Gastric H+-K+-ATPase in situ: relation to secretory state.

Isolated gastric glands from rabbit were used to measure the gastric H+-K+-adenosinetriphosphatase (ATPase) and its partial reaction, a K+ p-nitrophenyl phosphatase (pNPPase), in situ. Measurement of the enzyme activities required permeabilization of the cells with digitonin and the use of several ATPase inhibitors to reduce nonspecific activity. The enzyme activities were identified as the H+-K+-ATPase according to the following criteria: dependence on K+, association with parietal cells, insensitivity to ouabain and ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and inhibition by the specific inhibitors omeprazole and Sch 28080. K+-stimulated ATPase, but not K+-pNPPase, was enhanced by K+ ionophores, valinomycin and nigericin, with nigericin resulting in the greatest activity. Comparison of tissues that were preincubated to establish resting and stimulated states showed that prestimulation results in an increase in K+-stimulated ATPase activity with no change in the total activity. With the use of a two-step assay procedure, it could be shown that stimulation also results in an increase in the omeprazole-sensitive maximal ATPase activity. These results indicate that the major effects of stimulation are to enhance KCl activation of the enzyme and to increase the number of active enzyme molecules.