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EPR Detection of Glutathionyl and Protein-tyrosyl Radicals during the Interaction of Peroxynitrite with Macrophages (J774)
2001; Elsevier BV; Volume: 276; Issue: 43 Linguagem: Inglês
10.1074/jbc.m104012200
ISSN1083-351X
AutoresSilvia Lopes de Menezes, Ohára Augusto,
Tópico(s)Redox biology and oxidative stress
ResumoPeroxynitrite is one of the biological oxidants whose addition to cells has been shown to either activate signaling pathways or lead to cell injury, depending on cell type and oxidant concentration. The intermediacy of free radicals in these processes has been directly demonstrated only during the interaction of peroxynitrite with erythrocytes, a particular cell type, due to its high hemoglobin content. Here, we demonstrate that the addition of peroxynitrite to a macrophage cell line (J774) led to the production of glutathionyl and protein-tyrosyl radicals. The glutathionyl radical was characterized by EPR spin-trapping experiments with 5,5-dimethyl-1-pyrroline-N-oxide. Protein-tyrosyl radical formation was suggested by direct EPR spectroscopy and confirmed by EPR spin-trapping experiments with 3,5-dibromo-4-nitrosobenzenesulfonic acid and Western blot analysis of nitrated proteins in treated macrophages. Time dependence studies of free radical formation indicate that intracellular glutathione and unidentified proteins are the initial peroxynitrite targets in macrophages and that their derived radicals trigger radical chain reactions. The results are likely to be relevant to the understanding of the bioregulatory and biodamaging effects of peroxynitrite. Peroxynitrite is one of the biological oxidants whose addition to cells has been shown to either activate signaling pathways or lead to cell injury, depending on cell type and oxidant concentration. The intermediacy of free radicals in these processes has been directly demonstrated only during the interaction of peroxynitrite with erythrocytes, a particular cell type, due to its high hemoglobin content. Here, we demonstrate that the addition of peroxynitrite to a macrophage cell line (J774) led to the production of glutathionyl and protein-tyrosyl radicals. The glutathionyl radical was characterized by EPR spin-trapping experiments with 5,5-dimethyl-1-pyrroline-N-oxide. Protein-tyrosyl radical formation was suggested by direct EPR spectroscopy and confirmed by EPR spin-trapping experiments with 3,5-dibromo-4-nitrosobenzenesulfonic acid and Western blot analysis of nitrated proteins in treated macrophages. Time dependence studies of free radical formation indicate that intracellular glutathione and unidentified proteins are the initial peroxynitrite targets in macrophages and that their derived radicals trigger radical chain reactions. The results are likely to be relevant to the understanding of the bioregulatory and biodamaging effects of peroxynitrite. the sum of peroxynitrite anion (ONOO−, oxoperoxonitrate (−1)) and peroxynitrous acid (ONOOH, hydrogen oxoperoxonitrate) unless specified 3,5-dibromo-4-nitrosobenzenesulfonic acid 5,5-dimethyl-1-pyrroline-N-oxide phosphate-buffered saline Peroxynitrite (ONOO− + ONOOH),1 which is formed by the fast reaction between nitric oxide and superoxide anion, has been receiving increasing attention as a mediator of human diseases and as a toxin against invading microorganisms (1Beckman J.S. Beckman T.W. Chen J. Marshall P.A. Freeman B.