Hypoxia-reoxygenation increases O-2(-)center dot efflux which injures endothelial cells by an extracellular mechanism

The mechanisms by which superoxide anion (O-2(-.) injures reoxygenated vascular cells are not clearly understood. We hypothesized that O-2(-.) 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 Cr-51 upon reoxygenation. Evidence for an extracellular site of injury was the following. First, decreasing extracellular O-2(-.) levels (measured by cytochrome c reduction) with the anion channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulfo acid (DIDS) leads to decreased Cr-51 leak. In contrast to its effect on extracellular O-2(-.), DIDS increased intracellular O-2(-.) levels (measured by nitroblue tetrazolium reduction) following reoxygenation. Second, treatment with exogenous superoxide dismutase (SOD), while having no significant effect on intracellular O-2(-.), levels, also decreased Cr-51 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 O-2(-.). Finally, exposure of EC to extracellularly generated O-2(-.) (xanthine oxidase/hypoxanthine system) caused injury, which was decreased by SOD but not by blockade of O-2(-.) entry with 2 DIDS. The mechanism by which O-2(-.) injures EC may involve generation of . OH by surface-associated iron, since iron chelators and . OH scavengers of varying membrane permeability all decreased Cr-51 release to a similar extent. Furthermore, the iron chelators and . OH scavengers also decreased EC Cr-51 leak following exposure to exogenous xanthine oxidase/hypoxanthine but not following exposure to a O-2(-.)-independent agent (A23187). We conclude that hypoxia-reoxygenation injures EC in a manner that is at least in part dependent on the efflux of O-2(-.) into the extracellular space. Endogenous and exogenous strategies for protection against reoxygenation injury must target extracellular O-2(-.) as a potentially harmful species.