title: High-frequency field stimulation of primary neurons enhances ryanodine receptor-mediated Ca2+ release and generates hydrogen peroxide, which jointly stimulate NF-κB activity.
abstract: Neuronal electrical activity increases intracellular Ca(2+) concentration and generates reactive oxygen species. Here, we show that high frequency field stimulation of primary hippocampal neurons generated Ca(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(2+) entry through N-methyl-D-aspartate receptors and Ca(2+) release mediated by ryanodine receptors (RyR). Field stimulation also enhanced nuclear translocation of the NF-κB p65 protein and NF-κB -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(2+) signal component induced by electrical stimulation. Primary cortical cells behaved similarly as primary hippocampal cells. Exogenous hydrogen peroxide also activated NF-κB-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(2+) signals and hydrogen peroxide generation, which stimulated jointly NF-κB activity.
authors.affiliation: Center of Molecular Studies of the Cell, Institute of Biomedical Sciences Programs, Universidad de Chile, Santiago, Chile.
authors.forename: Denise
authors.initials: D
authors.lastname: Riquelme
authors.forename: Alvaro
authors.initials: A
authors.lastname: Alvarez
authors.forename: Nancy
authors.initials: N
authors.lastname: Leal
authors.forename: Tatiana
authors.initials: T
authors.lastname: Adasme
authors.forename: Italo
authors.initials: I
authors.lastname: Espinoza
authors.forename: Juan Antonio
authors.initials: JA
authors.lastname: Valdés
authors.forename: Natalia
authors.initials: N
authors.lastname: Troncoso
authors.forename: Steffen
authors.initials: S
authors.lastname: Hartel
authors.forename: Jorge
authors.initials: J
authors.lastname: Hidalgo
authors.forename: Cecilia
authors.initials: C
authors.lastname: Hidalgo
authors.forename: M Angélica
authors.initials: MA
authors.lastname: Carrasco
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D018929:Cell Culture Techniques; D004558:Electric Stimulation; D017930:Genes, Reporter; D006624:Hippocampus / Q000166:cytology; D006861:Hydrogen Peroxide / Q000378:metabolism*; D049410:Luciferases, Renilla / Q000096:biosynthesis / Q000235:genetics; D019255:NADPH Oxidases / Q000378:metabolism; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D009861:Onium Compounds / Q000494:pharmacology; D021381:Protein Transport; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*; D051996:Transcription Factor RelA / Q000378:metabolism; D014158:Transcription, Genetic; D015854:Up-Regulation
chemical_list: D016328:NF-kappa B; D009861:Onium Compounds; D016760:Proto-Oncogene Proteins c-fos; D017382:Reactive Oxygen Species; C498145:Rela protein, rat; D019837:Ryanodine Receptor Calcium Release Channel; D051996:Transcription Factor RelA; C007517:diphenyleneiodonium; D006861:Hydrogen Peroxide; D049410:Luciferases, Renilla; D019001:Nitric Oxide Synthase; D019255:NADPH Oxidases; D002118:Calcium; D000111:Acetylcysteine