Drug Development Research 22: 141-152 (1991)

Effect of Repeated Administration of Clonidine on Adrenergic, Cholinergic (Muscari nic), Dopami nerg ic, and Serotonergic Receptors in Brain Regions of Rats Anil Gulati, Ghazala Hussain, and Rikhab Chand Srimal

Department of Pharmacodynamics (m/c 865), The University of Illinois at Chicago, Chicago (A. G.); Division of Pharmacology, Central Drug Research Institute, India (G. H., R.C.S.)

ABSTRACT

Gulati, A., G. Hussain, and R.C. Srirnal: Effect of repeated administration of clonidine on adrenergic, cholinergic (muscarinic), dopaminergic, and serotonergic receptors in brain regions of rats. Drug Dev. Res. 22:141-152, 1991.

Clonidine (0.1 mg/kg) or its vehicle was administered orally to rats for 2 months. A signif- icant decrease in mean blood pressure was observed in clonidine treated rats. Chronic administration of clonidine in rats produced alterations in central receptors. In hypothalamus and medulla, clonidine treatment increased the B,,, value of 3H-dihydroergocryptine bind- ing to a-adrenergic receptors. In cortex, clonidine treatment increased the B,,, value of 3H-spiroperidol binding to dopaminergic receptors. Clonidine treatment produced a de- crease in the B,,, value in cortex while in hypothalamus and medulla, an increase in the B,,, value of 3H-quinuclydinyl benzylate (QNB) binding to muscarinic receptors was ob- served. In medulla, clonidine treatment increased the B,,, value of 3H-5-hydroxytryptamine (HT) binding to 5-HT, receptors. No change was observed in dissociation constant (Kd) values of any 3H-ligand in any brain region. In vitro studies showed interaction of clonidine with 3H-dihydroergocryptine and 3H-QNB. It can be concluded from the present study that chronic administration of clonidine produces an up-regulation of a-adrenergic, cholinergic (muscarinic), and serotonergic (5-HT,) receptors in brain regions mainly involved in car- diovascular regulation.

Received final version August 21, 1990: accepted September 10, 1990

Address reprint requests to Anil Gulati, M . B . , B .S . . M.D., Department of Pharmacodynamics (865), The University of Illinois at Chicago, 833, South Wood Street, Chicago, II 60612.

0 1991 Wiley-Liss, Inc.

142 Gulati et al.

Key words: receptor binding, B,,, value, receptor up-regulation

INTRODUCTION

Clonidine has been widely used as an antihypertensive drug [Schmitt and Schmitt, 1969; Srimal et al., 1977; Houston, 19811. It reduces blood pressure by decreasing the sympathetic tone [Houston, 19811 and noradrenaline release [Anden et al., 1970; Berthelsen and Pettinger, 19771. It is generally regarded that the main mechanism for the hypotensive action of clonidine is by stimulation of central a-adrenoceptors [Pettinger, 1975; Van Zwieten and Timmermanns, 19791. However, Srimal et al. [ 19771 have demonstrated that clonidine produces a fall in blood pressure by acting at cholinergic (muscarinic) receptors at the ventral surface of medulla in cats. Criscione et al. [1983] have proposed that cholinergic mechanism in the NTS tends to lower arterial pressure after clonidine administration and may modulate the baroreceptor reflex without being an integral part of the reflex arc. It is known that centrally administered cholinergic drugs decrease blood pressure [Porsius et al., 1978; Philippu, 19811 and the effect is explained by simultaneously occurring decrease in sympathetic out flow similar to cloni- dine. Central serotonergic receptors have also been involved in the hypotensive activity of clonidine. Antonaccio et al. [ 19751 demonstrated centrally induced hypotension and brady- cardia following the intracerebroventricular injection of methysergide in dogs and rats [An- tonaccio and Cote, 19761. The effect was associated with a reduced peripheral sympathetic activity. Similarly, as described for clonidine, methysergide has been shown to enhance vagal reflex bradycardia in cats after the injection of drug in the fourth ventricle [Tadepalli et al., 19791.

