The signal transduction of endothelin-1-induced circular smooth muscle cell contraction in cat esophagus.
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(beta) protein antibody inhibited the contraction. Neomycin phospholipase C (PLC) inhibitor inhibited the contraction, but 7,7-dimethyleicosadienoic acid (phospholipase A(2) inhibitor) and p-chloromercuribenzoic acid (phospholipase D inhibitor) had no effects. Incubation of permeabilized cells with PLC-beta(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-epsilon 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-alphabetagamma, -alpha, -delta, or -epsilon. myr-PKC-epsilon inhibited the contraction, confirming that PKC-epsilon 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-beta(3) isozyme, resulting in the activation of PKC-epsilon- or protein-tyrosine kinase-dependent pathway, subsequently mediating the activation of p44/p42 MAPK or p38 MAPK pathway.
Department of Pharmacology, College of Pharmacy, Chung Ang University, Seoul 156-756, Republic of Korea.
Chang Yell
CY
Shin
Yul Pyo
YP
Lee
Tai Sang
TS
Lee
Hyun Dong
HD
Je
Dong Seok
DS
Kim
Uy Dong
UD
Sohn
D000818:Animals; D015153:Blotting, Western; D002415:Cats; D002463:Cell Membrane Permeability / Q000187:drug effects; D019332:Endothelin-1 / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D004947:Esophagus / Q000187:drug effects*; D019204:GTP-Binding Proteins / Q000502:physiology; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000502:physiology; D008867:Microtomy; D019950:Mitogen-Activated Protein Kinase 1 / Q000502:physiology; D009119:Muscle Contraction / Q000187:drug effects; D009130:Muscle, Smooth / Q000187:drug effects*; D037342:Pertussis Toxin; D010740:Phospholipases / Q000502:physiology; D011493:Protein Kinase C / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000502:physiology; D011506:Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D019332:Endothelin-1; D007527:Isoenzymes; D011506:Proteins; D010566:Virulence Factors, Bordetella; D037342:Pertussis Toxin; D011505:Protein-Tyrosine Kinases; D011493:Protein Kinase C; D019950:Mitogen-Activated Protein Kinase 1; D010740:Phospholipases; D019204:GTP-Binding Proteins