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• W2053018171 abstract "The effects of nitric oxide (NO) on superoxide (O·̄2) generation of the NADPH oxidase in pig neutrophils were studied. NO dose-dependently suppressed O·̄2 generation of both neutrophil NADPH oxidase and reconstituted NADPH oxidase. Effects of NO on NADPH-binding site and the redox centers including FAD and low spin heme in cytochrome b 558 and the electron transfer rates from NADPH to heme via FAD were examined under anaerobic conditions. Both reaction rates and the K m value for NADPH were unchanged by NO. Visible and EPR spectra of cytochrome b 558showed that the structure of heme was unchanged by NO, indicating that NO does not affect the redox centers of the oxidase. In reconstituted NADPH oxidase system, NO did not inhibit O·̄2 generation of the oxidase when added after activation. The addition of NO to the membrane component or the cytosol component inhibited the activity by 24.0 ± 5.3 or 37.4 ± 7.1%, respectively. The addition of NO during the activation process or to the cytosol component simultaneously with myristate inhibited the activity by 74.0 ± 5.2 or 70.0 ± 8.3%, respectively, suggesting that cytosol protein(s) treated with myristate becomes susceptible to NO. Peroxynitrite did not interfere with O·̄2 generation. The effects of nitric oxide (NO) on superoxide (O·̄2) generation of the NADPH oxidase in pig neutrophils were studied. NO dose-dependently suppressed O·̄2 generation of both neutrophil NADPH oxidase and reconstituted NADPH oxidase. Effects of NO on NADPH-binding site and the redox centers including FAD and low spin heme in cytochrome b 558 and the electron transfer rates from NADPH to heme via FAD were examined under anaerobic conditions. Both reaction rates and the K m value for NADPH were unchanged by NO. Visible and EPR spectra of cytochrome b 558showed that the structure of heme was unchanged by NO, indicating that NO does not affect the redox centers of the oxidase. In reconstituted NADPH oxidase system, NO did not inhibit O·̄2 generation of the oxidase when added after activation. The addition of NO to the membrane component or the cytosol component inhibited the activity by 24.0 ± 5.3 or 37.4 ± 7.1%, respectively. The addition of NO during the activation process or to the cytosol component simultaneously with myristate inhibited the activity by 74.0 ± 5.2 or 70.0 ± 8.3%, respectively, suggesting that cytosol protein(s) treated with myristate becomes susceptible to NO. Peroxynitrite did not interfere with O·̄2 generation. Nitric oxide (NO) is now recognized as one of the key mediators in many physiological and pathological processes (see reviews in Refs. 1Nathan C.F. FASEB J. 1992; 6: 3051-3064Crossref PubMed Scopus (4144) Google Scholarand 2Stuehr D.J. Griffith O.W. Adv. Enzymol. Relat. Areas Mol. Biol. 1992; 65: 287-346PubMed Google Scholar). NO is also known to be a multi-functional molecule, one function of which is to inactivate biologically important enzymes such as mitochondrial respiratory enzymes and GAPDH, which play important roles in energy production (3Stuehr D.J. Nathan C.F. J. Exp. Med. 1989; 169: 1543-1555Crossref PubMed Scopus (1592) Google Scholar, 4Dimmeler S. Brüne B. FEBS Lett. 1993; 315: 21-24Crossref PubMed Scopus (27) Google Scholar), ribonucleotide reductase, which is the key enzyme for protein synthesis (5Lepoivre M. Fieschi F. Coves J. Biochem. Biophys. Res. Commun. 1991; 179: 442-448Crossref PubMed Scopus (294) Google Scholar), and the superoxide-generating enzymes, NADPH oxidase (6Clancy R.M. Leszczynska-Piziak J. Abramson S.B. J. Clin. Invest. 