protein-kinase-c and chelerythrine

1. Mechanisms involved in Ca(2+) sensitization of contractile elements induced by the activation of muscarinic receptors in membrane-permeabilized preparations of the rat proximal and distal colon were studied. 2. In alpha-toxin-permeabilized preparations from the rat proximal and distal colon, Ca(2+) induced a rapid phasic and subsequent tonic component. After Ca(2+)-induced contraction reached a plateau, guanosine 5'-triphosphate (GTP) and carbachol (CCh) in the presence of GTP further contracted preparations of both the proximal and distal colon (Ca(2+) sensitization). Y-27632, a rho-kinase inhibitor, inhibited GTP plus CCh-induced Ca(2+) sensitization more significantly in the proximal colon than in the distal colon. 3. Y-27632 at 10 microm had no effect on Ca(2+)-induced contraction or slightly inhibited phorbol-12,13-dibutyrate-induced Ca(2+) sensitization in either proximal or distal colon. Chelerythrine, a protein kinase C inhibitor, inhibited GTP plus CCh-induced Ca(2+) sensitization in the distal colon, but not in the proximal colon. The component of Ca(2+) sensitization that persisted after the chelerythrine treatment was completely inhibited by Y-27632. 4. In beta-escin-permeabilized preparations of the proximal colon, C3 exoenzyme completely inhibited GTP plus CCh-induced Ca(2+) sensitization, but PKC(19-31) did not. In the distal colon, C3 exoenzyme abolished GTP-induced Ca(2+) sensitization. It inhibited CCh-induced sensitization by 50 % and the remaining component was inhibited by PKC(19-31). 5. These results suggest that both protein kinase C and rho pathways in parallel mediate the Ca(2+) sensitization coupled to activation of muscarinic receptors in the rat distal colon, whereas the rho pathway alone mediates this action in the proximal colon.

Topics: ADP Ribose Transferases; Alkaloids; Amides; Animals; Bacterial Toxins; Benzophenanthridines; Botulinum Toxins; Calcium; Carbachol; Cell Membrane Permeability; Colon, Ascending; Colon, Descending; Escin; Guanosine Triphosphate; Hemolysin Proteins; Intracellular Signaling Peptides and Proteins; Japan; Male; Muscle Contraction; Myocytes, Smooth Muscle; Peptide Fragments; Phenanthridines; Phorbol 12,13-Dibutyrate; Protein Kinase C; Protein Serine-Threonine Kinases; Pyridines; Rats; Rats, Wistar; Receptors, Muscarinic; rho-Associated Kinases; Tritium; Type C Phospholipases

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.

Topics: Alkaloids; Animals; Benzophenanthridines; Clozapine; Drug Interactions; Electric Stimulation; Electrophysiology; Enzyme Activators; Enzyme Inhibitors; Evoked Potentials; Excitatory Amino Acid Agonists; Excitatory Postsynaptic Potentials; Fluorobenzenes; GABA Antagonists; gamma-Aminobutyric Acid; In Vitro Techniques; Mice; Mice, Knockout; Mice, Mutant Strains; N-Methylaspartate; Peptide Fragments; Phenanthridines; Phorbols; Piperidines; Protein Kinase C; Pyramidal Cells; Rats; Rats, Sprague-Dawley; Serotonin Antagonists; Tetradecanoylphorbol Acetate; Time Factors