J Korean Ophthalmol Soc.  2013 Sep;54(9):1429-1434.

A Study of the Pathway of Nitric Oxide Production by Nitroglycerin in Trabecular Meshwork Cells

• Yoon SH
• Kim JW

Abstract

PURPOSE
To investigate the effects of nitroglycerin on the production of nitric oxide and its related pathway in cultured human trabecular meshwork cells (HTMC).
METHODS
Primarily cultured HTMC were exposed to 10 nM nitroglycerin using 1% serum-containing media for 30 minutes. The production of nitric oxide was assessed with the Griess assay and expressions of eNOS mRNA was assessed with RT-PCR. Additionally, the cells were exposed to wortmanin and Akt1/2 kinase inhibitor to investigate the mechanism related to the production of nitric oxide.
RESULTS
Nitroglycerin increased the production of nitric oxide (p < 0.05) accompanied with increased expression of eNOS mRNA. The increased production of nitric oxide and eNOS mRNA was inhibited by wortmanin and Akt1/2 kinase inhibitor.
CONCLUSIONS
Low-dose nitroglycerin increased the production of nitric oxide accompanied by increased eNOS activity. Nitroglycerin drives eNOS activation via the phosphatidylinositol 3-kinase/protein kinase B pathway.

Keyword

eNOS; Nitroglycerin; Nitric oxide; Trabecular meshwork cells

MeSH Terms

Humans
Nitric Oxide
Nitroglycerin
Phosphatidylinositols
Phosphotransferases
RNA, Messenger
Trabecular Meshwork
Nitric Oxide
Nitroglycerin
Phosphatidylinositols
Phosphotransferases
RNA, Messenger

Figure

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  Figure 1. Effects of Nitroglycerin (GTN) and vascular endo-thelial growth factor (VEGF) on the production of nitric oxide in trabecular meshwork cells. Both GTN and VEGF increased production of nitric oxide significantly (* p < 0.05).

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  Figure 2. Effects of wortmanin and Akt1/2 kinase inhibitor on the nitroglycerin (GTN)-induced production of nitric oxide in trabecular meshwork cells. Akt1/2 kinase inhibitor and wort-manin inhibits GTN-induced production of nitric oxide (* p < 0.05).

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  Figure 3. Effects of nitroglycerin (GTN) on the activity of eNOS in trabecular meshwork cells. GTN and vascular growth factor (VEGF) increased eNOS expression, which were abolished by Akt1/2 kinase inhibitor and wortmanin compared to the non-exposed control.

Reference

References

1. Bond GS. Effect of various agents on the blood flow through the coronary arteries and veins. J Exp Med. 1910; 12:575–85.

2. Marsh N, Marsh A. A short history of nitroglycerine and nitric ox-ide in pharmacology and physiology. Clin Exp Pharmacol Physiol. 2000; 27:313–9.

3. Chen Z, Foster MW, Zhang J. . An essential role for mitochon-drial aldehyde dehydrogenase in nitroglycerin bioactivation. Proc Natl Acad Sci U S A. 2005; 102:12159–64.

4. Bonini MG, Stadler K, Silva SO. . Constitutive nitric oxide syn-thase activation is a significant route for nitroglycerin-mediated vasodilation. Proc Natl Acad Sci U S A. 2008; 105:8569–74.

5. Sydow K, Daiber A, Oelze M. . Central role of mitochondrial aldehyde dehydrogenase and reactive oxygen species in nitro-glycerin tolerance and cross-tolerance. J Clin Invest. 2004; 113:482–9.

6. Kleschyov AL, Oelze M, Daiber A. . Does nitric oxide mediate the vasodilator activity of nitroglycerin? Circ Res. 2003; 93:e104–12.

7. Millar TM, Stevens CR, Benjamin N. . Xanthine oxidor-eductase catalyses the reduction of nitrates and nitrite to nitric ox-ide under hypoxic conditions. FEBS Lett. 1998; 427:225–8.

8. Hill KE, Hunt RW Jr, Jones R. . Metabolism of nitroglycerin by smooth muscle cells: involvement of glutathione and glutathione S-transferase. Biochem Pharmacol. 1992; 43:561–6.

9. Abou-Mohamed G, Kaesemeyer WH, Caldwell RB, Caldwell RW. Role of L-arginine in the vascular actions and development of tol-erance to nitroglycerin. Br J Pharmacol. 2000; 130:211–8.

