Inhibition of renal Na +, K +-activated adenosine triphosphatase activity by ethacrynic acid

Abstract

The inhibition by ethacrynic acid (EA) of Na +,K +-activated, Mg 2+-dependent adenosine triphosphatase (ATPase) activity in a heavy microsomal preparation from guinea pig kidney was studied. The mean control activity of the preparation was 11.9 ± 0.5 μmoles P 1/mg protein/15 min at 37° in the presence of Na +, K + and Mg 2+, and 4.1 ±0.1 in the presence of Mg 2+ alone. The effect of EA, unlike the selective action of ouabain, was to decrease nonselectively both total NaKMg-ATPase and Mg-ATPase activity. Inhibition by EA was nearly two orders of magnitude less than that obtained by ouabain and p-hydroxymercuribenzoate (POMB). Cysteine added to the incubation medium partially blocked ATPase inhibition by either EA or the known sulfhydryl inhibitor, POMB. The extent of the block by cysteine increased as the cysteine:drug molar ratio increased. There was an apparent relationship between sulfhydryl reactivity assayed by Ellman's reagent (dithio-bis-nitrobenzoate) and ATPase inhibition for ethacrynic acid and its cyclic analogues. These data support the interaction between EA and enzyme sulfhydryl groups originally suggested by others. Preincubation of EA and the enzyme prior to dilution for the usual incubation produced an inhibition corresponding to the high drug level of the preincubation period rather than the final diluted concentration. This lack of dissociation of the EA-enzyme complex upon dilution, in contrast to the dissociation observed in similar experiments with POMB, a known reversible inhibitor, suggests an irreversible interaction between EA and ATPase in vitro.