Enhanced Cholinergic Activity in the Medulla Oblongata of DOCA-Salt Hypertensive and Renal Hypertensive Rats

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1 213 Enhanced Cholinergic Activity in the Medulla Oblongata of DOCA-Salt Hypertensive and Renal Hypertensive Rats Takao Kubo, Ryuji Fukumori, Midori Kobayashi, and Hiroaki Yamaguchi We previously demonstrated that cholinergic activity in the medulla oblongata is enhanced in adult spontaneously hypertensive rats (SHR), a genetically hypertensive rat model. In this study, we examined possible alterations of medulla oblongata cholinergic mechanisms in nongenetic forms of hypertension, using deoxycorticosterone acetate (DOCA)-salt hypertensive and renal hypertensive rats. At a fully developed stage of hypertension in DOCA-salt hypertensive and renal hypertensive rats, choline acetyltransferase (CAT) activity in the rostro-ventral medulla oblongata was enhanced, whereas there was no change in the activity of CAT in other parts of the medulla oblongata. There was no alteration of the medulla CAT activity in prehypertensive SHR or at an early stage of renal hypertension. Increases in blood pressure and plasma catecholamine levels induced by physostigmine (0.5 mg/kg, i.p.) were enhanced in DOCA-salt hypertensive and renal hypertensive rats. These findings suggest that cholinergic activities in the medulla oblongata are enhanced and that such activities are involved in enhancement of the sympathetic nervous system in non-genetically hypertensive rats. It seems unlikely that the altered Cholinergic activity in the rostral ventrolateral medulla of adult SHR occurs genetically. (Hypertens Res 1996; 19: ) Key Words: choline acetyltransferase, medulla oblongata, DOCA-salt hypertension, renal hypertension, plasma catecholamines Central cholinergic mechanisms have been implicated in the regulation of cardiovascular function (1). There is also evidence that responsiveness to cholinergic agents is altered in spontaneously hypertensive rats (SHR), a rat model of genetic hypertension. For example, intravenous injection of the centrally acting cholinesterase inhibitor physostigmine evokes an enhanced pressor response in SHR as compared with normotensive Wistar Kyoto rats (WKY) (2-4). The acetylcholine synthesis inhibitor hemicholinium-3, administered intracerebroventricularly, was found to lower blood pressure in SHR but not in WKY (S, 6). The rostral ventrolateral medulla (RVLM) plays a crucial role in central cardiovascular control (7-10). Anatomical and physiological studies have i ndicated that cardiovascular regulation by the RVLM is mediated through descending projections to the sympathetic preganglionic neurons in the intermediolateral cell column of the thoracic spinal cord (11-13). In the RVLM, there exist cholinergic mechanisms responsible for the regulation of the cardiovascular system (14-20). Since it has been demonstrated that physostigmine administered systemically causes a pressor response via activation of the RVLM cholinergic mechanism (21) and that this pressor response is enhanced in SHR (3), we could easily speculate that RVLM cholinergic activity in SHR is enhanced. Indeed, Lee et al. (22) reported that the pressor response to physostigmine injected into the RVLM was significantly greater in SHR than in WKY. In a previous study (23), we also confirmed the results of Lee et al. (22) and further demonstrated that ACh release in the RVLM was enhanced in SHR. In addition, CAT activity was increased only in the rostro-ventral part of the medulla, which contains the RVLM, but not in other parts of the medulla oblongata. Depressor responses produced by scopolamine injected into the RVLM were greater in SHR than in WKY. These lines of evidence suggest that RVLM cholinergic activities are enhanced in SHR and that the enhanced RVLM cholinergic activity in SHR contributes to the maintenance of hypertension in this strain. Although these findings indicate that RVLM cholinergic activity is surely enhanced in the genetically hypertensive rat, SHR, it is uncertain whether this alteration of RVLM cholinergic activity occurs genetically in SHR or whether it is a factor in the pathogenesis of hypertension, regardless of its specific cause. To determine the role of RVLM cho- From the Department of Pharmacology, Showa College of Pharmaceutical Sciences, Machida, Japan. Address for Reprints: Takao Kubo, Ph.D., Department of Pharmacology, Showa College of Pharmaceutical Machida 194, Japan. Received January 29, 1996; accepted in revised form May 22, Sciences,

