CYCLOSPORINE-A INHIBITS CA2+ CALMODULIN-DEPENDENT PROTEIN PHOSPHATASEAND SECRETION IN PANCREATIC ACINAR-CELLS/

The immunosuppressant cyclosporin A (CsA) was utilized as a highly spe cific inhibitor of the Ca2+/calmodulin-dependent protein phosphatase, PP2B in rat pancreatic acinar cells. Treatment of cells with CsA for 2 0 min resulted in a concentration-dependent inhibition of PP2B that wa s maximal (>90%) at 1 mu M and exhibited an IC50 of 65 nM. CsA also in hibited cholecystokinin-, 100 pM, or carbamylcholine-, 10 mu M, induce d amylase release in a concentration-dependent manner. A maximal inhib ition to 55% of stimulated control cells occurred at 1 mu M CsA with h alf-maximal inhibition occurring at approximately 200 nM. Secretion in response to 1 mu M 12-O-tetradecanoylphorbol-13-acetate (TPA) was une ffected by CsA treatment. Conversely, amylase release stimulated by th e Ca2+ mobilizing agent, thapsigargin, when added alone at 2 mu M or i n combination with TPA, was inhibited by CsA to 66 and 61% of control cells, respectively. These data indicate that CsA-mediated inhibition occurs only when stimulation involves an increase in intracellular Ca2 +. In addition, analogues of CsA, 6-methyl-alanine-CsA, and 11-methyl- leucine-CsA had no effect on PP2B activity or amylase secretion. The c hemically distinct immunosuppressant, FK506, produced only partial inh ibition of PP2B activity and did not significantly inhibit amylase sec retion at concentrations up to 1 mu M. Two-dimensional gel electrophor esis of proteins from P-32-labeled acinar cells revealed that CsA spec ifically blocked the cholecystokinin stimulated dephosphorylation of a 24-kDa protein in a concentration range similar to that seen for inhi bition of secretion. Using P-32-labeled cytosol and purified calcineur in, a Ca2+- and calmodulin-dependent dephosphorylation of the 24-kDa p rotein was also demonstrated in vitro. Collectively, these data indica te that the Ca2+/calmodulin-dependent protein phosphatase, PP2B, plays a significant role in stimulus-secretion coupling in pancreatic acina r cells.