Effects of Cyclic AMP, Sympathomimetic Amines, and Adrenergic Receptor Antagonists on Renin Secretion

Transcription

1 Effects of Cyclic AMP, Sympathomimetic Amines, and Adrenergic Receptor Antagonists on Renin Secretion By Nathaniel Winer, Deenbandhu S. Chokshi, and Walter G. Walkenhorst ABSTRACT Renal vein renin activity was measured by bioassay of plasma obtained from anesthetized dogs during renal arterial infusion of sympathomimetic amines, cyclic AMP, and other nucleotides. Renin secretion was calculated as the product of renal blood flow and the arterial-renal venous renin difference. Infusion of cyclic AMP significantly increased renin secretion, while administration of ATP, ADP, 5'AMP, and adenosine in quantities equimolar to cyclic AMP had no effect. Phentolamine, an rt-receptor antagonist, and dlpropranolol, a /3-receptor blocker, abolished the cyclic AMP-induced rise in renin secretion. Paradoxically, the increase in renin production induced by infusion of norepinephrine, a predominantly a-receptor agonist, was suppressed by propranolol administration, and the renin-stimluating effect of isoproterenol, a /3-receptor stimulator, was blocked by phentolamine administration. In addition, the d- and /-isomers of propranolol were equipotent in suppressing the isoproterenol-induced rise in renin secretion. Administration of AY-21,011, a B- receptor antagonist which lacks local anesthetic properties, also prevented the isoproterenol-induced rise in renin secretion, but lidocaine had no suppressive effect. The effects on renin secretion of cyclic AMP and sympathomimetic amines alone and in combination with adrenergic receptor antagonists were independent of changes in glomerular filtration rate, sodium excretion, urine flow, systemic blood pressure, and heart rate. The data suggest that cyclic AMP acts within the kidney as an intracellular mediator of renin secretion, and that phentolamine and propranolol suppress renin secretion at a site distal to cyclic AMP production, rather than by blockade of plasma membrane adrenergic receptors or inhibition of adenyl cyclase. KEY WORDS phentolamine nucleotides propranolol We have previously reported that the renin release provoked by upright posture and administration of agents such as diazoxide, ethacrynic acid, and theophylline could be inhibited by adrenergic receptor-blocking agents (1). These data suggested that many, if not all, stimuli for renin release might be mediated by a final common pathway, possi- From the Raymond H. Starr Hypertension Laboratory and the Department of Medicine, Menorah Medical Center, Kansas City, Missouri This investigation was supported by the Kansas City Henrt Association and the Woolford Foundation of Kansas City. Received February 5, Accepted for publication June 28, norepinephrine adenyl cyclase isoproterenol lidocaine bly involving a- and /3-receptor sites. Since catccholamines increase renin secretion when infused into the renal artery of the dog (2, 3) and also enhance adenyl cyclase activity (4), it seemed possible that intracellular accumulation of adenosine 3', 5' cyclic monophosphoric acid (cyclic AMP) might play a role in mediating the increased renin secretion induced by catecholamines and other stimuli. Cyclic AMP mediates a vast array of physiological phenomena (5, 6) and has been postulated to act as a "second messenger" in the intracellular action of several hormones (7). Moreover, cyclic AMP stimulates renin release in vitro (8). Circulation Research, Vol. XXIX, September

