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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38

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

BIBLIOGRAPHY

1. Ablad , B. (1963). A s tudy of mechanism of the haemodynamlc effects of hyd ra l az ine in man. Acta Pharmacol . Toxicol . ( S u p p l . l ) , 29, 1-53.

2. Anden, N . E . ; Cor rod i , H. ; Fuxe, K.; Hokfelt , B . ; Hokfelt , T. ; Rydin, C. & Svensson, T. (1970). Evidence for a cent ra l noradrena l ine r ecep to r s t imulat ion by c lonidine . Life Sci . , 9 , 513-523.

3. Aut re t , A.M.; Schmit t , H. ; Fenard , S. & P e t i l l o t , H. (1971) . Compa­r i son of haemodynamlc effects of ot-sympathomimetic d r u g s . Eur. J . Pha rmaco l . , 13 , 208-217.

4. Barne t t , A. & Benforado, J .M. (1966). The nicot inic effect of chol ine e s t e r s and of nicotine in guinea pig a t r i a . J . Pharmacol . E x p . T h e r . , 152, 29-36.

5. B a r r e t t , W.E . ; Pova l sk i , H. & Rutledge, R. (1965). A h y p o t h e s i s concerning the mechanism of action of hyd ra l az ine HU. Fed. Proc . , 24, 712.

6. Baum, T . ; S c h r o p s h i r e , A.T. & Verner , L . L . (1972) . Contr ibut ions of cent ra l nervous system to the act ions of seve ra l a n t i - h y p e r t e n ­s ive agents (methyl dopa , hydra l az ine and guane th id ine ) . J . Pharma­co l . Exp . T h e r . , 182, 135-144.

7. Beck, L . ; Ybar ra -Fa lcon , L. & Domino, E . F . (1957). Haemodynamlc re sponses to r e s e r p i n e in ' unanes the t ized dogs immobil ized wi th neuromuscular blocking agents . J . Pharmacol . Exp . T h e r . , 119, 133.

8. Bein, H . J . (1956). The pharmacology of rauwolf ia . Pharmacol . Rev. , 8, 435-483.

9. Bein, H . J . (1953). Zur Pharmakologie des Reserpineines neuen Alkalo ids aus Rauwo£^a 'i>^^p^ntina Benth. Exper ien t ia ( B a s e l ) , 9 , 107-110.

10. Bent ley , G.A.; Gullen, L .K. ; Reynotdson, J .A . & Widdope, E. (1986) . The effect of c lonidine on venous haemodynamics in ca t s and dogs . Br . J . P h a r m c o l . , 88(1) , 161-172.

11 . Berkowi tz , B .A . ; T a r v e r , J .H. & Spec to r , S. (1971). Norep inephr ine in blood v e s s e l s : concentra t ion, b ind ing , up take and dep l e t i on . J . Pharmacol . Exp . T h e r . , 117, 119-126.

12. B e r t l e r , A. ; Car l s son , A. & Rosengren, E. (1956). Release of r e s e r ­pine of catecholamines from r a b b i t s ' h e a r t s . Naturwissenschaf t e n , 43 , 521.

3 13. Bhargava , H.N. (1984). Effect of cyclo (Leucyl -g lyc ine) on ( H) s p i r o ­

pe r i do l binding in t h e corpus s t r i a tum and hypotha lamus of spon­taneously h y p e r t e n s i v e r a t s . Eur. J . Pha rmaco l . , 100, 109-112.

44

14. Bhargava , H.N. (1983). Effect of cyclo (Leucyl) on s u p e r s e n s i t i v i t y of dopamine r e c e p t o r s in spontaneously h y p e r t e n s i v e r a t s . Life S c i . , 32, 2131-2137.

15. Bhargava , K .P . (1973). Central Adrenergic and cho l inerg ic mechanisms in ca rd iovascu la r con t ro l . Ind. J . Pharmac. , 5 ( 2 ) , 293-312.

16. Boenner, G.; Beck, D. ; Deeg, M.; Marln-Grez, M. & Gross , F . (1982). Effect of d ihyd ra l az ine on the renal k a l l i k r e i n - k i n i n system of the r a t . Eur. J . Pha rmaco l . . 78(2) , 219-224.

