Myeloperoxidase Potentiates Nitric Oxide-mediated Nitrosation
2004; Elsevier BV; Volume: 280; Issue: 3 Linguagem: Inglês
10.1074/jbc.m411263200
ISSN1083-351X
AutoresVijaya M. Lakshmi, William M. Nauseef, Terry V. Zenser,
Tópico(s)Renin-Angiotensin System Studies
ResumoNitrosation is an important reaction elicited by nitric oxide (NO). To better understand how nitrosation occurs in biological systems, we assessed the effect of myeloperoxidase (MPO), a mediator of inflammation, on nitrosation observed during NO autoxidation. Nitrosation of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ; 10 μm) to 2-nitrosoamino-3-methylimidazo[4,5-f]quinoline (N-NO-IQ) was monitored by HPLC. Using the NO donor spermine NONOate at pH 7.4, MPO potentiated N-NO-IQ formation. The minimum effective quantity of necessary components was 8.5 nm MPO, 0.25 μm H2O2/min, and 0.024 μm NO/min. Autoxidation was only detected at ≥1.2 μm NO/min. MPO potentiation was not affected by a 40-fold excess flux of H2O2 over NO or less than a 2.4-fold excess flux of NO over H2O2. Potentiation was due to an 8.8-fold increased affinity of MPO-derived nitrosating species for IQ. Autoxidation was inhibited by azide, suggesting involvement of the nitrosonium ion, NO+. MPO potentiation was inhibited by NADH, but not azide, suggesting oxidative nitrosylation with NO2⋅ or an NO2⋅-like species. MPO nonnitrosative oxidation of IQ with 0.3 mm NO2− at pH 5.5 was inhibited by azide, but not NADH, demonstrating differences between MPO oxidation of IQ with NO compared with NO2−. Using phorbol ester-stimulated human neutrophils, N-NO-IQ formation was increased with superoxide dismutase and inhibited by catalase and NADH, but not NaN3. This is consistent with nitrosation potentiation by MPO, not peroxynitrite. Increased N-NO-IQ formation was not detected with polymorphonuclear neutrophils from two unrelated MPO-deficient patients. Results suggest that the highly diffusible stable gas NO could initiate nitrosation at sites of neutrophil infiltration. Nitrosation is an important reaction elicited by nitric oxide (NO). To better understand how nitrosation occurs in biological systems, we assessed the effect of myeloperoxidase (MPO), a mediator of inflammation, on nitrosation observed during NO autoxidation. Nitrosation of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ; 10 μm) to 2-nitrosoamino-3-methylimidazo[4,5-f]quinoline (N-NO-IQ) was monitored by HPLC. Using the NO donor spermine NONOate at pH 7.4, MPO potentiated N-NO-IQ formation. The minimum effective quantity of necessary components was 8.5 nm MPO, 0.25 μm H2O2/min, and 0.024 μm NO/min. Autoxidation was only detected at ≥1.2 μm NO/min. MPO potentiation was not affected by a 40-fold excess flux of H2O2 over NO or less than a 2.4-fold excess flux of NO over H2O2. Potentiation was due to an 8.8-fold increased affinity of MPO-derived nitrosating species for IQ. Autoxidation was inhibited by azide, suggesting involvement of the nitrosonium ion, NO+. MPO potentiation was inhibited by NADH, but not azide, suggesting oxidative nitrosylation with NO2⋅ or an NO2⋅-like species. MPO nonnitrosative oxidation of IQ with 0.3 mm NO2− at pH 5.5 was inhibited by azide, but not NADH, demonstrating differences between MPO oxidation of IQ with NO compared with NO2−. Using phorbol ester-stimulated human neutrophils, N-NO-IQ formation was increased with superoxide dismutase and inhibited by catalase and NADH, but not NaN3. This is consistent with nitrosation potentiation by MPO, not peroxynitrite. Increased N-NO-IQ formation was not detected with polymorphonuclear neutrophils from two unrelated MPO-deficient patients. Results suggest that the highly diffusible stable gas NO could initiate nitrosation