Brain nicotinic cholinoceptor binding in spontaneous hypertension

212

Brain Research, 410 (1987) 2 l 2-218 Elsevier

BRE 12527

Brain nicotinic cholinoceptor binding in spontaneous hypertension Shizuo Yamada, Yoshiyuki Kagawa, Hideto Ushijima, Noriyasu Takayanagi, Takako Tomita and Eiichi Hayashi Department of Pharmacology, Shizuoka Collegeof PharmaceuticalSciences, Shizuoka (Japan) (Accepted 16 September 1986) Key words: Spontaneous hypertension; Medulla oblongata; Nicotinic cholinoceptor; [3H]Nicotine

To study the role of central cholinergic mechanisms in hypertension, we have determined nicotinic and muscarinic agonist binding sites in the brain regions of stroke-prone spontaneously hypertensive rats (SHRSP), using [3H]nicotine and [3H]cismethyldioxolane (CD). There was a significant decrease in specific [3H]nicotine binding in the cerebral cortex, thalamus, midbrain, cerebellum and medulla oblongata of SHRSP at 16-24 weeks of age compared to that of age-matched Wistar Kyoto rats (WKY). Scatchard analysts revealed 35% decrease in the BmaXvalue for [3H]nicotine binding in the SHRSP medulla oblongata without a change in the K~ value, suggesting a change in the receptor density, Similar reduction of nicotinic cholinoceptor binding sites was also observed in the discrete brain regions of young (5-week-old) SHRSP. In contrast, there was no alteration in specific [3H1CDbinding in the SHRSP brain regions, except the hypothalamus which showed a significant increase. The SHRSP medulla oblongata showed no change in the ChAT activity. Thus, the present study suggests an important role for medullary nicotinic cholinoceptors in the pathogenesis of spontaneous hypertension.

INTRODUCTION It is known that the activation of central nicotinic receptors has a significant influence on the cardiovascular function in several species 1-3'7. Porsius and Van Zwieten 2° have found that an injection of nicotine into the vertebral artery in cats induced a hypotension and bradycardia possibly via an activation of medullary neurons. Kubo and Misu 13-15 have demonstrated that the microinjection of nicotinic cholinergic agonists into the dorsal area of medulla oblongata in rats produced a marked hypotension and bradycardia. These effects were abolished by hexamethonium but not by atropine, similarly injected 14. The medulla oblongata is known to contain a significant level of acetylcholine (ACh), choline acetyltransferase (CHAT) and acetylcholinesterase (ACHE) 1°'24. Thus, it is possible that the nicotinic cholinergic mechanism in the lower brainstem is involved in the physiological regulation of cardiovascular functions.

Previous studies have presented evidence that a central cholinergic mechanism may be involved in the pathogenesis of spontaneous hypertension 5'~°-12"16'27. The altered cardiovascular responsiveness to a stimulation of central cholinoceptors in spontaneously hypertensive rats (SHR) has been reported 5'12-16. We 27 and others II have found that there is a specific increase in muscarinic cholinoceptors and a decrease in the activity of ChAT in the hypothalamus of S H R and stroke-prone S H R (SHRSP), However, little information is available regarding the possible role of central nicotinic cholinergic mechanisms in the hypertension. Recently, we have characterized extensively central nicotinic cholinoceptors by investigating the inhibitory effect of neosurugatoxin (NSTX), a selective antagonist of nicotinic cholinoceptors, on specific [3H]nicotine binding in the rat brain 9~29. In the present study, we have measured specific [3H]nicotine binding in brain regions of S H R S P in comparison

Correspondence: S. Yamada, Department of Pharmacology, Shizuoka College of Pharmaceutical Sciences, 2-2-10shika, Shizuoka 422, Japan. 0006-8993/87/$03.50 (~) 1987 Elsevier Science Publishers B.V. (Biomedical Division)

213 with that in normotensive Wistar-Kyoto rats (WKY). Simultaneously, the specific binding of [3H]cismethyldioxolane (CD), which may selectively label muscarinic agonist sites 25, was measured in these animals.

