Protein tyrosine nitration in cytokine-activated murine macrophages. Involvement of a peroxidase/nitrite pathway rather than peroxynitrite.
Peroxynitrite, formed in a rapid reaction of nitric oxide (NO) and superoxide anion radical (O(2)), is thought to mediate protein tyrosine nitration in various inflammatory and infectious diseases. However, a recent in vitro study indicated that peroxynitrite exhibits poor nitrating efficiency at biologically relevant steady-state concentrations (Pfeiffer, S., Schmidt, K., and Mayer, B. (2000) J. Biol. Chem. 275, 6346-6352). To investigate the molecular mechanism of protein tyrosine nitration in intact cells, murine RAW 264.7 macrophages were activated with immunological stimuli, causing inducible NO synthase expression (interferon-gamma in combination with either lipopolysaccharide or zymosan A), followed by the determination of protein-bound 3-nitrotyrosine levels and release of potential triggers of nitration (NO, O(2)*, H(2)O(2), peroxynitrite, and nitrite). Levels of 3-nitrotyrosine started to increase at 16-18 h and exhibited a maximum at 20-24 h post-stimulation. Formation of O(2) was maximal at 1-5 h and decreased to base line 5 h after stimulation. Release of NO peaked at approximately 6 and approximately 9 h after stimulation with interferon-gamma/lipopolysaccharide and interferon-gamma/zymosan A, respectively, followed by a rapid decline to base line within the next 4 h. NO formation resulted in accumulation of nitrite, which leveled off at about 50 microm 15 h post-stimulation. Significant release of peroxynitrite was detectable only upon treatment of cytokine-activated cells with phorbol 12-myristate-13-acetate, which led to a 2.2-fold increase in dihydrorhodamine oxidation without significantly increasing the levels of 3-nitrotyrosine. Tyrosine nitration was inhibited by azide and catalase and mimicked by incubation of unstimulated cells with nitrite. Together with the striking discrepancy in the time course of NO/O(2) release versus 3-nitrotyrosine formation, these results suggest that protein tyrosine nitration in activated macrophages is caused by a nitrite-dependent peroxidase reaction rather than peroxynitrite.
Pfeiffer
S
S
Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität Graz, Universitätsplatz 2, A-8010 Graz, Austria.
Lass
A
A
Schmidt
K
K
Mayer
B
B
D000818:Animals; D002460:Cell Line; D016207:Cytokines / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016166:Free Radical Scavengers / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000378:metabolism; D015151:Immunoblotting; D007371:Interferon-gamma / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D009566:Nitrates / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D009573:Nitrites / Q000378:metabolism; D009584:Nitrogen / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D009195:Peroxidase / Q000037:antagonists & inhibitors; D011994:Recombinant Proteins / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism; D013997:Time Factors; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism*; D015054:Zymosan / Q000494:pharmacology
D016207:Cytokines; D004791:Enzyme Inhibitors; D016166:Free Radical Scavengers; D008070:Lipopolysaccharides; D009566:Nitrates; D009573:Nitrites; D011994:Recombinant Proteins; C039642:peroxynitric acid; D009569:Nitric Oxide; C002744:3-nitrotyrosine; D014443:Tyrosine; D007371:Interferon-gamma; D015054:Zymosan; D006861:Hydrogen Peroxide; D009195:Peroxidase; D009584:Nitrogen; D013755:Tetradecanoylphorbol Acetate; D010100:Oxygen