We investigated the mechanism of contraction induced by S1P in esophageal smooth muscle cells. Western blot analysis demonstrated that S1P1, S1P2, S1P3, and S1P5 receptors existed in the cat esophagus. Only penetration of EDG-5 (S1P2) antibody into permeabilized cells inhibited S1P-induced contraction. Pertussis toxin (PTX) also inhibited contraction, suggesting that it was mediated by S1P2 receptors coupled to a PTX-sensitive Gi protein. Specific antibodies to Gi2, Gq and Gb inhibited contraction, implying that the S1P-induced contraction depends on PTX-insensitive Gq and Gb dimers as well as the PTX-sensitive Gi2. Contraction was not affected by the phospholipase A2 inhibitor DEDA, or the PLD inhibitor ρ-chloromer-curibenzoate, but it was abolished by the PLC inhibitor U73122. Incubation of permeabilized cells with PLCb3 antibody also inhibited contraction. Contraction involved the activation of a PKC pathway since it was affected by GF109203X and chelerythrine. Since PKCe antibody inhibited contraction, PKCe 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 Gi2 proteins, and PTX-insensitive Gq and Gb proteins, and that the resulting activation of the PLCb3 and PKCe pathway leads to activation of a p44/p42 MAPK pathway.

Keywords: esophageal smooth muscle cell, ERK, S1P, contraction