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Hypoxia Inhibits Myosin Phosphatase in Pulmonary Arterial Smooth Muscle Cells

Role of Rho-Kinase

Department of Cellular/Integrative Physiology, and Department of Anatomy/Cell Biology, Indiana University School of Medicine, Indianapolis, Indiana

Address correspondence to: Dr. M. Carita Lannér, Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, IN 46202. E-mail: clanner{at}iupui.edu

Rho-kinase was recently found to phosphorylate the myosin-binding subunit (MBS) of myosin phosphatase (MP) and to regulate MP activity. Although myosin light chain (MLC) phosphorylation in pulmonary arterial smooth muscle cells (PASMCs) is thought to be the cellular/molecular basis for hypoxic pulmonary vasoconstriction (HPV), very little is known about the role that Rho-kinase/MP plays in HPV. Rat PASMCs were cultured and made hypoxic (PO₂ = 23 ± 2 mm Hg). Cells exposed to normoxia (PO₂ 148 mm Hg) served as controls. PASMCs exposed to hypoxia showed a significant increase in MLC and MBS phosphorylation, and a significant decrease in MP activity. Rho-kinase inhibitors (HA1077 or Y-27632) blocked hypoxia-induced MP inactivation and inhibited the hypoxia-induced MLC phosphorylation. Hypoxia was also found to induce stress fiber formation and actin polymerization in cultured PASMCs. In summary, these data show that MP inhibition in PASMCs is linked to activation of Rho-kinase, and that hypoxia inhibits the MP signaling pathway via Rho-kinase.

Abbreviations: Dulbecco's modified Eagle's medium, DMEM • dithiothreitol, DTT • fetal bovine serum, FBS • fluorescein isothiocyanate, FITC • hypoxic pulmonary vasoconstriction, HPV • immunoglobulin, IgG • lysophosphatidic acid, LPA • myosin-binding subunit, MBS • myosin light chain, MLC • MLC kinase, MLCK • myosin phosphatase, MP • okadaic acid, OA • pulmonary artery smooth muscle cell, PASMC • phosphate-buffered saline, PBS • Rho-associated serine/threonine kinase, Rho-kinase • sodium dodecyl sulfate, SDS • trichloroacetic acid, TCA