FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1967187479> ?p ?o ?g. }

• W1967187479 endingPage "12341" @default.
• W1967187479 startingPage "12328" @default.
• W1967187479 abstract "We studied protective effects of NO againsttert-butylhydroperoxide (t-BuOOH)-induced oxidations in a subline of human erythroleukemia K562 cells with different intracellular hemoglobin (Hb) concentrations.t-BuOOH-induced formation of oxoferryl-Hb-derived free radical species in cells was demonstrated by low temperature EPR spectroscopy. Intensity of the signals was proportional to Hb concentrations and was correlated with cell viability. Peroxidation of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and cardiolipin metabolically labeled with oxidation-sensitivecis-parinaric acid was induced byt-BuOOH. An NO donor, (Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)amino]-diazen-1-ium-1,2-diolate], produced non-heme iron dinitrosyl complexes and hexa- and pentacoordinated Hb-nitrosyl complexes in the cells. Nitrosylation of non-heme iron centers and Hb-heme protected againstt-BuOOH-induced: (a) formation of oxoferryl-Hb-derived free radical species, (b) peroxidation of cis-parinaric acid-labeled phospholipids, and (c) cytotoxicity. Since NO did not inhibit peroxidation induced by an azo-initiator of peroxyl radicals, 2,2′-azobis(2,4-dimethylvaleronitrile), protective effects of NO were due to formation of iron-nitrosyl complexes whose redox interactions with t-BuOOH prevented generation of oxoferryl-Hb-derived free radical species. We studied protective effects of NO againsttert-butylhydroperoxide (t-BuOOH)-induced oxidations in a subline of human erythroleukemia K562 cells with different intracellular hemoglobin (Hb) concentrations.t-BuOOH-induced formation of oxoferryl-Hb-derived free radical species in cells was demonstrated by low temperature EPR spectroscopy. Intensity of the signals was proportional to Hb concentrations and was correlated with cell viability. Peroxidation of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and cardiolipin metabolically labeled with oxidation-sensitivecis-parinaric acid was induced byt-BuOOH. An NO donor, (Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)amino]-diazen-1-ium-1,2-diolate], produced non-heme iron dinitrosyl complexes and hexa- and pentacoordinated Hb-nitrosyl complexes in the cells. Nitrosylation of non-heme iron centers and Hb-heme protected againstt-BuOOH-induced: (a) formation of oxoferryl-Hb-derived free radical species, (b) peroxidation of cis-parinaric acid-labeled phospholipids, and (c) cytotoxicity. Since NO did not inhibit peroxidation induced by an azo-initiator of peroxyl radicals, 2,2′-azobis(2,4-dimethylvaleronitrile), protective effects of NO were due to formation of iron-nitrosyl complexes whose redox interactions with t-BuOOH prevented generation of oxoferryl-Hb-derived free radical species. Nitric oxide (NO) 1The abbreviations used are: NO, nitric oxide; Hb, hemoglobin; oxy-Hb, ferrous (Fe(II)) form of Hb in complex with O2; met-Hb, ferric (Fe(III)) form of Hb; oxoferryl-Hb, oxoferryl (Fe(IV)=O) form of Hb; PnA, 9-cis,11-trans,13-trans,15-cis-octadecatetraenoic acid (cis-parinaric acid); HSA, human serum albumin;t-BuOOH, tert-butylhydroperoxide; AMVN, 2,2′-azobis(2,4-dimethylvaleronitrile); EPR, electron paramagnetic resonance; HIN, the hexacoordinated heme iron nitrosyl complexes; PIN, the pentacoordinated heme iron nitrosyl complexes; NOC-15 or PAPA NONOate, (Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)amino]-diazen-1-ium-1,2-diolate]); DPPC, l-α-phosphatidylcholine, dipalmitoyl (C18:1 [cis ]-9); PC, phosphatidylcholine; PS, phosphatidylserine; PEA, phosphatidylethanolamine; PI, phosphatidylinositol; CL, cardiolipin; mT, millitesla; HPLC, high performance liquid chromatography. 