Protein Tyrosine Nitration in Cytokine-activated Murine Macrophages

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-γ 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, H2O2, 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 ∼6 and ∼9 h after stimulation with interferon-γ/lipopolysaccharide and interferon-γ/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 μm 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. 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-γ 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, H2O2, 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 ∼6 and ∼9 h after stimulation with interferon-γ/lipopolysaccharide and interferon-γ/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 μm 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. high performance liquid chromatography N-acetyl 3-aminotyrosine dihydrorhodamine 123 lipopolysaccharide manganese (III) tetrakis(4-benzoic acid) porphyrin NG-nitro-l-arginine nitric oxide superoxide anion radical phosphate-buffered saline polyethylene glycol-labeled catalase PEG-labeled superoxide dismutase phorbol 12-myristate 13-acetate Tris-buffered saline containing Tween 20 zymosan A interferon The free radical nitric oxide (NO) is produced by constitutive and inducible nitric-oxide synthases and regulates numerous biological processes, including relaxation of blood vessels and neurotransmitter release in the brain. However, overproduction of NO appears to contribute essentially to tissue injury in inflammatory and ischemic conditions (1Mayer B. Hemmens B. Trends Biochem. Sci. 1997; 22: 453-498Abstract Full Text PDF PubMed Scopus (509) Google Scholar). One of the mechanisms by which excess NO can injure tissues is by its nearly diffusion-controlled reaction with O⨪2to give peroxynitrite, a potent oxidant thought to be a key mediator of NO-mediated tissue injury in atherosclerosis, congestive heart failure, glutamate excitotoxicity, and other disease states involving inflammatory oxidative stress (2Beckman J.S. Koppenol W.H. Am. J. Physiol. Cell Physiol. 1996; 40: C1424-C1437Crossref Google Scholar). There are several pieces of evidence implicating peroxynitrite as toxic agent in these pathologies as follows. (i) All of these diseases are associated with increased expression of inducible NO synthase, resulting in sustained formation of NO over relatively long periods of time, (ii) oxidative stress causes increased generation of O⨪2, (iii) authentic peroxynitrite triggers tyrosine nitration of a wide variety of proteins known to subserve important cellular functions that are lost upon nitration, and (iv) 3-nitrotyrosine levels have been observed in the injured tissues by both immunohistochemical techniques and quantitative analyses with HPLC1 or gas chromatography-mass spectrometry (3Ischiropoulos H. Arch. Biochem. Biophys. 1998; 356: 1-11Crossref PubMed Scopus (919) Google Scholar). Despite this apparently conclusive link between oxidative tissue injury, peroxynitrite, and tyrosine nitration, direct evidence for peroxynitrite-mediated nitration in vivo is still lacking (4Halliwell B. Zhao K. Whiteman M. Free Radical Res. 1999; 31: 651-669Crossref PubMed Scopus (264) Google Scholar, 5van der Vliet A. Eiserich J.P. Shigenaga M.K. Cross C.E. Am. J. Respir. Crit. Care Med. 1999; 160: 1-9Crossref PubMed Scopus (277) Google Scholar). This is of particular relevance because recent in vitro studies suggest that co-generation of NO and O⨪2, an obviously better approximation to the in vivo situation than bolus addition of concentrated peroxynitrite solutions, does not cause significant nitration of free tyrosine (6van der Vliet A. Eiserich J.P. O'Neill C.A. Halliwell B. Cross C.E. Arch. Biochem. Biophys. 1995; 319: 341-349Crossref PubMed Scopus (370) Google Scholar, 7Pfeiffer S. Mayer B. J. Biol. Chem. 1998; 273: 27280-27285Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 8Pfeiffer S. Schmidt K. Mayer B. J. Biol. Chem. 2000; 275: 6346-6352Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 9Goldstein S. Czapski G. Lind J. Merenyi G. J. Biol. Chem. 2000; 275: 3031-3036Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 10Hodges G.R. Marwaha J. Paul T. Ingold K.U. Chem. Res. Toxicol. 