W. P. Schilling: Rammelkamp Center, Rm R-322, MetroHealth Medical Center, 2500 MetroHealth Drive, Cleveland, OH 44109, USA.
J Physiol. 2012 Aug 1;590(15):3431-47. doi: 10.1113/jphysiol.2012.232645.
In non-excitable cells, thiol-oxidizing agents have been shown to evoke oscillations in cytosolic free Ca(2+) concentration (Ca(2+)) by increasing the sensitivity of the inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) to IP(3). Although thiol modification of the IP(3)R is implicated in this response, the molecular nature of the modification(s) responsible for changes in channel activity is still not well understood. Diamide is a chemical oxidant that selectively converts reduced glutathione (GSH) to its disulfide (GSSG) and promotes the formation of protein–glutathione (P-SSG) mixed disulfide, i.e. glutathionylation. In the present study, we examined the effect of diamide, and the model oxidant hydrogen peroxide (H(2)O(2)), on oscillations in Ca(2+) in fura-2-loaded bovine (BAECs) and human (HAECs) aortic endo-thelial cells using time-lapse fluorescence video microscopy. In the absence of extracellular Ca(2+), acute treatment with either diamide or H(2)O(2) increased the number of BAECs exhibiting asynchronous Ca(2+) oscillations, whereas HAECs were unexpectedly resistant. Diamide pretreatment increased the sensitivity of HAECs to histamine-stimulated Ca(2+) oscillations and BAECs to bradykinin-stimulated Ca(2+) oscillations. Moreover, in both HAECs and BAECs, diamide dramatically increased both the rate and magnitude of the thapsigargin-induced Ca(2+) transient suggesting that Ca(2+)-induced Ca(2+) release (CICR) via the IP(3)R is enhanced by glutathionylation. Similar to diamide, H(2)O(2) increased the sensitivity of HAECs to both histamine and thapsigargin. Lastly, biochemical studies showed that glutathionylation of native IP(3)R(1) is increased in cells challenged with H(2)O(2). Collectively our results reveal that thiol-oxidizing agents primarily increase the sensitivity of the IP(3)R to Ca(2+), i.e. enhanced CICR, and suggest that glutathionylation may represent a fundamental mechanism for regulating IP(3)R activity during physiological redox signalling and during pathologicalical oxidative stress.
在非兴奋细胞中，已证实通过增加三磷酸肌醇 (IP(3)) 受体 (IP(3)R) 对 IP(3) 的敏感性，硫醇氧化剂可引发细胞浆游离 Ca(2+) 浓度 (Ca(2+)) 的振荡。尽管 IP(3)R 的硫醇修饰与该反应有关，但负责通道活性变化的修饰（s）的分子性质仍未得到很好的理解。二酰胺是一种化学氧化剂，可选择性地将还原型谷胱甘肽 (GSH) 转化为其二硫化物 (GSSG)，并促进蛋白质-谷胱甘肽 (P-SSG) 混合二硫化物，即谷胱甘肽化的形成。在本研究中，我们使用延时荧光视频显微镜检查了二酰胺和模型氧化剂过氧化氢 (H(2)O(2)) 对牛 (BAECs) 和人 (HAECs) 主动脉内皮细胞中 Ca(2+) 振荡的影响。在没有细胞外 Ca(2+) 的情况下，急性处理二酰胺或 H(2)O(2) 均增加了表现出异步 Ca(2+) 振荡的 BAEC 数量，而 HAEC 出人意料地具有抗性。二酰胺预处理增加了 HAEC 对组胺刺激的 Ca(2+) 振荡和 BAEC 对缓激肽刺激的 Ca(2+) 振荡的敏感性。此外，在 HAEC 和 BAEC 中，二酰胺均极大地增加了 thapsigargin 诱导的 Ca(2+) 瞬变的速率和幅度，表明通过 IP(3)R 的 Ca(2+) 诱导的 Ca(2+) 释放 (CICR) 增强了谷胱甘肽化。与二酰胺类似，H(2)O(2) 增加了 HAEC 对组胺和 thapsigargin 的敏感性。最后，生化研究表明，在受到 H(2)O(2) 挑战的细胞中，天然 IP(3)R(1) 的谷胱甘肽化增加。总的来说，我们的结果表明，硫醇氧化剂主要通过增加 IP(3)R 对 Ca(2+) 的敏感性，即增强 CICR，来增加 IP(3)R 对 Ca(2+) 的敏感性，并且表明谷胱甘肽化可能是调节 IP(3)R 活性的基本机制在生理氧化还原信号和病理氧化应激过程中。
