- Email: [email protected]
Effects of papaverine on basal and on isoproterenol-stimulated renin secretion from rat kidney slices
Life Sciences, Vol. 27, pp. 1299-1305 Printed in the U.S.A.
Pergamon Press
EFFECTS OF PAPAVERINE ON BASAL AND ON ISOPROTERENOL-ST!MULATED RENIN SECRETION FROM RAT KIDNEY SLICES Paul C. Churchill, Franklin D. McDonald, and Monique C. Churchill Departments of Physiology and Internal Medicine Wayne State University School of Medicine and Hutzel Hospital Detroit, Michigan 48201 (Received in final form July 24, 1980) Summary Papaverine inhibited the basal renin secretion of rat kidney slices incubated in a physiological salt solution at 37°C. Inhibition was concentration-dependent; secretion was 99 + 0.2 % inhibited by 5 x 10 -4 M papaverine, and 8 x 10 -5 M was the ~stimated ED In 50" contrast, 2 x 10 -4 M IBMx (3-isobutyl-l-methyl-xanthine) increased the rate of secretion from 215 + 17 to 366 + 30 ng hr -I mg-i/20 min (p < 0.001). Isoproterenol (4--x 10 -7 , 8 x--10-7, and 5 x 10 -6 M) stimulated renin secretion in a concentration-dependent manner; the stimulatory effects were antagonized by papaverine but unaffected by IBMx. Thus, two known inhibitors of phosphodiesterase--IBMx and papaverine--produce sharply contrasting effects on basal and on isoproterenol-stimulated renin secretion from rat kidney slices. Papaverine has been used extensively in studies designed to elucidate the mechanisms which control renin secretion from the kidney (I-7). The rationale for its use in in vivo experiments is that papaverine dilates the renal vasculature, thereby blocking the baroreceptor mechanism for the control of renin secretion (8). Intrarenal arterial infusion of papaverine (in innervated and/or denervated non-filtering dog kidneys) blocks the stimulatory effects on renin secretion of a wide variety of stimulants including epinephrine (3), and the addition of papaverine to the perfusate blocks isoproterenol-stimulated renin secretion of the isolated-perfused rat kidney (i). That papaverine blocks the stimulatory effect of catecholamines is somewhat surprising. B4ta-adrenergic agonists presumably stimulate renin secretion by activation of adenylate cyclase, which results in increased intracellular adenosine 3',5'-cyclic monophosphate, or c-AMP (9-13). Papaverine is a potent inhibitor of phosphodiesterase activity (14,15), and according to the accepted criteria for establishing a second-messenger role for c-AMP, phosphodiesterase inhibitors should not only mimic, but potentiate, the effect of catecholamines. Due to their effects on the renal vasculature, and therefore on intrarenal pressures, the effects on in vivo renin secretion of substances such as papaverine and isoproterenol are difficult to interpret. Accordingly, it seemed worthwhile to examine their effects, singly and in combination, on renin secretion from rat kidney slices, a preparation in which the influences of renal hemodynamics are obviously excluded.
0024-3205/80/401299-07502.00/0 Copyright (c) 1980 Pergamon Press Ltd
1300
Papaverine,
Isoproterenol
and Renin Secretion
Vol. 27, No. 14, 1980
Methods Experiments were performed on adult (200-280 gram) Sprague-Dawley male rats. Prior to use, the animals were caged in a constant temperature room and given free access to tap water and Purina Lab Chow (0.45 % Na by weight). T e c h n i q u e s for studying renin secretion from rat kidney slices have been described in detail p r e v i o u s l y (16). Briefly, rats were a n e s t h e t i z e d with ether and b i l a t e r a l l y nephrectomized. Using a razor blade, four thin slices were cut from each kidney. Slices from five animals were pooled and randomized for each e x p e r i m e n t a l run. Slices were blotted gently and placed in flasks (two slices/ flask) which contained i0 ml of m e d i u m which had been equilibrated previously at 37oc with a m i x t u r e of 02 (95%) and CO 2 (5%). The flasks were placed in an o s c i l l a t i n g metabolic incubator at 37°C and gassed continuously throughout the incubation. Periodically, as described in the results section, the m e d i u m was sampled (200 ~i); samples were centrifuged at 4°C (8,000 g, 5 min), and the supernatants were frozen until renin assay. Following the incubations, the slices were dried to constant weight. The i n c u b a t i o n m e d i u m consisted of 124 NaCI, 19 NaHCO3, 4 KCI, 2.6 CaCI2, 1.2 NaH2PO4, and 0.8 MgSO 4 (in mM), and 0.2 grams % each of glucose and bovine albumin (Difco: Detroit, Michigan). P a p a v e r i n e HCI was obtained from the Amend Drug and Chemical Co. (Irvington, New Jersey), and d , l - i s o p r o t e r e n o l HCI and 3-isobutyl-l-methyl-xanthine (IBMx) from the Sigma Chemical Co. (St. Louis, Missouri). Vehicles for isoproterenol and for IBMx consisted of m e d i u m plus ascorbate (10 -5 M final) and m e d i u m plus ethylene glycol (0.2 %, V/V, final), respectively. Methods for m e a s u r i n g renin activity of the incubation m e d i u m have been