Abstract

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-α, -βII, and -γ isozymes in LES circular muscle, but only PKC-βII translocated from the cytosolic to the membrane fraction in response to ACh. In contrast, ESO contained PKC-βII, -γ, and -ε, and only PKC-ε 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-βII antiserum but not by other PKC antisera. In esophageal cells, contraction was inhibited by the PKC-ε 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 αβγ pseudosubstrate but not by the α, δ, or ε pseudosubstrate. Contraction of ESO cells was reduced by the ε pseudosubstrate but not by the α, δ, or αβγ 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-βII, whereas contraction of ESO is mediated by the calcium-independent PKC-ε.