DM

MATERIALS AND METHODS

DL-[N-methyl-3H]Nicotine (71.2 Ci/mmol, purity: 98%), [3H]CD (55.5 Ci/mmol) and [a4C]acetyl coenzyme A (CoA, 53.5 Ci/mmol) were purchased from New England Nuclear Corp., Boston, MA. [3H]Nicotine was stored frozen in the presence of a 4-fold molar excess of mercaptoacetic acid 18. NSTX was a gift from Professor T. Kosuge of this college. Other drugs and materials were obtained from commercial sources.

Animals WKY and SHRSP were originally provided by Professor K. Okamoto of Kinki University Medical School, and they were maintained by brother-sister breeding in our laboratory. The rats were housed in the laboratory with free access to food (normal rat chow) and water. The blood pressure in conscious rats was measured by tail plethysmographic methods 19.

FH]Nicotine and [3H]CD binding assays The brains from male WKY and SHRSP were dissected, and divided into the cerebral cortex, striatum, hippocampus, thalamus, hypothalamus, midbrain, medulla oblongata and cerebellum. In several experiments, the medulla oblongata was dissected into dorsomedial, dorsolateral, ventromedial and ventrolateral regions, using a razor blade as shown in Fig. 1. The wet weight of each region was similar and the regions from 2 or 3 rats were pooled for [3H]nicotine binding assays. Tissues were homogenized by using a Polytron (setting no. 11, Kinematica, Switzerland, 30 s) in 10 vols. of 20 mM Tris-HC1 buffer, containing (in raM): NaC1 118, KC! 4.8, CaCI 2 2.5 and MgSO4 1.2, pH 7.5. The preparation of brain membranes and binding assays of [3H]nicotine were performed as previously described 18,29. Briefly, the membranes (700-900/~g protein) of rat brain were incubated with [3H]nicotine, in a total volume of 250 /A of 20 mM Tris-HCl buffer at 37 °C. After 10 min,

VM

I

!

U

lmm Fig. 1. Schematic representations of dissection of the rat medulla oblongata. The rat medulla oblongata was dissected into dorsomedial (DM), dorsolateral (DL), ventromedial (VM) and ventrolateral (VL) regions using a razor blade.

the reaction was terminated by dilution and the subsequent rapid filtration under vacuum through Whatman GF/B glass fiber filters and each filter was rinsed 4 times with 4 ml of ice-cold buffer. The glass fiber filters were presoaked in an aqueous polyethylenimine solution (0.1%). Tissue-bound radioactivity was extracted from the filters overnight in 6 ml of scintillation fluid (2 liters of toluene, 1 liter of Triton X-100, 15 g of 2,5-diphenyloxazole and 0.3 g of 1,4-bis[2-(5phenyloxazolyl)]benzene) and counted by a liquid scintillation counter (Aloka, model LSC-602). Specific binding was defined as the difference in binding determined in the absence and presence of 10 ,uM (-)-nicotine. A similar amount of specific binding was obtained by 10ktM iobeline. [3H]CD binding assays in the rat brain were performed according to the method described by Vickroy et al. 25. The brain membranes (400-500 Bg protein) were incubated with 2 nM [3H]CD, in a total volume of 1 ml of 10 mM sodium-potassium phosphate buffer (pH 7.4) at 25 °C. After 120 min, the reaction was terminated by rapid filtration under vacuum as described in [3H]nicotine binding assays. The binding displayed by 1 ¢tM atropine was defined as specific binding. The concentrations of [3H]nicotine (12 nM) and [3H]CD (2 nM) were approximately K a values for the ligand, and they were chosen to improve the ratio of specific binding to non-specific binding. The assays were conducted in duplicate. In a typical study, specific binding was more than 70% of total binding at 12 nM [3H]nicotine and 2 nM [3H]CD. Proteins were deter-

214 mined by the method of Lowry et al. 17.