17. Bo i s s i e r , J . R . ; Guid ice l l i , J . F . ; F i c h e l l e , J . ; Schmi t t , H. & Schmit t , H. (1968). Card iovascular effects of 2 - ( 2 , 6 - d i c h l o r p h e n y l a m i n o ) -2-imidazoline h y d r o c h l o r i d e (St. 155) I p e r i p h e r a l sympa the t i c sys tem. Eur. J . Pha rmaco l . , 2 , 333-339.

18. Bolton, T .B. (1967). Intramural ne rves in the ven t r i cu l a r myocardium of the domestic fowl and o ther an imals . B r i t . J . Pha rmaco l . , 31 , 253-268.

19. Bousquet , P . : Rouot, B. & Schwar tz , J . (1983). The cent ra l a l p h a -ad renocep to r s . Some new a s p e c t s . Trends Pharmacol . S c i . , 206-208.

20. Bousquet, P . ; Feldman, J . ; Vel ly , J . & Block, R. (1975). Role of the vent ra l surface of the b ra in stem in the hypo tens ive action of c lonid ine . Eur. J . Pha rmaco l . , 34, 151-156.

2 1 . Bousquet , P. & Guertzenstein, P.G. (1973). Local izat ion of cent ra l ca rd iovascu la r act ion of c lonid ine . Br . J . Pharmacol . , 49, 573-579.

22. B ra l e t , J . & Rochett , L. (1973). Effect of c lonidine on the turnover r a t e of noradrenal ine in p e r i p h e r a l t i s sues of t h e r a t . Eur. J . Pha rmaco l . , 23 , 239-244.

23 . Brezenoff, H.E. & Jenden , D . J . (1969). Modification of a r t e r i a l blood p r e s s u r e in r a t s following micro-inject ion of drugs into the pos te r io r hypotha lamus . In t . J . Neuropharmacol. , 8, 593-600.

24. Brown, M.J. & Har land, D. (1984). Evidence for a p e r i p h e r a l compo­nent in the sympatho ly t i c effect of clonidine in r a t s . Br . J . Pharma­c o l . , 83(3) , 657-666.

25 . Brunner , H. ; Hedwall , P .R. & Meier , M. (1967). Influence of a d r e ­nergic be t a - r ecep to r b lockade on the acute c a r d i o v a s c u l a r effect of h y d r a l a z i n e . Br . J . Pharmacol . , 30, 123-133.

26. Burn, J .H . & Rand, M.J . (1965). Acetylcholine in ad rene rg ic t r ansmi ­ss ion . Ann. Rev. Pharmacol . , 5 , 163-182.

27. Ca la re su , F.R. & C i r i e l l o , J . (1980). Project ions to t h e hypothalamus from buffer nerves and nucleus s o l i t a r i u s in the c a t . Amer. J . P h y s i o l . , 239, R-130-R-136.

45

28. C e r r i t o , F . ; M a r t i r e , M. & P r e z i o s i , P. (1984). Long-term treatment with clonidine and o^- recep tors in the b ra in of normotensive r a t s . Brain R e s . , 321(1) , 45-54.

29. C i r i e l l o , J . & Caverson , M.M. (1986). Bid i rec t ional ca rd iovascu la r connections between Ventrolateral Medulla and Nucleus of the Sol i ­t a r y Trac t . Brain Res. . 367, 273-281.

30. Constant ine, J .W. & McShane, W.K. (1968) . Analysis of the c a r d i o ­vascular effects of 2 - (2 ,6 -d ich lo ropheny lamino) -2 - imidazo l ine h y d r o ­ch lo r ide ( C a t a p r e s ) . Eur. J . Pha rmaco l . , 4 , 109-123.

3 1 . Couper , I .M. & K i r b y , M.J . (1972). The effect of c lonidine on human i so la ted smooth muscle. Eur. J . Pharmacol . , 17, 50-58.

32. Cr i sc ione , L . ; Re is , D . J . & Talman, W.T. (1983). Chol inergic mecha­nisms in the nucleus t rac tus s o l i t a r r i i and ca rd iovascu la r regula t ion in the r a t . Eur. J . Pha rmaco l . , 88 (1 ) , 47-56.

33 . Cubeddu, L .X . ; Hoffmann, I . S . ; Davi la , J . ; B a r b e l l a , Y.R. & Ordaz , P. (1984). Clonidine reduces e l eva ted ce reb rosp ina l f luid ca techo la ­mine l eve l s in p a t i e n t s , wi th essen t i a l hype r t ens ion . Life Sci . , 35(13) , 1365-1372.

