(VanDongen & Peart, 1974) have been attributed to an increase in intracellular [Ca]

Transcription

1 J. Phygiol. (1981), 315, pp With 4 text-ftgures Printed in Great Britain CALCIUM DEPENDENCY OF THE INHIBITORY EFFECT OF ANTIDIURETIC HORMONE ON IN VITRO RENIN SECRETION IN RATS BY PAUL C. CHURCHILL From the Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan 48201, U.S.A. (Received 1 July 1980) SUMMARY 1. Synthetic arginine-vasopressin (ADH or antidiuretic hormone) inhibited renin secretary rate of rat renal cortical slices. The response was concentration-dependent and was maximal at 041 U. ml.-'. 2. Ca depletion (incubation of slices in medium containing Na2EGTA and no added CaCl2) stimulated renin secretion and eventually abolished the inhibitory effect of ADH. Both these effects were reversible. 3. The Ca antagonist D-600, at 0-5 pm, reversed the inhibitory effect of K depolarization on secretary rate but had no effect on secretary rate of non-depolarized slices. In the presence of 0 5 /M-D-600, ADH inhibited the secretary rate of either depolarized or non-depolarized slices. 4. These results confirm and extend previous observations suggesting that Ca plays an inhibitory coupling role in the control of renin secretion. Moreover they suggest that although Ca influx through voltage-sensitive Ca channels influences the secretary activity of juxtaglomerular cells, ADH activates an independent pathway for Ca mobilization. INTRODUCTION The granulated juxtaglomerular cells (JG cells), one of the three components of the renin-secreting juxtaglomerular apparatus, are considered to be the site of renin synthesis, storage and release (Davis & Freeman, 1976). It is believed that these secretary cells are derived from vascular smooth muscle (Barajas, 1979), and independently of whether or not the JG cells themselves are contractile, many agents and manipulations that affect the contractile state of vascular smooth muscle cells also affect the secretary activity of JG cells (Peart, 1978). For instance, it is well known that angiotensin II elicits contraction of vascular smooth muscle and inhibits the secretion of renin. Both effects appear to be receptor-mediated in that they are dose-dependent, have a plateau at high concentrations, and can be inhibited by angiotensin analogues such as saralasin; other analogues are agonists with respect to vascular smooth muscle contraction and the same ones inhibit the secretion of renin (VanDongen, Peart & Boyd, 1974; Freer, 1975; Bumpus, 1977; Naftilan & Oparil, 1978). Both the contraction (Goodman, 1978) and the inhibition of renin secretion (VanDongen & Peart, 1974) have been attributed to an increase in intracellular [Ca] /81/ $07.50 D 1981 The Physiological Society

2 22 P. C. CHURCHILL resulting from an influx of extracellular Ca and/or a mobilization of membrane-bound Ca. Neither angiotensin-induced vasoconstriction (Freer, 1975) nor angiotensininduced inhibition of renin secretion (Churchill, 1980) is blocked by 'specific' Ca antagonists such as verapamil and D-600 (methoxy verapamil), suggesting (Fleckenstein, 1977) that angiotensin-receptor interaction does not lead to a depolarizationinduced influx of extracellular Ca. Like angiotensin II, antidiuretic hormone (ADH) is both a vasoconstrictor and an inhibitor of the secretion of renin (Vander, 1968; Goodman, 1978). In the experiments described below, the inhibitory effect, and the Ca dependency of the inhibitory effect, of ADH on renin secretion from rat kidney slices were studied. METHODS Experimental procedures for studying renin secretion from rat kidney slices have been described previously (Churchill, 1979). Briefly, for each ofseveral experiments five adult male Sprague-Dawley rats were anaesthetized with ether and nephrectomized. The renal capsule was removed and four thin cortical slices cut from each kidney. The resulting forty slices were randomized and kept in incubation medium at room temperature for no longer than 20 min before beginning the incubations. Slices were placed in flasks (two slices per flask) each of which contained 10 ml. of medium which has been equilibrated previously at 37 0C with a 95%/5 % mixture of 02 and CO2. The flasks were stoppered, placed