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H. Oku, T. Sato, K. Tsuruma, M. Shimazawa, H. Hara, T. Sugiyama, T. Ikeda; Hypoxia Makes RGC-5 Cells Susceptible to Nitric Oxide. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4706.
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To determine whether retinal neurons become more susceptible to injury by nitric oxide (NO) under hypoxic conditions.
Cells from the RGC-5 line were exposed to different concentrations (0.1-100 µM) of S-nitroso-N-acetyl-penicillamine (SNAP), an NO donor, under normoxic and hypoxic (1.0% O2) conditions with 5.5 mM of glucose or with no glucose. In some experiments, carboxy-PTIO, a scavenger of NO, was added with the SNAP. The SNAP-induced cell injury was determined by the WST-8 assay and by the assessment of phosphatidylserine externalization and changes in hypodiploid DNAs as determined by flow cytometry. Alterations of the mitochondrial membrane potential, formation of superoxide anions, cellular ATP contents, and caspase activity were also determined after exposure to SNAP.
Exposure of RGC-5 cells to SNAP (100 µM) significantly decreased the number of living cells cultured under hypoxic conditions with or without glucose. Co-administration of carboxy-PTIO (1.0 µM) suppressed the SNAP-induced cell death. The SNAP-induced cell death in cells cultured under hypoxia with glucose was accompanied by increased expression of phosphatidylserine and hypodiploid DNAs. These findings indicated the death was mediated in part by apoptosis. In addition, we observed a loss of mitochondrial membrane potential, increase of superoxide formation, and activation of caspase. Cyclosporine A, TEMPOL, and Z-VAD-FMK suppressed the cell death. On the other hand, SNAP depleted the ATP contents of cells cultured under hypoxia without glucose causing mainly necrotic cell death.
These results indicate that RGC-5 cells become susceptible to SNAP under hypoxic conditions, where NO may have greater impact on mitochondrial function.
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