title: Mechanisms for cellular NO oxidation and nitrite formation in lung epithelial cells.
abstract: Airway lining fluid contains relatively high concentrations of nitrite, and arterial blood levels of nitrite are higher than venous levels, suggesting the lung epithelium may represent an important source of nitrite in vivo. To investigate whether lung epithelial cells possess the ability to convert NO to nitrite by oxidation, and the effect of oxygen reactions on nitrite formation, the NO donor DETA NONOate was incubated with or without A549 cells or primary human bronchial epithelial (HBE) cells for 24 h under normoxic (21% O2) and hypoxic (1% O2) conditions. Nitrite production was significantly increased under all conditions in the presence of A549 or HBE cells, suggesting that both A549 and HBE cells have the capacity to oxidize NO to nitrite even under low-oxygen conditions. The addition of oxyhemoglobin to the A549 cell medium decreased the production of nitrite, consistent with NO scavenging limiting nitrite formation. Heat-denatured A549 cells produced much lower nitrite and nitrate, suggesting an enzymatic activity is required. This NO oxidation activity was highest in membrane-bound proteins with molecular size <100kDa. In addition, 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one and cyanide inhibited formation of nitrite in A549 cells. It has been shown that ceruloplasmin (Cp) possesses an NO oxidase and nitrite synthase activity in plasma based on NO oxidation to nitrosonium cation. We observed that Cp is expressed intracellularly in lung epithelial A549 cells and secreted into the medium under basal conditions and during cytokine stimulation. However, an analysis of Cp expression level and activity measured via p-phenylenediamine oxidase activity assay revealed very low activity compared with plasma, suggesting that there is insufficient Cp to contribute to detectable NO oxidation to nitrite in A549 cells. Additionally, Cp levels were knocked down using siRNA by more than 75% in A549 cells, with no significant change in either nitrite or cellular S-nitrosothiol formation compared to scrambled siRNA control under basal conditions or cytokine stimulation. These data suggest that lung epithelial cells possess NO oxidase activity, which is enhanced in cell-membrane-associated proteins and not regulated by intracellular or secreted Cp, indicating that alternative NO oxidases determine hypoxic and normoxic nitrite formation from NO in human lung epithelial cells.
authors: Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA 15261, USA; Pulmonary, Allergy and Critical Care Medicine, University of Pittsburgh, Pittsburgh, PA 15261, USA.
authors: Xue-Jun
authors: XJ
authors: Zhao
authors: Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA 15261, USA.
authors: Ling
authors: L
authors: Wang
authors: Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA 15261, USA; Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA 15261, USA.
authors: Sruti
authors: S
authors: Shiva
authors: Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA 15261, USA; Pulmonary, Allergy and Critical Care Medicine, University of Pittsburgh, Pittsburgh, PA 15261, USA.
authors: Jesus
authors: J
authors: Tejero
authors: Pulmonary, Allergy and Critical Care Medicine, University of Pittsburgh, Pittsburgh, PA 15261, USA.
authors: Mike M
authors: MM
authors: Myerburg
authors: Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA 15261, USA.
authors: Jun
authors: J
authors: Wang
authors: Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA 15261, USA.
authors: Sam
authors: S
authors: Frizzell
authors: Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA 15261, USA; Pulmonary, Allergy and Critical Care Medicine, University of Pittsburgh, Pittsburgh, PA 15261, USA. Electronic address: gladwinmt@upmc.edu.
