title: Post-translational modification of manganese superoxide dismutase in acutely rejecting cardiac transplants: role of inducible nitric oxide synthase.
abstract: BACKGROUND
  Nitration of a critical tyrosine residue in the active site of manganese superoxide dismutase (MnSOD) can lead to enzyme inactivation. In this study, we examined the effect of inducible nitric oxide synthase (iNOS) on MnSOD expression, activity and nitration in acutely rejecting cardiac transplants.
  
  
  METHODS
  Lewis (isograft) or Wistar-Furth (allograft) donor hearts were transplanted into Lewis recipient rats. Some rats received L-N6-(1-iminoethyl) lysine (l-NIL), a specific iNOS inhibitor. Protein nitration was determined by immunohistochemical, Western blot and slot-blot analyses. MnSOD enzyme activity and gene expression were determined using Western, reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoprecipitation techniques.
  
  
  RESULTS
  MnSOD protein levels were decreased 50% by post-operative day 6 (POD 6), which was prevented by L-NIL. RT-PCR analysis indicated that this decrease could not be explained by any changes in MnSOD mRNA. MnSOD enzyme activity but not protein was decreased at POD 5 in untreated allografts. The loss of MnSOD activity at POD 5 was also prevented by L-NIL. Immunoreactive nitrotyrosine was apparent in untreated allografts at POD 6. Slot-blot analysis indicated that nitrotyrosine formation in allografts could be blocked by L-NIL. Nitration of MnSOD was evident upon immunoprecipitation of MnSOD followed by Western blotting for nitrotyrosine.
  
  
  CONCLUSIONS
  These results suggest that the decreased MnSOD enzyme activity in acutely rejecting cardiac allografts can be attributed to a post-translational modification related to nitration arising via an iNOS-dependent pathway. This could be a potential major source of amplified oxidative stress in acute graft rejection.
authors.affiliation: Division of Transplant Surgery, Department of Surgery, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA. vnilakan@mcw.edu
authors.forename: Vani
authors.initials: V
authors.lastname: Nilakantan
authors.forename: Nadine L N
authors.initials: NL
authors.lastname: Halligan
authors.forename: Thanh K
authors.initials: TK
authors.lastname: Nguyen
authors.forename: Gail
authors.initials: G
authors.lastname: Hilton
authors.forename: Ashwani K
authors.initials: AK
authors.lastname: Khanna
authors.forename: Allan M
authors.initials: AM
authors.lastname: Roza
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Johnson
authors.forename: Mark B
authors.initials: MB
authors.lastname: Adams
authors.forename: Owen W
authors.initials: OW
authors.lastname: Griffith
authors.forename: Galen M
authors.initials: GM
authors.lastname: Pieper
mesh_terms: D000208:Acute Disease; D000818:Animals; D004195:Disease Models, Animal; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D006084:Graft Rejection / Q000201:enzymology*; D016027:Heart Transplantation; D008239:Lysine / Q000031:analogs & derivatives / Q000494:pharmacology; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018384:Oxidative Stress; D030421:Peroxynitrous Acid / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000235:genetics / Q000502:physiology*; D051381:Rats; D011917:Rats, Inbred Lew; D011920:Rats, Inbred WF; D013482:Superoxide Dismutase / Q000235:genetics* / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; C090663:N(6)-(1-iminoethyl)lysine; D030421:Peroxynitrous Acid; C002744:3-nitrotyrosine; D014443:Tyrosine; D052247:Nitric Oxide Synthase Type II; D013482:Superoxide Dismutase; D008239:Lysine