in an oscillating incubator at 37 'C, and gassed with the mixture throughout the incubation. Periodically, as indicated in the Results section, 200,l. samples of incubation medium were withdrawn and centrifuged at 4 'C. The supernatants were frozen until determination of renin activity. Following the incubations, the slices were dried and weighed. The composition of the incubation medium was: 125 mm-nacl, 19 mm-nahco3, 4 mm-kc1, 2-65 mm-cacl2, 1M2 mm-nah2po4, 0-8 mm-mgso4, and 0-2 g per 100 ml. each of glucose and bovine albumin (United States Biochemical Corp). Deviations from this composition are described in the Results section. Na and Ca salts of EGTA (ethyleneglycol-bis[beta-aminoethyl ether]n,n'- tetraacetic acid) and ADH (synthetic arginine-vasopressin, grade VII) were obtained from the Sigma Chemical Co. D-600 (methoxy verapamil) was obtained from the Knoll Pharmaceutical Co. Methods for the determination of renin have been described previously (Churchill, 1979). Briefly, samples of supernatant were incubated with rat renin substrate at 37 'C for 30 min. Radioimmunoassay was used to measure the angiotensin I generated during the incubation. The renin activity of a sample was expressed in nanograms of angiotensin I generated per hour of incubation of the sample with renin substrate per millilitre of sample (ng hr-' ml.-'). Total renin secreted at a given time was calculated as the renin activity of the medium (ng hr-' ml.-') multiplied by the volume of the incubation medium (ml.) and divided by the tissue dry weight (mg), yielding the units ng hr-' mg-'. Renin secretary rate was calculated as the increment in the total amount of renin during a given interval of incubation of the slices, e.g. ng hr-' mg-' per 30 min. Results are presented as means +S.E.M.8. The paired and unpaired t tests were used to assess the statistical significance of observed changes and differences, respectively. RESULTS Dose-response relationship In the first series of experiments, slices were incubated in media containing 0-1 U. ml.-' ADH. The first 30 min of incubation were allowed for stabilization, and the increments in renin during the min period of incubation were taken as secretary rates. In Fig. 1 mean secretary rates are plotted versus the logarithm of the ADH concentration. The shaded area at the top represents the mean+ S.E.M. of the secretary rate in the absence of ADH (control rate). ADH concentrations from to 1 U. ml.-' suppressed secretion (P < 0-05 maximum). The response appeared

3 Ca, ADH AND RENIN SECRETION 23 to be maximal at 04 U. ml.-', as rates in media containing 0 1, 0 5 and 1 U. ml. were not significantly different from each other (p > 0-6). Rates in media containing these concentrations of ADH were , and % of the control rate, respectively. It is unlikely that this residual secretary rate can be accounted for by passive leak of renin from non-viable cells; as will be shown below, the release of renin from rat kidney slices can be completely suppressed by depolarization. 300 r > C') ED EI CD cn 0 oa I - Control~rate±SE. - TaI I TI I,I I ADH (U. ml-') Fig. 1. The effect of ADH on renin secretin from rat renal cortical slices. Secretory rates were measured during the min period of incubation. Means + s.e.m.s; n = 6 for each concentration ofadh, except n = 12 at 0 1 U. ml.-'. The shaded area at the top represents the mean control rate + 1 s.e.m. (rate in the absence of ADH). Effect of Ca depletion Slices were incubated in medium containing 2 mm-na2egta and no added CaCl2. So that any effect could be attributed specifically to a reduction in [Ca] and not to the presence of EGTA, other slices were incubated in medium containing 2 mm- CaEGTA plus the usual concentration of CaCl2 (2 65 mm). ADH (0-1 U. ml.