authors: Mark T
authors: MT
authors: Gladwin
mesh_terms: D045744:Cell Line, Tumor; D002570:Ceruloplasmin / Q000502:physiology; D004847:Epithelial Cells / Q000378:metabolism; D006801:Humans; D008168:Lung / Q000166:cytology / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000502:physiology; D009573:Nitrites / Q000378:metabolism*; D010084:Oxidation-Reduction
chemical_list: D009573:Nitrites; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; D002570:Ceruloplasmin
keywords: Ceruloplasmin; Human lung epithelial cells; Hypoxia; NO oxidase; Nitric oxide; Nitrite
references: Biochemistry. 1996 Jun 4;35(22):6976-83
references: 8679521
references: Life Sci. 1995;56(21):1749-58
references: 7739349
references: J Biol Chem. 2003 Nov 21;278(47):46349-56
references: 12952953
references: Proc Natl Acad Sci U S A. 2009 Mar 24;106(12):4671-6
references: 19261856
references: Acta Chem Scand. 1947;1(10):944-50
references: 18876909
references: J Biol Chem. 2008 Jun 27;283(26):17855-63
references: 18424432
references: Blood. 2008 Oct 1;112(7):2636-47
references: 18596228
references: J Am Soc Nephrol. 2006 May;17(5):1382-7
references: 16597684
references: J Biol Chem. 2011 Dec 9;286(49):42679-42689
references: 21965683
references: Nat Chem Biol. 2005 Nov;1(6):308-14
references: 16408064
references: Biochim Biophys Acta. 1999 May 5;1411(2-3):290-309
references: 10320664
references: J Immunol. 1997 Aug 15;159(4):1938-44
references: 9257859
references: Nat Chem Biol. 2006 Sep;2(9):486-93
references: 16906150
references: Arch Biochem Biophys. 1998 Sep 1;357(1):1-7
references: 9721176
references: Nat Rev Drug Discov. 2008 Feb;7(2):156-67
references: 18167491
references: J Pharmacol Exp Ther. 1996 Jan;276(1):30-3
references: 8558447
references: J Clin Invest. 2005 May;115(5):1232-40
references: 15841216
references: Arterioscler Thromb Vasc Biol. 2006 Apr;26(4):697-705
references: 16424350
references: Am J Physiol Lung Cell Mol Physiol. 2007 Jun;292(6):L1352-60
references: 17307814
references: J Chromatogr B Analyt Technol Biomed Life Sci. 2007 May 15;851(1-2):93-105
references: 17208057
references: Proc Natl Acad Sci U S A. 1986 Jul;83(14):5086-90
references: 2873574
references: Circ Res. 2007 Mar 16;100(5):654-61
references: 17293481
references: J Clin Invest. 2005 Aug;115(8):2099-107
references: 16041407
references: J Biol Chem. 1999 Sep 17;274(38):27069-75
references: 10480920
references: Circulation. 2012 Jul 17;126(3):325-34
references: 22685116
references: Proc Natl Acad Sci U S A. 2000 Oct 10;97(21):11482-7
references: 11027349
references: Nat Med. 2003 Dec;9(12):1498-505
references: 14595407
references: Acta Physiol Scand. 2001 Jan;171(1):9-16
references: 11350258
references: Blood. 2006 Jan 15;107(2):566-74
references: 16195332
references: J Biol Chem. 2006 Sep 15;281(37):26994-7002
references: 16845122
references: J Biol Chem. 2001 Jul 6;276(27):24482-9
references: 11312267
references: Am J Physiol Heart Circ Physiol. 2006 Nov;291(5):H2026-35
references: 16798825
references: Nat Chem Biol. 2009 Dec;5(12):865-9
references: 19915529
references: Am J Physiol Cell Physiol. 2001 Feb;280(2):C280-7
references: 11208522
references: Methods Enzymol. 2008;440:137-56
references: 18423215
references: J Neural Transm (Vienna). 2007;114(6):777-81
references: 17406962
references: Circulation. 2008 Feb 5;117(5):670-7
references: 18212289
references: J Biol Chem. 2005 Dec 9;280(49):40583-8
references: 16183646
references: Nat Med. 1995 Aug;1(8):804-9
references: 7585184
references: Biochemistry. 2012 Jul 3;51(26):5285-92
references: 22620259
references: J Biol Chem. 2011 May 20;286(20):18277-89
references: 21296891
references: Free Radic Res. 2003 Jan;37(1):1-10
references: 12653211
references: Am J Physiol Heart Circ Physiol. 2007 Jun;292(6):H3072-8
references: 17307993
references: Am J Physiol Lung Cell Mol Physiol. 2004 Sep;287(3):L467-74
references: 14672925
references: FEBS Lett. 1998 May 8;427(2):225-8
references: 9607316
references: Clin Chem. 1970 Nov;16(11):903-10
references: 5473551
references: J Biol Chem. 1997 Aug 8;272(32):20185-90
references: 9242695
references: J Biol Chem. 2005 Sep 2;280(35):31126-31
references: 15837788
references: Cardiovasc Res. 2007 Jul 15;75(2):327-38
references: 17568573