Post-Translational Modification of Manganese Superoxide Dismutase in Acutely Rejecting Cardiac Transplants: Role of Inducible Nitric Oxide Synthase

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• 作者：Vani Nilakantan ; Nadine L.N. Halligan ; Thanh K. Nguyen ; Gail Hilton ; Ashwani K. Khanna ; Allan M. Roza ; Christopher P. Johnson ; Mark B. Adams ; Owen W. Griffith and Galen M. Pieper
• 刊名：The Journal of Heart and Lung Transplantation
• 出版年：2005
• 出版时间：2005
• 年：2005
• 卷：24
• 期：10
• 页码：1591-1599
• 全文大小：367 K

文摘

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.