2 240 WINER, CHOKSHI, WALKENHORST In view of these observations we attempted to determine whether infusion of cyclic AMP and various syrnpathomirnetic amines would stimulate renin release in vivo. Further, we tried to define the site at which adrenergic, receptor-blocking agents inhibit renin release. Methods Mongrel dogs (15-20 kg) on an unrestricted dietary sodium intake were anesthetized with pentobarbital sodium, 30 mg/kg iv, with supplements given as necessary. Through either a subcostal or abdominal approach, 25-gauge needles attached to polyethylene catheters were inserted, into one or both renal arteries and tied into place with purse-string sutures. Indwelling catheters were placed in one or both renal veins and kept open by a constant infusion of normal saline (0.19 ml/min) and polyethylene cannulas (PE-240) were placed in one or both ureters. Blood pressure was monitored via a catheter in a femoral artery connected to a Sanbom transducer (Model 267PC) with a Hewlett Packard recorder. In three experiments, cyclic AMP dissolved in lactated Ringer's solution was given into one renal artery at a constant rate by an infusion pump, and lactated Ringer's solution alone was infused into the contralateral renal artery at the same rate. In all other experiments, only a single kidney was infused. ATP, ADP, 5'AMP, and adenosine were dissolved in Ringer's lactate and administered into the renal artery in quantities equimolar to cyclic AMP. Saline solutions of norepinephrine and isoproterenol were also infused intrarenally. In some experiments, saline solutions of phentolamine, AY-21,011 (4-[2-hydroxy-3-isopropylaminopropoxy] acetanalide), racemic propranolol and its dextro or levo isomers, and lidocaine were given intravenously. All agents infused either intravenously or into the renal artery were given over a 30-minute period in a volume of 10 ml or less and were preceded and followed by 30- minute infusions of an equal volume of normal saline. Plasma samples were obtained from the renal veins at 10-minute intervals for measurement of renal vein renin activity (RVRA) in our laboratory by the method of Gunnells et al. (9). This technique involves overnight dialysis of plasma samples to remove preformed angiotensin, and incubation of renin with endogenous renin substrate at ph 5.5 and 37 C for 1 hour. The blood pressure rise following injection of small aliquots of the incubation mixture into the pentolinium-blocked, nephrectomized rat is compared to the rise induced by a known concentration of synthetic angiotensin II. The results are expressed as ng of angiotensin II generated during a 1-hour incubation period. Six replicate determinations of a pooled plasma specimen gave a mean of 306 ng/100 ml with a standard deviation of 14 ng/100 ml. The possibility that the agents used might have interfered with the assay system was excluded by showing that the addition of an excess of the calculated circulating concentration of each agent to the renin-renin substrate incubation mixture did not affect the measured plasma renin activity. Moreover, the addition of 1 mg (4 renin pressor units/mg) of purified hog renin (Pentex) to the incubation mixture containing plasma from dogs treated with norepinephrine alone and norepinephrine in combination with either phentolamine, propranolol, or AY-21,011 increased the angiotensin II generated approximately sixfold in each instance, indicating that these agents had no effect on endogenous renin substrate or possible plasma activators or inhibitors. Renin secretion was calculated as the product of renal blood flow and the difference between arterial and RVRA in samples obtained at the end of the 30-minute infusion period. Renal blood flow was determined by measuring the clearance of PAH and correcting the value obtained for the extraction of PAH. PAH was measured by an automated modification of the technique of Bratton and Marshall. Urinary sodium concentration was measured by flame photometry. Serum and urinary creatinine W-TEST (KIDNEY 1CONTCOL KIDNEY FIGURE 1 Effects of renal artery infusion of cyclic AMP and norepinephrine and systemic administration of adrenergic receptor antagonists on renal vein renin activity (RVRA). RVRA of nucleotide-infused kidney (solid circles) rose during 30-minute infusion periods (shaded columns) while RVRA of the control kidney (open circles) did not change significantly. (See text.) Circulation Research, Vol XXIX, Sspltvibtr 1971