34. Dahls trom, A. & Fuxe , K. (1965). Evidence for the ex i s tence of mono­amine containing neuron»i in the cent ra l nervous sys tem. Acta Phy­s io l . Scand . , 62 ( S u p p l . ) , 247, 1-85.

35. Datar , S . ; L a v e r t y , W.H. & McNeill , J . R . (1986). Clonidine fa i l s to reduce p re s so r respons iveness of concious spontaneously h y p e r ­tens ive r a t s to va sop re s s in . Can. J . P h y s i o l . P h a r m a c o l . , 64 (3 ) , 284-289.

36. David , N .A . ; Welborn, W.S. & P i e r c e , H. I . (1975), Comparison of mult iple and combination t ab le t drug t h e r a p y in hype r t ens ion . Cu r r . Ther . R e s . , 18, 741-754.

37. Dhawan, B .N. ; J o h r i , M.B. ; Singh, G .B . ; S r imal , R.C. & Viswerman, D. (1975). Effect of clonidine on the e x c i t a b i l i t y of vasomotor loci in the ca t . Br . J . Pharmacol . , 54, 17-21 .

38. Doyle, A . E . ; McQueen, E.G. & Smirk , F.A. (1955). Treatment of h y p e r ­tension with r e s e r p i n e in combination with pen tapyr ro l id in ium and with r e s e r p i n e in combination with ve ra t rum a l k a l o i d s . C i rcu la t ion , 11, 170-181.

39. Eb inhara , A. & Mar t s , B.L. (1978). Comparat ive effects of c u r r e n t l y ava i l ab le a n t i h y p e r t e n s i v e agents of spontaneously and renal h y p e r ­tens ive r a t s . Am. J . Med. S c i . , 259, 257-261.

40. E h r r e i c h , S . J . (1978) . An t ihype r t ens ives : In New Drugs Discovery and development . Edi ted by A.A. Rubin, Marcel , Dekker , New York , Basel .

46

41 . E r iks son , L. & Tuomisto, L. (1983). Effect of naloxone on the h y p o ­tens ive action of clonidine in conscious normotensive goat. Acta Pharmacol . T o x i c o l . , 52 (4 ) , 241-245.

42. Farsang , C ; Varga, K.; Vajda, L . ; Alfoldi , S. & Kapocsi , J . (1984). Effects of clonidine and guanfacin in essent ia l h y p e r t e n s i o n , Cl in . Pharmacol . T h e r . . 36 (5 ) , 588-594.

43. F luck ige r , E. (1969). Pharmakologie der a n t i h y p e r t o n i k a . In : Hyper ­tonic Pathogenese, k l ink und T h e r a p i e , Edi ted by U. Sar re Scha t t -a u e r , Verlag, S tu t tgar t .

44. F r a n z , D.N. & Madsen, P.W.- (1982). Differential s e n s i t i v i t y of 4 cen­t r a l sympathet ic pa thways to depress ion by c lon id ine . Eur. J . Pharmacol . . 78(1) , 53-60.

45. Gaffney, T . E . ; C h i d s e y , C.A. & Braunwald, E. (1963) . Study of the r e l a t ionsh ip between the neuro t ransmi t te r s tore and ad rene rg i c nerve block induced by r e s e r p i n e and guani th id ine . C i r . Res. , 12, 264-268.

46. Golds te in , M.; Anagnoste, B . ; B a t t i s t a , A . P . ; Owen, W.S. & Naka tan i , 5 . (1969). Studies of amines in the s t r ia tum in monkeys with Nigral Les ions . J . Neurochem. , 16, 645-653.

47. Graham, R.M.; Compbel l , W.B. ; Jackson , E .K. ; Pe t t i nge r , W.A. & Lo i se l , D .P . (1979). Role of p ros tag landins in hyd ra l az ine - i nduced renin and catecholamine r e l e a s e in conscious r a b b i t s . Fed . Proc . , 38, 418.

48. Graubner , W. and Wolf, M. (1966) . Kr i t i sche Betrachtunger zum w i r -kungs mechanisms des ( 2 , 6 - d i c h l o r p h e n y l a m i n o ) - 2 - i m i d a z o l i n - h y d r o -c h l o r i d e s . Arzneime£ F o r s c h . , 16, 1055-1058.