2 214 Hypertens Res Vol. 19, No. 3 (1996) Table 1. Systolic Blood Pressure in DOCA-Salt Hypertensive Rats (DHR), Renal Hypertensive Rats (RHR), Prehypertensive Spontaneously Hypertensive Rats (SHR), and Their Respective Control Rats T l'/ 1 ~ TT \ linergic mechanisms in nongenetic forms of hypertension, we studied CAT activity in the RVLM area and physostigmine-induced increases in blood pressure and plasma catecholamine levels in deoxycorticosterone acetate (DOCA)-salt hypertensive and renal hypertensive rats. Materials and Methods Male Wistar rats initially weighing g were used to produce DOCA-salt hypertension and renal hypertension. Male 3-week-old SHR and agematched Wistar Kyoto rats (WKY) were obtained from Charles River Japan Inc., and they were used at 4 weeks of age. They were kept under alternative 12-h periods of dark and light and were given standard rat chow and tap water ad libitum unless otherwise noted. Induction o f Deoxycorticosterone-Salt Hypertension Rats were anesthetized with sodium pentobarbital (40 mg/kg, i.p.). The left kidney was removed, with care being taken to avoid adrenalectomy (24). Animals were given standard rat chow and a 1 % w/v NaCI solution as drinking water. DOCA was injected subcutaneously twice a week at a dose of 40 mg/kg. Sham-operated controls consisted of rats in which the left kidney was removed. The control rats were maintained on a standard diet and tap water. They were used in experiments 3 wk after surgery. One day before the experiments, the systolic blood pressure was measured indirectly in the conscious state by tail plethysmography (PE-300, Narco Bio- Systems, Inc.). Induction o f Renal Hypertension Wistar rats were anesthetized with pentobarbital and the left renal artery was occluded with a silver clip with a 0.2-mm slit (the two-kidney, one-clip Goldblatt model) (25). In the sham-operated controls, the left perirenal tissues were cleared. They were used in experiments 6 d or 3 wk after surgery. Determination o f Choline Acetyltrans f erase in Tissues Animals were sacrificed by decapitation. The brains were removed and placed on ice. The medulla oblongata was dissected into four parts (rostro-ventral, rostro-dorsal, caudo-ventral and caudo-dorsal) as described previously (23). Briefly, anterior and posterior coronal sections were made at the caudal end of the trapezoid body and the pyramidal decussation, respectively. In order to divide the medulla into two blocks, a coronal section was made at the middle between the trapezoid body and the pyramidal decussation. Further, both blocks were divided into two pieces at the midline between the dorsal surface and the ventral surface. CAT activity was measured as described by Kaneda and Nagatsu (26). Briefly, the tissues were homogenized in 25 mm ice-cold phosphate buffer solution containing 0.5% Triton X-100 (ph 7.4). The homogenate was left in an ice-water bath for 30 min and then centrifuged at 20,000 X g for 60 min at 4 C. The supernatant was mixed with a substrate containing 0.2 mm acetyl coenzyme A, 5 mm choline, 0.1 mm physostigmine, and 10 mm ethylene diaminetetracetic acid and then incubated at 37 C in a water for 20 min. After incubation, the reaction was stopped by adding 1 M perchloric acid in an icebath. CAT activity was measured by high-performance liquid chromatography with an electrochemical detector. Determination o f Plasma Catecholamines One day before the experiments, the rats were anesthetized with pentobarbital 40 mg/kg i.p., and a catheter filled with heparinized saline was inserted into the left femoral artery. The catheter was passed under the skin to emerge at the nape of the neck and was sealed with a stilette. During experiments, arterial pressure was recorded by connecting the catheter to a pressure transducer. Physostigmine sulfate (Wako Pure Chemicals, Osaka, Japan) (0.5 mg/kg) was injected intraperitoneally, and 10 min before and after the injection, blood samples (0.5 ml) were withdrawn from the arterial cannula into

