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Essential Role for Class II Phosphoinositide 3-kinase -Isoform in Ca²⁺-Induced, Rho- and Rho Kinase-Dependent Regulation of Myosin Phosphatase and Contraction in Isolated Vascular Smooth Muscle Cells

Department of Physiology (Y.K., N.S., N.T., Y.T.), Kanazawa University Graduate School of Medicine, Kanazawa, Japan; and Department of Health Sciences and Medicine, Ishikawa Prefectural Nursing University, Kanazawa, Japan (N.T.)

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 -isoform (PI3K-C2) 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-C2-specific short interfering RNA (siRNA) induced a selective and marked reduction in PI3K-C2 protein expression. The siRNA-mediated knockdown of PI3K-C2, but not class I PI3K p110, suppressed ionomycin-induced contraction without altering Ca²⁺-mobilization. PI3K-C2 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 p110. 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-C2 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-C2, and Rho-kinase. These observations indicate that PI3K-C2 is necessary for Ca²⁺-induced Rho- and Rho kinase-dependent negative regulation of MLCP and consequently MLC phosphorylation and contraction.

Address correspondence to: Dr. Yoh Takuwa, Department of Physiology, Kanazawa University Graduate School of Medicine, 13-1 Takara-machi, Kanazawa, Ishikawa 920-8640, Japan. E-mail: ytakuwa{at}med.kanazawa-u.ac.jp