Abstract
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 Gi3 or Gβ protein antibody inhibited the contraction. Neomycin phospholipase C (PLC) inhibitor inhibited the contraction, but 7,7-dimethyleicosadienoic acid (phospholipase A2 inhibitor) andp-chloromercuribenzoic acid (phospholipase D inhibitor) had no effects. Incubation of permeabilized cells with PLC-β3 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-ε 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-αβγ, -α, -δ, or -ε. myr-PKC-ε inhibited the contraction, confirming that PKC-ε 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 Gi3 protein and PLC-β3 isozyme, resulting in the activation of PKC-ε- or protein-tyrosine kinase-dependent pathway, subsequently mediating the activation of p44/p42 MAPK or p38 MAPK pathway.
Footnotes
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This research was supported by the Korean Science and Engineering Foundation (Grant 2000-1-21400-001-3).
- Abbreviations:
- ET-1, endothelin-1
- PLC, phospholipase C
- PLA2
- phospholipase A2
- PLD
- phospholipase D
- PKC
- protein kinase C
- myr-PKC
- myristoylated PKC
- PIP2
- phosphatidylinositol-4,5-bisphosphate
- IP3
- inositol triphosphate
- H-7
- 1-(5-isoquinolinesulfonyl)-2-methylpiperazine
- DAG
- diacylglycerol
- PTK
- protein-tyrosine kinase
- MAP
- mitogen-activated protein
- MAPK
- MAP kinase
- PD98059
- 2′-amino-3′-methoxyflavone
- DEDA
- 7,7-dimethyleicosadienoic acid
- R59949
- diacylglycerol kinase inhibitor II
- SB202190
- 4-(4-fluorophenyl) 2-(4-hydroxyphenyl)-5-(4-pyridyl) 1H-imidazole
- PBS
- phosphate-buffered saline
- PTX
- pertussis toxin
- PKI
- protein kinase inhibitor
- MEK
- mitogen-activated protein kinase kinase
- Received December 11, 2001.
- Accepted February 25, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
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