-', final concentration) was added at either 30, 60 or 90 min after beginning the incubations. Secretory rates of each slice were determined during a 20 min period just before the addition of ADH (initial rate) and during a 20 min period beginning just after the addition of ADH (final rate). Percentage of final/initial was calculated for each slice, and mean values versus time of ADH addition are plotted in Fig. 2. In medium containing CaCl2, percentage final/initial was significantly less than 100 (P < ), irrespective of the time of addition of ADH. Thus, the presence of EGTA did not prevent the inhibitory effect. In contrast, buffering extracellular [Ca] to a low value (approximately 10-8 M: Caldwell, 1970) prevented the inhibitory effect. Although ADH did not inhibit secretion when added at 30 min (percentage final/initial < 100: P < 0-001), the degree of inhibition was somewhat less than that found in medium with 2-5 mm-cacl2. The addition of ADH at either 60 or 90 min failed to inhibit secretion at all; indeed, percentage final/initial exceeded 100 (P < 0-005) when ADH was added at these times. In order to determine whether the effects of Ca depletion were reversible, slices were incubated in medium containing 1 mm-na2egta and no added CaCl2. After

4 24 P. C. CHURCHILL ' 200 to E 100 C C~~~~~~ o L Time at which ADH was added (min) Fig. 2. The effect of Ca depletion on inhibition of renin secretion by ADH. ADH was added (0-1 U. ml.-', final concentration) either 30, 60 or 90 min after incubation had begun in medium with low [Ca] (Na2EGTA and no added CaCI2; 0) or normal [Ca] (CaEGTA plus 2-65 mm-cacl2; A). Initial and final secretary rates were determined during a 20 min period before ADH addition and during a 20 min period beginning at the time of ADH addition, respectively. Means +S.E.M.S of percentage final/initial are shown; n = 12 for each point in the figure CaC12 ADH + CaC12 Ca N200 CL - 00 Fig. 3. The reversibility of the effect on renin secretion of Ca depletion. Incubation of slices began in medium containing 1 mm-na2egta and no added CaCl2. At 40 min ADH and/or CaCJ2 were added (2 65 mm-cacl2, 0 1 U. ml.-' ADH, final concentrations). Each pair of columns represents secretary rate before and after these additions. Means + S.E.M.S; n = 6 for each experiment. 30 min either CaCl2 or CaCl2 plus ADH was added (2-65 mm-cacl2, 0.1 U. ml.-' ADH, final concentrations) and the incubations continued. Initial and final secretary rates were taken as the increments in renin during the and the min periods, allowing the first 10 min of incubation and the first 10 min after the additions of ADH and/or CaCl2 for stabilization. Means are shown in Fig. 3. The means of initial secretary rates in these two experiments were not significantly different from each other (P > 0 7). Combining the results, initial secretary rate averaged ng hr-' mg-' per 20 min. Comparison of this value with the control rate shown in

5 Ca, ADH AND RENIN SECRETION 25 Fig. 1 ( ng hr-1 mg-' per 30 min: note difference in units) suggests that Ca depletion increases renin secretion. Secretory rate declined following the addition of CaCl2 (P < 0-01) or CaCl2 plus ADH (P < 0-001). Final secretary rate was lower after the addition of CaCl2 plus ADH than after the addition of CaCl2 alone (P < 0 05). Thus, the addition of CaCl2 reverses the stimulatory effect on secretion of Ca depletion and restores the inhibitory effect of ADH. Previous studies have shown that secretary rate is stable if no changes are made in the composition of the medium during an incubation (Churchill, 1979; Churchill & Churchill, 1980a, b; Fig. 4 below). Therefore using each slice as its own control, as in the experiments shown in Figs. 2 and 3, is justified. Diluent 0-1 U. ml-' ADH 200 _ i -E 100 C I o WD K (mm) D-600 (pm) 9 E 0-5 pm -D pm-d -600 C r Diluent 10200T 100 T K (mm) ADH (U. ml.') Fig. 4. The effects on renin secretion of D-600 and ADH. Each pair of columns represents the initial and final secretary rates in a given experiment. Concentrations of substances present throughout the incubations are indicated under the various columns; substances added 40 min after the incubations had begun (between periods during which initial and final secretary rates were measured) are indicated above. Means +S.E.M.S; n = 6 for each experiment shown. Effects of ADH and D-600 Several experiments were performed to determine whether D-600 antagonized the effect of ADH. First, slices were incubated for 80 min either in medium with [K] = 4 mm or in medium with [K] = 60 mm (54 mm-kcl was added to medium of otherwise normal composition). Secretory rates were determined during the and min periods. Results are