Yohimbine induces sympathetically mediated renin release in the conscious rat

European Journal of Pharmacology, 97 (1984) 247-255

247

Elsevier

YOHIMBINE INDUCES SYMPATHETICALLY MEDIATED RENIN RELEASE IN THE CONSCIOUS RAT * SANDRA L. PFISTER and T. KENT KEETON ** Division of Clinical Pharmacology, Department of Pharmacology, The University of Texas Health Science Center, San Antonio, Texas, 78284, U.S.A.

Received 30 August 1983, accepted 25 October 1983

S.L. PFISTER and T.K. KEETON, Yohimbine induces sympathetically mediated renin release in the conscious rat, European J. Pharmacol. 97 (1984) 247-255. The preferential aE-adrenergic antagonist yohimbine (4 m g / k g s.c.) caused a time-related increase in serum renin activity and heart rate in conscious Sprague-Dawley rats. Although mean arterial pressure was not decreased significantly over the 2-h period, heart rate was elevated significantly at 15 and 30 min post-injection. In contrast, serum renin activity remained elevated for up to 2 h with a 9-fold and 9.7-fold increase occurring at 30 and 60 min post-injection, respectively. Yohimbine (0.3, 1, 3 and 10 m g / k g s.c.) elicited a dose-related increase in serum renin activity and heart rate (30 min post-injection). The 1 m g / k g dose of yohimbine did not alter blood pressure whereas the 3 m g / k g dose caused a variable decrease in mean arterial pressure. The highest dose of yohimbine (10 mg/kg) significantly lowered blood pressure. The fl-adrenergic receptor antagonist propranolol (1.5 m g / k g s.c.), blocked the renin release and tachycardia caused by yohimbine (1 and 3 m g / k g s.c.), and the ganglionic blocking agent chlorisondamine partially inhibited the renin release elicited by 3 m g / k g (s.c.) of yohimbine. The prostaglandin synthetase inhibitors indomethacin (5 m g / k g s.c.) and meclofenamate (5 m g / k g s.c.) impaired the ability of yohimbine (3 mg/kg) to elevate SRA but did not alter the hemodynamic effects of yohimbine. Thus, the increase in renin release caused by yohimbine appears to be mediated by the sympathetic nervous system. Because the smaller doses of yohimbine increase renin release in the absence of a decrease in mean arterial pressure, it is unlikely that yohimbine stimulates renin release by baroreflex-mediated activation of the renal sympathetic nerves. We feel that yohimbine increases the rate of release of norepinephrine onto the granular juxtaglomerular cells of the kidney by blocking prejunctional a2-adrenergic receptors on the renal sympathetic nerves a n d / o r a centrally mediated activation of the peripheral sympathetic nervous system. a2-Adrenergic receptors Mean arterial pressure

Serum renin activity Propranolol

Yohimbine

I. Introduction T h e renal s y m p a t h e t i c nerves p l a y a n i m p o r t a n t role in the c o n t r o l of renin release in the conscious r a t ( K e e t o n a n d C a m p b e l l , 1980). M a n y * A portion of the results was presented at the 65th Annual Meeting of the Federation of American Societies of Experimental Biology held April 12-17, 1981, in Atlanta, Georgia, U.S.A. ** To whom all correspondence should be addressed: Department of Pharmacology, The University of Texas Health Science Center, San Antonio, Texas 78284, U.S.A. 0014-2999/84/$03.00 © 1984 Elsevier Science Publishers B.V.

Chlorisondamine

Indomethacin

v a s o d e p r e s s o r drugs elicit renin release in the conscious rat a n d h u m a n b y reflex activation, via the c a r o t i d baroreflex, of the renal s y m p a t h e t i c nerves ( K e e t o n a n d C a m p b e l l , 1980). F o r example, the renin release caused b y the p e r i p h e r a l v a s o d i l a t o r s h y d r a l a z i n e a n d m i n o x i d i l is b l o c k e d b y the fla d r e n e r g i c r e c e p t o r a n t a g o n i s t p r o p r a n o l o l even t h o u g h p r o p r a n o l o l p o t e n t i a t e s the v a s o d e p r e s s o r effects of these v a s o d i l a t o r s (Pettinger et al., 1973; Pettinger a n d K e e t o n , 1975). In a d d i t i o n , the decrease in b l o o d p r e s s u r e caused b y the non-selective a - a d r e n e r g i c r e c e p t o r a n t a g o n i s t s p h e n t o l a -

