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P. Vasantha Rao, Pei-Feng Deng, Janardan Kumar, David L. Epstein; Modulation of Aqueous Humor Outflow Facility by the Rho Kinase–Specific Inhibitor Y-27632. Invest. Ophthalmol. Vis. Sci. 2001;42(5):1029-1037.
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purpose. The goal of this study was to investigate the role of Rho
kinase in the modulation of aqueous humor outflow facility. Rho
kinase, a critical downstream effector of Rho GTPase is recognized to
control the formation of actin stress fibers, focal adhesions, and
methods. Expression of Rho GTPase, Rho kinase, and other downstream targets of
Rho GTPase were determined in human trabecular meshwork (HTM) and
Schlemm’s canal (SC) primary cell cultures by Western blot analysis.
The Rho kinase–specific inhibitor (Y-27632)-induced changes in actin
stress fibers, focal adhesions, and protein phosphotyrosine status were
evaluated by staining with rhodamine-phalloidin, anti-paxillin, and
anti-phosphotyrosine antibodies, respectively. Myosin light-chain
phosphorylation was determined by Western blot analysis.
Y-27632-induced changes in SC cell monolayer permeability were
quantitated using a colorimetric assay to evaluate horseradish
peroxidase diffusion through SC cell monolayers grown in transwell
chambers. Aqueous humor outflow facility was measured using enucleated
porcine eyes and a constant-pressure perfusion system.
results. Treatment of HTM and SC cells with Y-27632 (10 μM) led to significant
but reversible changes in cell shape and decreases in actin stress
fibers, focal adhesions, and protein phosphotyrosine staining. SC cell
monolayer permeability increased (by 80%) in response to Y-27632 (10μ
M) treatment, whereas myosin light-chain phosphorylation was
decreased in both HTM and SC cells. Aqueous humor outflow facility
increased (40%–80%) in enucleated porcine eyes perfused with Y-27632
(10–100 μM), and this effect was associated with widening of the
extracellular spaces, particularly the optically empty area of the
juxtacanalicular tissue (JCT). The integrity of inner wall of
aqueous plexi, however, was observed to be intact.
conclusions. Based on the Rho kinase inhibitor-induced changes in myosin light-chain
phosphorylation and actomyosin organization, it is reasonable to
conclude that cellular relaxation and loss of cell–substratum
adhesions in HTM and SC cells could result in either increased
paracellular fluid flow across Schlemm’s canal or altered flow pathway
through the JCT, thereby lowering resistance to outflow. This study
also suggests Rho kinase as a potential therapeutic target for the
development of drugs to modulate intraocular pressure in glaucoma
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