49. Green, A .F . (1962). Ant ihyper tens ive d rugs . Advanc. P h a r m a c o l . , 1, 161-225.

50. Greenberg , L.H. & Weiss , B. (1979). Abi l i ty of aged r a t s to a l t e r ^ - a d r e n e r g i c r ecep to r s of b r a in in response to r e p e a t e d a d m i n i s t r a ­t ion of r e se rp ine and desmethy l imipramine . J . Pharmaco. Exp . T h e r . 211(2) , 309-316.

51 . Greenberg , S. & Wilborn, W. (1982) . Effect of clonidine and p r o p r a n o ­lol on venous smooth muscle from spontaneously h y p e r t e n s i v e r a t s . Arch . In t . Pharmacodyn. T h e r . , 258(2) , 234-259.

52. Gross , F . ; Druey, J . & Meier , R. (1950). Eine neue Gruppe b l u l d r u c k -senkender substanzen von besonderem Wirkungscharakter . Exper i en t i a 6, 19-21 .

53 . Hawthorn, M.H, & Broad ley , K . J . (1982) . jb -adrenocep tor l igand binding and s u p e r s e n s i t i v i t y to i soprena l ine of ven t r i cu la r muscle af ter chronic r e se rp ine p r e t r ea tmen t . Naunyn-Schmiedeberg 's Arch . Pharma­c o l . , 320(2) , 240-245.

47

54. Hoefke, W. & Kobinger , W. (1966). Pharmakologische Wirkungen des Z-{Z , 6 - d i c h l o r p h e n y l a m i n o ) - 2 - i m i d a z o l i n h y d r o c h l o r i d e s , e iner neuen an t i hype r t ens iven subs tanz . Arzneimit: j I - F o r s c h . , 16, 1038-1050.

55. Hokfelt , B . ; Hedeland, H. & Hansson, B.G. (1975) . The effects of clonidine and penbuto lo l , r e s p e c t i v e l y on catecholamines in blood and u r i n e , plasma renin a c t i v i t y and u r i n a r y a ldos te rone in h y p e r ­tens ive p a t i e n t s . Arch. Int . Pharmacol . T h e r a p . , 213, 307-321.

56. Holzbauer, M. & Vogt, M. (1956). Depression by r e s e r p i n e of the noradrenal ine concentrat ion in the hypothalamus of the ca t . J . Neuro-chem. , 1, 8-11 .

57. Hukovic, S. (1959) . The action of sympathe t i c blocking agents on i so la ted and innerva ted a t r i a and v e s s e l s . Br . J . Pharmacol . , 15, 117-121.

58. Hull , C D . ; Lev ine , M.S . ; Buchwald, N .A . ; He l l e r , A. & Browning, R.A. (1974). The spontaneous fir ing pa t t e rn of f o r e - b r a i n neurons . I . The effects of dopamine and non-dopamine deple t ing les ions on caudate unit f i r ing p a t t e r n s . Brain Res. , 73(2) , 241-262.

59. I s r a i l e , 2.H. & Dayton, P.G. (1977). Metabolism of h y d r a l a z i n e . Drug Metab. R e v . , 6, 283-305.

60. de Jong, W. (1974). A central noradrenerg ic i n h i b i t o r y mechanism on blood p r e s s u r e and hear t r a t e in the nucleus t r ac tu s s o l i t a r i i of r a t medulla . Joint Meeting of the Dutch Medical and Biological Soc ie t ies . Nijmegen A b s t r . , p .219 .

61 . Juo r io , A.V. & Vogt, M. (1967). Monoamines and t h e i r metabol i tes in the av ian b r a i n . J . P h y s i o l . , 189, 489-518.

62. KannoJi, H. & Yamashi ta , H. (1985). Connections of neurons in the region of the nucleus t rac tus so l i t a r ius wi th the hypotha lamic p a r a ­ven t r i cu la r nuc leus : Their poss ib le involvement in neural control of the ca rd iovascu la r system in r a t s . Brain Res . , 329, 205-212.

63. Kark i , N .T . & Paasonen, M.K. (1959). Selec t ive dep le t ion of n o r a d r e ­naline and 5 -hydroxy t ryp tamine from r a t b r a i n , and in tes t ine by Rauwolfia a l k a l o i d s . J . Neurochem. , 3 , 352-357.

