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)
Dunlop
M
M
University of Melbourne, Department of Medicine, Royal Melbourne Hospital, Parkville, Victoria, Australia.
Clark
S
S
D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D003600:Cytosol / Q000201:enzymology*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005947:Glucose / Q000494:pharmacology*; D007515:Islets of Langerhans / Q000187:drug effects / Q000201:enzymology*; D008970:Molecular Weight; D010713:Phosphatidylcholines / Q000378:metabolism; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054467:Phospholipases A2; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D051381:Rats
D004791:Enzyme Inhibitors; D010713:Phosphatidylcholines; D011493:Protein Kinase C; D010741:Phospholipases A; D054467:Phospholipases A2; D005947:Glucose