at sites of neutrophil infiltration. Nitric oxide (NO) 1The abbreviations used are: NO, nitric oxide; 2-Cl-IQ, 2-chloro-3-methylimidazo[4,5-f]quinoline; DMPO, 5,5-dimethyl-1-pyrroline N-oxide; IQ, 2-amino-3-methylimidazo[4,5-f]quinoline; IQ dimer, 2-(5′-amino)-3,3′-dimethylimidazo[4,5-f]quinoline; MPO, myeloperoxidase; 2-N3-IQ, 2-azido-3-methylimidazo[4,5-f]quinoline; N-NO-IQ, 2-nitrosoamino-3-methylimidazo[4,5-f]quinoline; NO2-IQ1, 2-amino-5-nitro-3-methylimidazo[4,5-f]quinoline; NO2-IQ2, 2-nitroamino-3-methylimidazo[4,5-f]quinoline; PMA, β-phorbol 12-myristate 13-acetate; PMNs, polymorphonuclear neutrophils; RNOS, reactive nitrogen oxygen species; SpN, spermine NONOate; SOD, superoxide dismutase; HPLC, high pressure liquid chromatography; DETAPAC, diethylenetriaminepentaacetic acid. is an essential regulator for a variety of processes critical to normal functions in the cardiovascular, nervous, and immune systems (1Ignarro L.J. Nitric Oxide: Biology and Pathobiology. Academic Press, New York2000Google Scholar). Impaired responses are observed with excessive production of NO in pathological conditions associated with chronic inflammation. Effects of NO can be divided into direct and indirect (2Grisham M.B. Jourd'Heuil D. Wink D.A. Am. J. Physiol. 1999; 39: G315-G321Google Scholar). Direct effects of NO are mediated by low nanomolar concentrations of NO and are illustrated by its binding to guanylate cyclase and eliciting numerous effects, including smooth muscle relaxation. In contrast, indirect effects occur at higher concentrations of NO and result from the reaction of NO with either oxygen (autoxidation) or superoxide to produce reactive nitrogen oxygen species (RNOS). Indirect effects of NO elicited by RNOS include nitrosation, oxidation, and nitration reactions with numerous biological targets representing lipids, proteins, and DNA (2Grisham M.B. Jourd'Heuil D. Wink D.A. Am. J. Physiol. 1999; 39: G315-G321Google Scholar). This can cause lipid peroxidation, inhibition of enzymes, and deamination of DNA. Autoxidation of NO results in the formation of N2O3, which yields the nitrosonium ion, NO+ (3Stamler J.S. Singel D.J. Loscalzo J. Science. 1992; 258: 1898-1902Crossref PubMed Scopus (2459) Google Scholar, 4Lewis R.S. Deen W.M. Chem. Res. Toxicol. 1994; 7: 568-574Crossref PubMed Scopus (245) Google Scholar). The latter is a potent nitrosating agent. Autoxidation is slow and considered unlikely to occur in biological systems because NO can be rapidly inactivated. For example, NO is removed from the vascular compartment by near diffusion-limited interaction with erythrocyte oxyhemoglobin, yielding ferric hemoglobin and nitrate (5Eich R.F. Li T. Lemon D.D. Doherty D.H. Curry S.R. Aitken J.F. Mathews A.J. Johnson K.A. Smith R.D. Phillips G.N. Olson J.S. Biochemistry. 1996; 35: 6976-6983Crossref PubMed Scopus (574) Google Scholar). However, significant amounts of S-, N-, and heme-nitros(yl)ation are detected in vivo, suggesting that modes of nitrosation other than by autoxidation must exist (6Feelisch M. Rassaf T. Mnaimneh S. Singh N. Bryan N.S. Jourd'Heuil D. Kelm M. FASEB J. 2002; 16: 1775-1785Crossref PubMed Scopus (343) Google Scholar, 7Rassaf T. Bryan N.S. Kelm M. Feelisch M. Free Radic. Biol. Med. 2002; 33: 1590-1596Crossref PubMed Scopus (167) Google Scholar). NO is a physiologic substrate for several mammalian peroxidases, including myeloperoxidase (MPO) (8Abu-Soud H.M. Hazen S.L. J. Biol. Chem. 2000; 275: 37524-37532Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar). Direct spectroscopic and rapid kinetic studies support a facile reaction between peroxidases and NO (8Abu-Soud H.M. Hazen S.L. J. Biol. Chem. 2000; 275: 37524-37532Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar, 9Abu-Soud H.M. Hazen S.L. J. Biol. Chem. 2000; 275: 5425-5430Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Peroxidases may play an important role in attenuating direct effects of NO. Organ chamber studies with preconstricted vascular and tracheal rings have demonstrated that catalytic amounts of peroxidase hydrolyzed NO (10Abu-Soud H.M. Khassawneh M.Y. Sohn J.T. Murray P. Haxhiu M.A. Hazen S.L. Biochemistry. 