Stimulation of renin secretion from the intact kidney and from isolated glomeruli by the calcium ionophore A23187

20

Biochimica et Biophysica Acta,

583 (1979) 20--27 © Elsevier/North-Holland Biomedical Press

BBA 28813

STIMULATION OF RENIN SECRETION FROM THE INTACT KIDNEY AND FROM ISOLATED G L O M E R U L I BY THE CALCIUM IONOPHORE A23187

ETSUMORI HARADA

*, G A Y L E E. L E S T E R ** a n d R O N A L D P. R U B I N * * *

Department of Pharmacology, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298 (U.S.A.) (Received June 12th, 1978)

Key words: Ionophore A2318 7; Renin secretion; Ca2+ transport; (Kidney)

Summary The ionophore A23187 evoked a dose-dependent release of renin from the isolated perfused cat kidney, which was inhibited by calcium deprivation and adrenergic blockade. The latter finding indicates that the effects of A23187 on the intact kidney are mediated mainly by catecholamine release from sympathetic nerve endings. Ionophore also elicited a concentration-dependent enhancement of renin secretion from a pure preparation of glomeruli isolated from cat kidney; this stimulation was still manifest when the glomeruli were superfused with a calcium-free solution. These findings indicate that A23187 evokes renin secretion from juxtaglomerular cells by mobilizing cellular calcium and support the view that an increase in intracellular calcium is intimately involved in the mechanism of renin secretion.

Introduction

Previous studies from our laboratory provided evidence that renin release from the isolated perfused cat kidney does n o t directly involve extracellular calcium [1,2]. Thus, prolonged perfusion with calcium-free solution failed to depress catecholamine-induced renin secretion [1,2]. On the other hand, a role * During tenure as an A.D. Williams Distinguished Scholar: Medical College of Virginia. Permanent address: Department of Physiology, Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060, Japan. ** Present address: North Carolina Memorial Hospital, University of North Carolina School of Medicine, Chapel Hill, NC 27514, U.S.A. *** To wh om reprint requests should be addressed.

21 for intracellular calcium was suggested by the finding that enhanced secretion evoked by norepinephrine or by stimulation of renal sympathetic nerves was associated with a mobilization of intraceUular calcium which was temporally and quantitatively correlated with renin secretion [ 2]. A additional clue to the role of calcium was the observation that calcium alone was able to evoke renin release after a period of calcium deprivation [1,2]. These studies fostered the hypothesis that the physiological mechanism of renin secretion from the juxtaglomerular cells involves an increase in intracellular calcium derived from some cellular pool [1,2]. The calcium ionophore A23187 which has a high affinity for divalent cations and little or no affinity for monovalent cations [3] has provided a valuable tool for the study of the role of calcium in stimulus-secretion coupling. This ionophore apparently increases calcium permeability of the plasma membrane and thus activates hor:,~one and neurotransmitter release by increasing intracellular calcium [3,4]. It has previously been reported that A23187 enhances renin release from the isolated rat kidney in the absence of calcium and inhibits its release in the presence of calcium [ 5]. In another study, ionophore enhanced release from isolated rat glomeruli in the absence of external calcium [6]. These two groups concluded from their studies that calcium-stimulated exocytotic release of renin is not operative in the juxtaglomerular cell [5,6]. In order to help resolve the controversy concerning the role of calcium in renin secretion, the present investigation explores further the effects of A23187 on renin release from the isolated perfused kidney and isolated glomeruli of the cat to provide further information regarding the role of calcium in the events associated with renin secretion. Methods The cat kidney was perfused under constant pressure with Locke solution as previously described [1]. Flow rate varied from preparation to preparation (3--10 ml/min) but during the course of a given experiment, the constant pressure maintained a constant flow rate which was not altered by drug additions or by varying the ionic constituents. The Locke solution had the following basic composition: 154 mM NaC1, 5.6 mM KC1, 0.5 mM CaC12, 0.5 mM MgC12, 12 mM NaHCO3, 10 mM dextrose. In addition, the Locke solution also contained 3% dextran to maintain osmotic pressure and a mixture of amino acids to supply substrate for renin biosynthesis. The perfusion medium was equilibrated with 5% CO2 in oxygen, providing a pH of 7.1. In certain experiments CaC12 was omitted and ethyleneglycol-bis-{/~-aminoethylether)-N,N'-tetraacetic acid (EGTA) {0.4 mM) was added to chelate residual calcium. Two cats were administered reserpine {Serpasil-Ciba) (1.5 mg/kg intraperitoneally}, and 18 h later their kidneys were perfused in the usual manner. This reserpine concentration produces maximal depletion of peripheral catecholamine stores [7]. Glomeruli were isolated from feline kidney by a combination of graded sieving and magnetic iron oxide, which, while sacrificing yield, enables glomeruli to be separated from other tissue fragments in almost pure form [8]. Following perfusion of the kidney with Locke solution, a 40--60 ml suspension of magnetic iron oxide (15 mg/ml) was infused through the renal artery. The renal

