THE ROLE OF PROTEIN-KINASE-C IN THE DESENSITIZATION OF RAT PANCREATIC-ISLETS TO CHOLINERGIC STIMULATION

It is well known that protein kinase C (PKC) plays an important role i n mediating insulin secretion in response to cholinergic stimulation. In various cells PKC also mediates a desensitization process. The role of PKC for homologous desensitization of the insulin response to repe titive stimu lation with the muscarinic agonist carbachol (CCh) was in vestigated in perifusion experiments using isolated rat pancreatic isl ets. Repetitive (six times) stimulation with CCh (100 mu M) reduced in sulin secretion over time (up to 50% during the second challenge). Thi s was not a toxic effect since the desensitizing effect was mostly was hed out after 45 min. When PKC was downregulated by long term preincub ation (20 h) with 200 nM phorbol 12-myristate 13-acetate (TPA), the in itial stimulation of insulin release by CCh was reduced by 50%, and a desensitization by further CCh stimulation was no longer obvious. In c ontrast, when other compounds with different mechanisms of actions for inactivating PKC were used, i.e. PKC inhibitors such as staurosporin (100 nM), Ro 31-8220 (5 mu M) or PKC peptide (19-31), the insulin secr etion in response to CCh was reduced but the desensitization was not a bolished. When PKC was down-regulated or inhibited by the above method s, the PKC activator phorbol 12-myristate 13-acetate (TPA; 200 nM) was no longer able to evoke an increase in insulin secretion during stati c incubation, i.e. these control experiments indicate a real PKC inhib ition. When heparin (50 mu g/ml), an inhibitor of G-protein coupled re ceptor kinase (GRK), was used, the desensitization of the cholinergic stimulation of insulin release remained unchanged. The data indicate t hat PKC plays a role in CCh-mediated insulin secretion and also show a desensitization of this effect after repetitive stimulation with CCh. The data further indicate that specific PKC isoenzymes that are inhib ited by staurosporin or Ro 31-8220 do not take part in the desensitiza tion process, while isoenzymes that are downregulated by TPA are invol ved. It may be speculated that a hitherto unknown PKC isoenzyme that i s downregulated by TPA but not by the other used PKC inhibitors is inv olved in the desensitization process, or that a nonspecific effect of TPA is involved. Members of the GRK family are not involved in the des ensitization process of CCh.