抄録
Smooth muscle is generally resistant to the transduction of exogenous genes, which hampers gene manipulation-utilizing analysis of smooth muscle functions. Enzymatically dispersed rat aortic vascular smooth muscle (VSM) cells which are cultured under a serum-free condition on laminin-coated dishes can maintain differentiated phenotypes including high levels of contractile protein and receptor expression. The green fluorescent protein expression in these cells combined with observation using a fluorescent microscope allowed quantitative analysis of the contractile responses. We found that siRNA effectively knocks down gene expression in this differentiated cell system. We previously demonstrated that excitatory receptor agonists such as noradrenaline (NA) induce small GTPase Rho activation in a Ca2+-dependent manner, resulting in inhibition of myosin phosphatase (MP) through the mechanisms involving Rho kinase-mediated phosphorylation of its regulatory subunit MYPT1. In the search of signaling molecules involved in the Ca2+-dependent Rho activation, we found that silencing a PI3-kinase subtype inhibited NA-induced phosphorylation of MYPT1 and consequent 20-kDa myosin light chain (MLC) phosphorylation and contraction. These findings implicate the novel lipid kinase as an essential regulator of Ca2+-dependent, Rho/Rho kinase mediated VSM contraction. By taking advantage of the differentiated VSM cells and gene manipulation techniques, we can dissect signaling pathways for contraction. [Jpn J Physiol 55 Suppl:S15 (2005)]
