Significance of Nitric Oxide in Lens Damage. Implications in Cataractogenesis

Abstract

Purpose:: Oxides of nitrogen with unpaired electrons, such as NO and NO2, are recognized to be highly reactive biological entities. We hypothesize that their presence in the lens and aqueous can also become detrimental to the tissue physiology linked to cataract formation. We have investigated this possibility in rat lens culture studies using nitroaspirin (2-(acetyloxy)-3-[(nitrooxy)methyl] phenyl ester benzoic acid), a specific NO donor.

Methods:: The toxicity of NO was hence assessed by incubating rat lenses in medium containing ⁸⁶RbCl with or without nitroaspirin and determining their physiological status in terms of its ⁸⁶Rb⁺ transport activity and the contents of GSH, GSSG and ATP. The generation of NO was ascertained by diazotization of sulfanilamide followed by its condensation with naphthylethylene diamine generating the azo derivative with absorption maxima at 550nm. Measurements were done on media incubated with lens in the absence as well as presence of nitroaspirin. Rb⁺ transport was expressed as the ratio of its concentration in the lens and the medium post-incubation. ATP was determined by measuring chemiluminescence produced in the tissue extract by the firefly reaction. GSH and GSSG were determined by Ellman’s reaction, the latter after treating the sample with 1-methyl 4-vinyl-pyridinium trifluoromethane sulfonate.

Results:: That NO is indeed deleterious was proven by the inhibition of active transport of Rb+ reflected by its decreased uptake in the presence of nitroaspirin, the decrease being 50% and 66% of the controls in the older and younger lenses respectively, with similar decreases in the levels of ATP and GSH. The GSH/GSSG was also significantly depressed. That these effects are indeed related to NO generation was further authenticated by a substantial increase of NO₂^- (~0.4mM) in the medium incubated with the lens and Nitroaspirin, as compared to its barely detectable level (0.05mM) in the control medium incubated without nitroaspirin.

Conclusions:: The above findings, showing significant aberrations in transport and metabolic characteristics of the lenses incubated with nitroaspirin, a specific NO donor, are consistent with our hypothesis that excessive generation of this species either in the lens or its availability from the surrounding tissues and fluids such as the aqueous and vitreous could constitute a significant component of the battery of oxidative stress involved in cataractogenesis.

Keywords: cataract • nitric oxide • oxidation/oxidative or free radical damage