Glucose-induced phosphorylation and activation of a high molecular weight cytosolic phospholipase A2 in neonatal rat pancreatic islets.

Previous studies have shown that stimulus-secretion coupling for the release of insulin from the pancreatic islet is potentiated by phospholipase A2 activity. Several biochemically distinct phospholipase A2 activities have been described in the islet. A recently identified cytosolic high molecular weight phospholipase A2, which requires Ca2+ for association with cellular membranes but not for catalytic activity can be activated in a protein kinase C-dependent manner in other cell-types. We determined its phosphorylation and activation in response to phorbol ester and glucose in cultured islet cells from neonatal rats. Islet cell monolayers were labelled to equilibrium with [32P]orthophosphate. Following stimulation cytosolic phospholipase A2 was immunoprecipitated and, after electrophoretic separation and transfer to nitrocellulose membrane, 32P-labelled protein was detected by autoradiography. Phospholipase A2 activity of islet cell cytosol was determined by hydrolysis of exogenous I-stearyl- 2[14C]arachidonyl phosphatidylcholine substrate. It could be shown that phosphorylation of immunoprecipitated phospholipase A2 was augmented by prolonged glucose exposure (> 1 hr) in a protein kinase C-dependent manner. Phosphorylation occurred concomitant with a glucose-induced increase in total cellular phospholipase A2 activity (177 +/- 3 nmol substrate hydrolysed/mg protein at glucose 5.6 mM vs 267 +/- 32 (SEM, n = 4) at glucose 25 mM, P < 0.05). Both acute protein kinase C (459 +/- 71) and glucose-activated phospholipase A2 activities were reduced in the presence of a specific arachidonic acid analogue inhibitor of cytosolic phospholipase A2 (to 231 +/- 10 and 161 +/- 17, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)