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Role of the renin-angiotensin system in the blood pressure rebound to sodium nitroprusside in the conscious rat
European Journal o f Pharmacology, 58 (1979) 247--254 © Elsevier/North-Holland Biomedical Press
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ROLE OF THE RENIN-ANGIOTENSIN SYSTEM IN THE BLOOD PRESSURE REBOUND TO SODIUM NITROPRUSSIDE IN THE CONSCIOUS RAT MAHMOUD M. ABUKHRES, ROBERT J. ERTEL, BALWANT N. DIXIT and REGIS R. VOLLMER *
Department Pharmacology, School o f Pharmacy, University of Pittsburgh, Pittsburgh, PA 15261, U.S.A. Received 18 December 1978, revised MS received 24 April 1979, accepted 19 June 1979
M.M. ABUKHRES, R.J. ERTEL, B.N. DIXIT and R.R. VOLLMER, Role of the renin-angiotensin system in the blood pressure rebound to sodium nitroprusside in the conscious rat, European J. Pharmacol. 58 (1979) 247--254. Intravenous infusions of sodium nitroprusside (SNP) at doses of 20, 40 or 80 pg/kg min -1 for 30 rain produced dose-related decrements in blood pressure in conscious rats fitted with indwelling aortic and vena caval catheters. Immediately upon termination of SNP infusions, blood pressure rebounded to levels which were significantly above pre-SNP control values. The following evidence indicates that the rebound increase in blood pressure was due to increased activity of the renin-angiotensin system: (1) plasma renin activity was increased approximately four-fold by SNP, (2) rebound did not occur in nephrectomized rats, (3) rebound was markedly attenuated in animals treated with an angiotensin converting enzyme inhibitor, SQ14225, (D-3-mercapto-2-methylpropanoyl-Lproline) and (4) ~-adrenergic receptor blockade with propranolol reduced the rebound response. In addition, the magnitude of the rebound following SNP infusions was directly related to the dose of SNP infused. These results are consistent with the hypothesis that renin accumulates during SNP infusion more rapidly than it is metabolized. Consequently, the accumulated renin elicits a hypertensive response when SNP treatment is withdrawn. Angiotensin converting enzyme Renin-angiotensin system
Plasma renin activity Sodium nitroprusside
1. Introduction Sodium nitroprusside (SNP) is a potent vasodilator which acts directly upon vascular smooth muscle {Palmer and Lasseter, 1975). Infused intravenously, into man or experimental animals, SNP produces a controllable hypotension which may be rapidly established or reversed (Page and Yager, 1976; Gross and Kreye, 1977). It has been reported that sustained hypotension with SNP stimulates renin secretion. The resultant increase in the production of the vasoconstrictor, angiotensin II, seems to be an important compensatory * Address reprint requests and all correspondence to: Dr. Regis R. Vollmer, Department of Pharmacology, School of Pharmacy, University of Pittsburgh, Pittsburgh, P A 15261, U.S.A.
Blood pressure Conscious rats
mechanism that contributes to the maintenance of blood pressure (Kaneko et al. 1968; Miller et al., 1977). This ability of the reninangiotensin system to buffer the decrease in blood pressure induced by a vascular smooth muscle relaxant has also been clearly demonstrated with other longer acting vasodilators such as minoxidil and hydralazine (Ueda et al. 1970; Zacest et al., 1972; Pettinger et al., 1973; Pettinger and Keeton, 1975). In contrast to the long acting vasodilators, however, SNP's hypotensive effect is more rapid in onset. Because of this unique attribute, SNP was used in the present investigation in order to determine the temporal relationship between an acute reduction in blood pressure and the activation of the renin-angiotensin system. In addition, the present report describes a
248 rebound increase in blood pressure that was consistently observed following the cessation of intravenous infusions of SNP in conscious rats. The primary objective of this study was to characterize this hypertensive rebound and to evaluate the possible contribution of the renin-angiotensin system to this phenomenon.
2. Materials and methods
2.1. General Experiments were conducted with male Wistar rats, 300--400 g. The animals were anesthetized with pentobarbital (55 mg/kg, i.p.) and catheters were implanted in the lower abdominal aorta and inferior vena cava according to the m e t h o d of Weeks and Jones {1960). A minimum of three days were allowed for recovery from surgery. However, nephrectomized rats were used 24 h after surgery. All animals were used for a single experiment. Arterial blood pressures were continuously monitored in the conscious, unrestrained animals according to the m e t h o d of Laffan et al. (1972) and recorded on a Grass Model 7 polygraph. Heart rate was derived from the phasic blood pressure signal using a Grass Tachograph, Model 7P4D. Animals were allowed to stabilize for at least 90 min before experimental protocols were initiated. All drugs were prepared in 0.9% saline immediately before use. Sodium nitroprusside (SNP) solutions were constantly protected from light.
2.2. Hypotensive and rebound effects o f SNP The blood pressure and heart rate responses to SNP during 30 min infusions at rates of 20, 40 or 80/~g/kg min -1 were observed in three groups of animals (n = 6 rats/group). SNP was infused through the vena caval catheter at a rate of 0.02 ml/min using a Razel variable rate infusion pump, Model A-99. Following the cessation of SNP treatment, heart rate and blood pressure were monitored for an addi-
M.M. ABUKHRES ET AL. tional 30 min. During this latter time period, a rebound in blood pressure above pre-infusion levels was consistently noted and the rebound was maximal with the 40 pg/kg m i n - ' dose rate of SNP. Therefore, this dose of SNP was used for all further experiments designed to elucidate the mechanism of the rebound in blood pressure. In order to establish the relationship between the duration of the infusion and the magnitude of the rebound response, groups of 6 rats were infused for 1, 10, 30 or 60 min. The same group was used for the 10 and 60 min infusions with a 40 min stabilization interposed between the two infusions of SNP; separate groups were used for the 1 and 30 min infusions.
2.3. Effects o f SNP in nephrectomized rats The response to SNP in the absence of the renin-angiotensin system was studied in nephrectomized rats ( n = 6 ) . The kidneys were removed during the same surgical session in which the arterial and venous catheters were implanted. 24 h after surgery, the effects of SNP infusion (40 pg/kg m i n - ' for 30 min) were observed and compared to a group of intact rats (n = 6).
