Effects of Hydrogen Sulfide and Nitric Oxide on the Permeability of Cultured Trabecular Meshwork Cells

PURPOSE: To investigate the effects of hydrogen sulfide (H₂S) on the permeability of a cultured human trabecular meshwork cells (HTMC) monolayer and its interaction with nitric oxide (NO).METHODS: After exposing primary cultured HTMCs to 0, 50, 100, and 500 µM sodium hydrogen sulfide (NaHS) for 6 hours, the permeabilities through the HTMC monolayer were measured using a Transwell assay with carboxyfluorescein. The production of NO and eNOS mRNA expression were assessed using the Griess assay and reverse transcription-polymerase chain reaction, respectively. In addition, 0, 1, and 10 µM NaHS and 10 µM sodium nitroprusside (SN) were co-exposed to evaluate the possible synergistic effect of H₂S and NO.RESULTS: Greater than 100 µM NaHS increased the permeability through the HTMC monolayer in a dose-dependent manner (p < 0.05). These increased permeabilities were not accompanied by NO production or eNOS mRNA expression (p > 0.05). When 0, 1, and 10 µM NaHS and 10 µM SN were exposed together, there was no significant change of permeability, NO production, or eNOS mRNA expression (all, p > 0.05).CONCLUSIONS: NaHS at high concentrations increased the permeability of the HTMC monolayer, which was not affected by NO. NaHS at low concentrations did not show a synergistic effect with NO. Thus, H₂S at high concentrations may increase trabecular outflow, which may not be associated with NO.

Cytoprotective Effect of Rho Kinase Inhibitors against Oxidative Stress inTrabecular Meshwork Cells

Purpose@#We evaluated the protective effect of trabecular outflow drugs, Rho kinase (ROCK) inhibitors against oxidative stressin trabecular meshwork cells. @*Methods@#Primary-cultured human trabecular meshwork cells (HTMCs) were exposed to ROCK inhibitors at 10 and 20 μMY-27632, ripasudil or fasudil for 24 hours, after pretreatment with 200 μM hydrogen peroxide for 30 min. The cell viabilities andmetabolic activities were assessed using the Trypan Blue dye exclusion test and MTT assay, respectively. Reactive oxygen species(ROS) production was measured using the H2DCFDA assay, and the degree of apoptosis was measured with flow cytometryusing annexin-propidium iodide double staining. @*Results@#In HTMCs, Y-27632 suppressed ROS production. Ripasudil and fasudil increased the metabolic activities and decreasedthe degrees of apoptosis. Fasudil showed the most cytoprotective effects among the three ROCK inhibitors tested. @*Conclusions@#Against oxidative stress, ROCK inhibitors decreased apoptosis accompanied by decreased ROS production, especiallyfasudil. ROCK inhibitors may therefore have cytoprotective properties in addition to increasing trabecular outflow.

Effect of Valproic Acid on Nitric Oxide and Nitric Oxide Synthase in Trabecular Meshwork Cell

PURPOSE: To investigate the effects of valproic acid on the production of nitric oxide (NO) and expression of endothelial nitric oxide synthase (eNOS) in cultured human trabecular meshwork cells (HTMC). METHODS: Primarily cultured HTMC were exposed to 0.25, 0.5, and 1.0 mM valproic acid for 6, 12, and 24 hours. Expression of eNOS mRNA was assessed with Reverse transcription-polymerase chain reaction, and production of NO was assessed with Griess assay. Cellular survival was assessed with the 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay. RESULTS: Valproic acid at concentrations of 0.25, 0.5, 1.0 mM did not affect the cellular survival of HTMC significantly after exposure for 24 hours. Valproic acid increased NO production in a dose- and time-dependent manner. Also, valproic acid increased the degree of eNOS mRNA expression in a dose-dependent manner in HTMC. CONCLUSIONS: Valproic acid increases production of NO and expression of eNOS mRNA in HTMC. Thus, valproic acid might increase aqueous outflow through the trabecular meshwork.