Br. J. Pharmac. (1977), 61, 657-667

DIURETICS AND THE RENAL ADENYLATE CYCLASE SYSTEM J.K. DAWBORN, S. MACNEIL & T.J. MARTIN Department of Chemical Pathology, University of Sheffield Medical School, Beech Hill Road, Sheffield S 10 2RX

I The relationship between the diuretic effectiveness and the effect on the renal adenylate cyclase of three diuretics, acetazolamide, frusemide and ethacrynic acid, was examined. The hypothesis that acetazolamide and parathyroid hormone (PTH), inhibit renal carbonic anhydrase by a cyclic adenosine 3',5'-monophosphate (cyclic AMP)-dependent mechanism was also tested. 2 In vitro, acetazolamide, frusemide and ethacrynic acid at high concentrations (10-3OM) all produced some inhibition of basal and stimulated rat kidney plasma membrane adenylate cyclase. The effect of acetazolamide was much less than that of frusemide and ethacrynic acid. These plasma membrane effects were reproduced in studies of cyclic AMP formation in isolated kidney tubules of rats. 3 Intravenous injections of acetazolamide did not change the total cyclic AMP content of the kidneys of rats killed by microwave irradiation. 4 Acetazolamide produced a diuresis in the rat and a slight inhibition of the antidiuretic effect of Pitressin. Frusemide produced a diuresis and greatly reduced the antidiuretic response to Pitressin. Ethacrynic acid was ineffective as a diuretic in the rat and actually enhanced the antidiuretic response to Pitressin. 5 In investigating the possible influence of diuretics and PTH on the activity and state of phosphorylation of carbonic anhydrase it was found that: there was no correlation between the ability of diuretics to inhibit carbonic anhydrase activity and to inhibit carbonic anhydrase phosphorylation; neither PTH nor cyclic AMP (in the presence of adenosine triphosphate, Mg2+, K+ and incubation at 370C) inhibited rat cortex homogenate carbonic anhydrase activity. 6 It seems unlikely that any of the tested diuretics exerts its pharmacological effect by means of changes in kidney cyclic AMP metabolism.

Introduction

Several studies have suggested that certain diuretics, notably frusemide and ethacrynic acid, may produce some of their renal effects by inhibiting renal adenylate cyclase activity. (Ferguson & Twite, 1973; Ebel, 1974; Ebel & Sharp, 1975; Barnes, Hui & Dousa, 1975). The situation with acetazolamide is more complicated. Rodriguez, Walls, Yates & Klahr (1974) showed similar effects of acetazolamide and parathyroid hormone (PTH) on urinary cyclic adenosine 3',5'-monophosphate (cyclic AMP) and phosphate and found that, like PTH, acetazolamide stimulated the adenylate cyclase of the rat renal cortex. Beck, Kim, Wolak & Davis (1975), investigating the similar effects of acetazolamide and PTH on bicarbonate excretion, found that both PTH and cyclic AMP inhibited the activity of rat kidney carbonic anhydrase in vitro. Since acetazolamide is a potent inhibitor of carbonic anhydrase, this report together with that of Rodriguez et al. (1974) could be interpreted as suggesting that acetazolamide and PTH both increase cyclic AMP production by stimulating

the renal adenylate cyclase (Jacobsen & Kokko, 1976). The cyclic AMP generated, acting by a protein kinase, could then inhibit renal carbonic anhydrase, to produce the observed effects on phosphate and bicarbonate excretion. The present study was designed to evaluate the effects of acetazolamide, frusemide and ethacrynic acid on renal adenylate cyclase activity in vitro and to relate these to pharmacological effects of the drugs in vivo. The effects of PTH and cyclic AMP on carbonic anhydrase activity were also studied. Methods

Drugs and chemicals

(a-32P]-adenosine triphosohate (0.5-30 Ci/mmol) and [8-3H]-cyclic AMP (20-30 Ci/mmol) were obtained from the Radiochemical Centre, Amersham. Cyclic AMP (crystallized free acid) and the sodium

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J.K. DAWBORN, S. MACNEIL & T.J. MARTIN

salt of adenosine triphosphate (ATP) were obtained from Boehringer Mannheim, London. Acetazolamide (Crystalline) was obtained from Sigma Chemical Co. and the sodium salt of acetazolamide (Diamox), from Lederle Laboratories Division, American Cyanamid Company, Pearl River, N.Y., frusemide (20 mg ampoules for injection) from Heochst Pharmaceuticals and sodium ethacrynate (Edecrin) from Merck, Sharp and Dohme Ltd. All other chemicals were of A.R. grade.

Hormone preparations Parathyroid hormone (PTH) was a bovine preparation, (code name 69TP; Moseley, Martin, Robinson, Reit & Tregear (1975), of 1,000 iu/mg potency; Nahorski, Hunt, Rogers, Jones & Martin (1976)).