2.4. Effects o f SNP following blockade o f the renin-angiotensin system The blood pressure and heart rate responses to SNP ( 4 0 p g / k g min -1 for 30 min) were monitored in the same group of rats (n = 6) before and after blockade of the renin-angiotensin system with an angiotensin converting enzyme inhibitor (SQ14225 D-3-mercapto-2methylpropanoyl-L-proline, 5 mg/kg, i.p.). The two infusions of SNP were separated by an interval of at least 40 min in order to allow blood pressure and heart rate to stabilize. SQ14225 was administered five minutes before the end of the stabilization period. The effectiveness of the 5 mg/kg dose of inhibitor was tested previously in a separate group of 5 rats using the inhibition of angiotensin I
RENIN DEPENDENT BLOOD PRESSURE REBOUND TO SNP pressor responses as an indicator of the completeness of converting enzyme blockade. The responses to angiotensin I (300 ng/kg, i.v.) were inhibited 95% at 5 min and 90% at 30 min after SQ14225. These two time periods coincided with the interval during which the responses to SNP were studied. Pressor responses to angiotensin II (300 ng/kg, i.v.) were not significantly altered by SQ14225.
249
the presence of 8-hydroxyquinoline and dimercaprol (BAL). Following incubation, aliquots of plasmas were subjected to radioimmunoassay for angiotensin I using a Renin Assay Kit (New England Nuclear). PRA is expressed as ng of angiotensin I generated per ml of plasma per hour of incubation (ng/ml h-l).
2.7. Statistical analysis 2.5. Effect o f [Ladrenergic receptor blockade on responses to SNP Responses to SNP infusion (40 pg/kg min -~ for 30 min) were obtained in two groups of 6 rats before and after treatment with d,l-propranolol (1.5 mg/kg, or 5 mg/kg, s.c.). The two infusions of SNP, conducted in each animal, were separated by an interval of at least 40 min in order to allow blood pressure and heart rate to stabilize. Propranolol was administered 10 min before the end of the stabilization period.
2.6. Plasma renin activity (PRA ) The effects of SNP (40 pg/kg min -1 for 30 min) on PRA were studied in a group of 10 rats. Two 1.0 ml arterial blood samples, separated by an interval of 24 h in order to minimize the effects of blood loss on plasma renin activity, were taken from each rat. Each animal was used as its own control. In half of the rats, the control blood sample was taken first and the second blood sample was taken 24 h later, immediately following the SNP infusion. This procedure was reversed in the other half of the group, i.e., post-infusion samples were taken first and the control sample was taken 24 h later. Blood samples were drawn from the arterial catheter into chilled syringes and were rapidly transferred into chilled Vacutainer ® tubes containing Na2 EDTA. Plasma was separated in a refrigerated centrifuge and was frozen until being assayed. Prior to assay the plasma was thawed, buffered to pH 5.5 with citric acid (0.38 M ) a n d incubated for 30 min at 37°C (controls at 4°C) in
Data are reported as mean-+ S.E.M. Statistical differences within groups were determined using a t-test for paired comparisons. Between group differences were analyzed with Student's unpaired t-test.
3. Results
3.1. Hypotension and blood pressure rebound induced by sodium nitroprusside The intravenous administration of sodium nitroprusside (SNP) in three groups of conscious rats, at infusion rates of 20, 40 and 80 pg/kg min -1 for 30 min, resulted in doserelated decrements in blood pressure (fig. 1). The hypotensive response was rapid in onset and was sustained at a relatively constant level, during the 30 min infusions. Upon termination of SNP infusion, blood pressure recovered rapidly and eventually rose to values significantly higher than those observed prior to SNP infusion. This rebound in blood pressure was dose related and was maximal after the 40 pg/kg min -1 dose. Marked, reflexly mediated, increments in heart rate accompanied the hypotensive responses to SNP. As was the case with the rebound in blood pressure, the elevation of heart rate was maximal with the 40 pg/kg min -I dose rate of SNP even though the 80 pg/kg min -1 dose produced a greater fall in blood pressure. Because the 40 pg/kg min -~ dose rate produced the maximal responses in both hypertensive rebound and heart rate, this dose was selected to further investigate
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Fig. 1. Effects of intravenous infusion of SNP for 30 min on heart rate ± S E. (top panel) and m e a n arterial pressure ± S.E. ( b o t t o m panel). Dose rates o f SNP were: (O) 20 p g / k g rain -1, n = 6; (©) 40 p g / k g min -1, n = 6 and ( i ) 80 p g / k g min -1, n = 6. Three separate groups of animals were used to obtain these results.
the m e c h a n i s m of the b l o o d pressure r e b o u n d induced by SNP administration. The relationship b e t w e e n the duration of SNP administration and r e b o u n d in b l o o d pressure is s h o w n in fig. 2. N o measurable r e b o u n d in b l o o d pressure was observed after SNP (40 pg/kg min -1) infusion for 1 min, but the same dose rate infused for 10 min produced a significant r e b o u n d in pressure. There was n o further increase in the amplitude of the r e b o u n d in b l o o d pressure after the 30 or 60 min infusions.
Fig. 2. The relationship b e t w e e n the duration of an intravenous s o d i u m nitroprusside infusion ( 4 0 p g / k g min - ] ) and the magnitude of the blood pressure rebound. The key to s y m b o l s used to identifiy each infusion period are s h o w n at the b o t t o m of the figure. Vertical bars indicate standard errors. Groups of 6 animals were tested at each infusion period. The same group was used for the 10 and 60 rain infusions; separate groups were used for the 1 and 30 min infusions. 6OO -~ 500. 2 o 400.
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The effects o f SNP infusion (40 pg/kg min -1) in intact and n e p h r e c t o m i z e d rats are compared in fig. 3. N e p h r e c t o m y had n o significant effect on resting b l o o d pressure. In the intact rats, average mean arterial pressure was 1 0 7 _+ 11 m m Hg, and in n e p h r e c t o m i z e d rats, it was 1 0 7 +- 3 mm Hg. Heart rates were
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Fig. 3. Comparison of the effects of SNP infused intravenously at a dose rate of 40 p g / k g rain -1 on heart rate ± S.E. (upper panel) and m e a n arterial pressure-+ S.E. (lower panel) in intact ($), n = 6 and n e p h r e c t o m i z e d (©) rats, n = 6. Asterisks indicate significant differences b e t w e e n the t w o groups of rats, P < 0.05.
