title: Cyclosporine A-induced nitration of tyrosine 34 MnSOD in endothelial cells: role of mitochondrial superoxide.
abstract: OBJECTIVE
  Cyclosporine A (CsA) has represented a fundamental therapeutic weapon in immunosuppression for the past three decades. However, its clinical use is not devoid of side effects, among which hypertension and vascular injury represent a major drawback. Endothelial cells are able to generate reactive oxygen and nitrogen species upon exposure to CsA, including formation of peroxynitrite. This may result in endothelial cell toxicity and increased tyrosine nitration. We have now studied the subcellular origin of superoxide formation in endothelial cells treated with CsA and the biochemical consequences for the function of mitochondrial enzymes.
  
  
  RESULTS
  By using electron spin resonance and endothelial cells lacking functional mitochondria, we showed that superoxide anion is generated in mitochondria. This was associated with an effect of CsA on bioenergetic parameters: increased mitochondrial membrane potential and inhibition of cellular respiration. In addition, CsA inhibited the activity of the mitochondrial enzymes aconitase and manganese superoxide dismutase (MnSOD). The use of murine lung endothelial cells deficient in endothelial nitric oxide synthase (eNOS) and NOS/peroxynitrite inhibitors allowed us to establish that the presence of eNOS and concomitant NO synthesis and peroxynitrite formation were essential for CsA induced nitration and inhibition of MnSOD activity. As the latter has been shown to become inactivated by nitration, we sought to identify this modification by mass spectrometry analysis. We found that CsA induced specific MnSOD tyrosine 34 nitration both in the recombinant protein and in endothelial cells overexpressing MnSOD.
  
  
  CONCLUSIONS
  We propose that CsA induced endothelial damage may be related to increased mitochondrial superoxide formation and subsequent peroxynitrite-dependent nitroxidative damage, specifically targeting MnSOD. The inactivation of this key antioxidant enzyme by tyrosine nitration represents a pathophysiological cellular mechanism contributing to self-perpetuation and amplification of CsA-related vascular toxicity.
authors.affiliation: Departamento Medicina Celular y Molecular, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
authors.forename: Mariano
authors.initials: M
authors.lastname: Redondo-Horcajo
authors.forename: Natalia
authors.initials: N
authors.lastname: Romero
authors.forename: Pablo
authors.initials: P
authors.lastname: Martínez-Acedo
authors.forename: Antonio
authors.initials: A
authors.lastname: Martínez-Ruiz
authors.forename: Celia
authors.initials: C
authors.lastname: Quijano
authors.forename: Catia F
authors.initials: CF
authors.lastname: Lourenço
authors.forename: Nieves
authors.initials: N
authors.lastname: Movilla
authors.forename: Jose Antonio
authors.initials: JA
authors.lastname: Enríquez
authors.forename: Fernando
authors.initials: F
authors.lastname: Rodríguez-Pascual
authors.forename: Eduardo
authors.initials: E
authors.lastname: Rial
authors.forename: Rafael
authors.initials: R
authors.lastname: Radi
authors.forename: Jesús
authors.initials: J
authors.lastname: Vázquez
authors.forename: Santiago
authors.initials: S
authors.lastname: Lamas
mesh_terms: D000154:Aconitate Hydratase / Q000037:antagonists & inhibitors / Q000378:metabolism; D000818:Animals; D002417:Cattle; D019069:Cell Respiration / Q000187:drug effects; D002478:Cells, Cultured; D016572:Cyclosporine / Q000633:toxicity*; D004578:Electron Spin Resonance Spectroscopy; D042783:Endothelial Cells / Q000187:drug effects* / Q000201:enzymology; D007166:Immunosuppressive Agents / Q000633:toxicity*; D013058:Mass Spectrometry; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008928:Mitochondria / Q000187:drug effects* / Q000201:enzymology; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000172:deficiency / Q000235:genetics; D030421:Peroxynitrous Acid / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013482:Superoxide Dismutase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism*; D014162:Transfection; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D007166:Immunosuppressive Agents; D011994:Recombinant Proteins; D013481:Superoxides; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; C002744:3-nitrotyrosine; D014443:Tyrosine; D016572:Cyclosporine; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse; D013482:Superoxide Dismutase; D000154:Aconitate Hydratase