REGULATION OF CALCIUM-INDUCED EXOCYTOSIS FROM GASTRIC CHIEF CELLS BY PROTEIN PHOSPHATASE-2B (CALCINEURIN)

The molecular mechanisms whereby calcium stimulates secretion are unce rtain. In the present study, we used streptolysin O (SLO)-permeabilize d chief cells from guinea pig stomach to investigate whether protein p hosphatase-2B (calcineurin), a calcium/calmodulin-dependent, serine/th reonine phosphatase plays a role in mediating calcium-induced pepsinog en secretion. Preincubation of cells with alpha-naphthylphosphate, a n on-specific phosphatase inhibitor, decreased calcium-induced secretion . Likewise, specific inhibitors of protein phosphatase-2B (cyclosporin -A and FK-506) caused a dose-dependent reduction in calcium-induced pe psinogen secretion. Moreover, in intact cells, cyclosporin-A and FK-50 6 inhibited pepsinogen secretion caused by cholecystokinin, carbamylch oline and A23187, agonists known to increase chief cell cytosolic calc ium. Okadaic acid, an inhibitor of protein phosphatase-l and -2A, had no effect on secretion caused by these agonists. Chief cell calcium-de pendent phosphatase activity, measured using radiolabeled casein as su bstrate, was reduced selectively by inhibitors of protein phosphatase- 2B. Endogenous substrates for calcium/calmodulin-dependent phosphatase activity were identified by analyzing chief cell lysates using 2-dime nsional gel electrophoresis. Increasing the cytosolic calcium concentr ation resulted in dephosphorylation of a 55-kDa, acidic cytoskeletal p rotein. FK-506 inhibited dephosphorylation of this protein. Thus, in p ermeabilized chief cells, specific inhibitors of protein phosphatase-2 B inhibit calcium-induced pepsinogen secretion, calcium/calmodulin-dep endent phosphatase activity and calcium-induced dephosphorylation of a 55-kDa, acidic cytoskeletal protein. These results support the hypoth esis that protein phosphatase-2B (calcineurin) plays an important role in mediating calcium-induced exocytosis. (C) 1997 Elsevier Science B. V.