Effect of hypoxia on susceptibility of RGC-5 cells to nitric oxide.
OBJECTIVE
To determine whether retinal neurons become more susceptible to injury by nitric oxide (NO) under hypoxic conditions.


METHODS
Cells from the RGC-5 line were exposed to different concentrations (0.1-100 microM) of S-nitroso-N-acetyl-penicillamine (SNAP), an NO donor, under normoxic and hypoxic (1.0% O(2)) conditions with 5.5 mM glucose or with no glucose. In some experiments, carboxy-PTIO, a scavenger of NO, was added with 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. Alterations of mitochondrial membrane potential, superoxide anion formation, cellular adenosine triphosphate (ATP) contents, and caspase activity were also determined after exposure to SNAP.


RESULTS
Exposure of RGC-5 cells to SNAP (100 microM) significantly decreased the number of living cells cultured under hypoxic conditions with or without glucose. Coadministration of carboxy-PTIO (1.0 microM) suppressed SNAP-induced cell death. SNAP-induced cell death of cells cultured under hypoxia with glucose was accompanied by increased expression of phosphatidylserine and hypodiploid DNAs. These findings indicated that death was mediated in part by apoptosis. In addition, loss of mitochondrial membrane potential, increase of superoxide formation, and activation of caspase was observed. Cyclosporine A, TEMPOL, and Z-VAD-FMK suppressed cell death. On the other hand, SNAP depleted the ATP contents of cells cultured under hypoxia without glucose, causing mainly necrotic cell death.


CONCLUSIONS
These results indicate that RGC-5 cells become susceptible to SNAP under hypoxic conditions in which NO may have greater impact on mitochondrial function.
Sato
Takaki
T
Department of Ophthalmology, Osaka Medical College, Osaka, Japan.
Oku
Hidehiro
H
Tsuruma
Kazuhiro
K
Katsumura
Kozo
K
Shimazawa
Masamitsu
M
Hara
Hideaki
H
Sugiyama
Tetsuya
T
Ikeda
Tsunehiko
T
D000255:Adenosine Triphosphate / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects; D001565:Benzoates / Q000494:pharmacology; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D004247:DNA / Q000032:analysis; D004171:Diploidy; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D000860:Hypoxia / Q000378:metabolism*; D007093:Imidazoles / Q000494:pharmacology; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D020030:Nitric Oxide Donors / Q000633:toxicity*; D030421:Peroxynitrous Acid / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D012165:Retinal Ganglion Cells / Q000187:drug effects* / Q000378:metabolism; D026423:S-Nitroso-N-Acetylpenicillamine / Q000633:toxicity*; D013481:Superoxides / Q000378:metabolism
D001565:Benzoates; D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D007093:Imidazoles; D020030:Nitric Oxide Donors; D010718:Phosphatidylserines; D013481:Superoxides; C079393:1,3-dihydroxy-4,4,5,5-tetramethyl-2-(4-carboxyphenyl)tetrahydroimidazole; D030421:Peroxynitrous Acid; D026423:S-Nitroso-N-Acetylpenicillamine; D000255:Adenosine Triphosphate; D004247:DNA; D020169:Caspases; D005947:Glucose