Pieper Galen M, Nilakantan Vani, Halligan Nadine L N, Khanna Ashwani K, Hilton Gail, Vásquez-Vivar Jeannette
Division of Transplant Surgery, Department of Surgery, Medical College of Wisconsin, Milwaukee, WI, USA.
Biochem J. 2005 Nov 1;391(Pt 3):541-7. doi: 10.1042/BJ20050575.
Inducible nitric oxide synthase (iNOS) is a prominent component of the complex array of mediators in acute graft rejection. While NO production is determined by iNOS expression, BH4 (tetrahydrobiopterin), a cofactor of iNOS synthesized by GTP cyclohydrolase I, has been considered critical in sustaining NO production. In the present study, we examined time-dependent changes in iNOS and GTP cyclohydrolase I in rat cardiac allografts. The increase in iNOS protein and mRNA in allografts was similar at POD4 (post-operative day 4) and POD6. However, the peak increase in intragraft NO level at POD4 was not sustained at POD6. This disparity could not be explained by any decrease in iNOS enzyme activity measured ex vivo with optimal amounts of substrate and cofactors. Lower iNOS activity could be explained by changes in total biopterin levels in allografts at POD4 that was decreased to baseline at POD6. Changes in biopterin production correlated with lower GTP cyclohydrolase I protein levels but not by any change in GTP cyclohydrolase I mRNA. Functionally, allografts displayed bradycardia and distended diastolic and systolic dimensions at POD6 but not at POD4. Likewise, histological rejection scores were increased at POD4 but with a secondary increased stage at POD6. It is hypothesized that the dissimilar amounts of NO at early and later stages of rejection is due to uncoupling of iNOS arising from disproportionate synthesis of BH4. These findings provide insight into a potential pathway regulating NO bioactivity in graft rejection. Such knowledge may potentially assist in the design of newer strategies to prevent acute graft rejection.
诱导型一氧化氮合酶（iNOS）是急性移植排斥反应中复杂介质阵列的一个重要组成部分。虽然一氧化氮的产生由iNOS的表达决定，但四氢生物蝶呤（BH4），一种由GTP环化水解酶I合成的iNOS辅因子，被认为在维持一氧化氮的产生中起关键作用。在本研究中，我们检测了大鼠心脏同种异体移植中iNOS和GTP环化水解酶I随时间的变化。同种异体移植中iNOS蛋白和mRNA在术后第4天（POD4）和第6天（POD6）的增加相似。然而，POD4时移植内一氧化氮水平的峰值增加在POD6时并未持续。这种差异不能用在体外用最佳量的底物和辅因子测量的iNOS酶活性的任何降低来解释。较低的iNOS活性可以用POD4时同种异体移植中总生物蝶呤水平的变化来解释，该水平在POD6时降至基线。生物蝶呤产生的变化与较低的GTP环化水解酶I蛋白水平相关，但与GTP环化水解酶I mRNA的任何变化无关。在功能上，同种异体移植在POD6时出现心动过缓以及舒张期和收缩期尺寸增大，但在POD4时未出现。同样，组织学排斥评分在POD4时增加，但在POD6时出现继发性增加阶段。据推测，排斥反应早期和晚期一氧化氮量的差异是由于BH4合成不成比例导致iNOS解偶联。这些发现为移植排斥反应中调节一氧化氮生物活性的潜在途径提供了见解。此类知识可能有助于设计预防急性移植排斥反应的新策略。