In addition to the cardiovascular effects, clonidine possesses a wide spectrum of phar- macological activities, including behavioral effects. It is difficult to interpret the behavioral effects of clonidine in relation to the direction of effects on neurotransmitter systems. Cloni- dine is believed to act predominantly on noradrenergic transmitter systems. In humans, it selectively decreased urinary catecholamine excretion (Hokfelt et al., 19701 and cerebrospinal fluid (CSF) levels of 3-methoxy-4-hydroxyphenyl glycol (MHPG) [Bertilsson et al., 19771. The increases in plasma growth hormone after clonidine administration also indicate a central noradrenergic effect [Lal et al., 1975; Durrand et al., 19771. The drug may have inhibitory as well as excitatory effects on noradrenergic systems [Langer, 1974; Anden et al., 19761. Clonidine has little direct influence on dopamine systems [Anden et al., 1970; Rochette and Bralet, 19751. Some limited effects have also been reported on epinephrine [Bolme et al., 19741, histamine [Audigier et al., 1976; Karppanen et al., 19761, and cholinergic function [Maj et al., 19751. Clonidine increases central 5hydroxytryptamine (HT) turnover and firing rate of 5-HT raphe neurons [Anden et al., 1970; Svensson et al., 19751.

Although multiple receptors have been involved in the mechanism of action of clonidine which is one of the most widely used centrally acting antihypertensive drugs, no study has been performed to determine the effect of chronic administration of clonidine on various central neurotransmitter receptors. The clinical use of clonidine is almost entirely on a chronic basis; however, most of the data on its effects originate from acute studies. The aim of the present study is to investigate the effect of chronic administration (2 months) of clonidine on adrenergic, cholinergic (muscarinic), dopaminergic, and serotonergic receptors in brain re- gions of rats.

METHODS

Albino, male rats of the Charles Foster strain, bred in the animal house of the Central Drug Research Institute, Lucknow, were used. Rats weighing 100-150 g were kept in stan- dard laboratory conditions of temperature (23 k 1.5"C), humidity (50 -t 1 O%), and dark light

Clonidine and Central Receptors 143

TABLE 1. Summary of the Various Ligands Used for Radioreceptor Binding to Different Neurotransmitter Receptors Receptor Hot ligand Cold ligand a-adrenergic 3H-dihydroergocryptine Phentolamine

0.25-12 nM (Amersham) (1 PM) P-adrenergic

Muscarinic

Dopamine

5-HT,

5-HTZ

3H-dihydroalprenolol

3H-QNB

3H-spiroperidol

3H-5-HT

3H-spiroperidol

0.12-4 nM (Amersham)

0.1-2 nM (NEN)

0.1-5 nM (NEN)

0.25-15 nM (NEN)

0.15-10 nM INEN)

Propranolol ( 1 PW

Atropine ( 1 PM)

Haloperidol ( 1 PM)

(10 PM) Ketanserin

(1 LLM)

5-HT

(5.00-18.00 hr) cycle. The animals were allowed to acclimatize for 4 days before starting the experiment.

The antihypertensive drug, clonidine (0.1 mgikg) was dissolved in water and adminis- tered orally to rats for 2 months. The dose of this drug was selected on the basis of several reports, and preliminary work done in our laboratory, indicating the best dose which could be used chronically and effectively. The drug was prepared fresh everyday just before the ad- ministration.

The blood pressure was recorded indirectly in unanesthetized rats by the plethysmo- graphic tail cutoff method [Friedman and Freed, 19491 using a rat tail manometer system (IITC Model No. 45). The arterial pulses were detected photoelectrically by sensing cuffs and the output was recorded on a dual channel recorder to show the presence of tail pulses on one channel and the matching cuff pressure on the other channel. The IITC method to measure blood pressure determines systolic as well as the mean blood pressure of rodents. The first onset of pulses was taken as the systolic point and the first maximum amplitude was taken as the mean blood pressure point. The blood pressure using this system could be recorded at the room temperature without prewarming the rats to increase the blood flow to the tail.

