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Inhibition of serine/threonine protein phosphatases by secretagogues in insulin-secreting cells.

Reversible protein phosphorylation is considered to be an important and versatile mechanism by which cells transduce external signals into biological responses. Cellular levels of protein phosphorylation are determined by the balanced actions of both protein kinases and protein phosphatases (PPases). Compared with protein kinases, however, serine/threonine PPases have received less attention. In the present study, the effects of known insulin secretagogues and some intracellular second messengers on the activities of serine/threonine PPases in insulin-secreting RINm5F insulinoma cells were investigated. The stimulation of intact RINm5F cells with the insulin secretagogues L-arginine, L-glutamine, KCl, or ATP elicited time-dependent changes in PPase activities with an early (1 min) decrease in type 1-like and/or type 2A-like PPase activity that gradually returned to normal levels. Addition of cAMP, cGMP, or prostaglandins E2 and F1 alpha at widely different concentrations to RINm5F cell homogenates failed to affect PPase activities. In contrast, addition of physiological concentrations of adenine nucleotides, which are known to increase upon secretory stimulation, to cell homogenates inhibited type 2A-like and, to a lesser extent, type 1-like, PPase activity (ATP > ADP > AMP > adenosine). ATP and ADP IC50 values for type 2A-like PPase were approximately 75 and 250 microM, respectively. The inhibitory effect of ATP was reproduced and of comparable magnitude when purified PPases (types 1 and 2A) were used instead of RINm5F cell homogenates. It is concluded that insulin secretagogues cause time- and concentration-dependent inhibitory effects on RINm5F cell PPase activities, which may contribute to the increase in the phosphorylation state that occurs after stimulation of insulin release. Thus, inhibition of protein dephosphorylation may be a novel regulatory mechanism controlling the stimulus-secretion coupling in insulin-producing cells.