High-Frequency Field Stimulation of Primary Neurons Enhances Ryanodine Receptor-Mediated Ca2+ Release and Generates Hydrogen Peroxide, Which Jointly Stimulate NF-kB Activity

Abstract

Neuronal electrical activity increases intracellular Ca2+ concentration and generates reactive oxygen species. Here, we show that high frequency field stimulation of primary hippocampal neurons generated Ca2+ signals with an early and a late component, and promoted hydrogen peroxide generation via a neuronal NADPH oxidase. Hydrogen peroxide generation required both Ca2+ entry through N-methyl-D-aspartate receptors and Ca2+ release mediated by ryanodine receptors (RyR). Field stimulation also enhanced nuclear translocation of the NF-kB p65 protein and NF-kB -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 Ca2+ signal component induced by electrical stimulation. Primary cortical cells behaved similarly as primary hippocampal cells. Exogenous hydrogen peroxide also activated NF-kB-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 Ca2+ signals and hydrogen peroxide generation, which stimulated jointly NF-kB activity.