described in detail p r e v i o u s l y (16). Briefly, 50 ~I of the r e n i n - c o n t a i n i n g sample was incubated for 30 min at 37°C with 500 ~ i of rat renin substrate. The a n g i o t e n s i n I generated during this incubation was measured by radioimmunoassay. The renin activity of the m e d i u m (ng hr -I ml -I, i.e., ng angiotensin I per hour of i n c u b a t i o n with renin substrate per ml of medium) was m u l t i p l i e d by the total volume of the m e d i u m (ml) and divided by the tissue dry weight (mg), y i e l d i n g the units ng hr -I mg -I. The rate of renin secretion was calculated as the increment in total renin during a given interval of incubation of the slices. Results are given as means + SEMs. Both the paired and the unpaired t tests were used to assess the s ~ a t i s t i c a l s i g n i f i c a n c e of changes or differences. In some cases, three unpaired t tests were performed on the same set of data: therefore, p~0.04 can be considered significant at the 0.05 level (17). Results Rat kidney slices were incubated in media containing 0.0 to 5 x 10 -4 M papaverine, and the media were sampled at 30, 60, and 90 min for the determination of renin. The increments in renin during the three time periods, for several p a p a v e r i n e concentrations, are given in Table i. It can be seen that secretion rate was relatively stable over time in the absence of papaverine. P a p a v e r i n e inhibited secretion, and the extent of inhibition increased over time. The percent response was calculated as (i - V/Vo) x i00 %, where V = the secretory rate during the 30-60 min period, and V o = the secretory rate in the absence of p a p a v e r i n e during this same period. Selecting the second period rather than the first allowed 30 min for secretory rates to stabilize; at zero time, the slices had been transferred from room temperature to 37°C, and from medium without papaverine to media c o n t a i n i n g various concentrations of papaverine.
Vol. 27, No. 14, 1980
Papaverine,
Isoproterenol and Renin Secretion
1301
TABLE 1 Effect of Papaverine on Basal Renin Secretion Papaverine M
N
0.0 I x 10-4 2 x 10-4 5 x 10 -4
6 6 6 6
ng hr -I mg-i/30 min 0-30 30-60 60-90 278 122 81 82
+ $ + +
22 14 4 6
265 117 38 2
+ T ~ +
15 14 4 i
244 i01 24 5
+ + + +
13 i0 1 1
Total renin secreted by the slices was determined (ng hr-i ml -I times ml of medium) and factored by slice dry weight, yielding ng hr-I mg -I. Values are means + SEMs of increments in total renin during the 0-30, 30-60, and 60-90 min periods of incubation of slices. A complete dose-response curve for the effect of papaverine on renin secretion is shown in Figure i. Percent response was calculated as described above. From this figure, the ED50 (concentration at which the response was half-maximal) was estimated to be 8 x 10 -5 M.
~o0
Figure 1
75
25
oi
m SO x lO-S
Log dose-response curve of the effect of papaverine on renin secretion. Rates of secretion during the 30-60 min period of incubation were determined (V), and % response was calculated as (I - V/Vo) x i00 %, where V o = rate of secretion in the absence of papaverine. Means + SEMs with n = 6 determinations at each point.
Popovenr~ M
To determine if papaverine blocked isoproterenol-stimulated, as well as basal, renin secretion, slices were incubated in media containing 0.0, 8 x 10 -7 , and 5 x 10 -6 M isoproterenol. All media contained ascorbate. Samples were taken at 20 and 40 min for renin determinations, then either papaverine (2.5 x 10 -4 M final) or the same volume of diluent was added, the incubations were continued and samples were taken again at 60 and 80 min. Mean increments in renin during the 20-40 and the 60-80 min periods are shown as the first and second columns in each pair of columns in Figure 2. It can be seen that isoproterenol produced a concentration-dependent stimulation of secretion during the first period. Averaging the results of the two series of experiments (the papaverine and the diluent series), gave rates of 148 + 15, 217 + 12, and 286 + 12 ng hr -1 mg-1/20 min for 0, 8 x 10 -7 , and 5 x i0 -~ M isoproterenol, respectively. These rates differ significantly from each other (p <0.001). As can be seen in the top panel, rates did not decrease following the addition of diluent, and the concentration-dependent stimulation by isoproterenol is still evident during this period. In contrast, the results in the bottom panel show that papaverine antagonized basal and isoproterenol-stimulated renin secretion. The paired changes in each of the experiments shown were significant (p< 0.005 maximum). Whether isoproterenol had any effect at all in the presence of papaverine is equivocal; the difference between rates in media containing 0 and 8 x 10 -7 M isoproterenol is not significant at the 0.05 level whereas that between rates in media containing 0 and 5 x 10 -6 M isoproterenol is ( p ~ 0 . 0 3 ) .
1302
Papaverine,
Isoproterenol and Renin Secretion
Diluent
÷
500
Vol. 27, No. 14, 1980
+ 1
T
8 ,.