64. Keeler , J . R . ; S h u l t s , C ; Chase , T.N. & Helke , C . J . (1984) . - The vent ra l surface of the medulla in the r a t pharmacologic and auto­r ad io g raph i c loca l i sa t ion of GABA induced ca rd iovascu la r ef fec ts . Brain R e s . , 297(2 ) , 217-224.

65. Kobinger, W. & P i c h l e r , L. (1976). Cen t ra l ly induced reduct ion in sympathe t ic tone a post synapt ic oC-adrenoceptor st imulat ing act ion of imidazo l ins . Eur . J . Pharmacol . , 40, 311-320.

3 66. Kopin, I . J . & Gordon, E.K. (1962). Metabolism of norepinephr ine-H

r e l ea sed by tyramine and r e s e r p i n e . J . Pharmacol . Exp . T h e r . , 138, 351-359.

48

67. Laguzzi , R.; Reis , D.J , & William, T . T . (1984). Modulation of c a r ­d iovascular and e l ec t ro - co r t i c a l a c t i v i t y through sero tonerg ic mecha­nisms in the nucleus t rac tus s o l i t a r i u s of the r a t . Brain Res. , 304, 321-328.

68. Lim, R . K . S . ; Moffitt, R.L. & Glass , H.G, (1955). Observa t ions on the mechanism of cent ra l hype r t ens ion . J . Pharmacol . , 113, 33-34.

69. L i p s k i , J . ; P r z y b y l s k i , J . & Solnicka, E. (1976). Reduced hypo t ens ive effect of clonidine after les ions of the nucleus t r a c t u s s o l i t a r i i in r a t s . Eur. J . Pha rmaco l . , 38, 19-22.

70. Lorez , H . P . ; Kiss , D. ; P r a d a , M.Da. & Haeusler , G. (1983) . Effects of clonidine on the r a t e of noradrenal ine turnover in d i s c r e t e a r ea s of r a t central nervous sys tem. Naunyn-Schmiedberg 's Pharmacol . , 323(4) , 307-314.

71 . Luby , E. (1968). Reserpine l ike d rugs -c l in ica l e f f icacy , p p . 1077-1082. In , Psychopharmacology: A rev iew of p rogress 1957-19671.

72. Maltira, G.; Bonanno, G. & Ra i t e r i , M. (1985). Chronic c lonidine induces functional down-regulation of p r e s y n a p t i c OC -ad renocep to r s regulat ing t r i t i a t e d 5 -hydroxy t r ip t amine r e l ease in r a t b r a i n . Eur. J . Pha r ­maco l . , 112(1) , 105-110.

73. McAllen, R.M.; Nei l , J . J . & Loewy, A.D. (1982). Effects of Kainic ac id app l i ed to the ven t ra l surface of medulla oblongata on v a s o ­motor tone, the ba ro recep to r re f lex and hypotha lamic autonomic r e s p o n s e s . Brain R e s . , 238, 65-76.

74. McLean, A . J . ; DuSouich, P . ; Barron , K.W. &• Br iggs , A.H. (1978). In teract ion of hydra laz ine wi th tension development and mechanisms of calcium accumulation in K s t imulated r a b b i t ao r t i c s t r i p s . J . Pharmacol . Exp . T h e r . , 207, 40-48.

75. McRaven, D. R.; Kroetz, F .W.; Kioschos, J.W. & K i r k e n d a l l , W.M. (1971). The effect of c lonidine on haemodynamics in h y p e r t e n s i v e p a t i e n t s . Am. Heart . J . , 81 , 482-489.

76. Medgett , I . C . & Rohd, M.J . (1983). Effects of c lonidine on O t - a d r e -noceptors in th-e autoperfused h indqua te r s of p i t h e d r a t . Eur. J . Pha rmaco l . , 89 (3 /4 ) , 235-242.

77. Mel lander , S, (1960). Comparat ive s tud ies on t h e ad rene rg ic neuro­humoral control of r e s i s t ance and capaci tance blood v e s s e l s in ca t . Acta Phys io l . Scand. , 50, 1-86.

78. Mir kin , B .L. & Cervoni , P . (1962) . The adrenerg ic nature of neuro­humoral t ransmission in the cat n ic t i t a t ing membrane following t r e a t ­ment with r e s e r p i n e . J . Pharmacol . Exp . T h e r . , 138, 301-308.

