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.
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 alpha-isoform (PI3K-C2alpha) 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-C2alpha-specific short interfering RNA (siRNA) induced a selective and marked reduction in PI3K-C2alpha protein expression. The siRNA-mediated knockdown of PI3K-C2alpha, but not class I PI3K p110alpha, suppressed ionomycin-induced contraction without altering Ca2+-mobilization. PI3K-C2alpha 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 p110alpha. 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-C2alpha 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-C2alpha, and Rho-kinase. These observations indicate that PI3K-C2alpha is necessary for Ca2+-induced Rho- and Rho kinase-dependent negative regulation of MLCP and consequently MLC phosphorylation and contraction.
Yoshioka
Kazuaki
K
Department of Physiology, Kanazawa University Graduate School of Medicine, 13-1 Takara-machi, Kanazawa, Ishikawa 920-8640, Japan.
Sugimoto
Naotoshi
N
Takuwa
Noriko
N
Takuwa
Yoh
Y
D000818:Animals; D002118:Calcium / Q000502:physiology*; D002352:Carrier Proteins / Q000378:metabolism; D002478:Cells, Cultured; D058541:Class II Phosphatidylinositol 3-Kinases; D047908:Intracellular Signaling Peptides and Proteins / Q000502:physiology*; D015759:Ionomycin / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000201:enzymology* / Q000502:physiology; D032389:Myocytes, Smooth Muscle / Q000201:enzymology* / Q000502:physiology; D018994:Myosin Light Chains / Q000378:metabolism; D043263:Myosin-Light-Chain Phosphatase / Q000378:metabolism*; D009638:Norepinephrine / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D054645:Protein Phosphatase 1; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D014661:Vasoconstriction; D020741:rho GTP-Binding Proteins / Q000502:physiology*; D054460:rho-Associated Kinases
D002118:Calcium; D002352:Carrier Proteins; D058541:Class II Phosphatidylinositol 3-Kinases; D047908:Intracellular Signaling Peptides and Proteins; D015759:Ionomycin; D018994:Myosin Light Chains; D043263:Myosin-Light-Chain Phosphatase; D009638:Norepinephrine; D019869:Phosphatidylinositol 3-Kinases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D017346:Protein Serine-Threonine Kinases; D034741:RNA, Small Interfering; D020741:rho GTP-Binding Proteins; D054460:rho-Associated Kinases; C499850:Ppp1r12a protein, rat