TABLE I

Choline acetyltransferase assays

Blood pressure in WKY and SHRSP at 5 and 16-24 weeks of age

The activity of ChAT in the brain was determined 8'27. Frozen brains were homogenized in 20 vols. of 0.5% Triton X-100 with a Polytron for 30 s. The homogenate (5 ~tl) was incubated for 20 min at 37 °C in a reaction mixture (total volume: 30 ~tl) which contained (in mM): E D T A 1, sodium phosphate buffer 80, eserine 0.15, MgCI 2 5, choline 2, NaCI 300 and 100/~M acetyl CoA (23.3/~M [14C]acetyl-CoA). The reaction was stopped by placing the tubes into an ice bucket. After the addition of tetraphenyl boron, the tubes were shaken vigorously and centrifuged to break the emulsion; 50/~1 of the top layer was counted. Analysis o f data

The analysis of binding data was performed as previously described 26. The dissociation constant (Kd) and maximal binding sites (nmax) for specific [3H]nicotine binding were estimated by Scatchard analysis of the saturation data over a concentration range of 3-32 nM. The ability of cholinergic drugs to inhibit specific binding of [3H]nicotine and [3H]CD was estimated by IC50 values which are the molar concentrations of unlabeled drugs necessary for displacing 50% of the specific binding (determined by log probit analysis). K i values (inhibition constant) were calculated from the equation, K i = IC50/(1 + F/Ka) where the K d is the dissociation constant ([3H]nicotine: 12.5 nM, [3H]CD: 1.7 nM) for the ligand binding, and Fis the free concentration of the ligand. Statistical analysis of data was performed using a two-tailed Student's t-test. RESULTS Development o f spontaneous hypertension

The blood pressure in SHRSP at 5 weeks of age was slightly higher than that in age-matched WKY rats, but it was remarkably higher at 16-24 weeks of age (Table I). The systolic blood pressure at 5 and 16-24 weeks of age was 24 and 98 mm Hg respectively higher than that in age-matched WKY rats. Central nicotinic and muscarinic cholinoceptors

The specific binding of [3H]nicotine and [3H]CD in

The values are means + S.E.M. from 10-21 rats. Rat group

WKY SHRSP

Blood pressure (ram Hg) 5 weeks

16-24 weeks

112 -+ 3 128 _+_2*

13(~q~2 226 _+9**

Asterisks show a significant difference lrom age-matched WKY values, *P < 0.01, **P < 0.001. the brain of WKY was of high affinity and exhibited the expected pharmacological specificity of nicotinic receptors. Table II shows inhibitory effects (Ki values) of cholinergic drugs on the specific binding of [3H]nicotine and [3H]CD to the rat brain. The selective nicotinic agonists such as (-)-nicotine and lobeline showed much higher (1000-7000 times) affinity for brain [3H]nicotine binding sites than oxotremorine, a muscarinic agonist. NSTX, a specific antagonist of nicotinic receptors 9'21, was much more potent in competing with the [3H]nicotine binding sites than atropine. In contrast, binding of [3H]CD to the rat brain was displaced with approximately 20,000 times higher affinity by these muscarinic drugs than by (-)nicotine. Thus, these data suggest that specific binding of [3H]nicotine and [3H]CD in the rat brain under our assay conditions showed a pharmacological drug profile compatible with nicotinic and muscarinic cholinoceptors as previously reported 1s'25'29. The specific [3H]nicotine binding in 8 brain regions

TABLE II Inhibition of specific binding of [SH]nicotine and [3H]CD by cholinergic drugs in the whole brain of WKY

The inhibition of specificbinding of [3H]nicotine(12 nM) and [3H]CD (2 nM) by cholinergicdrugs was determined by incubating each ligand with 4-6 concentrations of these drugs. The values are means + S.E.M. from 3-5 rats; n.d., not determined. Cholinergic drugs

(-)-Nicotine Lobeline Oxotremorine Neosurugatoxin Atropine

Ki values (nM) [3H]nicotine

[3H]CD

9+ 2 67 + 4 63400 + 4800 42 + 5 137000 + 600(I

13500 + 1900 n.d. 0.6 _ 0.1 n.d. 0,7 +_0.1

215 TABLE 1II

Specific binding of [~H]nicotine and [3H]CD in brain regions of WKY and SHRSP at 16-24 weeks of age The concentrations of [3H]nicotine and [3H]CD were 12 nM and 2 nM respectively. The hypothalami and medulla oblongata from 2 or 3 rats were pooled in each experiment. The values are means -+ S.E.M. from 6 - 8 rats.

