Fish Physiology and Biochemistry vol. 7 nos 1-4 pp 323-329 (1989) Kugler Publications, Amsterdam/Berkeley

Signals controlling renin release in aglomerular toadfish Hiroko Nishimura and Margaret A. Madey Department of Physiology and Biophysics, University of Tennessee, Memphis, Tennessee 38163, USA Keywords: renin release, aglomerular teleost, toadfish, isoproterenol, cyclic AMP, cyclic GMP, calcium, calcium channel, K+ depolarization, baroreceptor, calcium channel antagonist

Abstract The toadfish, Opsanus tau, lacks renal glomeruli and macula densa, but has high renal renin activity and abundant granulated cells in renal arteries and arterioles. Reduction of blood pressure (BP) or blood volume by hemorrhage or vasodilatory drugs causes renin release, indicating that an intrarenal or extrarenal pressureor volume-sensitive mechanism exists for controlling renin release in the toadfish. Thus, we examined whether 1) 3-adrenergic receptor-mediated activation of renin release, and 2) calcium influx which may underlie the baroreceptor mechanism are involved in the cellular control of renin release. Acute injection of isoproterenol (1 jsg/kg, n = 6) decreased BP and increased plasma renin activity (PRA) 4-5 fold in unanesthetized toadfish. Propranolol abolished both effects, but did not decrease basal PRA levels. In vitro superfusion of renal slices with bicarbonate Ringer's solution showed a steady secretion of renin, and addition of 50 mM K+ (K + methylsulfate replacing NaCl, n = 10) to the superfusate markedly suppressed renin secretion. Nifedipine (10- 5 M, n = 8) completely restored the high K+-induced inhibition of renin secretion from renal slices, whereas isoproterenol (10- 4 M, n = 6) neither increased basal renin secretion nor restored K +-induced renin suppression. These results suggest that calcium influx may mediate inhibitory messages for renin secretion, while the -adrenoceptor-mediated activation of granulated cells appears absent in toadfish.

Introduction The renin-angiotensin system has been found in a variety of bony fishes and tetrapods and appears important in the control of cardiovascular function and in fluid-mineral homeostatis (Nishimura 1980, 1987; Wilson 1984). In mammals, four major mechanisms of control of renin release have been suggested: 1) an intrarenal baroreceptor, sensing the changes in renal arterial pressure at the juxtaglomerular (JG) cells; 2) the macula densa of the

distal tubule, perhaps sensing the rate of C1- or Na + transport; 3) sympathetic outflow via ,Badrenergic receptor activation of the JG cells; and 4) various humoral factors, including prostanoids, steroid hormones, angiotensin (ANG) and other peptide hormones, and electrolytes (Keeton and Campbell 1980). Although cellular mediators of renin secretion are not well understood, pharmacological observations in mammalian kidneys suggest that diverse first messengers or renin secretagogues alter the ac-

Correspondence to: Hiroko Nishimura, M.D., Department of Physiology and Biophysics, University of Tennessee, Memphis, 894 Union Avenue (NA 426), Memphis, Tennessee 38163, USA.

324 tivity of the renin secretory cells by influencing the activities of two intracellular mediators: 1) cyclic (c) AMP that acts as a stimulatory second messenger of the substances that activate adenylatecyclase, and 2) cytosol-free calcium ion (Ca 2 +i), which appears to be an inhibitory second messenger in renin secretion (Keeton and Campbell 1980; Churchill 1985, 1987; Fray et al. 1987). Kidneys of the aglomerular toadfish, Opsanus tau, lack functioning glomeruli and the macula densa, but have abundant granulated cells in the small arteries and arterioles. Toadfish release renin in response to the reduction of blood pressure (BP) and/or blood volume induced by hemorrhage and hypotensive drugs (Nishimura et al. 1979), suggesting that an intrarenal pressure- or volume-sensitive mechanism or extrarenal baroreceptor that transmits information by neural pathways exists to mediate renin relase in toadfish. Toadfish kidneys, however, do not exhibit monoamine-specific fluorescent nerve activity in relation to small arteries and arterioles (Madey et al. 1984). Thus, in the toadfish, a pressure-sensitive receptor, possibly located on the granulated cells, may be a primary mechanism for the control of renin release. In the present study, using in vivo unanesthetized toadfish and in vitro renal slices, we examined whether calcium influx and 3-adrenoceptor activation in granulated cells alter the control of renin secretion. Materials and methods Fish and maintenance Adult toadfish, Opsanus tau, of both sexes, 350-600g, were obtained from the Marine Biological Laboratory, Woods Hole, MA, U.S.A. They were kept in temperature-regulated aquaria (15°C) containing 50%0 sea water (Instant Ocean, synthetic salts, Aquarium Systems; Mentor, OH) and were fed fresh clams and shrimp twice a week. Surgical setup and experimental protocols in unanesthetized fish Three days before surgery, toadfish were placed in

