Effect of Various Diuretics on the Activity of Mg Activated Na, K-Dependent ATPase

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As already reported, thiazide diuretics are theoretically presumed to havea metal chelating action in the S-O part due to their similarity to the electronic state of the phosphate part of ATP. This study attempted to demonstrate experimentally the metal chelation of thiazide diuretics and to investigate the effects of this metal chelating action on the activity of Na, K-dependent ATPase which had been prepared from 0.1% DOC treated microsome fragments of the rat kidney cortex. Reduction potential of a mixed solution of 2mM thiazide diuretics (hydrochlorothiazide, acetothiazide, trichlormethiazide, polythiazide and benzthiazide) with 1mM CuCl2 was moved to more negative positions than that of Cu++ of ionic state. These half wave potentials were found to move to be more negative by pH change to alkaline side. Visible absorption spectra of a mixed solution with acetothiazide (20mM-60mM) and CuCl2 (20mM) were observed to have the maximum absorption at the wave length of 650mμ. And it was revealed by absorption spectra of various molar ratios of acetothiazide and Cu++ that the molar ratio of ligand (acetothiazide) to metal ion was 2: 1. Half wave potential was recognized polarographically in a diazoxide-CuCl2 solution, but not in quinethazone, furosemide and ethacrynic acid-CuCl2 solution. These findings suggest that thiazide diuretics and diazoxide have a metal chelating action in the SO2NH part of heterocyclic ring. The stability constant for thiazide-Cu chelate complex in 1N NaOH were calculated by the formula introduced from Heyrovsky and Ilkovic's using the polarographical and spectrophotometrical data. Magnitude of the stability constant showed that the chelating action of thiazide diuretics had a significant strength in vivo as compared with that of amino acids or antibiotics. Inhibitory effect of various diuretics on the activity of Na, K-dependent ATPase were investigated also in this study. The enzyme activity was inhibited by ethacrynic acid, and 50% inhibition of the enzyme activity was obtained in the concentration of 10-4M ethacrynic acid after one hour preincubation. 4×10-4M quinethazone resulted in a slight decrease of the activity of Na, K-dependent ATPase, but 10-3M hydrochlorothiazide, 5×10-4 M trichlormethiazide and 10-3M furosemide had no inhibition in vitro. Oral administration of trichlormethiazide 25mg/kg b. w. to the rats was found to have no effect on inhibition of enzyme activity. But even if thiazide diuretics (1mM)removed completly Mg++ (2mM)from the reaction media for assay of ATPase activity through their chelating action, only 0.5mM decrease of Mg++ could not have an effect on the enzyme activity, because Na, K-dependent ATPase had the same activity in the range of 0.5mM to 2mM Mg concentration of the media. Therefore, 0.5mM Mg concentration at the lowest of the media was employed to clarify the chelating action of thiazi dediuretics. By this study, an 18% inhibition of the activity of Na, K-dependent ATPase was found by hydrochlorothiazide (1mM) preincubated. The results suggest that thiazide diuretics would have a considerable degree of chelating action in vivo. But the effect of chelating action of thiazide diuretics on biometals could not be related directly to inhibition of the enzyme activity of Mg activated, Na, K-dependent ATPase. Membrane permeability of thiazide-biometal complex and the direct effect of ethacrynic acid on the enzyme protein of ATPase should be explored.
Japan Society of Clinical Chemistryの論文

Effect of Various Diuretics on the Activity of Mg Activated Na, K-Dependent ATPase

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