Brain regions

Cerebral cortex Hippocampus Thalamus Hypothalamus Striatum Midbrain Cerebellum Medulla oblongata

Specific [3H]nicotine binding (fmol/rng protein)

Specific [3H]CD binding (fmol/mg protein)

WKY

SHRSP

WKY

SHRSP

39.1 20.9 61.8 23.1 36.7 34.8 18.3 35.5

31.0 16.8 50.1 23.3 35.0 28.4 12.4 25.1

53.8 19.5 20.4 18.4 23.7 35.9 11.0 23.3

59.6 21.0 23.2 24.5 25.7 36.4 10.5 28.1

_+ 2.4 _+ 2.0 _+ 5.6 ± 1.5 + 2.7 _+ 1.7 + 1.4 + 3.9

± 2.2* ± 1.4 ± 4.5* ± 1.5 _+ 2.4 + 1.6" _+ 1.4" + 1.7"*

± 2.6 ± 0.8 ± 2.0 ± 1.4 _+ 2.8 _+ 2.2 ± 1.8 + 2.3

± 3.3 + 1.4 ± 2.0 + 1.6"* _+ 2.7 ± 2.5 + 1.3 ± 1.0

Asterisks show a significant difference from age-matched WKY value, *P < 0.05, **P < 0.01.

from WKY and SHRSP at 16-24 weeks of age was measured (Table III). The specific binding of [3H]nicotine in these rats was higher in the cerebral cortex, thalamus, striatum, midbrain and medulla oblongata than in the hippocampus, hypothalamus and cerebellum, suggesting a regional variation of central nicotinic cholinoceptors. Similar regional distribution of nicotinic receptor binding sites in the rat brain was previously described 29. As shown in Table III, the specific [3H]nicotine binding in SHRSP was significantly (18-32%) lower in the cerebral cortex, thala-

mus, midbrain, cerebellum and medulla oblongata than that in age-matched WKY rats. However, there was no change in the [3H]CD binding in most of brain regions of hypertensive rats, except the hypothalamus which showed a significant 33% increase.

Nicotinic cholinoceptors in the medulla oblongata Saturation isotherms for specific [3H]nicotine

TABLE IV

The Bm~x and K d values for specific [3H]nicotine binding in medulla oblongata and hypothalami of WKY and SHRSP at 5 and 16-24 weeks of age The Bmax and K d values were determined by saturation studies using 5 - 6 concentrations (3-32 nM) of [3H]nicotine. The medulla oblongata and hypothalami from 4 rats were pooled in each experiment. The values are means ± S.E.M. from 8-10 rats; n.d., not determined.

O

-.{ bWKY t

0

Specific [3H]nicotine binding, fmol/rng protein (K d, nM) Medulla oblongata WKY 25

,50

Specific [ 3H]Nicotine

75

Bound ,mo~.,,~ ~o.e,~

Fig. 2. Scatchard plot of specific [3H]nicotine binding in medulla oblongata of WKY (O) and SHRSP (Q) at 16-24 weeks of age. The abscissa is specific [3H]nicotine bound (fmol/mg protein) and the ordinate is bound/free (B/F:/A/mg protein). Each point represents the average of duplicate determinations from 8-10 determinations. The K d and Bmax a r e : WKY, 14.5 nM and 79.0 fmol/mg protein, correlation coefficient r = 0.92; SHRSP, 13.1 nM and 47.9 fmol/mg protein, r = 0.96.