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1620-1625Crossref PubMed Scopus (6718) Google Scholar, 2Squadrito G.L. Pryor W.A. Free Radical Biol. Med. 1998; 25: 392-403Crossref PubMed Scopus (730) Google Scholar, 3Ischiropoulos H. Arch. Biochem. Biophys. 1998; 356: 1-11Crossref PubMed Scopus (923) Google Scholar, 4Giorgio S. Linares E. Ischiropoulos H. Zuben F.J.V. Yamada A. Augusto O. Infect. Immun. 1998; 66: 807-814Crossref PubMed Google Scholar, 5Hurst J.K. Lymar S.V. Acc. Chem. Res. 1999; 32: 520-528Crossref Scopus (45) Google Scholar). The compound is a strong oxidant that is able to oxidize and nitrate a variety of biotargets by mechanisms that are presently being elucidated (6Augusto O. Gatti R.M. Radi R. Arch. Biochem. Biophys. 1994; 310: 118-125Crossref PubMed Scopus (169) Google Scholar, 7Gatti R.M. Alvarez B. Vásquez-Vivar J. Radi R. Augusto O. Arch. Biochem. Biophys. 1998; 349: 36-46Crossref PubMed Scopus (65) Google Scholar, 8Merényi G. Lind J. Goldstein S. Czapski G. Chem. Res. Toxicol. 1998; 11: 712-713Crossref PubMed Scopus (127) Google Scholar, 9Richeson C.E. Mulder P. Bowry V.W. Ingold K.U. J. Am. Chem. Soc. 1998; 120: 7211-7219Crossref Scopus (152) Google Scholar, 10Lymar S.V. Hurst J.K. Inorg. Chem. 1998; 37: 294-301Crossref Scopus (166) Google Scholar, 11Goldstein S. Czapski G. J. Am. Chem. Soc. 1999; 121: 2444-2447Crossref Scopus (50) Google Scholar, 12Coddington J.W. Hurst J.K. Lymar S.V. J. Am. Chem. Soc. 1999; 121: 2438-2443Crossref Scopus (235) Google Scholar, 13Gerasimov O.V. Lymar S.V. Inorg. Chem. 1999; 38: 4317-4321Crossref Scopus (97) Google Scholar, 14Lehnig M. Arch. Biochem. Biophys. 1999; 368: 303-318Crossref PubMed Scopus (66) Google Scholar, 15Bonini M.G. Radi R. Ferrer-Sueta G. Da C. Ferreira A.M. Augusto O. J. Biol. Chem. 1999; 274: 10802-10806Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 16Hodges G.R. Ingold K.U. J. Am. Chem. Soc. 1999; 121: 10695-10701Crossref Scopus (123) Google Scholar). Peroxynitrite-mediated oxidations are either bimolecular, first-order on peroxynitrite and target concentration or unimolecular, first order on peroxynitrite and independent of target concentration. Bimolecular processes can result in product yield either around stoichiometry and above, as is the case for thiol oxidation (17Radi R. Beckman J.S. Bush K.M. Freeman B.A. J. Biol. Chem. 1991; 266: 4244-4250Abstract Full Text PDF PubMed Google Scholar), or around 35%, as is the case for carbon dioxide oxidation (10Lymar S.V. Hurst J.K. Inorg. Chem. 1998; 37: 294-301Crossref Scopus (166) Google Scholar, 14Lehnig M. Arch. Biochem. Biophys. 1999; 368: 303-318Crossref PubMed Scopus (66) Google Scholar, 15Bonini M.G. Radi R. Ferrer-Sueta G. Da C. Ferreira A.M. Augusto O. J. Biol. Chem. 1999; 274: 10802-10806Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 16Hodges G.R. Ingold K.U. J. Am. Chem. Soc. 1999; 121: 10695-10701Crossref Scopus (123) Google Scholar). Presently, most investigators accept that product yields around 30% are characteristic of peroxynitrite-mediated free radical processes. Indeed, it has been established that peroxynitrite protonation (pK a = 6.6) leads to its fast decomposition (k = 0.17 s−1 and t 12 = 4.1 s at pH 7.4, 25 °C) to yield ∼70% nitrate and 30% hydroxyl radical and nitrogen dioxide (Reactions 1 and 2) (7Gatti R.M. Alvarez B. Vásquez-Vivar J. Radi R. Augusto O. Arch. Biochem. Biophys. 1998; 349: 36-46Crossref PubMed Scopus (65) Google Scholar, 8Merényi G. Lind J. Goldstein S. Czapski G. Chem. Res. Toxicol. 