1The abbreviations used are: NO, nitric oxide; Hb, hemoglobin; oxy-Hb, ferrous (Fe(II)) form of Hb in complex with O2; met-Hb, ferric (Fe(III)) form of Hb; oxoferryl-Hb, oxoferryl (Fe(IV)=O) form of Hb; PnA, 9-cis,11-trans,13-trans,15-cis-octadecatetraenoic acid (cis-parinaric acid); HSA, human serum albumin;t-BuOOH, tert-butylhydroperoxide; AMVN, 2,2′-azobis(2,4-dimethylvaleronitrile); EPR, electron paramagnetic resonance; HIN, the hexacoordinated heme iron nitrosyl complexes; PIN, the pentacoordinated heme iron nitrosyl complexes; NOC-15 or PAPA NONOate, (Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)amino]-diazen-1-ium-1,2-diolate]); DPPC, l-α-phosphatidylcholine, dipalmitoyl (C18:1 [cis ]-9); PC, phosphatidylcholine; PS, phosphatidylserine; PEA, phosphatidylethanolamine; PI, phosphatidylinositol; CL, cardiolipin; mT, millitesla; HPLC, high performance liquid chromatography. is an important physiological regulator of biological responses such as vasodilation, blood coagulation, neurotransmission, renal function, inflammation, and antitumor immune surveillance (1Palmer R.M.J. Ferrige A.G. Moncada S. Nature. 1987; 327: 524-526Crossref PubMed Scopus (9270) Google Scholar, 2Garthwaite J. Charles S.L. Chess-Willams R. Nature. 1988; 336: 385-388Crossref PubMed Scopus (2269) Google Scholar, 3Vallance P. Leone A. Calver A. Collier J. Moncada S. Lancet. 1992; 339: 572-575Abstract PubMed Scopus (1947) Google Scholar, 4Lancaster Jr., J.R. Hibbs Jr., J.B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1223-1227Crossref PubMed Scopus (488) Google Scholar, 5Drapier J.-C. Pellat C. Henry Y. J. Biol. Chem. 1991; 266: 10162-10167Abstract Full Text PDF PubMed Google Scholar, 6Stamler J.S. Singel D.J. Loscalzo J. Science. 1992; 258: 1898-1902Crossref PubMed Scopus (2438) Google Scholar). Paradoxically, it can simultaneously exert adverse effects on cells. Cytotoxic effects of NO are believed to be produced through three major pathways as follows: (i) direct modification of proteins by NO via nitrosylation of sulfhydryl groups, heme and non-heme sites, and possibly tyrosyl residues (e.g. modification of poly(ADP-ribose) synthetase, ribonucleotide reductase, and enzymes of mitochondrial electron transport) (4Lancaster Jr., J.R. Hibbs Jr., J.B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1223-1227Crossref PubMed Scopus (488) Google Scholar, 5Drapier J.-C. Pellat C. Henry Y. J. Biol. Chem. 1991; 266: 10162-10167Abstract Full Text PDF PubMed Google Scholar, 6Stamler J.S. Singel D.J. Loscalzo J. Science. 1992; 258: 1898-1902Crossref PubMed Scopus (2438) Google Scholar, 7Roy B. Lepoivre M. Henry Y. Fontecave M. Biochemistry. 1995; 34: 5411-5418Crossref PubMed Scopus (82) Google Scholar, 8Corbet J.A. Wang J.L. Hughes J.H. Wolf B.A. Sweetland M.A. Lancaster Jr., J.R. McDaniel M.L. Biochem. J. 1992; 287: 229-235Crossref PubMed Scopus (135) Google Scholar, 9Zhang J. Dawson V.L. Dawson T.M. Snyder S.H. Science. 1994; 263: 687-689Crossref PubMed Scopus (1076) Google Scholar, 10Takehara Y. Kanno T. Yoshioka T. Inoue M. Utsumi K. Arch. Biochem. Biophys. 1995; 323: 27-32Crossref PubMed Scopus (165) Google Scholar); (ii) NO-induced activation of enzymes involved in posttranscriptional regulation of protein expression (e.g.transferrin/ferritin pathway for iron mobilization) (11Richardson D.R. Neumannova V. Ponka P. Biochim. Biophys. Acta. 1995; 1266: 250-260Crossref PubMed Scopus (35) Google Scholar, 12Richardson D.R. Neumannova V. Nagy E. Ponka P. Blood. 1995; 86: 3211-3219Crossref PubMed Google Scholar); and (iii) oxidative damage to critical biomolecules such as nucleic acids, proteins, and lipids. The latter is mainly associated with the production of peroxynitrite (13Hogg N. Darley-Usmar V.M. Wilson M.T. Moncada S. FEBS Lett. 1993; 326: 199-203Crossref PubMed Scopus (139) Google Scholar, 14Beckman J.S. Carson M. Smith C.D. Koppenol W.H. Nature. 1993; 364: 584Crossref PubMed Scopus (786) Google Scholar, 15Beckman J.S. Beckman T.W. Chen J. Marshall P.A. Freeman B.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1620-1624Crossref PubMed Scopus (6671) Google Scholar, 16Radi R. Rodriguez M. Castro L. Telleri R. Arch. Biochem. Biophys. 1994; 308: 89-95Crossref PubMed Scopus (659) Google Scholar, 17Lipton S.A. Choi Y.B. Pan Z.H. Lei S.Z. Chen H.S. Sucher N.J. Loscalzo J. Singel D.J. Stamler J.S. Nature. 1993; 364: 626-632Crossref PubMed Scopus (2292) Google Scholar). It has been demonstrated recently that NO can also act as an antioxidant, thus protecting cells against oxidative damage (17Lipton S.A. Choi Y.B. Pan Z.H. Lei S.Z. Chen H.S. Sucher N.J. Loscalzo J. Singel D.J. Stamler J.S. Nature. 1993; 364: 626-632Crossref PubMed Scopus (2292) Google Scholar, 18Wink D.A. Cook J.A. Krishna M.C. Hanbauer J. DeGraff W. Gamson J. Mitchell J.B. Arch. Biochem. Biophys. 