79. M o r r i s , M . J . ; Devynck, M.; Woodcock, E .A . ; Johns tan , C . I . & Meyer , ?'. (1981). Specific changes in hypotha lamic OC-adrenocep tors in young spontaneously h y p e r t e n s i v e r a t s . Hyper tens ion, 3 , 516.

49

80. Muira, M. & Reis , D.J . (1969). Termination and secondary pro jec t ions of the ca ro t id s inus nerve in the cat bra in stem. Amer. J . P h y s i o l . 217, 142-153.

81 . Muira, M. & Reis , D.J . (1972). The role of the s o l i t a r y and p a r a ­median r e t i cu l a r nuclei in mediating ca rd iovascu la r r e f l ex r e sponses from ca ro t id baro and chemorecep to r s . J . Phys io l . , 223, 525-548.

82. Muira, M.; Onai, T. & Takayama, K. (1968). Project ions of upper s t ruc tu re to the spinal c a r d i o a c c e l e r a t o r y center in c a t s , an HP s tudy using a new microinject ion method. J . Aut. Nerv . Sys t . , 7, 119-139.

83. Muschel l , E. (1966), I nd i r ec t l y act ing sympathomimetic amines . P h a r ­macol. R e v . , 18, 551-559.

84. Nagaoka, A . ; Kikuchi , K. & Aramaki , Y. (1969). Depressor r e sponses to the spontaneously h y p e r t e n s i v e r a t s to t h e . a n t i h y p e r t e n s i v e agen t s . J a p . J . Pha rmaco l . , 19, 401.

85. Nay le r , W.G.; P r i c e , J . M . ; Swann, J . B . ; Mclnnes, L . ; Race, D. & Lowe, T .E . (1968). Effect of the hypotens ive drug St . 155 (Cata-p r e s ) on the hea r t and p e r i p h e r a l c i rcu la t ion . J . Pharmacol . Exp . T h e r . , 166, 364-373.

86. Nayle r , W.G. ; Roserbaum, M.; Mclnnes, I . & Lowe, T .E . (1966) . Effect of a new hypo tens ive drug , St. 155, on the sys temic c i r cu l a t i on . Am. Heart . , J . , 72, 764-770.

87. Ones t i , G.; Bock, K.D. ; Heimsoth, V . ; Kim, K.E. & Merguet, P. (1971) . Clonidine: A new a n t i h y p e r t e n s i v e agent . Am. J . C a r d i o l . , 28 , 74-83.

88. P a r k , Y . J . ; Lee, Y.W. & P a r k , C.W. (1983). An exper imenta l s tudy on the ad rene rg ic mechanism of c lon id ine . Seoul J . Med. , 2 3 ( 3 ) , 299-309.

89. Pegram, B . L . ; I s h i s e , S. & F r o h l i c h , E.D. (1982). Effect of me thy l -dopa , clonidine and hydra l az ine on ca rd iac mass and haemodynamics in Wistar Kyoto and spontaneously h y p e r t e n s i v e r a t s . C a r d i o v a s c . R e s . , 16 (1 ) , 40-46.

90. Pe r sson , T. & Waldeck, B. (1970). Fur the r s tud ies on the p o s s i b l e in te rac t ion between dopamine and noradrenal ine containing neurons in the b r a i n . Eur. J . Pha rmaco l . , 11, 315-320.

91 . P h i l i p p u , A . ; Roensberg, W. & Przun tek , H. (1973). Effects of a d r e ­nergic drugs on p r e s s o r responses to hypotha lamic s t imula t ion . Naunyn-Schmiedebergs. Arch. P h a r m a c o l . , 278, 373-386.

92. P h i l i p p u , A. & Kl t t e l , E. (1977). Presence of b e t a - a d r e n o c e p t o r s in the hypotha lamus ; t h e i r importance for the p r e s s o r r e sponse to hypotha lamic s t imula t ion . Naxmyn-Schmedebergs Arch . P h a r m a c o l . , 297, 219-225.

50

93. Ph i l i ppu , A. (1970) . Release of catecholamines from the hypothalamus by drugs and e l ec t r i ca l s t imula t ion. In: New a s p e c t s of s torage and r e l ea se mechanisms of ca techolamines . Schumann, H . J . ; Kroneberg, G. ( e d s . ) pp .258-267 , Berl in Heide lberg , New York, Spr inger .

