Vanadate-induced inhibition of renin secretion is unrelated to inhibition Na,K-ATPase activity

Life Sciences, Vol. 46, pp. 1953-1959 Printed in the U.S.A.

Pergamon Press

VANADATE-INDUCED INHIBITION OF RENIN SECRETION IS UNRELATED TO INHIBITION

Na,K-ATPase ACTIVITY Paul C. Churchill, Noreen F. Rossi, Monique C. Churchill and Virginia R. Ellis Departments of Physiology and Internal Medicine Wayne State University School of Medicine 540 East Canfield Detroit, Michigan 48201 (Received in final form April 23, 1990)

Summary There is evidence that three inhibitors of Na,K-ATPase activity--ouabain, K-free extracellular f l u i d , and vanadate--inhibit renin secretion by increasing Ca2+ concentration in juxtaglomerular cells, but in the case of vanadate, i t is uncertain whether the increase in Ca2+ is due to a decrease in Ca2+ efflux (inhibition of Ca-ATPase a c t i v i t y , or inhibition of Na,KATPase a c t i v i t y , followed by an increase in intrace]lular Na+ and a decrease in Na-Ca exchange) or to an increase in Ca~+ influx through potential operated Ca channels (inhibition of electrogenic Na,K transport, followed by membrane depolarization and activation of Ca channels). In the present experiments, the rat renal cortical slice preparation was used to compare and contrast the effects of ouabain, of K-free f l u i d , and of vanadate on renin secretion, in the absence and presence of methoxyverapamil, a Ca channel blocker. Basal renin secretory rate averaged 7.7 ± 0.3 GU/g/60 min, and secretory rate was reduced to nearly zero by 1 mM ouabain, by Kfree f l u i d , by 0.5 mM vanadate, and by K-depolarization (increasing extracellular K+ to 60 mM). Although 0.5 pM methoxyverapamil completely blocked the inhibitory effect of K-depolarization, i t failed to antagonize the inhibitory effects of ouabain, of K-free f l u i d , and of vanadate. A concentration of methoxyverapamil two hundred times higher (100 NM) completely blocked the inhibitory effects of vanadate, but s t i l l failed to antagonize the effects of ouabain and of K-free f l u i d . Collectively, these observations demonstrate that vanadate-induced inhibition of renin secretion cannot be attributed entirely to Na,K-ATPase inhibition, since in the presence of methoxyverapamil, the effect of vanadate differed from the effects of either ouabain (a specific Na,K-ATPase inhibitor) or K-free f l u i d . Moreover, i t cannot be attributed entirely to a depolarization-induced influx of Ca2+ through potentialoperated Ca channels, since methoxyverapamil antagonized Kdepolarization-induced inhibition of renin secretion much more effectively than i t antagonized vanadate-induced inhibition. Ouabain ( i ) , K-free extracellular f l u i d (2), and vanadate (3) i n h i b i t renin secretion in in vitro preparations, and ouabain (4) and vanadate (5) i n h i b i t renin secretion in vivo. There is evidence that the inhibitory effects of ouabain (6-10), of K-free f l u i d (2,11), aod of vanadate (3,5,12) are mediated by an increase in juxtaglomerular cell CaZ+ concentration, but the 0024-3205/90 $3.00 +.00 Copyright (c) 1990 Pergamon Press plc

