EFFECTS OF OKADAIC ACID INDICATE A ROLE FOR DEPHOSPHORYLATION IN PANCREATIC STIMULUS-SECRETION COUPLING

Okadaic acid completely inhibits phosphatase 2A at nanomolar concentrations, while complete inhibition of type 1 phosphatases occurs at 1 muM. Phosphatase 2B is significantly inhibited only at concentrations >1 muM. In rat pancreatic acini, 1 muM okadaic acid shifted the cholecystokinin (CCK) dose-response curve for stimulating amylase release to the right without reducing maximal secretion. At 3 muM, okadaic acid inhibited maximal CCK-induced amylase release to 78 +/- 7% of control, whereas the inactive analogue 1-Nor-okadaone had no effect. Three lines of evidence indicate that this inhibition by okadaic acid occurs at a late step in stimulus-secretion coupling: 1) intracellular Ca2+ signaling in response to agonist stimulation was not appreciably altered by okadaic acid; 2) stimulation with phorbol ester plus thapsigargin (thus by-passing receptor activation), which gave 85 +/- 4% of maximal CCK-induced amylase release, was inhibited 66 +/- 4% by 3 muM okadaic acid; and 3) Ca2+-induced amylase secretion in streptolysin O-permeabilized cells was also reduced by 85 +/- 7%. Two-dimensional polyacrylamide gel electrophoresis of P-32-labeled acini and autoradiography demonstrated that okadaic acid dose dependently increased overall protein phosphorylation. Correspondingly, okadaic acid also led to an inhibition of CCK-induced dephosphorylation. These results show that okadaic acid inhibits pancreatic acinar secretion at a step after generation of intracellular messengers and indicate a role for protein dephosphorylation in stimulus-secretion coupling.