To test whether in RINmSF rat insulinoma cells luminal acidity and the activity of a vacuolartype proton pump are involved in calcium sequestration by intracellular calcium stores sensitive to inositol 1,4,5-trisphosphate (InsP,) we examined the effects of various proton-conducting ionophores and ammonium chloride, and of bafilomycin, a specific inhibitor of vacuolar proton pumps, on this parameter. Bafilomycin in concentrations up to 1 pM did not affect calcium sequestration by nonmitochondrial, InsP,-sensitive stores at all; 50 pM carbonylcyanide m-chlorophenylhydrazone, 50 pM monensin and 30 mM NH,Cl, which are diverse ways to dissipate transmembrane pH gradients, did not inhibit calcium sequestration. This argues against significant involvement of internal acidity and vacuolar proton pumps in calcium sequestration by InsP,-sensitive stores in RINmSF cells. The proton-potassium-exchanging ionophore nigericin (20-100 pM), however, inhibited calcium sequestration by nonmitochondrial and InsP,-sensitive stores. This effect was dependent on the presence of potassium and could be reversed by inclusion of carbonylcyanide m-chlorophenylhydrazone or acetate in the incubation medium. Thus, the inhibitory effect of nigericin appears to be based on proton extrusion coupled to potassium influx across the membrane of calcium stores in RINm5F cells, creating an internal alkalinization of these stores. The effect of nigericin implies the continuous maintenance of an outside-to-inside potassium concentration gradient by nonmitochondrial calcium stores in RINmSF cells. This feature will be of potential interest in the identification of InsP,-sensitive calcium-storing organelles.

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