2001; 40: 11866-11875Crossref PubMed Scopus (73) Google Scholar). This prevented smooth muscle relaxation and NO-mediated ring dilation. A subsequent study further emphasized the role of MPO as a leukocyte-derived NO oxidase (11Eiserich J.P. Baldus S. Brennan M.L. Ma W. Zhang C. Tousson A. Castro L. Lusis A.J. Nauseef W.M. White C.R. Freeman B.A. Science. 2002; 296: 2391-2394Crossref PubMed Scopus (603) Google Scholar). The product of MPO metabolism of NO is thought to be NO+ (8Abu-Soud H.M. Hazen S.L. J. Biol. Chem. 2000; 275: 37524-37532Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar). Surprisingly, little consideration has been given to the possible role of MPO-derived NO+ in biologically important nitrosations. Cellular nitrosation has been demonstrated to be distinct from that catalyzed by NO autoxidation in aqueous solution. Experiments, using an oxidant to convert NO directly to NO2⋅, demonstrated that this RNOS facilitates nitrosation by combining with NO to form N2O3 (12Espey M.G. Miranda K.M. Thomas D.D. Wink D.A. J. Biol. Chem. 2001; 276: 30085-30091Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). NO2⋅-mediated nitrosation was shown to be similar to that observed in cells. We have recently demonstrated RNOS nitrosation of the colon carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), forming 2-nitrosoamino-3-methylimidazo[4,5-f]quinoline (N-NO-IQ) (13Lakshmi V.M. Hsu F.F. Zenser T.V. Chem. Res. Toxicol. 2002; 15: 1059-1068Crossref PubMed Scopus (13) Google Scholar). The latter binds DNA and is mutagenic. In this study, we have used IQ as a target for evaluating MPO potentiation of NO-mediated nitros(yl)ation. In view of the potential close temporal and spatial association between NO formation (inducible nitric-oxide synthase), hydrogen peroxide (H2O2) formation, and MPO during an inflammatory response, a better understanding of NO-mediated nitros(yl)ation may improve treatment and aid in prevention of inflammation-related diseases (i.e. certain cancers, atherosclerosis, and asthma). Materials—[2-14C]IQ (10 mCi/mmol, >98% radiochemical purity) was purchased from Toronto Research Chemicals (Toronto, ON). Hydrogen peroxide (H2O2), catalase (bovine liver), ascorbic acid, diethylenetriaminepentaacetic acid (DETAPAC), β-phorbol 12-myristate 13-acetate (PMA), NaCl, NaN3, NADH, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), d-glucose, glucose oxidase (Aspergillus niger; type X-S), and superoxide dismutase (SOD; bovine erythrocytes) were purchased from Sigma. Spermine NONOate (SpN), a NO donor, and MPO from human polymorphonuclear leukocytes (180–200 units/mg protein, ≥95% pure by SDS-PAGE, a Reinheit Zahl value of 0.85 (A430/A280)) were purchased from Calbiochem. SpN decomposes to release free NO into solution with a half-life of 42 min at pH 7.4 and 37 °C (14Maragos C.M. Morley D. Wink D.A. Dunams T.M. Saavedra J.E. Hoffman A. Bove A.A. Isaac L. Hrabie J.A. Keefer L.K. J. Med. Chem. 1991; 34: 3242-3247Crossref PubMed Scopus (702) Google Scholar). Stock solutions (10 mm) were prepared in 10 mm NaOH and were stored at –70 °C. Concentrations of SpN were determined from absorbance values at 250 nm (ϵ250 = 8,000 m–1) (14Maragos C.M. Morley D. Wink D.A. Dunams T.M. Saavedra J.E. Hoffman A. Bove A.A. Isaac L. Hrabie J.A. Keefer