Ud. Sohn et al., DIFFERENT PROTEIN-KINASE-C ISOZYMES MEDIATE LOWER ESOPHAGEAL SPHINCTER TONE AND PHASIC CONTRACTION OF ESOPHAGEAL CIRCULAR SMOOTH-MUSCLE, Molecular pharmacology, 51(3), 1997, pp. 462-470
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.