a Medical College of Wisconsin Department of Biomedical Engineering , Marquette University , Milwaukee , WI , USA.
c Division of Pulmonary and Critical Care Medicine , Medical College of Wisconsin , Milwaukee, WI , USA.
Free Radic Res. 2018 Sep;52(9):1052-1062. doi: 10.1080/10715762.2018.1511051. Epub 2018 Sep 28.
The objectives of this study were to develop a robust protocol to measure the rate of hydrogen peroxide (HO) production in isolated perfused rat lungs, as an index of oxidative stress, and to determine the cellular sources of the measured HO using the extracellular probe Amplex red (AR). AR was added to the recirculating perfusate in an isolated perfused rat lung. AR's highly fluorescent oxidation product resorufin was measured in the perfusate. Experiments were carried out without and with rotenone (complex I inhibitor), thenoyltrifluoroacetone (complex II inhibitor), antimycin A (complex III inhibitor), potassium cyanide (complex IV inhibitor), or diohenylene iodonium (inhibitor of flavin-containing enzymes, e.g. NAD(P)H oxidase or NOX) added to the perfusate. We also evaluated the effect of acute changes in oxygen (O) concentration of ventilation gas on lung rate of HO release into the perfusate. Baseline lung rate of HO release was 8.45 ± 0.31 (SEM) nmol/min/g dry wt. Inhibiting mitochondrial complex II reduced this rate by 76%, and inhibiting flavin-containing enzymes reduced it by another 23%. Inhibiting complex I had a small (13%) effect on the rate, whereas inhibiting complex III had no effect. Inhibiting complex IV increased this rate by 310%. Increasing %O in the ventilation gas mixture from 15 to 95% had a small (27%) effect on this rate, and this O-dependent increase was mostly nonmitochondrial. Results suggest complex II as a potentially important source and/or regulator of mitochondrial HO, and that most of acute hyperoxia-enhanced lung rate of HO release is from nonmitochondrial rather than mitochondrial sources.
本研究旨在建立一种稳健的方案，以测量分离灌注大鼠肺中过氧化氢 (HO) 的产生速率作为氧化应激的指标，并使用细胞外探针 Amplex red (AR) 确定所测量的 HO 的细胞来源。将 AR 添加到分离灌注的大鼠肺的再循环灌流液中。在灌流液中测量 AR 的高荧光氧化产物 Resorufin。进行了没有和有 Rotenone（复合物 I 抑制剂）、Thenoyltrifluoroacetone（复合物 II 抑制剂）、Antimycin A（复合物 III 抑制剂）、Potassium cyanide（复合物 IV 抑制剂）或 Dihydroethidium（黄素酶抑制剂，例如 NAD(P)H 氧化酶或 NOX）添加到灌流液中的实验。我们还评估了通气气体中氧气 (O) 浓度的急性变化对肺 HO 释放到灌流液中的速率的影响。基线肺 HO 释放速率为 8.45±0.31（SEM）nmol/min/g 干重。抑制线粒体复合物 II 将该速率降低了 76%，而抑制黄素酶将其降低了另外 23%。抑制复合物 I 对该速率的影响较小（13%），而抑制复合物 III 则没有影响。抑制复合物 IV 将该速率增加了 310%。将通气气体混合物中的 O 百分比从 15%增加到 95%对该速率的影响较小（27%），并且这种 O 依赖性增加主要是非线粒体来源的。结果表明，复合物 II 可能是线粒体 HO 的重要来源和/或调节剂，并且急性高氧增强肺 HO 释放的速率主要来自非线粒体而非线粒体来源。