1992; 90: 1116-1121Crossref PubMed Scopus (641) Google Scholar) and xanthine oxidase (7Fukahori M. Ichimori K. Ishida H. Nakazawa H. Okino H. Free Radical Res. 1994; 21: 203-212Crossref PubMed Scopus (71) Google Scholar). Particularly important is the effect of NO on NADPH oxidase, because under conditions such as inflammation, the accumulation of phagocytes is a common feature and the induction of NO synthase has been shown. It is plausible that increased formation of NO interferes with the activity of NADPH oxidase and reduces superoxide (O·̄2) production (8Rodenas J. Mitjavila M.T. Carbonell T. Free Radical Biol. Med. 1995; 18: 869-875Crossref PubMed Scopus (86) Google Scholar, 9Liu P. Hock C.E. Nagele R. Wong P.Y. Am. J. Phys. 1997; 272: H2327-H2336Crossref PubMed Google Scholar). Despite the importance of the NO effect on NADPH oxidase, no detailed study has been carried out since the initial report by Clancy et al. (6Clancy R.M. Leszczynska-Piziak J. Abramson S.B. J. Clin. Invest. 1992; 90: 1116-1121Crossref PubMed Scopus (641) Google Scholar) in which inhibition of O·̄2 generation by NO was demonstrated. The underlying mechanism was suggested to be the direct interaction of NO on the membrane components of NADPH oxidase (6Clancy R.M. Leszczynska-Piziak J. Abramson S.B. J. Clin. Invest. 1992; 90: 1116-1121Crossref PubMed Scopus (641) Google Scholar). The NADPH oxidase of phagocytes is a multi-component electron transport system, in which activation requires the assembly of three cytosolic regulatory proteins (p47 phox, p67 phox, and Rac1/Rac2) to membrane-bound cytochrome b 558 (10Chanock S.J. el Benna J. Smith R.M. Babior B.M. J. Biol. Chem. 1994; 269: 24519-24522Abstract Full Text PDF PubMed Google Scholar, 11Thrasher A.J. Keep N.H. Wientjes A. Segal A.W. Biochim. Biophys. Acta. 1994; 1227: 1-24Crossref PubMed Scopus (212) Google Scholar). Cytochrome b 558 is postulated to be a membrane-bound flavocytochrome with six-coordinated low spin heme and FAD as redox centers. The electrons provided by NADPH are thought to be transferred in a linear sequence, NADPH → FAD → heme (Fe3+) →O2. The heme in cytochrome b 558 is assumed to be the terminal electron donor in the production of O·̄2 from molecular oxygen due to its unusually low redox potential, −245 mV (12Cross A.R. Jones O.T.G. Harper A.M. Segal A.W. Biochem. J. 1981; 194: 599-606Crossref PubMed Scopus (138) Google Scholar). Although the most plausible site of NADPH oxidase attacked by NO was suggested to be in membrane protein(s) (6Clancy R.M. Leszczynska-Piziak J. Abramson S.B. J. Clin. Invest. 1992; 90: 1116-1121Crossref PubMed Scopus (641) Google Scholar), a detailed analysis of these effects has not been performed. Considering that nitrosyl-iron complex easily forms in heme-containing enzymes (3Stuehr D.J. Nathan C.F. J. Exp. Med. 1989; 169: 1543-1555Crossref PubMed Scopus (1592) Google Scholar), the heme structure of cytochrome b 558 and the electron flux from substrate (NADPH) to redox centers, FAD and low spin heme, in NADPH oxidase should be examined to clarify the effects of NO on its O·̄2-generating activity. In the present study, we examined the effects of NO on electron fluxes in neutrophil NADPH oxidase. Under aerobic conditions the effects of NO on O·̄2-generating activity of NADPH oxidase (reaction 1) was examined by the cytochrome c reduction method. In this study, we also employed the solubilized NADPH oxidase obtained from stimulated cells and measured its O·̄2-generating activity in the presence of NO. Under anaerobic conditions the effects of NO on the electron transfer reaction in each redox center was examined: NADPH → FAD → exogenous electron acceptor, cytochrome c (reaction 2) and NADPH → FAD → cytochrome b 558(reaction 3). Under both aerobic and anaerobic conditions, the binding ability of NO to the six-coordinated low spin heme (His-Fe3+-His) of cytochrome b 558(reaction 4) was examined by both visible absorption and EPR spectroscopy. We also studied the effects of NO on the activation of the oxidase (reaction 5), i.e. assembly of cytosolic and membrane components using the reconstituted NADPH oxidase system. Finally, we studied the effects of peroxynitrite (ONOO−) on NADPH oxidase to confirm that the results obtained above were caused by NO (and not by ONOO−) because addition of NO in the presence of O·̄2 produces ONOO− at nearly diffusion-limited rates (13Huie R.R. Padmaja S. Free Radical Res. Commun. 