Preparation of Membranes

At the end of 2 months of chronic drug treatment the animals were sacrificed and decapitated. Thereafter specific areas of the brain, corpus striatum, cerebral cortex, hypothal- amus, and medulla were dissected out in cold. Each brain area was weighed and then homog- enized in 30 volumes of 50 mM Tris-HC1 buffer (pH 7.7 at 25°C) using a motor driven teflon-glass tissue homogenizer. The tissue homogenate was centrifuged twice at 49,OOOg for 15 min in a refrigerated Sorvall centrifuge after resuspending in fresh Tris-HC1 buffer. The final pellet was suspended in 50 mM Tris buffer (pH 7.4 at 25°C) consisting of 0.1% ascorbic acid, 120 mM NaCl, 5 mM KCl, 2 mM CaCI,, and 10 y M pargyline to give a concentration of 10 mg wet tissue per milliliter of incubation buffer. The binding studies were performed essentially as described by Gulati et al. [1988a,b]. Details about the incubation conditions are given in Table 1 .

Binding of 3H-Dihydroergocryptine to a-Adrenergic Receptors

The binding of 3H-dihydroergocryptine to a-adrenergic receptors was performed as described by Williams and Lefkowitz [I9771 and Gulati et al. [1988b]. The standard assay mixture consisted of 0.2 ml of the homogenate, containing approximately 200 yg of tissue, 0.1 ml of 3H-dihydroergocryptine, 0.1 ml of competing agent (phentolamine), and buffer to make up the total volume of 1 ml. Incubation was carried out in triplicate in a shaking water

144 Gulati et al.

bath maintained at 37°C for 15 min. At the end of the 15 min period, the contents of the incubation tubes were rapidly filtered under partial vacuum using Millipore manifold filtration unit and Whatman GFK filters. It was followed by two 5 ml washes of ice cold SO mM Tris-HC1 buffer (pH 7.4). The filters were transferred to liquid scintillation vials containing 10 ml of cocktail. After an overnight equilibration period, the radioactivity was determined in a Packard Tricarb liquid scintillation spectrometer. The specific binding of 'H-dihydroergo- cryptine was defined as the difference in binding observed in the absence and in the presence of 1 pM unlabeled phentolamine.

Binding of 3H-Dihydroalprenolol to (3-Adrenergic Receptors

The binding of 'H-dihydroalprenolol to P-adrenergic receptors was performed as de- scribed by Alexander et al. [ 197.51 and Bylund and Snyder [1976]. The standard assay mixture consisted of 0.2 ml of the homogenate, containing approximately 200 pg of tissue, 0.1 ml of 3H-dihydroalprenolol, 0.1 ml of competing agent (propranolol), and buffer to make up the total volume of 1 nd. Incubation was carried out in triplicate in a shaking water bath main- tained at 37°C for 15 min. The samples were filtered and the radioactivity was determined as described earlier. The specific binding of 'H-dihydroalprenolol was defined as the difference in binding observed in the absence and in the presence of 1 pM unlabeled propranolol.

Binding of 3H-Quinuclydinyl Benzylate (QNB) to Cholinergic (Muscarinic) Receptors

The membranes from freshly dissected brain regions were prepared as described earlier. The binding of 'H-QNB to cholinergic (muscarinic) receptors was performed as described by Gulati et al. [1982, 1988bI. The standard assay mixture consisted of 0.2 ml of the homoge- nate, containing approximately 200 pg of tissue, 0. I mI of 'H-QNB, 0. I ml of competing agent (atropine), and buffer to make up the total volume of 1 ml. Incubation was carried out in triplicate in a shaking water bath maintained at 37°C for 15 min. At the end of the 15 rnin period, the contents of the incubation tubes were rapidly filtered and the radioactivity was determined. The specific binding of 'H-QNB was defined as the difference in binding observed in the absence and in the presence of 1 pM unlabeled atropine.

