Gastrin-releasing peptide (GRP) and its amphibian homolog, bombesin, are potent secretogogues in mammals. We determined the roles of intracellular free Ca2+ ([Ca2+](i)), protein kinase C (PKC), and mitogen-activated protein kinases (MAPK) in GRP receptor (GRP-R)-regulated secretion. Bombesin induced either [Ca2+](i) oscillations or a biphasic elevation in [Ca2+](i). The biphasic response was associated with peptide secretion. Receptor-activated secretion was blocked by removal of extracellular Ca2+, by chelation of [Ca2+](i), and by treatment with inhibitors of phospholipase C, conventional PKC isozymes, and MAPK kinase (MEK). Agonist- induced increases in [Ca2+](i) were also inhibited by dominant negative MEK-1 and the MEK inhibitor, PD89059, but not by an inhibitor of PKC. Direct activation of PKC by a phorbol ester activated MAPK and stimulated peptide secretion without a concomitant increase in [Ca2+](i). Inhibition of MEK blocked both bombesin- and phorbol 12-myristate 13-acetate-induced secretion. GRP-R-regulated secretion is initiated by an increase in [Ca2+](i); however, elevated [Ca2+](i) is insufficient to stimulate secretion in the absence of activation of PKC and the downstream MEK/MAPK pathways. We demonstrated that the activity of MEK is important for maintaining elevated [Ca2+](i) levels induced by GRP-R activation, suggesting that MEK may affect receptor-regulated secretion by modulating the activity of Ca2+- sensitive PKC.
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