250
~ ~ u
0
! T
"
I 0
8xlG 7
rr E 5 0 0
Popoverine
o~
2,5x10_4 M
o
c,- ~
250
0
5xlO-6
Isoproterenol, M
5xlO-6
Isoproterenol, M
T
0
8xlO -7
Figure 2
The effect of papaverine on basal and on isoproterenol-stimulated renin secretion. Slices were incubated in media containing isoproterenol, as indicated. Samples were taken at 20, 40, 60, and 80 min. Rates of secretion were taken as the increments in renin between 20-40 min (first columns in each pair) and between 60-80 min (second columns). Lower p a n e l , papaverine was added at 40 min. Upper panel, diluent was added at 40 min. Means + SEMs; n = 5 or 6 each experiment.
Finally, slices were incubated in media containing 2 x 10 -4 M IBM_x or its diluent, ethylene glycol. Samples for renin were taken at 20 and 40 min, then isoproterenol and/or vehicle were added such that final concentrations of isoproterenol were 0, 4 x 10 -7 , and 8 x 10-7 M (ascorbate present in all media). The incubations were continued, and samples were taken again at 60 and 80 min. Mean increments in renin during the 20-40 and the 60-80 min periods are shown as the first and second columns in each pair of columns in Figure 3. It is evident that renin secretion was stimulated by IBMx during the first period. Rates averaged 366 + 30 (n = 18) versus 215 + 17 (n = 18) ng hr -I mg-i/20 minutes for IBMx versus i~s control (p <0.001). --Renin secretion rate was not affected by the addition of ascorbate to either the control or the IBMx-containing media (left side of Figure 3). In contrast, the addition of either concentration of isoproterenol significantly stimulated renin secretion. The paired changes in secretory rates were significant in all four such experiments ( p ~ 0 . 0 5 maximum).
Vol. 27, No. 14, 1980
Papaverine,
Isoproterenol
and Renin Secretion
1303
Isoproterenol, M
6 600
4 x~O-7 t
t
-
8 x i0"7 t T
Control 300
T
o
T
c
o O
O-
c-"
Isoproterenol,M
~E
4 xlO-7
8xlO"7
t
t
i
rt-
~ ~ o
I
600 !BMx, 2x 10"4M
e-
:300
-
_
N Figure 3
The effect of IBMx on basal and on isoproterenol-stimulated renin secretion. Slices were incubated in media containing IBMx (lower panel) or its vehicle (upper panel, "control"). The flrst columns in each pair are the secretory rates during the 20-40 min period of incubation. At 40 min, isoproterenol and/or its vehicle were added. The second columns in each pair are the secretory rates during the 60-80 min periods of incubation. Means SEMs; n = 6 in each of the 6 experiments. The rationale for starting the incubations with IBMx then adding isoproterenol was as follows. As can be seen in Figure 3, secretion rates in the presence of isoproterenol increased between the first and second periods. Had th~ incubations begun with isoproterenol, and had I B ~ been added at 40 min, then any increase in rate between the two periods could have been attributed to greater stimulatory effect of isoproterenol (as seen in the experiments of Fi~qre 2) and/or to an effect of IBMx per se. In any case, the experiments sh~ i n Figures 2 and 3 demonstrate that papaverine inhibits basal as well as i$~ro~erenol-stimulated renin secretion whereas IBMx stimulates secretion and at least does not block the stimulatory effect of isoproterenol.
1304
Papaverine,
Isoproterenol and Renin Secretion
Vol. 27, No. 14, 1980
Discussion In the present experiments, papaverine inhibited the basal secretory rate of rat kidney slices in a concentration-dependent manner, suggesting at least some specificity of action. The observation that renin secretion averaged 2 + i ng hr -I mg -I during the 30-60 minute period of exposure to 5 x 10 -4 M papaverine (Table i) taken together with the previous observation that renin in unincubated rat kidney slices averaged 3702 ng hr -I mg -I (16), suggests that passive leak of renin from non-viable cells was minimal. Indeed, if papaverine were to have a toxic effect, one would expect to find increased rather than decreased rate of renin release from slices exposed to papaverine. These results confirm and extend the previous observation that papaverine inhibits renin secretion from isolated rat renal cortical cells (4). It can be calculated from the results in this previous report that secretion was lower than control during the first hour of exposure to 2.6 x 10 -4 M papaverine, and virtually zero thereafter. As documented in the introduction, infusion of papaverine into the renal artery blocks the effects of a wide variety of renin secretory stimulants. In in vivo experiments, the concentration of papaverine in renal arterial plasma ranged between 10 -4 and 5 x 10 -4 M, even assuming no recirculation (3, 5-7). These concentrations were approximately 50 and I00 % effective in blocking the basal secretory rate of rat kidney slices. Accordingly, one might expect papaverine to inhibit basal secretory rate in vivo. Although papaverine does block the elevated basal rate normally found in