Abstract
A Rho-kinase (ROK)-mediated Ca2+-sensitization plays an important role in the pathogenesis of abnormal vascular smooth muscle (VSM) contraction such as vasospasm. Previously we reported that sphingosylphosphorylcholine (SPC) induced ROK-mediated Ca2+-sensitization in VSM contraction via the activation of Src family tyrosine kinase (Src-TKs) and that SPC levels were elevated in vasospasm patients. However, the signal transduction mechanism by which Src-TKs can activate ROK is unknown. To identify the downstream signaling molecules of Src-TKs, we screened the molecules in which SPC and Src-TKs inhibitors can modulate the tyrosine phosphorylation in porcine coronary and rabbit mesenteric arteries, using anti-phosphotyrosine antibody (RC20). Arterial strips were stimulated with SPC to induce contraction and rapidly frozen-fractured at various time points and subjected to the immunoblot analysis against RC20. SPC modulated tyrosine phosphorylation in several molecules including p60, and PP2 and eicosapentaenoic acid (EPA), Src-TKs inhibitors, diminished the effect of SPC on tyrosine phosphorylation and the accompanying contraction. To identify those molecules, RC20-immunoprecipitated samples were separated by SDS-PAGE, transferred to PVDF membrane. The protein band correspond to p60 was excised, digested by Achromobacter Protease I and subjected to MALDI TOF-MS analysis for peptide mass fingerprinting. The result showed two tyrosine-phosphorylated proteins as the candidates for p60. [Jpn J Physiol 54 Suppl:S101 (2004)]