a plastic chamber through which 50% sea water (15°C) was circulated by a pump. They were anesthetized by immersion in tricaine methanesulfonate (Finquel, Ayerst, 0.03%) for 15 min. A clear vinyl expanded catheter (Dural Plastic & Engineering; Australia) was implanted in the gastric or splenic branch of the celiac artery and an intestinal branch of the hepatomesenteric artery for blood collection (or drug injection) and BP measurement, respectively. Blood samples for measuring plasma renin activity (PRA) were collected into chilled capillary tubes coated with ammonium EDTA. Isoproterenol (1 tgg/kg, n = 6) was injected intraarterially, and blood samples were collected before and 5-10, 30, 60, and 120 min after the drug injection. The time control study was done in the same fish before the experiment by injecting 0.9% saline instead of isoproterenol. To see the effect of -adrenergic blockade, propranolol (1 mg/kg/h, n = 5) was infused, and isoproterenol (1 and 2 ag/kg) was injected during propranolol infusion. Samples were collected at 15 and 30 min after isoproterenol injection.

Preparationof renal slices and experimental protocol in vitro Toadfish were anesthetized as above. Kidneys were excised promptly and cleaned of surrounding connective tissues. Renal slices (30-50 mg each; 2030 pieces; total, 0.8-1.0 g per column) pooled from 3-4 fish were divided into eight columns and superfused with bicarbonate Ringer's solution (Ringer superfusate, pH 7.4, 21-22°C, bubbled with 95% 02 and 5% CO2). Superfusion flow rate was controlled (0.35-0.50 ml/min), and collection flow rate was measured. After 90 min equilibration, three 15-min effluent samples were collected into preweighed plastic tubes. The columns were then superfused with Ringer's solution containing 50 mM K + , and another three 15-min collections were made. Drugs or hormones were then added to 50 mM K+ Ringer's, and four more samples were collected. To examine the effects of drugs on basal rate of renin release, the drugs were added to regular Ringer superfusate without 50 mM K+ during

325 the second three consecutive collection periods. Effluent samples were mixed with incubation medium (excluding plasma) immediately after collection and stored at -60°C till analysis. Renal slices were frozen (-60°C) for renin content analysis after the experiments were completed.

Solutions and drugs The Ringer superfusate (312 mOsm/kg/H 2 0) contained in mM: 120 NaCl, 5.0 KCI, 2 MgSO 4, 18 NaHCO 3, 10 Na acetate, 0.5 NaH 2PO 4 , 1.5 Na2 HPO4, 2.5 CaC12, 8.3 a-glucose, 5.0 -alanine. For high K + Ringer's solution, 50 mM K + methylsulfate was added, replacing NaCI. The Cl- concentration was reduced by substituting Na methylsulfate for part of the NaCl to prevent high extracellular Cl- from causing granulated cell swelling. Osmolality and pH of the high K + Ringer's solution were the same as that of regular Ringer's solution. Isoproterenol (Sigma), propranolol (d, 1; Ayerst Laboratories), nifedipine (Sigma), dibutyryl cAMP (Sigma), and 8-bromo cGMP (Sigma) were commercially purchased. Bay K was a gift from Miles, Inc. (West Haven, CT).