Binding of 3H-Spiroperidol to Dopamine Receptors

The membranes from freshly dissected brain regions were prepared as described earlier. The binding of 'H-spiroperidol to dopamine receptors was performed as described by Gulati et al. 11986, 1988al. The standard assay mixture consisted of 0.2 ml of the homogenate, containing approximately 200 pg of tissue, 0.1 ml of 3H-spiroperidol, 0.1 ml of competing agent (haloperidol), and buffer to make up the total volume of 1 ml. Incubation and filtration were carried out as described before. The filters were transferred to liquid scintillation vials and the radioactivity was determined in a Packard Tricarb liquid scintillation spectrometer. The specific binding of 'H-spiroperidol was defined as the difference in binding observed in the absence and in the presence of I pM unlabeled haloperidol.

Binding of 3H-5-HT to 5-HT, Receptors

The binding assays for 'H-5-HT was carried out as described by Fillion et al. [I9781 and Gulati and Bhargava [1988]. The tissue in each tube was about 300 pg. The incubation was carried out for 20 min in a shaking water bath maintained at 37°C. At the end of the incubation period, the contents of the incubation tubes were filtered rapidly under partial vacuum using Whatman GFK filter discs on a Millipore manifold filtration unit. The washing and counting of radioactivity were done as described before for other receptor studies. The specific binding was defined as the difference in binding of 'H-5-HT in the absence and presence of 10 p M 5-HT.

Clonidine and Central Receptors 145

Binding of 3H-Spiroperidol to 5-HT2 Receptors

The freshly dissected brain regions were homogenized and neuronal membranes pre- pared as described earlier. The binding of 3H-spiroperidol to 5-HT2 receptors was performed as described by Gulati et al. [1988a] and Gulati and Bhargava [1988]. The standard assay mixture consisted of 0.2 ml of the homogenate, containing approximately 200 kg of tissue, 0.1 ml of 'H-spiroperidol, 0.1 ml of competing agent (ketanserin), and buffer to make up the total volume of 1 ml. Incubation and filtration were done and radioactivity determined as described earlier. The specific binding of 3H-spiroperidol was defined as the difference in binding observed in the absence and in the presence of 1 pM unlabeled ketanserin.

The specific binding was expressed as pmoles of 3H-ligand bound per gram of tissue. The specific binding of the radioligand was determined at 8 to 10 concentrations in each case. This was followed by the Scatchard analysis to determine the dissociation constant (K,) and the receptor density (B,,J values. Five determinations, one in each rat, were made to compute the means and their standard errors. The data were analyzed by the ANOVA followed by Scheffe's S test.

RESULTS Effect of Chronic Administration of Clonidine on Blood Pressure of Rats

The mean blood pressure of rats before starting the treatment was 125.9 2 0.6 mmHg and 126.2 2 1.9 mmHg in control and treated groups, respectively. However, after 2 months of vehicle or clonidine treatment a significant decrease in mean blood pressure was observed and the blood pressure was 131.2 * 1.2 mmHg in control rats and 118.2 * 0.7 mmHg in clonidine treated rats (F[1,8] = 93.78; P < 0.0005).

Effect of Chronic Administration of Clonidine on a-Adrenoceptors

The population (B,,,) of the adrenoceptors in normal rat brain was found to be fairly constant in all the four areas. The B,,, values in different regions of the brain ranged from 30.21 k 1.38 pmol/g tissue to 41.49 & 2.19 pmol/g tissue, while the K, value ranged from 0.66 2 0.20 nM to 1.24 * 0.23 nM. Thus the binding of 'H-dihydroergocryptine was at a single high affinity binding site.

In cortex and corpus striatum of clonidine treated rats the B,,, and K, values of 'H-dihydroergocryptine binding remained unchanged as compared to control. In hypothala- mus, the B,,, value increased by 31.7% (F[ 1,8] = 15.35; P = 0.004) in clonidine treated rats while the K, value did not change as compared to control. Similarly, in the medulla of clonidine treated rats the B,,, value increased (58.9%) (F[1,8] = 60.93; P < 0.0005) and no change occurred in the K, value as compared to control (Table 2).