1998; 11: 712-713Crossref PubMed Scopus (127) Google Scholar, 9Richeson C.E. Mulder P. Bowry V.W. Ingold K.U. J. Am. Chem. Soc. 1998; 120: 7211-7219Crossref Scopus (152) Google Scholar, 11Goldstein S. Czapski G. J. Am. Chem. Soc. 1999; 121: 2444-2447Crossref Scopus (50) Google Scholar, 12Coddington J.W. Hurst J.K. Lymar S.V. J. Am. Chem. Soc. 1999; 121: 2438-2443Crossref Scopus (235) Google Scholar, 13Gerasimov O.V. Lymar S.V. Inorg. Chem. 1999; 38: 4317-4321Crossref Scopus (97) Google Scholar, 14Lehnig M. Arch. Biochem. Biophys. 1999; 368: 303-318Crossref PubMed Scopus (66) Google Scholar). These radicals are the species responsible for peroxynitrite-mediated unimolecular oxidations.ONOO−+H+⇄ONOOHONOOH→0.7NO3−+0.7H++0.3·+NO2+0.3·OHREACTIONS1AND2In biological systems, the half-life of peroxynitrite is expected to be much lower because of its reactions with biotargets, particularly carbon dioxide, hemoproteins, and thiol-containing compounds, all of which react fast with the oxidant in bimolecular reactions (2Squadrito G.L. Pryor W.A. Free Radical Biol. Med. 1998; 25: 392-403Crossref PubMed Scopus (730) Google Scholar, 18Lymar S.V. Hurst J.K. J. Am. Chem. Soc. 1995; 117: 8867-8868Crossref Scopus (495) Google Scholar, 19Denicola A. Freeman B.A. Trujillo M. Radi R. Arch. Biochem. Biophys. 1996; 333: 49-58Crossref PubMed Scopus (508) Google Scholar). The reaction with the biologically ubiquitous carbon dioxide is particularly fast (k = 2.6 × 104m−1 s−1 at pH 7.4, 25 °C) and produces ∼65% nitrate and 35% carbonate radical anion and nitrogen dioxide (Reaction 3) (10Lymar S.V. Hurst J.K. Inorg. Chem. 1998; 37: 294-301Crossref Scopus (166) Google Scholar, 14Lehnig M. Arch. Biochem. Biophys. 1999; 368: 303-318Crossref PubMed Scopus (66) Google Scholar, 15Bonini M.G. Radi R. Ferrer-Sueta G. Da C. Ferreira A.M. Augusto O. J. Biol. Chem. 1999; 274: 10802-10806Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 16Hodges G.R. Ingold K.U. J. Am. Chem. Soc. 1999; 121: 10695-10701Crossref Scopus (123) Google Scholar, 18Lymar S.V. Hurst J.K. J. Am. Chem. Soc. 1995; 117: 8867-8868Crossref Scopus (495) Google Scholar, 19Denicola A. Freeman B.A. Trujillo M. Radi R. Arch. Biochem. Biophys. 1996; 333: 49-58Crossref PubMed Scopus (508) Google Scholar). Relevantly, this reaction diverts biotarget oxidation by peroxynitrite at neutral pH values from two- to one-electron mechanisms (20Bonini M.G. Augusto O. J. Biol. Chem. 2001; 276: 9749-9754Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar).ONOO−+CO2→ONOOCO2−→0.65NO3−+0.65CO2+0.35·NO2+0.35CO3˙−REACTION3Peroxynitrite is capable of diffusing across biological membranes (21Marla S.S. Lee J. Groves J.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14243-14248Crossref PubMed Scopus (281) Google Scholar, 22Denicola A. Souza J.M. Radi R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3566-3571Crossref PubMed Scopus (369) Google Scholar, 23Romero N. Denicola A. Souza J.M. Radi R. Arch. Biochem. Biophys. 1999; 368: 23-30Crossref PubMed Scopus (95) Google Scholar, 24Khairutdinov R.F. Coddington J.W. Hurst J.K. Biochemistry. 2000; 39: 14238-14249Crossref PubMed Scopus (71) Google Scholar), a property that allows the interaction of extracellularly generated peroxynitrite with intracellular targets. There are many studies demonstrating that the addition of exogenous peroxynitrite to cells and cell cultures triggers oxidative events that either activate signaling pathways (see, for instance, Refs. 25Zouki C. Zhang S.L. Chan J.S. Filep J.G. FASEB J. 2001; 15: 25-27Crossref PubMed Scopus (87) Google Scholar, 26Lee C. Miura K. Liu X. Zweier J.L. J. Biol. Chem. 2000; 275: 38965-38972Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 27van der Vliet A. Hristova M. Cross C.E. Eiserich J.P. Goldkorn T. J. Biol. Chem. 1998; 273: 31860-31866Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 28Castro L.A. Robalinho R.L. Cayota A. Meneghini R. Radi R. Arch. Biochem. Biophys. 