248 mine and phenoxybenzamine results in a reflexlymediated increase in the activity of the renal sympathetic nerves which in turn stimulates renin release. Propranolol blocks the renin release caused by phentolamine and phenoxybenzamine (Keeton and Pettinger, 1979; Loeffler et al., 1972). These observations support the belief that the renin release caused by many vasodepressor drugs is mediated by a reflexly induced increase in the rate of release of norepinephrine onto/3-adrenergic receptors located on the granular juxtaglomerular cells of the kidney. In addition to postjunctional c~-adrenergic receptors located on effector cells, prejunctional a 2adrenergic receptors, which modulate the neuronal release of norepinephrine, have been identified (Starke and Endo, 1976; Doxey et al., 1977). Stimulation of prejunctional a2-adrenergic receptors by neuronally-released norepinephrine attenuates the further release of norepinephrine, especially at low frequencies of nerve stimulation. Conversely, blockade of these prejunctional c~2adrenergic receptors results in a greater release of norepinephrine with each nerve impulse. Yohimbine, unlike phentolamine and phenoxybenzamine, appears to act as a preferential c~2-adrenergic receptor antagonist (Doxey et al., 1977). The intravenous administration of yohimbine to conscious rats increases heart rate and plasma norepinephrine concentration even though blood pressure remains unchanged (Graham et al., 1980). These observations suggest that prejunctional a2adrenergic receptors in the rat are tonically stimulated by norepinephrine in vivo. Accordingly, the studies reported here were designed to determine the role of prejunctional a 2adrenergic receptors in the control of renin release by the renal sympathetic nerves. That is, we wanted to determine if yohimbine, by blocking prejunctional c~2-adrenergic receptors, would induce sympathetically-mediated renin release in the conscious rat in the absence of a decrease in blood pressure and the attendant reflex activation of the renal sympathetic nerves. We found that yohimbine caused a time- and dose-related increase in renin release which was blocked or attenuated by pretreatment with propranolol, chlorisondamine, indomethacin or

meclofenamate. The ability of yohimbine to stimulate renin release was not dependent on its ability to lower blood pressure. These data indicate that yohimbine induced sympatheticallymediated renin release in the conscious rat, but the actual site of action of yohimbine cannot be stated with certainty.

2. Materials and methods

2.1. Animals One hundred ninety-eight male Sprague Dawley rats (Simonsen Laboratories) weighing 260-300 g were housed in individual cages and exposed to light by an automated system from 7 : 0 0 a.m. to 7 : 00 p.m. The animals ingested rat chow containing 152 mEq of sodium per kg and tap water, ad libitum. 2.2. Drugs Yohimbine HC1 (Sigma), propranolol HCI (Ayerst) and chlorisondamine HC1 (Ciba-Geigy) were administered subcutaneously in distilled water. Indomethacin (Sigma) and meclofenamate sodium (Parke-Davis) were administered subcutaneously in an olive oil suspension. All drugs were injected in a volume of 0.5 ml. An equal volume of distilled water a n d / o r olive oil was administered as a placebo to the control rats. All drug dosages are given as mg free base or acid per kg. 2.3. Measurement of serum renin activity At specific times after injection, the rats were decapitated with a small animal guillotine (Harvards Instruments). Aortic blood was collected in siliconized glass tubes on ice during the first 4 s after decapitation. After the blood was allowed to clot, serum was collected by centrifugation at 1200 × g for 20 min at 4°C. Serum was stored at - 2 0 ° C prior to assay. The serum renin activity (SRA) of each sample was measured in triplicate by radioimmunoassay (Poulsen and Jorgensen, 1974), and the average of these three determinations was used to calculate

249

4~o]

the mean _+S.E.M. of the values of SRA for each group of six rats receiving treatment with a particular drug or combination of drugs.