R E N I N D E P E N D E N T B L O O D P R E S S U R E R E B O U N D TO SNP
410 +-17 beats/min in the intact rats and 361 ± 7 beats/min in the nephrectomized rats. Within the first minute of infusion of SNP into nephrectomized rats, the magnitude of the hypotensive response to SNP was significantly greater than that observed in intact animals. The ability of SNP to produce a greater hypotensive effect in nephrectomized animals was sustained for the duration of the SNP infusion. Also, the rebound in blood pressure, which occurred upon termination of SNP infusion in intact rats, was totally absent in the nephrectomized rats. Reflexly mediated heart rate responses were similar in both groups of rats during SNP infusion.
3.3. SNP effects following inhibition o f angiotensin converting enzyme The effects of SNP before and following treatment with an inhibitor of the angiotensin converting enzyme (SQ14225, 5 mg/kg, i.p.) are summarized in fig. 4. After treatment with the converting enzyme inhibitor, mean arterial blood pressure decreased slightly from 107 ± 2 mm Hg to 102 +- 2 mm Hg within 5 min. Heart rate increased from 410 + 17 to 439 + 14 bpm (not significant). After pretreatment with SQ14225, the hypotenisve response to SNP was significantly potentiated within the first minute of infusion, and the potentiation of the hypotension was sustained for the duration of the infusion. Although, SNP caused greater decrements in blood pressure after the animals were treated with SQ14225, the reflex tachycardia was not greater than that seen in the absence of SQ14225. The blood pressure rebound following SNP infusion was markedly attenuated by SQ14225 treatment.
3.4. Effects o f sodium nitroprusside in the presence o f a (J-adrenergic receptor blocking agent The effects of SNP alone and in the presence of propranolol (1.5 mg/kg or 5 mg/kg,
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s.c.) are shown in fig. 5. Both doses of propranolol caused a slight reduction in resting heart rate and a significant increase in blood pressure (P < 0.05) (table 1). Due to the significant effect of propranolol upon blood pressure, absolute changes in blood pressures and heart rates are given in fig. 5 so that the effects of SNP before and after propranolol can be compared. The control responses to SNP that were obtained in the two groups of animals before propranolol was administered were n o t statistically different, therefore, the results were pooled to construct fig. 5. Propranolol did n o t alter the hypotensive response to SNP; however, the rebound in blood pressure following SNP infusion was reduced in a dose-related manner by propranolol. The reflex tachycardia produced b y the hypotensive response to SNP was also greatly reduced b y propranolol.
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The major finding of this study is that blood pressure rebounds to hypertensive levels following the intravenous infusion of the vasodilator, sodium nitroprusside (SNP) in conscious rats and that this rebound appears to be mediated by the renin-angiotensin system. This conclusion is supported by several observations. First, plasma renin activity was markedly elevated at the time sodium nitroprusside infusion was discontinued in intact animals. Second, a rebound increase in blood pressure did not occur in nephrectomized rats indicating that the kidneys are required for a post-infusion hypertensive response. Third, rebound was significantly reduced in animals in which the formation of angiotensin II was prevented by pretreatment with an inhibitor of the angiotensin converting enzyme, SQ 14225. And finally, treatment of rats with a ~-adrenergic receptor blocking agent, propranolol, reduced the mag-
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Fig. 5. Effects o f ~-adrenergic b l o c k a d e w i t h p r o p r a n olol o n the changes in heart rate ± S.E. ( u p p e r p a n e l ) and mean arterial pressure ± S.E. (lower p a n e l ) induced by SNP infused intravenously at a dose rate of 40 p g / k g min -1 for 30 min. (o) c o n t r o l responses, n = 12, (©) responses obtained after p r o p r a n o l o l 1.5 mg/kg, s.c., n = 6 a n d (m) responses obtained after p r o p r a n o l o l 5.0 mg/kg, s.c., n = 6. S e p a r a t e groups of animals were used for the t w o doses o f p r o p r a n o l o l . C o n t r o l responses obtained in the t w o groups b e f o r e propranolol treatment were c o m b i n e d since t h e y were not statistically different from one another.
3.5. Effect of sodium nitroprusside on plasma renin activity Changes in plasma renin activity in response to SNP infusion were determined in TABLE 1
Effects o f p r o p r a n o l o l o n mean arterial blood pressure (MAP) and heart rate ( H R ) in conscious rats. H e m o d y n a m i c parameters were measured 10 rain after giving p r o p r a n o l o l .
