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

Abstract

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 µM CCh. With 0.5 mM EGTA [ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic 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 µM 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[γ-S]) was not inhibited by OAG with 0.5 mM [EGTA]_i. The pretreatment of gastric myocytes with PKC inhibitors, either 1 µM chelerythrine or 1 µM 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.