410 1

3901 3701

2.4. Hemodynamic measurements Hemodynamic measurements were made in a separate group of rats since the experiments involving the measurement of SRA were terminated by decapitating the rats. Mean arterial pressure (MAP) and heart rate were measured in conscious freely moving rats fitted with chronic indwelling catheters placed in the descending aorta via a femoral artery. A one-day recovery period was allowed prior to hemodynamic studies. Hemodynamic measurements were made with a Grass Model 7 polygraph recorder and Century CP-01 pressure transducers. In order to compare the hemodynamic effects of each drug or combination of drugs, the same group of animals received these drugs in a random fashion with one-day washout periods between each treatment.

2.5. Statistical analysis All data are expressed as mean +S.E.M. with six observations per group. One-way analysis of variance combined with Student-Newman-Keuls multiple range test was used to compare the results of the experiments in which SRA was measured and any other experiments in which measurements were made in separate groups of animals. When the same group of rats was used to determine the hemodynamic effects of several drugs or combination of drugs, statistical analysis was performed by two-way analysis of variance combined with Student-Neuman-Keuls multiple range test.

5501

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Minutes Post - Injection Fig. 1. The chronology of the increase in SRA and the changes in blood pressure and heart rate caused by yohimbine (4 m g / k g s.c.). Two separate groups of rats were used, one group (n = 30) for the measurement of SRA and one group (n = 6) for the measurement of MAP and heart rate. The control rats (zero time) in the studies of renin release were decapitated 30 min after sham injection. The number in the parenthesis is the ratio between the treatment and control values for SRA. Each point is the mean _+S.E.M. for 6 rats. In the hemodynamic studies, control measurements of MAP and heart rat were made for 1 h prior to the injection of yohimbine at zero time. Mean arterial pressure and heart rate were measured over the subsequent 120 rain. Each point is the mean +_S.E.M. for the determination of MAP and heart rate in 6 rats. Statistical comparisons are between each treatment value and the control value. * P < 0.05; ** P < 0.01.

3. Results

3.1. Chronology Yohimbine (4 m g / k g ) caused a rapid and sustained increase in SRA which peaked at 60 min and remained elevated above the control value for up to 2 h post-injection (fig. 1). In the accompanying hemodynamic studies, this same dose of

yohimbine had no effect on MAP, but a significant tachycardia was noted at 15 and 30 min post-injection (fig. 1). In contrast, the sham treatment of the same animals with distilled water caused only a transient elevation of MAP and heart rate which had dissipated within 5 min (data not shown).

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Fig. 2. The dose-response relationship of yohimbine-induced renin release. Blood samples for the determination of SRA were collected at 30 min post-injection. The number in parentheses is the ratio between the treatment and the control values for SRA. Each point is the mean _+S.E.M. for 6 rats. Statistical comparisons are between each value a n d the control value. * P < 0.01.

3.2. Dose-response curoe

Yohimbine elicited a dose-related increase in renin release when SRA was measured 30 win after injection (fig. 2). In hemodynamic studies in a separate group of rats, yohimbine caused a dose-related increase in heart rate with a significant increase in heart rate seen with the 3 and 10 m g / k g doses (table 1). In this experiment, only the 10 m g / k g dose of yohimbine caused a significant decrease ( - 2 0 % ) in MAP (table 1). 3.3. Propranolol

Propranolol and other fl-adrenergic receptor antagonists inhibit sympathetically mediated renin

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Propranolol(mg/kg) Yohimbine (mg/kg)

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Fig. 3. Inhibition of yohimbine-induced renin release by propranolol. Blood samples for the determination of SRA were collected 30 min after yohimbine and 40 min after propranolol. Propranolol was given 10 min prior to yohimbine. The number in the parenthesis is either the ratio between the treatment and control values for SRA or the percentage decrease in the treatment value relative to the control value for SRA. Each column is the mean 5: S.E.M. for 6 rats. Statistical P values are given in the middle of the line connecting the two groups compared. NS, not statistically significant.