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RENIN DEPENDENT BLOOD PRESSURE REBOUND TO SNP
nitude of the r e b o u n d increase in blood pressure. The magnitude of the hypertensive response was directly related to the dose of SNP infused. No measurable rebound was noted after infusions of one minute. However, after an infusion of 10 min a rebound in pressure was noted which was similar in magnitude to that observed when the infusion was continued for 30 or 60 min. These observations suggest that as the duration of administration of SNP is increased, renin may be accumulating in blood more rapidly than it is being destroyed. Consequently, when the hypotensive stimulus of SNP is terminated, the hypertensive effect of renin is unmasked. The plasma half-life of renin in rats has been reported to be between 10 and 16 min (Peters-Haefelli, 1971; Oates et al., 1974), whereas, less than three minutes were required for blood pressure to return to control levels or above following the termination of SNP infusion. Thus, it appears that significant amounts of renin are still present in the blood when sodium nitroprusside is no longer exerting its hypotensive effect. It is of interest that the dose of SNP which produced the maximal rebound in blood pressure also produced the maximal reflex tachycardic response. Furthermore, reflex tachycardia and the blood pressure rebound showed similar sensitivities to the antagonistic effect of the ~-adrenergic receptor blocking agent, propranolol. The parallel response characteristics of reflex tachycardia and rebound indirectly suggest the possibility that renin release may be due in large part to a neurally mediated reflex response. Substantial experimental evidence exists supporting the concept that one important factor controlling the release of renin is the activity of the renal sympathetic neurons (Davis and Freeman, 1976; Grandjean et al., 1978). Recent studies have proposed a positive correlation between sympathetic activity and plasma renin activity in hypertensive humans (De Quattro and Miura, 1976; Esler et al., 1977). In addition, it seems most likely
253
that the neural control of renin release is mediated via a /3-adrenergic receptor mechanism (Johnson et al., 1976; Osborn et al., 1977). Our data is consistent with the theory that the renin release induced b y the hypotensive response to SNP may be elicited via a neural reflex involving a/3-adrenergic receptor mechanism. However, the present experiments do not allow us to conclude whether the release of renin was due to the action of norepinephrine released from sympathetic neurons or to adrenal catecholamine release. The results of these experiments are also in agreement with the findings of other investigators who have demonstrated that increased activity of the renin-angiotensin system is important in the maintenance of blood pressure during the hypotension induced by SNP (Kaneko et al., 1968) and other vasodilators (Ueda et al., 1970; Zacest et al., 1972; Pettinger and Keeton, 1975). This conclusion is substantiated b y the observation that both n e p h r e c t o m y and angiotensin converting enzyme inhibition with SQ14225 significantly potentiated the hypotensive activity of SNP. Furthermore, the potentiated blood pressure response was noted during the first minute of SNP infusion which indicates that the reninangiotensin system was activated with extreme rapidity, a finding which is consistent with the hypothesis that neurally mediated release of renin may be of importance during rapid reductions in blood pressure. In conclusion, the present experiments have shown that the renin-angiotensin system is rapidly activated and contributes to the maintenance of blood pressure during the onset and sustained phases of hypotension to SNP. In addition we have described the characteristics of a renin dependent rebound in blood pressure which occurred consistently when SNP infusion was terminated.
Acknowledgements The authors are grateful for the assistance of Mrs. Andrea Tassick and Mrs. Geraldine Robinson for their
254 help in preparing this manuscript. This work was supported by a Pharmaceutical Manufacturers Association Foundation Starter Grant awarded to Regis R. Vollmer. The SQ14225 was kindly supplied by Z.P. Horovitz, E.R. Squibb and Sons, Inc.
References Davis, J.O. and R.H. Freeman, 1976, Mechanisms regulating renin release, Physiol. Rev. 56, 1. DeQuattro, V. and V. Miura, 1976, Plasma and tissue catecholamines in primary hypertension, in: The Nervous System in Arterial Hypertension, eds. S. Julius and M. Esler (Charles C. Thomas, Publisher, Springfield) p. 355. Esler, M., S. Julius, A. Sweifler, O. Randall, E. Harburg, M. Gardiner and V. DeQuattro, 1977, Mild high-renin essential hypertension-neurogenic human hypertension, New Engl. J. Med. 296,405. Grandjean, B., G. Annat, M. Vincent and J. Sassard, 1978, Influence of renal nerves on renin secretion in the conscious dog, Pfliigers Arch. 3 7 3 , 1 6 1 . Gross, F. and V.W. Kreye, 1977, Drugs acting on arteriolar smooth muscle (vasodilator drugs), in: Handbook of Experimental Pharmacology. XXXIX, ed. F. Gross (Springer-Verlag, New York) p. 397. Johnson, J.A., J.O. Davis, R.W. Gotshali, T.E. Lohmeier, J.L. Davis, B. Baverman and G.E. Tempel, 1976, Evidence for an intrarenal beta receptor in control of renin release, Amer. J. Physiol. 2 3 0 , 4 1 0 . Kaneko, Y., T. Ikeda, T. Takeda, G. Inoue, H. Tagawa and H. Ueda, 1968, Renin release in patients with benign essential hypertension, Circulation 38,353. Laffan, R.J., A. Peterson, S.W. Hitch and C. Jeunelot, 1972, A technique for prolonged, continuous
M.M. ABUKHRES ET AL. recording of blood pressure of unrestrained rats, Cardiovasc. Res. 6, 319. Miller, E.D., J.A. Ackerly, E.D. Vaughan, M.J. Peach and R.M. Epstein, 1977, The renin-angiotensin system during controlled hypotension with sodium nitroprusside, Anesthesiology 4 7 , 2 5 7 . Oates, H.F., J.A. Fretten and G.S. Stokes, 1974, Disappearance rate of circulating renin after bilateral nephrectomy in the rat, Clin. Exp. Pharmacol. Physiol. 1,547. Osborn, J.L., J.B. Hook and M.D. Baile, 1977, Control of renin release-effects of d-propranolol and renal denervation on furosemide-induced renin release in the dog, Circulation Res. 4 1 , 4 8 1 . Page, L.B. and H.M. Yager, 1976, Drugs in the management of hypertension. Part III, Amer. Heart J. 92, 252. Palmer, R.F. and K.C. Lasseter, 1975, Sodium nitroprusside, New Engl. J. Med. 292,294. Peters-Haefeli, L., 1971, Rate of inactivation of endogenous or exogenous renin in normal and in renin-depleted rats, Amer. J. Physiol. 221, 1339. Pettinger, W.A. and K. Keeton, 1975, Altered renin release and propranolol potentiation of vasodilatory drug hypotension, J. Clin. Invest. 55,236. Pettinger, W.A., W.B. Campbell and K. Keeton, 1973, Adrenergic component of renin release induced by vasodilating antihypertensive drugs in the rat, Circulation Res. 33, 82. Ueda, H., V. Kaneko, T. Takeda, T. Ikeda and S. Yagi, 1970, Observations on the mechanism of renin release by hydralazine in hypertensive patients, Circulation Res. 26, Suppl. II, 201. Weeks, J.R. and J.A. Jones, 1960, Routine direct measurement of arterial pressure in unanesthetized rats, Proc. Soc. Exp. Med. 104,646. Zacest, R., E. Gilmore and E. Koch-Weser, 1972, Treatment of essential hypertension with combined vasodilation and beta-adrenergic blockade, New Engl. J. Med. 2 8 6 , 6 1 7 .
