Transient activation of calcium-dependent phospholipase A sub(2) 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 Ca super(2+) 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 A sub(2), while glucose-induced arachidonic acid release is the result of diacylglycerol hydrolysis. In insulin-secreting clonal beta -cells RINm5F, HIT-T15, and beta -TC3, Ca super(2+)-independent phospholipase A sub(2) was mainly cytosolic, while in islets it was equally distributed between a crude membrane fraction and the cytosol. A membrane-associated Ca super(2+)-dependent phospholipase A sub(2) was found to be stimulated by millimolar Ca super(2+) concentrations, while a cytosolic Ca super(2+)-dependent activity was activated by micromolar Ca super(2+) levels. In order to determine whether phospholipase A sub(2) was activated in insulin secretion, we assessed whether pretreatment of intact islets with secretagogues affected phospholipase A sub(2) activity, which was subsequently measured in membrane or cytosolic fractions. The combination of glucose and carbachol transiently activated Ca super(2+)-dependent (but not Ca super(2+)-independent) phospholipase A sub(2) activity at 10 min, which corresponded to the peak of arachidonic acid release. No effect was seen with either agonist alone.