1998; 359: 215-224Crossref PubMed Scopus (93) Google Scholar) or lead to cell injury (see, for instance, Refs. 29Szabó C. Zingarelli B. O'Connor M. Salzman A.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1753-1758Crossref PubMed Scopus (625) Google Scholar and 30Bolaños J.P. Heales S.J. Land J.M. Clark J.B. J. Neurochem. 1995; 64: 1965-1972Crossref PubMed Scopus (473) Google Scholar), depending on cell type and oxidant concentration. The intermediacy of free radicals in these processes has been directly demonstrated only during the interaction of peroxynitrite with red blood cells, a particular cell type, because of its high hemoglobin content (31Minetti M. Scorza G. Pietraforte D. Biochemistry. 1999; 38: 2078-2087Crossref PubMed Scopus (50) Google Scholar). Accordingly, a long lived hemoglobin-tyrosyl radical has been detected by EPR in incubations of peroxynitrite with erythrocytes (31Minetti M. Scorza G. Pietraforte D. Biochemistry. 1999; 38: 2078-2087Crossref PubMed Scopus (50) Google Scholar). Free radical formation during the interaction of peroxynitrite with other cell types has not been examined. Here, we demonstrate that peroxynitrite addition to a macrophage cell line (J774) led to the EPR detection of glutathionyl and protein-tyrosyl radicals. All reagents were purchased from Sigma, Merck, or Fisher and were analytical grade or better. Peroxynitrite was synthesized from sodium nitrite (0.6 m) and hydrogen peroxide (0.65 m) in a quenched-flow reactor. To eliminate excess hydrogen peroxide, the peroxynitrite solution was treated with manganese dioxide. Synthesized peroxynitrite contained low levels of contaminating hydrogen peroxide ( 80%) by pretreatment with 100 µm BSO for 24 h (34Romão P.R. Fonseca S.G. Hothersall J.S. Noronha-Dutra A.A. Ferreira S.H. Cunha F.Q. Parasitology. 1999; 118: 559-566Crossref PubMed Scopus (36) Google Scholar) led to a much less intense EPR signal that was dominated by the DMPO/⋅OH radical adduct spectrum (Fig. 1, B andF). The above results suggest that intracellular glutathione is an important peroxynitrite target in macrophages. Accordingly, peroxynitrite led to a concentration-dependent depletion of macrophage soluble thiol but had marginal effects upon cell viability in the time scale of the experiments (TableI). Total soluble thiol (protein and nonprotein) was measured to prevent protein precipitation at low pH values, which produces nitric oxide from nitrite (39Zweier J.L. Samouilov A. Kuppusamy P. Biochim. Biophys. Acta. 1999; 1411: 250-262Crossref PubMed Scopus (318) Google Scholar), leading to thiol depletion as confirmed by control experiments. Nitrite is a peroxynitrite decomposition product at neutral pH values and a contaminant of peroxynitrite synthesis (40Pfeiffer 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 (304) Google Scholar, 41Nakao L.S. Ouchi D. Augusto O. Chem. Res. Toxicol. 