94. Reis , D . J . ; Granata, A.R. ; J o h , T .H . ; Ross, C . A . ; Ruggiero, D.A. & Pa rk , D.H. (1984). Brain stem catecholamine mechanisms in toxic and ref lex control of blood p r e s s u r e . Hypertension S u p p l . I I , 6, 5, I I - 7 - I I - 1 5 .

95. Robson, R.D. & Kaplan, H.R. (1969). An involvement of St.155 [2-(2 ,6 -d ich lorophenylamino) -2- imidazo l ine h y d r o c h l o r i d e c a t a p r e s ] in chol inergic mechanisms. Eur. J . Pha rmaco l . , 5 , 328-337.

96. Rogers, J . F . & Cubeddu, L.X. (1983). Naloxone does not antagonize the a n t i h y p e r t e n s i v e effect of clonidine in e s sen t i a l h y p e r t e n s i o n . Clin. Pharmacol . T h e r . , 34 (1 ) , 68-73.

97. Schlaefke , M.E. (1981) . Central chemosens i t iv i ty : a r e s p i r a t o r y d r i v e . Rev. Phys io l . Biochem. Pha rmaco l . , 90, 171-244.

98. Schmit t , H. & Schmit t , H. (1969). oC-Sympathomimetic drugs wi th cent­r a l l y mediated hypo tens ive effect . Fourth In ternat ional Congress on Pharmacology Abst . , 315-316.

99. Schmit t , H. & Gicquel , J . (1956). Action de I ' h y d r a z i n o ph tha laz ine (c5968) et la d i h y d r a z i n o - l ,4 -ph tha laz ine (c7448) sur les centers vasomoteurs . Arch. In t . Pharmacodyn. , 105, 269-278.

100. Schmit t , H. & Schmi t t , H. (1969). Localization of the hypo tens ive effect of 2 - (2 ,6 -d ich lo ropheny lamino) -2 - imidazo l ine h y d r o c h l o r i d e (St. 155 c a t a p r e s o n ) . Eur. J . Pharmaco l . , 6, 8-12.

101. Schmit t , H. & Schmitt H. (1970). In teract ions between 2 - ( 2 , 6 - d i c h l o -rophenylamino)-2- imidazol ine hydroch lo r ide (St . 155 Catapreson) and CJC-adrenergic blocking drugs . Eur. J . P h a r m a c o l . , 9 , 7-13.

102. Schul tz , H . S . ; Weber, M.A.; Brewer , D.D. & E l t o r a i , M.I . (1981). Cent ra l ly acting a n t i h y p e r t e n s i v e agents a comparison of lofexidine with c lonid ine . J . Cl in . Pha rmaco l . , 2 1 , 65-71 .

103. Shephe rd , A . ; Lin , M.S . ; Mcnay, J . ; Ludden, T. & Musgrave, G. (1981). Determinants of response to intravenous h y d r a l a z i n e in h y ­per tens ion . Cl in . Pharmacol . T h e r . , 30(6) , 773-781.

104. Sinha, J . N . ; Tangr i , K.K. ; Bhargava , K.P . & Schmi t t , H. (1975). Central s i t e s of sympa tho inh ib i to ry effects of c lonid ine and L-dopa . In: Recent Advances in Hyper tension, V o l . 1 , e d i t e d by Milliez & M. Safar, p p . 9 7 - 1 0 9 , Societe Aliena Reims.

105. Smialowska, M.; Ba l , A . ; So l tyz , Z. & Kaluza, J . (1985) . Monoamine d i s t r ibu t ion on t h e vent ra l surface of the r a t Medulla oblongata. J . Neural . Transmiss ion , 63 , 13-29.

51

106. Sr imal , R .C . ; Gulati , K. & Dhawan, B.N. (1977). On the mechanism of cent ra l hypotens ive act ion of c lon id ine . Can. J . P h y s i o l . Pharma­c o l . . 55 (5 ) , 1007-14.

107. S t a r k e , K.; Montel, H . ; Endo, T. & Taube , H.D. (1975) . Pharmaco­logical consequences of t h e p resynap t i c control of noradrena l ine r e l e a s e . In: Mi l l iez , P . ; Safar , M. ( E d s . ) ; Recent Advances in Hyper tens ion, 2 , 75-84.

