Transient activation of calcium-dependent phospholipase A2 by insulin secretagogues in isolated pancreatic islets.
Arachidonic acid is believed to be an important and necessary mediator of insulin secretion by beta-cells of islets of Langerhans, and it may regulate intracellular Ca2+ homeostasis. Insulin secretagogues, such as glucose and the muscarinic agonist carbachol, stimulate arachidonic acid accumulation, although the mechanisms involved are controversial: carbachol is believed to stimulate phospholipase A2, while glucose-induced arachidonic acid release is the result of diacylglycerol hydrolysis [Konrad, R. J., et al. (1992) Biochem. J. 287, 283-290]. In insulin-secreting clonal beta-cells RINm5F, HIT-T15, and beta-TC3, Ca(2+)-independent phospholipase A2 was mainly cytosolic, while in islets it was equally distributed between a crude membrane fraction and the cytosol. A membrane-associated Ca(2+)-dependent phospholipase A2 was found to be stimulated by millimolar Ca2+ concentrations, while a cytosolic Ca(2+)-dependent activity was activated by micromolar Ca2+ levels. In order to determine whether phospholipase A2 was activated in insulin secretion, we assessed whether pretreatment of intact islets with secretagogues affected phospholipase A2 activity, which was subsequently measured in membrane or cytosolic fractions. The combination of glucose and carbachol transiently activated Ca(2+)-dependent (but not Ca(2+)-independent) phospholipase A2 activity at 10 min, which corresponded to the peak of arachidonic acid release. No effect was seen with either agonist alone. Our results indicate that activation of Ca(2+)-dependent phospholipase A2 may be due to agonist-induced increases in intracellular Ca2+. We suggest that activation of islet Ca(2+)-dependent phospholipase A2 may be important in a distal process of insulin secretion, such as secretory granule exocytosis.
Jolly
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Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104.
Major
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Wolf
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BA
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D004789:Enzyme Activation; D005947:Glucose / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000201:enzymology* / Q000378:metabolism; D008297:Male; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D051381:Rats; D017207:Rats, Sprague-Dawley
D007328:Insulin; D016718:Arachidonic Acid; D002217:Carbachol; D010741:Phospholipases A; D054467:Phospholipases A2; D005947:Glucose; D002118:Calcium