Evidence for contribution by increased cytoplasmic Na+ to the insulinotropic action of PACAP38 in HIT-T15 cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) is localized to pancreatic nerve terminals and stimulates insulin secretion. The insulinotropic effect of PACAP38 in insulin-producing HIT-T15 cells is accompanied by increases in cellular cAMP and cytoplasmic Ca2+ ([Ca2+](cyt)). As also intracellular Na+ is important for insulin secretion after glucose and other cAMP forming peptides, we examined the Na+ dependence of the insulinotropic effect of PACAP38 in HIT-T15 cells. We found that PACAP38 (100 nM)-induced insulin secretion was diminished by approximately 50% by removal of extracellular Na+ (replaced by equimolar N-methyl-D-glucamine). In contrast, removal of Na+ did not diminish the formation of cellular cAMP (measured by radioimmunoassay) or the increase in [Ca2+](cyt) (measured in FURA-2AM-loaded cell suspensions) induced by PACAP38. Furthermore, PACAP-38 increased the cytoplasmic Na+ ([Na+](cyt)) in single HIT-T15 cells as measured by the fluorophore sodium-binding benzofran isophthalate. This increase was reduced by removal of extracellular Na+ and by inhibition of protein kinase A by H-89. We conclude that the insulinotropic action of PACAP38 is Na+-dependent. We propose that PACAP38 opens plasma membrane Na+ channels by an action partially mediated by cAMP and protein kinase A, and the subsequent raise in [Na+](cyt) elicits insulin secretion by an as yet unsolved mechanism.