Protein Kinase C 19-31

It is well known that protein kinase C (PKC) plays an important role in 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 repetitive stimulation with the muscarinic agonist carbachol (CCh) was investigated in perifusion experiments using isolated rat pancreatic islets. Repetitive (six times) stimulation with CCh (100 microM) reduced insulin secretion over time (up to 50% during the second challenge). This was not a toxic effect since the desensitizing effect was mostly washed out after 45 min. When PKC was downregulated by long term preincubation (20 h) with 200 nM phorbol 12-myristate 13-acetate (TPA), the initial stimulation of insulin release by CCh was reduced by 50%, and a desensitization by further CCh stimulation was no longer obvious. In contrast, 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 microM) or PKC peptide(19-31), the insulin secretion in response to CCh was reduced but the desensitization was not abolished. When PKC was downregulated or inhibited by the above methods, the PKC activator phorbol 12-myristate 13-acetate (TPA; 200 nM) was no longer able to evoke an increase in insulin secretion during static incubation, i.e. these control experiments indicate a real PKC inhibition. When heparin (50 microg/ml), an inhibitor of G-protein coupled receptor kinase (GRK), was used, the desensitization of the cholinergic stimulation of insulin release remained unchanged. The data indicate that 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 inhibited by staurosporin or Ro 31-8220 do not take part in the desensitization process, while isoenzymes that are downregulated by TPA are involved. It may be speculated that a hitherto unknown PKC isoenzyme that is downregulated by TPA but not by the other used PKC inhibitors is involved in the desensitization process, or that a nonspecific effect of TPA is involved. Members of the GRK family are not involved in the desensitization process of CCh.

1. CPI-17 has recently been identified as a novel protein in vascular smooth muscle. In vitro , its phosphorylation and thiophosphorylation by protein kinase C (PKC) specifically inhibits the type 1 class of protein phosphatases, including myosin light chain (MLC) phosphatase. 2. Both of the phosphorylated CPI-17 states dose-dependently potentiated submaximal contractions at constant [Ca2+] in beta-escin-permeabilized and Triton X-100-demembranated arterial smooth muscle, but produced no effect in intact and less intensely permeabilized (alpha-toxin) tissue. Thiophosphorylated CPI-17 (tp-CPI) induced large contractions even under Ca2+-free conditions and decreased Ca2+ EC50 by more than an order of magnitude. Unphosphorylated CPI-17 produced minimal but significant effects. 3. tp-CPI substantially increased the steady-state MLC phosphorylation to Ca2+ ratios in beta-escin preparations. 4. tp-CPI affected the kinetics of contraction and relaxation and of MLC phosphorylation and dephosphorylation in such a manner that indicates its major physiological effect is to inhibit MLC phosphatase. 5. Results from use of specific inhibitors in concurrence with tp-CPI repudiate the involvement of general G proteins, rho A or PKC itself in the Ca2+ sensitization by tp-CPI. 6. Our results indicate that phosphorylation of CPI-17 by PKC stimulates binding of CPI-17 to and subsequent inhibition of MLC phosphatase. This implies that CPI-17 accounts largely for the heretofore unknown signalling pathway between PKC and inhibited MLC phosphatase.

We have previously shown that the atypical antipsychotic drug clozapine facilitates N-methyl-D-aspartate (NMDA)- and electrically evoked responses in pyramidal cells of the medial prefrontal cortex (mPFC). In the present study, we investigated the role of protein kinase C (PKC) in the action of clozapine. Bath administration of the PKC activator phorbol-12-myristate 13-acetate (PMA), but not the inactive isomer 4alpha-PMA, significantly enhanced the NMDA-evoked inward current and electrically evoked excitatory postsynaptic currents. Chelerythrine, a selective blocker of PKC, completely prevented the potentiating action produced by either clozapine or PMA on these currents in the mPFC cells. Intracellular injection of the PKC inhibitor PKC-I, but not the control substance PKC-S, through the recording electrode totally blocked clozapine's potentiating effect, indicating that a post-synaptic expressed PKC is critically involved in the augmenting action of clozapine on NMDA-evoked currents. Of the PKC inhibitor PKC-I, but not the control substance PKC-S, through the recording electrode totally blocked clozapine's potentiating effect, indicating that a post-synaptic expressed PKC is critically involved in the augmenting action of clozapine on NMDA-evoked currents. To further test the role of PKC in mediating the augmenting action of clozapine, we performed experiments in PKCgamma mutant and wild-type mice. In contrast to results in pyramidal cells from rats or wild-type mice, neither clozapine nor PMA was able to potentiate NMDA-induced currents in the mPFC from the PKCgamma mutant mice. Taken together, these results suggest that the PKC signal transduction pathway is critically involved in the facilitating action of clozapine on the NMDA-induced responses in pyramidal cells of the mPFC.