Native low-density lipoprotein increases endothelial cell nitric oxide synthase generation of superoxide anion

Abstract

To examine mechanisms by which native low-density 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, n-LDL, did not alter EC NO synthase (cNOS) enzyme activity as measured by the [3H]citrulline assay, N(w)-Nitro- L-arginine methyl ester, a specific inhibitor of cNOS activity, increases C- EC release of O2 by >300% but decreases LDL-treated EC (LDL-EC) release by >95%. L-Arginine inhibits the release of O2 from LDL-ECs by >95% but did not effect C-EC release of O2. Indomethacin and SKF 525A partially attenuates LDL-induced increases in O2 production by ≃50% and 30%, respectively. Thus, n-LDL increases O2 and NO production, which increases the likelihood of the formation of peroxynitrite (ONOO), a potent oxidant. n-LDL increases the levels of nitrotyrosine, a stable oxidation product of ONOO, and tyrosine by ≃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 cNOS generation of O2. Such changes may represent one of the earliest EC perturbations in atherogenesis.