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

Abstract

Abstract To examine mechanisms by which native low-density lipoprotein (n-LDL) perturbs endothelial cell (EC) release of superoxide anion (O 2 − ) 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 O 2 − 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 [ 3 H]citrulline assay. N ω -Nitro- l -arginine methyl ester, a specific inhibitor of eNOS activity, increases C-EC release of O 2 − by >300% but decreases LDL-treated EC (LDL-EC) release by >95%. l -Arginine inhibits the release of O 2 − from LDL-ECs by >95% but did not effect C-EC release of O 2 − . Indomethacin and SKF 525A partially attenuate LDL-induced increases in O 2 − production by ≈50% and 30%, respectively. Thus, n-LDL increases O 2 − 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 eNOS generation of O 2 − . Such changes may represent one of the earliest EC perturbations in atherogenesis.