Post-translational modification of manganese superoxide dismutase in acutely rejecting cardiac transplants: role of inducible nitric oxide synthase.
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.
Nilakantan
Vani
V
Division of Transplant Surgery, Department of Surgery, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA. vnilakan@mcw.edu
Halligan
Nadine L N
NL
Nguyen
Thanh K
TK
Hilton
Gail
G
Khanna
Ashwani K
AK
Roza
Allan M
AM
Johnson
Christopher P
CP
Adams
Mark B
MB
Griffith
Owen W
OW
Pieper
Galen M
GM
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
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