1999; 12: 1010-1018Crossref PubMed Scopus (51) Google Scholar). In these control experiments, total nonprotein thiol content of macrophages (mostly reduced glutathione) was determined to be 4.5 ± 0.6 nmol/106 cells. Cell viability and cell lysis were marginally affected by peroxynitrite, but some cell lysis (∼20–30%, depending on incubation time) occurred in modified PBS (Table I). Cell lysis was minimized by the addition of 5 mm glucose or by the use of Dulbecco's modified Eagle's medium, but these media alone treated with peroxynitrite produced radicals (data not shown) and could not be used in the EPR experiments. Consequently, it became important to obtain further evidence that peroxynitrite was reacting with intracellular GSH under our experimental conditions. Cell suspensions were then treated with peroxynitrite, centrifuged, resuspended in PBS, and examined by EPR. As shown in Fig. 2, the EPR signal detected in the resuspended pellet 7 min after treatment (Fig. 2 B) was similar to the one detectable in the whole suspension (Fig. 2 A), indicating that most of the radical adducts were produced inside the cells. When the cells were first lysed by 30-min incubation with 1 m phosphate buffer and then treated with peroxynitrite, they showed an intense spectrum dominated by the DMPO/⋅OH adduct (Fig. 2 C). This result is similar to those previously reported in incubations of glutathione solutions with peroxynitrite in the presence of DMPO (6Augusto O. Gatti R.M. Radi R. Arch. Biochem. Biophys. 1994; 310: 118-125Crossref PubMed Scopus (169) Google Scholar). In this case, DMPO/⋅OH is produced by trapping of the hydroxyl radical produced during spontaneous peroxynitrite decomposition and by the decay of the DMPO-OOH adduct. The latter is produced from the superoxide anion resulting from a chain reaction whereby the radicals produced from peroxynitrite (hydroxyl radical and nitrogen dioxide) oxidize glutathione to the glutathionyl radical (Reactions 2, 4, and 5) (6Augusto O. Gatti R.M. Radi R. Arch. Biochem. Biophys. 1994; 310: 118-125Crossref PubMed Scopus (169) Google Scholar). The latter reacts with excess glutathione, producing the glutathione disulfide radical anion that reacts with oxygen to produce superoxide anion (Reactions 6–R9) (6Augusto O. Gatti R.M. Radi R. Arch. Biochem. Biophys. 1994; 310: 118-125Crossref PubMed Scopus (169) Google Scholar, 20Bonini M.G. Augusto O. J. Biol. Chem. 2001; 276: 9749-9754Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar).⋅OH+GSH→GS⋅+H2O⋅NO2+GSH→GS⋅+NO2−+H+GS⋅+GS−→GSSG⨪GSSG⨪+O2→GSSG+O⨪2DMPO+O⨪2+H+→DMPO/⋅OOHDMPO/⋅OOH→→DMPO/⋅OHREACTIONS4–9Indeed, we have previously demonstrated that superoxide dismutase inhibits by about 40% the yield of the DMPO-OH adduct produced in incubations of glutathione solutions with peroxynitrite (6Augusto O. Gatti R.M. Radi R. Arch. Biochem. Biophys. 1994; 310: 118-125Crossref PubMed Scopus (169) Google Scholar). In the case of macrophage suspensions where most glutathione is inside the cells, the dominant adduct was always DMPO/⋅SG (Figs. 1 and 2), and its yield was little affected by the presence of superoxide dismutase (data not shown). Moreover, the presence of 1 mmcarbon dioxide that reacts fast with peroxynitrite, greatly diminishing its half-life (from 4.1 to 0.027 s at pH 7.4, 25 °C) and its opportunity to permeate cell membranes, strongly inhibited the DMPO/⋅SG radical adduct yield (data not shown). It should be emphasized that CO2 increases thiyl radical production from the oxidation of thiol solutions by peroxynitrite at neutral pH values due to the formation of carbonate radical anion and nitrogen dioxide (Reaction 3) (15Bonini M.G. Radi R. Ferrer-Sueta G. Da C. Ferreira A.M. Augusto O. J. Biol. Chem. 1999; 274: 10802-10806Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 20Bonini M.G. Augusto O. J. Biol. Chem. 2001; 276: 9749-9754Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Peroxynitrite and nitrogen dioxide can permeate biological membranes, but the carbonate radical anion cannot (23Romero N. Denicola A. Souza J.M. Radi R. Arch. Biochem. Biophys. 