Na-deprived dogs and in dogs with thoracic caval constriction (7,8), papaverine does not seem to decrease secretion below the normal basal level (3,5,6). Why this is the case is not clear. Although speculative, one explanation is suggested by the oft-reported observation that basal in vivo renin secretion rate is so low that further reductions are difficult to detect (18-20). Namely, at the normal renal perfusion pressure in vivo, the baroreceptor mechanism must be exerting a tonic inhibitory effect on renin secretion. Such tonic inhibition is clearly absent in the slice preparation, perhaps explaining why basal secretory rates are higher in vitro than in vivo (21). Moreover, since papaverine eliminates the stimulatory effect of decreases in pressure on in vivo renin secretion (9), papaverine would be expected to eliminate the stimulatory effect of a lack of "pressure" on in vitro renin secretion. There is now reasonable accord that papaverine antagonizes beta-adrenergic stimulation of renin secretion. Although Johnson et al. (3) reported that it blocked the effects of epinephrine but not the effects of norepinephrine, the dose of norepinephrine was 50 % larger than the dose of epinephrine; papaverine might well have blocked a lower, more comparable, dose of norepinephrine. Papaverine also blocks isoproterenol-stimulated renin secretion from the isolated-perfused rat kidney (i). It is significant that papaverine blocked the effects of isoproterenol in the slice preparation, in that changes in renal hemodynamics cannot be invoked to explain the effect. Papaverine has a spectrum of diverse actions, including inhibition of oxidative phosphorylation (14), inhibition of cellular uptake of adenosine (2223), and inhibition of phosphodiesterase activity (14,15). It follows that its effects on renin secretion could have a number of explanations. Clearly, much remains to be done to establish the mechanism of action, but explanations based on phosphodiesterase inhibition seem unlikely in view of the observations that papaverine is intermediate in potency between IBMx and theophylline (14,15,20), that IBMx (present study) and theophylline stimulate renin secretion per se and/
Vol. 27, No. 14, 1980
Papaverine,
Isoproterenol
and Renin Secretion
1305
or potentiate the stimulatory effect of catecholamines (10,11) and that papaverine inhibits renin secretion per se and blocks beta-adrenergic stimulation.* Acknowledsements This work was supported by a grant from NIH support from the BMA Management Company.
(HL
24880-01),
and
salary
References
i. 2. 3. 4.
5. 6. 7. 8. 9. i0. II. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
J.C.S. FRAY, J. Physiol. 282:207-217 (1978). K. GAAL, J. SIKLOS, T. MOZES AND G.F. TOTH, Acta Physiol. Acad. Sci. Hung. 51:305-314 (1978). J.A. JOHNSON, J.O. DAVIS AND R.T. WITTY, Circ. Res. 29:646-653 (1971). H.J. LYONS AND P.C. CHURCHILL, Proc. Soc. exp. Biol. Med. 160:237-240 (1979). A.A. SEYMOUR AND J.E. ZEHR, Circ. Res. 45:13-25 (1979). R.T. WITTY, J.O. DAVIS, J.A. JOHNSON AND R.L. PREWITT, Am. J. Physiol. 221:1666-1671 (1971). R.T. WITTY, J.O. DAVIS, R.E. SHADE, J.A. JOHNSON AND R.L. PREWITT, Circ. Res. 31:339-347 (1972). J.O. DAVIS AND R.H. FREEMAN, Physiol. Rev. 56:1-56 (1976). A.M. MICHELAKIS, J. CAUDLE AND G.W. LIDDLE, Proc. Soc. exp. Biol. Med. 130:748-753 (1969). H.L. NOLLY, I.A. REID AND W.F. GANONG, Circ. Res. 35:575-579 (1974). R.J. VISKOPER, M.H. MAXWELL, A.N. LUPU AND S. ROSENFELD, Am. J. Physiol. 232:F248-F253 (1977). N. WINER, D.S. CHOKSHI, M.S. YOON AND A.D. FREEDMAN, J. Clin. Endocrinol. Metab. 29:1168-1175 (1969). N. WINER, D.S. CHOKSHI AND W.G. WALKENHORST, Circ. Res. 29:239-248 (1971). M. FERRARI, Pharmacol. Res. Com. 6:97-115 (1974). L. TRINER, Y. VULLIEMOZ AND M. VEROSKY, Eru. J. Pharmacol. 41:37-46 (1977). P.C. CHURCHILL, J. Physiol. 294:123-134 (1979). C.W. DUNNET, Statistics in Endocrinolosy, edited by J.W. McARTHUR AND T. COLTON, pp. 79-103, MIT Press, Cambridge, Mass. (1970). A.J. VANDER AND R. MILLER, Am. J. Physiol. 207:537-546 (1964). H. TAGAWA AND A.J. VANDER, Circ. Res. 26:327-338 (1970). P.C. CHURCHILL AND F.D. McDONALD, J. Physiol. 242:635-646 (1974). K. BLENDSTRUP, P.P. LEYSSAC, K. POULSEN AND S.L. SKINNER, J. Physiol. 246:653-672 (1975). M. HUANG AND G.I. DRUMMOND, Biochem. Pharmacol. 25:2713-2719 (1976). J.W. PHILLIS, J.P. EDSTROM AND G.K. KOSTOPOULOS, Can. J. Physiol. Pharmacol. 57:1289-1312 (1979).
Seymour and Zehr (5) found that papaverine increased the efflux of c-AMP from dog kidney, which suggests that papaverine increased renal tissue levels of c-AMP in their experiments. Gaal et al. (2) measured tissue levels of c-AMP and found a significant increase following papaverine administration. Unfortunately, the heterogeneity of cell populations in the kidney precludes any estimate of intracellular c-AMP in the renin-secreting population, a small fraction of the total.
