Vol. 302, Issue 3, 924-934, September 2002

The Signal Transduction of Endothelin-1-Induced Circular Smooth Muscle Cell Contraction in Cat Esophagus

Chang Yell Shin, Yul Pyo Lee, Tai Sang Lee, Hyun Dong Je, Dong Seok Kim and Uy Dong Sohn

Department of Pharmacology, College of Pharmacy, Chung Ang University, Seoul, Republic of Korea

It has been known that endothelin-1 (ET-1) exerts important actions in gastrointestinal smooth muscle motility, but its precise mechanism remains unsolved. We investigated the intracellular mechanism of ET-1-induced circular smooth muscle cell contraction in cat esophagus. ET-1 produced contraction of smooth muscle cells isolated by enzymatic digestion. The contraction in response to ET-1 was concentration-dependent. Pertussis toxin (PTX) blocked contraction induced by ET-1 in intact cells. To identify the specific G protein involved in the contraction, muscle cells were permeabilized with saponin. The G_i3 or G protein antibody inhibited the contraction. Neomycin phospholipase C (PLC) inhibitor inhibited the contraction, but 7,7-dimethyleicosadienoic acid (phospholipase A₂ inhibitor) and p-chloromercuribenzoic acid (phospholipase D inhibitor) had no effects. Incubation of permeabilized cells with PLC-₃ isozyme antibody inhibited the contraction. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine, chelerythrine [protein kinase C (PKC) inhibitor], or genistein (protein tyrosine kinase inhibitor) inhibited the contraction, but not by diacylglycerol (DAG) kinase inhibitor, R59949. To test whether the contraction may be PKC isozyme-specific, we examined the effect of PKC isozymes antibodies on the contraction. PKC- antibody inhibited the contraction. To characterize further the specific PKC isozymes that mediate the contraction, we used, as an inhibitor, N-myristoylated peptides (myr-PKC) derived from the pseudosubstrate sequences of PKC-, -, -, or -. myr-PKC- inhibited the contraction, confirming that PKC- isozyme is involved in the contraction. To examine whether mitogen-activated protein kinases (MAPKs) mediate the contraction, specific MAPK inhibitors [MAPK kinase inhibitor, PD98059, (2'-amino-3'-methoxy-flavone), and p38 MAPK inhibitor, SB202190 (4-4-fluorophenyl) 2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole)] were used. PD98059 or SB202190 blocked the contraction. ET-1 increased the intensity of the detection bands identified by immunological methods as MAPK monoclonal p44/p42 peptides. PD98059 decreased the intensity of the detection bands compared with ET-1. In conclusion, ET-1-induced contraction in cat esophageal circular muscle cells depends on PTX-sensitive G_i3 protein and PLC-₃ isozyme, resulting in the activation of PKC-- or protein-tyrosine kinase-dependent pathway, subsequently mediating the activation of p44/p42 MAPK or p38 MAPK pathway.