2000; 13: 1287-1293Crossref PubMed Scopus (44) Google Scholar). Although all of those studies, performed in four independent laboratories with a number of different NO/O⨪2-generating systems including pulse radiolysis, gave essentially identical results, Sawa et al. (11Sawa T. Akaike T. Maeda H. J. Biol. Chem. 2000; 275: 32467-32474Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar) recently reported on highly efficient tyrosine nitration by low fluxes of NO/O⨪2. The reason for this discrepancy is unclear. The striking difference between peroxynitrite generated in situ at relatively low fluxes and bolus addition of authentic peroxynitrite appears to be a consequence of the different steady-state concentrations that are achieved with the two experimental protocols; at low (submicromolar) steady-state concentrations, the reaction of peroxynitrite with tyrosine was found to give almost exclusively dityrosine, i.e. the product of tyrosyl radical dimerization, whereas 3-nitrotyrosine is the major product at the fairly high concentrations of peroxynitrite that occur upon bolus addition of the authentic compound (8Pfeiffer S. Schmidt K. Mayer B. J. Biol. Chem. 2000; 275: 6346-6352Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Recent studies have revealed an alternative mechanism of tyrosine nitration with potential in vivo relevance (4Halliwell B. Zhao K. Whiteman M. Free Radical Res. 1999; 31: 651-669Crossref PubMed Scopus (264) Google Scholar). Heme peroxidases such as myeloperoxidase or eosinophil peroxidase have been shown to utilize H2O2 to oxidize nitrite to a reactive nitrogen oxide species that triggers nitration of protein tyrosine residues and other phenolic compounds (12van der Vliet A. Eiserich J.P. Halliwell B. Cross C.E. J. Biol. Chem. 1997; 272: 7617-7625Abstract Full Text Full Text PDF PubMed Scopus (716) Google Scholar, 13Eiserich J.P. Hristova M. Cross C.E. Jones A.D. Freeman B.A. Halliwell B.,. van der Vliet A. Nature. 1998; 391: 393-397Crossref PubMed Scopus (1353) Google Scholar, 14Wu W. Chen Y. Hazen S.L. J. Biol. Chem. 1999; 274: 25933-25944Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). Since inflammatory processes are typically associated with an infiltration of phagocytes, which contain high levels of heme peroxidases, this pathway has to be considered as a possible alternative to peroxynitrite in mediating protein tyrosine nitration in vivo. Intriguingly, the dependence of peroxidase-catalyzed nitration on the local levels of NO2− and H2O2suggests that the peroxidase pathway operates under exactly the conditions that favor formation of peroxynitrite, i.e.increased formation of both NO and O⨪2, the reactive precursors of NO2− and H2O2, respectively. Thus, the experimental evidence currently available does not allow a decision as to which of the two pathways is responsible for tyrosine nitration in vivo. Although several in vitrostudies with NO/O⨪2-generating systems (see above) argue against peroxynitrite as a mediator of nitration, it should be emphasized that those studies were performed with highly artificialin vitro systems not necessarily reflecting the in vivo situation. As a first approach in addressing this issue, we attempted to clarify the cellular pathways mediating protein tyrosine nitration in cultured macrophages activated with established immunological stimuli. As a model system we used the murine macrophage RAW 264.7 cell line. These cells are known to express high levels of inducible NO synthase and 3-nitrotyrosine-like immunoreactivity in response to immunological challenge with IFN-γ in combination with LPS or zymosan (15Shigenaga M.K. Lee H.H. Blount B.C. Christen S. Shigeno E.T. Yip H. Ames B.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3211-3216Crossref PubMed Scopus (174) Google Scholar). Upon cell activation, we measured several key parameters of the NO/O⨪2/peroxynitrite pathway as a function of time and compared the data with protein tyrosine nitration. These experiments revealed a striking discrepancy in the time course of NO formation and nitration and showed that peroxidase inhibitors as well as catalase attenuated the formation of 3-nitrotyrosine, whereas peroxynitrite scavengers had no significant effects. Based on these results we suggest that tyrosine nitration in cytokine-activated macrophages is mediated by a peroxidase/nitrite pathway rather than NO/O⨪2-derived peroxynitrite. DHR and 3-nitrotyrosine were from Fluka (Vienna, Austria). Recombinant mouse IFN-γ and pronase were from Roche Molecular Biochemicals (Vienna, Austria). MnTBAP was from Alexis (Vienna, Austria). Rabbit anti-human myeloperoxidase antibody was from DAKO (Vienna, Austria). Human myeloperoxidase was from Planta Naturstoffe (Vienna, Austria). The 3-nitrotyrosine antibody (clone 1A6, mouse monoclonal IgG, 100 μg/100 μl) was from Upstate Biotechnology (Lake Placid, NY). Penicillin, amphotericin, and fetal calf serum were from PAA Laboratories GmbH (Linz, Austria). The ECL Western blotting detection system was obtained from Amersham Pharmacia Biotech.Centrifuge tube filters (0.22 μm cellulose acetate) were from Szabo (Vienna, Austria). Lipopolysaccharide was from Salmonella typhosa; bovine erythrocytes SOD, horse heart cytochromec (type VI), and all other chemicals were from Sigma. PEG-Cat and PEG-SOD were prepared according to Beckman et al. (16Beckman J.S. Minor R.L. White C.W. Repine J.E. Rosen G.M. Freeman B.A. J. Biol. Chem. 1988; 263: 6884-6892Abstract Full Text PDF PubMed Google Scholar). All solutions were prepared freshly each day. Water was from a Milli-Q reagent water system from Millipore (Vienna, Austria; resistance ≥ 18 megaohms × cm−1). DHR was dissolved in acetonitrile to 10 mm and kept in the dark until use. MnTBAP was dissolved in methanol to 0.1 m. PBS was 8 mmNa2HPO4, 1.5 mmKH2PO4, 137 mm NaCl, 2.7 mm KCl, pH 7.4. Krebs-Ringer phosphate buffer was 129 mm NaCl, 4.86 mm KCl, 0.54 mmCaCl2, 1.22 mm MgSO4, 15.8 mm NaH2PO4, pH 7.35. TBST was 20 mm Tris/HCl, 137 mm NaCl, 0.05% (w/v) Tween 20, pH 7.7. RAW 264.7 macrophages were cultured in Petri dishes (diameter, 90 mm) at 37 °C and 5% CO2 in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) heat-inactivated fetal calf serum, penicillin (100 units/ml), amphotericin (1.25 μg/ml), and NaHCO3 (3.7 g/l) as described (17Garthwaite J. Southam E. Boulton C.L. Nielsen E.B. Schmidt K. Mayer B. Mol. Pharmacol. 1995; 48: 184-188PubMed Google Scholar). Cells were grown to confluence (∼5 × 107 cells/dish) and incubated for up to 48 h in the presence of IFN-γ (50 units/ml) and either LPS (0.5 μg/ml) or zymosan A (0.5 mg/ml) in fresh phenol red-free Dulbecco's modified Eagle's medium. At the time points indicated in the text and graphs, the activated cells were assayed for the following parameters: nitrite accumulation in the culture medium, release of NO, O⨪2, and H2O2, DHR oxidation, and intracellular levels of protein-bound 3-nitrotyrosine. The concentration of nitrite in the cell culture supernatants was determined photometrically with the Griess assay as described previously (18Pfeiffer S. Gorren A.C.F. Schmidt K. Werner E.R. Hansert B. Bohle D.S. Mayer B. J. Biol. Chem. 1997; 272: 3465-3470Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). The culture medium was removed, and the cells (one Petri dish for each measurement) were washed with PBS, harvested, centrifuged, and resuspended in 0.5 ml of PBS. NO release was continuously monitored with a Clark-type NO-sensitive electrode (Iso-NO, World Precision Instruments, Berlin, Germany) at 37 °C in disposable tubes (19Schmidt K. Mayer B. Titherage M.A. Nitric Oxide Protocols. Humana Press Inc., Totowa, NJ1997: 101-109Google Scholar). After 1 min, 5 μl of a 0.1m solution of l-arginine (final concentration, 1 mm) was injected. NO formation was quantified from the initial release rates obtained after injection ofl-arginine using the Macintosh CHART software. Rates of O⨪2release were measured as PEG-SOD-inhibitable reduction of acetylated cytochrome c as described (20Lass A. Argawal S. Sohal R.S. J. Biol. Chem. 1997; 272: 19199-19204Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). At the indicated time points, the cells were washed three times and equilibrated for 30 min in PBS followed by incubation with 10 μm acetylated cytochrome c for 45 min with and without 150 units of PEG-SOD/ml. The cell supernatants were centrifuged at 1,300 ×g for 3 min followed by the determination of the absorbance at 550 nm against PEG-SOD-containing blanks. PEG-SOD inhibited total cytochrome c reduction by ∼80%. O⨪2 release was calculated using an extinction coefficient of 27,700m−1 × cm−1 at 550 nm (21Ernster L. Dallner G. Biochim. Biophys. Acta. 1995; 1271: 195-204Crossref PubMed Scopus (1097) Google Scholar). Formation of H2O2 was measured as horseradish peroxidase-catalyzed oxidation of fluorescent scopoletin as described (22De la Harpe J. Nathan C.F. J. Immunol. Methods. 