Pergamon Press
EFFECTS OF PAPAVERINE ON BASAL AND ON ISOPROTERENOL-ST!MULATED RENIN SECRETION FROM RAT KIDNEY SLICES Paul C. Churchill, Franklin D. McDonald, and Monique C. Churchill Departments of Physiology and Internal Medicine Wayne State University School of Medicine and Hutzel Hospital Detroit, Michigan 48201 (Received in final form July 24, 1980) Summary Papaverine inhibited the basal renin secretion of rat kidney slices incubated in a physiological salt solution at 37°C. Inhibition was concentration-dependent; secretion was 99 + 0.2 % inhibited by 5 x 10 -4 M papaverine, and 8 x 10 -5 M was the ~stimated ED In 50" contrast, 2 x 10 -4 M IBMx (3-isobutyl-l-methyl-xanthine) increased the rate of secretion from 215 + 17 to 366 + 30 ng hr -I mg-i/20 min (p < 0.001). Isoproterenol (4--x 10 -7 , 8 x--10-7, and 5 x 10 -6 M) stimulated renin secretion in a concentration-dependent manner; the stimulatory effects were antagonized by papaverine but unaffected by IBMx. Thus, two known inhibitors of phosphodiesterase--IBMx and papaverine--produce sharply contrasting effects on basal and on isoproterenol-stimulated renin secretion from rat kidney slices. Papaverine has been used extensively in studies designed to elucidate the mechanisms which control renin secretion from the kidney (I-7). The rationale for its use in in vivo experiments is that papaverine dilates the renal vasculature, thereby blocking the baroreceptor mechanism for the control of renin secretion (8). Intrarenal arterial infusion of papaverine (in innervated and/or denervated non-filtering dog kidneys) blocks the stimulatory effects on renin secretion of a wide variety of stimulants including epinephrine (3), and the addition of papaverine to the perfusate blocks isoproterenol-stimulated renin secretion of the isolated-perfused rat kidney (i). That papaverine blocks the stimulatory effect of catecholamines is somewhat surprising. B4ta-adrenergic agonists presumably stimulate renin secretion by activation of adenylate cyclase, which results in increased intracellular adenosine 3',5'-cyclic monophosphate, or c-AMP (9-13). Papaverine is a potent inhibitor of phosphodiesterase activity (14,15), and according to the accepted criteria for establishing a second-messenger role for c-AMP, phosphodiesterase inhibitors should not only mimic, but potentiate, the effect of catecholamines. Due to their effects on the renal vasculature, and therefore on intrarenal pressures, the effects on in vivo renin secretion of substances such as papaverine and isoproterenol are difficult to interpret. Accordingly, it seemed worthwhile to examine their effects, singly and in combination, on renin secretion from rat kidney slices, a preparation in which the influences of renal hemodynamics are obviously excluded.
0024-3205/80/401299-07502.00/0 Copyright (c) 1980 Pergamon Press Ltd
1300
Papaverine,
Isoproterenol
and Renin Secretion
Vol. 27, No. 14, 1980
Methods Experiments were performed on adult (200-280 gram) Sprague-Dawley male rats. Prior to use, the animals were caged in a constant temperature room and given free access to tap water and Purina Lab Chow (0.45 % Na by weight). T e c h n i q u e s for studying renin secretion from rat kidney slices have been described in detail p r e v i o u s l y (16). Briefly, rats were a n e s t h e t i z e d with ether and b i l a t e r a l l y nephrectomized. Using a razor blade, four thin slices were cut from each kidney. Slices from five animals were pooled and randomized for each e x p e r i m e n t a l run. Slices were blotted gently and placed in flasks (two slices/ flask) which contained i0 ml of m e d i u m which had been equilibrated previously at 37oc with a m i x t u r e of 02 (95%) and CO 2 (5%). The flasks were placed in an o s c i l l a t i n g metabolic incubator at 37°C and gassed continuously throughout the incubation. Periodically, as described in the results section, the m e d i u m was sampled (200 ~i); samples were centrifuged at 4°C (8,000 g, 5 min), and the supernatants were frozen until renin assay. Following the incubations, the slices were dried to constant weight. The i n c u b a t i o n m e d i u m consisted of 124 NaCI, 19 NaHCO3, 4 KCI, 2.6 CaCI2, 1.2 NaH2PO4, and 0.8 MgSO 4 (in mM), and 0.2 grams % each of glucose and bovine albumin (Difco: Detroit, Michigan). P a p a v e r i n e HCI was obtained from the Amend Drug and Chemical Co. (Irvington, New Jersey), and d , l - i s o p r o t e r e n o l HCI and 3-isobutyl-l-methyl-xanthine (IBMx) from the Sigma Chemical Co. (St. Louis, Missouri). Vehicles for isoproterenol and for IBMx consisted of m e d i u m plus ascorbate (10 -5 M final) and m e d i u m plus ethylene glycol (0.2 %, V/V, final), respectively. Methods for m e a s u r i n g renin activity of the incubation m e d i u m have been described in detail p r e v i o u s l y (16). Briefly, 50 ~I of the r e n i n - c o n t a i n i n