1999; 368: 23-30Crossref PubMed Scopus (95) Google Scholar, 24Khairutdinov R.F. Coddington J.W. Hurst J.K. Biochemistry. 2000; 39: 14238-14249Crossref PubMed Scopus (71) Google Scholar). In agreement, exogenous CO2 inhibited DMPO/⋅SG but increased DMPO/⋅OH adduct yield in a process that was little affected by superoxide dismutase addition (data not shown). In this case, DMPO/⋅OH is likely to be produced by the reaction of the impermeable carbonate radical anion with extracellular DMPO (42Zhang H. Joseph J. Felix C. Kalyanaraman B. J. Biol. Chem. 2000; 275: 14038-14045Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar).Table IEffects of peroxynitrite on macrophage viability and soluble thiol depletionMacrophage treatmentViabilitySoluble thiol%nmol/106 cellsPBS926.9 ± 0.51 mm decomposed ONOO−NM6.5 ± 0.60.25 mm ONOO−875.1 ± 0.60.5 mm ONOO−784.6 ± 0.51.0 mm ONOO−853.4 ± 0.2Macrophages were resuspended in modified PBS (108 cells/ml) and treated as described. After a 10-min incubation, viability and soluble thiol were measured as described under "Experimental Procedures." Although cell viability was high, some cell lysis (20–30%) occurred, including in the controls (see "Results"). All values correspond to the mean of three independent experiments; S.D. are shown in the case of soluble thiol values. NM, not measured. Open table in a new tab Macrophages were resuspended in modified PBS (108 cells/ml) and treated as described. After a 10-min incubation, viability and soluble thiol were measured as described under "Experimental Procedures." Although cell viability was high, some cell lysis (20–30%) occurred, including in the controls (see "Results"). All values correspond to the mean of three independent experiments; S.D. are shown in the case of soluble thiol values. NM, not measured. The addition of 1 mm peroxynitrite to a macrophage suspension (1 × 108 cells/ml) in PBS in the absence of DMPO did not produce EPR signals detectable with a modulation amplitude of 1 G, which is the instrumental parameter usually employed for the detection of spin trap radical adducts. Using modulation amplitudes of 5 G and over, it was possible to clearly detect a one-line EPR signal in the absence of DMPO (Fig. 3). No signal was detected in the absence of peroxynitrite or macrophages or in reverse addition experiments (data not shown). There are very few biomolecule-derived radicals that can be detected by direct EPR in aerobic solutions at room temperature. Among the exceptions are protein-bound tyrosyl (31Minetti M. Scorza G. Pietraforte D. Biochemistry. 1999; 38: 2078-2087Crossref PubMed Scopus (50) Google Scholar,43Barr D.P. Gunther M.R. Deterding L.J. Tomer K.B. Mason R.P. J. Biol. Chem. 1996; 271: 15498-15503Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar) and protein-bound semiquinone radicals (44van der Mer R.A. Duine J.A. FEBS Lett. 1988; 235: 194-200Crossref Scopus (32) Google Scholar). The latter has EPR parameters close to those of the one-line signal detected in peroxynitrite-treated macrophages (g = 2.005; line width = 7.4 G) (Fig. 3 A). Most of reported protein-tyrosyl radicals have g values around 2.004 and line widths higher than 20 G (45DeGray J.A. Lassmann G. Curtis J.F. Kennedy T.A. Marnett L.J. Eling T.E. Mason R.P. J. Biol. Chem. 1992; 267: 23583-23588Abstract Full Text PDF PubMed Google Scholar), although one-line protein signals of about 10 G have been attributed to protein-tyrosyl radicals by parallel spin trapping experiments with nitroso compounds (31Minetti M. Scorza G. Pietraforte D. Biochemistry. 