Formation of nitric oxide-derived oxidants by myeloperoxidase in monocytes- Pathways for monocyte-mediated protein nitration and lipid peroxidation in vivo

Protein nitration and lipid peroxidation are implicated in the pathogenesis of atherosclerosis; however, neither the cellular mediators nor the reacti on pathways for these events in vivo are established, In the present study, we examined the chemical pathways available to monocytes for generating re active nitrogen species and explored their potential contribution to the pr otein nitration and lipid peroxidation of biological targets, Isolated huma n monocytes activated in media containing physiologically relevant levels o f nitrite (NO2-), a major end product of nitric oxide ((NO)-N-.) metabolism , nitrate apolipoprotein B-100 tyrosine residues and initiate LDL lipid per oxidation. LDL nitration (assessed by gas chromatography-mass spectrometry quantification of nitrotyrosine) and lipid peroxidation (assessed by high-p erformance liquid chromatography with online tandem mass spectrometric quan tification of distinct products) required cell activation and NO2-; occurre d in the presence of metal chelators, superoxide dismutase (SOD), and scave ngers of hypohalous acids; and was blocked by myeloperoxidase (MPO) inhibit ors and catalase, Monocytes activated in the presence of the exogenous (NO) -N-. generator PAPA NONOate (Z-[N-{3-aminopropyl}-N-{n-propyl}amino]diazen- 1-ium-1,2-diolate) promoted LDL protein nitration and lipid peroxidation by a combination of pathways. At low rates of (NO)-N-. flux, both protein nit ration and lipid peroxidation were inhibited by catalase and peroxidase inh ibitors but not SOD, suggesting a role for MPO. As rates of (NO)-N-. flux i ncreased, both nitrotyrosine formation and 9-hydroxy-10,12-octadecadienoate /9-hydroperoxy-10,12-octadecadienoic acid production by monocytes became in sensitive to the presence of catalase or peroxidase inhibitors, but they we re increasingly inhibited by SOD and methionine, suggesting a role for pero xynitrite. Collectively, these results demonstrate that monocytes use disti nct mechanisms for generating (NO)-N-.-derived oxidants, and they identify MPO as a source of nitrating intermediates in monocytes.