NATIVE LOW-DENSITY-LIPOPROTEIN INCREASES ENDOTHELIAL-CELL NITRIC-OXIDE SYNTHASE GENERATION OF SUPEROXIDE ANION

To examine mechanisms by which native low-density lipoprotein (n-LDL) perturbs endothelial cell (EC) release of superoxide anion (O-2(-)) an d nitric oxide (NO), ECs were incubated with n-LDL at 240 mg cholester ol per deciliter for 4 days with media changes every 24 hours. n-LDL i ncreases EC release of O-2(-) by more than fourfold and increases nitr ite production by 57%. In the conditioned media from day-4 incubations , n-LDL increases total nitrogen oxides 20 times control EC (C-EC) lev els. However, n-LDL did not alter EC NO synthase (eNOS) enzyme activit y as measured by the [H-3]citrulline assay. N-omega-Nitro-L-arginine m ethyl ester, a specific inhibitor of eNOS activity, increases C-EC rel ease 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 app roximate to 50% and 30%, respectively. Thus, n-LDL increases O-2(-) an d NO production, which increases the likelihood of the formation of pe roxynitrite (ONOO-), a potent oxidant. n-LDL increases the levels of n itrotyrosine, a stable oxidation product of ONOO-, and tyrosine by app roximate to 50%. In spite of this increase in oxidative metabolism, an alysis of thiobarbituric acid substances reveals that no significant c hanges 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 rele ase to increase eNOS generation of O-2(-). Such changes may represent one of the earliest EC perturbations in atherogenesis.