Zweier J L, Kuppusamy P, Lutty G A
Department of Medicine, Johns Hopkins Medical Institutions, Baltimore, MD 21224.
Proc Natl Acad Sci U S A. 1988 Jun;85(11):4046-50. doi: 10.1073/pnas.85.11.4046.
Oxygen free radicals have been demonstrated to be important mediators of postischemic reperfusion injury in a broad variety of tissues; however, the cellular source of free radical generation is still unknown. In this study, electron paramagnetic resonance measurements with the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) demonstrate that bovine endothelial cells subjected to anoxia and reoxygenation become potent generators of superoxide and hydroxyl free radicals. A prominent DMPO-OH signal aN = aH = 14.9 G is observed on reoxygenation after 45 min of anoxic incubation. Quantitative measurements of this free radical generation and the time course of radical generation are performed. Both superoxide dismutase and catalase totally abolish this radical signal, suggesting that O2 is sequentially reduced from O2-. to H2O2 to OH.. Addition of ethanol resulted in trapping of the ethoxy radical, further confirming the generation of OH.. Endothelial radical generation was shown to cause cell death, as evidenced by trypan blue uptake. Radical generation was partially inhibited and partially scavenged by the xanthine oxidase inhibitor allopurinol. Marked inhibition of radical generation was observed with the potent xanthine oxidase inhibitor oxypurinol. These studies demonstrate that endothelial cells subjected to anoxia and reoxygenation, conditions observed in ischemic and reperfused tissues, generate a burst of superoxide-derived hydroxyl free radicals that in turn cause cell injury and cell death. Most of this free radical generation appears to be from the enzyme xanthine oxidase. Thus, endothelial cell free radical generation may be a central mechanism of cellular injury in postischemic tissues.
氧自由基已被证明是多种组织缺血后再灌注损伤的重要介质；然而，自由基产生的细胞来源仍不清楚。在本研究中，使用自旋捕捉剂5,5'-二甲基-1-吡咯啉-N-氧化物（DMPO）进行电子顺磁共振测量表明，经历缺氧和复氧的牛内皮细胞成为超氧阴离子和羟基自由基的强大产生者。在缺氧孵育45分钟后的复氧过程中，观察到一个突出的DMPO-OH信号（aN = aH = 14.9 G）。对这种自由基产生进行了定量测量，并测定了自由基产生的时间进程。超氧化物歧化酶和过氧化氢酶都完全消除了这种自由基信号，表明O2依次从O2-还原为H2O2再到OH-。加入乙醇导致乙氧基自由基被捕捉，进一步证实了OH-的产生。内皮细胞自由基的产生被证明会导致细胞死亡，台盼蓝摄取证明了这一点。自由基的产生被黄嘌呤氧化酶抑制剂别嘌呤醇部分抑制和部分清除。用强效黄嘌呤氧化酶抑制剂氧嘌呤醇观察到自由基产生受到明显抑制。这些研究表明，经历缺氧和复氧（在缺血和再灌注组织中观察到的情况）的内皮细胞会产生一阵由超氧阴离子衍生的羟基自由基，进而导致细胞损伤和细胞死亡。这种自由基产生似乎大部分来自黄嘌呤氧化酶。因此，内皮细胞自由基的产生可能是缺血后组织细胞损伤的核心机制。
