The involvement of Fyn tyrosine kinase and an in vitro assay to visualize the downstream signaling molecules of signal pathway in abnormal contraction of vascular smooth muscle induced by sphingosylphosphorylcholine

Abstract

Whereas the Ca²⁺-dependent contraction of vascular smooth muscle (VSM) regulates physiological vascular tone, the Rho-kinase (ROK)-mediated Ca²⁺-sensitization of VSM contraction contributes to abnormal VSM contraction such as vasospasm. We previously found that sphingosylphosphorylcholine (SPC) is an upstream messenger for the ROK-mediated Ca²⁺-sensitization and those inhibitors of Src family tyrosine kinase (Src-TKs) blocked the SPC-induced contraction and activation of ROK. Among Src-TKs, Fyn and c-Src were expressed in VSM, and SPC translocated Fyn, but not c-Src, from the cytosol to the cell membrane in cultured human coronary artery smooth muscle cells (CASMCs). In the present study, we attempted to determine, which molecule in CASMCs contributes to the Ca²⁺-sensitization mediated by a SPC/ROK pathway. The siRNA-mediated knockdown of Fyn inhibited the SPC-induced contraction of CASMCs. In addition, Fyn constructs [wild type, and constitutively active form (ca-Fyn), and dominant negative form (dn-Fyn)] were transfected to CASMCs with high efficiency (> 50%), although CASMCs were well-differentiated contractile cells. Overexpression of ca-Fyn induced remarkable CASMCs contraction. But, dn-Fyn did not induce any contraction. We have also attempted to design an in vitro assay to visualize the downstream molecules of the signal pathway, which will be discussed. [J Physiol Sci. 2007;57 Suppl:S124]