release (Keeton and Campbell, 1980). Pretreatment with propranolol (1.5 mg/kg) completely blocked the 2.3-fold increase in SRA caused by 1 m g / k g of yohimbine (fig. 3). In fact, combined treatment with propranolol and yohimbine actually resulted in a 35% reduction in SRA below the values seen in the control rats (fig. 3). Propranolol alone suppressed basal SRA by 52%. Propranolol also impaired a major portion of the 8.3-fold in-

TABLE 1 Dose-response of yohimbine-induced vasodepression and tachycardia in conscious rats. All values are the mean 5: S.E.M. for 6 rats. All of the values were obtained from the same 6 animals with the doses of yohimbine given in a random fashion with one-day wash-out periods interposed between each dose. All statistical comparisons are between the treatment (30 min post-injection) and the control values for each individual dose of yohimbine. * P < 0.05; ** P < 0.01. Doses of yohimbine

Mean arterial pressure (mmHg)

Heart rate (b.p.m.)

( m g / k g s.c.)

Control

Treatment

Control

Treatment

Sham injection 0.3 1.0 3.0 10.0

122 5:5 124 -I-4 1215:5 1215:6 122 5:5

122 + 6 123 + 6 112+7 1165:6 102 5:5 *

340 5 : 9 338 + 13 3385:8 3375:10 326 + 11

332 + 22 350 + 13 366+19 388+ 9 * 426 + 18 **

251 - - N S Ol

TABLE 2

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Impairment of yohimbine-induced renin release by indomethacin. Blood samples for the determination of SRA were collected 30 min after yohimbine (3 m g / k g s.c.) and 120 min after indomethacin (5 m g / k g s.c.). Indomethacin was given 90 min before yohimbine. The number in parenthesis is either the ratio between the treatment and control values for SRA or the percentage decrease in the treatment value relative to the control value for SRA. Each value in the mean + S.E.M. for 6 rats.

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Treatment

Serum renin activity (ng A I / m l per h)

Control (water and olive oil) Indomethacin Yohimbine Indomethacin, then yohimbine

5.0+ 0.4" 2.8 +_ 0.4 ( - 44%) b 57.5 ± 10.9 (11.4 x ) ~ 22.0 _+ 2.4 (4.4 × ) d

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Propronolol (mg/kg) Yohimbine (mg/kg)

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Fig. 4. Inhibition of yohimbine-induced renin release by propranolol. Blood samples for the determination of SRA were collected 30 min after yohimbine and 40 min after propranolol. Propranolol was given 10 min prior to yohimbine. The number in the parenthesis is either the ratio between the treatment and control values for SRA or the percentage decrease in the treatment value relative to the control value for SRA. Each column is the mean + S.E.M. for 6 rats. Statistical P values are given in the middle of the line connecting the two groups compared. NS, not statistically significant.

crease in SRA caused by a larger dose of yohimbine (3 mg/kg) (fig. 4). After pretreatment with propranolol, this dose of yohimbine caused only a 1.9-fold increase in SRA relative to the control values. In the accompanying hemodynamic studies, yohimbine (1 and 3 m g / k g ) caused a slight insignificant increase in heart rate, but had no significant effect on MAP (data not shown). After combined treatment with propranolol and yohimbine, neither MAP or heart rate were changed relative to the control values (data not shown).

fects of indomethacin and meclofenamate on yohimbine-induced renin release. Indomethacin (5 m g / k g ) suppressed SRA by 44% relative to the control values whereas yohimbine (3 mg/kg) elevated SRA from 5.0 to 57.5 ng A I / m l per h (table 2). Although yohimbine elicited an 11.4-fold increase in SRA in the absence of indomethacin, only a 4.4-fold rise in SRA was seen when yohimbine was given after indomethacin. Thus, in-

TABLE 3 Attenuation of yohimbine-induced renin release by meclofenamate. Blood samples for the determination of SRA were collected 30 min after yohimbine (3 m g / k g s.c.) and 120 min after meclofenamate (5 m g / k g s.c.). Meclofenamate was given 90 min before yohimbine. The number in parenthesis is either the ratio between the treatment and control value for SRA or the percentage decrease in the treatment value relative to the control value for SRA. Each value is the mean + S.E.M. for 6 rats. Treatment