3 Fig. 1. Choline acetyltransferase (CAT) activity in the rostro-ventral (RV), rostro-dorsal (RD), caudo-ventral (CV) and caudo-dorsal (CD) parts of the medulla oblongata of DOCA hypertensive (DHR) and sham-operated (Control) rats (A), and of renal hypertensive (RHR) and sham-operated (Control) rats (B). DHR, RHR and corresponding control rats were used 3 wk after surgery. The CAT activity was determined as described in Materials and Methods. Values are the mean ± SEM from 8 animals in DHR experiments and from 10 animals in RHR experiments. * p < 0. 05, compared with corresponding controls. # p<0.05, compared with DHR. syringes containing EGTA and reduced glutathion and replaced with an equal volume of physiologic saline solution. Plasma was separated in a refriger ated centrifuge (3000 X g for 10 min). A 200-p1 ali quot of the plasma supernatant of each sample wa frozen and stored. Plasma noradrenaline and adren aline were measured radioenzymatically using catecholamine [3H] research assay system (Amen sham) (27). The results are expressed as mean ± SEM. Sta tistical studies, using analysis of variance and Stu dent's t-test for individual differences, were per formed. Differences were considered significant at <0.05. Results Systolic Blood Pressure Systolic blood pressure was greater in DOCA-sal hypertensive rats than in sham-operated control rat Fig. 2. Choline acetyltransferase (CAT) activity in the rostro-ventral (RV), rostro-dorsal (RD), caudo-ventral (CV) and caudo-dorsal (CD) parts o f the medulla oblongata of 4-week-old SHR and age-matched WKY (A), and of renal hypertensive (RHR) and sham-operated (Control) rats 6 d after surgery (B). Values are the mean ± SEM from 8-10 animals in RHR experiments and from 10 animals in SHR experiments. 3 wk after surgery (Table 1). In renal hypertensive rats, the systolic blood pressure rose in a time-dependent manner, 5 d and 3 wk after surgery. There was no difference in systolic blood pressure between 4-week-old SHR and age-matched WKY. CAT Activity in the Medulla Oblongata Figure 1 shows the activity of CAT in the rostroventral, rostro-dorsal, caudo-ventral, and caudodorsal parts of the medulla oblongata in DOCA-salt hypertensive and renal hypertensive rats. In DOCA-salt hypertensive rats 3 wk after surgery as compared with control rats, a significant activation of CAT was detected in the rostro-ventral part of the medulla, whereas no significant change was detected in the other three parts of the medulla oblongata (Fig. 1A). Also in renal hypertensive rats 3 wk after surgery as compared with control rats, increased CAT activity was detected in the rostroventral part of the medulla (Fig. 1B). In the other three parts of the medulla oblongata, there was no significant change in the activity of CAT in the renal hypertensive rats as compared with control rats. CAT activities in the rostro-ventral medulla were

4 216 Hypertens Res Vol. 19, No. 3 (1996) Fig. 3. A, B: plasma noradrenaline and adrenaline levels in DOCA-salt hypertensive (DHR) and sham-operated (Control) rats (A), and in renal hypertensive (RHR) and sham-operated (Control) rats (B) 10 min before and after injection of physostigmine (0.5 mg/kg, i. p.). Basal mean BP was 173 ± 5 mmhg and 111 ± 2 mmhg in DHR and sham-operated control rats, respectively, and 170±5 mmhg and 113±2 mmhg in RHR and sham-operated control rats, respectively. DHR, RHR and corresponding control rats were used 3 wk after surgery. Values are the mean ± SEM from 7 animals in DHR experiments and 8 animals in RHR experiments. * p < 0. 05, compared with before physostigmine. tp < 0. 05, hypertensive rats vs. control rats. p<0.05, compared with RHR. greater in renal hypertensive and sham-operated control rats than in DOCA-salt hypertensive and sham-operated control rats, but the difference between hypertensive and control rats in CAT activities in this region tended to be greater in DOCAsalt hypertensive rats (0.46 nmol/mg protein/min) than that in renal hypertensive rats (0.40 nmol/mg protein/mm). Next, we determined CAT activity in prehypertensive SHR in order to examine whether SHR have enhanced CAT activity before the onset of hypertension. However, no significant difference between 4-week-old SHR and age-matched WKY in CAT activity was detected in any part of the medulla oblongata (Fig. 2A). We then measured CAT activity at an early stage of hypertension in renal hypertensive rats to study whether the enhanced CAT activity in the medulla oblongata plays a role in the initiation of experimental hypertension. As shown in Fig. 2B, no significant change was detected in the rostro-ventral part of the medulla in renal hypertensive rats 6 d after surgery, although they were already hypertensive (Table 1). Plasma Catecholamine Levels In DOCA-salt hypertensive rats, plasma noradrenaline and adrenaline levels before physostigmine were greater than those in sham-operated control rats (Fig. 3A). Ten minute after physostigmine

