Cyclosporin A inhibits Ca2+/calmodulin-dependent protein phosphatase and secretion in pancreatic acinar cells.
The immunosuppressant cyclosporin A (CsA) was utilized as a highly specific inhibitor of the Ca2+/calmodulin-dependent protein phosphatase, PP2B in rat pancreatic acinar cells. Treatment of cells with CsA for 20 min resulted in a concentration-dependent inhibition of PP2B that was maximal (> 90%) at 1 microM and exhibited an IC50 of 65 nM. CsA also inhibited cholecystokinin-, 100 pM, or carbamylcholine-, 10 microM, induced amylase release in a concentration-dependent manner. A maximal inhibition to 55% of stimulated control cells occurred at 1 microM CsA with half-maximal inhibition occurring at approximately 200 nM. Secretion in response to 1 microM 12-O-tetradecanoylphorbol-13-acetate (TPA) was uneffected by CsA treatment. Conversely, amylase release stimulated by the Ca2+ mobilizing agent, thapsigargin, when added alone at 2 microM or in 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 chemically distinct immunosuppressant, FK506, produced only partial inhibition of PP2B activity and did not significantly inhibit amylase secretion at concentrations up to 1 microM. Two-dimensional gel electrophoresis of proteins from 32P-labeled acinar cells revealed that CsA specifically blocked the cholecystokinin-stimulated dephosphorylation of a 24-kDa protein in a concentration range similar to that seen for inhibition of secretion. Using 32P-labeled cytosol and purified calcineurin, a Ca(2+)- and calmodulin-dependent dephosphorylation of the 24-kDa protein was also demonstrated in vitro. Collectively, these data indicate that the Ca2+/calmodulin-dependent protein phosphatase, PP2B, plays a significant role in stimulus-secretion coupling in pancreatic acinar cells.
Groblewski
G E
GE
Department of Physiology, University of Michigan, Ann Arbor 48109.
Wagner
A C
AC
Williams
J A
JA
D000595:Amino Acid Sequence; D000681:Amylases / Q000378:metabolism; D000818:Animals; D019703:Calcineurin; D002118:Calcium / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000037:antagonists & inhibitors*; D016572:Cyclosporine / Q000494:pharmacology*; D008297:Male; D008969:Molecular Sequence Data; D010179:Pancreas / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley
D002148:Calmodulin-Binding Proteins; D016572:Cyclosporine; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D000681:Amylases; D002118:Calcium