Effects of Hydrogen Peroxide on Endothelial

Interactions of granulocytes with vascular endothelial cells, a prominent feature of the inflammatory response, can result in endothelial damage. Hydrogen peroxide, released during the respiratory burst associated with granulocyte activation, has been implicated in causing lethal damage on the basis of experiments that demonstrated catalase-inhibitable release of 5~Cr from endothelial cells (1, 2). Activated granulocytes can also have sublethal effects on endothelium (for example, inducing transient increases in microvascular permeability [3]), but the functional responses of endothelium in such circumstances have not been investigated or quantified. To do this, it would be desirable to define sensitive indices of endothelial functions and to monitor such functions simultaneously with conventional measures of cell damage. We therefore measured K ÷ efflux (using 86Rb), prostaglandin production, and the release of cytoplasmic purines from endothelial cells, as well as measuring cytotoxic effects by monitoring adenine uptake, 51Cr release, and the uptake of vital dye. The damaging stimuli we used were reagent H202 and xanthine plus xanthine oxidase, which has been previously used as a cell-free model of the superoxide-generating system in the granulocyte (4). It is perhaps worth noting that the neutrophil respiratory burst can produce active agents other than those generated by xanthine oxidase (for example, products of myeloperoxidase and any compounds resulting from interactions between activated oxygen species and other neutrophil secretory products), although the major role in neutrophilmediated endothelial destruction has been ascribed to H202 (2). We confirmed that H202 was the product generated by xanthine plus xanthine oxidase that was mainly responsible f,~r the effects on endothelium and we found that the first three of the parameters of cell function listed above were all affected by concentrations of H~O2 at least 30-fold lower than those causing lethal damage to the cells. In addition, exposure to even lower concentrations of H202 (<0.1 #M) prevented endothelial cells from responding to vasoactive agents.