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 exami ned 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 com pletely abolished under these conditions. However, in the presence of 12-O-tetradecanoylphorbol-13-acetate or forskolin, 10 mM glucose signi ficantly enhanced insulin release, even in the presence of 5 mu M nitr endipine or 150 mu M diazoxide. The [Ca2+](i) was not increased under these conditions. Even under Ca2+-deprived conditions, achieved by 60- min preincubation in Ca2+-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-tetradecano ylphorbol-13-acetate and forskolin. Because of these findings, additio nal studies were performed with pituitary adenylate cyclase-activating peptide (PACAP) and carbachol to see whether physiological stimulatio n via receptor activation could stimulate insulin release in the absen ce of a rise in [Ca2+](i). Under normal Ca2+-containing conditions, PA CAP and carbachol stimulated insulin release and markedly potentiated glucose-stimulated release. In the presence of nitrendipine and thapsi gargin, glucose failed to stimulate insulin release. Also, neither glu cose 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 un able to elevate [Ca2+](i). Thus, simultaneous activation of the beta c ell by PACAP, carbachol, and glucose can stimulate insulin release eve n when [Ca2+](i) is not elevated.