J. Phyeiol. (1978), 274, pp. 367-379 With 6 text-ftgure8

367

Printed in Great Britain

STIMULATION OF RENIN SECRETION AND CALCIUM EFFLUX FROM THE ISOLATED PERFUSED CAT KIDNEY BY NORADRENALINE AFTER PROLONGED CALCIUM DEPRIVATION

BY E. HARADA* AND R. P. RUBINt From the Department of Pharmacology, Medical College of Virginia, Virginia Commonwealth Univereity, Richmond, Virginia 23298 U.S.A.

(Received 1 Auguwt 1977) SUMMARY

1. The effects of noradrenaline on the secretion of rein and on the efflux of Ca have been investigated in the isolated cat kidney perfused with Locke solution containing the a-adrenergic blocking agent, phenoxybenzamine, to block increases in renal vascular resistance. 2. Noradrenaline elicited a marked increase in renin secretion in the absence of any discernible alterations in renal arterial pressure, and prolonged perfusion with Ca-free Locke solution failed to depress noradrenaline-evoked renin secretion. 3. Noradrenaline caused an increase in the efflux of 45Ca from prelabelled kidneys perfused with Ca-free solution. Increasing the noradrenaline concentration produced graded increases in "iCa efflux and renin release, and the peak rise in efflux preceded or coincided with peak renin secretion. 4. DL-Propranolol inhibited the increase in 45Ca efflux and rein release resulting from noradrenaline stimulation. 5. Electrical stimulation of the renal nerve enhanced 45Ca efflux and renin release from prelabelled kidneys perfused with normal Locke solution. 6. These findings provide further support for the view that the process of catecholamine-induced renin secretion involves mobilization of Ca from a cellular site. INTRODUCTION

The concept of Ca as a mediator in stimulus-secretion coupling was originally developed from studies carried out on the adrenal medulla (Douglas & Rubin, 1961, 1963). One of the many pieces of evidence accumulated in favour of this concept was the relationship established between the extracellular Ca concentration and the rate of acetylcholine-induced catecholamine release. The seemingly universal role of Ca in stimulus-secretion coupling has been extended to many other secretary systems (see Rubin, 1974), although in certain tissues, such as the rat parotid gland (Schramm & Selinger, 1975) and human blood platelet (Feinstein & Fraser, 1975), a significant * During tenure as a A. D. Williams Distinguished Scholar, Virginia Commonwealth University, Medical College of Virginia. Permanent Address: Department of Physiology, Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060, Japan. t Send reprint requests to: Dr R. P. Rubin, Department of Pharmacology, Medical College of Virginia, Richmond, Virginia 23298 USA.

E. HARADA AND R. P. RUBIN degree of evoked release persists in the absence of Ca in the extracellular medium. In such tissues the utilization of Ca from intracellular stores may be critical for activating secretion. One of the factors which controls renin secretion from the juxtaglomerular cells of the kidney is the sympathetic nervous system (see Davis & Freeman, 1976). Recent experiments carried out in our laboratory demonstrated that perfusion of the feline kidney for several hours with a Ca-free medium failed to inhibit isoprenaline-induced renin release, although synthesis and/or mobilization of this enzyme was depressed (Lester & Rubin, 1977). Since Ca alone evoked a large increase in renin secretion after prolonged Ca deprivation, it was tentatively concluded that the same Ca-sensitive mechanism which controls most other secretary processes also controls renin secretion, but that catecholamine-induced renin secretion is mediated by the translocation of a cellular pool of Ca. If the activation of a cellular store of Ca constitutes an important event in triggering renin release, then it should be possible to demonstrate the mobilization of Ca from the juxtaglomerular cells by analysis of radiocalcium efflux. The results of such experiments provide the basis of this study. 368

