Sphingosine 1-phosphate-induced signal transduction in cat esophagus smooth muscle cells

Abstract

We investigated the mechanism of contraction induced by SIP in esophageal smooth muscle cells. Western blot analysis demonstrated that SIPI, S1P(2), S1P(3), and S1P(5) receptors existed in the cat esophagus. Only penetration of EDG-5 (S1P(2)) antibody into permeabilized cells inhibited S1P-induced contraction. Pertussis toxin (PTX) also inhibited contraction, suggesting that it was mediated by S1P(2) receptors coupled to a PTX-sensitive G(i) protein. Specific antibodies to G(i2), G(q) and G(beta) inhibited contraction, implying that the S1P-induced contraction depends on PTX-insensitive G(q) and G(beta) dimers as well as the PTX-sensitive G(i2) Contraction was not affected by the phospholipase A(2) inhibitor DEDA, or the PLD inhibitor p-chloromercuribenzoate, but it was abolished by the PLC inhibitor U73122. Incubation of permeabilized cells with PLC beta 3 antibody also inhibited contraction. Contraction involved the activation of a PKC pathway since it was affected by GF109203X and chelerythrine. Since PKC epsilon antibody inhibited contraction, PKC epsilon, may be required. Preincubation of the muscle cells with the MEK inhibitor PD98059 blocked S1P-induced contraction, but the p38 MAP kinase inhibitor SB202190 did not. In addition, co-treatment of cells with GF 109203X and PD98059 did not have a synergistic effect, suggesting that these two kinases are involved in the same signaling pathway. Our data suggest that S1P-induced contraction in esophageal smooth muscle cells is mediated by S1P2 receptors coupled to PTX-sensitive G(i2) proteins, and PTX-insensitive Gq and G(beta) proteins, and that the resulting activation of the PLC beta 3 and PKC epsilon pathway leads to activation of a p44/p42 MAPK pathway.