3 INTRACELLULAR MEDIATION OF RENIN SECRETION 241 levels were measured by an automated modification of the method of Folin and Wu (10). Statistical significance was determined by Student's t-test. Results Bilateral Infusion. Figure 1 illustrates a representative experiment in an anesthetized dog with bilateral renal arterial and venous catheters. During infusion of cyclic AMP (22 fig kg" 1 mkr 1 ) into the left renal artery, there was a definite increase in RVEA, the peak occurring in 30 minutes, whereas RVRA from the contralateral kidney showed no change. In contrast, when either phentolamine (8.8 p-g kg~ ] min" 1 ) or propranolol (2.2 fig kg- 1 mnr 1 ) was administered systemically in combination with intrarenal cyclic AMP infusion, the rise in RVRA was completely suppressed. In addition, norepinephrine (0.06 fig kg 1 mnr 1 ) also increased RVRA, an effect which was inhibited by systemic administration of propranolol. Similar results were observed in two other experiments with bilateral renal artery infusions. In each case RVRA rose with infusion of cyclic AMP, and the rise was blocked by simultaneous administration of either phentolamine or propranolol. No significant change in RVRA was observed in the control kidney. Effects of Nucleotides, Adenosine, and Adrenergic Receptor Antagonists on Renin Activity. The effects of renal artery infusion of various nucleotides and adenosine on RVRA and renin secretion are summarized in Table 1. Infusion of cyclic AMP increased mean RVRA in 14 experiments from ± 27.2 (SD) ng/100 ml to a maximum of ±26.2 ng/100 ml, a highly significant difference (P < 0.001) when evaluated by a paired t-test. The peak rise in RVRA was usually observed at the end of the 30-minute infusion of cyclic AMP. During the recovery phase following cyclic AMP infusion, mean RVRA fell to ±27.8 ng/100 ml (P< 0.001). In nine additional studies (Table 1 and Fig. 2), mean renin secretion rose from 35.5 ±36.7 to a maximum of ± ng/min during infusion of cyclic AMP (P<0.01) and fell to 27.8 ± 26.7 ng/min during the recovery period (P < 0.01). When intrarenal infusion of cyclic AMP was accompanied by systemic administration of HMEPIKEPHRIPE KMEP1HCPW HE + + PTOPBArtOLCH. PHEBTOLAmNE FIGURE 2 Effect of infusion of cyclic AMP, norepinephrine, and adrenergic receptor-blocking agents on renin secretion. Circles represent values for renin secretion during 30-mimrte infusion periods, and squares designate those during 30-minute control and recovery periods. Circulation RcsMrcb, Vol. XXIX. Stptember 1971

4 242 WINER, CHOKSHI, WALKENHORST Effects of Nrtdeotides, A denosine, and Adrenergic Antagonists on Rcnin and Renal Hrmodyruiniics Agent Cyclic AMP Cyclic AMP + phentolamine Cyclic AMP + propranolol ATP ADP 5' AMP Adenosine RVRA (ng/100ml) ± =±= 26.2* ±27.8* ± ± 1S ± =t ± * ± ± ± ± =*= ± =t =t ± ± ± ± 30.0 Art. rtnin activity (ng/looml) ± = = 20.3* ± 20.4* ± ± ± ± ± * ± ± =t = = ± 30.S ± * ± =t ± ± ± 30.4 Renin secretion (ng/min) 35.5 ± ± 27.8 =t 14.6 =*= 16.9 ± 29.2 ± 31.4 =*= 35.4 ± 15.S =>= 17.2 ± 12.5 =<= 36.1 ± 15.7 ± 11.8 =<= 39.6 ± 15.9 * 29.4 ± 26.9 =t 26.1 =*= 20.8 =<= 16.0 ± 36.7 ]21.3t 26.7t TABLE RBF (ml/min) 1S4.0 ± =*= ± ± =t ± ± ± * ± ± =t ± ± =* ± ± ± ± ± ± Values are means ="= SD of four or more observations. P values by paired (-test: *<0.001; f<0.01; t<0.05. RVRA = renal vein reuin activity; RBF = renal blood flow; C or = creatinine clearance; MABP = mean arterial blood pressun phentolamine (8.8 jig kg" 1 mkr 1 ), there was no significant- change in mean RVRA or mean renin secretion. The rise in mean RVRA and mean renin secretion with infusion of cyclic AMP alone differed significantly