Serum renin activity (ng A I / m l per h)

Control (water and olive oil) Meclofenamate Yohimbine Meclofenamate, then yohimbine

4.8 ± 0.6" 3.5 ± 0.8 ( - 28%) b 47.4 _+5.4 (9.9 × ) ~ 30.3 _+4.1 (6.3 × ) d

3.4. Indomethacin and meclofenamate Because Campbell et al. (1979) found that many types of sympathetically mediated renin release in the conscious rat are blocked by inhibitors of prostaglandin synthetase, we determined the ef-

a-c p = 0.01. a-d p = 0.01. b-c p = 0.01. b-a p = 0.01. c~ p = 0.01.

252

vent yohimbine-induced renin release. Chlorisondamine (10 mg/kg) alone increased SRA by 37% whereas yohimbine (3 mg/kg) elevated SRA by 6.6-fold relative to the control value (table 4). Pretreatment with chlorisondamine followed by yohimbine resulted in a 2.8-fold increase in SRA. Therefore, chlorisondamine prevented 67% of yohimbine-induced renin release. In a separate group of rats, yohimbine (3 mg/kg) lowered MAP by 14% (P < 0.01) and increased heart rate by 11% (table 5). Chlorisondamine (10 mg/kg) lowered MAP by 28% in the absence of yohimbine and 34% in the presence of yohimbine. Chlorisondamine prevented the increase in heart rate caused by yohimbine (table 5).

TABLE 4 Impairment of yohimbine-induced renin release by chlorisondamine. Blood samples for the determination of SRA were collected 30 min after yohimbine (3 m g / k g s.c.) and 40 min after chlorisondamine (10 m g / k g s.c.). Chlorisondamine was given 10 min before yohimbine. The number in parenthesis is the ratio between the treatment and control values. Each value is the mean + S.E.M. for 6 rats. Treatment

Serum renin activity (ng A i / m l per h)

Control (water) Chlorisondamine Yohimbine Chlorisondamine, then yohimbine

6.9±0.9 a 9.4±0.9(0.4×) b 45.6±5.9(6.6×) c 19.6±2.7(2.8×) d

~-c p = 0.01. ~-d p = 0.05. b-¢ p = 0.01. b-d p = 0.05. c-d p = 0.01.

4. Discussion

domethacin impaired 68% of yohimbine-induced renin release. In like fashion, meclofenamate (5 mg/kg) attenuated 40% of the increase in SRA caused by yohimbine (3 mg/kg) (table 3). In a separate group of rats, neither indomethacin alone nor indomethacin plus yohimbine had a significant effect on MAP or heart rate (data not shown). 3.5. Chlorisondamine

Because yohimbine appeared to elicit renin release via the sympathetic nervous system (figs. 3 and 4), it was reasoned that prior blockade of sympathetic ganglionic transmission would pre-

Although yohimbine has been used extensively as a preferential c~2-adrenergic receptor antagonist in studies conducted in anesthetized animals or in vitro, its hemodynamic and neuroendocrine effects in conscious animals have not been studied in detail. Although no other researchers have studied the effects of preferential a2-adrenergic antagonists on renin release, the effects of yohimbine on MAP and heart rate in conscious rats have been reported (Lang et al., 1975; Gomes et al., 1980; Rockhold and Gross, 1981). Taken collectively, these data (Lang et al., 1975; Gomes et al., 1980; Rockhold and Gross, 1981) indicate that: (1)

TABLE 5 The hemodynamic effects of treatment with chlorisondamine (10 m g / k g s.c.) a n d / o r yohimbine (3 m g / k g s.c.) in conscious rats. All the values are the mean + S.E.M. for 6 rats. All of the values were obtained from the same 6 animals with the various treatment given in a random fashion with one-day wash-out periods interposed between each treatment. The treatment measurements were made 30 min after yohimbine and 40 rain after chlorisondamine. Chlorisondamine was given 10 min prior to the administration of yohimbine. All statistical comparisons are between the control values and the treatment values on that day. * P < 0.01. Treatment

Control (water) Yohimbine Chlorisondamine Chlorisondamine, then yohimbine

Mean arterial pressure (mmHg)

Heart rate (b.p.m.)

