第71回 日本循環器学会 2007年6月15日〜17日(神戸)


Class II
α phosphoinositide 3-kinase is an essential regulator of Ca2+-dependent Rho GTPase activation, myosin phosphatase and contraction in vascular smooth muscle cells

Kazuaki Yoshioka, Noriko Takuwa, Naotoshi Sugimoto, and Yoh Takuwa
Department of Physiology, Kanazawa University Graduate School of Medicine, 13-1 Takara-machi, Kanazawa, Ishikawa 920-8640, Japan.

ABSTRACT
We previously demonstrated that phosphoinositide 3-kinase (PI3K) inhibitors suppressed Rho activation and myosin phosphatase (MLCP) inhibition in vascular smooth muscle (VSM) strips, resulting in inhibition of 20-kDa myosin light chain (MLC20) phosphorylation and contraction. Based upon the analyses of PI3K isoform expression in VSM and the compatibility of PI3K inhibitor, we hypothesized that class II PI3K, PI3K-C2, might be a responsible isoform that is involved in the regulation of the Rho signaling pathway and contraction in VSM. To test this hypothesis, we adopted a differentiated VSM cell culture, which maintains a contractile phenotype, and siRNA-mediated knockdown of PI3K-C2 expression. The siRNA-mediated knockdown of PI3K-C2α, but not class I PI3K p110α, suppressed ionomycin-induced contraction without altering Ca2+-mobilization. Ionomycin-induced contraction was inhibited only by a relatively high concentration of LY294002. Consistent with our previous observations, ionomycin-induced contraction was dependent on Rho and Rho-kinase in a Ca2+-dependent manner. Ionomycin induced phosphorylation of the MLCP-regulatory subunit MYPT1 at Thr850/Thr695 and the MLC20 in a Rho kinase-dependent manner. Knockdown of PI3K-C2α suppressed phosphorylation of both MYPT1 and MLC. The receptor agonist noradrenaline, which induced a rapid increase in the [Ca2+]i and Ca2+-dependent contraction, stimulated phosphorylation of MYPT1 and MLC in a Ca2+-, PI3K-C2α-, and Rho kinase-dependent manner. These observations indicate that PI3K-C2α mediates Ca2+-induced, Rho- and Rho kinase-dependent negative regulation of MLCP, and consequently MLC phosphorylation and contraction in VSMCs.