1999; 38: 2078-2087Crossref PubMed Scopus (50) Google Scholar). Likewise, in the presence of 20 mmDBNBS, the one-line signal detected in macrophages treated with peroxynitrite was replaced by an EPR signal that is apparently composed of those of an immobile (2a Nzz = 58.60 G) and a relatively isotropic radical adduct signal (a N = 13.51 G) (Fig. 4A). Both of these adducts have EPR parameters that are consistent with DBNBS/⋅tyrosyl radical adducts (31Minetti M. Scorza G. Pietraforte D. Biochemistry. 1999; 38: 2078-2087Crossref PubMed Scopus (50) Google Scholar, 43Barr D.P. Gunther M.R. Deterding L.J. Tomer K.B. Mason R.P. J. Biol. Chem. 1996; 271: 15498-15503Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). The presence of two radical adducts was also suggested by the fact that the mobile signal decreased with incubation time, whereas the immobilized signal increased (Fig. 4 D). This increase indicates the occurrence of radical chain reactions (also see below), because the added peroxynitrite should have decomposed in less than 1 min (t12 = 4.1 s in the absence of cells). The presence of two major radical adducts was also evidenced by the effects of CO2 clearly inhibiting the isotropic signal intensity and having a minor influence on the immobilized radical adduct (Fig. 4 D). Of note, CO2 also decreased the yield of the one-line signal detected by direct EPR (Fig. 3). The DBNBS/⋅tyrosyl radical adducts were not detectable in control experiments, although other, less intense, EPR signals were observed. Incubation of DBNBS with macrophages led to the detection of a highly immobilized signal that results from DBNBS addition to biomolecules such as lipids and proteins (Fig. 4, C and F) (46Hiramoto K. Hasegawa Y. Kikugawa K. Free Radical Res. 1994; 21: 341-349Crossref PubMed Scopus (21) Google Scholar). However, this signal intensity was too low to become a considerable contribution to the spectra obtained in the presence of peroxynitrite on the time scale of our experiments (Fig. 4). The addition of peroxynitrite to DBNBS alone produced an isotropic radical adduct (a N= 12.60 G) (data not shown) that has been previously attributed to the DBNBS/⋅OH radical adduct (47Kohno M. Yamada M. Mitsuta K. Mizuta Y. Yoshikawa T. Bull. Chem. Soc. Jpn. 1991; 64: 1447-1453Crossref Google Scholar).Figure 4Representative EPR spectra of DBNBS adducts obtained during the interaction of macrophages (J774) with peroxynitrite. The spectra were obtained after the addition of 1 mm peroxynitrite to a macrophage (1 × 108cells/ml) suspension in modified PBS containing 20 mmDBNBS, pH 7.4, at room temperature. A, control incubation after 1 min; B, same as A in the presence of 1 mm CO2; C, macrophages treated with DBNBS after 1 min; D, same as A after 7-min incubation; E, same as B after 7-min incubation; F, same as C after 7-min incubation. The composite spectrum of A is labeled to show its components: immobile DBNBS/⋅tyrosyl-protein (2a Nzz = 58.60 G) (●) and isotropic DBNBS/⋅tyrosyl-protein (a N = 13.51 G) (○) radical adducts. D, the appearance of hyperfine structures is labeled as ×. Instrumental conditions were as follows: microwave power, 20 milliwatts; time constant, 327.7 ms; scan rate, 0.3 G/s; modulation amplitude, 2.5 G; gain, 1.0 × 106.