Measurement of PRA and superfusion effluent PRA was determined as the rate of ANG I generation as reported previously (Nishimura et al. 1977, 1979). ANG I was determined by radioimmunoassay using [His 9 ]ANG I antibody (New England Nuclear Radioimmunoassay Kit), which crossreacts with toadfish ANG I (Nishimura et al. 1977). PRA was expressed as nanograms of ANG I equivalent formed from 1 ml of plasma during lh of incubation. The incubation mixture for measurement of renin content in the superfusion effluent contained: toadfish plasma, 20 tl; superfusion effluent, 100 1l; 2 M ammonium acetate buffer (pH7.4, 42 /1l); 3.8% EDTA-NH 4 (pH 7.4, 20 l); 25 mg/ml phenylmethylsulfonylfluoride (Sigma, 8 /l); and 1% neomycin sulfate (Sigma)-1% thimerosal (Sigma) solution (pH 7.4, 10 /al). Incubation was conducted at 20°C for 3h, which yields ANG I

linear to the duration of incubation. Toadfish plas0.40 ma has high angiotensinogen levels (4.16 /g ANG I equivalent/ml plasma, n = 9) and low renin activity. ANG formation was negligible in incubation mixtures from which either plasma or superfusion effluent was removed. Renin secretion from renal slices was expressed in two ways: 1) ng of ANG I equivalent formed/ml of superfusion effluent/mg of fresh kidney/h of incubation; 2) renin secretion as shown in 1) during lh of superfusion.

Results Effect of /3-adrenergicdrugs on BP and PRA A single injection of isoproterenol reduced BP and increased PRA 4-5 fold immediately after injection, and PRA gradually returned to the preinjection level when BP was restored (Fig. 1). Neither BP nor PRA changed during the 2-h time control study. Infusion of propranolol (Prop, n = 5) tended to decrease BP slightly (mm Hg) (control, 1.2), but did not signi19.8 + 1.3; Prop, 18.6 ficantly alter PRA (ng angiotensin I/ml/h) (con0.05). Injection trol, 0.28 + 0.03; Prop, 0.35 of isoproterenol (2 pxg/kg) during propranolol infusion (Prop + Isop) neither increased PRA (Prop + Isop: 0.37 + 0.04) nor decreased BP (Prop + Isop: 19.2 + 1.2).

Renin releasefrom renal slices and effect of K + -depolarization The time course and handling controls for renin secretion from renal slices are shown in Fig. 2. During the 150-min experimental course, the effluent flow rate remained stable, while renin secretion tended to decrease. This gradual decline of renin secretion is not due to the depletion of renin in the kidney, however, since the renin content of renal slices is more than 1,000 times higher than the total amount of renin released into the effluent during 4h of superfusion. Addition to the superfusate of 50 mM of K + , that presumably depolarizes granulated cell membranes (Fishman 1976), markedly decreased renin secretion (Fig. 3, upper panel).

326 EFFECT OF ISOPROTERENOL ON PLASMA RENIN ACTIVITY

RINGER RINGER

ISOPROTERENOL fg/kg

RINGER

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60

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90

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120

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150

TIME (min)

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3 3

Fig. 2. Time course and handling control study of renin release from renal slices of toadfish superfused with regular Ringer's solution at pH 7.4, 21-230 C, bubbled with 95%o 02 and 5% CO 2 . Effluent flow rates shown here are the same, at steady state, as the superfusion rate. Data are mean + SEM (n = 6).

o

PRE-

0

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INJECTION LTIME AFTER INJECTION (min) PLASMA

RENIN ACTIVITY

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RINGER 150 mM KI 50 mM K+

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TIME CONTROL

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ISOPROTERENOL

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ISOPROTERENOL

Opsanus iaou 50% Seawater

Fig. . Plasma renin activity (PRA) (dotted column) and blood pressure (BP) (closed circle) responses to an intraarterial injection of isoproterenol in the unanesthetized toadfish (mean + SEM, n = 6). Time control PRA (open column) and BP (open circle) were measured in the same fish before the experiments (n = 4). Significance of the differences in PRA between the time control and isoproterenol studies was determined by a paired t test (4 pairs), using the logs of the data (transformed).