Effect of Chronic Administration of Clonidine on p-Adrenoceptors

The binding of 'H-dihydroalprenolol to (3-adrenoceptors was at a single high affinity binding site. The B,,, value ranged from 24.80 k 2.11 pmol/g tissue to 38.76 5 2.57 pmolig tissue, while the K, ranged from 0.58 2 0.04 nM to 1.76 ? 0.07 nM.

In the cortex, corpus striatum, hypothalamus, and medulla of clonidine treated rats the B,,, and K, values of 'H-dihydroalprenolol binding did not differ as compared to the control rats (Table 3).

Effect of Chronic Administration of Clonidine on Dopaminergic Receptors

The binding of 'H-spiroperidol to dopaminergic sites in the corpus striatum was found to be at a single high affinity binding site with a B,,, value of 58.68 k 2.09 pmol/g tissue and a K, value of 0.03 2 0.01 nM. In other brain regions the B,,,, values ranged from 25.23 k

146 Gulati et al.

TABLE 2. Effect of Repeated Administration of Clonidine on 3H-Dihydroergocryptine Binding to a-Adrenergic Receptors in Rat Brain Regions

Cerebral cortex Treatment B,,, (pmol/g tissue) K, (nM)

Vehicle 30.21 ? 1.38 0.81 f 0.12 Clonidine 30.53 i 1.54 0.83 i 0.14

Corpus striatum Vehicle Clonidine

Hypothalamus Vehicle Clonidine

41.49 i 2.19 45.24 ? 1.99

32.89 f 2.19 43.31 t 1.92*

1.24 i 0.23 1.43 f 0.14

0.68 t 0.1 1 0.87 i 0.16

Me d u 11 a Vehicle 31.09 i 1.30 0.66 f 0.20 Clonidine 49.41 * 2.29* 0.87 i 0.16

The values are in Mean t S.E.M., N=5. ' P < 0.05 as compared to vehicle treated control.

TABLE 3. Effect of Repeated Administration of Clonidine on 'H-Dihydroalprenolol Binding to 6-Adrenergic Receptors in Rat Brain Regions

Treatment B,,, (pmolig tissue) K, (nM) Cerebral cortex

Vehicle 38.76 i 2.57 0.80 ? 0.05 Clonidine 38.37 2 2.30 0.72 ? 0.11

Corpus striatum Vehicle Clonidine

35.77 i 1.44 35.21 t 1.11

1.76 i 0.07 1.89 t 0.05

Hypothalamus Vehicle 24.80 i 2.11 0.58 f 0.04 Clonidine 25.74 i 1.41 0.52 t 0.05

Vehicle 31.77 t 1.73 0.68 5 0.06 Clonidine 32.27 f 2.29 0.66 i 0.05

Medulla

The values are in Mean t S.E.M. , N=5.

0.92 pmolig tissue to 46.63 2 1.70 pmolig tissue, whereas the K, value ranged from 0.31 5 0.04 nM to 0.43 i 0.09 nM.

In cortex, clonidine treatment produced a 17.6% increase (F[ 1,8] = 11.64; P = 0.009) in the B,,,, value while no change occurred in the K, value as compared to control. In corpus striatum, hypothalamus, and medulla of clonidine treated rats no change occurred in either the B,,, or the K, value of 'H-spiroperidol binding as compared to control (Table 4).

Effect of Chronic Administration of Clonidine on Cholinergic (Muscarinic) Receptors

The binding of 'H-QNB to muscarinic receptor sites was at a single high affinity binding site. The B,,, value ranged from 36.53 2 1.63 pmolig tissue to 63.10 L 2.20 pmolig tissue, while the K, value ranged from 0.73 2 0.06 nM to 1.42 rt 0.09 nM.

