Phorbol ester desensitization of clonal insulin-releasing cell response to carbachol involves depletion of an intracellular calcium pool

Abstract

The mechanism by which 12-o-tetradecanoylphorbol-13-acetate (TPA) desensitizes carbachol mobilization of glucose-incorporated calcium (Ca2+) was studied in clonal insulin-releasing cells (RINm5F) using colour indicators and dual wavelength spectrophotometry. The net uptake of Ca2+ stimulated by 20 mM glucose reached saturation after 19 +/- 2 min when it corresponded to 1.21 +/- 0.09 mmol calcium kg-1 protein. Carbachol then induced a release of 0.21 +/- 0.03 mmol calcium kg-1 protein. Half of the remaining Ca2+ was liberated by antimycin A and the rest with the Ca2+ ionophore A-23187. When 0.1 microM TPA was added initially, the cells lost 0.29 +/- 0.08 mmol calcium kg-1 protein within 10 min. The subsequent addition of glucose resulted in a sluggish uptake of only 0.58 +/- 0.09 mmol calcium kg-1 protein reaching equilibrium after 35 +/- 3 min. Carbachol now failed to induce any Ca2+ release. The actions of TPA were essentially unchanged by previous exposure to glucose, removal of Na+ from the medium and even when some of the glucose-incorporated Ca2+ had been liberated with carbachol. The results indicate that TPA desensitization of carbachol-induced mobilization of Ca2+ in the RINm5F cells is due to the disappearance of Ca2+ from the sensitive pool, an effect which may depend on stimulated extrusion of Ca2+ from the cells by the (Ca2+-Mg2+)-ATPase.