypertensive subjects or the change
following antihypertensive therapy. With the development and use of ligand
binding studies, it is now possible to precisely determine the state of
the receptors including their affinity and number in any particular area
of the brain.
The use of ligand binding technique to study neurotransmitter rece
ptor sites has led to a broad range of important biological advances.
It is now possible to obtain information about regional distribution of
neurotransmitter receptors in brain, the pharmacological, biochemical and
developmental characteristics of these s i t es , and the functional inter-rela
tionship between neuronal cell types . The main limitation of radioreceptor
assays is their specificity since any substance having an appreciable affi
nity for the neurotransmitter receptor site will displace the specifically
bound ligand. Thus before routinely using a radio receptor assay, precau
tions must be taken to ensure that the only substance in the sample that
will interfere with the ligand binding is the substance being analysed.
Because of the high specific activity of neurotransmitter radioligands,
the radioreceptor assay procedure is very sensit ive, being able to readily
detect picomole quantities of the compound.
Radio receptorassay. is also simple, inexpensive and rapid in that
hundreds of samples can be analysed in a single day. Studies using radio
receptor assays have been reported as analytical tools indicating the ver-
satali ty and usefulness of this procedure in neuro-chemical investigations.
In the present investigation radio ligand binding technique has
been used to determine the differences in the receptor affinity and popula
tion between normal rats and those treated with the antihypertensive drugs.
It is farily well established that receptor population a l ters in an inverse
manner to changes in neural impulse flow (Morris et_ al_., 1981). It i s ,
therefore, clear that the change in the radioreceptor binding will indirectly
reflect the neuronal ac t iv i ty .
39
The drugs investigated in the present study are some ant i -hyper
tensive drugs like centhaquin, clonidine, hydralazine and reserpine on
the adrenergic and dopaminergic receptors . The selection of the drugs
has been guided by an effort to ensure that they act by different mechani
sms so that changes in receptors will probably reflect as much effect
of reduced blood pressure as the effect of drugs themselves.
Centhaquin has been reported to lower blood pressure by acting
mainly in the brain (Srimal e^ aj_. , 1978). The results indicate that it
has i ts action mainly on the of-adrenergic receptors of the medulla and
the hypothalamus. These areas are involved in cardiovascular regulation
(Muira et_ aj_. , 197i). and i t could be possible that centhaquin produces
hypotension by acting on the oC^adrenergic receptors in these a reas . Several
studies indicate that oC-adrenergic receptors in the nucleus tractus solita-
rious (dejong, 1974) and ventral surface of medulla (Zhang and Cir ie l lo ,
1982; McAllen et_ aj_. , 1982) are involved in the cardiovascular regulation.
In this study no attempt was made to dissect out different areas of the
medulla for preparing membranes and therefore, it is not possible to detect
changes in individual areas of the medulla. Only the overall changes have
been detected.
Centhaquin caused an increase in the population of alpha-adreno-
ceptors of medulla and hypothalamus but their affinity had gone down.
Such a situation can arise only if there is a reduced amouht of the neuro
transmitter available under the effect of the drug. It is possible that
centhaquin may be acting on the presynaptic alpha-adrenergic receptors
and decreasing the release of adrenergic neurotransmitter which has resulted
in an increase in the density of post-synaptic of-adrenergic receptors .
Centhaquin has not produced any change in the adrenergic and
dopaminergic receptors of cortex and the caudate suggesting that these
areas are not involved in its action.
Clonidine has been claimed to stimulate the central oC-adrenoceptors
to produce hypotension (Hausler, 1974). It has also been reported by
Medgett and Rand (1983) that the cardiovascular effects of clonidine are
not related to decrease in central noradrenaline turnover, due to the fact
that cardiovascular effects of this drug can be elicited even in the absence
of neural noradrenaline.
40
The hypotension resulting after the chronic administration of small
doses of clonidine is associated with a reduction in the preganglionic splan
chnic nerve discharge indicating a reduction in the central sympathetic
tone. Transection experiments indicate that the medullary and the hypotha
lamic centres are involved in this response (Schmitt and Schmitt, 1969).
