title: Nitric oxide potentiates TNF-α-induced neurotoxicity through suppression of NF-κB.
abstract: Modulation of enzyme activity through nitrosylation has recently been identified as a new physiological activity of nitric oxide (NO). We hypothesized that NO enhances the TNF-α-induced death of retinal neurons through a suppression of nuclear factor-κB (NF-κB) by nitrosylation. In this study, cells from the RGC-5 line were exposed to different concentrations (2.0, 10, and 50 ng/ml) of TNF-α, and the degree of TNF-α-induced cell death was determined by the WST-8 assay and by flow cytometric measurements of the externalization of phosphatidylserine. The effects of etanercept, a soluble TNFR-Fc fusion protein, and S-nitroso-N-penicillamine (SNAP), an NO donor, on the toxicity were determined. Experiments were also performed to determine whether nitric oxide synthase (NOS) was associated with the toxicity of TNF-α. The activation of NF-κB was determined by the detection of the p65 subunit in the nuclear extracts. Our results showed that exposure of RGC-5 cells to different concentrations of TNF-α significantly decreased the number of living cells in a dose-dependent way. The death was partially due to apoptosis with an externalization of phosphatidylserine, and the death was suppressed by etanercept. Exposure to TNF-α increased the activation of NF-κB and the expression of iNOS. Although NF-κB inhibitors suppressed the increase of iNOS, they also potentiated the TNF-α-induced death. Both L-NAME and aminoguanidine, both NOS inhibitors, rescued the cells from death. In contrast, addition of SNAP caused nitrosylation of the inhibitory κB kinase, and suppressed the NF-κB activation and potentiated the TNF-α-induced neurotoxicity. These results indicate that NO potentiates the neurotoxicity of TNF-α by suppressing NF-κB.
authors.affiliation: Department of Ophthalmology, Osaka Medical College, 2-7 Daigaku-machi, Takatsuki, Osaka, 569-8686, Japan.
authors.forename: Atsuko
authors.initials: A
authors.lastname: Nakaizumi
authors.forename: Taeko
authors.initials: T
authors.lastname: Horie
authors.forename: Teruyo
authors.initials: T
authors.lastname: Kida
authors.forename: Takuji
authors.initials: T
authors.lastname: Kurimoto
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Sugiyama
authors.forename: Tsunehiko
authors.initials: T
authors.lastname: Ikeda
authors.forename: Hidehiro
authors.initials: H
authors.lastname: Oku
mesh_terms: D000818:Animals; D001921:Brain / Q000187:drug effects / Q000502:physiology; D016923:Cell Death / Q000187:drug effects; D002478:Cells, Cultured; D015536:Down-Regulation / Q000187:drug effects / Q000235:genetics; D004357:Drug Synergism; D051379:Mice; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000378:metabolism; D009474:Neurons / Q000187:drug effects* / Q000502:physiology; D009569:Nitric Oxide / Q000378:metabolism / Q000494:pharmacology*; D052248:Nitric Oxide Synthase Type I / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D015538:Nitrosation / Q000187:drug effects; D026423:S-Nitroso-N-Acetylpenicillamine / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D026423:S-Nitroso-N-Acetylpenicillamine; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; C496321:Nos1 protein, mouse; C496317:Nos2 protein, mouse