RT Journal Article SR Electronic T1 Essential Role for Class II Phosphoinositide 3-kinase α-Isoform in Ca2+-Induced, Rho- and Rho Kinase-Dependent Regulation of Myosin Phosphatase and Contraction in Isolated Vascular Smooth Muscle Cells JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP 912 OP 920 DO 10.1124/mol.106.032599 VO 71 IS 3 A1 Kazuaki Yoshioka A1 Naotoshi Sugimoto A1 Noriko Takuwa A1 Yoh Takuwa YR 2007 UL http://molpharm.aspetjournals.org/content/71/3/912.abstract AB The laser confocal fluorescent microscope-based observation of contractile responses in green fluorescent protein-expressing differentiated vascular smooth muscle cells, combined with the RNA interference-mediated gene-silencing technique, allowed us to determine the role of phosphoinositide 3-kinase (PI3K) class II α-isoform (PI3K-C2α) as a novel, Ca2+-dependent regulator of myosin light-chain phosphatase (MLCP) and contraction. The Ca2+-ionophore ionomycin induced a robust contractile response with an increase in the intracellular free Ca2+ concentration ([Ca2+]i). The PI3K-C2α-specific short interfering RNA (siRNA) induced a selective and marked reduction in PI3K-C2α protein expression. The siRNA-mediated knockdown of PI3K-C2α, but not class I PI3K p110α, suppressed ionomycin-induced contraction without altering Ca2+-mobilization. PI3K-C2α is uniquely less sensitive to the PI3K inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) than the other PI3K members, including p110α. Ionomycin-induced contraction was inhibited only by a relatively high concentration of LY294002. Consistent with our previous observations showing that ionomycin and membrane depolarization induced Rho activation in vascular smooth muscle tissues in a Ca2+-dependent manner, ionomycin-induced contraction was dependent on Rho and Rho-kinase. Ionomycin induced phosphorylation of the MLCP-regulatory subunit myosin targeting protein 1(MYPT1) at Thr850 and the 20-kDa myosin light chain (MLC) 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, which was also dependent on Ca2+, PI3K-C2α, and Rho-kinase. These observations indicate that PI3K-C2α is necessary for Ca2+-induced Rho- and Rho kinase-dependent negative regulation of MLCP and consequently MLC phosphorylation and contraction. The American Society for Pharmacology and Experimental Therapeutics