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mechanism by which vanadate in particular increases Ca2+ concentration is uncertain. On the one hand, Na,K-ATPase a c t i v i t y is inhibited by ouabain (specifically), and by K-free extracellular f l u i d and vanadate (13,14). This effect would decrease the rate of CaZ+ efflux via Na-Ca exchang?, since i t would decrease the transmembrane concentratio~ gradient for Na÷ influx. In addition, vanadate could decrease active CaZ+ efflux, since i t inhibits Ca-ATPase a c t i v i t y in addition to Na,K-ATPase a c t i v i t y (14). On the other hand, Na,KATPase inhibitors depolarize cells since active Na,K transpQrt is electrogenic. Depolarization could increase the rate of CaZ+ influx through potential-operated Ca channels, In accord with such a mechanism of action, Ca channel blockers antagonize some vanadate-induced and Ca-mediated cellular responses, including vanadate-induced inhibition of renin secretion (5,12) and vanadate-induced contraction of vascular smooth muscle (12,15; but see 16) and other smooth muscle (17,18; but see 19). The purpose of the present studies was to compare and contrast the effects of ouabain, K-free extracellular f l u i d , and vanadate on renin secretion in the absence and presence of methoxyverapamil, a Ca channel blocker. Methods Adult male Sprague Dawley rats were used for these experiments. They had free access to tap water and Purina Rodent Chow, and were cared for in accordance with the principles of the Guide for the Care and Use of Laboratory Animals (Department of Health, Education and Welfare No. NIH 80-23). For each of several experiments, five or six rats were anesthetized with ether and nephrectomized. After removing the renal capsule, four thin slices were cut from each kidney using a razor blade. The slices were placed in flasks (two slices per flask) which contained 12 ml of incubation medium which had been equilibrated previously at 370 with a mixture of Op (95%) and COp (5%). The flasks were stoppered, placed in an oscillating ~ncubator at 37°C, and gassed continuously throughout the incubation. At 30, 60 and 90 min, 1 ml samples were withdrawn and centrifuged at 4°C. The supernatants were stored frozen until renin concentration was determined. After the incubations, tissue dry weight was determined. Tissue dry weight (two slices) averaged 3.14 ± 0.06 mg (n = 144). The composition of the standard incubation medium was 125 mM NaCl, 19 mM NaHC03, 4 mM KCl, 2.5 mM CaCl2, 1.2 mM NaH2P04, 0.8 mM MgS04, and 0.2 g% each of gIGcose and bovine serum aTbumin. In some experiments, 4 mM KCI was replaced by 4 mM NaCl (K-free medium), and in others, 56 mM of NaCl was replaced by 56 mM KCl (60 mM KCI t o t a l ) . Ouabain (Strophanthin G) was obtained from the Sigma Chemical Company, and methoxyverapamil (D600) from Knoll Pharmaceutical. Renin concentration of the supernatant was determined using a previously described method (7) as subsequently modified (20). Renin standards (ICN Nutritional Biochemicals) and renin-containing supernatants were incubated with rat renin substrate for 30 min at 37°C. The angiotensin I generated during this incubation was determined by radioimmunoassay. Renin concentration was calculated as nanograms of angiotensin I generated per m i l l i l i t e r of renin-containing sample per hour of incubation with renin substrate, then converted to Goldblatt Units per l i t e r as previously described (20). The total amount of renin secreted at a given time during the incubation of the slices was calculated as the renin concentration of the medium (Goldblatt