from that associated with the cyclic AMP-phentolamine combination (P < and F<0.05, respectively). Likewise, systemic administration of fzz-propranolol completely suppressed the cyclic AMP-induced rise in RVRA and renin secretion. Again, the difference between the rise of mean RVRA or mean renin secretion with nucleotide infusion alone compared with that of nucleotide-dz-propranolol administration was significant (P < and P = 0.05). In contrast to the results obtained with cyclic nucleotides, infusion of ATP (41 /xg kg" 1 min- 1 ), ADP (33 /zg kg- 1 min" 1 ), 5'AMP (24 /u,g kg" 1 min" 1 ), and adenosine (18 yu,g kg" 1 min" 1 ) produced no significant change in mean RVRA or mean renin secretion (Table 1). Effects of Sympathomimetic Amines and Adrenergic Receptor Antagonists on Renin Activity. As shown in Table 2 and Figures 2 and 3, with infusion of norepinephrine (0.06 fjig kg" 1 min" 1 ) into the renal artery in seven experiments, mean RVRA increased from ±18.0 ng/100 ml to a maximum of ±41.8 ng/100 ml (P< 0.001), and returned to ± 19.8 ng/100 ml during the recovery phase (P< 0.001). In six other experiments with norepinephrine infusion, mean renin secretion rose from 16.9 ± 12.5 to ± 56.5 ng/min (P < 0.02) and fell to 32.8 ± 31.7 ng/min during the recovery period (P<0.05). With simultaneous systemic administration of either phentolamine (8.8 tig kg" 1 min- 3 ) or rfz-propranolol (2.2 ^u.g kg" 1 mill" 1 ), intrarenal norepinephrine infusion produced no rise in mean RVRA or mean renin secretion. The difference between the rise in mean RVRA with norepinephrine infusion alone, and that with the norepinephrine-phentolamine or norepinephrine-dz-propranolol combination was highly significant (P < 0.001). A similar difference was observed in mean renin secretion (P < 0.02 and P= 0.02, respectively). Circulation Resetrcb, Vol. XXIX, September 1971

5 INTRACELLULAR MEDIATION OF RENIN SECRETION 243 Cor Til kg" 1 mill" 1 ) 1.7 ± = = ± ± ± ± * ± = = ± ± =t ± l.l 2.2 ± ± ± =fc ± ± ± ± 1.7 Urine flow OJiter kg" 1 min" 1 ) 37.8 ± S.4 ± ± ± ± ± =*= ± =*= =t =*= ± ± ± ± =>= ± ± =t ± ± 22.1 Urine Na OiEq kg" 1 min" 1 ) 6.3 ± 6.3 =»= 6.7 ± 6.7 ± 5.8 ± 4.6 ± 3.7 =*= 3.8 ± 4.0 ± 12.4 ± 12.2 ± 12.2 ± 11.6 ± 12.1 ± 10.5 ± 7.3 =t 6.2 ± 6.2 ± 6.0 ± 7.1 ± 6.8 ± MABP (mm Hg) ± ± ± ± ± ± 21.9 ± ± ± ± ± ± ± ± ± ± ± =fc =t =t ± 5.3 Heart rate (beau/min) ± ± ± ± ± ± ± ± =t ± ± ± ± =fc ± * ± ± ± ± ± 8.3 ISOPKOTEREXOl ISWROTERCHOL + D-PROPRANOLOL ISOnoTEREKL AY ISOrftOTEUKOL LIDOCAKE FIGURE 3 Effect of infmion of isoproterenol, adrenergic receptor-blocking agents, and lidocaine on renin secretion. Circles represent values for renin secretion during 30-minute infusion periods, and squares designate those during 30-minute control and recovery periods. Circulation Roiearcb, Vol. XXIX, September 1$>71

6 244 WINER, CHOKSHI, WALKENHORST Effects of Sympalhomimetic Amines and Adrenergic Receptor Antagonists on Renin and Renal Hemodynamics Agent Norepinephrine Norepinephrine + phentolamine Norepinephrine + propranolol Isoproterenol Isoproterenol + phentolamine Isoproterenol + J-propranolol Isoproterenol 4- d-propranolol Iaoproterenol + AY-21,011 Iaoproterenol + lidocaine RVRA (n«/100 ml) ± ± 41.8* =t 19.8* ± ± ± ± ± =t ± ± 64.7* =* 29.1* ± ± ± ± ± ± ± ± ± ± ± ± ± ± 54.7* =±= 27.1* Art. renin»ctivity (ng/100 ml) ± ± 36.4f ± 28.4f ± ± ± ± ± = = ± =t 72.3* ± 42.8* ± ± ± = = ± ± S ± ± ± =t S ± =t ± =fc ± 17.5$ Kenln iecretion (ng/min) 16.9 ± ± 56.5$ 32.8 = = 31.7$ 30.5 ± =t ± ± =<= ± d= ± 67.4f 23.4 ± 15.8f 17.0 ± ± ± ± ± ± ± ± ± ± ± * ± ± ± 15.7$ TABLE 2 RBF (ml/min) =t ± 50.6} ± 37.6J ± ± ± ± ± =* ± ± ± ± ± ± ± =t ± ± ± ± ± ± ± * * ± 55.2 Values are means = = SD of four or more observations. P values by paired (-test: *P < 0.001; fp < 0.01; JP < With intrarenal administration of isoproterenol (0.01 fig kg" 1 mkr 1 ), mean RVRA rose from a control level of ± 27.6 ng/100 ml to a maximum of ± 64.7 ng/100 ml (P< 0.001) and fell significantly to ± 29.1 ng/100 ml during the recovery period (P < 0.001). Mean renin secretion rose from 31.4 ± 26.3 to ± 67A ng/min (P < 0.005) and fell to 23 ± 16 ng/100 ml (P<0.01). When phentolamine (8.8 /xg kg" 1 mkr 1 ) was given systemically, there was a significant suppression of the isoproterenol-induced rise in both mean RVRA and renin secretion compared with the effect of isoproterenol infusion alone (P< and P < 0.01, respectively). Similarly, systemic administration of either the dextro or levo isomer of propranolol in doses identical to that of dz-propranolol produced a comparable degree of suppression of the isoproterenol-stimulated renin release. With either the dextro or levo isomer the rise in mean RVRA was significantly less than with isoproterenol infusion alone, although complete suppression was not achieved. A similar suppression in mean renin secretion was observed with d- and Z-propranolol. Systemic infusion of AY- 21,011 (8.8 fxg kg" 1 min" 1 ), an agent which specifically blocks /3-receptor sites and lacks the local anesthetic or quinidinelike effect of propranolol (11), completely blocked the isoproterenol-induced rise in RVRA (P< 0.001) and renin secretion (P < 0.005). In contrast, lidocaine (3.3 mg kg" 1 mkr 1 ), a local anesthetic, had no significant inhibitory effect on the isoproterenol-stimulated rise in either RVRA or renin secretion, although the effects of lidocaine on renin secretion were somewhat variable. Circulation Research, Vol. XXIX, September 1971

7 INTRACELLULAR MEDIATION OF RENIN SECRETION 245 Car ll kg"' mln-') 2.1 ± = = 0.4 L.5 ± ± 0.1 i.o ± ± ± ± ± 0.9 ).6 ± 0.4 ).5 ± 0.3 ).6 ± ± ± =>= 0.3 L.9 ± ± =* ± ± ± ± ± ± ± ± ± 0.9 Urine flow (filter kg" 1 min-i) 20.9 ± ± * ± ± * =*= ± ± =t =t ="= ± =<= ± ± ± * ± =t =t =t * ± =fc ± ± 28.8 Urine Na (^i q kg" L Tnin -1 ) 2.3 ± ± ± ± ± =fc ± ± ± ± ± ± ± =t ± ± =t ± ± ± ± =t ± ± ± =»= ± 3.6 Effects on Renal Function and Hemodynamics As shown in Table 1, mean renal blood flow fell during renal artery infusion of cyclic AMP from a control value of ± 64.9 to ± 66.0 ml/min (P < 0.05) and failed to rise during the recovery phase. Renal blood flow was not significantly changed by cyclic AMP-phentolamine or cyclic AMP-propranolol infusion; similarly, no significant alterations in renal blood flow were observed during infusion of ATP, ADP, 5'AMP, or adenosine. In addition, creatinine clearance, urine flow, urinary sodium excretion, mean arterial blood pressure, and heart rate were not significantly changed during infusion of cyclic AMP alone or in combination with adrenergic receptor-blocking agents, or during administration of ATP, ADP, 5'AMP, and adenosine. Infusion of norepinephrine into the renal CircuUsio* Research, Vol. XXIX, Sepl+mbtr 1971 MABP (mm Hg) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± * ± ± ± =t ± =t * 13.5 Heart rate (beats/mln) ± ± ± ± ± ± ± ± =<= ± =*= ± ± ± ± ± ± ± ± =* ± ± ± ± =t ± * 28.4 artery (Table 2) decreased mean renal blood flow from a control level of ±56.1 to ±50.6 ml/min (P<0.05). Renal blood flow rose nearly to the control value after the norepinephrine infusion. With simultaneous administration of norepinephrine and phentolamine, the flow was unchanged, whereas during norepinephrine-propranolol infusion it tended to decrease, although the fall was not statistically significant. Isoproterenol, when administered alone or with either adrenergic blocking agents or lidocaine, produced no significant change in mean renal blood flow. No significant alterations in creatinine clearance, urine flow, urinary sodium excretion, mean arterial blood pressure, or heart rate were observed during infusion of norepinephrine or isoproterenol alone, or with administration of these agents in combination with adrenergic blocking agents or lidocaine.

