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.
Raufman
J P
JP
Division of Gastroenterology, University of Arkansas for Medical Sciences, Little Rock 72205-7199, USA.
Malhotra
R
R
Raffaniello
R D
RD
D000818:Animals; D001426:Bacterial Proteins; D019703:Calcineurin; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002148:Calmodulin-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016572:Cyclosporine / Q000494:pharmacology; D003598:Cytoskeletal Proteins / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005089:Exocytosis; D005753:Gastric Mucosa / Q000187:drug effects* / Q000378:metabolism; D006168:Guinea Pigs; D010435:Pepsinogens / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054645:Protein Phosphatase 1; D013301:Streptolysins; D016559:Tacrolimus / Q000494:pharmacology
D001426:Bacterial Proteins; D002148:Calmodulin-Binding Proteins; D003598:Cytoskeletal Proteins; D004791:Enzyme Inhibitors; D010435:Pepsinogens; D013301:Streptolysins; C025964:streptolysin O; D016572:Cyclosporine; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D002118:Calcium; D016559:Tacrolimus