Abstract
Rho-kinase (ROK)-mediated Ca2+-sensitization plays an essential role in abnormal VSM contraction such as vasospasm. We identified sphingosylphosphorylcholine (SPC) as upstream signaling molecule of ROK-mediated Ca2+-sensitization of VSM. Moreover, we found that SPC required activation of Src family tyrosine kinases (Src-TKs) to induce ROK-mediated Ca2+-sensitization. Among Src-TKs, Fyn and c-Src were found to be expressed in VSM tissue and cultured cells, and SPC induced the translocation of Fyn, but not of c-Src, from cytosol to plasma membrane of VSM cells. In order to obtain the direct evidence for the involvement of Fyn, we made recombinant Fyn proteins either in active or inactive form, using a baculovirus system and applied them to the cytosol of VSM whose membrane was permeabilized with β-escin. The constitutively active Fyn induced Ca2+-sensitization in VSM permeabilized with β-escin, but not with α-toxin, which was inhibited by a ROK inhibitor Y27632. The dominant negative Fyn (dn-Fyn) inhibited the contractions induced by SPC or a GPCR agonist U46619 +GTP. In addition, the SPC-induced contraction was about 50% of the maximum force and it was remarkably diminished to about 18% by applying dn-Fyn (n=4, P<0.01). These findings suggest that Fyn tyrosine kinase plays a pivotal role in the ROK-mediated Ca2+-sensitization of VSM contraction induced by SPC or U46619. [J Physiol Sci. 2007;57 Suppl:S92]