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ROLE OF THE RENIN-ANGIOTENSIN SYSTEM IN THE BLOOD PRESSURE REBOUND TO SODIUM NITROPRUSSIDE IN THE CONSCIOUS RAT MAHMOUD M. ABUKHRES, ROBERT J. ERTEL, BALWANT N. DIXIT and REGIS R. VOLLMER *
Department Pharmacology, School o f Pharmacy, University of Pittsburgh, Pittsburgh, PA 15261, U.S.A. Received 18 December 1978, revised MS received 24 April 1979, accepted 19 June 1979
M.M. ABUKHRES, R.J. ERTEL, B.N. DIXIT and R.R. VOLLMER, Role of the renin-angiotensin system in the blood pressure rebound to sodium nitroprusside in the conscious rat, European J. Pharmacol. 58 (1979) 247--254. Intravenous infusions of sodium nitroprusside (SNP) at doses of 20, 40 or 80 pg/kg min -1 for 30 rain produced dose-related decrements in blood pressure in conscious rats fitted with indwelling aortic and vena caval catheters. Immediately upon termination of SNP infusions, blood pressure rebounded to levels which were significantly above pre-SNP control values. The following evidence indicates that the rebound increase in blood pressure was due to increased activity of the renin-angiotensin system: (1) plasma renin activity was increased approximately four-fold by SNP, (2) rebound did not occur in nephrectomized rats, (3) rebound was markedly attenuated in animals treated with an angiotensin converting enzyme inhibitor, SQ14225, (D-3-mercapto-2-methylpropanoyl-Lproline) and (4) ~-adrenergic receptor blockade with propranolol reduced the rebound response. In addition, the magnitude of the rebound following SNP infusions was directly related to the dose of SNP infused. These results are consistent with the hypothesis that renin accumulates during SNP infusion more rapidly than it is metabolized. Consequently, the accumulated renin elicits a hypertensive response when SNP treatment is withdrawn. Angiotensin converting enzyme Renin-angiotensin system
Plasma renin activity Sodium nitroprusside
1. Introduction Sodium nitroprusside (SNP) is a potent vasodilator which acts directly upon vascular smooth muscle {Palmer and Lasseter, 1975). Infused intravenously, into man or experimental animals, SNP produces a controllable hypotension which may be rapidly established or reversed (Page and Yager, 1976; Gross and Kreye, 1977). It has been reported that sustained hypotension with SNP stimulates renin secretion. The resultant increase in the production of the vasoconstrictor, angiotensin II, seems to be an important compensatory * Address reprint requests and all correspondence to: Dr. Regis R. Vollmer, Department of Pharmacology, School of Pharmacy, University of Pittsburgh, Pittsburgh, P A 15261, U.S.A.
Blood pressure Conscious rats
mechanism that contributes to the maintenance of blood pressure (Kaneko et al. 1968; Miller et al., 1977). This ability of the reninangiotensin system to buffer the decrease in blood pressure induced by a vascular smooth muscle relaxant has also been clearly demonstrated with other longer acting vasodilators such as minoxidil and hydralazine (Ueda et al. 1970; Zacest et al., 1972; Pettinger et al., 1973; Pettinger and Keeton, 1975). In contrast to the long acting vasodilators, however, SNP's hypotensive effect is more rapid in onset. Because of this unique attribute, SNP was used in the present investigation in order to determine the temporal relationship between an acute reduction in blood pressure and the activation of the renin-angiotensin system. In addition, the present report describes a
248 rebound increase in blood pressure that was consistently observed following the cessation of intravenous infusions of SNP in conscious rats. The primary objective of this study was to characterize this hypertensive rebound and to evaluate the possible contribution of the renin-angiotensin system to this phenomenon.
2. Materials and methods
2.1. General Experiments were conducted with male Wistar rats, 300--400 g. The animals were anesthetized with pentobarbital (55 mg/kg, i.p.) and catheters were implanted in the lower abdominal aorta and inferior vena cava according to the m e t h o d of Weeks and Jones {1960). A minimum of three days were allowed for recovery from surgery. However, nephrectomized rats were used 24 h after surgery. All animals were used for a single experiment. Arterial blood pressures were continuously monitored in the conscious, unrestrained animals according to the m e t h o d of Laffan et al. (1972) and recorded on a Grass Model 7 polygraph. Heart rate was derived from the phasic blood pressure signal using a Grass Tachograph, Model 7P4D. Animals were allowed to stabilize for at least 90 min before experimental protocols were initiated. All drugs were prepared in 0.9% saline immediately before use. Sodium nitroprusside (SNP) solutions were constantly protected from light.
2.2. Hypotensive and rebound effects o f SNP The blood pressure and heart rate responses to SNP during 30 min infusions at rates of 20, 40 or 80/~g/kg min -1 were observed in three groups of animals (n = 6 rats/group). SNP was infused through the vena caval catheter at a rate of 0.02 ml/min using a Razel variable rate infusion pump, Model A-99. Following the cessation of SNP treatment, heart rate and blood pressure were monitored for an addi-
M.M. ABUKHRES ET AL. tional 30 min. During this latter time period, a rebound in blood pressure above pre-infusion levels was consistently noted and the rebound was maximal with the 40 pg/kg m i n - ' dose rate of SNP. Therefore, this dose of SNP was used for all further experiments designed to elucidate the mechanism of the rebound in blood pressure. In order to establish the relationship between the duration of the infusion and the magnitude of the rebound response, groups of 6 rats were infused for 1, 10, 30 or 60 min. The same group was used for the 10 and 60 min infusions with a 40 min stabilization interposed between the two infusions of SNP; separate groups were used for the 1 and 30 min infusions.
2.3. Effects o f SNP in nephrectomized rats The response to SNP in the absence of the renin-angiotensin system was studied in nephrectomized rats ( n = 6 ) . The kidneys were removed during the same surgical session in which the arterial and venous catheters were implanted. 24 h after surgery, the effects of SNP infusion (40 pg/kg m i n - ' for 30 min) were observed and compared to a group of intact rats (n = 6).
