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Molecular Pharmacology

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Research ArticleArticle

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

Kazuaki Yoshioka, Naotoshi Sugimoto, Noriko Takuwa and Yoh Takuwa
Molecular Pharmacology March 2007, 71 (3) 912-920; DOI: https://doi.org/10.1124/mol.106.032599
Kazuaki Yoshioka
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Naotoshi Sugimoto
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Noriko Takuwa
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Yoh Takuwa
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Abstract

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.

Footnotes

  • This work was supported by grants from the Ministry of Education, Science, Sports and Culture of Japan, and Novartis Pharma.

  • Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

  • doi:10.1124/mol.106.032599.

  • ABBREVIATIONS: MLCK, myosin light-chain kinase; MLCP, myosin light-chain phosphatase; MLC, 20-kDa myosin light chain; PI3K-C2α, phosphoinositide 3-kinase class II α isoform; MYPT1, myosin targeting protein 1; CPI-17, 17-kDa protein kinase C-potentiated inhibitory protein of PP1; GFP, enhanced green fluorescent protein; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; BAPTA-AM, 1,2-bis(o-aminophenoxy)ethane-N,N,N,N′-tetraacetic acid tetra (acetoxymethyl) ester; VSMC, vascular smooth muscle; PI3K, phosphoinositide 3-kinase; siRNA, short interfering RNA; C2α-siRNA, phosphoinositide 3-kinase-C2α-specific short interfering RNA; EGFP, enhanced green fluorescent protein; sc-siRNA, scrambled short interfering RNA; Y27632, N-(4-pyridyl)-4-(1-aminoethyl)cyclohexanecarboxamide dihydrochloride.

  • ↵ Embedded Image The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.

    • Received November 13, 2006.
    • Accepted December 19, 2006.
  • The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 71 (3)
Molecular Pharmacology
Vol. 71, Issue 3
1 Mar 2007
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Research ArticleArticle

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

Kazuaki Yoshioka, Naotoshi Sugimoto, Noriko Takuwa and Yoh Takuwa
Molecular Pharmacology March 1, 2007, 71 (3) 912-920; DOI: https://doi.org/10.1124/mol.106.032599

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Research ArticleArticle

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

Kazuaki Yoshioka, Naotoshi Sugimoto, Noriko Takuwa and Yoh Takuwa
Molecular Pharmacology March 1, 2007, 71 (3) 912-920; DOI: https://doi.org/10.1124/mol.106.032599
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