L.K. J. Med. Chem. 1991; 34: 3242-3247Crossref PubMed Scopus (702) Google Scholar) directly prior to use. The rate of SpN decomposition was unaltered in reaction mixtures demonstrating potentiation of N-NO-IQ formation by MPO (Fig. 1, lower panel). Rather than adding a bolus of reagent H2O2, it was generated in situ, using d-glucose and glucose oxidase. H2O2 was measured by MPO and 3,5,3′,5′-tetramethylbenzidine (ϵ652 = 3.9 × 104 m–1) (15Josephy P.D. Eling T. Mason R.P. J. Biol. Chem. 1982; 257: 3669-3675Abstract Full Text PDF PubMed Google Scholar). H2O2 generation, assessed by increased absorbance at 652 nm, was calculated from a standard curve made with titrated reagent H2O2 solutions. 2-(5′-Amino)-3,3′-dimethylimidazo[4,5-f]quinoline (IQ dimer) was prepared by HOCl oxidation of IQ in pH 7.4 phosphate buffer (16Tsuda M. Negishi C. Makino R. Sato S. Yamaizumi Z. Hirayama T. Sugimura T. Mutat. Res. 1985; 147: 335-341Crossref PubMed Scopus (41) Google Scholar). The nitration products of IQ metabolism, 2-amino-5-nitro-3-methylimidazo[4,5-f]quinoline (NO2-IQ1) and 2-nitroamino-3-methylimidazo[4,5-f]quinoline (NO2-IQ2), were prepared as previously described (13Lakshmi V.M. Hsu F.F. Zenser T.V. Chem. Res. Toxicol. 2002; 15: 1059-1068Crossref PubMed Scopus (13) Google Scholar). Ultima-Flo AP was purchased from PerkinElmer Life Sciences. Metabolism of IQ by RNOS—To assess NO-mediated nitrosation, [14C]IQ (0.01 mm) was incubated in 100 mm potassium phosphate buffer, pH 7.4, containing 0 to 0.1 mm SpN, 0.1 mm DETAPAC, 1 mm glucose, 0–20 milliunits/ml glucose oxidase, and 0–150 nm MPO in a total volume of 0.1 ml for 40 min at 37 °C. The reaction was started by the addition of SpN, and tubes capped. Blank values were obtained in the absence of SpN. The reactions were stopped by adding 10 mm ascorbic acid in dimethylformamide (0.05 ml). Samples were immediately frozen and kept at –70 °C for analysis by HPLC mobile phase 1 (13Lakshmi V.M. Hsu F.F. Zenser T.V. Chem. Res. Toxicol. 2002; 15: 1059-1068Crossref PubMed Scopus (13) Google Scholar). To assess MPO nonnitrosative oxidation of IQ, [14C]IQ (0.06 mm) was incubated in 100 mm potassium phosphate buffer, pH 5.5, containing 0.1 mm DETAPAC, 1 mm glucose, 15 milliunits/ml glucose oxidase, 0.3 mm NaNO2, and 85 nm MPO in a total volume of 0.1 ml for 60 min at 37 °C (13Lakshmi V.M. Hsu F.F. Zenser T.V. Chem. Res. Toxicol. 2002; 15: 1059-1068Crossref PubMed Scopus (13) Google Scholar). Reactions were started by the addition of [14C]IQ. Blank values were obtained in the absence of glucose oxidase. The reactions were stopped by adding 10 mm ascorbic acid in dimethylformamide (0.05 ml). Samples were immediately frozen and kept at –70 °C for analysis by HPLC mobile phase 1. HPLC Analysis of Metabolites—Metabolites were assessed using a Beckman HPLC with System Gold software and a 5-μm, 4.6 × 150-mm C-18 ultrasphere column attached to a guard column. The mobile phase contained either 20 mm ammonium formate buffer (pH 3.1; mobile phase 1) or 20 mm ammonium acetate buffer (pH 5.0; mobile phase 2) in 8% acetonitrile, 0–2 min; 8–16%, 2–10 min; 16–21%, 13–18 min; 21–35%, 18–23 min; 35–8%, 30–35 min; flow rate 1 ml/min. Radioactivity in HPLC eluents was measured using a FLO-ONE radioactive flow detector. Data are expressed as a percentage of total radioactivity or pmol recovered by HPLC. Preparation of Human Polymorphonuclear Neutrophils (PMNs)— Human blood from healthy donors was mixed with EDTA (0.2% final concentration) and immediately layered over an equal volume of neutrophil isolation medium from Robins Scientific Corp. (Sunnyvale, CA). Neutrophils were isolated by centrifugation using the manufacturer's specifications. Red blood cell contamination was eliminated by