shown in the upper and lower left-hand panels of Fig. 4. In neither of these experiments was there a significant difference between initial and final secretary rates (P > 0 9). Comparisons of rates in the upper versus the lower panels demonstrate that K depolarization almost abolishes renin secretion, as has been reported previously (Churchill, 1980). In the third experiment the 80 min incubation was carried out in medium with [K] = 60 mm, and D-600 was added at 40 min. Initial and final secretary rates are presented in the lower middle panel. The addition of D-600 stimulated secretion significantly (P < 0 001), and the final rate in this experiment was not significantly different from that shown in the upper left-hand panel (P > 0 8), demonstrating that the inhibitory effect of K depolarization is completely antagonized by 0 5,tM-D-600. The fourth experiment, the results of

6 26 P. C. CHURCHILL which are shown in the lower right-hand panel, was carried out in medium with [K] = 60 mm and 0-1 U. ml.-' of ADH. The addition of D-600 at 40 min (0-5gM, final concentration) increased the final secretary rate in comparison with the initial rate (P < 0-01). However, the final rate in this experiment was suppressed in comparison with final rates shown either in the upper left-hand (P < 0 005) or in the lower middle (P < 0-01) panels. Therefore, ADH suppresses renin secretion even after all the effects of depolarization have been blocked with D-600. The fifth and sixth experiments were carried out using medium with [K] = 4 mm and 0-5 /SM-D-600. Either diluent or ADH was added at 40 min. Initial and final secretary rates are shown in the upper middle and right-hand panels. It can be seen that 0 5 #um-d-600 had no significant stimulatory or inhibitory effects in medium with [K] = 4 mm (P > 0-2, comparing upper left-hand and middle panels), and nor did this concentrationn of D-600 prevent a decrease in renin secretion when ADH was added (P < 0-001, comparing initial and final secretary rates in upper right-hand panel). DISCUSSION The intravenous or the intrarenal-arterial administration of ADH inhibits renin secretion in dogs and rats (Bunag, Page & McCubbin, 1967; Vander, 1968; Churchill, Churchill & McDonald, 1973). Although it has been suggested that this effect is mediated by a direct action of ADH on JG cells, several factors complicate the interpretation of ADH-induced changes in renin secretion in vivo. ADH may cause changes in arterial blood pressure, in intrarenal vascular pressures, in intrarenal distribution of blood flow, and in the filtration, transport and excretion of electrolytes, all of which are believed to affect renin secretion, either directly or indirectly (Davis & Freeman, 1976). Only some of these factors were excluded by using a non-filtering kidney, perfused at a constant pressure, to demonstrate an inhibitory effect (Shade, Davis, Johnson, Gotshall & Spielman, 1973). Many more complicating factors are excluded in the kidney slice preparation and, to our knowledge, this is the first report ofan inhibitory effect ofadh in such a preparation. Over the range U. ml.-i synthetic arginine-vasopressin, renin secretion was inhibited in a concentrationdependent manner. Both the 'saturation' of the response at high concentrations and the sigmoid nature of the dose-response relationship are consistent with a receptormediated event (Goodman & Gilman, 1975). In dogs, ADH inhibition of renin secretion is probably an important control mechanism. Unstimulated endogenous plasma levels of ADH are of the order of 1-2 1zU. ml.-' (Tagawa, Vander, Bonjour & Malvin, 1971), and infusions of pitressin (a mixture of arginine and lysine-vasopressin), in amounts calculated to raise plasma ADH by as little as 1-5uU. ml.-', produce detectable inhibitory effects on renin secretion (Bunag, et al. 1967; Vander, 1968; Tagawa et al. 1971; Shade et al. 1973). It should be noted, however, that in all these experiments the basal level of renin secretion was elevated (due variously to anaesthesia, prior Na deprivation or depletion, reduced renal perfusion pressure) in order to facilitate the detection of an inhibitory effect. Although ADH does decrease plasma renin and inhibit renin secretion in rats, the physiological relevance of the effect may be questioned. Based on the observation that plasma renin is elevated in rats lacking ADH, Gutman & Benzakein (1971, 1974) have suggested that endogenous ADH in normal rats may