The affinity of clonidine for presynaptic oC-adrenergic receptors
is atleast ten times higher than i ts affinity for postsynaptic flC-adrenocep-
tors (Starke ei_ aj_. , 1974). Therefore, it could be possible that it may
be acting on the presynaptic alpha-adrenergic receptors , thus decreasing
the release of adrenergic neurotransmitter resulting in the increase in
the density of the post-synaptic alpha-adrenergic receptors , as observed
in this study. Like centhaquin, clonidine also decreased the affinity of
the receptors located in the medulla and the hypothalamus and increased
their population, indicating a decrease in the neural transmission. An inc
rease in the population of dopaminergic receptors in the cerebral cortex
by clonidine treatment may be unrelated to i ts cardiovascular actions since
it is also known to cause sedation and affect release of other neurotrans
mitters like acetylcholine etc.
Reserpine depletes the stores of catecholamines and 5-hydroxy-
tryptamine in the brain as well as peripherally and most of i ts pharma
cological effects have been attributed to this action (Juoria and Vogt,
1967). In the present study reserpine was found to decrease the density
of the e<-adrenergic, ^6-adrenergic as well as dopaminergic receptors of
the medulla and the of-adrenergic and dopaminergic receptors of the cortical
region of the brain. The oC-adrenoceptors of the hypothalamus were also
decreased but ^-adrenoceptors and dopaminergic receptors were increased.
The action of reserpine on the central nervous system (CNS) app
ears not to play a major role in the reduction of sympathetic nerve act ivi ty .
There may even be an increase in sympathetic out flow (Iggo and Vogt,
1960). Reserpine prevents the storage of catecholamines without effecting
their synthesis by adrenergic neurons. It i s , therefore, assumed that this
leaves more free noradrenaline to react with the receptor . This could
also par t ia l ly explain the central hypotensive action of reserpine.
The effect of reserpine is not confined to catecholamines alone,
it also depletes 5-HT in the brain and depression and suicidal tendency
41
in the patients has been attributed to this effect (Doyle et a]_, , 1955).
Antidepressants are now known to down regulate vS-adrenoceptors and upre-
gulate 5-HT receptors in the cortex. The present study has demonstrated
an increase in the population of ^-adrenoceptors in the cortex, the hypo
thalamus and the caudate which could part ial ly explain the depressant
effect of reserpine. The effect of reserpine on 5-HT receptors has not
been studied but presumably they should be upregulated like oc-adrenergic
receptors .
After chronic administration of hydralazine, there is a fall in
blood pressure , with an increase in the heart rate and the cardiac output.
This occurs as a result of reflex enhanced peripheral sympathetic act ivi ty
following fall of blood pressure due to peripheral vasodilation (Brunner
et a l . , 1967). The increase in myocardial contractility could be inhibited
by oC-adrenergic antagonists (Barrett et £1^., 1965). The present study
has shown that the density of the oC-adrenergic receptors increased in
the caudate and the medulla and decreased in hypothalamus and cortex.
In case of the ^-adrenergic receptors , their density increased in the cortex
and caudate and decreased in medulla and hypothalamus. In the dopaminer
gic receptors a decrease in the density has been observed in the cortex,
medulla and the hypothalamus. The varied effect of hydralazine on Ct-
and J^ -adrenoceptors and dopaminergic receptors in different areas of
the brain could be viewed as the response of the brain to the chronic
hypotensive stage. Hydralazine presumably does not effect specifically
any of the central neurotransmitters and lowers the blood pressure by
peripheral effects alone (Ablad, 1963). It will be interesting to study
the effect of some other direct vasodilators which . lower blood pressure
by peripheral mechanisms only. The changes in the receptor population
are an indirect reflection on the state of neurotransmitter avai labi l i ty
of the receptor site in response to lowered blood pressure which the
cardiovascular centers will t ry to bring back to normal. It must, however,
be emphasised that no drug affects only one function and therefore changes
in the receptor population in the medulla and the hypothalamus may reflect
changes secondary to hypotension while changes in cortex and caudate
may reflect changes in other body functions.
Thus it seems that centhaquin and clonidine have a central mode
of action, while reserpine and hydralazine, which are per ipheral ly acting
42
drugs, produce a generalised effect. Also that centhaquin and clonidine
produce the effect primarily in the medulla and in the hypothalamus,
whereas reserpine and hydralazine do not have a limited effect on a pa r t i
cular area of the brain.
13
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