Ca-induced contraction of cat esophageal circular smooth muscle cells

Cao, W., Q. Chen, U. D. Sohn, N. Kim, M. T. Kirber, K. M. Harnett, J. Behar, and P. Biancani. Ca-induced contraction of cat esophageal circular smooth muscle cells. Am J Physiol Cell Physiol 280: C980–C992, 2001.— ACh-induced contraction of esophageal circular muscle (ESO) depends on Ca influx and activation of protein kinase Ce (PKCe). PKCe, however, is known to be Ca independent. To determine where Ca is needed in this PKCe-mediated contractile pathway, we examined successive steps in Ca-induced contraction of ESO muscle cells permeabilized by saponin. Ca (0.2–1.0 mM) produced a concentration-dependent contraction that was antagonized by antibodies against PKCe (but not by PKCbII or PKCg antibodies), by a calmodulin inhibitor, by MLCK inhibitors, or by GDPbs. Addition of 1 mM Ca 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). Ca-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 Ca may directly activate phospholipases, producing DAG and arachidonic acid (AA), and PKCe, which may indirectly cause phosphorylation of MLC. In addition, direct G protein activation by GTPgS augmented Ca-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 Ca and G protein activation), producing DAG and AA, and activating PKCedependent mechanisms to cause contraction.