1993; 18: 195-199Crossref PubMed Scopus (2009) Google Scholar) and because ONOO− is known to be a potent oxidant. Myristic acid and arachidonic acid from Wako Pure Chemical (Tokyo, Japan) were dissolved in ethanol. Heptylthioglucoside was purchased from Dojindo Laboratories (Kumamoto, Japan). NADPH was from Oriental Yeast (Tokyo, Japan). Superoxide dismutase, cytochrome c (type VI from horse heart), and phorbol myristate acetate (PMA) 1The abbreviations used are: PMA, phorbol myristate acetate; SH3, Src homology 3. were purchased from Sigma. Other reagents were of analytical grade. Saturated NO solution was prepared according to the methods published elsewhere (7Fukahori M. Ichimori K. Ishida H. Nakazawa H. Okino H. Free Radical Res. 1994; 21: 203-212Crossref PubMed Scopus (71) Google Scholar, 14Ignarro L.J. Byrns R.E. Buga G.M. Wood K.S. Circ. Res. 1987; 61: 866-879Crossref PubMed Scopus (944) Google Scholar). Briefly, nitrogen gas was bubbled through 50 mm phosphate buffer (pH 7.0) for 20 min to remove dissolved oxygen. Then authentic NO gas (99%, Nippon Sanso Co. Ltd., Tokyo) that had passed through 1 m KOH to remove nitrogen dioxide was bubbled into the solution for 20 min. The concentration of NO was measured spectrophotometrically as previously reported (15Doyle M.P. Hoekstra J.W. J. Inorg. Biochem. 1981; 14: 351-358Crossref PubMed Scopus (533) Google Scholar). Peroxynitrite was synthesized by the reaction of acidified H2O2and NaNO2 in a quenched flow reactor with a subsequent stabilization induced by 1.5 m NaOH as described previously (16Reed J.W. Ho H.H. Jolly W.L. J. Am. Chem. Soc. 1974; 96: 1248-1249Crossref Scopus (137) Google Scholar). The purity and concentration of ONOO− was checked by its absorbance at 302 nm. The production of ONOO− from NO and O·̄2 was also confirmed by the increase in absorbance at 302 nm. Neutrophils were obtained from pig blood as reported previously (17Wakeyama H. Takeshige K. Takayanagi R. Minakami S. Biochem. J. 1982; 205: 593-601Crossref PubMed Scopus (89) Google Scholar) by the Conray-Ficoll differential density configuration method. The cell pellet was frozen and thawed in the presence of phenylmethylsulfonyl fluoride at a final concentration of 1 mm and sonicated in ice-cold Krebs-Ringer phosphate buffer containing 0.34 m sucrose. Membrane vesicles and cytosol were obtained from sonicated cells by centrifugation (100,000 ×g for 60 min) (18Kakinuma K. Fukuhara Y. Kaneda M. J. Biol. Chem. 1987; 262: 12316-12322Abstract Full Text PDF PubMed Google Scholar). The membrane component of the NADPH oxidase was solubilized from the membrane vesicle with heptylthioglucoside (19Fujii H. Kakinuma K. Biochim. Biophys. Acta. 1991; 1095: 201-209Crossref PubMed Scopus (20) Google Scholar) and used for further purification of cytochrome b 558. The cytosol and solubilized NADPH oxidase from resting membrane vesicles were used in the reconstituted NADPH oxidase. All procedures were applied to both resting and stimulated cells. Cell stimulation was induced by myristate as reported previously (19Fujii H. Kakinuma K. Biochim. Biophys. Acta. 1991; 1095: 201-209Crossref PubMed Scopus (20) Google Scholar). Cytochrome b 558 was purified from the membrane component (20Fujii H. Johnson M.K. Finnegan M.G. Miki T. Yoshida L.S. Kakinuma K. J. Biol. Chem. 1995; 270: 12685-12689Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar) with slight modifications to avoid denaturation of heme in cytochrome b 558. A buffer composed of 50 mm phosphate buffer (pH 7.0), 50 mm NaCl, 10% glycerol, and 0.6% heptylthioglucoside was used in the heparin-Sepharose column. O·̄2-generating activities of both neutrophils and solubilized NADPH oxidase were measured at the rate of cytochrome c reduction subtracting the rate in the presence of superoxide dismutase from that in the absence of superoxide dismutase in an assay medium containing 1.5 mm MgCl2 and 50 mm phosphate buffer (pH 7.4) (21Babior B.M. Curnutte J.T. McMurrich B.J. J. Clin. Invest. 