10. Schwarz M, Katz SD, Demopoulos L. . Enhancement of endo-thelium-dependent vasodilation by low-dose nitroglycerin in patients with congestive heart failure. Circulation. 1994; 89:1609–14.

11. Forster C, Main JS, Armstrong PW. Endothelium modulation of the effects of nitroglycerin on blood vessels from dogs with pacing-induced heart failure. Br J Pharmacol. 1990; 101:109–14.

12. Gruhn N, Aldershvile J, Boesgaard S. Tetrahydrobiopterin improves endothelium-dependent vasodilation in nitroglycerin-tolerant rats. Eur J Pharmacol. 2001; 416:245–9.

13. Gruhn N, Boesgaard S, Andersen C, Aldershvile J. Nitroglycerin tolerance: different mechanisms in vascular segments with or with-out intact endothelial function. J Cardiovasc Pharmacol. 2002; 40:201–9.

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

15. Rohen JW, LÜtjen-drecoll E, FlÜgel C. . Ultrastructure of the trabecular meshwork in untreated cases of primary open-angle glaucoma. Exp Eye Res. 1993; 56:683–92.

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

17. Wiederholt M, Stumpff F. Civan MM, editor. The trabecular meshwork and aqueous humor reabsorption. Current topics in membranes. The eye's aqueous Humor: from secretion to glaucoma. vol. 45. 1998. San Diego: Academic Press;p. 163–202.

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

19. 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.

20. Mosmann T. Rapid colorimetric assay for cellular growth and sur-vival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983; 65((1-2)):55–63.

21. Freimoser FM, Jakob CA, Aebi M, Tuor U. The MTT [3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay is a fast and reliable method for colorimetric determination of fungal cell densities. Appl Environ Microbio. 1999; 65:3727–9.

22. Green LC, Wagner DA, Glogowski J. . Analysis of Nitrate, Nitrite and [15N]nitrate in biologic fluids. Analytical Biochem. 1982; 126:131–8.

23. Stamler JS, Jaraki O, Osborne J. . Nitric oxide circulates in mammalian plasma primarily as an S-nitroso adduct of serum albumin. Proc Natl Acad Sci U S A. 1992; 89:7674–7.

24. Schneider AJ, Teule GJ, Groeneveld AB. . The immediate ef-fect of nitroglycerin on total body blood volume distribution in patients with congestive heart failure: A noninvasive study. Eur Heart J. 1987; 8:1119–25.

25. Laslett LJ, Baker L. Sublingual nitroglycerin administered by spray versus tablet: Comparative timing of hemodynamic effects. Cardiology. 1990; 77:303–10.

26. Bauer JA, Fung HL. Arterial versus venous metabolism of nitro-glycerin to nitric oxide: A possible explanation of organic nitrate venoselectivity. J Cardiovasc Pharmacol. 1996; 28:371–4.

27. Dinerman JL, Lawson DL, Mehta JL. Interactions between nitro-glycerin and endothelium in vascular smooth muscle relaxation. Am J Physiol. 1991; 260((3 Pt 2)):H698–701.

28. Bonini MG, Stadler K, Silva SO. . Constitutive nitric oxide synthase activation is a significant route for nitroglycerinmediated Vasodilation. Proc Natl Acad Sci U S A. 2008; 105:8569–74.

29. Mao M, Sudhahar V, Ansenberger-Fricano K. . Nitroglycerin drives endothelial nitric oxide synthase activation via the phospha-tidylinositol 3-kinase/protein kinase B pathway. Free Radic Biol Med. 2012; 52:427–35.

30. Bouloumié A, Schini-Kerth VB, Busse R. Vascular endothelial growth factor up-regulates nitric oxide synthase 1 expression in en-dothelial cells. Cardiovasc Res. 1999; 41:773–80.

31. Wang H, Keiser JA. Vascular endothelial growth factor upregulates the expression of matrix metalloproteinases in vascular smooth muscle cells: Role of flt-1. Circ Res. 1998; 83:832–40.

32. Laursen JB, Boesgaard S, Poulsen HE, Aldershvile J. Nitrate toler-ance impairs nitric oxide-mediated vasodilation in vivo. Cardiovasc Res. 1996; 31:814–9.

33. Gori T, Parker JD. Nitrate tolerance: A unifying hypothesis. Circulation. 2002; 106:2510–13.