DFG Research Center Molecular Physiology of the Brain (CMPB), Zentrum für Physiologie und Pathophysiologie, Abteilung Neuro- und Sinnesphysiologie, Georg-August-Universität Göttingen, Humboldtallee 23, 37073, Göttingen, Germany.
Pflugers Arch. 2009 Sep;458(5):937-52. doi: 10.1007/s00424-009-0672-0. Epub 2009 May 10.
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.
活性氧（ROS）从功能失调的线粒体中释放出来，通过调节细胞内的氧化还原状态和氧化还原敏感蛋白，对正常和病理生理细胞信号转导起作用。为了鉴定这种信号转导中涉及的可能的氧化还原靶标，我们将海马神经元暴露于过氧化氢（H2O2）中。氧化还原敏感染料表明，外源性 H2O2 可能氧化细胞培养物和急性组织切片中的细胞内靶标。在培养的神经元中，H2O2（EC50 为 118μM）诱导细胞内 Ca2+ 升高，即使在 Ca2+ 耗尽和线粒体解偶联后，仍能引起 Ca2+ 升高。然而，它被 thapsigargin、dantrolene、2-氨基乙氧基二苯硼酸盐和高浓度的ryanodine 拮抗，这表明内质网（ER）是涉及的细胞内 Ca2+ 库。通过抑制谷胱甘肽过氧化物酶，内源性产生的 H2O2 引起的细胞内积累也从 ER 释放 Ca2+，而超氧化物的细胞外产生也如此。PLC 介导的代谢型信号在 H2O2 存在下被抑制，但细胞溶质环磷酸腺苷（cAMP）水平不受影响。H2O2（0.2-5mM）适度去极化线粒体，以 Ca2+和 cAMP 非依赖性方式停止其细胞内运输，并直接氧化细胞烟酰胺腺嘌呤二核苷酸（NADH）和黄素腺嘌呤二核苷酸（FADH2）。部分线粒体去极化反映了先前从 ER 释放的 Ca2+的摄取。我们得出结论，H2O2 通过ryanodine 和三磷酸肌醇受体从 ER 释放 Ca2+。即使是毫摩尔浓度的 H2O2，线粒体功能也不会明显受损。ROS 对 Ca2+信号转导和细胞器相互作用的这种调节会影响 PLC 介导的代谢型信号转导的效率，并可能有助于神经元功能对氧化还原条件和代谢供应的调整。
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