Abstract

Effect on insulin release, cyclic AMP content and protein phosphorylation of agents modifying cyclic AMP levels have been tested in intact rat islets of Langerhans. Insulin release induced by glucose was potentiated by dibutyryl cyclic AMP, 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 μM; higher concentrations, up to 1 mM, were resistant to inhibition by trifluoperazine. IBMX-potentiated insulin release was also inhibited by 2-deoxyadenosine, and 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 μM-IBMX. Islet cyclic AMP was increased by OBMX, with the maximum rise occurring with 100 μM-IBMX The increase in cyclic AMP elicited by IBMX was more rapid than that induced by cholera toxin. Trifluoperazine did not significantly effect islet cyclic AMP levels under any of the conditions tested. When islets were incubated with 32PP(i), radioactivity was incorporated into islet ATP predominantly in the γ-position. The rate of equilibration of label was dependent on medium P(i) and glucose concentration and at optimal concentration of these 100% equilibration of internal 32PATP with external 32PP(i) required a period of 3 h. Radioactivity was incorporated into islet protein and, in response to an increase in islet cyclic AMP, the major effect was on a protein of M(r) 15 000 on sodium dodecyl sulphate/polyacrylamide gels. The extent of phosphorylation of the M(r)-15 000 protein was correlated with the level of cyclic AMP: phosphorylation in response to IBMX was inhibited by 2-deoxyadenosine but not by trifluoperazine. Fractionation of islets suggested that the M(r)-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 cyclic AMP: the major species had M(r) values of 18 000, 25 000, 34 000, 38 000 and 48 000. Culture of islets with IBMX incrased the rate of 3H-thymidine incorporation. These data indicated that the potentiation of insulin release by agents elevating cyclic AMP is accompanied by activation of cyclic AMP-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 cyclic AMP 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 cyclic AMP rather than in the regulation of cyclic AMP concentration.