Different protein kinase C isozymes mediate lower esophageal sphincter tone and phasic contraction of esophageal circular smooth muscle.

Circular muscle of the esophagus (ESO) is normally relaxed and contracts phasically in response to neural stimuli. In contrast, lower esophageal sphincter (LES) circular muscle maintains spontaneous tone and relaxes in response to neural stimuli. We have previously shown that in vitro, spontaneous LES tone and contraction of ESO in response to acetylcholine (ACh) are antagonized by protein kinase C (PKC) inhibitors, suggesting that PKC activation is responsible for these functions. In the current study, Western blot analysis of LES and ESO revealed PKC-alpha, -betaII, and -gamma isozymes in LES circular muscle, but only PKC-betaII translocated from the cytosolic to the membrane fraction in response to ACh. In contrast, ESO contained PKC-betaII, -gamma, and -epsilon, and only PKC-epsilon translocated to the membrane fraction in response to ACh. In LES single cells isolated by enzymatic digestion and permeabilized by saponin, 1-2-dioctanoylglycerol-mediated contraction was inhibited by preincubation with PKC-betaII antiserum but not by other PKC antisera. In esophageal cells, contraction was inhibited by the PKC-epsilon antiserum but not by antisera against other PKC isozymes. N-Myristoylated peptides derived from the pseudosubstrate sequences of PKC isozymes were used to inhibit saponin, 1-2-dioctanoylglycerol-induced contraction of LES and ESO smooth muscle cells. Contraction of LES cells was reduced by the alpha beta gamma pseudosubstrate but not by the alpha, delta, or epsilon pseudosubstrate. Contraction of ESO cells was reduced by the epsilon pseudosubstrate but not by the alpha, delta, or alpha beta gamma pseudosubstrate. We conclude that different types of contractile activity in the ESO and LES are mediated by different PKC isozymes. LES contraction is mediated by the calcium-dependent PKC-betaII, whereas contraction of ESO is mediated by the calcium-independent PKC-epsilon.