Terada L S
Webb-Waring Institute for Biomedical Research, University of Colorado Health Sciences Center, Denver 80262, USA.
Am J Physiol. 1996 Mar;270(3 Pt 2):H945-50. doi: 10.1152/ajpheart.1996.270.3.H945.
The mechanisms by which superoxide anion (O2-.) injures reoxygenated vascular cells are not clearly understood. We hypothesized that O2-. formed in an intracellular compartment during reoxygenation may egress through plasmalemmal anion channels and mediate injury from an extracellular site. Bovine pulmonary artery endothelial cells (EC) kept hypoxic for 48 h had increased release of preloaded 51Cr upon reoxygenation. Evidence for an extracellular site of injury was the following. First, decreasing extracellular O2-. levels (measured by cytochrome c reduction) with the anion channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) leads to decreased 51Cr leak. In contrast to its effect on extracellular O2-., DIDS increased intracellular O2-. levels (measured by nitroblue tetrazolium reduction) following reoxygenation. Second, treatment with exogenous superoxide dismutase (SOD), while having no significant effect on intracellular O2-. levels, also decreased 51Cr leak. Furthermore, cotreatment of EC with DIDS did not abrogate the protective effects of exogenous SOD, suggesting that SOD decreased injury by decreasing extracellular and not intracellular O2-. Finally, exposure of EC to extracellularly generated O2-. (xanthine oxidase/hypoxanthine system) caused injury, which was decreased by SOD but not by blockade of O2-. entry with DIDS. The mechanism by which O2-. injures EC may involve generation of .OH by surface-associated iron, since iron chelators and .OH scavengers of varying membrane permeability all decreased 51Cr release to a similar extent. Furthermore, the iron chelators and .OH scavengers also decreased EC 51Cr leak following exposure to exogenous xanthine oxidase/hypoxanthine but not following exposure to a O2(-.)-independent agent (A23187). We conclude that hypoxia-reoxygenation injures EC in a manner that is at least in part dependent on the efflux of O2-. into the extracellular space. Endogenous and exogenous strategies for protection against reoxygenation injury must target extracellular O2-. as a potentially harmful species.
超氧阴离子（O2-.）损伤复氧血管细胞的机制尚不清楚。我们推测，复氧过程中在细胞内区室形成的O2-.可能通过质膜阴离子通道流出，并介导细胞外部位的损伤。对牛肺动脉内皮细胞（EC）进行48小时缺氧处理后，复氧时预加载的51Cr释放增加。损伤发生在细胞外部位的证据如下。首先，用阴离子通道阻滞剂4,4'-二异硫氰基芪-2,2'-二磺酸（DIDS）降低细胞外O2-.水平（通过细胞色素c还原测量）会导致51Cr泄漏减少。与它对细胞外O2-.的作用相反，DIDS在复氧后增加了细胞内O2-.水平（通过硝基蓝四氮唑还原测量）。其次，用外源性超氧化物歧化酶（SOD）处理，虽然对细胞内O2-.水平没有显著影响，但也减少了51Cr泄漏。此外，用DIDS对EC进行联合处理并没有消除外源性SOD的保护作用，这表明SOD通过降低细胞外而非细胞内的O2-.来减少损伤。最后，将EC暴露于细胞外产生的O2-.（黄嘌呤氧化酶/次黄嘌呤系统）会导致损伤，SOD可降低这种损伤，但用DIDS阻断O2-.进入则不能。O2-.损伤EC的机制可能涉及表面相关铁产生.OH，因为不同膜通透性的铁螯合剂和.OH清除剂均能在相似程度上降低51Cr释放。此外，铁螯合剂和.OH清除剂在EC暴露于外源性黄嘌呤氧化酶/次黄嘌呤后也能降低51Cr泄漏，但在暴露于不依赖O2(-.)的试剂（A23187）后则不能。我们得出结论，缺氧-复氧以至少部分依赖于O2-.外流进入细胞外空间的方式损伤EC。针对复氧损伤的内源性和外源性保护策略必须将细胞外O2-.作为潜在有害物质加以靶向。
