Native Low-Density Lipoprotein Increases Endothelial Cell Nitric Oxide Synthase Generation of Superoxide Anion

To examine mechanisms by which native lowdensity lipoprotein (n-LDL) perturbs endothelial cell (EC) release of superoxide anion (O2) and nitric oxide (NO), ECs were incubated with n-LDL at 240 mg cholesterol per deciliter for 4 days with media changes every 24 hours. n-LDL increases EC release of O2 by more than fourfold and increases nitrite production by 57%. In the conditioned media from day-4 incubations, n-LDL increases total nitrogen oxides 20 times control EC (C-EC) levels. However, n-LDL did not alter EC NO synthase (eNOS) enzyme activity as measured by the [sup 3 H]citrulline assay. N -Nitro-L-arginine methyl ester, a specific inhibitor of eNOS activity, increases C-EC release of O2 by more than 300% but decreases LDL-treated EC (LDL-EC) release by more than 95%. L-Arginine inhibits the release of O2 from LDL-ECs by more than 95% but did not effect C-EC release of O2. Indomethacin and SKF 525A partially attenuate LDL-induced increases in O2 production by approximate equals 50% and 30%, respectively. Thus, n-LDL increases O2 and NO production, which increases the likelihood of the formation of peroxynitrite (ONOO sup minus), a potent oxidant. n-LDL increases the levels of nitrotyrosine, a stable oxidation product of ONOO, and tyrosine by approximate equals 50%. In spite of this increase in oxidative metabolism, analysis of thiobarbituric acid substances reveals that no significant changes in the oxidation of n-LDL occur during the 24-hour incubations with ECs. These data indicate that n-LDL directly perturbs endothelial oxidative metabolism and uncouples L-arginine metabolism from NO release to increase eNOS generation of O2. Such changes may represent one of the earliest EC perturbations in atherogenesis.(Cir Res. 1995;77:510-518.)