METHODS

Kidney perfusion. Under i.P. pentobarbitone anaesthesia, the left or right kidney of the cat (2-3 kg) was prepared for perfusion in 8itu at room temperature by a modification of the previously described technique (Lester & Rubin, 1977). After perfusion was begun in situ by means of a cannula inserted into the abdominal aorta, the kidney was removed and placed in a plexiglass chamber. A perfusion cannula was inserted in the renal artery and perfusion fluid was transported through the kidney by means of a pulsatile pump (Harvard); collection of the effluent was carried out by means of a cannula placed in the renal vein. The flow rate varied

from preparation to preparation but was generally 6 ml./min. All perfusions were carried out at room temperature with normal or modified bicarbonatebuffered solution containing dextran (3 %) to maintain osmotic pressure and a mixture of amino acids as a substrate source. The composition of the Locke solution in m-mole/l. was: NaCl 154; KCl 5-6; CaCl2 2-0; MgC12 0 5; NaHCO3 12, dextrose 10. In certain experiments the CaCl2 (2 mM) normally present in Locke solution was omitted. Noradrenaline, the sympathetic neurotransmitter, is a potent a-adrenergic agonist and therefore produces an increase in renal sympathetic tone by vasoconstriction of the renal vasculature. Thus, the a-adrenergic blocking agent, phenoxybenzamine (1-2 FM), was added to perfusion solutions to block any increase in renal vascular resistance elicited by the administration of noradrenaline or by nerve stimulation. All solutions were equilibrated with 95 % oxygen and 5 % carbon dioxide with a pH of 7 0. Perfusion pressure was monitored using a Statham pressure transducer (P23AC) and recordings made by a Grass (Model 7) polygraph. At the onset of perfusion, pressure was variable but stabilized during the 60 min of perfusion with normal Locke solution at a level of 70-90 mm Hg. Nerve stimulation. With the aid of a dissecting microscope, the renal nerves were separated from the renal artery and laid across Pt stimulating electrodes, approximately 1-2 cm from the hilus of the kidney. Biphasic stimuli were applied from a Grass (Model SD5) stimulator. The parameters of stimulation chosen (20 V; 20 Hz; 5 msec) were greater than those which produced marked stimulation of renin secretion by the cat kidney in vivo (Coote, Johns, MacLeod &

Singer, 1972).

Estimation of renin. Perfusate samples collected over various intervals (2-10 min) were converted to angiotensin I and renin activity determined by radioimmunoassay as previously described (Lester & Rubin, 1977). Values were corrected for flow and expressed as ,tg/min or as percent of basal rates of secretion. 4"Ca-effiux studies. In this series of experiments kidneys were initially perfused with normal Locke solution for 60 min and then with Ca-free Locke solution plus phenoxybenzamine (1-2 /zM). After 30 min of Ca-free perfusion, "Ca (1 #c/ml.) was perfused at a constant rate for

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60 min. During this period of labelling, noradrenaline (10-8 M) was added every tenth minute for six 2 min periods and then perfusion was continued with nonradioactive Ca-free solution. Drugs were added at various times during washout (see Results). In the experiments which employed nerve stimulation, perfusion was switched to normal Locke solution after 90 min of Ca-free perfusion. Perfusate was collected in 2-10 min samples. Aliquots of perfusate (500 /4.) were added to ACS scintillation cocktail (Amersham/Searle) and the 45Ca content determined by scintillation spectrometry. The radioactivity in the kidneys at the end of the experiment was also determined by homogenizing randomly selected pieces (1-5-2-5 g) of kidney in 4 ml. CaCl2 (3 mm). The homogenate was diluted with 4 ml. trichloroacetic acid (10 %, v/v), mixedthoroughly, and allowed to stand overnight at room temperature. Aliquots (100-200 #1.) were added to scintillation solution and radioactivity determined. Washout curves were plotted from a summation of the "Ca present in each sample and the amount of 45Ca remaining in the kidney at the end of the washout period. The computed curves were expressed as the 45Ca efflux rate coefficient (percent/min), which is a measure of the amount of radioactivity leaving the kidney as a percentage of the radioactivity in the organ at any given time. When statistical analysis is employed results are expressed as the mean + s.E. Substances used. Phenoxybenzamine (HCl salt) was generously supplied by Smith, Kline & French; and DL-propranolol (HCl salt) and dexpropranolol (AY-20,694) were generously donated by Layerst Laboratories. Noradrenaline bitartrate was obtained from Sigma; [125I]angiotensin was obtained from New England Nuclear; and the amino acid mixture came from Grand Island Biological Company. Stock solutions of all reagents were freshly prepared and diluted with saline to desired concentrations. RESULTS