1976; 58: 989-996Crossref PubMed Scopus (223) Google Scholar). The reaction was started by adding PMA for cells (37 °C) or 0.1 mm NADPH for both the stimulated and reconstituted NADPH oxidase (25 °C) as reported previously (22Miki T. Yoshida L.S. Kakinuma K. J. Biol. Chem. 1992; 267: 18695-18701Abstract Full Text PDF PubMed Google Scholar). The increase in absorption at 550–540 nm was monitored in a Hitachi model 556 spectrophotometer. The measurements were performed in neutrophils, reconstituted NADPH oxidase, and solubilized NADPH oxidase obtained from stimulated cells in the presence or the absence of NO or ONOO−. In reconstituted NADPH oxidase, NO was added to membrane or cytosol or to both components before and after activation of the oxidase with myristate. The electron fluxes through two redox centers, FAD and cytochrome b 558, in the electron transport chain of the NADPH oxidase system were examined. The electron flux from NADPH to FAD was evaluated by measuring the reduction rate of cytochrome c, which was utilized as an exogenous electron acceptor (reaction 2) according to the previously reported method (19Fujii H. Kakinuma K. Biochim. Biophys. Acta. 1991; 1095: 201-209Crossref PubMed Scopus (20) Google Scholar). The electron flux to cytochrome b 558 from NADPH catalyzed by FAD was measured by following the absorbance at 558 nm spectrophotometrically (reaction 3) (19Fujii H. Kakinuma K. Biochim. Biophys. Acta. 1991; 1095: 201-209Crossref PubMed Scopus (20) Google Scholar). The extent of reduction of cytochrome b 558 was calculated using the extinction coefficient at 558–540 nm, 21.6 × 103 liter mol−1 cm−1 (23Cross A.W. Higson F.K. Jones O.T.G. Harper A.M. Segal A.W. Biochem. J. 1982; 204: 479-485Crossref PubMed Scopus (154) Google Scholar). Spectral changes of cytochrome b 558 were observed every minute over the 400–600 nm range. Strictly anaerobic conditions were achieved by using glucose (10 mm)/glucose oxidase (40 units/ml) in an airtight cuvette (19Fujii H. Kakinuma K. Biochim. Biophys. Acta. 1991; 1095: 201-209Crossref PubMed Scopus (20) Google Scholar). All data were presented as the means ± S.D. of at least three experiments. Nonlinear least squares regression was used to calculate K m value. Oxygen consumption was measured with a Clark-type oxygen electrode (Yellow Spring Instrument, Yellow Spring, OH) in neutrophils (1 × 107 cells in 1 ml of Krebs-Ringer phosphate buffer with 5 mm glucose) and solubilized NADPH oxidase obtained from stimulated neutrophils (30 μg of protein in 1 ml of Krebs-Ringer phosphate buffer). To determine whether NO binds to the heme of cytochrome b 558, i.e. formation of nitrosyl heme, purified cytochrome b 558 was reduced with dithionite, and its spectrum was observed in the presence or the absence of NO. EPR spectra were recorded in a JEOL JES-FE X-band ESR spectrometer at 77–100 K. To examine the effects of the change in the spin state of the heme on NO binding, intact five-coordinated low spin cytochrome b 558 was denatured at 40 °C for 120 min to form six-coordinated high spin heme. Typical EPR conditions were: microwave power, 5 mW; modulation amplitude, 10 gauss at 100 kHz; response, 0.3 s; sweep time, 4 min. Fig. 1shows the time course of cytochrome c reduction by PMA-stimulated neutrophils in the absence (trace B) or the presence of 25 μm NO (trace C). As a control,trace A was measured in the absence of neutrophils. NO was added 2 min before the addition of cytochrome c to avoid the possible binding of NO to cytochrome c, because NO is reported to bind to a variety of hemoproteins such as hemoglobin (24Shiga K. Hwang K.-J. Tyuma I. Biochemistry. 