SK-AND-F-96067 IS A REVERSIBLE, LUMENALLY ACTING INHIBITOR OF THE GASTRIC (H+ + K+)-ATPASE

SK&F 96067 [3-butyryl-4-(2-methylphenylamino)-8-methoxyquinoline] has been identified, from a novel class of 4-aminoquinolines, as a reversible inhibitor of the gastric (H+ + K+)-ATPase. This compound has been studied in gastric membrane vesicle preparations enriched in the (H+ + K+)-ATPase. At pH 7.0, SK&F 96067 inhibited K+-stimulated ATPase activity competitively with respect to the activating cation K+, with a K(i) value of 0.39 +/- 0.05-mu-M. Under comparable conditions, SK&F 96067 was 32 times more potent as an inhibitor of the gastric (H+ + K+)-ATPase relative to the closely related (Na+ + K+)-ATPase. Studies in intact gastric vesicles showed that SK&F 96067 also inhibited hydrogen ion transport. Using the initial rate of acridine orange quenching as the index of acidification, an IC50 of 0.84 +/- 0.24-mu-M was observed. Steady state acidification, as measured by aminopyrine accumulation, was inhibited with greater potency (IC50 = 0.06 +/- 0.01-mu-M) consistent with the accumulation of this inhibitor into the intravesicular acidic space to a site of action on the inside (lumenal) face of the enzyme. Inhibition of ATPase activity in the presence of both SK&F 96067 and the K+-competitive (H+ + K+)-ATPase inhibitor, SCH 28080, indicated that their binding was mutually exclusive, consistent with SK&F 96067 acting at the same lumenal binding site as does SCH 28080. The steady-state inhibition kinetics of SK&F 96067 against K+-stimulated ATPase activity were followed as a function of pH. At pH 6.6 and 7.0 the inhibition was competitive with respect to the activating cation K+. At pH 7.5 and 8.1 a mixed pattern of inhibition was detected. Thus, at alkaline pH values, the binding of SK&F 96067 and K+ were no longer mutually exclusive. The potency of SK&F 96067 decreased as pH rose, consistent with the protonated form of the inhibitor being the preferred inhibitory species. A kinetic model is discussed, in which, at acidic pH, the protonated form of SK&F 96067 binds to the enzyme competitively with respect to K+, whereas, at alkaline pH, the neutral form of SK&F 96067 can bind simultaneously with K+.