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Units per l i t e r ) , multiplied by the volume of the incubation medium at the time of sampling (e.g., 0.012 l i t e r s at 30 min, etc.) and divided by the tissue dry weight in grams, yielding the units Goldblatt Units per gram (GU/g). Renin secretory rates were calculated as the increment in the total amount of renin secreted during the final hour of incubation of the slices. The results are expressed as means ± S.E.M.'s, with n taken as the number of flasks, each of which contained two slices. Analysis of variance and Scheffe contrasts (21) were used to assess the s t a t i s t i c a l significance of differences in means. P-values < 0.05 were considered s t a t i s t i c a l l y s i g n i f i cant. Results Basal renin secretory rate, shown as the horizontal bar in each panel of Figure 1, averaged 7.7 ± 0.3 GU/g/60 min. When slices were incubated in Kfree extracellular f l u i d or in the presence of either 1 mM ouabain or 0.5 mM vanadate, renin secretory rates were s i g n i f i c a n t l y lower (p < 0.00005 maximum). In fact, they were nearly zero. The inhibitory effects of K-free f l u i d , of ouabain, and of vanadate were not s i g n i f i c a n t l y affected by 0.5 pM methoxyverapamil. In contrast, 100 pM methoxyverapamil completely blocked the effect of vanadate but failed to antagonize the effects of K-free f l u i d or of ouabain. As can be seen in Figure 2 below, renin secretory rates were reduced to nearly zero by 60 mM KCl alone or in combination with 1 mM ouabain or 0.5 mM vanadate. These rates were a l l s i g n i f i c a n t l y lower than the basal renin secretory rate of 7.7 ± 0.3 GU/g/60 min (p < 0.00005 maximum). Methoxyverapamil at 0.5 pM completely blocked the inhibitory effect of 60 mM KCl alone (upper panel), but failed to antagonize the inhibitory effects of combinations of 60 mM KCl with ouabain (middle panel) or vanadate (lower panel). At 100 pM, methoxyverapamil blocked the inhibitory effect of a combination of 60 mM KCl and vanadate (secretory rate was 7.4 ± 0.7 GU/g/60 min, compared with the basal secretory rate of 7.7 ± 0.3 GU/g/60 min) and actually stimulated renin secretion in the presence of 60 mM KCl alone (secretory rate was 14.2 ± 1.0 GU/g/60 min, compared with the basal secretory rate of 7.7 ± 0.3 GU/g/60 min). Discussion We have shown previously that decreasing extracellular K+ below 2 mM (2,11), ouabain (11), and vanadate (3) i n h i b i t renin secretion concentrationdependently in the rat renal cortical slice preparation. In the present studies, we used maximally effective inhibitory concentrations (e.g., 0 mM KCl, 1 mM ouabain, and 0.5 mM vanadate), and the results confirm our previous results. The inhibitory effect of 60 mM KCI and the blockade of this effect by 0.5 ~M methoxyverapamil also confirm previous results (22). Increasing + extracellular K progressively depolarizes renin-secreting juxtaglomerular cells (23), and increasing extracellular K+ over the range 4 to 60 mM inhibits renin secretion in a concentration-dependent manner, independently of changes in Na+, Cl-, or osmolality (24). Four structurally different Ca channel blockers antagonize the inhibitory effect of K-depolarization in concentration-dependent manner: methoxyverapamil (22), verapamil (24), diltiazem (25), and nifedipine (26). The observed order of potency and the IC50s of these drugs for blocking the inhibitory effect of K-depolarization constitute strong pharmacological evidence for potential-operated Ca channels in juxtagomerular cells (26,27).

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Effect of Vanadate on Renin Secretion

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Fig. i Effects of methoxyverapamil (D600) on renin secretion of rat renal cortical slices incubated in K-free extracellular fluid (upper

panel) and in media containing i mM ouabain (middle panel) and 0.5 mM vanadate (V04; lower panel). Means ± S.E.M.'s; n = 6 for each incubation condition. Basal rate ± S.E.M. (n = 12) is depicted as the X - f i l l e d rectangle in each panel. The p-value and NS (p > 0.05) are in comparison with the absence of D600; basal secretory rate was s i g n i f i c a n t l y higher than a l l others except for the rate in the presence of VO4 plus 100 pM D600. There is evidence that increased Ca2+ concentrations in juxtaglomerular cells mediates the inhibitory effects on renin secretion of K-depolarization (6), ouabain (6), K-free extracellular f l u i d (2,11), and vanadate (3). However, whereas Kzdepolarization is believed to increase Caz+ concentration by increasing Caz+ i n f l u x (22,24), the other thcee inhibitors are believed to increase Ca2+ concentration by decreasing Ca2+ efflux: ouabain and K-free extracellular f l u i d by inhibiting Na,K-ATPase a c t i v i t y and therefore decreasing Na-Ca exchange, and vanadate either by this same mechanism or by i n h i b i t ing Ca-ATPase a c t i v i t y and therefore decreasing active Caz+ efflux and sequestration (27). I t follows that i f the inhibitory effects are indeed

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mediated by decreased Ca2+ e f f l u x rather than by increased Ca2+ influx, then they should be immune to antagonism by Ca channel blockers. This was true for ouabain and for K-free extracellular f l u i d , but not for vanadate.