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The above results (Figs. 3 and 4) demonstrate that, together with glutathione (Figs. 1 and 2), protein-tyrosine residues are important peroxynitrite targets in macrophages. The EPR results, however, cannot provide information on the proteins that are oxidized. Both the immobilized and isotropic signals (Fig. 4) may be due to tyrosine residues from different proteins, to different tyrosine residues of the same protein, or even to the same tyrosine residue of one protein in different conformations (45DeGray J.A. Lassmann G. Curtis J.F. Kennedy T.A. Marnett L.J. Eling T.E. Mason R.P. J. Biol. Chem. 1992; 267: 23583-23588Abstract Full Text PDF PubMed Google Scholar). Under our experimental conditions, at least three macrophage proteins (∼76, 69, and 32 kDa) are likely to be oxidized by peroxynitrite to tyrosyl radicals (24Khairutdinov R.F. Coddington J.W. Hurst J.K. Biochemistry. 2000; 39: 14238-14249Crossref PubMed Scopus (71) Google Scholar, 48Santos C.X.C. Bonini M.G. Augusto O. Arch. Biochem. Biophys. 2000; 377: 146-152Crossref PubMed Scopus (101) Google Scholar) because they were shown to be nitrated by nitrotyrosine Western blot analysis (Fig.5). It is not possible to exclude minor amounts of DBNBS-lipid radical adducts in the spectra shown in Fig. 4,A and B. This possibility appears unlikely, however, because reported DBNBS/lipid radical adducts show some hyperfine structure (46Hiramoto K. Hasegawa Y. Kikugawa K. Free Radical Res. 1994; 21: 341-349Crossref PubMed Scopus (21) Google Scholar, 49Kalyanaraman B. Joseph J. Kondratenko N. Parthasarathy S. Biochim. Biophys. Acta. 1992; 1126: 309-313Crossref PubMed Scopus (10) Google Scholar) in contrast with the spectra shown in Fig.4, A and B. Hyperfine structures start to appear in the spectra scanned 7 min after peroxynitrite addition (Fig. 4,D and E; labeled as X), suggesting that lipid oxidation may be a secondary event resulting from free radical chain reactions triggered by peroxynitrite. Our results demonstrate that the addition of peroxynitrite to a macrophage suspension promotes the oxidation of intracellular glutathione and proteins with the production of glutathionyl and protein-tyrosyl radicals, respectively (Figs. Figure 1, Figure 2, Figure 3, Figure 4, Figure 5; Table I). The glutathionyl radical was identified by EPR spin-trapping experiments (Figs. 1 and 2), whereas production of protein-tyrosyl radicals was suggested by direct EPR spectroscopy (Fig. 3) and confirmed by EPR spin-trapping experiments (Fig. 4) and Western blot analysis of nitrated proteins in treated macrophages (Fig. 5). Time dependence studies of free radical production indicate that glutathione (Fig. 1,A and E) and unidentified proteins (Figs. 3 and4, A and D) are likely to be the initial peroxynitrite targets in macrophages. The radicals produced from them trigger radical chain reactions because the yield of the immobilized DBNBS/tyrosyl-protein radical adduct (labeled as ● in Fig.4 A) keeps increasing after complete peroxynitrite decomposition (Fig. 4 D). This late process appears to be accompanied by some lipid oxidation, because hyperfine structures characteristic of DBNBS/lipid radical adducts (labeled as × in Fig. 4 D) (46Hiramoto K. Hasegawa Y. Kikugawa K. Free Radical Res. 1994; 21: 341-349Crossref PubMed Scopus (21) Google Scholar, 49Kalyanaraman B. Joseph J. Kondratenko N. Parthasarathy S. Biochim. Biophys. Acta. 1992; 1126: 309-313Crossref PubMed Scopus (10) Google Scholar) appear in spectra scanned 7 min after peroxynitrite addition to macrophages. 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