In cortex, clonidine treatment produced a decrease (25.4%) (F[1,8] = 34.24; P < 0.0005) in the B,,, value while the K, value of 3H-QNB binding was not affected. In corpus striatum, no change was observed in the B,,, or K, values of clonidine treated rats as

Clonidine and Central Receptors 147

TABLE 4. Effect of Repeated Administration of Clonidine on 3H-Spiroperidol Binding to Dopaminergic Receptors in Rat Brain Regions Treatment B,,, (prnol/g tissue) K, (nM) Cerebral cortex

Vehicle Clonidine

Corpus striatum Vehicle Clonidine

Hypothalamus Vehicle Clonidine

Vehicle Medulla

46.63 * 1.70 54.84 f 1.96*

58.68 * 2.09 58.05 * 1.26

25.23 * 0.92 26.66 * 0.97

44.49 f 1.68

0.43 2 0.09 0.48 * 0.09

0.03 t 0.01 0.03 * 0.01

0.31 * 0.04 0.32 2 0.07

0.39 2 0.05 Clonidine 43.84 2 1.49 0.43 * 0.07

The values are in Mean 2 S.E.M., N = 5 . *P < 0.05 as compared to vehicle treated control.

compared to control. In hypothalamus, clonidine treatment increased (50.6%) (F[ 1,8] = 55.74; P < 0.0005) the B,,, value while the K, value of 3H-QNB binding was not affected. Similarly in medulla, clonidine treatment increased (42.0%) (F[ 1,8] = 47.05; P < 0.0005) the B,,, value while the K, value of 3H-QNB binding was unaltered as compared to control (Table 5) .

Effect of Chronic Administration of Clonidine on 5-HT, Receptors

The 3H-5-HT binding to 5-HT, receptors is fairly constant and at a single high affinity binding site. The B,,, value ranged from 23.74 2 2.1 1 pmolig tissue to 31.46 5 1.35 pmolig tissue, while the K, value ranged from 0.86 t 0.05 nM to 1.54 t 0.07 nM. The cortex, corpus striatum, and hypothalamus of clonidine treated rats did not show any change in either the B,,, value or the K, value of 3H-5-HT binding. In medulla, clonidine treatment increased (47.1 %) (F[l,8] = 18.26; P = 0.003) the B,,, value while no change occurred in the K, value of 3H-5-HT binding (Table 6).

Effect of Chronic Administration of Clonidine on 5-HT2 Receptors

The binding of 3H-spiroperidol to 5-HT, receptors was at a single high affinity binding site. The B,,, value ranged from 23.36 t 1.25 pmol/g tissue to 34.86 t 2.61 pmolig tissue, while the K, value ranged from 0.78 t 0.02 nM to 1.38 t 0.07 nM.

In cortex, corpus striatum, hypothalamus, and medulla of clonidine treated rats no change in the B,,, and K, values of the 3H-spiroperidol binding was observed as compared to control (Table 7).

In Vitro Interactions of Clonidine

Interaction of clonidine (1 pM) with above mentioned ligands was studied in whole brain membranes. It was found that clonidine displaced the specific binding of 3H-dihydro- ergocryptine (46.1%) and 'H-QNB (20.9%) but did not affect the specific binding of 3H- dihydroalprenolol, 3H-spiroperidol, and 3H-5-HT.

DISCUSSION

The results of the present investigation show that chronic administration of clonidine in rats produced a decrease in blood pressure and alterations in central a-adrenergic, dopamin-

148 Gulati et al.

TABLE 5. Effect of Repeated Administration of Clonidine on 3H-QNB Binding to Cholinergic (Muscarinid ReceDtors in Rat Brain Regions

Treatment B,,, (pmolig tissue) K, (nM) Cerebral cortex

Vehicle 51.13 t 1.92 0.78 t 0.09 Clonidine 38.13 t 1.53" 0.96 t 0.06

Corpus striatuni Vehicle Clonidine

Hypothalamus Vehicle Clonidine

63.10 2 2.20 60.10 t 2.95

36.53 2 1.63 55.00 t 2.06*

0.77 t 0.11 0.76 t 0.09

1.42 t 0.09 1.13 t 0.18

Medulla Vehicle 39.93 2 1.83 0.73 t 0.06 Clonidine 56.70 t 2.12* 0.59 t 0.09

The values are in Mean 2 S . E . M . , N=5. * P < 0.05 as compared to vehicle treated control.

