Abstract
Purpose: We previously found two mechanisms for the dysfunction in Ca2+ regulation caused by excessive nitric oxide (NO) using the lenses of hereditary cataract model rats: the first is that NO causes a decrease in Adenosine-5′-triphosphate (ATP) level via cytochrome c oxidase (CCO), resulting in a decrease in ATPase function; the second is that NO causes enhanced lipid peroxidation, resulting in the oxidative inhibition of Ca2+-ATPase. In this study, we demonstrate the effect of excessive NO on lipid peroxidation and ATP production in human lens using a human lens epithelial cell line, SRA 01/04 (human lens epithelial (HLE) cells).Methods: Excessive NO via inducible NO synthase (iNOS) was induced by stimulating cells with a combination of interferon-gamma (1000 IU IFN-γ) and lipopolysaccharide (100 ng/mL LPS). CCO activity was measured using a Mitochondrial Isolation kit and Cytochrome c Oxidase Assay kit, and ATP levels were determined using a Sigma ATP Bioluminescent Assay Kit and a luminometer AB-2200.Results: Cytochrome c oxidase activity and ATP levels were decreased in HLE cells stimulated with IFN-γ and LPS, and aminoguanidine (AG) and diethyldithiocarbamate (DDC) added 6 h before cell collection significantly attenuated these decreases in cells stimulated with the IFN-γ and LPS for 24–30 h. However, the lower CCO activity and ATP levels in HLE cells stimulated with the IFN-γ and LPS for 30 h were not changed by treatment with AG or DDC for 6–12 h, while the CCO activity and ATP levels in HLE cells treated with AG or DDC for 18 were recovered.Conclusion: Excessive NO causes a decrease in CCO activity and ATP levels, and the recovery time for CCO activity is related to exposure time to NO in HLE cells.
Published Version
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