5 Kubo et al: Central Acetylcholine and Hypertension 217 levels after physostigmine were 0.61 ± 0.06 in DOCA-salt hypertensive rats and 0.67 ± 0.08 in renal hypertensive rats. Blood Pressure Responses Intraperitoneal injection of physostigmine produced an increase in blood pressure. The increase in blood pressure induced by physostigmine (0.5 mg/kg, i.p.) was significantly greater in both DOCA-salt hypertensive and renal hypertensive rats 3 wk after surgery than in the respective controls (Fig. 4A, B). In contrast, there was no difference in the physostigmine (0.5 mg/kg, i.p.)-induced increase in blood pressure either between 4-week-old SHR and agematched WKY or between renal hypertensive and control rats 6 d after surgery (Fig. 4C, D). Fig. 4. Pressor responses (zibp) to physostigmine (0.5 mg/kg, i.p.) in DHR and sham-operated (Control) rats 3 wk after surgery (A), RHR and sham-operated (Control) rats 3 wk (3W) after surgery (B), 4-week-old SHR and age-matched WKY (C), and RHR and sham-operated (Control) rats 6 d (6D) after surgery (D). Values are the mean ± SEM from 6-8 animals. tp < 0.05, hypertensive rats vs. control rats. treatment (0.5 mg/kg, i.p.), plasma catecholamine levels were markedly increased in both hypertensive and control rats. The ratios of plasma levels after to before physostigmine in DOCA-salt hypertensive and control rats were 6.7 ± 1.2 (n = 7) and 3.0 ± 0.4 (n = 7), respectively (p < 0.05), for noradrenaline, and 15.3±3.1 (n = 7) and 7.2±1.1 (n=7), respectively, (p<0.05), for adrenaline. In renal hypertensive rats 3 wk after surgery, plasma catecholamine levels before physostigmine tended to be slightly but not significantly higher than in sham-operated control rats (Fig. 3B). Ten minutes after physostigmine (0.5 mg/kg, i.p. ), plasma noradrenaline and adrenaline levels were markedly increased in renal hypertensive and control animals. The ratios of plasma levels after to before physostigmine in renal hypertensive and control rats were 7.0 ± 1.5 (n = 8) and 3.1 ± 0.5 (n = 8), respectively, (p < 0.05), for noradrenaline, and 10.0 ± 1.5 (n = 8) and 5.6 ± 1.1 (n = 8), respectively, for adrenaline. Plasma noradrenaline and adrenaline levels after physostigmine were greater in DOCAsalt hypertensive rats than in renal hypertensive rats. Ratios of plasma noradrenaline to adrenaline Discussion In the present study, the activity of CAT, an enzyme responsible for the synthesis of ACh, was increased in the medulla oblongata of DOCA-salt hypertensive and renal hypertensive rats. Since the activity of CAT increases in parallel with an increase in impulse flow in cholinergic nerves (28), the results of the present study suggest that cholinergic activities are enhanced in the medulla oblongata in both forms of hypertension in rats. CAT activity was increased only in the rostro-ventral part, which contains the RVLM, but not in other three parts of the medulla oblongata in DOCA-salt hypertensive and renal hypertensive rats, suggesting that cholinergic activity is specifically enhanced in the RVLM or an area surrounding the RVLM. Cholinergic mechanisms in the rostro-ventral medulla are responsible for blood pressure regulation (20, 21). Peripheral administration of physostigmine, a centrally acting cholinesterase inhibitor, causes pressor responses via activation of rostroventral cholinergic mechanisms, since microinjections of tetrodotoxin, a sodium channel blocker, lidocaine, a local anesthetic, and scopolamine into the RVLM abolish the pressor action of physostigmine (21). The pressor response to peripheral physostigmine is induced via activation of the sympatho-adrenal medulla system (29). Thus, it could be speculated that if cholinergic activities are increased in the rostro-ventral medulla of DOCAsalt hypertensive and renal hypertensive rats, sympathetic activation and thus pressor responses induced by peripheral administration of physostigmine would be enhanced in both hypertensive animals. In the present study, indeed, the ratio of plasma catecholamine levels after to before intraperitoneal physostigmine and the pressor response to physostigmine were greater in DOCA-salt hypertensive and renal hypertensive rats than in their respective control rats. These findings are compatible with the notion that cholinergic activities are increased in the rostro-ventral medulla, especially in the RVLM, and that this increased activity is involved in enhancement of the sympathetic nervous system in both types of experimentally hypertensive rats.