1969; 8: 378-383Crossref PubMed Scopus (64) Google Scholar) or iron-sulfur centers (25Lancaster J.R. Hibbs J.B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1223-1227Crossref PubMed Scopus (489) Google Scholar). As shown in Fig. 1 A, cytochrome c reduction was not observed when NO was added to the assay buffer, indicating that there is no direct interaction of NO with cytochrome c. (These results were consistent with the report that the rate of NO reaction with cytochrome c is severalfold slower than that with oxygen or proteins under aerobic conditions (26Yoshimura T. Suzuki S. Inorg. Chim. Acta. 1988; 152: 241-249Crossref Scopus (28) Google Scholar).) 2 min after the addition of NO to the cell suspension, the residual concentration of NO was less than 1% of its initial concentration, which excludes the quenching of O·̄2through direct reaction of NO to O·̄2. Neutrophils without NO produced O·̄2 at a rate of 85 nmol/min/107 cells (trace B), whereas O·̄2 generation was decreased to about 70% of that in the presence of NO. These inhibitory effects by NO on O·̄2 generation in neutrophils were also supported by the decrease in oxygen consumption in the presence of NO (21 ± 3.4% reduction). When neutrophils were preincubated with 25 μmnitrite or nitrate (which are end products of NO), no change in O·̄2 generation was observed. Dose dependence of NO on the inhibition of O·̄2 generation is shown in Fig.2 for neutrophils and solubilized NADPH oxidase. In this comparison, we adjusted the cell number of neutrophils and sample volume of the solubilized oxidase to equalize O·̄2production, i.e. 6.1 nmol of O·̄2/min. The inhibitory effect of NO in solubilized NADPH oxidase was less than that in neutrophils. The plausible reason for the difference became clear through further experiments to investigate the mechanisms with which NO inactivates the O·̄2-generating activity of NADPH oxidase.Figure 2Dose dependence of NO-induced inhibition of O·̄2-forming activities of neutrophils and solubilized NADPH oxidase from stimulated cells. Neutrophils (○, 5 × 105 cells) were preincubated with NO at 0–35 μm for 2 min before addition of 30 μmcytochrome c at 37 °C and then stimulated by PMA (4 ng in Me2SO). Solubilized NADPH oxidase prepared from stimulated cells (▵, 35 μg of protein) was preincubated with NO at 0–35 μm 2 min before the addition of 30 μmcytochrome c at 25 °C. Superoxide generation was started by addition of 0.1 mm NADPH.View Large Image Figure ViewerDownload Hi-res image Download (PPT) First, we examined whether NO impairs the binding ability of NADPH to oxidase (reaction 1). K m values for NADPH of the solubilized NADPH oxidase were determined in the presence or the absence of NO. As shown in Table I, the K m obtained with NO was almost the same as that of the oxidase in the absence of NO (35.0 ± 4.2 versus30.6 ± 3.4 μm, respectively), indicating that NO treatment does not affect the NADPH-binding site of the oxidase.Table IKinetic parameters of solubilized NADPH oxidase obtained from stimulated neutrophils in the absence or the presence of NOKm for NADPHV maxμmnmol/min/mg protein−NO30.6 ± 3.4315.5 ± 27.4+NO35.0 ± 4.2223.8 ± 18.5 Open table in a new tab Electron transfer reactions from NADPH to FAD (reaction 2) and from NADPH to cytochrome b 558 (reaction 3) were studied using solubilized NADPH oxidase from stimulated neutrophils under anaerobic conditions (TableII). The cytochrome c-reducing activities of the NADPH oxidase obtained from both stimulated and resting cells were in the same range, either in the presence or the absence of 25 μm NO, indicating that the electron flux from NADPH to FAD is not affected by NO. The interdomain electron transfer from NADPH through FAD to heme in cytochrome b 558 was measured by the reduction of cytochrome b 558. The reduction rates of cytochrome b 558 of both stimulated and resting NADPH oxidase did not differ in the presence or the absence of NO, indicating that the electron flux