Control

Treatment

Control

Treatment

107 + 102 + 106 + 119 +

100 + 88 + 76 + 78 +

372 +_24 371 + 23 346,+ 11 356 + 18

398 + 40 413 + 23 322 + 16 324 ___16

9 5 5 4

9 5* 7* 5*

253 yohimbine given i.v. causes a dose-related decrease in MAP and increase in heart rate of short duration (less than 5 min), (2) the intracerebroventricular injection of yohimbine increases blood pressure and (3) the hemodynamic effects of yohimbine are altered by anesthesia. When these results are compared to our data, it is apparent that the magnitude of the changes in MAP and heart rate seen after the subcutaneous administration of yohimbine, although of longer duration, is smaller than the transient changes observed after i.v. injection. Both ~1- and a2-adrenergic receptors have been identified postjunctionally in the vasculature of the rat (Timmermans and Van Zwieten, 1981), and presumably the decrease in MAP caused by yohimbine results from the fact that either these post-junctional ct2-adrenergic receptors receive tonic sympathetic stimulation or yohimbine, at the higher doses given here, looses its selectively for vascular ,~2-adrenergic receptors and begins to block post-junctional al-adrenergic receptors. The data presented here indicate that the preferential ~2-adrenergic receptor antagonist yohimbine, in doses that have little effect on MAP, increases renin release in the conscious rat via activation of the sympathetic nervous system. This conclusion is supported by several observations. First, propranolol, a B-adrenergic receptor antagonist, prevented or attenuated the increase in SRA caused by yohimbine. Second, like many other forms of sympathetically mediated renin release in the conscious rat (Campbell et al., 1979), the rise in SRA caused in yohimbine was impaired by two inhibitors of prostaglandin synthetase, indomethacin and meclofenemate. Last, when sympathetic ganglionic transmission was blocked with chlorisondamine, the ability of yohimbine to increase renin release was greatly attenuated. Even though our data indicate that the renin release caused by yohimbine is sympathetically mediated, the exact site(s) of action of yohimbine remains to be determined. At least five sites of action can be considered based on the observations of previous investigators. First, yohimbine, by blocking prejunctional negative-feedback ~2adrenergic receptors, may augment nerve-stimulated norepinephrine release onto the fl-adrenergic receptors located on the granular juxtaglomerular

cells of the kidney. This site of action is consistent with the findings of Graham et al. (1980) who noted that yohimbine (4 m g / k g i.v.) elicited a tachycardia and a 3.6-fold increase in plasma norepinephrine concentration in conscious rats even though blood pressure was not changed. We also observed that yohimbine caused a mild tachycardia which was prevented by prior treatment with propranolol or chlorisondamine. Both the tachycardia and renin release caused by yohimbine are consistent with the known ability of this drug to amplify noradrenergic neurotransmission by blocking prejunctional a2-adrenergic receptors in the rat heart (Docherty and McGrath, 1979) and the existence of functional prejunctional a-adrenergic receptors on the renal sympathetic nerves of the rat (Ekas et al., 1982). Next, the slight decrease in MAP caused by yohimbine (1 and 3 mg/kg) may cause reflex activation, via the carotid baroreflex, of the renal sympathetic nerves. For example, 1 m g / k g (s.c.) of yohimbine lowered MAP by 7% and increased heart rate by 8% (table 1), and 3 m g / k g (s.c.) of yohimbine decreased MAP by 4 to 14% and elevated heart rate by 11 to 15% (tables 1 and 5). In contrast to these minor and usually statistically insignificant changes in MAP and heart rate, the 1 and 3 m g / k g doses of yohimbine increased SRA by 2- to 7-fold (figs. 2 and 3) and 8- to 11-fold (figs. 2 and 4, tables 2-4), respectively, By comparison, Graham and Pettinger, 1979) found that 0.3 m g / k g (i.v.) of phentolamine, a non-selective ~-adrenergic antagonist, increased SRA by only 1.2-fold when MAP was reduced by 7% and heart rate increased by 7% in conscious rats. At a dose of 1 m g / k g (i.v.), phentolamine lowered MAP by 17% and elevated heart rate by 27%, and yet SRA rose only 2.5-fold. Thus, with comparable vasodepressor responses, the preferential c~2-antagonist yohimbine caused a greater increase in renin release than did the nonselective alpha-antagonist phentolamine. Therefore, we feel it is unlikely that the smaller doses of yohimbine stimulate renin release solely by baroreflex-mediated activation ot the renal sympathetic nerves. As mentioned previously, several groups of researchers (Lang et al., 1975; Gomes et al., 1980; Rockhold and Gross, 1981) have reported that the