7 Ca, ADH AND RENIN SECRETION 27 be sufficiently high tonically to inhibit renin secretion. However, a concentration of ADH of 1 mu. ml.-' (which is three orders of magnitude greater than endogenous plasma levels in dogs) failed to inhibit basal secretary rate of rat kidney slices (Fig. 1) or of the isolated-perfused rat kidney (VanDongen, 1975). Moreover, the doses of ADH found to decrease plasma renin or renin secretary rate in intact rats (5 mu. ml.-' i.v.: Churchill et al. 1973; 0A4-1'0 U. I.M. or s.c.: Gutman & Benzakein 1971,1974) are orders of magnitude greater, on a body weight basis, than those found to decrease plasma renin or renin secretary rate in dogs ( mu. min-' i.v.: Tagawa et al. 1971). Possibly only pharmacological levels of ADH inhibit renin secretion in rats. The results of the present study are in accord with previous reports that chelation of extracellular Ca with EGTA stimulates renin secretion from rat kidney slices (Churchill, 1979; Churchill & Churchill, 1980a, b). A stimulatory effect of Ca chelation has also been found on renin release from the isolated-perfused rat kidney (VanDongen & Peart, 1974; VanDongen, 1975), from isolated superfused rat glomeruli (Baumbach & Leyssac, 1977), and from pig kidney slices (Park & Malvin, 1978). It could be argued that Ca chelation enhances, not the secretion, but the passive leak, of renin from damaged cells. However, in previous experiments (Churchill, 1979; Churchill & Churchill, 1980 a, b) and in the experiments shown in Fig. 3, increasing extracellular concentration of free Ca from 10-8 to 10-3 M decreased renin _ secretion. Moreover, abruptly changing extracellular [Ca] from 10-8 to 10-3 M - or from 10-3 to 10-8 M produced opposite effects on renin secretion but did not - - alter the rate of accumulation of lactate dehydrogenase in the incubation medium (Churchill, 1979). Presumably, a generalized increase in the permeability of cell membranes would have been reflected by an increased leak of lactate dehydrogenase, an intracellular protein. Influx and/or mobilization of Ca appears to be a prerequisite for the inhibitory effect of ADH on renin secretion, as buffering extracellular [Ca] to a low value with EGTA not only stimulated the basal secretary rate but eventually abolished the inhibitory effect of ADH. The time course of this effect was similar to that found previously: Ca chelation only gradually blocked the inhibitory effect on renin secretion of angiotensin II (Churchill, 1980). Such a time course is not unexpected, as the addition of chelating agents to extracellular fluid only gradually reduces the amount of Ca which is available for influx and/or mobilization (Weiss, 1978). Smooth muscle cells and many secretary cells are stimulated to contract/secrete when depolarized by high extracellular [K], and it is well known that these responses are dependent upon an influx of extracellular Ca (Rubin, 1974; Fleckenstein, 1977; Rosenberger & Triggle, 1978). It is believed that K depolarization opens voltagesensitive Ca channels through which influx occurs; influx is blocked by drugs such as verapamil and D-600, and because of the apparent specificity of this effect the blocking of Ca-independent activities by verapamil or D-600 is taken as evidence for the existence of voltage-sensitive Ca channels (Fleckenstein, 1977; Rosenberger & Triggle, 1978). One might postulate the existence ofjg cell voltage-sensitive channels on the basis of the secretary activity of these cells and on the basis of their derivation from vascular smooth muscle (Barajas, 1979). As shown in Fig. 4 above, and as has been shown previously (Fray, 1978; Park & Malvin, 1978; Churchill, 1979), K depolarization blocks renin secretion. The inhibitory effect can in turn be blocked

