Regulation of calcium-induced exocytosis from gastric chief cells by protein phosphatase-2B (calcineurin).

The molecular mechanisms whereby calcium stimulates secretion are uncertain. In the present study, we used streptolysin O (SLO)-permeabilized chief cells from guinea pig stomach to investigate whether protein phosphatase-2B (calcineurin), a calcium/calmodulin-dependent, serine/threonine phosphatase plays a role in mediating calcium-induced pepsinogen secretion. Preincubation of cells with alpha-naphthylphosphate, a non-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 pepsinogen secretion. Moreover, in intact cells, cyclosporin-A and FK-506 inhibited pepsinogen secretion caused by cholecystokinin, carbamylcholine and A23187, agonists known to increase chief cell cytosolic calcium. Okadaic acid, an inhibitor of protein phosphatase-1 and -2A, had no effect on secretion caused by these agonists. Chief cell calcium-dependent phosphatase activity, measured using radiolabeled casein as substrate, 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-dimensional gel electrophoresis. Increasing the cytosolic calcium concentration resulted in dephosphorylation of a 55-kDa, acidic cytoskeletal protein. FK-506 inhibited dephosphorylation of this protein. Thus, in permeabilized chief cells, specific inhibitors of protein phosphatase-2B inhibit calcium-induced pepsinogen secretion, calcium/calmodulin-dependent phosphatase activity and calcium-induced dephosphorylation of a 55-kDa, acidic cytoskeletal protein. These results support the hypothesis that protein phosphatase-2B (calcineurin) plays an important role in mediating calcium-induced exocytosis.