title: Nitrosative stress and redox-cycling agents synergize to cause mitochondrial dysfunction and cell death in endothelial cells.
abstract: Nitric oxide production by the endothelium is required for normal vascular homeostasis; however, in conditions of oxidative stress, interactions of nitric oxide with reactive oxygen species (ROS) are thought to underlie endothelial dysfunction. Beyond canonical nitric oxide signaling pathways, nitric oxide production results in the post-translational modification of protein thiols, termed S-nitrosation. The potential interplay between S-nitrosation and ROS remains poorly understood and is the focus of the current study. The effects of the S-nitrosating agent S-nitrosocysteine (CysNO) in combination with redox-cycling agents was examined in bovine aortic endothelial cells (BAEC). CysNO significantly impairs mitochondrial function and depletes the NADH/NAD(+) pool; however, these changes do not result in cell death. When faced with the additional stressor of a redox-cycling agent used to generate ROS, further loss of NAD(+) occurs, and cellular ATP pools are depleted. Cellular S-nitrosothiols also accumulate, and cell death is triggered. These data demonstrate that CysNO sensitizes endothelial cells to redox-cycling agent-dependent mitochondrial dysfunction and cell death and identify attenuated degradation of S-nitrosothiols as one potential mechanism for the enhanced cytotoxicity.
authors.affiliation: Department of Biophysics, Redox Biology Program, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226 USA.
authors.forename: Anne R
authors.initials: AR
authors.lastname: Diers
authors.forename: Katarzyna A
authors.initials: KA
authors.lastname: Broniowska
authors.forename: Neil
authors.initials: N
authors.lastname: Hogg
mesh_terms: D000818:Animals; D001011:Aorta / Q000166:cytology* / Q000187:drug effects; D002417:Cattle; D016923:Cell Death; D002478:Cells, Cultured; D003545:Cysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D004357:Drug Synergism; D042783:Endothelial Cells / Q000187:drug effects* / Q000473:pathology; D008928:Mitochondria / Q000187:drug effects* / Q000502:physiology; D009285:Naphthoquinones / Q000494:pharmacology*; D015538:Nitrosation; D017382:Reactive Oxygen Species; D026403:S-Nitrosothiols / Q000494:pharmacology*
chemical_list: D009285:Naphthoquinones; D017382:Reactive Oxygen Species; D026403:S-Nitrosothiols; C063002:2,3-dimethoxy-1,4-naphthoquinone; C021315:S-nitrosocysteine; D003545:Cysteine
keywords: BAEC, Bovine aortic endothelial cells; BSO, Buthioninesulphoximine; CysNO, S-nitrosocysteine; DMNQ, 2,3-dimethoxy-1,4-naphthoquinone; DMSO, Dimethyl sulfoxide; DPBS, Dulbecco’s phosphate buffered saline; DTPA, Diethylenetriaminepentaacetic acid; DTT, Dithiothreitol; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; GSHee, Glutathione Ethyl Ester; LDH, Lactate Dehydrogenase; Mitochondria; N.D., Not detectable; NAC, N-acetyl cysteine; NOS, Nitric oxide synthase; Nitric oxide; OCR, Oxygen consumption rate; ROS, Reactive oxygen species; Reactive oxygen species; S-nitrosation; S-nitrosylation; SEM, Standard error of the mean.; Thiol; cGMP, Cyclic guanosine monophosphate
references.citation: J Biol Chem. 2006 Nov 10;281(45):33835-41
references.pmid: 16893892
references.citation: Antioxid Redox Signal. 2011 Jun;14(11):2151-62
references.pmid: 21083422
references.citation: Front Pharmacol. 2012 Nov 16;3:193
references.pmid: 23162467
references.citation: Biochem Biophys Res Commun. 1987 Sep 30;147(3):1095-104
references.pmid: 3499150
references.citation: Free Radic Biol Med. 2002 Dec 1;33(11):1590-6
references.pmid: 12446216
references.citation: Free Radic Biol Med. 2002 Dec 1;33(11):1465-74
references.pmid: 12446203
