Dysfunction in Cytochrome C Oxidase Caused by Excessive Nitric Oxide in Human Lens Epithelial Cells Stimulated With interferon-γ and Lipopolysaccharide

Curr Eye Res. 2012 Oct;37(10):889-97. doi: 10.3109/02713683.2012.689070. Epub 2012 May 25.

Abstract

Purpose: 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.

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.

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Calcium / metabolism
  • Cataract / enzymology*
  • Cataract / pathology
  • Cell Line
  • Electron Transport Complex IV / metabolism*
  • Enzyme Activation / drug effects
  • Epithelial Cells / cytology
  • Epithelial Cells / drug effects
  • Epithelial Cells / enzymology*
  • Humans
  • Interferon-gamma / pharmacology*
  • Lens, Crystalline / cytology
  • Lens, Crystalline / drug effects
  • Lens, Crystalline / enzymology*
  • Lipopolysaccharides / pharmacology*
  • NF-kappa B / metabolism
  • Nitric Oxide / metabolism*
  • Nitric Oxide Synthase Type II / metabolism

Substances

  • Lipopolysaccharides
  • NF-kappa B
  • Nitric Oxide
  • Interferon-gamma
  • Adenosine Triphosphate
  • NOS2 protein, human
  • Nitric Oxide Synthase Type II
  • Electron Transport Complex IV
  • Calcium