beta-escin 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

Muscarinic receptor stimulation increases Ca2+ sensitivity, i.e., the amount of force produced at a constant submaximal cytosolic Ca2+ concentration ([Ca2+]i), in permeabilized smooth muscle preparations. It is controversial whether this increase in Ca2+ sensitivity is in part mediated by protein kinase C (PKC). With the use of a beta-escin permeabilized canine tracheal smooth muscle (CTSM) preparation, the effect of four putative PKC inhibitors [calphostin C, chelerythrine chloride, a pseudosubstrate inhibitor for PKC [PKC peptide-(19-31)], and staurosporine] on Ca2+ sensitization induced by acetylcholine (ACh) plus GTP was determined. Preincubation with each of the inhibitors did not affect subsequent Ca2+ sensitization induced by muscarinic receptor stimulation in the presence of a constant submaximal [Ca2+]i, neither did any of these compounds reverse the increase in Ca2+ sensitivity induced by ACh plus GTP. Administration of a 1,2-diacylglycerol analog, 1-oleoyl-2-acetyl-sn-glycerol, did not induce Ca2+ sensitization at a constant submaximal [Ca2+]i. Thus we found no evidence that PKC mediates increases in Ca2+ sensitivity produced by muscarinic receptor stimulation in permeabilized CTSM.

Topics: Acetylcholine; Alkaloids; Animals; Benzophenanthridines; Calcium; Cell Membrane Permeability; Cytosol; Diglycerides; Dogs; Enzyme Inhibitors; Escin; Female; Guanosine Triphosphate; In Vitro Techniques; Kinetics; Male; Muscle Contraction; Muscle, Smooth; Naphthalenes; Peptide Fragments; Phenanthridines; Protein Kinase C; Receptors, Muscarinic; Staurosporine; Trachea