2.4. Effects o f SNP following blockade o f the renin-angiotensin system The blood pressure and heart rate responses to SNP ( 4 0 p g / k g min -1 for 30 min) were monitored in the same group of rats (n = 6) before and after blockade of the renin-angiotensin system with an angiotensin converting enzyme inhibitor (SQ14225 D-3-mercapto-2methylpropanoyl-L-proline, 5 mg/kg, i.p.). The two infusions of SNP were separated by an interval of at least 40 min in order to allow blood pressure and heart rate to stabilize. SQ14225 was administered five minutes before the end of the stabilization period. The effectiveness of the 5 mg/kg dose of inhibitor was tested previously in a separate group of 5 rats using the inhibition of angiotensin I
RENIN DEPENDENT BLOOD PRESSURE REBOUND TO SNP pressor responses as an indicator of the completeness of converting enzyme blockade. The responses to angiotensin I (300 ng/kg, i.v.) were inhibited 95% at 5 min and 90% at 30 min after SQ14225. These two time periods coincided with the interval during which the responses to SNP were studied. Pressor responses to angiotensin II (300 ng/kg, i.v.) were not significantly altered by SQ14225.
249
the presence of 8-hydroxyquinoline and dimercaprol (BAL). Following incubation, aliquots of plasmas were subjected to radioimmunoassay for angiotensin I using a Renin Assay Kit (New England Nuclear). PRA is expressed as ng of angiotensin I generated per ml of plasma per hour of incubation (ng/ml h-l).
2.7. Statistical analysis 2.5. Effect o f [Ladrenergic receptor blockade on responses to SNP Responses to SNP infusion (40 pg/kg min -~ for 30 min) were obtained in two groups of 6 rats before and after treatment with d,l-propranolol (1.5 mg/kg, or 5 mg/kg, s.c.). The two infusions of SNP, conducted in each animal, were separated by an interval of at least 40 min in order to allow blood pressure and heart rate to stabilize. Propranolol was administered 10 min before the end of the stabilization period.
2.6. Plasma renin activity (PRA ) The effects of SNP (40 pg/kg min -1 for 30 min) on PRA were studied in a group of 10 rats. Two 1.0 ml arterial blood samples, separated by an interval of 24 h in order to minimize the effects of blood loss on plasma renin activity, were taken from each rat. Each animal was used as its own control. In half of the rats, the control blood sample was taken first and the second blood sample was taken 24 h later, immediately following the SNP infusion. This procedure was reversed in the other half of the group, i.e., post-infusion samples were taken first and the control sample was taken 24 h later. Blood samples were drawn from the arterial catheter into chilled syringes and were rapidly transferred into chilled Vacutainer ® tubes containing Na2 EDTA. Plasma was separated in a refrigerated centrifuge and was frozen until being assayed. Prior to assay the plasma was thawed, buffered to pH 5.5 with citric acid (0.38 M ) a n d incubated for 30 min at 37°C (controls at 4°C) in
Data are reported as mean-+ S.E.M. Statistical differences within groups were determined using a t-test for paired comparisons. Between group differences were analyzed with Student's unpaired t-test.
3. Results
3.1. Hypotension and blood pressure rebound induced by sodium nitroprusside The intravenous administration of sodium nitroprusside (SNP) in three groups of conscious rats, at infusion rates of 20, 40 and 80 pg/kg min -1 for 30 min, resulted in doserelated decrements in blood pressure (fig. 1). The hypotensive response was rapid in onset and was sustained at a relatively constant level, during the 30 min infusions. Upon termination of SNP infusion, blood pressure recovered rapidly and eventually rose to values significantly higher than those observed prior to SNP infusion. This rebound in blood pressure was dose related and was maximal after the 40 pg/kg min -1 dose. Marked, reflexly mediated, increments in heart rate accompanied the hypotensive responses to SNP. As was the case with the rebound in blood pressure, the elevation of heart rate was maximal with the 40 pg/kg min -I dose rate of SNP even though the 80 pg/kg min -1 dose produced a greater fall in blood pressure. Because the 40 pg/kg min -~ dose rate produced the maximal responses in both hypertensive rebound and heart rate, this dose was selected to further investigate
250
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Fig. 1. Effects of intravenous infusion of SNP for 30 min on heart rate ± S E. (top panel) and m e a n arterial pressure ± S.E. ( b o t t o m panel). Dose rates o f SNP were: (O) 20 p g / k g rain -1, n = 6; (©) 40 p g / k g min -1, n = 6 and ( i ) 80 p g / k g min -1, n = 6. Three separate groups of animals were used to obtain these results.
the m e c h a n i s m of the b l o o d pressure r e b o u n d induced by SNP administration. The relationship b e t w e e n the duration of SNP administration and r e b o u n d in b l o o d pressure is s h o w n in fig. 2. N o measurable r e b o u n d in b l o o d pressure was observed after SNP (40 pg/kg min -1) infusion for 1 min, but the same dose rate infused for 10 min produced a significant r e b o u n d in pressure. There was n o further increase in the amplitude of the r e b o u n d in b l o o d pressure after the 30 or 60 min infusions.
Fig. 2. The relationship b e t w e e n the duration of an intravenous s o d i u m nitroprusside infusion ( 4 0 p g / k g min - ] ) and the magnitude of the blood pressure rebound. The key to s y m b o l s used to identifiy each infusion period are s h o w n at the b o t t o m of the figure. Vertical bars indicate standard errors. Groups of 6 animals were tested at each infusion period. The same group was used for the 10 and 60 rain infusions; separate groups were used for the 1 and 30 min infusions. 6OO -~ 500. 2 o 400.
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The effects o f SNP infusion (40 pg/kg min -1) in intact and n e p h r e c t o m i z e d rats are compared in fig. 3. N e p h r e c t o m y had n o significant effect on resting b l o o d pressure. In the intact rats, average mean arterial pressure was 1 0 7 _+ 11 m m Hg, and in n e p h r e c t o m i z e d rats, it was 1 0 7 +- 3 mm Hg. Heart rates were
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Fig. 3. Comparison of the effects of SNP infused intravenously at a dose rate of 40 p g / k g rain -1 on heart rate ± S.E. (upper panel) and m e a n arterial pressure-+ S.E. (lower panel) in intact ($), n = 6 and n e p h r e c t o m i z e d (©) rats, n = 6. Asterisks indicate significant differences b e t w e e n the t w o groups of rats, P < 0.05.
