High-Frequency Field Stimulation of Primary Neurons Enhances Ryanodine Receptor-Mediated Ca super(2+) Release and Generates Hydrogen Peroxide, Which Jointly Stimulate NF- Kappa B Activity

Neuronal electrical activity increases intracellular Ca super(2+) concentration and generates reactive oxygen species. Here, we show that high frequency field stimulation of primary hippocampal neurons generated Ca super(2+) signals with an early and a late component, and promoted hydrogen peroxide generation via a neuronal NADPH oxidase. Hydrogen peroxide generation required both Ca super(2+) entry through N-methyl-D-aspartate receptors and Ca super(2+) release mediated by ryanodine receptors (RyR). Field stimulation also enhanced nuclear translocation of the NF- Kappa B p65 protein and NF- Kappa B -dependent transcription, and increased c-fos mRNA and type-2 RyR protein content. Preincubation with inhibitory ryanodine or with the antioxidant N-acetyl L-cysteine abolished the increase in hydrogen peroxide generation and the late Ca super(2+) signal component induced by electrical stimulation. Primary cortical cells behaved similarly as primary hippocampal cells. Exogenous hydrogen peroxide also activated NF- Kappa B-dependent transcription in hippocampal neurons; inhibitory ryanodine prevented this effect. Selective inhibition of the NADPH oxidase or N-acetyl L-cysteine also prevented the enhanced translocation of p65 in hippocampal cells, while N-acetyl L-cysteine abolished the increase in RyR2 protein content induced by high frequency stimulation. In conclusion, the present results show that electrical stimulation induced reciprocal activation of ryanodine receptor-mediated Ca super(2+) signals and hydrogen peroxide generation, which stimulated jointly NF- Kappa B activity.