Pituitary adenylate cyclase-activating peptide, carbachol, and glucose stimulate insulin release in the absence of an increase in intracellular Ca2+

Insulin secretion from the pancreatic beta cell line HIT-T15 was examined under conditions in which the elevation of intracellular free Ca2+ concentration (Ca2+]i) was inhibited by nitrendipine or diazoxide or by severe Ca2+ deprivation. Glucose-induced insulin release was completely abolished under these conditions. However, in the presence of 12-O-tetradecanoyl-phorbol-13-acetate or forskolin, 10 mM glucose significantly enhanced insulin release, even in the presence of 5 microM nitrendipine or 150 microM diazoxide. The Ca2+]i was not increased under these conditions. Even under Ca(2+)-deprived conditions, achieved by 60-min preincubation in Ca(2+)-free buffer containing 1 mM ethylene glycol bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), glucose in the complete absence of extracellular Ca2+ significantly enhanced insulin release when the cells were treated also with 12-O-tetradecanoylphorbol-13-acetate and forskolin. Because of these findings, additional studies were performed with pituitary adenylate cyclase-activating peptide (PACAP) and carbachol to see whether physiological stimulation via receptor activation could stimulate insulin release in the absence of a rise in Ca2+]i. Under normal Ca(2+)-containing conditions, PACAP and carbachol stimulated insulin release and markedly potentiated glucose-stimulated release. In the presence of nitrendipine and thapsigargin, glucose failed to stimulate insulin release. Also, neither glucose in combination with PACAP nor glucose with carbachol was able to stimulate release. However, under the same conditions, the combination of glucose, PACAP, and carbachol did stimulate release while being unable to elevate Ca2+]i. Thus, simultaneous activation of the beta cell by PACAP, carbachol, and glucose can stimulate insulin release even when Ca2+]i is not elevated.