between FAD and the low spin heme of cytochrome b 558 is not the site of impairment by NO.Table IIEffects of NO on the electron flux through FAD and heme of cytochrome b558 in the NADPH oxidase under anaerobic conditionsCytochrome c reductionCytochrome b 558 reductionStimulatedRestingStimulatedRestingnmol/min/mg of protein−NO55.0 ± 8.245.7 ± 6.40.20 ± 0.030.21 ± 0.05+NO52.4 ± 5.443.3 ± 3.50.21 ± 0.040.19 ± 0.03 Open table in a new tab To examine whether NO binds to the heme of cytochrome b 558 or changes the spin state of the heme, nitrosyl-heme formation was measured by both visible absorption and EPR spectra of solubilized NADPH oxidase. Fig.3 shows the difference spectra of cytochrome b 558 in NADPH oxidase from resting cells obtained by subtracting the oxidized spectrum from the dithionite-reduced spectrum in the absence (Fig. 3 A) and the presence (Fig. 3, B and C) of NO. Spectra were measured after incubating the oxidase with NO for 5 and 15 min at 4 °C (Fig. 3, B and C, respectively). No apparent difference was found in the α-peak (558 nm) or γ-peak (427 nm) in the presence of NO. The same results were obtained again, when solubilized NADPH oxidase from stimulated cells was examined, suggesting that the structure of heme was not affected by NO. However, a very high and unphysiological concentration of NO (1 mm) changed the cytochrome b 558 conformation slightly; the spectral intensity was reduced by about 10%, and the γ-peak was shifted about 0.6 nm. To investigate whether NO is trapped at the heme site, nitrosyl-iron complex formation was examined by EPR for purified cytochrome b 558 reduced with dithionite in the presence of NO at 77–100 K. As shown in Fig.4 A, signals characteristic of nitrosyl-iron complex were not observed. On the other hand, when five-coordinated high spin cytochrome b 558,i.e. denatured cytochrome b 558, was incubated with NO, a triplet hyperfine signal characteristic of heme group nitrosylation was observed (Fig. 4 B). The results indicate that NO does not bind nor is trapped at the six-coordinated low spin heme site in intact cytochrome b 558 and that neither the fifth or sixth histidine ligand (27Fujii H. Finnegan M.G. Miki T. Crouse B.R. Kakinuma K. Johnson M.K. FEBS Lett. 1995; 377: 345-348Crossref PubMed Scopus (18) Google Scholar) is displaced by NO. From the results of Figs. 3 and 4, and Table II, we can conclude that the redox centers in the electron transfer chain of the NADPH oxidase remain intact after incubation of the oxidase with NO. The effects of NO on the activation and assembly processes were examined in reconstituted NADPH oxidase (Fig. 5). When the membrane and cytosol components were mixed with myristate for 5 min in the absence of NO, the O·̄2-generating activity was 87 mol/s/mol of cytochrome b 558. When either the membrane or cytosol component was pretreated with NO for 5 min before myristate activation, the O·̄2-generating activity was decreased to 76.0 ± 5.3 and 62.6 ± 7.1% of the control, respectively (A and B in Fig. 5). When a mixture of membrane and cytosol components was activated with myristate simultaneously with NO, the O·̄2-forming activity was decreased to 26.0 ± 5.3% of control (C in Fig. 5). In contrast, when NO was added after the activation, its inhibitory effect was much weaker (D and E in Fig. 5). These results suggest that the main site of NO-induced inhibition is the impairment of activation, such as the assembly of cytosol protein(s) and the membrane. The inhibition of C in Fig. 5, which was more pronounced than the sum of inhibitions of each component (A and Bin Fig. 5), suggests that myristate is the important factor for NO-induced inhibition in addition to the impairment of each component. To further clarify the role of myristate, the cytosol was pretreated with NO in the presence of myristate before mixing with membrane components (D in Fig. 6). The O·̄2-generating activity was markedly inhibited to the