Somers M J, Burchfield J S, Harrison D G
Division of Cardiology, Emory University School of Medicine and Atlanta VA Hospital, Atlanta, GA 30322, USA.
Antioxid Redox Signal. 2000 Winter;2(4):779-87. doi: 10.1089/ars.2000.2.4-779.
A growing body of evidence has suggested that a membrane-bound NADH/NADPH oxidase is the predominant source of reactive oxygen species (ROS) in vascular cells. Prior studies have used indirect assessments of superoxide including lucigenin-enhanced chemiluminescence, cytochrome c, and fluorescent dye techniques. The present study was performed to determine if NADH/NADPH oxidase function could be detected human endothelial cells using electron spin resonance. Human umbilical vein endothelial cells (HUVEC) were homogenized and fractionated into cytosolic and membrane components. Cell fractions were incubated in buffer containing either NADH or NADPH (100 microM for each) and the spin trap 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO). EPR signals were obtained in a Bruker EMX spectrometer. Cytoplasmic fractions were devoid of activity. In contrast, incubation of membrane fractions with NADH produced a signal with a total peak intensity of 1,038 +/- 64, which was significantly greater than that observed with NADPH (540 +/- 101). The signal was completely inhibited by either manganese superoxide dismutase (MnSOD, 100 U/ml) or the flavoprotein inhibitor diphenylene iodinium (DPI, 100 microM). Rotenone (100 microM) did not significantly alter the signal intensity, (833 +/- 88). These data demonstrate direct evidence for a functional NADH/NADPH oxidase in human endothelial cells and show that electron spin resonance is a useful tool for study of this enzyme system.
越来越多的证据表明，膜结合的NADH/NADPH氧化酶是血管细胞中活性氧（ROS）的主要来源。先前的研究使用了间接评估超氧化物的方法，包括光泽精增强化学发光、细胞色素c和荧光染料技术。本研究旨在确定是否可以使用电子自旋共振在人内皮细胞中检测到NADH/NADPH氧化酶的功能。将人脐静脉内皮细胞（HUVEC）匀浆并分离为胞质和膜成分。将细胞组分在含有NADH或NADPH（各100μM）以及自旋捕获剂5-（二乙氧基磷酰基）-5-甲基-1-吡咯啉N-氧化物（DEPMPO）的缓冲液中孵育。在布鲁克EMX光谱仪中获得电子顺磁共振（EPR）信号。胞质组分没有活性。相比之下，膜组分与NADH孵育产生的信号总峰强度为1,038±64，显著大于与NADPH孵育时观察到的信号（540±101）。该信号被锰超氧化物歧化酶（MnSOD，100 U/ml）或黄素蛋白抑制剂二亚苯基碘鎓（DPI，100μM）完全抑制。鱼藤酮（100μM）并未显著改变信号强度（833±88）。这些数据证明了人内皮细胞中功能性NADH/NADPH氧化酶的直接证据，并表明电子自旋共振是研究该酶系统的有用工具。
