Cyclic AMP-dependent protein phosphorylation and insulin secretion in intact islets of Langerhans

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Abstract
Effects of insulin release, cAMP content and protein phosphorylation of agents modifying cAMP levels were tested in intact rat islets of Langerhans. Insulin release induced by glucose was potentiated by dibutyryl cAMP, glucagon, cholera toxin and 3-isobutyl-1-methylxanthine (IBMX); the calmodulin antagonist trifluoperazine reversed these potentiatory effects. Inhibition by trifluoperazine of IBMX-potentiated release was, however, confined to concentrations of IBMX below 50 .mu.M; higher concentrations, up to 1 mM, were resistant to inhibition by trifluoperazine. IBMX-potentiated insulin release was also inhibited by 2-deoxyadenosine, an inhibitor of adenylate cyclase. In the absence of glucose, IBMX at concentrations up to 1 mM did not stimulate insulin release and in the presence of 3.3 mM glucose IBMX was effective only at a concentration of 1 mM; under the latter conditions trifluoperazine again did not inhibit insulin secretion. The maximum effect on insulin release was achieved with 25 .mu.M IBMX. Islet [cAMP] was increased by IBMX, with the maximum rise occurring with 100 .mu.M IBMX. The increase in [cAMP] elicited by IBMX was more rapid than that induced by cholera toxin. Trifluoperazine did not significantly affect islet cAMP levels under any of the conditions tested. When islets were incubated with [32P]Pi, radioactivity was incorporated into islet ATP predominantly in the .gamma.-position. The rate of equilibration of label was dependent on medium Pi and glucose concentration and at optimal concentrations of these 100% equilibration of internal [32P]ATP with external [32P]Pi required 3 h. Radioactivity was incorporated into islet protein and, in response to an increase in islet [cAMP], the major effect was on a protein of MW 15,000 on sodium dodecyl sulfate/polyacrylamide gels. The extent of phosphorylation of the MW 15,000 protein was correlated with the level of cAMP: phosphorylation in response to IBMX was inhibited 2-deoxyadenosine but not by trifluoperazine. Fractionation of islets suggested that the MW 15,000 protein was of nuclear origin: the protein co-migrated with histone H3 on acetic acid/urea/Triton gels. In the islet cytosol a number of proteins were phosphorylated in response to elevation of islet [cAMP]: the major species had MW values of 18,000, 25,000, 34,000, 38,000 and 48,000. Culture of islets with IBMX increased the rate of [3H]thymidine incorporation. The potentiation of insulin release by agents elevating [cAMP] is accompanied by activation of cAMP-dependent protein kinase and phosphorylation of islet proteins. However, the major substrate was tentatively identified as histone H3; this phosphorylation is unlikely to be related to exocytosis but could be relevant to effects of cAMP on cell division. The findings with trifluoperazine suggest that calmodulin is more likely to play a role in secretion in mediating Ca2+-dependent steps modulated by cAMP rather than in the regulation of cAMP concentration.