hypotonic lysis at 4 °C. Cells were resuspended at 2 × 106 cells/ml in Hanks' balanced salt solution with calcium, magnesium, and bicarbonate. All protocols were in accordance with institutional guidelines on research involving human subjects of either the St. Louis VA Medical Center or the University of Iowa. All participants gave written informed consent. For experiments with MPO-deficient patients, blood was collected in lithium heparin tubes and shipped overnight for analysis the next day. A normal healthy control sample of blood was drawn and included in the shipment. Neutrophils were isolated by a two-step procedure. First red blood cells were removed by dextran sedimentation, Mr 200,000–300,000 (MP-Biomedicals, Irvine, CA). Neutrophils were isolated from the supernatant following Ficoll-Hypaque Plus (Amersham Biosciences) gradient centrifugation. Neutrophils were resuspended in Hanks' balanced salt solution with calcium, magnesium, and bicarbonate. Molecular characteristics of the two MPO-deficient patients have been reported (17Nauseef W.M. Cogley M. McCormick S. J. Biol. Chem. 1996; 271: 9546-9549Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 18Nauseef W.M. Cogley M. Bock S. Petrides P.E. J. Leukocyte Biol. 1998; 63: 264-269Crossref PubMed Scopus (30) Google Scholar). Metabolism of IQ by Neutrophils—Neutrophils (0.3 × 106 cells in 0.3 ml) were incubated with 0.01 mm [14C]IQ in 12 × 75-mm polypropylene tubes at 37 °C for 36 min in Hanks' balanced salt solution containing calcium, magnesium, and bicarbonate without phenol red. Where indicated, 0.054 mm PMA, 1.2 μm NO/min (0.05 mm SpN), 1 mm NaN3, and 0.1 mm NADH were added at 5, 6, 8, and 8 min, respectively, whereas 66 μg/ml catalase and 66 μg/ml SOD were present at zero time. Blank values were obtained in the absence of SpN. The reaction was stopped by placing on ice and freezing at –70 °C. Samples were sonicated for 15 s three times, and 0.3 ml of dimethylformamide containing 2 mm ascorbic acid was added. Samples were spun at 1,500 × g for 10 min. The supernatant was evaporated, and residue was dissolved in 0.1 ml of methanol/dimethylformamide (8:2) and analyzed by HPLC with mobile phase 1. The oxidant burst response was measured for each PMN preparation (19Babior B.M. Kipnes R.S. Curnutte J.T. J. Clin. Invest. 1973; 52: 741-744Crossref PubMed Scopus (2135) Google Scholar). Superoxide generation was activated by the addition of PMA. Superoxide-specific reduction of cytochrome c was determined spectrophotometrically (ϵ550 = 21.1 mm–1 cm–1) and was inhibited by superoxide dismutase (10 μg/ml). Values observed with cells in the absence of PMA were considered as blanks. To assess the authenticity of neutrophil-derived N-NO-IQ, selected supernatants were pooled after HPLC analysis, evaporated to dryness, and converted to either 2-azido-3-methylimidazo[4,5-f]quinoline (2-N3-IQ) or 2-chloro-3-methylimidazo[4,5-f]quinoline (2-Cl-IQ) derivatives. For 2-N3-IQ, pH 2.0, ammonium formate was added along with 10 mm NaN3. After 60 min at 37 °C, the pH was adjusted to pH 7.0–7.4. For 2-Cl-IQ, the residue was dissolved in 0.18 ml of acetonitrile, and 0.02 ml of 1 n HCl was added for 30 min at 37 °C and then neutralized with 1 n NH4OH. Authentic N-NO-IQ (98% pure) was treated in a similar manner to prepare standards. A recent study has verified the structure of these compounds (20Lakshmi V.M. Hsu F.F. Zenser T.V. Chem. Res. Toxicol. 