SHRSP

Hypothalamus WKY SHRSP

B max,

5 weeks

16-24 weeks

72.2 (16.4 52.9 (14.8

74.8 (15.0 48.9 (13.5

± 8.4 +_ 1.2) ± 2.8** + 1.2)

36.4 (11.5 38.7 (13.0

+ + + ±

n.d. n.d.

± 8.2 _+ 1.0) + 4.7* + 1.4)

3.8 1.2) 7.1 1.8)

Asterisks show a significant difference from age-matched WKYvalue, *P
216 binding and subsequent Scatchard analysis were carried out in the medulla oblongata and hypothalamus from WKY rats and S H R S P at 5 and 16-24 weeks of age. As shown in Fig. 2 and Table IV, the Scatchard analysis revealed that maximal [3H]nicotine binding sites (Bmax) in the medulla oblongata were significantly (35%) lower in 16-24-week-old S H R S P than in age-matched WKY rats. A similar decrease in the Bm,,~ value for [3H]nicotine binding was observed in the medulla oblongata of 5-week-old SHRSP. On the other hand, there was no significant difference in the dissociation constant (K d values) between two groups at both ages. In addition, in both WKY rats and SHRSP, there was no significant difference in the K d and B .... values for [3H]nicotine binding between ages. The K a and B ..... values for hypothalamic [3H]nicotine binding in S H R S P were identical to those in W K Y rats (Table IV). The specific [3H]nicotine binding was measured in dorsomedial, dorsolateral, ventromedial and ventrolateral regions of the medulla oblongata of WKY rats and S H R S P at 16-24 weeks of age. As shown in Table V, the specific binding of [3H]nicotine (12 nM) was highest in the dorsomedial region, followed by dorsolateral, ventrolateral and ventromedial regions. As compared to WKY rats, there was a significant decrease in the [3H]nicotine binding in dorsomedial, dorsolateral and ventrolateral regions of the medulla oblongata of SHRSP, and the decrease in each region was 31%, 29% and 28% respectively.

TABLE V Specific [3Hlnicotine binding in dorsomedial, dorsolateral, ventromedial and ventrolateral regions of medulla oblongata oJ WKY and SHRSP at 16-24 weeks of age

The concentration of [3Hlnicotine was 12 nM. The regions of medulla oblongata from 2-3 rats were pooled in each experiment. The values are means _+S.E.M. from 5-8 rats. Regions

Dorsomedial Dorsolateral Ventromedial Ventrolateral

Specific [3Hlnicotine binding (fmol/mg protein) WKY

SHRSP

41.7 _+ 1. I 35.8 + 2.5 21.5 + 2.1 34.1 _+2.4

28.6 +__1.6* 25.3 + 2.0* 19.3 + 2.4 24.5 _+[).6*

Asterisks show a significant difference from age-matched WKY values, *P < 0.01.

Choline acetyltransferase activit)

The activity of CHAT, an enzyme responsible for the synthesis of acetylcholine, m the medulla oblongata of W K Y rats and S H R S P at 16-24 weeks of age was measured. It was found that there was no difference in the activity of ChAT in the medulla oblongata between these two groups (CHAT activity in W K Y vs SHRSP: 99.5 _+ 5.7 vs 95.5 +_ 1.9 nmo] ACh formed/g tissue/min, mean +_ S.E.M., n :~ ~), DISCUSSION Characteristics of central nicotinic and muscarinic cholinoceptors in spontaneous hypertension were examined by measuring specific binding of [~H]nicotine and [3H]CD. We found that as compared to normotensive age-matched WKY rats, there was a significant decrease in specific [3H]nicotine binding in the cerebral cortex, thalamus, midbrain, cerebellum and medulla oblongata of S H R S P at the established stage of hypertension, whereas there was no change in specific binding of the same ligand in other brain regions of hypertensive rats. Scatchard analysis demonstrated that a decrease in specific [3H]nicotine binding in the medulla oblongata of S H R S P might be due mainly to a lowered density of nicotinic receptors. Similar reduction of nicotinic receptor binding sites in the medulla oblongata was also observed in young SHRSP, thereby indicating that it may not be a change which occurs as a consequence of hypertension. The dorsomedial region of the medulla oblongata contains several critical nuclei for the control of cardiovascular functions. They include nucleus tractt, s solitarii (nts) and dorsal motor nucleus of the vagus nerve (dmv). In the present study, specific [~H]nicotine binding was relatively high in the dorsomedial region of medulla oblongata. This finding appears to coincide with the physiological observation by Kubo and Misu is who have shown that a nicotine-sensitive area in inducing the cardiovascular effect exists around the area postrema on the dorsal surface of the rat medulla oblongata. There was a significant decrease in specific [3H]nicotine binding in the dorsomedial region of the medulla obkmgata of S H R S P as compared to W K Y rats, suggesting a loss of nicotinic receptors in the critical brain area for control of cardiovascular functions. In contrast to the decrease in