108. S t a r k e , K.; Montel, H. ; Gayk, W. & Merker , R. (1974) . Comparison of the effects of c lonidine on p re and post synap t i c ad renocep to r s in the r a b b i t pulmonary a r t e r y , oC-sympathomimetic i nh ib i t i on of neurogenic vasocons t r ic t ion . Naunyn-Schmiedeberg 's Arch, P h a r m a k o l . , 285, 133-150.

109. S t ruyker Boudier , H.A.J . & van Rossum, J .M. (1972). Clonid ine- induced card iovascu la r effects af ter s t e r eo tax ic app l i ca t ion in the h y p o t h a ­lamus of r a t s . J . Pharm. Pha rmaco l . , 24, 410-411.

110. Sved , A . F . ; Bless ing, W.W. & Donald, J .R . (1985). Caudal ven t ro l a ­t e ra l medulla can a l t e r v a s o p r e s s i n and a r t e r i a l p r e s s u r e . Brain Res. B u l l . , 14, 227-232.

111. T i t e l e r , M. & Seeman, P. (1982), Effects of a n t i h y p e r t e n s i v e c lonidine congeners on a lpha -ad rene rg i c r e c e p t o r s . Can. J . P h y s i o l . P h a r m a c o l . , 60(3 ) , 342-344.

112. Vak i l , R . J . (1949). Clinical t r i a l of rauwolfia serpent ina in e s sen t i a l hype r t ens ion . Br . Heart J . , I I , 350-355.

113. Velasco , M.; Urbina-Quintana, A . ; Mori l lo , J . ; Vizcar rondo , H. ; Ramirez A. ; Hernandez, E. & Hernandez -P ie re t t i , 0 . (1985) . Systemic and ca rd iac haemodynamic in te rac t ions between guanfacin and h y d r a l a z i n e in hype r t ens ive p a t i e n t s . Eur . J . Cl in . Pha rmaco l . , 2 7 ( 4 ) , 393-396.

• 114. Waite, R. (1975). Inh ib i t ion of sympathe t i c nerve a c t i v i t y resul t ing

from centra l a lpha -ad renocep to r s t imulat ion. In: Mi l l i ez , P . ; Safar , M. ( E d s . ) : Recent Advances in Hyper tens ion, p p . 2 7 - 3 6 .

115. Werner, U. ; S t a rke , K. & Schumann, H . J . (1972). Actions of c lonidine and 2 - (2 -methy l -6 -e thy lcyc lohexy lamino) -2 -oxazo l ine on pos t ganglio­nic autonomic n e r v e s . Arch. In t . Pharmacodyn. T h e r . , 195, 282-290.

116. Wi lk ins , R.W. & Judson, W.E. (1953). The use of Rauwolfia se rpent ina in hypotens ive agents . New Engl. J . Med. , 248, 48-53 .

117. Wi l l e t t e , R.N.; Ba rcas , P . P . ; Kr ieger , A . J . & Sapru , H.N. (1984) . Endogenous GABAergic mechanism in Medulla and regu la t ion of blood pressure . - J . Pharmacol . & E x p . T h e r a p . , 230(1) , 34-39.

52

118. Yamada, K.A.; Norman, W.P . ; Hamosh, P. & G i l l i s , R.A. (1982). Medullary ven t ra l surface GABA r e c e p t o r s affect r e s p i r a t o r y and ca rd iovascu la r function. Brain R e s . , 248, 71-78.

119. Zacest , R.; Gilmore, E. & Koch-Weser, J . (1972). Treatment of e s sen ­t ia l hype r t ens ion with combined vasod i la t ion and be t a - ad rene rg i c b lockade . N. Engl. J . Med. , 286, 617-622.

120. Zhang, T.X. & C i r i e l l o , J . (1982). Lesions of p a r a v e n t r i c u l a r nucleus r e v e r s e the e l eva ted a r t e r i a l p r e s su re after ao r t i c ba ro recep to r denervat ion in the r a t . Soc. Neurosci . Abs t r . , 8, 484.

121. van Zwieten, A. (1968). Pharmakologis und biochemische Wirkungen von Hydralazin und Dihydra laz in . Arzneineo^^Zjorsch. , 18, 79-84.