Effect of pH upon Ca(2+)-ATPase activity of rat pancreatic islets: its possible contribution to the inhibitory effect of different insulin secretagogues.

Abstract

This work was undertaken in an attempt to elucidate the possible mechanism by which insulin secretagogues produce a fast and transient drop in the Ca(2+)-ATPase activity of the pancreatic islet membrane. For this purpose, the enzyme activity was measured in either homogenates or partially purified membranes of islets previously incubated under different experimental conditions. Ca(2+)-ATPase activity measured in homogenates of islets preincubated with 8 mM glucose decreased significantly compared to control islets incubated with 2.8 mM glucose. The inhibition was also observed when the enzyme activity was measured in homogenates of islets preincubated with 2.8 mM glucose plus 20 mM propionic acid as well as with glucose 2.8 mM in a buffer equilibrated with a gas mixture of O2 and either 12% or 30% CO2. Ca(2+)-ATPase activity decreased significantly in partially purified islet membranes preincubated for 3 min with glucose (2 and 8 mM), 15 mM KCl and 2 mM tolbutamide. These substances did not affect the Ca(2+)-ATPase activity when added directly to the enzyme assay medium. The enzyme activity also decreased when measured in membranes preincubated at pH 6.5. The addition of 1 mM ATP to the preincubation medium protected the Ca(2+)-ATPase activity from the inhibition induced by glucose, KCl and tolbutamide as well as from the one produced by acidic pH in the medium. On account of these results, we suggest that insulin secretagogues, as well as either acidification of B-cell cytosol or islet membrane incubation medium, produce changes at the islet membrane level which promote a decrease in the Ca(2+)-ATPase activity. A shift of the E1-E2 equilibrium of the phosphoenzyme towards E1 may account for such decreased activity. Changes in Ca(2+)-ATPase activity could either favour the decrease or the increase in the cytosolic concentration of Ca2+ in B-cells. Therefore, negative and positive modulation of its activity might allow Ca(2+)-ATPase to play a role in the switch-on and -off mechanism for intracellular Ca2+ signal regulation of B-cell secretion of insulin.