217 the nicotinic receptor binding, specific [3H]CD binding was unaltered in most brain regions of SHRSP except the hypothalamus which showed a significant increase. The specific enhancement of hypothalamic muscarinic receptors in S H R and S H R S P was also demonstrated by using an antagonist ligand, [-SH]quinuclidinyl benzilate (QNB) I 1.27 ACh synthesis in cholinergic neurons is regulated by ChAT activity as well as by concentrations of ACh, CoA and choline at the site of synapses <22, and the activity of ChAT parallels the cholinergic neuronal activity. The medulla oblongata is known to contain significant levels of ACh, ChAT and A C h E m'24. In the present study, the ChAT activity in the medulla oblongata of SHRSP was identical to that in WKY rats, suggesting no change in the ACh turnover rate in the cholinergic neurons. Compatible with these resuits, Helke et al. m have reported no alteration in the ChAT activity and ACh content in several medullary nuclei of SHR. Additionally, in this discrete region, there was no change in the muscarinic receptor binding sites which were measured either by [3H]CD (in the present study) or by [3H]QNB~J'27. Thus, it is unlikely that a loss of nicotinic receptors in the medulla oblongata of SHRSP is a compensatory down regulation by an increase in the cholinergic neuronal activity as previously observed in the brain of chronically diisopropylfluorophosphate (DFP, an organophosphorus anticholinesterase)-treated rats 6,23'2s. Previous physiological studies suggest that a nicotinic cholinergic mechanism in the lower brainstem plays a physiological role in the central regulation of cardiovascular functions. Porsius and Van Zwieten > found that an injection of a low dose of nicotine into the vertebral artery of anesthetized cats induced hypotension and bradycardia. These workers have also

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shown that nicotine, when injected into the left vertebral artery in radioactive form, mainly accumulated in the pons-medullary area, whereas the hypothalamus contained only insignificant amounts. The hypotension and bradycardia in rats were also demonstrated by a microinjection of physostigmine and nicotine into the dorsal medial area of medulla oblongata in rats r3 15, and these effects were abolished by hexamethonium but not by atropine, injected into the same sites 14. The abolition of cardiovascular effects of nicotine by hexamethonium appears to coincide with our biochemical observation that there may be a closer pharmacological resemblance of brain nicotinic receptors to the ganglionic type of nicotinic receptors >. The injection of hexamethonium into the medullary sites in rats caused a significant rise in blood pressure 1~. Thus, it is conceivable that a nicotinic cholinergic mechanism in the medulla oblongata is tonically involved in lowering blood pressure. Taken together, an observed loss of nicotinic receptor binding sites in the SHRSP medulla oblongata may cause the dysfunction of a 'normal depressor mechanism', which consequently leads to a rise in the blood pressure. In conclusion, the present study provides the first biochemical evidence to suggest that a central nicotinic cholinergic mechanism may be inw)lved in the important pathophysiological process of spontaneous hypertension.

ACKNOWLEDGEMENT The authors wish to thank Professor T. Kosuge, Shizuoka College of Pharmaceutical Sciences, for kindly supplying neosurugatoxin.

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