Protein kinase C mediates the desensitization of CCh-activated nonselective cationic current in guinea-pig gastric myocytes

The possibility of the protein kinase C (PKC) pathway being a mechanism underlying the desensitization of carbachol-(CCh-)activated nonselective cationic current (I-CCh) was investigated in a study of guinea-pig gastric myocytes. Using the conventional whole-cell patch-clamp technique with symmetrical CsCl-rich solution in pipette and bath, I-CCh was induced by bath application of 50 mu M CCh. With 0.5 mM EGTA [ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetra-acetic acid] in the pipette solution (0.5 mM [EGTA](i)), I-CCh decayed spontaneously (desensitization of I-CCh) to around 20% within 10 min. Desensitization of I-CCh was significantly attenuated with 2 mM [EGTA](i). At a concentration of 20 mu M OAG (1-oleoyl-2-acetyl-sn-glycerol), a PKC activator, inhibited I-CCh at 0.5 mM [EGTA](i) but far less at 2 mM [EGTA](i) (18% and 81% of control, respectively). The same cationic current induced by intracellular guanosine-5'-O-(3-thiotriphosphate) (GTP[gamma-S]) was not inhibited by OAG with 0.5 mM [EGTA](i). The pretreatment of gastric myocytes with PKC inhibitors, either 1 mu M chelerythrine or 1 mu M peptide inhibitor, attenuated the desensitization of I-CCh. [Ca2+](i) was also measured by single cell microfluorometry using fura-2, Under CCh stimulation with 2 mM [EGTA](i), [Ca2+](i) did not increase above 100 nM while it increased to around 260 nM with 0.5 mM [EGTA](i). These results suggest that the desensitization of I-CCh is partly due to the Ca2+-dependent PKC pathway in guinea-pig gastric myocytes.