2004; 17: 709-716Crossref PubMed Scopus (7) Google Scholar). Samples were analyzed by HPLC on mobile phase 1 and 2. Statistical Analysis—Data are expressed as a mean ± S.E., and significant differences were evaluated using Student's paired t test with p < 0.05. MPO Potentiation of NO-mediated Nitrosation—MPO was evaluated to determine its effect on NO-mediated nitrosation of IQ (Fig. 1). Previous studies have demonstrated significant transformation of IQ to a stable N-nitroso product, N-NO-IQ, during autoxidation of NO, using a high flux of NO (∼30 μm NO/min, 0.1 mm diethylamine NONOate) (13Lakshmi V.M. Hsu F.F. Zenser T.V. Chem. Res. Toxicol. 2002; 15: 1059-1068Crossref PubMed Scopus (13) Google Scholar). However, using a flux of NO more likely to occur during inflammation (2.4 μm/min, 0.1 mm SpN), N-NO-IQ formation due to autoxidation (0.2 μm) was near the limit of detection, 1.6% of the total radioactivity recovered by HPLC (Fig. 1, top). In the absence of NO, MPO (85 nm) and H2O2 (10 μm/min H2O2) did not catalyze N-NO-IQ formation or metabolize IQ (Fig. 1, middle). When incubation mixtures contained NO, MPO, and H2O2, significant N-NO-IQ formation (3.1 μm) was detected, 31% of the total radioactivity recovered by HPLC (Fig. 1, bottom). N-NO-IQ formation during the autoxidation of NO was not affected by 10 μm H2O2/min. However, H2O2 is required for the potentiation of N-nitroso formation observed with NO/MPO/H2O2. Using conditions illustrated in the bottom panel, N-NO-IQ formation was linear for at least 60 min. Thus, catalytic amounts of MPO potentiate NO-mediated IQ nitrosation. Effect of MPO Concentration, Fluxes of H2O2 and NO, and Concentrations of IQ on NO Nitrosation—MPO concentration dependent potentiation of NO-mediated IQ nitrosation was evaluated with a constant flux of NO (2.4 μm/min) and H2O2 (10 μm/min) (Fig. 2). Using the conditions illustrated in Fig. 1 (bottom), a range of MPO concentrations from 0 to 170 nm was evaluated. N-NO-IQ formation by autoxidation was significantly increased from 16 ± 2to53 ± 5 pmol (p < 0.05) with 8.5 nm MPO. A linear increase in N-NO-IQ formation was observed from 2.8 to 170 nm MPO. Thus, in the presence of a low flux of NO, MPO elicits a concentration-dependent increase in IQ nitrosation. To investigate MPO potentiation of NO-mediated IQ nitrosation in more detail, N-NO-IQ formation was evaluated with increasing fluxes of H2O2, 0–20 μm/min, in the presence of a constant flux of NO (0.48 μm/min, 0.02 mm SpN) (Fig. 3). Significant increases in N-NO-IQ formation were observed at 0.25 μm H2O2/min (p < 0.05), with maximum increases observed at 10 μm/min. Fluxes of H2O2 (20 μm/min) 40-fold greater than NO did not decrease N-NO-IQ formation. Thus, MPO potentiation of nitrosation was observed at high as well as low fluxes of H2O2. IQ nitrosation was also assessed at varied fluxes of NO (0–0.48 μm/min) in the presence of constant H2O2 production (10 μm/min) (Fig. 4). With MPO and H2O2, N-NO-IQ formation was not detected in the absence of NO or with 0.012 μm NO/min. N-NO-IQ formation was detected with 0.024 μm NO/min and continued to increase from 0.012 to 0.48 μm NO/min. Formation of N-NO-IQ was similar, with fluxes of NO from 0.48 to 2.4 μm/min (not shown). At 0.24 μm NO/min, the efficiency of N-NO-IQ formation (0.038 μm/min) was 16%. Nitrosation of IQ elicited by NO autoxidation was not detected with the fluxes of NO evaluated. Thus, with MPO and H2O2, low fluxes of NO exhibited a dose-responsive increase in IQ nitrosation. MPO potentiation was evaluated with NO fluxes in excess of H2O2 (Fig. 5). N-NO-IQ formation was assessed with increasing fluxes of NO (0.6 to 9.6 μm/min) in the presence and absence of MPO and H2O2 (1 μm/min). From 0.6 and 9.6 μm NO/min, a general increase in N-NO-IQ formation was observed with MPO. N-NO-IQ formation due to autoxidation was observed at fluxes of NO ≥1.2 μm. MPO potentiation is expressed in Fig. 5 as the difference between values observed with MPO minus values observed in its absence (control). Potentiation