DIFFERENTIAL INHIBITORY EFFECTS OF SERINE THREONINE PHOSPHATASE INHIBITORS AND A CALMODULIN ANTAGONIST ON PHOSPHOINOSITOL CALCIUM AND CYCLIC ADENOSINE ONOPHOSPHATE-MEDIATED PANCREATIC AMYLASE SECRETION/

Background: Protein phosphorylation and dephosphorylation events are c onsidered to be kev steps in the control of agonist-induced pancreatic enzyme release. This study was designed to characterize the role of s erine/threonine phosphatases in phosphoinositol/calcium- and cyclic ad enosine monophosphate (cAMP)-mediated stimulus-secretion coupling in r at pancreatic acini. Methods: Isolated rat pancreatic acini were incub ated with either the serine/threonine phosphatase inhibitors okadaic a cid, calyculin A, and cyclosporin A or the calmodulin antagonist W-7. Amylase secretion was stimulated with cholecystokinin (CCK)-8. secreti n, vasoactive intestinal polypeptide (VIP) or pituitary adenylate cycl ase-activating polypeptide (PACAP). and the intracellular second messe ngers calcium and cAMP were determined. Results: Okadaic acid or calyc ulin A reduced secretagogue-stimulated amylase release to near-basal l evels. Inhibition of cAMP-mediated secretion (by VIP, secretin, or PAC AP) occurred at lower concentrations than with inositol triphosphate ( IP3)/Ca2+-dependent enzyme release (via CCK). Cyclosporin A diminished CCK-8-stimulated secretion by 35%, whereas secretion in response to c AMP-mediated secretagogues was not affected. W-7 completely inhibited acinar secretion in response to cAMP- or IP3/Ca2+-mediated secretagogu es. Binding of I-125-CCK-8- or I-125-PACAP-(1-27) to acini was not inf luenced by the phosphatase inhibitors or W-7. Okadaic acid and calycul in A affected neither CCK-8-stimulated intracellular Ca2+ release nor PACAP-(1-27)-stimulated cAMP synthesis, whereas W-7 inhibited by 50% a nd 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/Ca2+-dependent stimulus-secretion coupling. The calmodulin antagonist W-7 interferes at multiple steps of intracellular signal-transduction pathways.