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 contrac ts phasically in response to neural stimuli. In contrast, lower esopha geal sphincter (LES) circular muscle maintains spontaneous tone and re laxes in response to neural stimuli. We have previously shown that in vitro, spontaneous LES tone and contraction of ESO in response to acet ylcholine (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-a lpha, -beta II, and -gamma isozymes in LES circular muscle, but only P KC-beta II translocated from the cytosolic to the membrane fraction in response to ACh. In contrast, ESO contained PKC-PII, -gamma, and -eps ilon, and only PKC-epsilon translocated to the membrane fraction in re sponse 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-beta II 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 isozym es. N-Myristoylated peptides derived from the pseudosubstrate sequence s of PKC isozymes were used to inhibit saponin, 1-2-dioctanoyl-glycero l-induced contraction of LES and ESO smooth muscle cells. Contraction of LES cells was reduced by the alpha beta gamma pseudosubstrate but n ot 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 differe nt types of contractile activity in the ESO and LES are mediated by di fferent PKC isozymes. IES contraction is mediated by the calcium-depen dent PKC-beta II, whereas contraction of ESO is mediated by the calciu m-independent PKC-epsilon.