OKADAIC ACID INDICATES A MAJOR FUNCTION FOR PROTEIN PHOSPHATASES IN STIMULUS-RESPONSE COUPLING OF RINM5F RAT INSULINOMA CELLS

Stimulus-induced insulin secretion involves the activation of several protein kinases within the beta cell. Most prominent are protein kinas e A, protein kinase C and calcium/calmodulin-dependent protein kinases . Protein kinase action is functionally antagonized by protein phospha tases. The four ubiquious serine/threonine protein phosphatases are te rmed PP-I, PP-2A, -2B and -2C. PP-1 and PP-2A are in vivo parts of maj or protein complexes. These complexes presumably regulate the phosphat ase activity and direct the enzyme to its site of action. Therefore, P P-I and -2A could play an important role in controlling intracellular signal transmission. Two different toxins, okadaic acid and calyculin A, both from marine invertebrates, were recently discovered and identi fied as potent and highly specific inhibitors of PP-1 and PP-2A. Both compounds emerged as very useful tools for studying intracellular phos phorylation events. We took advantage of these substances to investiga te the significance of protein phosphatase action in stimulus-induced insulin secretion. To avoid major complexity, we confined our study to the cAMP and the phosphoinositide signal pathway. Okadaic acid alone evoked virtually no secretory response. cA1MP-dependent secretion was markedly enhanced by I mu M okadaic acid. The stimulatory effect of ok adaic acid was strongly dependent on the concentration of cAMP analoga . Tn contrast, insulin release caused by the cholinergic agonist carba chol was not influenced by okadaic acid. Calyculin A (10 nM) slightly increased cAMP-induced secretion, but its high toxicity prohibited acc urate interpretation of the data. Our findings support the idea that s erine/threonine phosphatases act as important regulators in stimulus r esponse coupling. Firstly, by affecting cAMP-induced insulin liberatio n, protein phosphatases could, theoretically, modify the magnitude of response to a given stimulus. Secondly, the cAMP pathway is obviously controlled by phosphatases other than the phosphoinositide pathway sin ce the latter is not influenced by okadaic acid. This argues for a mor e differentiated function of protein phosphatases than just switching off any protein kinase activation.