Hypoxia-reoxygenation increases superoxide anion efflux which injures endothelial cells by an extracellular mechanism. Terada, Lance S. Webb-Waring Institute for Biomedical Research and the Department of Medicine at the University of Colorado Health Sciences Center, Denver, CO
APStracts 2:0346H, 1995.
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: 1) decreasing extracellular O2__ levels (measured by cytochrome c reduction) with the anion channel blocker 4,4'-diisocyanatos tilbene -2,2'-disulfonic acid (DIDS) lead to decreased 51Cr leak. In contrast to its effect on extracellular O2__, DIDS increased intracellular O2__ levels (measured by nitroblue tetrazolium reduction) following reoxygenation. 2) Treatment with exogenous SOD, while having no significant effect on intracellular O2__ levels, also decreased 51Cr leak. Further, 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__. 3) 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. Further, 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 which is at least in part dependent upon 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. 

Received 31 March 1995; accepted in final form 3 August 1995.
APS Manuscript Number H311-5.
Article publication pending Am. J. Physiol. (Heart Circ. Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 14 August 1995.