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Effects of calcium channel blocker and calcium channel agonist on renin secretion from renal slices The addition of nifedipine (10- 5 M), a voltagesensitive calcium channel blocker, to the 50 mM K + superfusate restored renin secretion suppressed by K+ to the control levels (Fig. 3, lower panel). Furthermore, Bay K 8644 (10- 5 M, n = 4), a calcium channel agonist which presumably prolongs the opening time of the channels, reversed the nifedipine effect on K+-induced renin suppression (Table 1).

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TIME(min) Fig. 3. Suppression of renin secretion from toadfish renal slices by 50 mM K+ in the superfusate (upper panel, n = 10) and the inhibitory effect of nifedipine on K+ -induced renin suppression (lower panel, n = 8). Superfusate was exchanged without interrupting superfusion. Flow rate: effluent flow rate; data are mean + SEM.

327 Table 1. Effects of drugs on renin release from toadfish renal slices Renin secretion (ng ANG I/mg fresh kidney/h) No. exp.

Control

Bay K 8644, 10- 5 M + Nifedipine, 10- 5 M

4

0.71

Dibutyryl cAMP, 10- 4 M

Drugs

50 mM K

+

50 mM K+ plus drug

0.15

0.26 + 0.09

0.27 + 0.09

4

0.74 + 0.18

0.17 + 0.03

0.11 + 0.02

4

8 bromo cGMP, 10 M

4

0.99 ± 0.20

0.32 + 0.07

0.19 + 0.05

Acetylcholine, 10 - 4 M

2

0.53

0.20

0.09

Data shown are means ± SEM of two or three consecutive collections. Kidneys from 3-4 fish were pooled for each experiment.

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Fig. 4. Effects of isoproterenol (10- 4 M) on K+ -induced renin suppression (n = 6). Details are the same as in Fig. 3.

Effects of isoproterenol, dibutyryl cAMP, 8-bromo cGMP, and acetylcholine on renin secretion Isoproterenol (10 - 4 M) neither increased basal renin secretion (ng ANG I/mg kidney/h) from renal slices superfused with Ringer's solution (Ringer, 0.55; drug, 0.45, n = 2) nor stimulated renin release from the slices in which renin secretion was suppressed by 50 mM K+ (Fig. 4). Furthermore, preliminary studies indicate that neither dibutyryl cAMP (10- 4 M) nor 8-bromo cGMP (10- 4 M) restored renin release suppressed by 50 mM K+ (Table 1). Acetylcholine (Ach, 10- 4 M, n = 4) did not stimulate either basal renin release (Ringer, 0.59 + 0.14; Ach, 0.63 + 0.2) or restore suppressed renin release (Table 1).

The present results suggest that high extracellular K + inhibits renin secretion from toadfish renal slices, and inhibition of Ca 2 + influx by a voltagesensitive calcium channel antogonist prevents this effect. It has been suggested that in mammalian kidneys, increasing extracellular potassium depolarizes JG cells and opens voltage-sensitive calcium channels, resulting in the inhibition of renin release (Fishman 1976; Churchill and Churchill 1982b). This inhibitory effect is blocked by Ca2 + chelation and calcium channel blockers (Churchill 1987), suggesting that Ca 2 + influx mediates the renin inhibitory effect of depolarization. Although the presence of voltage-sensitive calcium channels on granulated cells in the toadfish kidney is not known, present observations suggest that Ca2+ influx may mediate inhibitory messages for renin release. Moreover, preliminary study indicates that Bay K 8644, a calcium channel agonist (Churchill and Churchill 1987), reverses the nifedipine effect in toadfish renal slices. This further supports the above-mentioned concept. Furthermore, Fray (1980a), and Fray and Lush (1984) proposed that voltage-dependent changes in Ca 2 + influx underlie the baroreceptor mechanism for controlling renin secretion. Mechanical distortion and stretch depolarize JG cells; depolarization increases Ca 2 + influx through calcium channels, which leads to inhibition of renin secretion. De-