Synthetic salmon calcitonin (CT), 4,700 u/mg was obtained from Dr J.W. Bastian, Armour Pharmaceuticals, Kankakee, Illinois. Pitressin, 20 u/ml, was obtained from Parke-Davis & Co. Water-loaded, alcohol-anaesthetized rat preparations Male CFY, Remote Sprague-Dawley rats (120-150g) East Anglia Laboratory Animals Ltd., were used in these experiments. In this preparation the animal is anaesthetized with alcohol and water-loaded to produce a state of constant water diuresis which is very responsive to exogenous ADH. Surgery was carried out as described by Harris & Jenner (1972). Animals were infused at 0.2ml/min with a modified Czaczkes solution (Czaczkes, Kleeman & Koenig, 1964) containing 51.2 mM NaCl, 92.7 mM glucose and 2.5% v/v ethanol. The diuretics under study were added to this infusion. The bladder cannula was connected to a drop counter with a digital display. The antidiuretic response to intravenous injections of Pitressin was calculated as the percentage reduction in urine flow in the 10 min following injection compared to the 10 min before injection. The same calculation was used to measure the effect of single intravenous injections of diuretics on urine output. The effects of sustained infusion of the diuretics on the response to Pitressin were also examined. Injections of 40, 80 and 120±u Pitressin were given at 20 min intervals during control periods and during infusion of diuretics.

Kidney tubule preparation Rat kidney cortex tubules were made by the method of Larkins, MacAuley, Rappoport, Martin, Tulloch, Byfield, Matthews & MacIntyre, (1974) resuspended in the required volume of oxygenated buffer (Larkins et al. 1974) containing 10 mM theophylline and 0. 13% bovine serum albumin (BSA) and incubated for 20 min at 37°C. The accumulation of unlabelled cyclic AMP was measured in incubations of final volume 2ml containing 10mM theophylline, 0.13% BSA and the test substances. The incubations were started by the addition of 500tl1 cell suspension and after 20 min at 37°C were terminated by placing the glass vials in a boiling water bath for 2 to 3 minutes. Supernatants from these incubations were stored for cyclic AMP determinations.

Cyclic AMP determinations Kidneys from animals killed by microwave irradiation were homogenized in 2ml/ 100mg w/w of 4:1, ethanol:water, using a Polytron model PCU-2 speed 4, 30 seconds. After centrifugation (5,OOOg for 10 min) the pellet was re-extracted as before. The combined supernatants were dried in air and resuspended in assay buffer. The cyclic AMP content of rat kidneys and rat tubule supernatant was assayed by the protein binding method of Brown, Albano, Elkins & Sgherzi (1971). Acetazolamide, frusemide and ethacrynic acid at concentrations from 1O-5M to 10-3M did not affect the binding of cyclic AMP to the binding protein. A denylate cyclase determinations

Administration of diuretics to intact animals

Kidneys removed for assay of adenylate cyclase were cut longitudinally and the medulla and papilla removed from the cortex. Plasma membrane preparations were then made of the cortex and medulla. The tissue was homogenized in cold 25mM Tris-HCI (pH 7.4) containing 0.25M sucrose and lmM tetrasodium edetate, using 3 strokes of a loose and 3 strokes of a tight Dounce pestle. The homogenate was then spun to 2200 g (Sorvall RC2-B at 4°C) and stopped immediately. The supernatant was poured off and then spun at 2200 g for 10 min and the resultant pellet resuspended in 25 mM Tris-HCI (pH 7.8) buffer containing 0.013% (w/v) BSA, 30 mM KCI and 4.5 mM MgCl2.

Injections of 0.4 ml 0.9% w/v NaCl solution (saline) or of saline containing 0.4 mg frusemide or sodium acetazolamide (Diamox) were given via the tail vein to male Wistar rats of 40 g weight, which were killed by microwave irradiation (Nahorski et al., 1976). Kidneys were removed and stored at -200C for total cyclic AMP determinations.

Adenylate cyclase reaction mixtures contained, in 1001il, 25 mM Tris-HCI (pH 7.8); 1 mM [a32PJ-ATP (10 ct min-' pmol-1); 1 mM cyclic AMP; 2.5 mM phosphoenolpyruvate; 1.5 iu pyruvate kinase; 0.013% (w/v) BSA; 30 mM KCI; 4.5 mM MgCl2; and the appropriate hormone and/or diuretics. The reaction was started by addition of 50gl of the plasma membrane suspension containing 100 to 300mg proa volume of

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Br. J. Pharmac. (1977), 61, 657-667 DIURETICS AND THE RENAL ADENYLATE CYCLASE SYSTEM J.K. DAWBORN, S. MACNEIL & T.J. MARTIN Department of Chemical Pa...
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