The effect of prolonged Ca deprivation on noradrenaline-evoked rein release. During prolonged perfusion with normal Locke solution a period of steady-state spontaneous renin release was attained; during this period the average rate of secretion was 211 ± 30 ng/min (n = 12). A 10 min exposure to noradrenaline elicited a dosedependent increase in renin secretion, reaching and maintaining peak levels during the 6th-lOth min (Fig. 1A), and then gradually returning to basal levels after the stimulus was withdrawn. As the concentration of noradrenaline was increased, the secretary response developed more rapidly and returned to basal levels more slowly after cessation of stimulation. After prolonged Ca-free perfusion the average rate of basal renin secretion (548 ± 113 ng/min) (n = 22) was significantly enhanced (P < 0-05). A previous study demonstrated that prolonged periods of Ca deprivation failed to depress the initial burst of renin release that followed exposure to isoprenaline (Lester & Rubin, 1977). Similarly, prolonged perfusion with Ca-free Locke solution failed to depress the secretary response to varying concentrations of noradrenaline (Fig. 1 B). In fact, a significant enhancement of renin secretion was observed with noradrenaline 10-7 M after 144 min of Ca-free perfusion (P < 0-01). In two other experiments, kidneys were perfused for 80 min with Ca-free Locke solution plus ethyleneglycol-bis (,f-amino ethyl ether)-N,N'-tetraacetic acid (EGTA) (0-4 mM) to chelate any residual Ca. After 80 min of perfusion, exposure to noradrenaline 3 x 10-8 and 3 x 107 M augmented renin secretion 3-2 and 6-3-fold, respectively; these increases compare favourably with average increases of 2-9 and 5-9-fold, respectively, obtained by these same noradrenaline concentrations after prolonged Ca-free perfusion in the absence of EGTA (Fig. 1 B). In several experiments the renal arterial pressure was monitored prior to and during noradrenaline stimulation. The results of such experiments conducted during

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370

perfusion with normal and Ca-deprived Locke solution are depicted in Fig. 1A and B. In the presence of phenoxybenzamine, the noradrenaline-induced increase in renin release was not accompanied by any discernable change in the renal arterial pressure. However, these experiments would not detect any existing autoregulation of renal perfusate flow within the microcirculation. B Ca-free Locke solution

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140 144 148 152 156 160 148 152 156 160 (min) (min) Fig. 1. Pattern of noradrenaline-induced renin secretion from the cat kidney during perfusion with (A) normal or (B) Ca-free Locke solution. Kidneys were perfused with normal Locke solution or Ca-free Locke solution and varying concentrations of noradrenaline were added to the perfusion fluid for 10 min during the 144th-154th min of perfusion. Phenoxybenzamine (1-2 /M) was also present in the perfusion medium. Each point represents the mean output ( + s.E.) during each 10 min collection period (n = 4), expressed as percent of basal values obtained from perfusate collected immediately before stimulation. The lower panels depict mean values obtained for renal perfusion pressure during corresponding collection periods with 10-7 M-noradrenaline (n = 9). 140 144

Pattern of spontaneous 45Ca efflux. The pattern of 45Ca efflux from the perfused kidney was first determined after preloading the tissue with '5a for 60 min. One such washout curve is depicted in Fig. 2. It shows a multiphasic pattern with an initial rapid phase which was followed at about 30 min and 60 min by slower phases.

