Secretion of cholecystokinin (CCK) from the endocrine cells of small intestinal mucosa and the murine intestinal tumor cell line STC-1 is known to involve both adenosine 3',5'-cyclic monophosphate (cAMP)-and Ca(2+)-dependent signal transduction pathways. However, the endogenous stimulant(s) that acts through the cAMP-dependent cascade has not been identified. We determined the effect of pituitary adenylate cyclase-activating polypeptide (PACAP) on CCK secretion and cAMP production and its interaction with other CCK secretagogues in STC-1 cells. At concentrations > 10 nM, PACAP-27 stimulated the release of large intestinal CCK from STC-1 cells in a time- and dose-dependent manner. The stimulatory effect of PACAP-27 was enhanced by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). PACAP-27, PACAP-38, and vasoactive intestinal polypeptide (VIP), with or without IBMX, were equally effective and potent to elicit CCK release with similar half-maximal doses and maximal levels of stimulation. Both forms of PACAP and VIP stimulated a transient but not significant increase in the cellular cAMP level. In the presence of IBMX, all three peptides increased significantly the cellular cAMP level between 2 and 5 min, but PACAP produced a two times higher level than VIP. The stimulatory effect of PACAP-27 on CCK release was also potentiated by bombesin and KCl but without a synergistic production of cAMP. With or without IBMX, PACAP-27-stimulated CCK secretion was not affected by the Ca2+ channel blocker diltiazem (1 microM), the cell-permeable Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM; 25 microM), or by downregulation of protein kinase C. The stimulatory effects of KCl and bombesin were either reduced or abolished by these treatments. The synergistic effect of bombesin with PACAP was abolished by diltiazem and BAPTA-AM but not by downregulation of protein kinase C, whereas KCl remained synergistic with PACAP after these treatments. Taken together, these results indicate that PACAP may be a neuromodulator of CCK secretion that acts through activation of adenylate cyclase and may function as a coregulator with other CCK secretagogues that are known to increase intracellular Ca2+ concentration.