AJP - Cell Physiology, Vol 263, Issue 6 C1172-C1180, Copyright © 1992 by American Physiological Society
Effects of okadaic acid indicate a role for dephosphorylation in pancreatic stimulus-secretion coupling
A. C. Wagner, M. J. Wishart, D. I. Yule and J. A. Williams
Department of Physiology, University of Michigan, Ann Arbor 48109-0622.
Okadaic acid completely inhibits phosphatase 2A at nanomolar
concentrations, while complete inhibition of type 1 phosphatases occurs at
1 microM. Phosphatase 2B is significantly inhibited only at concentrations
> 1 microM. In rat pancreatic acini, 1 microM okadaic acid shifted the
cholecystokinin (CCK) dose-response curve for stimulating amylase release
to the right without reducing maximal secretion. At 3 microM, 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 microM okadaic acid; and 3)
Ca(2+)-induced amylase secretion in streptolysin O-permeabilized cells was
also reduced by 85 +/- 7%. Two-dimensional polyacrylamide gel
electrophoresis of 32P-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.