371 45Ca EFFLUX AND RENIN RELEASE A 4th phase was delineated beginning at about 100 min. Similar curves were obtained in all experiments. The most rapid phase presumably reflects loss from the extracellular space. The origin of the intermediate phases cannot be defined although they probably reflect washout from various tissue components of the kidney. Since the 900 r

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slowest phase presumably represents loss from the intracellular compartments, it was decided to test the effects of noradrenaline on 45Ca efflux after 144 min of washout. 45Ca efflux during enhanced secretary activity. After the 4Ca efflux had become relatively constant, and presumably represented Ca released from intracellular components, the addition of noradrenaline induced a rapid and well-defined dose-related

E. HARADA AND R. P. RUBIN 372 increase in 4JCa release (Fig. 3A) which paralleled the increase in renin secretion (Fig. 3B). 45Ca released reached a peak within the first 2 min with noradrenaline 10- M and within the first 4 min with noradrenaline 3 x 10-8 M and then slowly 120 r-

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373 45Ca EFFLUX AND RENIN RELEASE declined (Fig. 3A). Although it is more clearly seen with noradrenaline 10-7 M, peak "Ca efflux preceded peak renin release and returned to below basal levels while secretion was still clearly enhanced (Fig. 3A and B). The fl-adrenergic receptor blocking agent, DL-propranolol 10-6 M, produced a parallel inhibition of "5Ca efflux and renin release induced by noradrenaline 10-7 M (Fig. 4). Thus in the presence of DL-propranolol, noradrenaline augmented 45Ca efflux and renin secretion by only 3 and 22 %, respectively, as compared to 15 % and 600

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rsneo propranolol -fold~ ~-nrae ~DL-propranolol ~~~~~~~~~~D Lpopaoo.I h(A) "Ca Ofnteasneo efflux and (B) renin Fig. 4. Effect on noradrenahne-evoked secretion. Kidneys prelabelled with3'Ca were perfused with Ca-free Locke solution and noradrenaline (10-d m) was added as indicated for 10 mm. In certain experiments DLhpropranolol (10s m) was present from the 120th min of perfusion. Each point represents

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5-5-fold increases in the absenceofDL-propranolol. In the presence of dexpropranolol ( 06 M), which is devoid of fl-receptor blocking activity, noradrenaline 10co m increased peak45Ca efflux and renin secretion by 11(±+ 2-0) 0/ and 4-5 (± 0.5)-fold, respectively (n = 4); these increases are not significantly different from their corresponding controls (P > 0.05). Although our primary interest concerned the action of noradrenaline, glucagon was utilized as a stimulating agent in 3 different preparations. The results of one such experiment are depicted in Fig. 5. Glucagon caused a peak rise in 5Ca efflux within the first 2 min, which gradually declined toward basal levels during continued exposure to the hormone. Renin secretion peaked at 6 min and returned to near basal levels by the time the stimulus was removed. Response to nerve stimulation. Due to the well known effects of Ca on nerve conduction and transmitter release (Rubin, 1974) it was not surprising to find that initial experiments which attempted to elicit renin secretion by nerve stimulation were generally unsuccessful when perfusion was carried out with Ca-free Locke solution.

E. HARADA AND R. P. RUBIN 374 So subsequent experiments were conducted during perfusion with normal Locke solution containing phenoxybenzamine. Nerve stimulation elicited a prompt rise in renin secretion which peaked during the 2nd-4th min of stimulation and then very gradually declined even after stimulation ceased (Fig. 6B). During stimulation no change in renal arterial pressure was discernible (Fig. 6C). Altering the frequency of stimulation did not elicit marked changes in the secretary rate, although the rate could be quantitatively correlated with the pulse duration over the range of 1-lOmsec. 115 r A

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45Ca EFFLUX AND RENIN RELEASE 375 can be noted by comparing Figs. 3 and 6, that nerve stimulation and noradrenaline 3 x 10-8 M, which elicited "Ca release of a similar magnitude, also evoked similar peak levels of renin secretion. 110 r A

