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Background: Protein phosphorylation and dephosphorylation events are considered to be key steps in the control of agonist-induced pancreatic enzyme release. This study was designed to characterize the role of serine/threonine phosphatases in phosphoinositol/calcium- and cyclic adenosine monophosphate (cAMP)-mediated stimulus-secretion coupling in rat pancreatic acini., Methods: Isolated rat pancreatic acini were incubated with either the serine/threonine phosphatase inhibitors okadaic acid, calyculin A, and cyclosporin A or the calmodulin antagonist W-7. Amylase secretion was stimulated with cholecystokinin (CCK)-8, secretin, vasoactive intestinal polypeptide (VIP) or pituitary adenylate cyclase-activating polypeptide (PACAP), and the intracellular second messengers calcium and cAMP were determined., Results: Okadaic acid or calyculin A reduced secretagogue-stimulated amylase release to near-basal levels. Inhibition of cAMP-mediated secretion (by VIP, secretin, or PACAP) occurred at lower concentrations than with inositol triphosphate (IP3)/Ca(2+)-dependent enzyme release (via CCK). Cyclosporin A diminished CCK-8-stimulated secretion by 35%, whereas secretion in response to cAMP-mediated secretagogues was not affected. W-7 completely inhibited acinar secretion in response to cAMP-or IP3/Ca(2+)-mediated secretagogues. Binding of 125I-CCK-8- or 125I-PACAP-(1-27) to acini was not influenced by the phosphatase inhibitors or W-7. Okadaic acid and calyculin A affected neither CCK-8-stimulated intracellular Ca2+ release nor PACAP-(1-27)-stimulated cAMP synthesis, whereas W-7 inhibited by 50% and 40%, respectively., Conclusions: The inhibitory profiles of okadaic acid, calyculin A, cyclosporin A, and W-7 indicate that phosphatases 1 and 2A play a relevant role in cAMP-mediated enzyme release, whereas phosphatases 1 and 2B are predominantly involved in IP3/Ca(2+)-dependent stimulus-secretion coupling. The calmodulin antagonist W-7 interferes at multiple steps of intracellular signal-transduction pathways.

The gut-brain neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) is a novel highly conserved member of the secretin-glucagon-VIP peptide family comprising 38 or 27 amino acid residues. In this study, we investigate the actions of PACAP 1-27 or PACAP 1-38 on jejunal and caecal muscle strips from pig or guinea pig and demonstrate the neutralizing effect of two PACAP-specific monoclonal antibodies of the IgG1 subtype, RSP27II and RSP38. These antibodies were used to set up assay systems specific for PACAP 1-27 or PACAP 1-38. Monoclonal antibody RSP27II recognizes exclusively PACAP 1-27, whereas RSP38 binds only PACAP 1-38. PACAP 1-27 and PACAP 1-38 relax taenia caeci dose-dependently in the presence of guanethidine and scopolamine. Both peptides inhibit the spontaneous contractions of porcine jejunal muscle strips equipotently. Monoclonal antibodies RSP27II and RSP38 specifically neutralize the actions of either exogenously applied or endogenously released PACAP. Thus, they represent processing-specific tools to examine the physiological role of both molecular forms of PACAP in the gastrointestinal tract.

The binding and biological effect of pituitary adenylate cyclase activating polypeptide (PACAP), a novel hypothalamic peptide with high sequence homology to vasoactive intestinal polypeptide (VIP), were studied in rat AR 4-2 J pancreatic carcinoma cells and isolated rat pancreatic acini. PACAP(1-27) and analogue PACAP(1-23, VIP-24-28), but not VIP, displaced potently and reversibly 125I-PACAP(1-27) from binding to an abundantly expressed high affinity PACAP-preferring receptor on AR 4-2 J cells, referred to as "PACAP-1 receptor." High affinity binding was dependent on N-terminal and C-terminal residues of PACAP(1-27): PACAP(1-24,Cys-25) (7.3 +/- 1.6 microM), PACAP(1-23) (8.2 +/- 1.5 microM), VIP (> 30 microM), PACAP(3-27), PACAP(1-19), PACAP(3-19), PACAP(1-12), and PACAP(18-38) (all > 50 microM) showed low or no binding potency. In contrast, high and low affinity binding of 125I-VIP to AR 4-2 J cells was displaced equipotently by PACAP(1-27) and VIP, thus defining on these cells, in addition, two scarcely expressed binding sites, designated "VIP/PACAP-2 receptor," similar or identical to the previously described high and low affinity acinar VIP receptor. Binding of 125I-PACAP(1-27) to a high and low affinity binding site on rat pancreatic acini was inhibited equipotently by PACAP(1-27) and VIP, identifying these sites as VIP/PACAP-2 receptors. PACAP(1-23) recognized both type 2 binding sites with only slightly lower affinity. PACAP(1-27), PACAP(1-38), PACAP(1-23, VIP-24-28), and PACAP(1-23) equipotently stimulated acinar lipase release and cyclic AMP production in pancreatic acini. Co-incubation of PACAP(1-27) or VIP with cholecystokinin-8 or carbachol revealed additive effects on enzyme secretion. Our results suggest the predominant expression of VIP/PACAP-2 receptors on rat pancreatic acini, whereas AR 4-2 J cells express mainly PACAP-1 receptors. PACAP is a potent ligand for both receptor types and has to be regarded as a novel VIP-like pancreatic secretagogue.