328 creased stretch, conversely, causes hyperpolarization, which results in decreased Ca 2 + i and enhanced renin secretion. Indeed, calcium channel antagonists block the inhibitory effects of both depolarization and increased perfusion pressure on renin secretion, respectively, in rat renal slices (Churchill and Churchill 1980b) and in isolated perfused rat kidneys (Fray 1980b; Fray and Lush 1984). In toadfish, hemorrhage- or vasodilatorinduced hypotension caused a marked renin release (Nishimura et al. 1979), whereas macula densa and adrenergic innervation to the granulated cells may be absent. The pressure-sensitive mechanism of granulated cells and Ca 2 + influx-mediated renin suppression observed in the present study may be closely linked in the toadfish. It has been shown that renin secretion in vivo or in vitro from mammalian renal slices is stimulated by substances that activate adenylate cyclase and are inhibited by substances that suppress adenylate cyclase (Keeton and Campbell 1980; Churchill and Churchill 1982a). Substances known to stimulate renin release by activation of the adenylate cyclasecAMP synthesis include -adrenergic receptor agonists, A2 -adenosine receptor agonists, H2-histamine receptor agonists, glucagon, PGE 2, PGI 2, forskolin, and others. Conversely, renin secretion in mammalian kidneys is inhibited by a-adrenoceptor agonists and A,-adenosine receptor agonists (Churchill 1985, 1987). Renin secretion is also stimulated by phosphodiesterase inhibitors and by exogenous cAMP and dibutyryl cAMP (Churchill 1987; Yamamoto et al. 1973). Although in the present study isoproterenol increased PRA several-fold in conscious toadfish, this may be due to the stimulation of a pressuresensitive mechanism located in the granulated cells, by the reduction in renal perfusion pressure. We found that depletion of catecholamines from adrenergic nerve endings by 6-hydroxydopamine did not alter basal levels of PRA or BP (Madey et al. 1984). Furthermore, monoamine-specific nerve fluorescent activity was not demonstrated in relation to small arteries or arterioles in kidneys from toadfish loaded with epinephrine or norepinephrine and with monoamine oxidase inhibitor (Madey et al. 1984), suggesting that adrenergic innervation

in granulated cells, if any, may be scarce. These results and the lack of stimulation of renin release from renal slices -by isoproterenol and dibutyryl cAMP suggest that in toadfish kidneys, stimulation of renin release by activation of a 0-adrenergic receptor mechanism and/or cAMP production may be absent. In mammals, cholinergic innervation in renal arteries and the influence of parasympathetic nerves on renin release has not been clearly demonstrated (Keeton and Campbell 1980). Although cholinergic innervation of the blood vessels appears dominant in more primitive vertebrates (Burnstock 1969), acetylcholine neither stimulated basal renin release nor restored K+-induced renin suppression in the toadfish. A recent study suggests that cGMP enhances the sequestration of cytosolic Ca 2 + by the sarcoplasmic reticulum in cultured rat aortic smooth muscle cells (Twort and van Breeman 1988). Renin secretion is not increased, however, by cGMP in renal slices from toadfish. In mammalian JG cells, cAMP and Ca 2 + i systems appear to interact. Increased Ca 2 + i concentration leads to a decrease in cAMP by inhibiting adenylate cyclase activity and/or by stimulating phosphodiesterase, whereas increased cAMP production induces the reduction of Ca 2 + i by increasing Ca 2 + efflux through the stimulation of Na-K-ATPase, Na + -Ca 2 + exchange, and/or Ca 2 +-ATPase (Churchill 1985, 1987). Thus, the renin secretion of toadfish granulated cells may be operated only by the Ca2 + i system linked by membrane depolarization and voltage-sensitive calcium channels without interference from a stimulatory cAMP mechanism. The toadfish provides a simple and useful model in which to examine the role of Ca 2 + as an inhibitory second messenger and the cellular mechanism that underlies a stretch or baroreceptor in the granulated cells in the control of renin release.