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376

E. HARADA AND R. P. RUBIN DISCUSSION

The present investigation supports previous findings from our laboratory (Lester & Rubin, 1977) that extracellular Ca is not directly involved in the mechanism of catecholamine-evoked renin release from the perfused cat kidney. One important piece of evidence offered to substantiate this postulate derives from the observation that perfusion with a Ca-free medium for up to 3 hr failed to depress the secretary response to noradrenaline, even though as previously reported (Fray, 1977) calciumfree perfusion enhanced basal secretion. However, the fact that evoked secretion is unimpaired in a Ca-free medium need not necessarily imply that extracellular Ca is not involved, especially if only a small amount of Ca is needed to activate the secretary process. Indeed, the kidney contains high levels of Ca relative to other tissues (see Rubin, 1974), so perfusion with a Cafree medium, which would unquestionably increase cell membrane permeability, could cause leaching of Ca from tissues to the medium to sustain secretion. But the fact that the Ca antagonist, D-600, was unable to block catecholamine-evoked renin secretion (Lester & Rubin, 1977), even in concentrations as high as 500 /M (E. Harada & R. P. Rubin, unpublished), lends strong support for the view that an intracellular source of Ca is utilized for renin release. On the other hand, the juxtaglomerular cell can utilize extracellular Ca when the nature of the stimulus so dictates, as evidenced by the fact that the readdition of Ca to the perfusion medium after a period of Ca deprivation causes a marked enhancement of renin secretion (Chen & Poisner, 1976; Lester & Rubin, 1977). Under these conditions of Ca-free perfusion the resulting increase in cell membrane permeability presumably enables the restored Ca to enter the cell and stimulate secretion (see Douglas, 1975). The noradrenaline-evoked increase in 4'Ca efflux from the perfused kidney also identifies an effect on cellular Ca. A modest increase in 45Ca efflux was demonstrable, amounting to only 15 %, but the magnitude of this effect is consistent with the small number of juxtaglomerular cells in the kidney which would be responsive to stimulation by noradrenaline. This enhanced efflux was demonstrable in Ca-free medium, which reflects a primary release of Ca from cellular stores rather than an exchange effected by an influx of extracellular Ca. Measuring Ca fluxes in response to noradrenaline in such a heterogeneous tissue as the kidney could be fraught with potential difficulties. However, careful analysis of this effect leads to the conclusion that the juxtaglomerular cells are the source of the enhanced 45Ca efflux and that this Ca may be involved in renin secretion. A temporal and quantitative correlation was established between 45Ca efflux and renin secretion. Peak Ca efflux preceded or coincided with peak renin release and graded concentrations of noradrenaline produced parallel increases in 'RCa efflux and renin secretion. Moreover, the adrenergic f-receptor blocking agent, DL-propranolol, produced a concomitant inhibition of 45Ca efflux and renin secretion. The fact that dexpropranolol, which is as potent a local anaesthetic agent as L-propranolol (Dohadwalla, Freedberg & Vaughan Williams, 1969; Langslet, 1970) but has no ,f-receptor blocking action (Barrett & Cullum, 1968), was ineffective in inhibiting catecholamine-induced renin release supports the proposition that DL-propranolol blocked 45Ca effiux by antagonizing the interaction of noradrenaline with the

