Effects of phosphatase inhibitors and a protein phosphatase on norepinephrine secretion by permeabilized bovine chromaffin cells.

A protein phosphatase and phosphatase inhibitors were used to examine the role of protein phosphorylation in the regulation of norepinephrine secretion in digitonin-permeabilized bovine chromaffin cells. Addition of okadaic acid, a potent inhibitor of type 1 and type 2A protein phosphatases, or 1-naphthylphosphate, a more general phosphatase inhibitor, to digitonin-permeabilized chromaffin cells caused about a 100% increase in the amount of norepinephrine secreted in the absence of Ca2+ (in 5 mM EGTA) without affecting the amount of norepinephrine secreted in the presence of 10 microM free Ca2+. This stimulation of norepinephrine secretion by protein phosphatase inhibitors suggests that in the absence of Ca2+ there is a slow rate phosphorylation and that this phosphorylation triggers secretion. Addition of an exogenous type 2A protein phosphatase caused almost a 50% decrease in Ca(2+)-dependent norepinephrine secretion. Thus, the amounts of norepinephrine released both in the absence of Ca2+ and in the presence of Ca2+ appear to depend upon the level of protein phosphorylation.