Dysfunction in cytochrome c oxidase caused by excessive nitric oxide in human lens epithelial cells stimulated with interferon-γ and lipopolysaccharide.
OBJECTIVE
We previously found two mechanisms for the dysfunction in Ca(2+) 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 Ca(2+)-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.


CONCLUSIONS
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.
School of Pharmacy and Pharmaceutical Research and Technology Institute, Kinki University, Higashi-Osaka, Osaka, Japan.
Noriaki
N
Nagai
Yoshimasa
Y
Ito
D000255:Adenosine Triphosphate / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002386:Cataract / Q000201:enzymology* / Q000473:pathology; D002460:Cell Line; D003576:Electron Transport Complex IV / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology*; D007908:Lens, Crystalline / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D016328:NF-kappa B / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism
D008070:Lipopolysaccharides; D016328:NF-kappa B; D009569:Nitric Oxide; D007371:Interferon-gamma; D000255:Adenosine Triphosphate; C496319:NOS2 protein, human; D052247:Nitric Oxide Synthase Type II; D003576:Electron Transport Complex IV; D002118:Calcium