1985; 78: 323-336Crossref PubMed Scopus (137) Google Scholar). At the indicated time points, macrophages were washed three times with PBS and incubated for 45 min with an assay mixture containing 30 μm scopoletin, 1 mmNaN3, and 10 units/ml horseradish peroxidase in Krebs-Ringer phosphate buffer. Supernatants were centrifuged at 1,300 × g for 3 min followed by the determination of the fluorescence at excitation and emission wavelengths of 305 and 470 nm, respectively. The fluorescence of the assay mixture without cells was subtracted as the blank. The method was calibrated with standard solutions of H2O2 adjusted photometrically using an extinction coefficient of 40 m−1 × cm−1 at 240 nm. Oxidation of DHR was determined as a measure of peroxynitrite formation (23Crow J.P. Nitric Oxide. 1997; 1: 145-157Crossref PubMed Scopus (552) Google Scholar). At the indicated time points, the cells were washed three times with PBS and incubated for 45 min in PBS containing 0.1 mm DHR and 0.1 mm of the metal chelator diethylenetriaminepentaacetic acid. The cell supernatants were centrifuged at 1,300 ×g for 3 min followed by determination of the absorbance at 500 nm against blank samples obtained by incubation of the assay mixture without cells. DHR oxidation was calculated using an extinction coefficient of 78,800 m−1 × cm−1at 500 nm (23Crow J.P. Nitric Oxide. 1997; 1: 145-157Crossref PubMed Scopus (552) Google Scholar). Protein-bound 3-nitrotyrosine was determined by HPLC with electrochemical detection after derivatization toN-AcATyr following a protocol described recently (24Shigenaga M.K. Methods Enzymol. 1999; 301: 27-40Crossref PubMed Scopus (40) Google Scholar). The cells were homogenized in 0.1 m phosphate buffer, pH 7.4, and adjusted to a protein concentration of 16–30 mg of protein/ml. Protein was determined with the Bradford method using bovine serum albumin as a standard (25Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (214351) Google Scholar). Homogenates (0. 5 ml) were precipitated with 0.6 ml of HPLC grade acetonitrile, thoroughly vortexed and centrifuged (1000 × g), followed by resuspension of the precipitates in 0.1 m phosphate buffer, pH 7.4, and sonication for ∼10 s at 50 watts. This procedure was repeated three times to efficiently wash out non-protein material. The final suspensions were incubated overnight (16–20 h) at 50 °C with 1–2 mg of pronase and 0.5 mm CaCl2. Subsequently, 350-μl aliquots of the samples were centrifuged (20,000 × g), and an equal volume of 3 mphosphate buffer, pH 9.6, was added followed by the addition of 25 μl of acetic anhydride. After 10 min of incubation at ambient temperature, ethyl acetate (1 ml) and formic acid (0.2 ml) were added. The samples were thoroughly vortexed for 30 s and then centrifuged at 20,000 × g for 1 min. The ethyl acetate phase was concentrated to dryness under a gentle stream of N2 at 50 °C. For deacetylation of the phenolic acetate group, the samples were resuspended in 1 n NaOH (60 μl). After 30 min of incubation at 37 °C, 60 μl of 1 m phosphate buffer, pH 6.5, was added followed by the addition of 0.1 m sodium dithionite (10 μl) to reduce the nitro substituent to the corresponding amine. The samples were incubated for 10 min at ambient temperature, acidified by addition of concentrated hydrochloric acid (20 μl), and centrifuged at 20,000 × g for 10 min in centrifuge tube filters. Aliquots (100 μl) were injected onto a 250 × 4 mm C18 reversed phase HPLC column (LiChrospher 100 RP-18, 5-μm particle size, Merck) and eluted with 10 mm H3PO4 at 0.7 ml/min. The performance of the column decreased gradually over time. This loss in resolution was overcome by supplementing the solvent with up to 2% (v/v) methanol. N-AcATyr was detected electrochemically with an ESA Coulochem II detector. The potentials of the two electrodes were set to −70 mV and +70 mV (versus palladium), respectively. The method was calibrated daily with authentic N-AcATyr (5–500 nm) prepared as described (24Shigenaga M.K. Methods Enzymol. 