We hypothesize that Rho/Rho kinase regulated, actomyosin-mediated contraction and relaxation events in trabecular meshwork (TM), juxtacanalicular tissue (JCT) and Schlemm's canal (SC) cells modulate aqueous humor outflow resistance by changing the geometry of the flow pathway for aqueous humor, and/or by affecting paracellular fluid flow through the inner wall of Schlemm's canal. Strong support for this hypothesis derives from our recent studies, which demonstrate that augmentation and inhibition of Rho/Rho kinase function have contrasting effects not only upon cytoskeletal changes and myosin light chain (MLC) phosphorylation in human trabecular meshwork (HTM) and Schlemm's canal (HSC) cells, but on SC cell monolayer permeability characteristics and outflow facility as well. This convincing correlation between biochemical, morphological and functional attributes argues that Rho kinase dependent signaling pathway(s) represent a potential target for therapeutic modulation of outflow facility in glaucoma. To define the mechanistic basis(es) by which Rho/Rho kinase modulates aqueous outflow, we propose to study in detail, 1. The regulation of MLC phosphorylation in HTM, HSC and human ciliary muscle cells, and cytoskeletal organization (actin stress fibers, focal adhesions and adherens junctions) in HTM and HSC cells by using physiological (endothelin-1 and thromboxane A2, mimetic-U46619) and pharmacological (Y-27632 and HA-1077) modulators of Rho kinase activity and overexpression of a dominant negative mutant of Rho kinase (DNRK), 2. Modulation of paracellular permeability in HSC cell monolayers and of outflow facility in organ cultured post-mortem human eyes, using physiological agonists and pharmacological inhibitors of Rho kinase activity and overexpression of a DNRK. Changes in morphological integrity of outflow tissues will also be evaluated in perfused porcine and human eyes, 3. The ability of physiological factors and elevated ocular pressure to regulate Rho kinase expression in HTM and SC cells, and in perfused eyes, respectively, will be studied at the level of RNA and protein. The relevance of such changes for contractile function in TM will be also investigated. The completion of this work should provide significant insight into the role of the Rho/Rho kinase signaling pathway(s) in particular, and myosin phosphorylation and cellular contraction and relaxation in general, in regulating trabecular outflow facility. Identification of specific signaling mechanisms regulating function of outflow pathway tissues could enable the design of target-specific approaches to the treatment of glaucoma.
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