Received for publication November 14, 2006.
Revised December 18, 2006.
Accepted for publication December 19, 2006.

Essential role for class II phosphoinositide 3-kinase alpha-isoform in Ca²⁺-induced, Rho- and Rho kinase- dependent regulation of myosin phosphatase and contraction in isolated vascular smooth muscle cells

Abstract

The laser confocal fluorescent microscope-based observation of contractile responses in green fluorescent protein-expressing differentiated vascular smooth muscle cells (VSMCs), 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 siRNA (C2-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 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 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.

Key words: G12,13;other G's, Calcium (G Protein Coupled Signals), Protein Kinases (other), Protein ser/thr Phosphatases, G protein regulation