6 218 Hypertens Res Vol. 19, No. 3 (1996) In prehypertensive SHR, a genetically hypertensive rat model, as compared with age-matched WKY, there was no difference in CAT activity in the rostro-ventral part of the medulla oblongata. In addition, as stated above, the CAT activity in the medulla oblongata was enhanced in the non-genetically hypertensive rat models, i. e., DOCA-salt hypertensive rats and renal hypertensive rats. Thus, it seems unlikely that the altered RVLM cholinergic activity previously observed in adult SHR (23) is genetically determined. Although the enhanced cholinergic activity in the medulla oblongata of hypertensive rats seems to occur secondarily to sustained hypertension, it may next play an important role in the maintenance of hypertension in experimentally hypertensive rats. In the present study, in fact, the peripheral administration of the cholinesterase inhibitor physostigmine caused an enhanced pressor response and a markedly enhanced increase in plasma catecholamine levels in both DOCA-salt hypertensive and renal hypertensive rats. In addition, we previously demonstrated that in adult SHR, cholinergic activities were enhanced in the rostro-ventral medulla and that the pressor response to physostigmine injected intraperitoneally was enhanced (23). These findings suggest that the enhancement of cholinergic activity in the medulla oblongata may be an expression of a common mechanism that is integral to the pathogenesis of hypertension regardless of its cause. Similarly, Giuliano and Brezenoff (6) have shown that intracerebroventricular injection of hemicholinium-3, a drug inhibiting the synthesis of ACh, causes an enhanced depressor response in SHR, DOCA-salt hypertensive rats, and Grollman renal hypertensive rats. In the present study, no change was found in the medulla oblongata CAT activity at an early stage of hypertension in renal hypertensive rats or at the prehypertensive stage in SHR. Consistently with this finding, no difference was found in the intraperitoneally injected physostigmine-induced increase in blood pressure between 4-week-old SHR and age-matched WKY or between renal hypertensive rats and control rats 6 d after surgery. Thus, we obtained no evidence that altered cholinergic activity in the medulla oblongata is involved in the initiation of hypertension in experimentally hypertensive rats. In the present study, the ratios of noradrenaline to adrenaline in plasma after intraperitoneal physostigmine were 0.61 in DOCA-salt hypertensive rats and 0.67 in renal hypertensive rats. The ratios were similar to that (0.66) in rats undergoing electrical stimulation of the RVLM (9). These results are compatible with studies (20, 21) showing that peripheral administration of physostigmine produces pressor responses via activation of RVLM cholinergic mechanisms. It is interesting that although the peripheral renin-angiotensin system is enhanced in the two-kidney, one-clip Goldblatt model and inhibited in DOCA-salt hypertensive rats (30, 31), the medulla cholinergic activity is enhanced in both of these hypertensive rats. In the present study, plasma noradrenaline and adrenaline levels after physostigmine were greater in DOCA-salt hypertensive rats than in renal hypertensive rats. Consistent with this finding, the difference between hypertensive and control rats in rostro-ventral medulla CAT activity tended to be greater in DOCA-salt hypertensive rats than in renal hypertensive rats. However, the rostro-ventral medulla CAT activity in DOCA-salt hypertensive rats was lower than that in renal hypertensive rats. These results suggest that rostro-ventral medulla CAT activity itself does not necessarily parallel sympathetic nervous activity. It is possible that CAT activities not only responsible for but also not responsible for blood pressure regulation in this brainstem region are changed in DOCA-salt hypertensive, renal hypertensive rats, or both. It has been demonstrated that cholinergic inputs to the RVLM area arise from cholinergic neurons intrinsic to the RVLM or from other cholinergic neurons in the laterodorsal tegmental nucleus, the nucleus tractus solitarii, the spinal cord, and the nucleus ambiguus (17). Mechanisms for the enhancement of the rostroventral medulla cholinergic activity in experimentally hypertensive rats remain to be settled. In conclusion, the present study demonstrates that cholinergic activity in the medulla oblongata is enhanced and that such activity is involved in enhancement of the sympathetic nervous system in DOCA-salt hypertensive rats and renal hypertensive rats, two non-genetic models of hypertension. It seems unlikely that the altered RVLM cholinergic activity in adult SHR is genetic in origin. This enhanced cholinergic activity may play a role in the maintenance of hypertension in experimentally hypertensive rats. Acknowledgements This study was supported by a grant (No. scientific research from the Department Sciences and Culture, Japan. References ) for of Education, 1. Brezenoff HE, Giuliano R: Cardiovascular control by cholinergic mechanisms in the central nervous system. Annu Rev Pharmacol Toxicol 1982; 22: Buccafusco JJ, Spector S: Role of central cholinergic neurons in experimental hypertension. 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