TABLE 6. Effect of Repeated Administration of Clonidine on 3H-5-HT Binding to 5-HT, ReceDtors in Rat Brain Regions Treatment B,,, (pmolig tissue) K, (nM) Cerebral cortex

Vehicle 31.46 t 1.35 1.34 t 0.11 Clonidine 30.82 t 1.80 1.28 f 0.22

Corpus striatum Vehicle Clonidine

23.74 2 2.11 25.25 t 1.98

0.86 t 0.05 0.76 t 0.06

Hypothalamus Vehicle 26.02 t 1.82 1.54 t 0.07 Clonidine 2.5.74 2 1.88 1.43 k 0.15

Vehicle 24.99 t 1.66 1.35 t 0.07 Clonidine 36.76 % 2.60* 1.16 t 0.10

Medulla

The values are in Mean t S.E.M., N=5. *P < 0.05 as compared to vehicle treated control.

ergic, cholinergic (muscarinic), and serotonergic (5-HT,) receptors. In the cerebral cortex and corpus striatum of clonidine treated rats, the density and affinity of 3H-dihydroergocryptine binding did not change, while in hypothalamus and medulla clonidine treatment increased the density but did not change the affinity. Clonidine treatment did not affect the density and affinity of 3H-dihydroalprenolol binding to 6-adrenoceptors in any of the four brain regions. In the cortex, clonidine treatment increased the density but did not change the affinity of the dopaminergic receptors, while in the striatum, hypothalamus and medulla clonidine did not affect either the density or the affinity of 'H-spiroperidol binding to dopamine receptors. In cortex, clonidine treatment produced a decrease in the density of the muscarinic receptors; in corpus striatum, no change was observed in the density or affinity by clonidine treatment; in hypothalamus and medulla, clonidine treatment increased the density and had no effect on the affinity of 3H-QNB binding to muscarinic receptors. Cerebral cortex, corpus striaturn, and hypothalamus of clonidine treated rats did not show any change in the density or affinity of 3H-5-HT binding to 5-HT, receptors; in medulla, clonidine increased the density of HT,

Clonidine a n d Central Receptors 149

TABLE 7. Effect of Repeated Administration of Clonidine on 'H-Spiroperidol Binding to 5-HT, Receptors in Rat Brain Regions Treatment B,,, (pmolig tissue) K, (nM) Cerebral cortex

Vehicle 34.86 t 2.61 0.86 +- 0.04 Clonidine 35.21 t 1.90 0.87 t 0.02

Vehicle 33.80 2 2.25 0.78 t 0.02 Clonidine 34.43 +- 1.92 0.81 * 0.01

Corpus striatum

Hypothalamus Vehicle Clonidine

Vehicle Clonidine

Medulla

23.36 t 1.25 24.35 * 2.30

27.19 +- 1.40 27.06 ? 1.48

0.86 t 0.06 0.96 * 0.08

1.38 2 0.07 1.30 t 0.11

The values are in Mean ? S.E.M.. N = 5 .

receptors but had no affect on its affinity. Clonidine treatment did not show any change in the density and affinity of 3H-spiroperidol binding to 5-HT, receptors in any region of the brain.

The up-regulation of a-adrenergic receptors in hypothalamus and medulla support the presynaptic action of clonidine. The affinity of clonidine for presynaptic a-adrenoceptors is at least 10 times higher than its affinity for postsynaptic a-adrenoceptors [Starke, 19771. It could be possible that repeated administration of clonidine decreases the release of norepinephrine in the hypothalamus and medulla by acting on presynaptic receptors. The decreased concentra- tion of norepinephrine in the synapse will result in an increase in the density of postsynaptic a-adrenoceptors. Svensson and Strombom [I9771 showed in mice that a reduction in brain catecholamine synthesis persisted in various brain regions during chronic clonidine treatment. In the rat Tang et al. [ 19781 reported that clonidine produced a consistent decrease in brain total 3-MHPG. These results support a presynaptic action of clonidine and a reduction in noradren- aline turnover [Rochette et al., 19821 that would produce an up-regulation of postsynaptic a-adrenergic receptors.

