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Am J Physiol Cell Physiol 280: C980-C992, 2001;
0363-6143/01 $5.00
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Vol. 280, Issue 4, C980-C992, April 2001

Ca2+-induced contraction of cat esophageal circular smooth muscle cells

W. Cao1, Q. Chen1, U. D. Sohn2, N. Kim3, M. T. Kirber1, K. M. Harnett1, J. Behar1, and P. Biancani1

1 Department of Medicine, Rhode Island Hospital and Brown Medical School, Providence, Rhode Island 02903; 2 Department of Pharmacology, College of Pharmacy, Chung Ang University, Seoul 156-756, and 3 Department of Internal Medicine, Kangnam General Hospital, Public Corporation, Seoul 135-090, Korea

ACh-induced contraction of esophageal circular muscle (ESO) depends on Ca2+ influx and activation of protein kinase Cepsilon (PKCepsilon ). PKCepsilon , however, is known to be Ca2+ independent. To determine where Ca2+ is needed in this PKCepsilon -mediated contractile pathway, we examined successive steps in Ca2+-induced contraction of ESO muscle cells permeabilized by saponin. Ca2+ (0.2-1.0 µM) produced a concentration-dependent contraction that was antagonized by antibodies against PKCepsilon (but not by PKCbeta II or PKCgamma antibodies), by a calmodulin inhibitor, by MLCK inhibitors, or by GDPbeta s. Addition of 1 µM Ca2+ to permeable cells caused myosin light chain (MLC) phosphorylation, which was inhibited by the PKC inhibitor chelerythrine, by D609 [phosphatidylcholine-specific phospholipase C inhibitor], and by propranolol (phosphatidic acid phosphohydrolase inhibitor). Ca2+-induced contraction and diacylglycerol (DAG) production were reduced by D609 and by propranolol, alone or in combination. In addition, contraction was reduced by AACOCF3 (cytosolic phospholipase A2 inhibitor). These data suggest that Ca2+ may directly activate phospholipases, producing DAG and arachidonic acid (AA), and PKCepsilon , which may indirectly cause phosphorylation of MLC. In addition, direct G protein activation by GTPgamma S augmented Ca2+-induced contraction and caused dose-dependent production of DAG, which was antagonized by D609 and propranolol. We conclude that agonist (ACh)-induced contraction may be mediated by activation of phospholipase through two distinct mechanisms (increased intracellular Ca2+ and G protein activation), producing DAG and AA, and activating PKCepsilon -dependent mechanisms to cause contraction.

calcium; smooth muscle; protein kinase C; phospholipase C; phospholipase D; myosin phosphorylation


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