Effect of Rho Kinase Inhibitor on the Production of Nitric Oxide in Trabecular Meshwork Cells

Jae Woo Kim; Keun Hae Kim; Seok Jin Hwang

doi:10.3341/jkos.2016.57.4.650

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

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

Original Article

J Korean Ophthalmol Soc 2016;57(4):650-656.

Published online: 3 September 2016

DOI: https://doi.org/10.3341/jkos.2016.57.4.650

Effect of Rho Kinase Inhibitor on the Production of Nitric Oxide in Trabecular Meshwork Cells

Jae Woo Kim, MD, PhD, Keun Hae Kim, MD, Seok Jin Hwang, MD

Department of Ophthalmology, Catholic University of Daegu College of Medicine, Daegu, Korea

Address reprint requests to Jae Woo Kim, MD, PhD Department of Ophthalmology, Daegu Catholic University Medical Center, #33 Duryugongwon-ro 17-gil, Nam-gu, Daegu 42472, Korea Tel: 82-53-650-4728, Fax: 82-53-627-0133 E-mail: jwkim@cu.ac.kr

Received 17 December 2015 Revised 15 January 2016 Accepted 25 February 2016

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.

Keywords: Endothelial nitric oxide synthase (eNOS), Nitric oxide, Rho kinase (ROCK) inhibitor, Trabecular meshwork, Y-276320

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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).

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).

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.

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.

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.

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.

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