8 246 WINER, CHOKSHI, WALKENHORST Discussion In the present study, cyclic AMP consistently stimulated renin secretion when infused into the renal artery of the dog. That the site of action of cyclic AMP was within the kidney is indicated by the lack of any change in RVRA from the contralateral kidney. Norepinephrine in the dose used also increased RVRA only in the catecholamine-infused kidney, suggesting that its action, likewise, was mediated within the kidney. The absence of a significant rise in blood pressure during norepinephrine infusion also indicates that relatively little of the catecholamine entered the systemic circulation. The rise in RVRA during infusion of cyclic AMP and norepinephrine was probably not due to decreased renal blood flow, since renin secretion, calculated as the product of renal blood flow and arterial-renal venous renin difference, also increased with cyclic AMP and norepinephrine infusion. Although infusion of ATP, ADP, 5'AMP, and adenosine had no effect on renal blood flow in the present study, evidence has been presented that renal vascular dilation occurs during ATP administration (12, 13) as a result of efferent dilation (14) and, conversely, that total renal vascular resistance increases with infusion of either adenosine or 5'AMP (13) presumably because of afferent arteriolar constriction (14). However, the discrepancy between the findings of the present study and those of others may be related to the higher doses of ATP used (13), the use of a single injection technique (14), and the use of salt-depleted dogs (15). In any event, the failure of ATP, ADP, 5'AMP, and adenosine to stimulate renin secretion, when administered in doses of the same molar quantities as cyclic AMP, would suggest that cyclic AMP is specific among adenine compounds in stimulating renin secretion. Moreover, administration of norepinephrine, as shown in this and other studies (15, 16), causes renal vasoconstriction, whereas injection of isoproterenol into the renal artery either increases or does not alter renal blood flow (16). In the present study, cyclic AMP and norepinephrine increased renin secretion and reduced renal blood flow, whereas isoproterenol enhanced renin secretion and adrenergic receptor antagonists suppressed isoproterenol-stimulated renin secretion without affecting renal blood flow; therefore, it would appear that renin secretion is independent of changes in renal blood flow. The observation that mean arterial renin activity rose during infusion of cyclic AMP, norepinephrine, and isoproterenol (Tables 1 and 2) also suggests that these agents increased renin secretion, rather than merely increasing renal venous renin concentration. Our data also indicate that renin secretion is dissociated from alterations in urinary sodium excretion, glomenilar filtration rate, and urine flow, heart rate, and blood pressure. The recent report of Michelalds, et al (8) showing that cyclic AMP and catecholamines stimulate renin production in a renal cell suspension likewise suggests that these agents stimulate renin secretion independently of changes in renal hemodynamics. We have previously shown that theophylline, ethacrynic acid, and diazoxide stimulate renin secretion in man (1). While the effect of these agents on cyclic AMP levels in the kidney has not yet been determined, theophylline (17), and possibly ethacrynic acid (19) and diazoxide (18, 19) may act by inhibiting cyclic nucleotide phosphodiesterase. Thus, their effect on renin release may be mediated, in part, by intracellular accumulation of cyclic AMP. The data presented here show that the stimulatory effect of cyclic AMP on renin secretion can be blocked by either a- or J3- receptor antagonists (1). If these physiologic and pharmacologic stimuli are causing renin release by an action at the plasma membrane adrenergic receptor site, it is surprising that both a- and /3-receptor-blocking agents inhibit their effect on renin secretion. In fact, the suppression of cyclic AMP-induced renin release by phentolamine or propranolol suggests that the latter agents are acting at a locus distal to cyclic AMP production, rather than at plasma membrane receptor sites. A similar site of action has been suggested to explain the inhibition of dibutyryl cyclic AMP- Citculation Research, Vol. XXIX, September 1971

