TRANSIENT ACTIVATION OF CALCIUM-DEPENDENT PHOSPHOLIPASE-A(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 ma y regulate intracellular Ca2+ homeostasis. Insulin secretagogues, such as glucose and the muscarinic agonist carbachol, stimulate arachidoni c acid accumulation, although the mechanisms involved are controversia l: carbachol is believed to stimulate phospholipase A2, while glucose- induced arachidonic acid release is the result of diacylglycerol hydro lysis [Konrad, R. J., et al. (1992) Biochem. J. 287, 283-290]. In insu lin-secreting clonal beta-cells RINm5F, HIT-T15, and beta-TC3, Ca2+-in dependent phospholipase A2 was mainly cytosolic, while in islets it wa s equally distributed between a crude membrane fraction and the cytoso l. A membrane-associated Ca2+-dependent phospholipase A2 was found to be stimulated by millimolar Ca2+ concentrations, while a cytosolic Ca2 +-dependent activity was activated by micromolar Ca2+ levels. In order to determine whether phospholipase A2 was activated in insulin secret ion, we assessed whether pretreatment of intact islets with secretagog ues affected phospholipase A2 activity, which was subsequently measure d in membrane or cytosolic fractions. The combination of glucose and c arbachol transiently activated Ca2+-dependent (but not Ca2+-independen t) phospholipase A2 activity at 10 min, which corresponded to the peak of arachidonic acid release. No effect was seen with either agonist a lone. Our results indicate that activation of Ca2+-dependent phospholi pase A2 may be due to agonist-induced increases in intracellular Ca2+. We suggest that activation of islet Ca2+-dependent phospholipase A2 m ay be important in a distal process of insulin secretion, such as secr etory granule exocytosis.