Hypoxia-reoxygenation increases O₂^-· efflux which injures endothelial cells by an extracellular mechanism

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 ³¹Cr 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.