Nitric Oxide Potentiates TNF-α-Induced Neurotoxicity Through Suppression of NF-κB

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-alpha-induced death of retinal neurons through a suppression of nuclear factor-kappa B (NF-kappa B) 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-alpha, and the degree of TNF-alpha-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-alpha. The activation of NF-kappa B 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-alpha 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-alpha increased the activation of NF-kappa B and the expression of iNOS. Although NF-kappa B inhibitors suppressed the increase of iNOS, they also potentiated the TNF-alpha-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 kappa B kinase, and suppressed the NF-kappa B activation and potentiated the TNF-alpha-induced neurotoxicity. These results indicate that NO potentiates the neurotoxicity of TNF-alpha by suppressing NF-kappa B.