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.
Signal transduction involved in lipoxin A4-induced protection of tubular epithelial cells against hypoxia/reoxygenation injury