1995; 319: 402-407Crossref PubMed Scopus (119) Google Scholar, 19Wink D.A. Hanbauer I. Krishna M.C. DeGraff W. Gamson J. Mitchell J.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9813-9817Crossref PubMed Scopus (733) Google Scholar). In Chinese hamster V79 lung fibroblasts and human umbilical vein endothelial cells, this antioxidant effect of NO was associated with its ability to scavenge lipid alkoxyl and peroxyl radicals (18Wink D.A. Cook J.A. Krishna M.C. Hanbauer J. DeGraff W. Gamson J. Mitchell J.B. Arch. Biochem. Biophys. 1995; 319: 402-407Crossref PubMed Scopus (119) Google Scholar, 19Wink D.A. Hanbauer I. Krishna M.C. DeGraff W. Gamson J. Mitchell J.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9813-9817Crossref PubMed Scopus (733) Google Scholar, 20Rubbo H. Radi R. Trujillo M. Telleri R. Kalyanaraman B. Barnes S. Kirk M. Freeman B.A. J. Biol. Chem. 1994; 269: 26066-26075Abstract Full Text PDF PubMed Google Scholar). The balance between intracellular antioxidant and pro-oxidant effects of NO in vivo remains to be elucidated. It has been suggested that the interaction of NO with hemoglobin and myoglobin may prevent hydroperoxide-induced formation of oxoferryl hemoproteins, thus blocking subsequent generation of oxygen-derived reactive species and oxidative damage (18Wink D.A. Cook J.A. Krishna M.C. Hanbauer J. DeGraff W. Gamson J. Mitchell J.B. Arch. Biochem. Biophys. 1995; 319: 402-407Crossref PubMed Scopus (119) Google Scholar, 19Wink D.A. Hanbauer I. Krishna M.C. DeGraff W. Gamson J. Mitchell J.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9813-9817Crossref PubMed Scopus (733) Google Scholar, 21Kerwin Jr., J.F. Lancaster Jr., J.R. Feldman P.L. J. Med. Chem. 1995; 38: 4343-4362Crossref PubMed Scopus (535) Google Scholar, 22Juckett M. Weber M. Balla J. Jacob H.S. Vercellotti G.M. Free Radical Biol. & Med. 1996; 20: 63-73Crossref PubMed Scopus (37) Google Scholar, 23Kanner J. Harel S. Granit R. Arch. Biochem. Biophys. 1991; 289: 130-136Crossref PubMed Scopus (389) Google Scholar, 24Gorbunov N.V. Osipov A.N. Day B.W. Zayas-Rivera B. Kagan V.E. Elsayed N.M. Biochemistry. 1995; 34: 6689-6699Crossref PubMed Scopus (120) Google Scholar, 25Hogg N. Kalyanaraman B. Joseph J. Struck A. Parthasarathy S. FEBS Lett. 1993; 334: 170-174Crossref PubMed Scopus (351) Google Scholar). In line with this, our previous studies demonstrated that NO was capable of inhibiting oxoferryl-induced oxidation in simple model systems such astert-butylhydroperoxide (t-BuOOH)/hemoglobin ort-BuOOH/myoglobin (24Gorbunov N.V. Osipov A.N. Day B.W. Zayas-Rivera B. Kagan V.E. Elsayed N.M. Biochemistry. 1995; 34: 6689-6699Crossref PubMed Scopus (120) Google Scholar). The proposed antioxidant mechanism of NO involves reduction of oxoferryl-derived radicals (24Gorbunov N.V. Osipov A.N. Day B.W. Zayas-Rivera B. Kagan V.E. Elsayed N.M. Biochemistry. 1995; 34: 6689-6699Crossref PubMed Scopus (120) Google Scholar). Whether this mechanism operates in cells remained unclear. In the present work, we attempted to elucidate the antioxidant role of NO against intracellular oxoferryl hemoglobin-induced oxidative stress. We studied the effects of NO on t-BuOOH-induced, heme and non-heme iron-dependent oxidation of membrane phospholipids and cytotoxicity in a subline of human erythroleukemic K562 cells in which concentrations of endogenous hemoglobin can be easily manipulated. Human hemoglobin (Hb), tert-butylhydroperoxide, sodium hydrosulfite (dithionite), hemin, andl-α-phosphatidylcholine, dipalmitoyl-(C18:1 (cis)-9) (DPPC) were obtained from Sigma. Potassium phosphate (monobasic) was purchased from Fisher. 9-cis,11-trans13-trans15-cis-octadecatetraenoic acid (cis-parinaric acid, PnA) was purchased from Molecular Probes, Inc. (Eugene, OR). NOC-15, (Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)amino]-diazen-1-ium-1,2-diolate] (PAPA NONOate) was from Cayman Chemical Co. (Ann Arbor, MI). AMVN, 2,2′-azobis(2,4-dimethylvaleronitrile) was obtained from Polysciences, Inc. (Warrington, PA); Sephadex G-25 columns were obtained from Pharmacia LKB (Uppsala, Sweden). K/VP.5 cells used for most of the experiments described are a subline of human erythroleukemia K562 cells selected for resistance to the anticancer agent etoposide (26Ritke M.K. Roberts D. Allan W.P. Raymond J. Bergoltz V.V. Yalowich J.C. Br. J. Cancer. 