8 28 P. C. CHURCHILL either by Ca chelation (Park & Malvin, 1978) or by D-600, in a concentrationdependent manner (Churchill, 1980). At 0-5,#M, D-600 restored the secretary rate of depolarized slices to the level found in non-depolarized slices. This same concentration had no effect on the secretary rate of non-depolarized slices. Since ADH decreased the secretary rate in the presence of 0 5/tM-D-600, whether or not the slices were depolarized, it is concluded that ADH does not act by depolarization of the cell membrane followed by Ca influx through voltage-sensitive Ca channels. The possibility that cyclic 3':5'-adenosine monophosphate (camp) plays a role should be mentioned. The second-messenger role of camp in the ADH responses of several transporting epithelia (including mammalian renal tubular cells) is well established: ADH-sensitive adenylate cyclase has been demonstrated, tissue levels of camp rise in response to ADH, and both exogenous camp and phosphodiesterase inhibitors mimic and/or potentiate the effect of ADH (reviewed by Dousa & Valtin, 1976). On the other hand, there are grounds for believing that camp does not mediate other effects ofadh, in particular the effects on vascular smooth muscle contractility and on the secretary activity of JG cells, which are derived from vascular smooth muscle (Barajas, 1979). The vascular effect is mediated by increased intracellular [Ca], whereas increased intracellular camp is usually associated with decreased intracellular [Ca] and relaxation of vascular smooth muscle. Similarly, the inhibitory effect on secretary activity appears to be mediated by increased intracellular [Ca] also, whereas increased intracellular camp is thought to stimulate renin secretion, although the evidence is less than compelling at the present time (reviewed by Oparil, 1976): beta-adrenergic agonists stimulate secretion, presumably by activating adenylate cyclase, although neither a hormone-sensitive adenylate cyclase associated with JG cell membranes nor an increase in JG levels of camp in response to catecholamines has been demonstrated. Exogenous camp and phosphodiesterase inhibitors mimicked or potentiated the stimulatory effects of beta-adrenergic agonists in some studies, but exactly the opposite effects were found in other studies. In conclusion, ADH may be added to a growing list of substances which inhibit the secretion of renin, most probably by increasing intracellular [Ca]. It has been shown that extracellular Ca is required for the inhibitory effects of angiotensin II (VanDongen & Peart, 1974; Churchill, 1980), of ouabain (Park & Malvin, 1978; Churchill, 1979), of vanadate (Churchill & Churchill, 1980b), of low extracellular [K] (Churchill & Churchill, 1980a) and of high extracellular [K] (Park & Malvin, 1978; Churchill, 1980). It is not yet clear whether all the substances which stimulate renin secretion act by decreasing intracellular [Ca], but there is evidence that stimulatory effects can be blocked by agents believed to increase intracellular [Ca]. For example, the stimulatory effect of beta-adrenergic agonists can be blocked by angiotensin II (VanDongen & Peart, 1974; Capponi, Gourjon & Vallotton, 1977), by ADH (VanDongen, 1975), by ouabain or vanadate (Churchill & Churchill, 1980b) and by low or high extracellular [K] (Churchill & Churchill, 1980c). Thus, it would appear that intracellular [Ca] plays an inhibitory coupling role in the control of renin secretion, which is opposite to its role in most other secretary cells (Rubin, 1974). The author wishes to thank M. C. Churchill for expert technical assistance and Professor Dr Oberdorf, of the Knoll Pharmaceutical Company, for a generous supply of D-600. This research was supported by the National Institutes of Health (grant HL ).

9 Ca, ADH AND RENIN SECRETION 29 REFERENCES BARAJAS, L. (1979). Anatomy of the juxtaglomerula apparatus. Am. J. Physiol. 237, F333-F343. BAUMBACH, L. & LEYSSAC, P. P. (1977). Studies on the mechanism of renin release from isolated superfused rat glomeruli: effects of calcium, calcium ionophore and lanthanum. J. Physiol. 