references.citation: Nat Rev Mol Cell Biol. 2005 Feb;6(2):150-66
references.pmid: 15688001
references.citation: Free Radic Biol Med. 2005 May 15;38(10):1278-95
references.pmid: 15855047
references.citation: Free Radic Res. 2003 Jan;37(1):1-10
references.pmid: 12653211
references.citation: Biochim Biophys Acta. 1999 May 5;1411(2-3):334-50
references.pmid: 10320667
references.citation: Chem Rev. 2011 Oct 12;111(10):5997-6021
references.pmid: 21928855
references.citation: Anal Biochem. 1998 May 1;258(2):322-30
references.pmid: 9570848
references.citation: J Biol Chem. 2003 Dec 19;278(51):51360-71
references.pmid: 14551203
references.citation: Biochim Biophys Acta. 1980 Jun 5;630(1):119-30
references.pmid: 6248123
references.citation: Front Physiol. 2012 Sep 14;3:369
references.pmid: 23049513
references.citation: Drug Discov Today. 2008 Mar;13(5-6):268-74
references.pmid: 18342804
references.citation: Biochem J. 2006 Mar 15;394(Pt 3):627-34
references.pmid: 16371007
references.citation: Am J Physiol Heart Circ Physiol. 2010 Oct;299(4):H1212-9
references.pmid: 20675567
references.citation: J Immunol Methods. 1983 Dec 16;65(1-2):55-63
references.pmid: 6606682
references.citation: Biochem J. 2010 May 13;428(2):255-67
references.pmid: 20331438
references.citation: J Mol Cell Cardiol. 2009 Jun;46(6):804-10
references.pmid: 19285082
references.citation: Am J Physiol Heart Circ Physiol. 2011 Sep;301(3):H803-12
references.pmid: 21685262
references.citation: Proc Natl Acad Sci U S A. 2004 May 25;101(21):7891-6
references.pmid: 15148403
references.citation: Arch Biochem Biophys. 2003 Mar 1;411(1):145-57
references.pmid: 12590933
references.citation: Biochem J. 2012 Jun 15;444(3):561-71
references.pmid: 22458763
references.citation: Trends Neurosci. 2007 Jan;30(1):37-45
references.pmid: 17137643
references.citation: Biochem Pharmacol. 1995 Jan 18;49(2):127-40
references.pmid: 7530954
references.citation: J Biol Chem. 2007 May 11;282(19):14186-93
references.pmid: 17389593
references.citation: Antioxid Redox Signal. 2008 Aug;10(8):1343-74
references.pmid: 18522489
references.citation: Free Radic Biol Med. 2006 Feb 1;40(3):459-68
references.pmid: 16443161
references.citation: J Cardiovasc Pharmacol. 1988;12 Suppl 5:S115-8
references.pmid: 2469867
references.citation: Antioxid Redox Signal. 2012 Oct 1;17(7):969-80
references.pmid: 22468855
references.citation: Biochem J. 2012 Feb 15;442(1):191-7
references.pmid: 22070099
references.citation: J Mol Cell Cardiol. 2007 Apr;42(4):812-25
references.pmid: 17350035
references.citation: Antioxid Redox Signal. 2008 Jun;10(6):1045-59
references.pmid: 18321201
references.citation: Biochem J. 2010 Jan 27;426(1):31-41
references.pmid: 19916962
references.citation: Biochemistry. 1999 Nov 23;38(47):15407-16
references.pmid: 10569923
references.citation: Annu Rev Physiol. 1995;57:707-36
references.pmid: 7539994
references.citation: Proc Natl Acad Sci U S A. 2004 Mar 23;101(12):4003-8
references.pmid: 15020765
references.citation: Free Radic Biol Med. 2010 Apr 1;48(7):905-14
references.pmid: 20093177
references.citation: J Physiol. 2009 Dec 1;587(Pt 23):5767-81
references.pmid: 19841000
references.citation: Free Radic Biol Med. 2007 Oct 1;43(7):1086-94
references.pmid: 17761304
references.citation: Free Radic Biol Med. 2009 Aug 1;47(3):269-74
references.pmid: 19409484
references.citation: Proc Natl Acad Sci U S A. 2009 Jun 30;106(26):10764-9
references.pmid: 19528654
references.citation: Free Radic Biol Med. 2010 Dec 15;49(12):1925-36
references.pmid: 20937380
references.citation: Cell Mol Life Sci. 2008 Dec;65(24):3950-60
references.pmid: 19011746
references.citation: Free Radic Biol Med. 2005 Apr 1;38(7):831-8
references.pmid: 15749378
references.citation: J Inorg Biochem. 2011 May;105(5):745-53
references.pmid: 21481816