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

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 G i3 or G ␤ 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-␤ 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, Nmyristoylated 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, (2Ј-amino-3Ј-methoxyflavone), 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.

Signaling mechanisms of sphingosine 1-phosphate-induced ERK1/2 activation in cultured feline esophageal smooth muscle cells

“…S1P stimulation activates PTK via G proteins involved in MAPK activation (Tanimoto et al, 2004;Waters et al, 2005). The activation of PTK is blocked by tyrosine kinase inhibitors, such as genistein and tyrphostin, suggesting that tyrosine phosphorylation is involved in MAPK pathways (Laniyonu et al, 1994;Shin et al, 2002). Here, however, genistein partially decreased the activation of ERK1/2 by S1P, suggesting the involvement of PTK, but tyrphostin 51, an EGFR tyrosine kinase inhibitor did not, suggesting there was no cross talk between growth factor receptors and S1P receptors.…”

Regulation of lysophosphatidic acid-induced COX-2 expression by ERK1/2 activation in cultured feline esophageal epithelial Cells

“…LPA also activates PKC in airway epithelial cells and bronchial epithelial cells (He et al, 2008;Kassel et al, 2008). PKC is present in the cell cytoplasm and upon stimulation, it translocates to the membrane fraction or particulate (Shin et al, 2002). Our data indicate that PKC plays a pivotal role in ERK1/2 activation and finally leads to COX-2 expression.…”

“…PKC is present in the cell cytoplasm and, upon stimulation, translocates to the membrane fraction or particulate (Shin et al, 2002). PKC plays a pivotal role in cell signaling by relaying information from lipid mediators to protein substrates (Violin and Newton 2003).…”

Hydrogen Peroxide-Induced Extracellular Signal-Regulated Kinase Activation in Cultured Feline Ileal Smooth Muscle Cells

“…To examine whether MEK is involved in H 2 O 2 -induced ERK activation, a specific MEK inhibitor was used in this study. Growth-arrested cells were treated with 10 M 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059) Shin et al, 2002) for 40 min prior to 1 mM H 2 O 2 stimulation for 30 min. Preincubation of PD98059 decreased the ERK activation by H 2 O 2 in cultured ISMC (Fig.…”