R E N I N D E P E N D E N T B L O O D P R E S S U R E R E B O U N D TO SNP
410 +-17 beats/min in the intact rats and 361 ± 7 beats/min in the nephrectomized rats. Within the first minute of infusion of SNP into nephrectomized rats, the magnitude of the hypotensive response to SNP was significantly greater than that observed in intact animals. The ability of SNP to produce a greater hypotensive effect in nephrectomized animals was sustained for the duration of the SNP infusion. Also, the rebound in blood pressure, which occurred upon termination of SNP infusion in intact rats, was totally absent in the nephrectomized rats. Reflexly mediated heart rate responses were similar in both groups of rats during SNP infusion.
3.3. SNP effects following inhibition o f angiotensin converting enzyme The effects of SNP before and following treatment with an inhibitor of the angiotensin converting enzyme (SQ14225, 5 mg/kg, i.p.) are summarized in fig. 4. After treatment with the converting enzyme inhibitor, mean arterial blood pressure decreased slightly from 107 ± 2 mm Hg to 102 +- 2 mm Hg within 5 min. Heart rate increased from 410 + 17 to 439 + 14 bpm (not significant). After pretreatment with SQ14225, the hypotenisve response to SNP was significantly potentiated within the first minute of infusion, and the potentiation of the hypotension was sustained for the duration of the infusion. Although, SNP caused greater decrements in blood pressure after the animals were treated with SQ14225, the reflex tachycardia was not greater than that seen in the absence of SQ14225. The blood pressure rebound following SNP infusion was markedly attenuated by SQ14225 treatment.
3.4. Effects o f sodium nitroprusside in the presence o f a (J-adrenergic receptor blocking agent The effects of SNP alone and in the presence of propranolol (1.5 mg/kg or 5 mg/kg,
251
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s.c.) are shown in fig. 5. Both doses of propranolol caused a slight reduction in resting heart rate and a significant increase in blood pressure (P < 0.05) (table 1). Due to the significant effect of propranolol upon blood pressure, absolute changes in blood pressures and heart rates are given in fig. 5 so that the effects of SNP before and after propranolol can be compared. The control responses to SNP that were obtained in the two groups of animals before propranolol was administered were n o t statistically different, therefore, the results were pooled to construct fig. 5. Propranolol did n o t alter the hypotensive response to SNP; however, the rebound in blood pressure following SNP infusion was reduced in a dose-related manner by propranolol. The reflex tachycardia produced b y the hypotensive response to SNP was also greatly reduced b y propranolol.
252
M.M. A B U K H R E S E T AL.
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a separate group of 10 rats. The SNP dose rate of 40 pg/kg min -~ was investigated since this dose was utilized in the majority of the hemodynamic experiments. Control PRA was 16.3 i 2.8 ng AI/ml h-'. Following 30 min of SNP infusion, PRA was 80.9 ± 7 ng/ml h -~ (P < 0.001, paired t-test). The PRA at this time reflected the plasma levels of renin present when SNP infusion was terminated.
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The major finding of this study is that blood pressure rebounds to hypertensive levels following the intravenous infusion of the vasodilator, sodium nitroprusside (SNP) in conscious rats and that this rebound appears to be mediated by the renin-angiotensin system. This conclusion is supported by several observations. First, plasma renin activity was markedly elevated at the time sodium nitroprusside infusion was discontinued in intact animals. Second, a rebound increase in blood pressure did not occur in nephrectomized rats indicating that the kidneys are required for a post-infusion hypertensive response. Third, rebound was significantly reduced in animals in which the formation of angiotensin II was prevented by pretreatment with an inhibitor of the angiotensin converting enzyme, SQ 14225. And finally, treatment of rats with a ~-adrenergic receptor blocking agent, propranolol, reduced the mag-
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Fig. 5. Effects o f ~-adrenergic b l o c k a d e w i t h p r o p r a n olol o n the changes in heart rate ± S.E. ( u p p e r p a n e l ) and mean arterial pressure ± S.E. (lower p a n e l ) induced by SNP infused intravenously at a dose rate of 40 p g / k g min -1 for 30 min. (o) c o n t r o l responses, n = 12, (©) responses obtained after p r o p r a n o l o l 1.5 mg/kg, s.c., n = 6 a n d (m) responses obtained after p r o p r a n o l o l 5.0 mg/kg, s.c., n = 6. S e p a r a t e groups of animals were used for the t w o doses o f p r o p r a n o l o l . C o n t r o l responses obtained in the t w o groups b e f o r e propranolol treatment were c o m b i n e d since t h e y were not statistically different from one another.
3.5. Effect of sodium nitroprusside on plasma renin activity Changes in plasma renin activity in response to SNP infusion were determined in TABLE 1
Effects o f p r o p r a n o l o l o n mean arterial blood pressure (MAP) and heart rate ( H R ) in conscious rats. H e m o d y n a m i c parameters were measured 10 rain after giving p r o p r a n o l o l .
Dose of p r o p r a n o l o l (mg/kg, s.c.)