g sample was incubated for 30 min at 37°C with 500 ~ i of rat renin substrate. The a n g i o t e n s i n I generated during this incubation was measured by radioimmunoassay. The renin activity of the m e d i u m (ng hr -I ml -I, i.e., ng angiotensin I per hour of i n c u b a t i o n with renin substrate per ml of medium) was m u l t i p l i e d by the total volume of the m e d i u m (ml) and divided by the tissue dry weight (mg), y i e l d i n g the units ng hr -I mg -I. The rate of renin secretion was calculated as the increment in total renin during a given interval of incubation of the slices. Results are given as means + SEMs. Both the paired and the unpaired t tests were used to assess the s ~ a t i s t i c a l s i g n i f i c a n c e of changes or differences. In some cases, three unpaired t tests were performed on the same set of data: therefore, p~0.04 can be considered significant at the 0.05 level (17). Results Rat kidney slices were incubated in media containing 0.0 to 5 x 10 -4 M papaverine, and the media were sampled at 30, 60, and 90 min for the determination of renin. The increments in renin during the three time periods, for several p a p a v e r i n e concentrations, are given in Table i. It can be seen that secretion rate was relatively stable over time in the absence of papaverine. P a p a v e r i n e inhibited secretion, and the extent of inhibition increased over time. The percent response was calculated as (i - V/Vo) x i00 %, where V = the secretory rate during the 30-60 min period, and V o = the secretory rate in the absence of p a p a v e r i n e during this same period. Selecting the second period rather than the first allowed 30 min for secretory rates to stabilize; at zero time, the slices had been transferred from room temperature to 37°C, and from medium without papaverine to media c o n t a i n i n g various concentrations of papaverine.
Vol. 27, No. 14, 1980
Papaverine,
Isoproterenol and Renin Secretion
1301
TABLE 1 Effect of Papaverine on Basal Renin Secretion Papaverine M
N
0.0 I x 10-4 2 x 10-4 5 x 10 -4
6 6 6 6
ng hr -I mg-i/30 min 0-30 30-60 60-90 278 122 81 82
+ $ + +
22 14 4 6
265 117 38 2
+ T ~ +
15 14 4 i
244 i01 24 5
+ + + +
13 i0 1 1
Total renin secreted by the slices was determined (ng hr-i ml -I times ml of medium) and factored by slice dry weight, yielding ng hr-I mg -I. Values are means + SEMs of increments in total renin during the 0-30, 30-60, and 60-90 min periods of incubation of slices. A complete dose-response curve for the effect of papaverine on renin secretion is shown in Figure i. Percent response was calculated as described above. From this figure, the ED50 (concentration at which the response was half-maximal) was estimated to be 8 x 10 -5 M.
~o0
Figure 1
75
25
oi
m SO x lO-S
Log dose-response curve of the effect of papaverine on renin secretion. Rates of secretion during the 30-60 min period of incubation were determined (V), and % response was calculated as (I - V/Vo) x i00 %, where V o = rate of secretion in the absence of papaverine. Means + SEMs with n = 6 determinations at each point.
Popovenr~ M
To determine if papaverine blocked isoproterenol-stimulated, as well as basal, renin secretion, slices were incubated in media containing 0.0, 8 x 10 -7 , and 5 x 10 -6 M isoproterenol. All media contained ascorbate. Samples were taken at 20 and 40 min for renin determinations, then either papaverine (2.5 x 10 -4 M final) or the same volume of diluent was added, the incubations were continued and samples were taken again at 60 and 80 min. Mean increments in renin during the 20-40 and the 60-80 min periods are shown as the first and second columns in each pair of columns in Figure 2. It can be seen that isoproterenol produced a concentration-dependent stimulation of secretion during the first period. Averaging the results of the two series of experiments (the papaverine and the diluent series), gave rates of 148 + 15, 217 + 12, and 286 + 12 ng hr -1 mg-1/20 min for 0, 8 x 10 -7 , and 5 x i0 -~ M isoproterenol, respectively. These rates differ significantly from each other (p <0.001). As can be seen in the top panel, rates did not decrease following the addition of diluent, and the concentration-dependent stimulation by isoproterenol is still evident during this period. In contrast, the results in the bottom panel show that papaverine antagonized basal and isoproterenol-stimulated renin secretion. The paired changes in each of the experiments shown were significant (p< 0.005 maximum). Whether isoproterenol had any effect at all in the presence of papaverine is equivocal; the difference between rates in media containing 0 and 8 x 10 -7 M isoproterenol is not significant at the 0.05 level whereas that between rates in media containing 0 and 5 x 10 -6 M isoproterenol is ( p ~ 0 . 0 3 ) .
1302
Papaverine,
Isoproterenol and Renin Secretion
Diluent
÷
500
Vol. 27, No. 14, 1980
+ 1
T
8 ,.
