Critical O2 and NO concentrations in NO-induced cell death in a rat liver sinusoidal endothelial cell line

Nitric oxide (NO) plus oxygen (O2) are known to cause cell damage via formation of reactive nitrogen species. NO itself directly inhibits cytochrome oxidase of the mitochondrial respiratory chain in competition with O2, thus inducing a hypoxic-like injury. To assess the critical NO and O2 concentrations for both mechanisms of NO-induced cell injury, cells of a rat liver sinusoidal endothelial cell line were incubated in the presence of the NO donor spermineNONOate at different O2 concentrations, and their loss of viability was determined by the release of lactate dehydrogenase. Protection by ascorbic acid was used as indication for the involvement of reactive nitrogen species, whereas a hypoxic-like injury was indicated by the protective effects of glycine and glucose and the increase in NAD(P)H fluorescence. High concentrations of NO (approx. 10 microM NO) and O2 (21% O2) were required to induce endothelial cell death mediated by formation of reactive nitrogen species. On the other hand, pathophysiologically relevant NO concentrations at low but physiological O2 concentrations (ca. 2 microM NO at 5% O2 and about 1 microM NO at 2% O2) induced hypoxic-like cell death in the endothelial cells that was prevented by the presence of glucose.