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Fig. 2 Effects of methoxyverapamil (D600) on renin secretion of rat renal cortical slices incubated in K-depolarizing medium (upper panel) and in K-depolarizing medium containing 1 mM ouabain (middle panel),and 0.5 mM vanadate (V04; lower panel). Means ± S.E.M. s; n = 6 - 12 for each Incubation condition. The p values and NS (not significant, p > 0.05) are in comparison with the absence of D600. At a concentration that completely blocked the inhibitory effect of depolarization (0.5 pM) and at a concentration mere than two orders of magnitude higher, methoxyverapamil failed to even antagonize the inhibitory effects of ouabain and K-free extracellular f l u i d (Figure 1). Moreover, ouabain s t i l l inhibited renin secretion after the inhibitory effects of depolarization per se were blocked by methoxyverapamil (Figure 2). Collectively, these results demonstrate that the i n h i b i t o r y effect of ouabain is independent of membrane potential, and that the inhibitory effects of bQth ouabain and K-free extrac e l l u l a r f l u i d are not mediated by increased CaZ+ i n f l u x through potential-

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operated Ca channels. In contrast, although a high concentration was required, methoxyverapamil completely blocked the inhibitory effect of vanadate, in the presence of either 4 mM (Figure 1) or 60 mM KCI (Figure 2). This result is in accord with two previous reports: nifedipine antagonized vanadate-induced inhibition of renin secretion in anesthetized dogs (5) and verapamil antagonized vanadateinduced decreases in plasma renin a c t i v i t y in conscious dogs (12). Although these observations tempt one to conclude that vanadate-induced inhibition of renin secretion is mediated entirely by a depolarization-induced influx of CaZ+ through potential-operated Ca channels, there are two c o n f l i c t ing observations. First, although 0.5 NM methoxyverapamil completely blocked depolarization-induced inhibition of renin secretion (Figure 2), i t did not significantly antagonize vanadate-induced inhibition (Figure 1). Second, the presence of vanadate prevented 0.5 NM methoxyverapamil from completely blocking the effects of depolarization on renin secretion (Figure 2). Moreover, in the presence of 60 mM KCl and 100 NM methoxyverapamil, secretory rate was lower when vanadate was present than when i t was absent (Figure 2). Consistent with the in vivo results (5,12), vanadate appears to activate potential-operated Ca channels, but by a mechanism differing from both Kdepolarization or depolarization secondary to inhibition of Na,K-ATPase activity. Taken together, these observations suggest that vanadate-induced inhibition of renin secretion cannot be exclusively attributed either to Na,K-ATPase inhibition or to a depolarization-induced Ca2+ influx through potentialoperated Ca channels. Rather, both mechanisms may be involved in mediating the effects of vanadate on renin secretion. A role for Ca-ATPase a c t i v i t y cannot be excluded. In conclusion, vanadate-induced inhibition of renin secretion cannot be attributed entirely either to Na,K-ATPase inhibition (since its effects in the presence of methoxyverapamil d i f f e r from thQse of ouabain, a specific Na,KATPase inhibitor) or to a depolarization Ca2+ influx through potentialoperated Ca channels (since methoxyverapamil blocked depolarization-induced inhibition of renin secretion much more effectively than i t blocked vanadateinduced inhibition). Perhaps both these mechanisms, and possibly vanadateinduced inhibition of Ca-ATPase a c t i v i t y as well, are operative. Finally, the mechanism by which vanadate activates potential-operated Ca channels remains to be elucidated. Clearly i t differs from K-depolarization. Moreover, since the effects of vanadate and ouabain were so different in the presence of methoxyverapamil, i t does not appear to involve depolarization resulting from inhibition of Na,K-ATPase a c t i v i t y and therefore of electrogenic Na,Ktransport. Acknowledgements This research was supported by the National Institutes of Health (HL 24880) and by Biomedical Applications of Detroit. 1.

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