gradually decreased over the range of NO fluxes assessed with significant decreases observed at all fluxes of NO ≥2.4 μm/min. Thus, MPO potentiation is sensitive to fluxes of NO greater than H2O2 with a ≥2.4-fold excess inhibitory. Nitrosation of IQ illustrated in Fig. 1 (top and bottom) was evaluated kinetically over a range of IQ concentrations (0.005–0.080 mm) (Fig. 6). Keeping the flux of NO constant (2.4 μm/min), N-NO-IQ formation was evaluated in the presence and absence of MPO (85 nm) and H2O2 (10 μm/min). The affinities of nitrosating species produced by NO for IQ were greater in the presence of MPO/H2O2 (–1/Xint = 21 + 2 μm) than with NO alone by autoxidation (–1/Xint = 189 + 3 μm). By contrast at infinite concentrations of IQ, the maximal rates of N-NO-IQ formation were similar with MPO/H2O2 (1/Yint = 24 + 3 pmol/min) and autoxidation (1/Yint = 28 + 1 pmol/min). Thus, potentiation of NO-mediated IQ nitrosation is due to increased affinity of MPO-derived RNOS for IQ. Inhibition of NO and MPO Potentiation of NO Nitrosation—To assess N-NO-IQ formation mediated by NO autoxidation, a high NO flux (9.6 μm/min) was necessary to test different agents (Table I). Autoxidation of IQ was not significantly altered by 100 mm NaCl, 33 μg/ml catalase, or 33 μg/ml SOD. Complete inhibition of N-NO-IQ formation was observed with 0.3 mm ascorbic acid. NaN3 (1 mm), NADH (0.1 mm), and DMPO (30 mm) decreased N-NO-IQ formation 70, 73, and 56%, respectively. With 0.48 μm NO/min and 10 μm/min H2O2, these same agents characterized MPO potentiation of N-NO-IQ formation. MPO potentiation was not significantly altered by 1 mm NaN3, 100 mm NaCl, or 33 μg/ml SOD. Complete inhibition was observed with 0.1 mm NADH, 0.3 mm ascorbic acid, and 33 μg/ml catalase. DMPO (30 mm) decreased N-NO-IQ formation 78%. Test agents did not affect the half-life of SpN. Thus, N-NO-IQ formation mediated by NO and potentiated by MPO have different sensitivities to the selected test agents, suggesting different mechanisms of N-NO-IQ formation.Table IEffect of test agents on N-NO-IQ formation by NO with and without MPOConditionN-NO-IQPercentage of totalpmol%SpN99 ± 5100 + 1 mm NaN330 ± 4bp < 0.03 versus SpN.30 + 0.1 mm NADH27 ± 6bp < 0.03 versus SpN.27 + 100 mm NaCl83 ± 584 + 0.3 mm ascorbic acidNDaND, not detected.0 + 33 μg/ml catalase80 ± 181 + 33 μg/ml SOD104 ± 10105 + 30 mm DMPO44 ± 6bp < 0.03 versus SpN.44SpN + MPO210 ± 15100 + 1 mm NaN3187 ± 389 + 0.1 mm NADHND0 + 100 mm NaCl185 ± 388 + 0.3 mm ascorbic acidND0 + 33 μg/ml catalaseND0 + 33 μg/ml SOD138 ± 1266 + 30 mm DMPO47 ± 1cp < 0.03 versus SpN + MPO.22a ND, not detected.b p < 0.03 versus SpN.c p < 0.03 versus SpN + MPO. Open table in a new tab Inhibition of MPO Nonnitrosative Oxidation—To help interpret the effects of test agents in Table I, the same test agents were used to evaluate MPO nonnitrosative oxidation of IQ (Table II). This oxidation of IQ at pH 5.5 requires not only H2O2, but also NO2− (13Lakshmi V.M. Hsu F.F. Zenser T.V. Chem. Res. Toxicol. 2002; 15: 1059-1068Crossref PubMed Scopus (13) Google Scholar). A previous study identified these products of IQ oxidation by MPO as NO2-IQ1, NO2-IQ2, and IQ dimer. NaN3 (0.3 mm) and ascorbic acid (0.3 mm) completely inhibited the formation of all three products, whereas DMPO (30 mm) significantly reduced their formation. With NADH (0.1 mm), the formation of NO2-IQ1 and NO2-IQ2 was reduced, but not significantly. NaCl (100 mm) reduced the formation of all three products, but only NO2-IQ1 and NO2-IQ2 were significantly reduced. Catalase elicited complete inhibition of all products (not shown). These test agents reveal important differences in the mechanism by which MPO oxidizes IQ with NO (Table I) and oxidizes IQ with NO2−, an