273, BUMPUS, F. M. (1977). Mechanisms and sites ofaction ofnewer angiotensin agonists and antagonists in terms of activity and receptor. Fedn Proc. 36, BUNAG, R. D., PAGE, I. H. & MCCUBBIN, J. W. (1967). Inhibition of renin release by vasopressin and angiotensin. Cardiova8c. Res. 1, CALDWELL, P. C. (1970). Calcium chelation and buffers. In Calcium and Cellular Function, ed. CUTHBERT, A. W., pp New York: St Martins Press. CAPPONI, A. M., GOURJON, M. & VALLOTTON, M. D. (1977). Effect of f-blocking agents and angiotensin II on isoproterenol-stimulated renin release from rat kidney slices. Circulation Res. 40, CHURCHILL, M. C. & CHURCHILL, P. C. (1980a). Separate and combined effects of ouabain and extracellular potassium on renin secretion from rat renal cortical slices. J. Phy8iol. 300, CHURCHILL, P. C. (1979). Possible mechanism of the inhibitory effect of ouabain on renin secretion from rat renal cortical slices. J. Phy8iol. 294, CHURCHILL, P. C. (1980). Effect of D-600 on inhibition of in vitro renin release in the rat by high extracellular potassium and angiotensin II. J. Physiol. 304, CHURCHILL, P. C. & CHURCHILL, M. C. (1980b). Vanadate inhibits renin secretion from rat kidney slices. J. Pharmac. exp. Ther. 213, CHURCHILL, P. C. & CHURCHILL, M. C. (1980c). Biphasic effect of extracellular K on isoproterenolstimulated renin secretion from rat kidney slices. J. Pharmac. exp. Ther. 214, CHURCHILL, P. C., CHURCHILL, M. C. & MCDONALD, D. D. (1973). Renin release in anaesthetized rats. Kidney Int. 4, DAVIS, J. 0. & FREEMAN, R. H. (1976). Mechanisms regulating renin release. Physiol. Rev. 56, DOUSA, T. P. & VALTIN, H. (1976). Cellular actions of vasopressin in the mammalian kidney. Kidney Int. 10, FLECKENSTEIN, A. (1977). Specific pharmacology of calcium in myocardium, cardiac pacemakers, and vascular smooth muscle. A. Rev. Pharmac. Tox. 17, FRAY, J. C. S. (1978). Stretch receptor control of renin release in perfused rat kidney: effect of high perfusate potassium. J. Physiol. 282, FREER, R. J. (1975). Calcium and angiotensin tachyphylaxis in rat uterine smooth muscle. Am. J. Physiol. 228, GOODMAN, F. R. (1978). Calcium related basis of action of vascular agents: cellular approaches. In Calcium and Drug Action, ed. WEISS, G. B., pp New York: Plenum Press. GOODMAN, L. W. & GILMAN, A. (1975). The Pharmacological Basis of Therapeutics, 5th edn. New York: Macmillan. GUTMAN, Y. & BENZAKEIN, F. (1971). Effect of an increase and of lack of antidiuretic hormone on plasma renin activity in the rat. Life Sci. Oxford 10, GUTMAN, Y. & BENZAKEIN, F. (1974). Antidiuretic hormone and renin in rats with diabetes insipidus. Eur. J. Pharmac. 28, NAFTILAN, A. J. & OPARIL, S. (1978). Inhibition of renin release from rat kidney slices by the angiotensins. Am. J. Physiol 235, F62-F68. OPARIL, S. (1976). Renin 1976: Annual Research Reviews. Montreal: Eden Press. PARK, C. S. & MALVIN, R. L. (1978). Calcium in the control of renin release. Am. J. Physiol. 235, F22-F25. PEART, W. S. (1978). Intra-renal factors in renin release. Contr. Nephrol. 12, ROSENBERGER, L. & TRIGGLE, D. J. (1978). Calcium, calcium translocation, and specific calcium antagonists. In Calcium and Drug Action, ed. WEISS, G. B., pp New York: Plenum Press. RUBIN, R. P. (1974). Calcium and the Secretory Process. New York: Plenum Press. SHADE, R. E., DAVIS, J. O., JOHNSON, J. A., GOTSHALL, R. W. & SPIELMAN, W. S. (1973). Mechanism of action of angiotensin II and antidiuretic hormone on renin secretion. Am. J. Physiol. 224, TAGAWA, H., VANDER, A. J., BONJOUR, J.-P. & MALVIN, R. L. (1971). Inhibition of renin secretion by vasopressin in unanesthetized sodium-deprived dogs. Am. J. Physiol. 220,

10 30 P. C. CHURCHILL VANDER, A. J. (1968). Inhibition of renin release in the dog by vasopressin and vasotocin. Circulation Res. 23, VANDER, A. J. & GEELHOED, G. W. (1965). Inhibition of renin secretion by angiotensin II. Proc. Soc. exp. Biol. Med. 120, VANDoNGEN, R. (1975). Inhibition of renin secretion in the isolated rat kidney by antidiuretic hormone. Clin. Sci. 49, VANDONGEN, R. & PEART, W. S. (1974). Calcium dependence of the inhibitory effect of angiotensin on renin secretion in the isolated perfused kidney of the rat. Br. J. Pharmac. 50, VANDONGEN, R., PEART, W. S. & BOYD, G. W. (1974). Effect of angiotensin II and its nonpressor derivatives on renin secretion. Am. J. Phyaiol. 226, WEIss, G. B. (1978). Quantitative measurement of binding sites and washout components for calcium ion in vascular smooth muscle. In Calcium and Drug Action, ed. WEISs, G. E., pp New York: Plenum Press.