1999; 301: 27-40Crossref PubMed Scopus (40) Google Scholar). The recovery of authentic 3-nitrotyrosine added to homogenates of resting RAW 264.7 macrophages was 69.3 ± 12.8%. To visualize 3-nitrotyrosine formation, RAW 264.7 macrophages were subjected to immunostaining with a monoclonal antibody. The cells were grown to confluence onl-polylysine-treated cover slides followed by activation with IFN-γ/Zy in phenol red-free Dulbecco's modified Eagle's medium for 24 h. After 14 h of activation the test compounds (methionine, 0.25 mm; MnTBAP, 50 μm; KCN, 0, 25 mm; NaN3, 0, 25 mm, and PEG-Cat, 2000 units/ml) were added followed by incubation for a further 10 h. As a negative control, non-activated macrophages were incubated under identical conditions for 24 h. For positive control, the cells were treated with authentic peroxynitrite (1 mm) for 1 h. After incubation, the cover slides were gently rinsed three times with PBS and fixed for 1 h with a solution, pH 6.5, containing Na2HPO4 (6.5 g/liter), NaH2PO4 (4 g/liter), (v/v) 15 ml/liter methanol (15 ml/liter; v/v), and formaldehyde (100 ml of 37%/liter, v/v). Thereafter, cover slides were gently rinsed three times with PBS. The 3-nitrotyrosine antibody was diluted to 10 μg/ml in PBS containing 1% bovine serum albumin (w/v). 50 μl of this solution were carefully applied to each cover slide to cover the entire surface and incubated for 1 h at 37 °C under humidified atmosphere. After gentle rinsing of the cover slides three times with PBS, 50 μl of biotinylated goat anti-mouse IgG (part of the mouse ExtrAvidin peroxidase staining kit obtained from Sigma diluted 1/20 in PBS containing 1% bovine serum albumin) were applied on the cover slides and incubated for 30 min at 37 °C under a humidified atmosphere. Then cover slides were again gently rinsed three times with PBS followed by the application of 100 μl of ExtrAvidin peroxidase (10 μg/ml in PBS) on each cover slide and incubation for 30 min at 37 °C under humidified atmosphere and rinsing of the slides with PBS. The staining solution was prepared by mixing 0.2 ml of 20 mg of 3-amino-9-ethylcarbazole in 2.5 ml of dimethylformamide with 3.8 ml of 0.05 m acetate buffer, pH 5.0. Before use, 20 μl of 3% (v/v) H2O2 were added to the staining solution, and 100 μl were applied on the cover slides until the appropriate color development (3–4 min). Reactions were terminated by rinsing the slides gently with distilled water. Cell homogenates were subjected to SDS-polyacrylamide gel electrophoresis on 12% slab gels (26Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206602) Google Scholar) and transferred onto nitrocellulose membranes in 25 mmTris/HCl, pH 8.3, containing 192 mm glycine, 0.02% (w/v) SDS, and 20% (v/v) methanol at 250 mA for 90 min. Unspecific binding sites were saturated by overnight incubation of the membranes at 4 °C in TBST containing 3% (w/v) ovalbumin. Subsequently, the membranes were washed twice for 5 min followed by incubation for 2 h with the anti-myeloperoxidase antibody diluted 1:500 in TBST containing 0.3% (w/v) ovalbumin. Subsequently, the membranes were washed twice for 15 min with TBST and incubated for 1 h with horseradish peroxidase-labeled anti-rabbit-IgG antibody that had been diluted 1:5000 in TBST buffer containing 0.3% (w/v) ovalbumin. Finally, the membranes were washed three times for 20 min with TBST buffer and processed with the ECL Western-blotting detection system according to the recommendations of Amersham Pharmacia Biotech. Unless otherwise indicated, release rates are expressed as amounts of product (pmol or pg)/min/mg of total cell protein. Results are the mean values ± S.E. ofn experiments as indicated in the figure legends. The statistical significance of the data shown in Fig. 6 was evaluated by analysis of variance using Fisher's protected least significant difference test. Activation of RAW 264.7 macrophages with either IFN-γ/LPS or IFN-γ/Zy led to a pronounced release of NO accompanied by an accumulation of nitrite in the cell culture media. As shown in Fig.1 A, the maximal rates of NO release were 116.2 ± 15.0 and 90.9 ± 11.5 pmol × min−1 × mg−1 at 7 and 9 h after stimulation with IFN-γ/LPS and IFN-γ/Zy, respectively. Note that with both stimuli, NO release was virtually back to base line after 14 h of incubation. The inset in Fig. 1 Ashows that NO release was markedly increased upon the addition ofl-arginine, an observation that agrees well with previous studies reporting on a pronounced dependence of macrophage NO synthesis on extracellular substrate supply (27Hibbs J.B. Vavrin Z. Taintor R.R. J. Immunol. 