end product of NO metabolism (Table II).Table IIEffect of test agents on MPO nonnitrosative oxidation of IQConditionsNO2-IQ1NO2-IQ2IQ dimerpmolComplete413 ± 23227 ± 1580 ± 6 + 0.3 mm NaN3NDaND, not detected.NDND + 0.1 mm NADH337 ± 35200 ± 680 ± 6 + 100 mm NaCl143 ± 8bp < 0.02 versus complete.85 + 5bp < 0.02 versus complete.63 ± 13 + 0.3 mm ascorbic acidNDNDND + 30 mm DMPO52 ± 6bp < 0.02 versus complete.43 ± 5bp < 0.02 versus complete.NDa ND, not detected.b p < 0.02 versus complete. Open table in a new tab NO-mediated Nitrosation with Human PMNs—To assess the significance of MPO potentiation of NO-mediated IQ nitrosation, N-NO-IQ formation by human PMNs was assessed (Table III). Cells were incubated with 0.01 mm IQ and 1.2 μm NO/min (Fig. 7, top). In the absence of PMA, the amount of N-NO-IQ formed was similar to that observed in the absence of cells (not shown). PMA increased N-NO-IQ formation 2.7-fold. This increase was prevented by 66 μg/ml catalase. Values observed with PMA were further increased by the presence of 66 μg/ml SOD (2-fold) (Fig. 7, middle). Catalase and NADH, but not azide, suppressed the increase observed with PMA and SOD. To assess the authenticity of N-NO-IQ, neutrophil-derived N-NO-IQ was converted to 2-N3-IQ by pH 2.0/10 mm NaN3 treatment. As illustrated in Fig. 7 (bottom), this treatment resulted in the disappearance of the N-NO-IQ peak observed in Fig. 7 (middle panel) at 10.2 min and the appearance of a new peak at 15.5 min, 2-N3-IQ. Synthetic 2-N3-IQ standard also eluted at 15.5 min. The yield of 2-N3-IQ was 52%. The elution time of neutrophil-derived and synthetic 2-N3-IQ was identical on a different HPLC solvent system. In addition, treatment of neutrophil-derived N-NO-IQ with 1 n HCl produced 2-Cl-IQ. Neutrophil-derived and synthetic 2-Cl-IQ were shown to have identical elution times on two different HPLC solvent systems. This confirms the formation of N-NO-IQ by PMA-stimulated neutrophils exposed to a constant low flux of NO. Results with human neutrophils are consistent with MPO potentiation of NO-mediated IQ nitrosation.Table IIIFormation of N-NO-IQ by human PMNsConditionsN-NO-IQpmolNeutrophils18 ± 3 + 0.05 mm PMA48 ± 5ap < 0.001 versus neutrophils. + 0.05 mm PMA + 66 μg/ml catalase22 ± 9bp < 0.003 versus PMA. + 0.05 mm PMA + 66 μg/ml SOD99 ± 14bp < 0.003 versus PMA. + 0.05 mm PMA + 66 μg/ml SOD + 66 μg/ml catalase54 ± 5cp < 0.003 versus PMA + SOD. + 0.05 mm PMA + 66 μg/ml SOD + 1.0 mm NaN397 ± 9 + 0.05 mm PMA + 66 μg/ml SOD + 0.1 mm NADH20 ± 2cp < 0.003 versus PMA + SOD.a p < 0.001 versus neutrophils.b p < 0.003 versus PMA.c p < 0.003 versus PMA + SOD. Open table in a new tab To more directly demonstrate a catalytic role for MPO in IQ nitrosation by PMNs, two unrelated patients with MPO-deficient PMNs were evaluated. Normal PMNs incubated with 0.01 mm IQ and 1.2 μm NO/min for 36 min produced 26 ± 3 pmol of N-NO-IQ. In maximally stimulated neutrophils, PMA + SOD produced significantly more N-NO-IQ, 42 ± 3 pmol (p < 0.01). This increase in N-NO-IQ formation was completely inhibited by catalase. As demonstrated in Fig. 7 (bottom), the N-NO-IQ HPLC peak was converted to 2-N3-IQ by incubation of PMN-derived material under acidic conditions with 10 mm NaN3. In the MPO-deficient PMNs, N-NO-IQ production observed for one patient was 27 ± 1 and 26 ± 2 pmol for neutrophils and neutrophils with PMA + SOD, respectively. For the second patient, N-NO-IQ production was 26 ± 2 and 30 ± 2 pmol for neutrophils and neutrophils with PMA + SOD, respectively. The oxidant bursts observed with PMNs from the normal control and MPO-deficient patients were similar. The inability of MPO-deficient PMNs to increase N-NO-IQ forma
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