EFFECT OF A RAB3A EFFECTOR DOMAIN-RELATED PEPTIDE, CCK, AND EGF IN PERMEABILIZED PANCREATIC ACINI

We report here that a synthetic peptide of the effector domain of the small-molecular-weight GTP-binding protein Rab3A (ED(Rab3AL)) is a potent stimulator of inositol 1,4,5-trisphosphate [Ins(1,4,5)P-3] production and amylase secretion in digitonin-permeabilized pancreatic acini. Moreover, the Rab3A effector domain peptide caused phosphatidylinositol 4,5-bisphosphate breakdown, indicating that the observed increase in Ins(1,4,5)P-3 is due to stimulation of a phosphoinositide-specific phospholipase C (PLC). The dose-response curve for ED(Rab3AL)-induced amylase release was biphasic, showing a maximum at 0.3 nM ED(Rab3AL) and a decline at higher peptide concentrations. By contrast, the dose-response curve for ED(Rab3AL)-induced Ins(1,4,5)P-3 production was monophasic, showing stimulation with increasing ED(Rab3AL) concentrations. A peptide of the effector domain of Rab1A, ED(Rab1AL), had no effect, indicating that the response to ED(Rab3AL) is specific. Cholecystokinin octapeptide (CCK-8) and ED(Rab3AL) had additive effects on the acinar Ins(1,4,5)P-3 level. Epidermal growth factor (EGF), which has recently been shown to inhibit CCK-8-induced Ins(1,4,5)P-3 production in pancreatic acinar cells, also decreased ED(Rab3AL)-induced Ins(1,4,5)P-3 production. These results suggest that ED(Rab3AL) and CCK-8 act on the same EGF-inhibitable PLC by independent mechanisms. CCK-8 increased and EGF decreased amylase release in response to submaximal ED(Rab3AL) concentrations. By contrast, at supramaximal ED(Rab3AL) concentrations EGF increased and CCK-8 decreased ED(Rab3AL)-stimulated amylase release. ED(Rab3AL) had no effect in intact acini, indicating that the site of action of ED(Rab3AL) is intracellular. We conclude that ED(Rab3AL) regulates phosphoinositide-specific PLC activity and thereby amylase secretion in an analogous fashion to CCK-8, but from within the cell. This indicates that Rab-like proteins might be involved in the regulation of phospholipase C.