sphingosine-1-phosphate and fasudil

Sphingosine-1-phosphate (S1P) has been implicated recently in the physiology and pathology of the cardiovascular system including regulation of vascular tone. Pilot experiments showed that the vasoconstrictor effect of S1P was enhanced markedly in the presence of phenylephrine (PE). Based on this observation, we hypothesized that S1P might modulate α

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Amides; Animals; Drug Synergism; Lysophospholipids; Mice, Inbred C57BL; Mice, Knockout; Phenylephrine; Pyridines; Receptors, Adrenergic, alpha-1; rho-Associated Kinases; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Vasoconstriction; Vasoconstrictor Agents; Vasodilator Agents

We previously reported that sphingosine 1-phosphate induces heat shock protein 27 (HSP 27) via activation of phosphatidylinositol 3-kinase (PI3K)/Akt and p38 mitogen-activated protein (MAP) kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether Rho-kinase is implicated in sphingosine 1-phosphate-stimulated induction of HSP27 in MC3T3-E1 cells. Sphingosine 1-phosphate time-dependently induced the phosphorylation of myosin phosphatase targeting subunit (MYPT-1), a Rho-kinase substrate. Y27632, a specific Rho-kinase inhibitor, significantly reduced sphingosine 1-phosphate-stimulated HSP27 induction, as well as MYPT-1 phosphorylation. Fasudil, another inhibitor of Rho-kinase, also suppressed sphingosine 1-phosphate-stimulated HSP27 induction. Y27632, as well as fasudil, attenuated sphingosine 1-phosphate-induced phosphorylation of p38 MAP kinase. However, Akt phosphorylation induced by sphingosine 1-phosphate was not affected by either Rho-kinase inhibitor. These results strongly suggest that Rho-kinase regulates sphingosine 1-phosphate-stimulated induction of HSP27 at a point upstream of p38 MAP kinase in osteoblasts.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Amides; Animals; Cell Line; HSP27 Heat-Shock Proteins; Lysophospholipids; Mice; Myosin-Light-Chain Kinase; Myosin-Light-Chain Phosphatase; Osteoblasts; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Pyridines; rho-Associated Kinases; Sphingosine

The lysophospholipid mediator sphingosine-1-phosphate (S1P) activates G protein-coupled receptors (GPCRs) to induce potent inhibition of platelet-derived growth factor (PDGF)-induced Rac activation and, thereby, chemotaxis in rat vascular smooth muscle cells (VSMCs). We explored the heterotrimeric G protein and the downstream mechanism that mediated S1P inhibition of Rac and cell migration in VSMCs.. S1P inhibition of PDGF-induced cell migration and Rac activation in VSMCs was abolished by the selective S1P(2) receptor antagonist JTE-013. The C-terminal peptides of Galpha subunits (Galpha-CTs) act as specific inhibitors of respective G protein-GPCR coupling. Adenovirus-mediated expression of Galpha(12)-CT, Galpha(13)-CT, and Galpha(q)-CT, but not that of Galpha(s)-CT or LacZ or pertussis toxin treatment, abrogated S1P inhibition of PDGF-induced Rac activation and migration, indicating that both G(12/13) and G(q) classes are necessary for the S1P inhibition. The expression of Galpha(q)-CT as well as Galpha(12)-CT and Galpha(13)-CT also abolished S1P-induced Rho stimulation. C3 toxin, but not a Rho kinase inhibitor or a dominant negative form of Rho kinase, abolished S1P inhibition of PDGF-induced Rac activation and cell migration. The angiotensin II receptor AT(1), which robustly couples to G(q), did not mediate either Rho activation or inhibition of PDGF-induced Rac activation or migration, suggesting that activation of G(q) alone was not sufficient for Rho activation and resultant Rac inhibition. However, the AT(1) receptor fused to Galpha(12) was able to induce not only Rho stimulation but also inhibition of PDGF-induced Rac activation and migration. Phospholipase C inhibition did not affect S1P-induced Rho activation, and protein kinase C activation by a phorbol ester did not mimic S1P action, suggesting that S1P inhibition of migration or Rac was not dependent on the phospholipase C pathway.. These observations together suggest that S1P(2) mediates inhibition of Rac and migration through the coordinated action of G(12/13) and G(q) for Rho activation in VSMCs.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; ADP Ribose Transferases; Animals; Botulinum Toxins; Calcium; Cell Movement; Cells, Cultured; Dose-Response Relationship, Drug; GTP-Binding Protein alpha Subunits, G12-G13; GTP-Binding Protein alpha Subunits, Gq-G11; Lysophospholipids; Male; Muscle, Smooth, Vascular; Protein Kinase C; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-sis; Pyrazoles; Pyridines; rac GTP-Binding Proteins; Rats; Rats, Wistar; Receptor, Angiotensin, Type 1; Receptors, Lysosphingolipid; Recombinant Fusion Proteins; rho GTP-Binding Proteins; rho-Associated Kinases; Signal Transduction; Sphingosine; Transfection