9 INTRACELLULAR MEDIATION OF RENIN SECRETION 247 induced insulin secretion by sotalol (MJ 1999), a /3-receptor-blocking agent (20). In addition, a- and y3-receptor-blocking agents inhibit adipose tissue lipolysis induced by cyclic AMP (21, 22). Also, epinephrine and cyclic AMP induced hyperglycemia in intact rats (23) and in the isolated perfused rat liver (24) could be prevented by administration of dihydroergotamine, an a-receptor-blocking agent. Thus, in vivo and in vitro evidence from experiments with other organ systems indicates that adrenergic antagonists may exert an inhibitory action at a site beyond adenyl cyclase. However, a site of action as yet undefined and remote from the cyclic AMP sequence, cannot be excluded. The possibility that phentolamine and propranolol might also be inhibiting renin secretion by blocking adrenergic receptor sites on the plasma membrane is unlikely in view of other data presented in this report. While both norepinephrine, a predominantly a-receptor stimulator, and isoprotercnol, a /3-receptor agonist, enhanced renin secretion, their stimulatory effects were reversed by /3- and a- receptor-blocking agents, respectively. Such effects are not explainable on the basis of the presently accepted concepts of a~ and /3- receptors (25), unless one postulates the presence of an "indifferent" receptor. Moreover, it is unlikely that the /3-receptor or the adenyl cyclase system or both may be serving as the site of action of the adrenergic receptorblocking agents in view of the equipotent inhibitory effect of the dextro and levo isomers of propranolol on isoproterenol-induced renin secretion. Studies of several /3-receptor-blocking agents have shown that the blocking effect of adenyl cyclase is manifested by the levo form, whereas the dextro form is relatively ineffective (27,28). It is possible that the inhibitory effects of dlpropranolol and its isomers might have been due to the local anesthetic property of these agents, especially since the dextro and levo forms were equally active in suppressing renin secretion (27). Since propranolol-induced inhibition of catecholamine release from the CircuUtwn Research, Vol. XXIX, September 1971 perfused cat adrenal medulla can be reversed by increasing the calcium concentration of the perfusate, it has been suggested that the inhibitory action of propranolol may be due to its local anesthetic property, which in turn reduces membrane permeability to calcium. However, the ability of AY-21,011, a /3-receptor-blocking agent which lacks a local anesthetic effect (10), to suppress completely the isoproterenol-induced rise in renin secretion suggests that propranolol and its isomers inhibit renin secretion by a mechanism other than a local anesthetic or quinidinelike effect. The failure of large doses of lidocaine, a local anesthetic agent, to suppress isoproterenolinduced renin secretion is consistent with this hypothesis. Whether other types of pharmacologic antagonists, such as autimuscarinic agents, ganglionie blockers, or antihistamines, might inhibit renin secretion was not examined in the present study. Another possibility is that the adrenergic receptor antagonists might have suppressed renin activity by inhibiting endogenous renin substrate production or by promoting the release of activators or inhibitors of the reninangiotensin system. These possibilities would seem unlikely since the addition of exogenous renin to the assay system produced the expected rise in angiotensin generation in samples obtained during infusion of adrenergic receptor antagonists. Recently, Tagawa and Vander (14) reported that renal venous renin was either suppressed or unchanged by cyclic AMP, in contrast to the findings of the present study and those of Michelakis et al (8). However, it is possible that since Tagawa and Vander studied salt-depleted dogs, further increases in renal venous renin were obscured by levels of renin activity that were already high. In addition, they found inhibition of renin secretion only with doses of cyclic AMP that were 3- to 15-fold higher than were used in the present study, raising the possibility that the suppression of renin secretion they observed might represent a nonspecific toxic effect of the nucleotide.

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