Distinctive G protein-dependent signaling in smooth muscle by sphingosine 1-phosphate receptors S1P1and S1P2

Abstract

We examined expression of sphingosine 1-phosphate (S1P) receptors and sphingosine kinase (SPK) in gastric smooth muscle cells and characterized signaling pathways mediating S1P-induced 20-kDa myosin light chain (MLC20) phosphorylation and contraction. RT-PCR demonstrated expression of SPK1 and SPK2 and S1P1 and S1P2 receptors. S1P activated Gq, G13, and all Gi isoforms and stimulated PLC-β1, PLC-β3, and Rho kinase activities. PLC-β activity was partially inhibited by pertussis toxin (PTX), Gβ or Gαq antibody, PLC-β1 or PLC-β3 antibody, and by expression of Gαq or Gαi minigene, and was abolished by a combination of antibodies or minigenes. S1P-stimulated Rho kinase activity was partially inhibited by expression of Gα13 or Gαq minigene and abolished by expression of both. S1P stimulated Ca2+ release that was inhibited by U-73122 and heparin and induced concentration-dependent contraction of smooth muscle cells (EC50 1 nM). Initial contraction and MLC20 phosphorylation were abolished by U-73122 and MLC kinase (MLCK) inhibitor ML-9. Initial contraction was also partially inhibited by PTX and Gαq or Gβ antibody and abolished by a combination of both antibodies. In contrast, sustained contraction and MLC20 phosphorylation were partially inhibited by a PKC or Rho kinase inhibitor (bisindolylmaleimide and Y-27632) and abolished by a combination of both inhibitors but not affected by U-73122 or ML-9. These results indicate that S1P induces 1 ) initial contraction mediated by S1P2 and S1P1 involving concurrent activation of PLC-β1 and PLC-β3 via Gαq and Gβγi, respectively, resulting in inositol 1,4,5-trisphosphate-dependent Ca2+ release and MLCK-mediated MLC20 phosphorylation, and 2 ) sustained contraction exclusively mediated by S1P2 involving activation of RhoA via Gαq and Gα13, resulting in Rho kinase- and PKC-dependent MLC20 phosphorylation.