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RENIN DEPENDENT BLOOD PRESSURE REBOUND TO SNP
nitude of the r e b o u n d increase in blood pressure. The magnitude of the hypertensive response was directly related to the dose of SNP infused. No measurable rebound was noted after infusions of one minute. However, after an infusion of 10 min a rebound in pressure was noted which was similar in magnitude to that observed when the infusion was continued for 30 or 60 min. These observations suggest that as the duration of administration of SNP is increased, renin may be accumulating in blood more rapidly than it is being destroyed. Consequently, when the hypotensive stimulus of SNP is terminated, the hypertensive effect of renin is unmasked. The plasma half-life of renin in rats has been reported to be between 10 and 16 min (Peters-Haefelli, 1971; Oates et al., 1974), whereas, less than three minutes were required for blood pressure to return to control levels or above following the termination of SNP infusion. Thus, it appears that significant amounts of renin are still present in the blood when sodium nitroprusside is no longer exerting its hypotensive effect. It is of interest that the dose of SNP which produced the maximal rebound in blood pressure also produced the maximal reflex tachycardic response. Furthermore, reflex tachycardia and the blood pressure rebound showed similar sensitivities to the antagonistic effect of the ~-adrenergic receptor blocking agent, propranolol. The parallel response characteristics of reflex tachycardia and rebound indirectly suggest the possibility that renin release may be due in large part to a neurally mediated reflex response. Substantial experimental evidence exists supporting the concept that one important factor controlling the release of renin is the activity of the renal sympathetic neurons (Davis and Freeman, 1976; Grandjean et al., 1978). Recent studies have proposed a positive correlation between sympathetic activity and plasma renin activity in hypertensive humans (De Quattro and Miura, 1976; Esler et al., 1977). In addition, it seems most likely
253
that the neural control of renin release is mediated via a /3-adrenergic receptor mechanism (Johnson et al., 1976; Osborn et al., 1977). Our data is consistent with the theory that the renin release induced b y the hypotensive response to SNP may be elicited via a neural reflex involving a/3-adrenergic receptor mechanism. However, the present experiments do not allow us to conclude whether the release of renin was due to the action of norepinephrine released from sympathetic neurons or to adrenal catecholamine release. The results of these experiments are also in agreement with the findings of other investigators who have demonstrated that increased activity of the renin-angiotensin system is important in the maintenance of blood pressure during the hypotension induced by SNP (Kaneko et al., 1968) and other vasodilators (Ueda et al., 1970; Zacest et al., 1972; Pettinger and Keeton, 1975). This conclusion is substantiated b y the observation that both n e p h r e c t o m y and angiotensin converting enzyme inhibition with SQ14225 significantly potentiated the hypotensive activity of SNP. Furthermore, the potentiated blood pressure response was noted during the first minute of SNP infusion which indicates that the reninangiotensin system was activated with extreme rapidity, a finding which is consistent with the hypothesis that neurally mediated release of renin may be of importance during rapid reductions in blood pressure. In conclusion, the present experiments have shown that the renin-angiotensin system is rapidly activated and contributes to the maintenance of blood pressure during the onset and sustained phases of hypotension to SNP. In addition we have described the characteristics of a renin dependent rebound in blood pressure which occurred consistently when SNP infusion was terminated.
Acknowledgements The authors are grateful for the assistance of Mrs. Andrea Tassick and Mrs. Geraldine Robinson for their
254 help in preparing this manuscript. This work was supported by a Pharmaceutical Manufacturers Association Foundation Starter Grant awarded to Regis R. Vollmer. The SQ14225 was kindly supplied by Z.P. Horovitz, E.R. Squibb and Sons, Inc.
References Davis, J.O. and R.H. Freeman, 1976, Mechanisms regulating renin release, Physiol. Rev. 56, 1. DeQuattro, V. and V. Miura, 1976, Plasma and tissue catecholamines in primary hypertension, in: The Nervous System in Arterial Hypertension, eds. S. Julius and M. Esler (Charles C. Thomas, Publisher, Springfield) p. 355. Esler, M., S. Julius, A. Sweifler, O. Randall, E. Harburg, M. Gardiner and V. DeQuattro, 1977, Mild high-renin essential hypertension-neurogenic human hypertension, New Engl. J. Med. 296,405. Grandjean, B., G. Annat, M. Vincent and J. Sassard, 1978, Influence of renal nerves on renin secretion in the conscious dog, Pfliigers Arch. 3 7 3 , 1 6 1 . Gross, F. and V.W. Kreye, 1977, Drugs acting on arteriolar smooth muscle (vasodilator drugs), in: Handbook of Experimental Pharmacology. XXXIX, ed. F. Gross (Springer-Verlag, New York) p. 397. Johnson, J.A., J.O. Davis, R.W. Gotshali, T.E. Lohmeier, J.L. Davis, B. Baverman and G.E. Tempel, 1976, Evidence for an intrarenal beta receptor in control of renin release, Amer. J. Physiol. 2 3 0 , 4 1 0 . Kaneko, Y., T. Ikeda, T. Takeda, G. Inoue, H. Tagawa and H. Ueda, 1968, Renin release in patients with benign essential hypertension, Circulation 38,353. Laffan, R.J., A. Peterson, S.W. Hitch and C. Jeunelot, 1972, A technique for prolonged, continuous
M.M. ABUKHRES ET AL. recording of blood pressure of unrestrained rats, Cardiovasc. Res. 6, 319. Miller, E.D., J.A. Ackerly, E.D. Vaughan, M.J. Peach and R.M. Epstein, 1977, The renin-angiotensin system during controlled hypotension with sodium nitroprusside, Anesthesiology 4 7 , 2 5 7 . Oates, H.F., J.A. Fretten and G.S. Stokes, 1974, Disappearance rate of circulating renin after bilateral nephrectomy in the rat, Clin. Exp. Pharmacol. Physiol. 1,547. Osborn, J.L., J.B. Hook and M.D. Baile, 1977, Control of renin release-effects of d-propranolol and renal denervation on furosemide-induced renin release in the dog, Circulation Res. 4 1 , 4 8 1 . Page, L.B. and H.M. Yager, 1976, Drugs in the management of hypertension. Part III, Amer. Heart J. 92, 252. Palmer, R.F. and K.C. Lasseter, 1975, Sodium nitroprusside, New Engl. J. Med. 292,294. Peters-Haefeli, L., 1971, Rate of inactivation of endogenous or exogenous renin in normal and in renin-depleted rats, Amer. J. Physiol. 221, 1339. Pettinger, W.A. and K. Keeton, 1975, Altered renin release and propranolol potentiation of vasodilatory drug hypotension, J. Clin. Invest. 55,236. Pettinger, W.A., W.B. Campbell and K. Keeton, 1973, Adrenergic component of renin release induced by vasodilating antihypertensive drugs in the rat, Circulation Res. 33, 82. Ueda, H., V. Kaneko, T. Takeda, T. Ikeda and S. Yagi, 1970, Observations on the mechanism of renin release by hydralazine in hypertensive patients, Circulation Res. 26, Suppl. II, 201. Weeks, J.R. and J.A. Jones, 1960, Routine direct measurement of arterial pressure in unanesthetized rats, Proc. Soc. Exp. Med. 104,646. Zacest, R., E. Gilmore and E. Koch-Weser, 1972, Treatment of essential hypertension with combined vasodilation and beta-adrenergic blockade, New Engl. J. Med. 2 8 6 , 6 1 7 .