level when myristate and NO were simultaneously added to membrane and cytosol components (E in Fig. 6). Membrane components were pretreated with NO in a similar way as cytosol, but the inhibitory effect of NO was not changed in the presence of myristate (Aand B in Fig. 6). From the results in Table II and Figs. 3and 4, it becomes clear that NO does not impair redox centers in NADPH oxidase, suggesting that sulfhydryl groups in proteins are candidates to be attacked by NO, because NO is known to react with tissue sulfhydryls to form S-nitrosothiol compounds, such as S-nitrosocysteine (28Mendelsohn M.E. O'Neill S. George D. Loscalzo J. J. Biol. Chem. 1990; 265: 19028-19034Abstract Full Text PDF PubMed Google Scholar).Figure 6Effect of NO on myristate pretreated cytosol or membrane in reconstituted NADPH oxidase system. The amounts of cytosol, membrane, myristate, and NO used in this experiment were the same as in Fig. 5. A, membrane was incubated with NO without pretreatment with myristate. B, membrane was pretreated with myristate and then incubated with NO for 5 min. C, cytosol was incubated with NO without pretreatment with myristate.D, cytosol was pretreated with myristate and then incubated with NO for 5 min. E, same as C in Fig. 5.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Some insight for an explanation of the effect of myristate and plausible impaired site appears to be shown by a recent report on the role of Src homology 3 (SH3) domain in p47 phox during activation (29Sumimoto H. Kage Y. Nunoi H. Sasaki H. Nose T. Fukumaki Y. Ohno M. Minakami S. Takeshige K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5345-5349Crossref PubMed Scopus (255) Google Scholar). It demonstrated that specifically SH3 domain is folded, masked, and localized at its C-terminal region in dormant cells or in the resting cytosol but opens up upon activation and then binds to the membrane protein(s), mainly cytochrome b 558. There is one cysteine residue (Cys-196) in the SH3 domain that is masked in the resting state of the oxidase, but once the SH3 domain opens up upon activation by myristate, the thiol group of Cys-196 may become accessible to NO. Because there are three other cysteine residues in the SH3 domain of p47 phox and 8 cysteine residues in the cytosolic p67 phox protein, NO-induced oxidation of these sulfhydryl groups may be attributable to the 30–35% inhibition seen in the treatment of cytosol with NO (B in Fig. 5). Similarly the 20–25% inhibition upon treatment of the membrane component with NO may also be a result of sulfhydryl group oxidation (A in Fig. 5). This answered the observation shown in Fig. 2 in which the inhibitory effect of NO in neutrophils was more pronounced than that in the solubilized NADPH oxidase, because, in solubilized NADPH oxidase from stimulated cells, the activation process was already completed and all components were correctly assembled, whereas in neutrophils the activation process was initiated in the presence of NO. Because NO was added to the O·̄2-generating system under aerobic conditions (leading to the formation of ONOO− through the reaction of NO and O·̄2), we examined the contributions of ONOO−. The production of ONOO− was measured by the increase in absorbance at 302 nm. Upon addition of 25 μm NO to a solubilized NADPH oxidase preparation forming 310 nmol O·̄2/min/mg of protein, the increase in absorbance at 302 nm was 0.03, which corresponds to the generation of 18 nmol of ONOO−. Fig.7 shows the NADPH-dependent reduction of cytochrome c with O·̄2 produced by solubilized NADPH oxidase obtained from stimulated cells. When NO was added to an oxidase preparation generating O·̄2, the cytochrome c reduction suddenly stopped and then restarted after a lag time. As seen in Fig. 7, this lag time increased with increasing NO concentration. There are at least three explanations for the abrupt cessation of the cytochrome c reduction: (i) The