J Korean Ophthalmol Soc.  2016 Apr;57(4):650-656. 10.3341/jkos.2016.57.4.650.

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

• Kim JW
• Kim KH
• Hwang SJ

• KMID: 2212801
• DOI: http://doi.org/10.3341/jkos.2016.57.4.650

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.

Keyword

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

MeSH Terms

Collagen
Humans
Nitric Oxide Synthase Type III
Nitric Oxide*
Permeability
rho-Associated Kinases*
RNA, Messenger
Trabecular Meshwork*
Collagen
Nitric Oxide
Nitric Oxide Synthase Type III
RNA, Messenger
rho-Associated Kinases

Figure

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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).

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  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.

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  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.

Reference

References

1. Alvarado J, Murphy C, Juster R. Trabecular meshwork cellularity in primary open-angle glaucoma and nonglaucomatous normals. Ophthalmology. 1984; 91:564–79.

2. Rohen JW, Lütjen-drecoll E, Flügel C, et al. Ultrastructure of the trabecular meshwork in untreated cases of primary open-angle glaucoma (POAG). Exp Eye Res. 1993; 56:683–92.

3. Schmidl D, Schmetterer D, Garhöfer G, Popa-Cherecheanu A. Pharmacotherapy of glaucoma. J Ocul Pharmacol Ther. 2015; 31:63–77.

4. Kopczynski CC, Epstein DL. Emerging trabecular outflow drugs. J Ocul Pharmacol Ther. 2014; 30:85–7.

5. Inoue T, Tanihara H. Rho-associated kinase inhibitors: a novel glaucoma therapy. Prog Retin Eye Res. 2013; 37:1–12.

6. Hein TW, Rosa RH Jr, Yuan Z, et al. Divergent roles of nitric oxide and rho kinase in vasomotor regulation of human retinal arterioles. Invest Ophthalmol Vis Sci. 2010; 51:1583–90.

7. Okamura N, Saito M, Mori A, et al. Vasodilator effects of fasudil, a Rho-kinase inhibitor, on retinal arterioles in stroke-prone spontaneously hypertensive rats. J Ocul Pharmacol Ther. 2007; 23:207–12.

8. Meyer-ter-Vehn T, Sieprath S, Katzenberger B, et al. Contractility as a prerequisite for TGF-beta-induced myofibroblast transdifferentiation in human tenon fibroblasts. Invest Ophthalmol Vis Sci. 2006; 47:4895–904.

9. Honjo M, Tanihara H, Kameda T, et al. Potential role of Rho-associated protein kinase inhibitor Y-27632 in glaucoma filtration surgery. Invest Ophthalmol Vis Sci. 2007; 48:5549–57.

10. Thieme H, Nuskovski M, Nass JU, et al. Mediation of calcium-independent contraction in trabecular meshwork through protein kinase C and rho-A. Invest Ophthalmol Vis Sci. 2000; 41:4240–6.

11. Rao PV, Deng PF, Kumar J, Epstein DL. Modulation of aqueous humor outflow facility by the Rho kinase-specific inhibitor Y-27632. Invest Ophthalmol Vis Sci. 2001; 42:1029–37.

12. Honjo M, Tanihara H, Inatani M, et al. Effects of rho-associated protein kinase inhibitor Y-27632 on intraocular pressure and outflow facility. Invest Ophthalmol Vis Sci. 2001; 42:137–44.

13. Novack GD. Rho kinase inhibitors for the treatment of glaucoma. Drugs Future. 2013; 38:107–13.

14. Wiederholt M, Dörschner N, Groth J. Effect of diuretics, channel modulators and signal interceptors on contractility of the trabecular meshwork. Ophthalmologica. 1997; 211:153–60.

15. Wiederholt M, Stumpff F. The trabecular meshwork and aqueous humor reabsorption. Civan MM, editor. Current topics in membranes. The eye's aqueous Humor: from secretion to glaucoma. 1st ed.San Diego: Academic Press;1998. 45:chap. 7.

16. Wiederholt M, Sturm A, Lepple-Wienhues A. Relaxation of trabecular meshwork and ciliary muscle by release of nitric oxide. Invest Ophthalmol Vis Sci. 1994; 35:2515–20.

17. Behar-Cohen FF, Goureau O, D'Hermies F, Courtois Y. Decreased intraocular pressure induced by nitric oxide donors is correlated to nitrite production in the rabbit eye. Invest Ophthalmol Vis Sci. 1996; 37:1711–5.

18. Dismuke WM, Mbadugha CC, Ellis DZ. NO-induced regulation of human trabecular meshwork cell volume and aqueous humor outflow facility involve the BKCa ion channel. Am J Physiol Cell Physiol. 2008; 294:C1378–86.

19. Sanka K, Maddala R, Epstein DL, Rao PV. Influence of actin cytoskeletal integrity on matrix metalloproteinase-2 activation in cultured human trabecular meshwork cells. Invest Ophthalmol Vis Sci. 2007; 48:2105–14.

20. Ming X-F, Viswambharan H, Barandier C, et al. Rho GTPase/Rho kinase negatively regulates endothelial nitric oxide synthase phosphorylation through the inhibition of protein kinase B/Akt in human endothelial cells. Mol Cell Biol. 2002; 22:8467–77.

21. Takemoto M, Sun J, Hiroki J, et al. Rho-kinase mediates hypoxia-induced downregulation of endothelial nitric oxide synthase. Circulation. 2002; 106:57–62.

22. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983; 65:55–63.

23. Liu XD, Skold CM, Umino T, et al. Sodium nitroprusside augments human lung fibroblast collagen gel contraction independently of NO-cGMP pathway. Am J Physiol Lung Cell Mol Physiol. 2000; 278:L1032–8.

24. Kameda T, Inoue T, Inatani M, et al. The effect of Rho-associated protein kinase inhibitor on monkey Schlemm's canal endothelial cells. Invest Ophthalmol Vis Sci. 2012; 53:3092–103.

25. Green LC, Wagner DA, Glogowski J, et al. Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem. 1982; 126:131–8.

26. Polansky JR, Weinreb RN, Baxter JD, Alvarado J. Human trabecular cells. I. Establishment in tissue culture and growth characteristics. Invest Ophthalmol Vis Sci. 1979; 18:1043–9.

27. Alvarado JA, Wood I, Polansky JR. Human trabecular cells. II. Growth pattern and ultrastructural characteristics. Invest Ophthalmol Vis Sci. 1982; 23:464–78.

28. Rikitake Y, Liao JK. Rho GTPases, statins, and nitric oxide. Circ Res. 2005; 97:1232–5.

29. Noma K, Oyama N, Liao JK. Physiological role of ROCKs in the cardiovascular system. Am J Physiol Cell Physiol. 2006; 290:C661–8.

30. Nakajima E, Nakajima T, Minagawa Y, et al. Contribution of ROCK in contraction of trabecular meshwork: proposed mechanism for regulating aqueous outflow in monkey and human Eyes. J Pharm Sci. 2005; 94:701–8.

31. Kim HY, Kim JW. Effect of nitric oxide on the permeability of trabecular meshwork cell monolayer. J Korean Ophthalmol Soc. 2015; 56:771–5.