Distinctive G protein-dependent signaling in smooth muscle by sphingosine 1-phosphate receptors S1P1 and S1P2.
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 (MLC(20)) phosphorylation and contraction. RT-PCR demonstrated expression of SPK1 and SPK2 and S1P(1) and S1P(2) receptors. S1P activated G(q), G(13), and all G(i) isoforms and stimulated PLC-beta1, PLC-beta3, and Rho kinase activities. PLC-beta activity was partially inhibited by pertussis toxin (PTX), Gbeta or Galpha(q) antibody, PLC-beta1 or PLC-beta3 antibody, and by expression of Galpha(q) or Galpha(i) minigene, and was abolished by a combination of antibodies or minigenes. S1P-stimulated Rho kinase activity was partially inhibited by expression of Galpha(13) or Galpha(q) minigene and abolished by expression of both. S1P stimulated Ca(2+) release that was inhibited by U-73122 and heparin and induced concentration-dependent contraction of smooth muscle cells (EC(50) 1 nM). Initial contraction and MLC(20) phosphorylation were abolished by U-73122 and MLC kinase (MLCK) inhibitor ML-9. Initial contraction was also partially inhibited by PTX and Galpha(q) or Gbeta antibody and abolished by a combination of both antibodies. In contrast, sustained contraction and MLC(20) 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 S1P(2) and S1P(1) involving concurrent activation of PLC-beta1 and PLC-beta3 via Galpha(q) and Gbetagamma(i), respectively, resulting in inositol 1,4,5-trisphosphate-dependent Ca(2+) release and MLCK-mediated MLC(20) phosphorylation, and 2) sustained contraction exclusively mediated by S1P(2) involving activation of RhoA via Galpha(q) and Galpha(13), resulting in Rho kinase- and PKC-dependent MLC(20) phosphorylation.
Zhou
Huiping
H
Depts. of Physiology and Medicine, Medical College of Virginia Campus, Virginia Commonwealth Univ., Richmond, VA 23298, USA.
Murthy
Karnam S
KS
D000067956:Adenylyl Cyclase Inhibitors; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000502:physiology*; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000378:metabolism; D008246:Lysophospholipids / Q000494:pharmacology; D008970:Molecular Weight; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology; D009130:Muscle, Smooth / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D018994:Myosin Light Chains / Q000737:chemistry / Q000378:metabolism; D054799:Phospholipase C beta; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011817:Rabbits; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D049312:Receptors, Lysophospholipid; D015398:Signal Transduction / Q000502:physiology*; D013110:Sphingosine / Q000031:analogs & derivatives / Q000494:pharmacology; D013270:Stomach; D010738:Type C Phospholipases / Q000378:metabolism; D054460:rho-Associated Kinases; D000099157:Sphingosine Kinase
D000067956:Adenylyl Cyclase Inhibitors; D002118:Calcium; D019204:GTP-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D008246:Lysophospholipids; D018994:Myosin Light Chains; D054799:Phospholipase C beta; D017853:Phosphotransferases (Alcohol Group Acceptor); D017346:Protein Serine-Threonine Kinases; D043562:Receptors, G-Protein-Coupled; D049312:Receptors, Lysophospholipid; D013110:Sphingosine; D010738:Type C Phospholipases; D054460:rho-Associated Kinases; D000099157:Sphingosine Kinase; C060506:sphingosine 1-phosphate