H(2)O(2)-mediated modulation of cytosolic signaling and organelle function in rat hippocampus

Reactive oxygen species (ROS) released from (dys-)functioning mitochondria contribute to normal and pathophysiological cellular signaling by modulating cytosolic redox state and redox-sensitive proteins. To identify putative redox targets involved in such signaling, we exposed hippocampal neurons to hydrogen peroxide (H(2)O(2)). Redox-sensitive dyes indicated that externally applied H(2)O(2) may oxidize intracellular targets in cell cultures and acute tissue slices. In cultured neurons, H(2)O(2) (EC(50) 118 microM) induced an intracellular Ca(2+) rise which could still be evoked upon Ca(2+) withdrawal and mitochondrial uncoupling. It was, however, antagonized by thapsigargin, dantrolene, 2-aminoethoxydiphenyl borate, and high levels of ryanodine, which identifies the endoplasmic reticulum (ER) as the intracellular Ca(2+) store involved. Intracellular accumulation of endogenously generated H(2)O(2)-provoked by inhibiting glutathione peroxidase-also released Ca(2+) from the ER, as did extracellular generation of superoxide. Phospholipase C (PLC)-mediated metabotropic signaling was depressed in the presence of H(2)O(2), but cytosolic cyclic adenosine-5'-monophosphate (cAMP) levels were not affected. H(2)O(2) (0.2-5 mM) moderately depolarized mitochondria, halted their intracellular trafficking in a Ca(2+)- and cAMP-independent manner, and directly oxidized cellular nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH(2)). In part, the mitochondrial depolarization reflects uptake of Ca(2+) previously released from the ER. We conclude that H(2)O(2) releases Ca(2+) from the ER via both ryanodine and inositol trisphosphate receptors. Mitochondrial function is not markedly impaired even by millimolar concentrations of H(2)O(2). Such modulation of Ca(2+) signaling and organelle interaction by ROS affects the efficacy of PLC-mediated metabotropic signaling and may contribute to the adjustment of neuronal function to redox conditions and metabolic supply.