254 central injection of yohimbine increased blood pressure via activation of the peripheral sympathetic nervous system. Central activation of the renal sympathetic nerves is known to increase renin release (Keeton and Campbell, 1980). In support of a central site of action of yohimbine in increasing renin release, And~n et al. (1976) demonstrated that peripherally administered yohimbine (0.3-10 m g / k g , i.p.) increased the central turnover of norepinephrine and dopamine in the rat. We did observe that the larger doses of yohimbine (3 and 10 m g / k g ) caused an increase in spontaneous motor activity, but the smaller doses (0.3 and 1 m g / k g ) had no such behavioral effect. It should be pointed out that the smaller doses of yohimbine reproducibly elevated SRA by 2- to 7-fold even though they did not elicit overt central excitation. A fourth mechanism which may account for the ability of yohimbine to increase renin release is the stimulation, either directly or indirectly, of catecholamine release from the adrenal medulla. Although G r a h a m et al. (1980) did not detect a change in plasma epinephrine concentration after yohimbine (4 m g / k g i.v.), it is still possible that thd subcutaneous administration of yohimbine leads to an increase in the release of norepinephrine and epinephrine from the adrenal medulla. Circulating catecholamines do have the ability to stimulate renin release (Keeton and Campbell, 1980). Moreover, a-adrenergic receptors, which function as a negative feedback system, have been identified in the adrenal medulla of the rat (Gutman and Boonyaviorj, 1977), and yohimbine has the potential to amplify adrenomedullary secretion by blocking these receptors. Last, based on in vivo studies with clonidine (Pettinger et al., 1976) and tyramine (Meyer and Herrman, 1978) and in vitro studies with norepinephrine (Desaulles et al., 1975; Capponi and Vallotton, 1976; Morris et al., 1979), it has been hypothesized that the granular juxtaglomerular cells possess az-adrenergic receptors which mediate an inhibition of renin release. If renin release was tonically inhibited by direct a-adrenergic stimulation, then yohimbine could increase renin release by blockade of these post-junctional a2-adrenergic receptors. We consider this possibil-

ity highly unlikely for several reasons. First, and foremost, it is a well-known fact that stimulation of the renal sympathetic nerves, in frequencies within the physiological range, results in an increase, rather than a decrease, in renin release (Keeton and Campbell, 1980). In addition, the concentrations of norepinephrine required to inhibit renin release in vitro far exceed the concentrations which would be encountered in vivo (Desaulles et al., 1975; Capponi and Vallotton, 1976; Morris et al., 1979). At present, we feel that the smaller doses of yohimbine, which do not alter MAP, increase renin release by an action at prejunctional a2-adrenergic receptors located on the renal sympathetic nerves a n d / o r a centrally mediated increase in peripheral sympathetic tone. The larger doses of yohimbine, which decrease MAP, probably increase renin release by an additional baroreflexmediated activation of the renal sympathetic nerves. Each of these mechanisms would mutually enhance the sympathetically-mediated renin release caused by each individual mechanism.

Acknowledgements The authors thank Ana Biediger and Diane Lopez for technical assistance and Ruth Janz and Kathy Lynn for secretarial assistance. These studies were supported by grants from the Texas Affiliate of the American Heart Association and the National Institute of Health (HL29441).

References

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