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P. Deng, Y. Sasaki, D.L. Epstein, P.V. Rao; Regulation of Trabecular Meshwork Myosin Light Chain Phosphorylation by Rho/Rho Kinase Pathway and its Influence on Aqueous Humor Outflow Facility . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4421.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:The goal of this study was to investigate the effects of physiological and pharmacological modulation of Rho/Rho kinase-mediated trabecular meshwork (TM) myosin light chain phosphorylation (MLC) on aqueous humor outflow facility. Methods:The effects of physiological agonists endothelin-1 and thromboxane A2 on activation of Rho/Rho kinase was monitored in human TM cells using a pull down assay. The effects of Rho kinase inhibition on human TM cell morphology, cytoskeletal organization and myosin light chain phosphorylation were tested using a novel and potent inhibitor (H1152) of Rho kinase. To evaluate Rho kinase as a potential therapeutic target for lowering intraocular pressure, aqueous outflow facility was monitored in cadaver eyes perfused with H1152 using a Grant perfusion system. Results:Treatment of serum starved human TM cells with endothelin-1 (0.1 µM) and thromboxane A2 (1.0 µM), known physiological agonists of G-protein coupled receptors, increased activation of Rho GTPase as determined by GTP loading of Rho in pull down assays. On the other hand, treatment of human TM cells with Rho kinase inhibitor-H1152 (0.1 to 2 µM) in the presence of serum caused a dose-dependent change in cell shape, decreased actin stress fiber and focal adhesion staining, and decreased MLC phosphorylation. Perfusion of cadaver porcine eyes with H1152 (10 µM) increased outflow facility by 40% (n=6). Conclusions:This study has identified potential physiological regulators of Rho/Rho kinase signaling in human TM cells and supports the possible role of Rho kinase-mediated MLC phosphorylation in the modulation of aqueous outflow facility. Based on this and previous studies, it is becoming increasingly likely that the Rho/Rho kinase signaling pathway represents an important molecular target for lowering intraocular pressure in glaucoma patients.
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