250
~ ~ u
0
! T
"
I 0
8xlG 7
rr E 5 0 0
Popoverine
o~
2,5x10_4 M
o
c,- ~
250
0
5xlO-6
Isoproterenol, M
5xlO-6
Isoproterenol, M
T
0
8xlO -7
Figure 2
The effect of papaverine on basal and on isoproterenol-stimulated renin secretion. Slices were incubated in media containing isoproterenol, as indicated. Samples were taken at 20, 40, 60, and 80 min. Rates of secretion were taken as the increments in renin between 20-40 min (first columns in each pair) and between 60-80 min (second columns). Lower p a n e l , papaverine was added at 40 min. Upper panel, diluent was added at 40 min. Means + SEMs; n = 5 or 6 each experiment.
Finally, slices were incubated in media containing 2 x 10 -4 M IBM_x or its diluent, ethylene glycol. Samples for renin were taken at 20 and 40 min, then isoproterenol and/or vehicle were added such that final concentrations of isoproterenol were 0, 4 x 10 -7 , and 8 x 10-7 M (ascorbate present in all media). The incubations were continued, and samples were taken again at 60 and 80 min. Mean increments in renin during the 20-40 and the 60-80 min periods are shown as the first and second columns in each pair of columns in Figure 3. It is evident that renin secretion was stimulated by IBMx during the first period. Rates averaged 366 + 30 (n = 18) versus 215 + 17 (n = 18) ng hr -I mg-i/20 minutes for IBMx versus i~s control (p <0.001). --Renin secretion rate was not affected by the addition of ascorbate to either the control or the IBMx-containing media (left side of Figure 3). In contrast, the addition of either concentration of isoproterenol significantly stimulated renin secretion. The paired changes in secretory rates were significant in all four such experiments ( p ~ 0 . 0 5 maximum).
Vol. 27, No. 14, 1980
Papaverine,
Isoproterenol
and Renin Secretion
1303
Isoproterenol, M
6 600
4 x~O-7 t
t
-
8 x i0"7 t T
Control 300
T
o
T
c
o O
O-
c-"
Isoproterenol,M
~E
4 xlO-7
8xlO"7
t
t
i
rt-
~ ~ o
I
600 !BMx, 2x 10"4M
e-
:300
-
_
N Figure 3
The effect of IBMx on basal and on isoproterenol-stimulated renin secretion. Slices were incubated in media containing IBMx (lower panel) or its vehicle (upper panel, "control"). The flrst columns in each pair are the secretory rates during the 20-40 min period of incubation. At 40 min, isoproterenol and/or its vehicle were added. The second columns in each pair are the secretory rates during the 60-80 min periods of incubation. Means SEMs; n = 6 in each of the 6 experiments. The rationale for starting the incubations with IBMx then adding isoproterenol was as follows. As can be seen in Figure 3, secretion rates in the presence of isoproterenol increased between the first and second periods. Had th~ incubations begun with isoproterenol, and had I B ~ been added at 40 min, then any increase in rate between the two periods could have been attributed to greater stimulatory effect of isoproterenol (as seen in the experiments of Fi~qre 2) and/or to an effect of IBMx per se. In any case, the experiments sh~ i n Figures 2 and 3 demonstrate that papaverine inhibits basal as well as i$~ro~erenol-stimulated renin secretion whereas IBMx stimulates secretion and at least does not block the stimulatory effect of isoproterenol.
1304
Papaverine,
Isoproterenol and Renin Secretion
Vol. 27, No. 14, 1980
Discussion In the present experiments, papaverine inhibited the basal secretory rate of rat kidney slices in a concentration-dependent manner, suggesting at least some specificity of action. The observation that renin secretion averaged 2 + i ng hr -I mg -I during the 30-60 minute period of exposure to 5 x 10 -4 M papaverine (Table i) taken together with the previous observation that renin in unincubated rat kidney slices averaged 3702 ng hr -I mg -I (16), suggests that passive leak of renin from non-viable cells was minimal. Indeed, if papaverine were to have a toxic effect, one would expect to find increased rather than decreased rate of renin release from slices exposed to papaverine. These results confirm and extend the previous observation that papaverine inhibits renin secretion from isolated rat renal cortical cells (4). It can be calculated from the results in this previous report that secretion was lower than control during the first hour of exposure to 2.6 x 10 -4 M papaverine, and virtually zero thereafter. As documented in the introduction, infusion of papaverine into the renal artery blocks the effects of a wide variety of renin secretory stimulants. In in vivo experiments, the concentration of papaverine in renal arterial plasma ranged between 10 -4 and 5 x 10 -4 M, even assuming no recirculation (3, 5-7). These concentrations were approximately 50 and I00 % effective in blocking the basal secretory rate of rat kidney slices. Accordingly, one might expect papaverine to inhibit basal secretory rate in vivo. Although papaverine does block the elevated basal rate normally found in