4'Ca EFFLUX AND RENIN RELEASE 377 Preceptor. Additionally, the kidneys were perfused with the a-receptor blocking agent, phenoxybenzamine, so that little or no changes in renal arterial pressure were discernible during the infusion of noradrenaline. Taken together these data suggest that noradrenaline evokes "Ca efflux and renin secretion by a direct action on the ,8-adrenergic receptor of the juxtaglomerular cell and not via alterations in pressure changes in the renal vasculature (Schrier, 1974). Even though it is not known what proportion of the kidney consists of smooth muscle cells, these cells were not likely to be a major source of the "Ca efflux. It has previously been noted that catecholamines cause little or no changes in 45Ca efflux in smooth muscle cells (LUllmann, 1970; Deth & Van Breemen, 1977), especially in a Ca-deprived medium; and when such an effect is demonstrable, it is blocked by a-receptor blocking agents rather than f-receptor blocking agents (Deth & Van Breemen, 1977). The juxtaglomerular cells are innervated by postganglionic sympathetic fibres (Davis & Freeman, 1976), and a previous study has shown that nerve stimulation causes renin release from the feline kidney in vivo (Coote et al. 1972), although these effects were attributed to an accompanying sharp decrease in renal blood flow. In the present investigation electrical stimulation of the renal nerve elicited a concomitant enhancement of 4Ca efflux and renin release in the absence of any measurable change in perfusion pressure. These data make it tempting to speculate that the physiological mechanism of renin secretion involves the release of noradrenaline from sympathetic nerve endings which acts directly on the juxtaglomerular cell to effect an intracellular mobilization of bound Ca. However, conclusions drawn from these experiments must be qualified since they were conducted during perfusion with a medium containing Ca; this modification in the experimental procedure was necessitated by the Ca requirement for neurotransmitter release from sympathetic nerve endings (see Rubin, 1974). Thus, these results do not preclude the possibility that a Ca-Ca exchange rather than a primary mobilization of intracellular Ca was the critical event during renin secretion elicited by nerve stimulation. In an earlier study, glucagon was found to be a potent stimulant of renin secretion from the perfused cat kidney by a mechanism not mediated by fl-adrenergic receptor activation (Lester & Rubin, 1977). The fact that the secretary action of glucagon is accompanied by a prior increase in "Ca efflux indicates that the increase in 45Ca release is not restricted to agents which augment renin release by fireceptor activation but may be a general property of agents which are capable of triggering renin secretion. Although the evidence marshalled up to now suggests a key role for cellular Ca in the mechanism of renin release, the exact source of this cation remains to be defined. Secretory organelles are known to contain relatively large amounts of Ca (Borowitz, Fuwa & Weiner, 1965; Wallach & Schramm, 1971; Clemente & Meldolesi, 1975) which may be released pari pass with other secretary products. However, the renin-containing secretary granule does not appear to be the principal locus of the released Ca since M5Ca efflux preceded rather than paralleled renin secretion and under certain conditions fell to below basal levels while renin secretion was still enhanced. Some light might be shed on this aspect of the problem by extrapolating from studies conducted on catecholamine-facilitated amylase release from

E. HARADA AND R. P. RUBIN the rat parotid gland, which is also mediated by Preceptor activation and does not require extracellular Ca (Schramm & Selinger, 1975). In this tissue, Dormer & Ashcroft (1974) have reported that adrenaline releases Ca from mitochondrial and microsomal fractions of the cell. If, indeed, catecholamines elicit release of Ca from mitochondrial and microsomal compartments of the cell, the question arises as to how these effects are mediated. Evidence derived from other tissues provides strong support for the concept that expression of the effects of catecholamines through fl-receptor activation is mediated by cyclic AMP (Robison, Butcher & Sutherland, 1971). Although the results of the present and previous studies (see Davis & Freeman, 1976) strongly support the notion that catecholamine-induced renin secretion involves activation of fl-receptors, the presently available evidence regarding the role of cyclic AMP in renin secretion is sparse and contradictory (Winer, Chokshi & Walkenhorst, 1971; Peart, Quesada & Tenyi, 1975). A better understanding of the disposition of Ca in the juxtaglomerular cell during activation of renin release by catecholamines may be arrived at by precise definition of the role of not only cyclic AMP, but the prostaglandins as well; both of these putative mediators appear capable of acting as Ca ionophores (Rasmussen, Jensen, Lake, Friedman & Goodman, 1975; Rubin & Laychock, 1977).

378

This work was supported by United States Public Health Service Research Grant AM-18066.

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BAmREn,

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