22 capsule was removed and the kidneys hemisected. The renal cortex was minced in ice-cold Locke solution and forced through a stainless steel sieve (30 mesh, 500 gm) with a spatula. The tissue suspension was then passed sequentially through a 60 mesh (250 pm), 100 mesh (155 um), and a 230 mesh (63 pm) sieve. The tissue which was retained on the 230-mesh sieve was washed into a beaker which was placed atop a 2 kg magnet. The suspended tissue was swirled, allowed to settle, and the solution decanted and resuspended several times with additional washings. The final tissue precipitate which remained by virtue of its strong attraction to the magnet was a 99--100% pure preparation of glomeruli as observed by light microscopy. More than 90% of the glomeruli were completely devoid of renal arterioles which contain the main adrenergic nerve supply [9]. The intact glomeruli were resuspended in Locke solution at a concentration of 5 . 1 0 s glomeruli per 10 ml, placed in a 1 ml plastic syringe plugged with glass wool, and superfused with Locke solution containing 0.2% bovine serum albumin using a peristaltic pump (LKB-Varioperpex). The flow rate was 0.4--0.6 ml/min. The renal perfusate and glomerular superfusate were analyzed for renin activity by radioimmunoassay of angiotensin I as previously described [1], using renin substrate obtained from plasma of anesthetized cats whose renal vessels had been ligated for 6 h prior to extraction of blood. Values were corrected for flow and were expressed as ng/min or as percent of basal secretion. The ionophore A23187 which was generously supplied by Dr. R.L. Hamill of the Eli Lilly Co., Indianapolis, IN, U.S.A., was dissolved in ethanol and the final concentration of ethanol in the medium perfusing the intact kidney did not exceed 0.01%. Although ethanol alone had no discernible effect on renin release from isolated glomeruli, it was added to all control solutions to give the same ethanol concentration (0.3%) as those solutions which contained A23187. Results

Intact kidney The addition of ionophore A23187 to Locke solution evoked a dose-dependent increase in renin secretion from the isolated perfused cat kidney (Fig. 1). The threshold concentration of A23187 was 10 nM; peak effects were observed with 80 nM and higher concentrations manifested diminishing secretory activity (Fig. 1). No significant change in the secretory rate was discernible in control experiments in which ethanol alone (0.01%) was present in the perfusion solution (97 _+ 13%) (n = 4). Analysis of the pattern of the secretory response showed a maximum effect during the 10 min exposure to the ionophore and secretion gradually decreased after the stimulus was withdrawn (Fig. 2). However, 30 min after the ionophore was removed, a second phase of elevated secretion was sometimes observed, which also rapidly declined to basal levels (Fig. 2). Exposure to A23187 longer than 10 min did not further augment secretion, but in fact depressed it. Thus, during the 10--20th min of exposure to A23187 (300 nM) the secretory rate fell to 79 _+18% (n = 3) of that observed during the first 10 min exposure, and continued perfusion with the ionophore diminished o u t p u t to below basal levels.

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Renin release induced by catecholamines or sympathetic nerve stimulation can be blocked by fl-adrenergic blocking agents [2,10]. In three of five experiments, the ~-receptor blocking agent, propranolol (1 gM) markedly depressed or completely abolished the stimulant effects of A23187. However, in the other two experiments which employed propranolol, A23187 increased renin secretion by 124 and 67%, respectively. In light of the variable effects of propranolol, the stimulant action of A23187 was studied in kidneys from two reserpinized cats. Under these conditions A23187 (80 nM) failed to stimulate renin secretion, although isoproterenol (100 nM) was still an effective secretagogue (Fig. 3).

Isolated glomeruli Following a 60 min period of superfusion, spontaneous renin secretion from isolated glomeruli attained a fairly stable level (2.6 + 0.5 ng/min) (n = 17). The subsequent addition of A 2 3 1 8 7 elicited a dose-dependent facilitation of renin release. A transient enhancement of release was observed with 1 ~M A23187,

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and 10/~M A 2 3 1 8 7 produced a more sustained response which extended beyond the period of exposure to the ionophore (Fig. 4). A 20 min exposure to 5 pM A 2 3 1 8 7 enhanced renin secretion by 86%, but peak levels were reached during the following 30 min after the stimulus was removed (148%) 1.50

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and release was still enhanced by 124% after 50 min (Fig. 5). The stimulant action of an equimolar concentration of the fl-agonist isoproterenol is compared with that of the ionophore (Fig. 5). Although during exposure to isoproterenol renin release approached that of the ionophore, secretion fell more rapidly after isoproterenol was removed, reaching control levels after 50 min, while the stimulating effect of A23187 was still clearly manifest. When the calcium concentration was increased above 0.5 mM, the effect of A23187 was abolished. Thus, during and subsequent to the 20 min exposure to A23187 (5 ~M) in the presence of 2 mM calcium, renin release was 92 _+7 and 94 + 11% of basal, respectively (n = 8). When isolated glomeruli were perfused with calcium-free Locke solution basal renin release was enhanced. In two experiments renin release was increased b y an average of 92%. Ionophore (5 pM) further augmented renin release in the calcium-deprived medium. During a 20 min exposure to A23187 there was an average increase in renin release of 39%; and a 139 and 104% increase 30 and 50 min after removal of the ionophore, respectively. These increases compare favorably with average increases obtained by ionophore in the presence of calcium.

