Inhibition of adenyl cyclase preparations from rat kidney by calcium ions and various diuretics

Abstract

Antidiuretic hormone (ADH) increases the permeability to water of certain epithelial membranes. This effect, found in the urinary bladder of the toad and in the distal tubules and the collecting ducts of kidney, is mediated intracellularly by adenosine 3′∶5′-monophosphate (Ado-3′∶5′-P). Calcium ions and the diuretic ethacrynic acid are known to inhibit the ADH-induced increase in water permeability of the toad bladder. In adenyl cyclase preparations from rat renal cortex and medulla, the influence of these substances as well as of other diuretics added in vitro has been studied. Adenyl cyclase activity has been determined, excepted as noted, by measuring Ado-3′∶5′-P formed from 1 mM 14C-ATP in the presence of 10 mM Mg++, an ATP regenerating system, and 5 mM unlabeled Ado-3′∶5′-P to reduce the enzymatic degradation of the labeled Ado-3′∶5′-P. Calcium ions reduced the rate of Ado-3′∶5′-P formation by particles from renal cortex and medulla when the activity was measured in the presence of either Mg++ or Mn++. With 10 mM Mg++, 1 mM Ca++ decreased adenyl cylase activity by about 50%. Activities of cortical adenyl cyclase stimulated by parathyroid hormone, thyrocalcitonin or ADH and of medullary adenyl cyclase stimulated by ADH were also reduced by about 50% in the presence of 1 mM Ca++. The inhibition was independent of the ATP concentration, but was influenced by the Mg++ content of the incubation medium. Adenyl cyclase activities of cortical and medullary membrane preparations were reduced by about 50% by 0.2 mM ethacrynic acid. The extent of this inhibition was essentially the same whether the enzymatic activity was determined in the absence or presence of stimulating hormones. The inhibitory action of ethacrynic acid was partially prevented by simultaneous addition of dithioerythritol (DTE). A derivative of ethacrynic acid, L 589420-0-2, also inhibited renal adenyl cyclase, but its action was not influenced by the addition of DTE. Adenyl cyclase from both parts of the kidney was inhibited by about 90% by 0.2 mM mersalyl. This action was almost completely prevented by the addition of 1 mM DTE. The pharmacological significance of adenyl cyclase inhibition by these diuretics is still uncertain since the role of Ado-3′∶5′-P in the regulation of sodium transport is as yet unclear. Other diuretics, hydrochlorothiazide, furosemide, mefruside, amiloride, and the non-diuretic benzothiadiazine, diazoxide, had essentially no effect on cortical and medullary adenyl cyclase preparations when they were added in 0.1–0.5 mM concentration. The methylxanthines, theophylline and caffeine, which are known to inhibit nucleoside 3′∶5′-monophosphate phosphodiesterase, reduced the rate of Ado-3′∶5′-P formation. The unstimulated and the hormone-stimulated adenyl cyclases were inhibited to the same extent by theophylline. When adenyl cyclases was stimulated by fluoride, however, we found only a very small inhibition by theophylline. Inhibition of the medullary adenyl cyclase was greater than that of the enzyme prepared from renal cortex. At a concentration of 1 mM these methylxanthines significantly inhibited the medullary enzyme, but the inhibition became asymptotic at about 50% when concentrations up to 20 mM were used. Therefore, it is likely that inhibition by these substances varies in different cell types and tissues. Instead of phosphodiesterase inhibitors, unlabeled Ado-3′∶5′-P can be used in the assay of adenyl cyclase activity to reduce the degradation of enzymatically formed labeled Ado-3′∶5′-P. This addition, though, can also influence adenyl cyclase activity. In a medullary enzyme preparation 0.2 mM Ado-3′∶5′-P reduced the adenyl cyclase activity by 13%, 5 mM Ado-3′∶5′-P by 35%.