1994; 69: 687-697Crossref PubMed Scopus (65) Google Scholar). Cells were grown in continuous culture in Dulbecco's modified Eagle's medium in the presence of 7.5% iron-supplemented calf serum. K/VP.5 cells were chosen for study because it was found that intracellular Hb was 25 pmol/106 cell compared with 5 pmol/106cell in parental K562 cells. This permitted us to discriminate a Hb-dependent t-BuOOH toxicity to the cells from the total iron-catalyzed toxicity. To additionally increase the intracellular amount of Hb in K/VP.5 cells, the growth medium was supplemented with hemin (25 μm) for 24 h (27Kawasaki N. Morimoto K. Tanimoto T. Hayakaea T. Arch. Biochem. Biophys. 1996; 382: 289-294Crossref Scopus (30) Google Scholar). Extracellular hemin in the growth medium obtained after sedimentation of cells (1,500 × g for 5 min) had a characteristic maximum in the Soret band at 395 nm (Fig. 1 A,spectrum 4). In contrast, spectra of intracellular content (obtained by treatment of cells with alamethicin, see below) as well as spectra of solubilized cell debris (membranes) displayed maxima in the Soret band characteristic of hemoglobin (414 nm) (Fig. 1, Aand B, spectrum 1). Hence, only hemoglobin was present in cells, and excess extracellular hemin was effectively removed by centrifugation. (It is likely that hemin in the growth medium was predominantly residing in complexes with calf serum albumin since the dissociation constant for hemin-albumin complex isKd = 10−8m (28Muller-Eberhard U. Nikkila H. Semin. Hematol. 1989; 26: 86-104PubMed Google Scholar, 29Barnard M.L. Muller-Eberhard U. Turrens J.F. Biochem. Biophys. Res. Commun. 1993; 192: 82-87Crossref PubMed Scopus (20) Google Scholar).) Log phase cells were separated from growth medium by centrifugation (1,500 × g for 5 min). The cell pellet was rinsed twice with L1210 buffer containing 115 mm NaCl, 5 mm KCl, 5 mm NaH2PO4, 1 mm MgCl2, 10 mm glucose, and 25 mm Hepes, pH 7.4. To prevent redox effects of adventitious iron, all aqueous solutions were treated with Chelex-100 (Bio-Rad). The cells diluted to a density of 1.0 × 106 cell/ml (80 × 106 cell/ml for EPR measurements) were incubated for 10 min in the absence or presence of NOC-15 (20 and 80 nmol/106 cells) releasing NO with a half-life of 76 min, following which t-BuOOH (100 nmol/106 cells) was added to incubate for an additional 60 min. Aliquots of a cell suspension (1.0 × 106 cell/ml) were taken to assess cell viability using trypan blue dye exclusion. Ten million cells were incubated (for 10 min at room temperature) in the presence of an amphiphilic channel-forming peptide, alamethicin (50 μm) to release intracellular Hb (30He K. Ludtke S.J. Huang H.W. Warcester D.L. Biochemistry. 1995; 34: 15614-15618Crossref PubMed Scopus (158) Google Scholar). After centrifugation (100,000 ×g for 20 min), the supernatant was collected, and pelleted cell debris were solubilized in 100 mm phosphate buffer, pH 7.4, containing 1% of Triton X-100. Spectra of Hb in the supernatant and of the solubilized membrane pellet were recorded in the range 350–700 nm using a Shimadzu 160U UV-VIS spectrophotometer. Three maxima at 414 nm (Soret band of oxyhemoglobin) as well as at 542 and 576 nm (characteristic of oxyhemoglobin) were observed in spectra of both the supernatants and the solubilized membranes (Fig. 1,A and B). The concentration of Hb in cells was estimated using the molar extinction coefficient for the Hb Soret band (414 nm) of 125,000 m−1 × cm−1. Under the conditions used, no more than 7% total intracellular Hb remained bound to cell membranes (debris) after treatment of cells with alamethicin and release of hemoglobin. No additional maximum characteristic of hemin (at 395 nm) was detectable in the spectra of supernatants or solubilized membranes (Fig. 1, A andB). Additional evidence for the lack of hemin in K/VP.5 cells comes from our EPR measurements. The EPR spectra (and hyperfine splitting constants) for nitrosylated Hb in a model system are similar to that in Hb-enriched K/VP.5 cells (see Figs. 2 and 3). Both of these spectra are dissimilar from that of hemin in liposomes (see