Acknowledgements We thank Mr. Sylvester Bowens for his technical assistance. Helpful suggestions from Drs. Edward G. Schneider and Arthur A. Manthey are greatly

329 appreciated. This investigation was supported by National Science Foundation grant DCB 8616261 and, in part, by National Heart, Lung and Blood Institute grant HL 29364.

References cited Burnstock, G. 1969. Evolution of the autonomic innervation of visceral and cardiovascular systems in vertebrates. Pharmacol. Rev. 21: 247-324. Churchill, P.C. 1985. Second messengers in renin secretion. Am. J. Physiol. 249: F175-F184. Churchill, P.C. 1987. Calcium channel antagonists and renin release. Am. J. Nephrol. 7: 32-38. Churchill, P.C. and Churchill, M.C. 1982a. Isoproterenolstimulated renin secretion in the rat: second messenger roles of Ca and cyclic AMP. Life Sci. 30: 1313-1319. Churchill, P.C. and Churchill, M.C. 1982b. Ca-dependence of the inhibitory effect of K-depolarization on renin secretion from rat kidney slices. Arch. Int. Pharmacodyn. 258: 300312. Churchill, P.C. and Churchill, M.C. 1987. Bay K 8644, a calcium channel agonist, inhibits renin secretion in vitro. Arch. Int. Pharmacol. Therap. 285: 87-97. Fishman, M.C. 1976. Membrane potential of juxtaglomerular cells. Nature, Lond. 260: 542-544. Fray, J.C.S. 1980a. Stimulus-secretion coupling of renin. Role of hemodynamic and other factors. Circ. Res. 47: 485-492. Fray, J.C.S. 1980b. Mechanism by which renin secretion from perfused rat kidneys is stimulated by isoprenaline and inhibited by high perfusion pressure. J. Physiol., Lond. 308: 1-13. Fray, J.C.S. and Lush, D.J. 1984. Stretch receptor hypothesis

for renin secretion: the role of calcium. J. Hypertension 2: 19-23. Fray, J.C.S, Park, C.S. and Valentine, A.N.D. 1987. Calcium and the control of renin secretion. Endocr. Rev. 8: 53-93. Keeton, T.K. and Campbell, W.B. 1980. The pharmacologic alteration of renin release. Pharmacol. Rev. 31: 81-227. Madey, M.A., Nakamura, Y., Nishimura, H., Cagen, L.M. and Barajas, L. 1984. Control of renin release in the aglomerular toadfish. Fed. Proc. 43: 1076. Nishimura, H. 1980. Comparative endocrinology of renin and angiotensin. In The Renin Angiotensin System. pp. 29-77. Edited by J.A. Johnson and R.R. Anderson. Plenum, New York. Nishimura, H. 1987. Role of the renin-angiotensin system in osmoregulation. In Vertebrate Endocrinology: Fundamentals and Biomedical Implications. Vol. 2, pp. 157-187. Edited by P.K.T. Pang and M. Schreibman. Academic Press, New York. Nishimura, H., Crofton, J.T., Norton, V.M. and Share, L. 1977. Angiotensin generation in teleost fish determined by radioimmunoassay and bioassay. Gen. Comp. Endocrinol. 32: 236-247. Nishimura, H., Lunde, L.G. and Zucker, A. 1979. Renin response to hemorrhage and hypotension in the aglomerular toadfish Opsanus tau. Am. J. Physiol. 6: H105-HlIL. Twort, C.H.C. and van Breemen, C. 1988. Cyclic guanosine monophosphate-enhanced sequestration of Ca2 + by sarcoplasmic reticulum in vascular smooth muscle. Circ. Res. 62: 961-964. Wilson, J.X. 1984. The renin-angiotensin system in nonmammalian vertebrates. Endocr. Rev. 5: 45-61. Yamamoto, K., Okahara, T., Abe, Y., Ueda, J., Kishimoto, T. and Morimoto, S. 1973. Effects of cyclic AMP and dibutyryl cyclic AMP on renin release in vivo and in vitro. Jap. Circ. J. 37: 1271-1276.