Discussion The present investigation has demonstrated the stimulant action of A23187 on renin secretion from the intact kidney and from isolated glomeruli. How-

26 ever, the basis for this stimulant action appears fundamentally different in these two preparations. Foremost among the factors controlling renin secretion by the juxtaglomerular cells are intrarenal baroreceptors and catecholamines released from renal sympathetic nerves [10]. The dose-dependent activation of renin secretion by ionophore in the perfused kidney appears to be indirect through the release of catecholamines as suggested by the inconsistent blockade produced by propranolol and confirmed by the inability of A23187 to stimulate renin release from perfused kidneys of reserpinized cats. The calciumdependent catecholamine-releasing properties of A23187 are well-documented and in adrenergically innervated organs such as the heart, many, if not all of the ionophore effects are mediated by catecholamine release [3,11]. Despite its inability to stimulate directly the juxtaglomerular cells of the perfused kidney, A23187 provided to be an effective stimulus of renin release from a pure preparation of glomeruli which almost completely lacked arteriolar attachments and was thus devoid of adrenergic nerve terminals [9]. In fact, in this preparation the ionophore elicited a greater peak response with a more prolonged duration than the response to an equimolar concentration of isoproterenol. It appears as though A23187 must penetrate intracellularly to exert its effect since removal of extracellular calcium failed to depress renin release from isolated glomeruli. A23187 is fully capable of facilitating the transfer of Ca 2÷ across intracellular membranes as well as the plasma membrane [3,4], and there are several lines of evidence that A23187 can stimulate secretory systems by an action on intracellular calcium stores [4,12--14]. The refractoriness of the effect of ionophore to calcium deprivation is analogous to the action of the catecholamines, where previous studies from our laboratory have provided evidence that prolonged calcium deprivation fails to depress catecholamineinduced renin release, although the intracellular receptor responsible for triggering renin secretion is, like other secretory systems, calcium sensitive [ 1,2]. The increase in spontaneous renin secretion from isolated cat glomeruli observed during calcium deprivation was also previously reported by Baumbach and Leyssac [6] using isolated rat glomemli; although this effect of calcium deprivation was n o t manifest when we perfused the feline kidney under constant pressure. The fact that A23187 enhanced the facilitatory effect of calcium deprivation prompted Baumbach and Leyssac to conclude that calcium, rather than exerting some positive modulatory role, plays some inhibitory role in renin secretion. Peart and his colleagues [5,15] have drawn similar conclusions from their studies on the intact rat kidney. Such conclusions are supported by the blunting of the secretory action of ionophore at high concentrations or in the presence of elevated calcium concentrations as observed in previous [5,6] and in the present studies. However, another interpretation of these data previously offered [16,17] is that secretion induced by A23187 is the result of a complex interaction between ionophore and calcium; and excessively high levels of free calcium in the cell produced by high concentrations of ionophore and/or calcium may be inhibitory. And in regard to the stimulatory effects of calcium deprivation on renin secretion, it must be noted that in addition to its action on the secretory process, calcium can regulate cell volume by virtue of its effects on the permeability properties of the cell. Yet a distinction must be made between effects on spontaneous secretion and on release evoked

27

by catecholamines, sympathetic nerve stimulation, A23187 or any other stimulating agent. Enhanced renin release under the highly unphysiologic stimulus of complete calcium deprivation might merely represent leakage through a more permeable membrane. In support of this view is the recent finding that glucagon release evoked by calcium deprivation, in contrast to release evoked by the more physiological secretagogue, arginine, is not an exocytotic process

[18]. It has previously been shown that calcium alone can evoke renin secretion [1,19] as it does in other secretory systems and renin secretion evoked by catecholamines or sympathetic nerve stimulation is associated with an increased efflux of radiocalcium from the perfused cat kidney [2]. Thus, the stimulant action of ionophore demonstrated in the present study should be construed as further supportive evidence that calcium provides the link between membrane activation and the secretory response in the juxtaglomerular cell just as it does in other secretory systems. In the juxtaglomerular cell it is a cellular pool of calcium that appears to play a principal role in this process. Acknowledgement The work was supported by U.S.P.H.S. Research Grant AM-18066 and Pharmacology Training Grant GM-0711. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

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