Nilakantan Vani, Halligan Nadine L N, Nguyen Thanh K, Hilton Gail, Khanna Ashwani K, Roza Allan M, Johnson Christopher P, Adams Mark B, Griffith Owen W, Pieper Galen M
Division of Transplant Surgery, Department of Surgery, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
J Heart Lung Transplant. 2005 Oct;24(10):1591-9. doi: 10.1016/j.healun.2005.01.009.
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.
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.
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.
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.
锰超氧化物歧化酶（MnSOD）活性位点关键酪氨酸残基的硝化作用可导致酶失活。在本研究中，我们检测了诱导型一氧化氮合酶（iNOS）对急性排斥反应的心脏移植中MnSOD表达、活性及硝化作用的影响。
将Lewis（同基因移植）或Wistar-Furth（异基因移植）供体心脏移植到Lewis受体大鼠体内。部分大鼠接受L-N6-（1-亚氨基乙基）赖氨酸（L-NIL），一种特异性iNOS抑制剂。通过免疫组织化学、蛋白质印迹和狭缝印迹分析测定蛋白质硝化作用。使用蛋白质印迹、逆转录聚合酶链反应（RT-PCR）和免疫沉淀技术测定MnSOD酶活性和基因表达。
术后第6天（POD 6）时，MnSOD蛋白水平降低了50%，L-NIL可防止这种降低。RT-PCR分析表明，这种降低不能用MnSOD mRNA的任何变化来解释。在未处理的异基因移植中，POD 5时MnSOD酶活性降低，但蛋白质水平未降低。L-NIL也可防止POD 5时MnSOD活性的丧失。POD 6时，未处理的异基因移植中免疫反应性硝基酪氨酸明显可见。狭缝印迹分析表明，L-NIL可阻断异基因移植中硝基酪氨酸的形成。对MnSOD进行免疫沉淀，随后针对硝基酪氨酸进行蛋白质印迹时，MnSOD的硝化作用明显。
这些结果表明，急性排斥反应的心脏异基因移植中MnSOD酶活性降低可归因于通过iNOS依赖性途径产生的与硝化作用相关的翻译后修饰。这可能是急性移植物排斥反应中氧化应激放大的一个潜在主要来源。
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