1987; 138: 550-565PubMed Google Scholar, 28Stuehr D.J. Gross S.S. Sakuma I. Levi R. Nathan C.F. J. Exp. Med. 1989; 169: 1011-1020Crossref PubMed Scopus (374) Google Scholar). NO release was not significantly affected by the addition of SOD (1000 units/ml). The signal rapidly declined to zero upon the addition of the NO scavenger hemoglobin, demonstrating the specificity of the Clark-type NO electrode. The time course of nitrite accumulation in the cell culture supernatant was virtually identical with both combinations of stimuli (Fig.1 B). Nitrite levels progressively increased from 4 to 15 h of incubation followed by a plateau corresponding to nitrite concentrations of about 50 μm. Conversion of the rates of NO release from macrophages activated with either cytokine combination (Fig. 1 A) to accumulating concentrations revealed that the decrease in the rates of NO release is in good accordance with the observed reduction in the rate of nitrite accumulation; based on the nitrite data, the apparent recovery of NO detected with the Clark electrode was ∼50% (not shown). These results indicate that macrophage NO synthesis ceased after about 15 h of cell activation, presumably due to inducible NO synthase inactivation and/or limiting cofactor supply. Interestingly, the small but significant rightward shift of the time course of NO release from macrophages activated with IFN- γ/Zy- as compared with that from IFN-γ/LPS-stimulated cells was not paralleled by a significant difference in the time course of nitrite accumulation, suggesting that specific intracellular pathways may affect net NO release from activated macrophages. The differences in the kinetics of O⨪2release were considered as an obvious explanation. Activation of macrophages with IFN-γ/Zy led to a burst of O⨪2 production (26.5 ± 3.5 pmol × min−1 × mg−1) during the first hour of stimulation followed by a steady decline that reached basal rates (∼2 pmol × min−1 × mg−1) after 7 h (Fig. 2 A). Release of O⨪2 from cells stimulated with IFN-γ/LPS was much less pronounced. The maximal rate of 12.8 ± 1.6 pmol × min−1 × mg−1 observed 2 h post-stimulation had declined to basal rates 4 h after stimulation. As shown in Fig. 2 B, the time course of H2O2 formation was similar to that of O⨪2 with both stimulation protocols, but the overall fluxes were ∼1000-fold higher (note the different scales in the twoy axes of Fig. 2 B). We considered the possibility that the apparent decrease in O⨪2formation was a consequence of a rapid reaction of O⨪2 with NO to yield peroxynitrite and carried out two sets of experiments to test this hypothesis. First, we repeated the experiments shown in Fig.2 A using cells treated with a high concentration of a non-selective NO synthase inhibitor (L-NNA; 1 mm). L-NNA almost completely inhibited nitrite accumulation in the cell culture supernatant (data not shown) but had no effect on the rates of O⨪2 release measured 7 h after cell stimulation (inset to Fig. 2 A). Secondly, we determined the time course of DHR oxidation as a measure for peroxynitrite formation. Neither of the two protocols of macrophage activation (IFN-γ/LPS and IFN-γ/Zy) resulted in a considerable increase in the rates of DHR oxidation (1–3 pmol × min−1 × mg−1), which was insensitive to L-NNA (data not shown). Together, these results argue against peroxynitrite as a major reactive nitrogen species formed by activated macrophages. Protein-bound 3-nitrotyrosine was measured in the cell extracts as the N-acetyl-amino derivative (N-AcATyr). As expected, treatment of macrophages with authentic peroxynitrite (1 mm final) resulted in a pronounced increase in tyrosine nitration from 19.4 ± 17.3 to 855.9 ± 270.2 pg of N-AcATyr/mg of cellular protein (n = 3 each). Fig. 3shows that a significant increase in nitration was also observed upon activation of the macrophages with either IFN-γ/Zy or IFN-γ/LPS. The time course of N-AcATyr formation was similar with both combinations of stimuli, although IFN-γ/Zy led to about a 3-fold higher product formation than IFN-γ/LPS (385.3 ± 77.8 and 127.9 ± 8.7 pg × mg−1, respectively). Nitration occurred with a pronounced lag phase of 6 (IFN-γ/Zy) to 18 h (IFN-γ/LPS), was maximal 24 h post-stimulation, and slowly declined during the next 24 h. Thus, we observed a pronounced difference in the time course of protein tyrosine nitration and NO/O⨪2 formation such that nitration started to increase at a time when the rates of NO/O⨪2 had already declined close to basal levels. These results argue against peroxynitrite as a mediator of tyrosine nitration in activated macrophages. However, because of the apparent lack of peroxynitrite formation, as evident from the lack of significant D