Na-deprived dogs and in dogs with thoracic caval constriction (7,8), papaverine does not seem to decrease secretion below the normal basal level (3,5,6). Why this is the case is not clear. Although speculative, one explanation is suggested by the oft-reported observation that basal in vivo renin secretion rate is so low that further reductions are difficult to detect (18-20). Namely, at the normal renal perfusion pressure in vivo, the baroreceptor mechanism must be exerting a tonic inhibitory effect on renin secretion. Such tonic inhibition is clearly absent in the slice preparation, perhaps explaining why basal secretory rates are higher in vitro than in vivo (21). Moreover, since papaverine eliminates the stimulatory effect of decreases in pressure on in vivo renin secretion (9), papaverine would be expected to eliminate the stimulatory effect of a lack of "pressure" on in vitro renin secretion. There is now reasonable accord that papaverine antagonizes beta-adrenergic stimulation of renin secretion. Although Johnson et al. (3) reported that it blocked the effects of epinephrine but not the effects of norepinephrine, the dose of norepinephrine was 50 % larger than the dose of epinephrine; papaverine might well have blocked a lower, more comparable, dose of norepinephrine. Papaverine also blocks isoproterenol-stimulated renin secretion from the isolated-perfused rat kidney (i). It is significant that papaverine blocked the effects of isoproterenol in the slice preparation, in that changes in renal hemodynamics cannot be invoked to explain the effect. Papaverine has a spectrum of diverse actions, including inhibition of oxidative phosphorylation (14), inhibition of cellular uptake of adenosine (2223), and inhibition of phosphodiesterase activity (14,15). It follows that its effects on renin secretion could have a number of explanations. Clearly, much remains to be done to establish the mechanism of action, but explanations based on phosphodiesterase inhibition seem unlikely in view of the observations that papaverine is intermediate in potency between IBMx and theophylline (14,15,20), that IBMx (present study) and theophylline stimulate renin secretion per se and/
Vol. 27, No. 14, 1980
Papaverine,
Isoproterenol
and Renin Secretion
1305
or potentiate the stimulatory effect of catecholamines (10,11) and that papaverine inhibits renin secretion per se and blocks beta-adrenergic stimulation.* Acknowledsements This work was supported by a grant from NIH support from the BMA Management Company.
(HL
24880-01),
and
salary
References
i. 2. 3. 4.
5. 6. 7. 8. 9. i0. II. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
J.C.S. FRAY, J. Physiol. 282:207-217 (1978). K. GAAL, J. SIKLOS, T. MOZES AND G.F. TOTH, Acta Physiol. Acad. Sci. Hung. 51:305-314 (1978). J.A. JOHNSON, J.O. DAVIS AND R.T. WITTY, Circ. Res. 29:646-653 (1971). H.J. LYONS AND P.C. CHURCHILL, Proc. Soc. exp. Biol. Med. 160:237-240 (1979). A.A. SEYMOUR AND J.E. ZEHR, Circ. Res. 45:13-25 (1979). R.T. WITTY, J.O. DAVIS, J.A. JOHNSON AND R.L. PREWITT, Am. J. Physiol. 221:1666-1671 (1971). R.T. WITTY, J.O. DAVIS, R.E. SHADE, J.A. JOHNSON AND R.L. PREWITT, Circ. Res. 31:339-347 (1972). J.O. DAVIS AND R.H. FREEMAN, Physiol. Rev. 56:1-56 (1976). A.M. MICHELAKIS, J. CAUDLE AND G.W. LIDDLE, Proc. Soc. exp. Biol. Med. 130:748-753 (1969). H.L. NOLLY, I.A. REID AND W.F. GANONG, Circ. Res. 35:575-579 (1974). R.J. VISKOPER, M.H. MAXWELL, A.N. LUPU AND S. ROSENFELD, Am. J. Physiol. 232:F248-F253 (1977). N. WINER, D.S. CHOKSHI, M.S. YOON AND A.D. FREEDMAN, J. Clin. Endocrinol. Metab. 29:1168-1175 (1969). N. WINER, D.S. CHOKSHI AND W.G. WALKENHORST, Circ. Res. 29:239-248 (1971). M. FERRARI, Pharmacol. Res. Com. 6:97-115 (1974). L. TRINER, Y. VULLIEMOZ AND M. VEROSKY, Eru. J. Pharmacol. 41:37-46 (1977). P.C. CHURCHILL, J. Physiol. 294:123-134 (1979). C.W. DUNNET, Statistics in Endocrinolosy, edited by J.W. McARTHUR AND T. COLTON, pp. 79-103, MIT Press, Cambridge, Mass. (1970). A.J. VANDER AND R. MILLER, Am. J. Physiol. 207:537-546 (1964). H. TAGAWA AND A.J. VANDER, Circ. Res. 26:327-338 (1970). P.C. CHURCHILL AND F.D. McDONALD, J. Physiol. 242:635-646 (1974). K. BLENDSTRUP, P.P. LEYSSAC, K. POULSEN AND S.L. SKINNER, J. Physiol. 246:653-672 (1975). M. HUANG AND G.I. DRUMMOND, Biochem. Pharmacol. 25:2713-2719 (1976). J.W. PHILLIS, J.P. EDSTROM AND G.K. KOSTOPOULOS, Can. J. Physiol. Pharmacol. 57:1289-1312 (1979).
Seymour and Zehr (5) found that papaverine increased the efflux of c-AMP from dog kidney, which suggests that papaverine increased renal tissue levels of c-AMP in their experiments. Gaal et al. (2) measured tissue levels of c-AMP and found a significant increase following papaverine administration. Unfortunately, the heterogeneity of cell populations in the kidney precludes any estimate of intracellular c-AMP in the renin-secreting population, a small fraction of the total.