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

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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‐1protein. Carbachol then induced a release of 0.21 ± 0.03 mmol calcium kg‐1protein. Half of the remaining Ca2+was liberated by antimycin A and the rest with the Ca2+ionophore A‐23187. When 0.1 /μm TPA was added initially, the cells lost 0.29 ± 0.08 mmol calcium kg‐1protein within ro min. The subsequent addition of glucose resulted in a sluggish uptake of only 0.58 ± 0.09 mmol calcium kg‐1protein 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.

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