Fig. 3,c and d). In addition, EPR spectra obtained from K/VP.5 cells exposed to t-BuOOH contained features typical of protein-centered free radical species (Fig. 4). In contrast, only a signal of t-BuO⋅-alkoxyl radical was detected in the EPR spectrum of hemin integrated into DPPC liposomes (Fig. 4). Thus a part of hemin from the growth medium was integrated into intracellular hemoglobin, and the rest of it (bound to serum proteins in the medium) was removed by centrifugation; no “loose” membrane-bound hemin was present in the cells used in the experiments performed.Figure 3Low temperature EPR spectra of PIN and HIN complexes obtained from the following. a, 20 μm Hb with 20 μm NOC-15 incubated in 100 mm phosphate buffer, pH 7.4, for 10 min. b, Hb-enriched K/VP.5 cells incubated with 20 nmol of NOC-15/106 cells (obtained by a subtraction of the reconstructed EPR spectrum (A4, Fig. 2) from the spectrum (B2, Fig. 2)). c, 25 μm hemin incorporated into DPPC liposomes (see “Materials and Methods”) incubated with 20 μm NOC-15. d, 25 μm hemin incorporated into DPPC liposomes (see “Materials and Methods”) incubated with 80 μm NOC-15.e, Hb-enriched K/VP.5 cells incubated with 80 nmol of NOC-15/106 cells obtained by a subtraction of the reconstructed EPR spectrum (A4, Fig. 2) from the spectrum (B3, Fig. 2). Incubation, spectrometer conditions, and computer manipulations were the same as in Fig. 2. Spectraa, b, and e are magnified versions of the same spectra as 5a, 5b, and 4a, respectively, shown on Fig. 2.View Large Image Figure ViewerDownload (PPT)Figure 4Comparison of the low temperature EPR spectra obtained from K/VP.5 cells (solid line) and hemin in DPPC liposomes (dashed line) incubated witht-BuOOH. Incubation conditions were the same as in Fig. 3. Spectrometer conditions were the same as in Fig. 2.View Large Image Figure ViewerDownload (PPT) PnA was incorporated into cells by addition of its HSA complex (PnA·HSA) to cell suspensions (31Ritov V.B. Banni S. Yalowich J.C. Day B.W. Claycamp H.G. Corongiu F.P. Kagan V.E. Biochim. Biophys. Acta. 1996; 1283: 127-140Crossref PubMed Scopus (71) Google Scholar). Cells in log phase growth were rinsed twice with L1210 buffer, diluted to a density of 1.0 × 106cells/ml, and then incubated with PnA·HSA complex (final concentration of PnA 4 μg/ml) in L1210 buffer at 37 °C for 2 h. After incubation, cells were pelleted by centrifugation and then washed twice with isotonic buffer with HSA. The total amount of PnA metabolically incorporated into membrane phospholipids was less than 1% of fatty acid residues. PnA-treated cells were incubated in the presence of t-BuOOH (100 nmol/106 cells) and/or NOC-15 (20 and 80 nmol/106 cells) as described above. Total lipid extracts from the cells were obtained using a Folch procedure (32Folch J. Lees M. Sloane-Stanley G.H. J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar). The lipid extract was dissolved in 3:4:0.16 (v/v) hexane/isopropyl alcohol/water (0.15 ml). Lipid extracts were separated by a normal phase HPLC as described previously (31Ritov V.B. Banni S. Yalowich J.C. Day B.W. Claycamp H.G. Corongiu F.P. Kagan V.E. Biochim. Biophys. Acta. 1996; 1283: 127-140Crossref PubMed Scopus (71) Google Scholar). A 5-μm Supelcosil LC-Si column (4.6 × 250 mm) was employed with the following mobile phase flowing at 1 ml/min: solvent A (57:43:1 isopropyl alcohol/hexane/H2O), solvent B (57:43:10 isopropyl alcohol/hexane, 40 mm aqueous ammonium acetate, pH 7.0), 0–3 min linear gradient from 10% B to 37% B, 3–15 min isocratic at 37% B, 15–23 min linear gradient to 100% B, 23–45 min isocratic at 100% B. A Shimadzu HPLC system (model LC-600) equipped with a fluorescence detector (model RF-551) was used. Fluorescence of PnA in eluates was monitored by emission at 420 nm after excitation at 324 nm. Fluorescence data were processed and stored in digital form with Shimadzu EZChrom software. Commercial Hb was mainly in the met (ferric) form. We reduced met-Hb (1 mm solution in 100 mm phosphate buffer, pH 7.4) to its ferrous (oxy-Hb) form using 4-fold excess of sodium dithionite. Pure oxy-Hb was obtained by separation on a Sephadex G-25 column preequilibrated with 100 mm phosphate buffer, pH 7.4. The concentrations of oxy-Hb/met-Hb were calculated as described previously by Winterbourn (33Winterbourn C.C. Methods Enzymol. 