Reactive oxygen species (ROS) play a vital role in cell signaling and redox regulation, but when present in excess, lead to numerous pathologies. Detailed quantitative characterization of mitochondrial superoxide anion (O[Formula: see text] production in fetal pulmonary artery endothelia cells (PAECs) has never been reported. The aim of this study is to assess mitochondrial O[Formula: see text] production in cultured PAECs over time using a novel quantitative optical approach. The rate, the sources, and the dynamics of O[Formula: see text] production were assessed using targeted metabolic modulators of the mitochondrial electron transport chain (ETC) complexes, specifically an uncoupler and inhibitors of the various ETC complexes, and inhibitors of extra-mitochondrial sources of O[Formula: see text]. After stabilization, the cells were loaded with nanomolar mitochondrial-targeted hydroethidine (Mito-HE, MitoSOX) online during the experiment without washout of the residual dye. Time-lapse fluorescence microscopy was used to monitor the dynamic changes in O[Formula: see text] fluorescence intensity over time in PAECs. The transient behaviors of the fluorescence time course showed exponential increases in the rate of O[Formula: see text] production in the presence of the ETC uncoupler or inhibitors. The most dramatic and the fastest increase in O[Formula: see text] production was observed when the cells were treated with the uncoupling agent, PCP. We also showed that only the complex IV inhibitor, KCN, attenuated the marked surge in O[Formula: see text] production induced by PCP. The results showed that mitochondrial respiratory complexes I, III and IV are sources of O[Formula: see text] production in PAECs, and a new observation that ROS production during uncoupling of mitochondrial respiration is mediated in part via complex IV. This novel method can be applied in other studies that examine ROS production under stress condition and during ROS-mediated injuries in vitro.

中文翻译：

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肺动脉内皮细胞线粒体超氧化物动力学的定量光学测量

活性氧 (ROS) 在细胞信号传导和氧化还原调节中起着至关重要的作用，但当过量存在时，会导致许多病理。从未报道过胎儿肺动脉内皮细胞 (PAECs) 中线粒体超氧阴离子 (O[Formula: see text] 产生的详细定量表征。本研究的目的是评估培养的 PAECs 中线粒体 O[Formula: see text] 的产生随着时间的推移，使用一种新的定量光学方法。使用线粒体电子传递链 (ETC) 复合物的靶向代谢调节剂，特别是解偶联剂和抑制剂来评估 O[公式：见文本] 生产的速率、来源和动力学各种 ETC 复合物，以及 O[公式：见正文] 的线粒体外源抑制剂。稳定后，细胞在实验过程中在线加载纳摩尔线粒体靶向氢乙啶（Mito-HE，MitoSOX），而不会洗掉残留的染料。延时荧光显微镜用于监测 PAECs 中 O[公式：见文本] 荧光强度随时间的动态变化。在 ETC 解偶联剂或抑制剂存在的情况下，荧光时间过程的瞬态行为显示出 O[公式：见正文] 生成速率呈指数增长。当用解偶联剂 PCP 处理细胞时，观察到 O[公式：见正文] 产量的最显着和最快的增加。我们还表明，只有复合 IV 抑制剂 KCN 可以减弱 PCP 诱导的 O[公式：见正文] 产生的显着激增。结果表明，线粒体呼吸复合物 I、III 和 IV 是 PAECs 中 O[公式：见文本] 产生的来源，以及一个新的观察结果，即线粒体呼吸解偶联过程中 ROS 的产生部分通过复合物 IV 介导。这种新方法可应用于其他研究，检查应激条件下和 ROS 介导的体外损伤期间的 ROS 产生。