The involvement of cholinergic (muscarinic) receptors in the mechanism of action of clonidine has been demonstrated. Srimal et al. [ 19771 suggested that intact cholinergic link in the brain stem is essential for the hypotensive action of clonidine. Criscione et al. [ 19831 have proposed that cholinergic mechanism in the NTS tends to lower arterial pressure after clonidine administration and may modulate the baroreflex without being an integral part of the reflex arc. The up-regulation of cholinergic (muscarinic) receptors in hypothalamus and medulla could play an important role in the mechanism of action of clonidine.

Clonidine does not seem to stimulate the dopamine receptors in the corpus striatum of rat [Anden et al., 19701, and this is in agreement with the results of the present study. However, if a high dose of clonidine (3,000 pg/kg, intraperitoneally [i.p.]) is given in com- bination with the dopamine receptor stimulating agent apomorphine, a marked potentiation of the apomorphine-induced increase in motor activity is observed [Anden et al., 19731. Endog- enous concentrations of dopamine are not significantly changed by clonidine though there is a tendency to an elevation after the high dose [Anden et al., 19701. Clonidine had no effect on turnover of dopamine when given in low doses (0.1 mg/kg); however, at a high dose ( 1 mg/kg) dopamine turnover was found to be reduced [Rochette et al., 19821. Besides, the changes in turnover of dopamine following clonidine administration have been shown to be secondary to its effect on adrenergic receptors [Anden and Grabowska, 1976; Strombom, 19761. These findings support the results of the present study where no change in dopamine receptors was

150 Gulati et al.

observed in corpus striatum, hypothalamus, and medulla. Whether or not the increase in the density of cortical dopamine receptors at 0.1 mgikg dose is responsible for some of the behavioral actions of clonidine is not known at present.

Clonidine has been found to decrease serotonin (5-HT) turnover; this is indicated by a' deceleration in the disappearance of serotonin in the rat brain after inhibition of its synthesis [Anden et a]. , 1970; Rochette et al., 19741. Clonidine also reduces the concentration of 5-HT metabolites 5-hydroxy-3-indoleacetic acid (5-HIAA) in the rat brain [Scheel-Kruger and Has- selager, 19741. In contrast to the effect of clonidine on noradrenergic neurons, the inhibitory effect of clonidine on 5-HT neurons is probably indirect. Maura et al. [ 19851 have shown that the presynaptic action of clonidine at a-adrenergic receptors mediates inhibition of 3H-nor- adrenaline and 3H-5-HT release. The decrease in release of 5-HT will result in up-regulation of 5-HT receptors as observed in the present study.

In vitro studies showed that clonidine interacts with 3H-dihydroergocryptine and 3H- QNB binding, where clonidine could displace the specific binding of these ligands. The direct interaction of clonidine with these receptors is not possible because the membranes were washed with buffer three times to wash out the clonidine administered systemically. Thus the increase in the density of a-adrenergic and cholinergic (muscarinic) receptors is not due to direct in vitro interaction of clonidine with these receptors but due to in vivo action of clonidine on adrenergic, cholinergic, and serotonergic systems.

It is well known that major cardiovascular regulatory centers are located in the hypo- thalamus and medulla and we have observed selective changes induced by clonidine in the neurotransmitter receptors of the hypothalamus and medulla. It could be possible that these changes in receptors might be important in the hypotensive action of clonidine. On the other hand, the changes in cerebral cortical receptors by clonidine could be responsible for the sedative side effects.

Finally, it can be concluded from the present study that clonidine acts on several neurotransmitter receptors. The brain regions mainly involved in cardiovascular regulation, hypothalamus and medulla, show up-regulation of a-adrenergic, cholinergic (muscarinic), and serotonergic (5-HT,) receptors and may play a role in the mechanism of antihypertensive action of clonidine.

ACKNOWLEDGMENTS

The Dean of the College of Pharmacy, University of Illinois at Chicago is gratefully acknowledged for providing partial financial support. Vandana Gulati is acknowledged for help in the preparation of this manuscript.

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