1990; 186: 265-272Crossref PubMed Scopus (396) Google Scholar) using oxy-Hb extinction coefficient at 577 nm 15.0 mm−1 × cm−1 (33Winterbourn C.C. Methods Enzymol. 1990; 186: 265-272Crossref PubMed Scopus (396) Google Scholar). Deoxygenation of oxy-Hb was performed by incubating it in a nitrogen atmosphere. Liposomes were prepared from a stock solution of 2 mm DPPC in CHCl3/CH3OH (1:1) containing 0.2 mmhemin. The solvent was evaporated under a stream of N2. Phosphate buffer (100 mm, pH 7.4) was added, and the resulting suspension was sonicated (three 30-s bursts at 65 watts) with a Cole-Parmer Instrument Co. 4710 Series Ultrasonicator (Chicago, IL). To reduce ferric form of hemin to its ferrous form we used sodium dithionite (0.8 mm). Removal of excess dithionite was achieved by gel filtration of liposomes through a Sephadex G-25 column preequilibrated with 100 mm phosphate buffer. Thus prepared hemin-containing liposomes were used for EPR and spectrophotometric measurements. Log phase cells were separated from the growth media as mentioned above. Two different sets of EPR measurements were performed. Cells adjusted to either 1 × 106cell/ml or 80 × 106 cell/ml were incubated for 10 min in the absence or presence of NOC-15 (20 and 80 nmol/106cells, releasing NO with a half-life of 76 min), following which 100 nmol of t-BuOOH/106 cells was added for an additional 60 min. Aliquots of cell suspensions (3 × 106 cells or 20 × 106 cell in 250 μl) were withdrawn, placed into a Teflon tube (3.7 mm internal diameter), frozen in liquid nitrogen, and then removed from the tube to perform EPR measurements. For spectrum recording, each sample was placed in an EPR quartz tube (5 mm internal diameter) in such a way that the entire sample was within the effective microwave irradiation area. To obtain discernible EPR signals from low concentrations of cells and reagents in some measurements we used multiple spectra acquisitions (10 per sample) and their computer-assisted averaging (as indicated in the figure legends). EPR measurements were performed on a JEOL-RE1X spectrometer with a variable temperature controller (Research Specialists, Chicago, IL). The spectra were recorded at −170 °C, 320 mT center field, 10 mW power, 0.1 mT field modulation, 25 mT sweep width, 0.1-s time constant. The g factor values were determined relative to external standards, containing Mn2+ (in MgO). Analog signals were converted into digital form and imported to an IBM computer. Intensity of the signals was calculated using a program developed by Duling (34Duling D.R. J. Magn. Reson. 1994; 104: 105-110Crossref Scopus (892) Google Scholar). The EPR spectra of both control K/VP.5 cells (25 pmol Hb/106 cells) and K/VP.5 cells enriched with Hb (90 pmol Hb/106 cells) displayed only nonspecific free radical signals at g = 2.004 (Fig. 2,A1 and B1), probably resulting from components of the mitochondrial respiratory chain (35Lepoivre M. Flaman J.M. Henry Y. J. Biol. Chem. 1992; 267: 22994-23000Abstract Full Text PDF PubMed Google Scholar, 36Stadler J. Bergonia H.A. Di Silvio M. Sweetland M.A. Billiar T.R. Simmons R.L. Lancaster Jr., J.R. Arch. Biochem. Biophys. 1993; 302: 4-11Crossref PubMed Scopus (115) Google Scholar). Incubation of control (nontreated with hemin) K/VP.5 cells with two different concentrations of NOC-15 (20 and 80 nmol/106 cells) resulted in EPR signals with identical profiles (Fig. 2, A2 and A3). We registered four-line anisotropic spectra with (i) principal features atg∥ = 2.04 and g⊥ = 2.015; (ii) additional features at g = 2.07 (a maximun), and g = 1.989 (a trough); (iii) a free radical signal at g = 2.004 (Fig. 2, A2 andA3). Similar spectra, with an axial anisotropic feature atg∥ 2.04 and g⊥ = 2.015, have been previously observed upon exposure of various types of cells to NO (5Drapier J.-C. Pellat C. Henry Y. J. Biol. Chem. 1991; 266: 10162-10167Abstract Full Text PDF PubMed Google Scholar, 8Corbet J.A. Wang J.L. Hughes J.H. Wolf B.A. Sweetland M.A. Lancaster Jr., J.R. McDaniel M.L. Biochem. J. 1992; 287: 229-235Crossref PubMed Scopus (135) Google Scholar, 35Lepoivre M. Flaman J.M. Henry Y. J. Biol. Chem. 1992; 267: 22994-23000Abstract Full Text PDF PubMed Google Scholar, 36Stadler J. Bergonia H.A. Di Silvio M. Sweetland M.A. Billiar T.R. Simmons R.L. Lancaster Jr., J.R. Arch. Biochem. Biophys. 