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. However, n-LDL did not alter EC NO synthase (eNOS) enzyme activity as measured by the [3H]citrulline assay. N omega-Nitro-L-arginine methyl ester, a specific inhibitor of eNOS 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 attenuate LDL-induced increases in O2- production by approximately 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 approximately 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.(ABSTRACT TRUNCATED AT 250 WORDS)