Dorette Z. Ellis

Endogenous regulation of human Schlemm's canal cell volume by nitric oxide signaling.

PURPOSE There is a time-course correlation between nitric oxide (NO)-induced decreases in trabecular meshwork (TM) cell volume and NO-induced increases in outflow facility. The Schlemms canal (SC) cells may also provide resistance to aqueous humor outflow; therefore, this study tests the involvement of the nitric oxide synthase (NOS) and NO signaling pathway and the BK(Ca)-channel in mediating SC cell volume decreases. METHODS Cell volume was measured in low-passage human SC cells using calcein AM fluorescent dye; images were captured with a confocal microscope, and data were quantified using NIH ImageJ software. RESULTS Inhibition of endogenous NOS resulted in a 7% increase in SC cell volume. Exposure of SC cells to DETA-NO resulted in a 12% to 16% decrease in cell volume that was abolished by the soluble guanylyl cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (5 μM), the protein kinase G (PKG) inhibitor (RP)-8-Br-PET-cGMP-S (50 μM), and the high-conductance calcium-activated potassium channel (BK(Ca) channel) inhibitor iberiotoxin (50 nM). Hypertonic media significantly decreased SC cell volume by 14%, whereas hypotonic media significantly increased cell volume by 11.2%. CONCLUSIONS These data suggest that endogenous NOS regulates steady state cell volume and the involvement of the NOS/NO/sGC/cGMP/PKG pathway and the BK(Ca)-channel in mediating NO-induced reductions in SC cell volume. These decreases in cell volume correlated with the time-course for NO-induced increases in outflow facility, suggesting that the NO-induced reduction in SC cell volume may also influence outflow facility.

Characterization of soluble guanylate cyclase in NO-induced increases in aqueous humor outflow facility and in the trabecular meshwork.

PURPOSE Nitric oxide (NO) increases the rate at which aqueous humor exits the eye; however, the involvement of soluble guanylate cyclase (sGC) is unknown. This study investigated the role of sGC in mediating the NO-induced increases in outflow facility. METHODS Outflow facility was measured in porcine eyes using the anterior segment organ culture perfusion system. sGC activity was assessed by cGMP production in low-passage porcine and human trabecular meshwork (TM) cells and transformed human TM cells, as measured by enzyme immunoassay. sGCalpha and sGCbeta isoform expression were determined using Western blot analysis. RESULTS Activation of sGC is necessary for the NO-induced increases in outflow facility (0.3215 microL/min per mm Hg [baseline outflow facility]+/-0.0837 [SEM]). NO resulted in increased sGC activity that was abolished by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-1 (ODQ). Western blot analysis of total protein demonstrated an equivalent ratio of sGCalpha and sGCbeta subunit expression. In transformed cell fractions, however, the level of cytoplasmic sGCalpha subunit expression was decreased compared with low-passage human TM cells. CONCLUSIONS Activation of sGC is involved in the NO-induced increases in outflow facility. The expression of alpha and beta sGC subunits in an equivalent ratio would suggest a functional sGC heterodimer because DETA-NO increased cGMP levels in low-passage human and porcine TM cells. However, the inability of DETA-NO to cause increases in cGMP levels in transformed TM cells suggests that though the sGC heterodimer is necessary, it is not sufficient and may require other factors not present in transformed cells.

Guanylate Cyclase Activators, Cell Volume Changes and IOP Reduction

Glaucoma afflicts millions of people worldwide and is a major cause of blindness. The risk to develop glaucoma is enhanced by increases in IOP, which result from deranged flow of aqueous humor. Aqueous humor is a fluid located in the front of the eye that gives the eye its buoyancy and supplies nutrients to other eye tissues. Aqueous humor is secreted by a tissue called ciliary processes and exits the eye via two tissues; the trabecular meshwork (TM) and Schlemm’s canal. Because the spaces through which the fluid flows get smaller as the TM joins the area of the Schlemm’s canal, there is resistance to aqueous humor outflow and this resistance creates IOP. There is a correlation between changes in TM and Schlemm’s canal cell volume and rates of aqueous humor outflow; agents that decrease TM and Schlemm’s canal cell volume, increase the rate of aqueous humor outflow, thus decreasing IOP. IOP is regulated by guanylate cyclase activators as shown in humans, rabbits and monkeys. There are two distinct groups of guanylate cyclases, membrane guanylate cyclase and soluble guanylate cyclase (sGC); activation of both have been shown to decrease IOP. Members of the membrane guanylate cyclase family of receptors bind to peptide ligands, while the sGC responds to gases (such as NO and CO2) and compounds (such as YC1, [3-(5’-hydroxymethyl-2’furyl)-1-benzyl indazole), a benzyl indazole derivative, and BAY-58-2667); activation of either results in formation of cyclic GMP (cGMP) and activation of protein kinase G (PKG) and subsequent phosphorylation of target proteins, including the high conductance calcium activated potassium channel (BKca channel). While activators of both membrane guanylate cyclase and sGC have the ability to lower IOP, the IOP lowering effects of sGC are noteworthy because sGC activators can be topically applied to the eye to achieve an effect. We have demonstrated that activators of sGC increase the rate at which aqueous humor exits the eye in a time course that correlates with the time course for sGC-induced decreases in TM and Schlemm’s canal cell volume. Additionally, sGC-induced decrease in cell volume is accompanied by both K+ and Cl- efflux induced by activation of K+ and Cl- channels, including the BKca channel and/or K+Cl- symport. This suggests that parallel K+Cl- efflux, and resultant H2O efflux result in decreases in cell volume. These observations suggest a functional role for sGC activators, and suggest that the sGC/cGMP/PKG systems are potential therapeutic targets in the treatment of glaucoma.