1993; 302: 4-11Crossref PubMed Scopus (115) Google Scholar, 37Chamulitrat W. Jordan S.J. Mason R.P. Litton A.L. Wilson J.G. Wood E.R. Wolberg G. Molina y Vedia L. Arch. Biochem. Biophys. 1995; 316: 30-37Crossref PubMed Scopus (46) Google Scholar), and were assigned to the characteristic EPR signals of protein- and nonprotein nonheme iron-dinitrosyl complexes (35Lepoivre M. Flaman J.M. Henry Y. J. Biol. Chem. 1992; 267: 22994-23000Abstract Full Text PDF PubMed Google Scholar, 36Stadler J. Bergonia H.A. Di Silvio M. Sweetland M.A. Billiar T.R. Simmons R.L. Lancaster Jr., J.R. Arch. Biochem. Biophys. 1993; 302: 4-11Crossref PubMed Scopus (115) Google Scholar, 37Chamulitrat W. Jordan S.J. Mason R.P. Litton A.L. Wilson J.G. Wood E.R. Wolberg G. Molina y Vedia L. Arch. Biochem. Biophys. 1995; 316: 30-37Crossref PubMed Scopus (46) Google Scholar, 38Woolum J.C. Tiezzi E. Commoner B. Biochim. Biophys. Acta. 1968; 160: 311-320Crossref PubMed Scopus (139) Google Scholar, 39Vanin A.F. Stud. Biophys. 1975; 49: 13-25Google Scholar). We and others (7Roy B. Lepoivre M. Henry Y. Fontecave M. Biochemistry. 1995; 34: 5411-5418Crossref PubMed Scopus (82) Google Scholar, 12Richardson D.R. Neumannova V. Nagy E. Ponka P. Blood. 1995; 86: 3211-3219Crossref PubMed Google Scholar, 40Geng Y. Hellstrand K. Wennmalm Å. Hansson G.K. Cancer Res. 1996; 56: 866-874PubMed Google Scholar) have detected EPR signals with similar features at g∥ = 2.04 andg⊥ = 2.015 in parental K562 cells (with a very low level of endogenous Hb, 5 pmol/106 cells) treated with NO (spectra not shown). In Hb-enriched K/VP.5 cells, treatment with the NO donor (20 and 80 nmol/106 cells) caused EPR spectra with profiles different from those of control K/VP.5 cells. A five-line anisotropic signal with incompletely resolved superfine structure was detected (Fig. 2, B2 andB3). The features g⊥ = 2.04 andg∥ = 2.015 were assigned to non-heme iron dinitrosyl complexes (35Lepoivre M. Flaman J.M. Henry Y. J. Biol. Chem. 1992; 267: 22994-23000Abstract Full Text PDF PubMed Google Scholar, 36Stadler J. Bergonia H.A. Di Silvio M. Sweetland M.A. Billiar T.R. Simmons R.L. Lancaster Jr., J.R. Arch. Biochem. Biophys. 1993; 302: 4-11Crossref PubMed Scopus (115) Google Scholar, 38Woolum J.C. Tiezzi E. Commoner B. Biochim. Biophys. Acta. 1968; 160: 311-320Crossref PubMed Scopus (139) Google Scholar, 39Vanin A.F. Stud. Biophys. 1975; 49: 13-25Google Scholar, 41Reddy D. Lancaster Jr., J.R. Cornforth D.P. Science. 1983; 221: 769-770Crossref PubMed Scopus (199) Google Scholar) similar to that in the control K/VP.5 cells. The signals in Hb-enriched cells, however, also exhibited additional multiplet signals at g = 2.07,g = 2.025, g = 2.004 andg = 1.989. The features at g = 2.07 and g = 1.989 in both control- and Hb-enriched K/VP.5 cells can be assigned to nitrosyl complexes of heme iron, apparently resulting from nitrosylation of intracellular Hb. Indeed, these spectral components in the Hb-enriched K/VP.5 cells were much more intense than in control cells, although partly obscured by signals from non-heme iron dinitrosyl complexes at g⊥ = 2.04 andg∥ = 2.015 (Fig. 2, B2 andB3) Similar signals of nitrosyl complexes of hemoglobin have been shown previously in model systems (42Taketa F. Antholine W.E. Chen J.Y. J. Biol. Chem. 1978; 253: 5448-5451Abstract Full Text PDF PubMed Google Scholar, 43Hille R. Olson J.S. Palmer G. J. Biol. Chem. 1979; 254: 12110-12120Abstract Full Text PDF PubMed Google Scholar, 44Henry Y. Ducrocq C. Drapier J.-C. Servent D. Pellat C. Guissani A. Eur. Biophys. J. 1991; 20: 1-15Crossref PubMed Scopus (187) Google Scholar). Moreover, essentially the same spectra were observed in erythrocytes upon exposure to NO (37Chamulitrat W. Jordan S.J. Mason R.P. Litton A.L. Wilson J.G. Wood E.R. Wolberg G. Molina y Vedia L. Arch. Biochem. Biophys. 1995; 316: 30-37Crossref PubMed Scopus (46) Google Scholar,45Eriksson L.E. Biochem. Biophys. Res. Commun. 1994; 203: 176-181Crossref PubMed Scopus (15) Google Scholar). To confirm this assignment of the features g = 2.07 and g = 1.989 to the signal of nitrosylated Hb in K/VP.5 cells, and to identify the additional features atg" @default.
