Journal List > J Korean Ophthalmol Soc > v.57(4) > 1010566

Kim, Kim, and Hwang: Effect of Rho Kinase Inhibitor on the Production of Nitric Oxide in Trabecular Meshwork Cells

Abstract

Purpose

To investigate the effects of Rho kinase (ROCK) inhibitor 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 µM, 10 µM or 100 µM Y-27632 for 3 days and NO production was assessed using Griess assay. After 24 hours, the effect of Y-27632 on the contraction of collagen matrix and the permeability of the HTMC monolayer was determined. The expression of eNOS mRNA was assessed using reverse transcription-polymerase chain reaction (RT-PCR) and cellular survival with the 3-(4, 5 –dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay.

Results

In HTMC, 10 µM and 100 µM Y-27632 significantly increased NO production after 1 day and 3 days (p = 0.020 and 0.001, respectively). At 1 day after exposure, Y-276320 significantly relaxed the collagen matrix and increased the permeability of the HTMC monolayer (all p = 0.001) and the eNOS mRNA expression (p = 0.039).

Conclusions

Increased NO production may play a role in the mechanism of increased trabecular outflow associated with ROCK inhibitor.

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Figure 1.
Effects of Y-27632 on the survival of trabecular meshwork cells. Y-27632 did not affect the survival significantly compared to non-exposed controls (p > 0.05).
jkos-57-650f1.tif
Figure 2.
Effects of Y-26732 on the contraction of collagen gels embedded with trabecular meshwork cells. Both 10 μM (Y10) and 100 μM (Y100) Y-26732 increased the diameter of collagen gels up to 120% compared with non-exposed controls (*p and p represent Y10 and Y100, respectively).
jkos-57-650f2.tif
Figure 3.
Effects of Y-27632 on the permeability of carboxyfluorescin through the trabecular meshwork cell monolayer. Exposure to 10 μM and 100 μM Y-27632 increased the permeability of carboxyfluorescin significantly compared with non-exposed controls. Carboxyfluorescin intensity of outer chamber normalized to the mean value obtained using non-exposed control (permeability 100%). *p = 0.001.
jkos-57-650f3.tif
Figure 4.
Effects of Y-27632 on the production of nitric oxide (NO) in trabecular meshwork cells after exposure for 24 hours. Both 10 μM and 100 μM Y-27632 increased production of NO significantly in a dose-dependent manner. *p = 0.020 and 0.001, respectively.
jkos-57-650f4.tif
Figure 5.
Effects of Y-27632 on the production of nitric oxide (NO) in trabecular meshwork cells after exposure for 3 days. Both 10 μM and 100 μM Y-27632 increased production of NO significantly in a dose-dependent manner. *p = 0.046 and 0.021, respectively.
jkos-57-650f5.tif
Figure 6.
Expression of eNOS mRNA after exposure to Y-27632 (Y) in trabecular meshwork cells (A). Exposure to 100 μM Y-27632 for 1 day (B) increased the degree of eNOS mRNA expression significantly (*p = 0.039). β-actin used as internal standard. eNOS = endothelial nitric oxide synthase.
jkos-57-650f6.tif
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