O·̄2-generating activity of the NADPH oxidase is completely inhibited by ONOO− formed from the reaction of endogenously generated O·̄2 and the added NO. (ii) ONOO− oxidized the reduced cytochrome c by O·̄2. (iii) The generated O·̄2 was quenched by NO much faster than it reacted with cytochrome c. The first explanation is unlikely, because the addition of ONOO− (50 μm) to the reaction mixture or pretreatment of the NADPH oxidase with ONOO− (50 μm) did not inhibit O·̄2 generation in the oxidase (E and F in Fig. 7, respectively). The second explanation is also unlikely, because the oxidation of reduced cytochrome c by ONOO− is as slow as that with hydrogen peroxide (30Turrens J.F. McCord J.M. FEBS Lett. 1988; 227: 43-46Crossref PubMed Scopus (31) Google Scholar). Therefore, we conclude that the temporary lag in cytochrome c reduction is caused by the “quenching” of O·̄2by NO. Actually the reaction of O·̄2 with NO is much faster than the reduction reaction of cytochrome c by O·̄2. Note that after the lag time the reduction rate of cytochrome creturned to nearly the initial value. The slight decrease in the reduction rate of cytochrome c after NO addition is consistent with the slight decrease in the O·̄2 generation shown in the reconstituted NADPH oxidase (D and E in Fig. 5). This decrease in the slopes in Fig. 7 was also concentration-dependent. These results suggest that ONOO− formed by the addition of NO to O·̄2 is not a major factor in the inhibition of the O·̄2 generation in NADPH oxidase. This conclusion was also supported by findings in neutrophils treated with ONOO− (data now shown). In summary, we have found that NO suppresses the O·̄2-generating activity of neutrophil NADPH oxidase. The suppression is more pronounced when NO attacks the oxidase during the activation process. The suppression is trivial after the activation (assembly) is completed. The affected site is a protein itself, either membrane or cytosol proteins, and the binding site of NADPH and redox centers are not impaired by NO. The results are of significant importance in various inflammatory conditions because a large amount of NO can be produced by inducible NO synthase and also a large amount of O·̄2 is produced by infiltrating phagocytes. Furthermore, the evidence that NO inhibited the O·̄2-generating activity of the NADPH oxidase suggests a new mechanism for modulating the O·̄2-generating activity of neutrophils by endogenous NO produced by constitutive NO synthase, because expression of constitutive NO synthase and NO generation by constitutive NO synthase in neutrophils have already been shown (31Carreras M.C. Pargament G.A. Catz S.D. Poderoso J.J. Boveris A. FEBS Lett. 1994; 341: 65-68Crossref PubMed Scopus (334) Google Scholar, 32Riesco A. Caramelo C. Blum G. Monton M. Gallego M.J. Casado S. Farre A.L. Biochem. J. 1993; 292: 791-796Crossref PubMed Scopus (40) Google Scholar, 33Fukuyama N. Ichimori K. Su Z. Ishida H. Nakazawa H. Biochem. Biophys. Res. Commun. 1996; 1041: 414-419Crossref Scopus (66) Google Scholar). This mechanism might protect tissues and cells from oxidative stress at inflammation sites in vivo by the attenuation of the O·̄2concentration by NO. Peroxynitrite does not play a role in this physiological process. We are very grateful to Prof. Lawrence J. Berliner of Ohio State University for critical reading of our manuscript." @default.
• W2053018171 created "2016-06-24" @default.
• W2053018171 creator A5004901883 @default.
• W2053018171 creator A5042749170 @default.
• W2053018171 creator A5049029382 @default.
• W2053018171 creator A5052770369 @default.
• W2053018171 date "1997-12-01" @default.
• W2053018171 modified "2023-10-18" @default.
• W2053018171 title "Nitric Oxide Inactivates NADPH Oxidase in Pig Neutrophils by Inhibiting Its Assembling Process" @default.
• W2053018171 cites W1512142111 @default.
• W2053018171 cites W1517162073 @default.
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