• W1967187479 created "2016-06-24" @default.
• W1967187479 creator A5002009822 @default.
• W1967187479 creator A5034176813 @default.
• W1967187479 creator A5037992586 @default.
• W1967187479 creator A5038216324 @default.
• W1967187479 creator A5042993045 @default.
• W1967187479 creator A5049450190 @default.
• W1967187479 creator A5070091746 @default.
• W1967187479 creator A5080340455 @default.
• W1967187479 date "1997-05-01" @default.
• W1967187479 modified "2023-10-11" @default.
• W1967187479 title "Nitric Oxide Prevents Oxidative Damage Produced bytert-Butyl Hydroperoxide in Erythroleukemia Cells via Nitrosylation of Heme and Non-heme Iron" @default.
• W1967187479 cites W1489860413 @default.
• W1967187479 cites W1501279037 @default.
• W1967187479 cites W1525976258 @default.
• W1967187479 cites W1535540127 @default.
• W1967187479 cites W1548245551 @default.
• W1967187479 cites W1555898132 @default.
• W1967187479 cites W1565072410 @default.
• W1967187479 cites W1598472648 @default.
• W1967187479 cites W1605002548 @default.
• W1967187479 cites W1634873152 @default.
• W1967187479 cites W1734082729 @default.
• W1967187479 cites W1857086613 @default.
• W1967187479 cites W1964634019 @default.
• W1967187479 cites W1964729357 @default.
• W1967187479 cites W1967556520 @default.
• W1967187479 cites W1969184431 @default.
• W1967187479 cites W1970499396 @default.
• W1967187479 cites W1971242876 @default.
• W1967187479 cites W1973724603 @default.
• W1967187479 cites W1973812004 @default.
• W1967187479 cites W1974809944 @default.
• W1967187479 cites W1974936925 @default.
• W1967187479 cites W1977700675 @default.
• W1967187479 cites W1977954884 @default.
• W1967187479 cites W1979713155 @default.
• W1967187479 cites W1985811388 @default.
• W1967187479 cites W1987165240 @default.
• W1967187479 cites W1991608880 @default.
• W1967187479 cites W1992491531 @default.
• W1967187479 cites W1996068858 @default.
• W1967187479 cites W1999781532 @default.
• W1967187479 cites W2002334319 @default.
• W1967187479 cites W2003281598 @default.
• W1967187479 cites W2004893165 @default.
• W1967187479 cites W2005706069 @default.
• W1967187479 cites W2005896707 @default.
• W1967187479 cites W2007607715 @default.
• W1967187479 cites W2007737411 @default.
• W1967187479 cites W2011233979 @default.
• W1967187479 cites W2011450860 @default.
• W1967187479 cites W2011888550 @default.
• W1967187479 cites W2013302223 @default.
• W1967187479 cites W2015341133 @default.
• W1967187479 cites W2016718505 @default.
• W1967187479 cites W2019814232 @default.
• W1967187479 cites W2020089086 @default.
• W1967187479 cites W2020914812 @default.
• W1967187479 cites W2024423358 @default.
• W1967187479 cites W2024862570 @default.
• W1967187479 cites W2026547315 @default.
• W1967187479 cites W2027259889 @default.
• W1967187479 cites W2027968050 @default.
• W1967187479 cites W2028474406 @default.
• W1967187479 cites W2029067920 @default.
• W1967187479 cites W2032960989 @default.
• W1967187479 cites W2035690567 @default.
• W1967187479 cites W2036245306 @default.
• W1967187479 cites W2040540764 @default.
• W1967187479 cites W2040572237 @default.
• W1967187479 cites W2058137293 @default.
• W1967187479 cites W2058385357 @default.
• W1967187479 cites W2060026323 @default.
• W1967187479 cites W2060621718 @default.
• W1967187479 cites W2061295485 @default.
• W1967187479 cites W2062384458 @default.
• W1967187479 cites W2065168167 @default.
• W1967187479 cites W2066033513 @default.
• W1967187479 cites W2066786384 @default.
• W1967187479 cites W2067493659 @default.
• W1967187479 cites W2073046293 @default.
• W1967187479 cites W2073909911 @default.
• W1967187479 cites W2075824657 @default.
• W1967187479 cites W2076205773 @default.
• W1967187479 cites W2079229575 @default.
• W1967187479 cites W2084826163 @default.
• W1967187479 cites W2085014479 @default.
• W1967187479 cites W2085366528 @default.
• W1967187479 cites W2088327605 @default.
• W1967187479 cites W2090899269 @default.
• W1967187479 cites W2091309716 @default.
• W1967187479 cites W2091418004 @default.
• W1967187479 cites W2095115132 @default.
• W1967187479 cites W2120199681 @default.
• W1967187479 cites W2124459218 @default.
• W1967187479 cites W2153434041 @default.

• next