Pittman K M, MacMillan-Crow L A, Peters B P, Allen J B
Department of Anatomy, Physiological Sciences and Radiology, College of Veterinary Medicine, North Carolina State University, 4700 Hillsborough Street, Raleigh, NC 27606, USA.
Exp Eye Res. 2002 Apr;74(4):463-71. doi: 10.1006/exer.2002.1141.
Reactive nitrogen species, in particular, peroxynitrite (ONOO(-)) have been proposed to play an important role in the pathogenesis of endotoxin-induced uveitis (EIU). Tyrosine nitration by ONOO(-) has been shown in other model systems to inhibit the activity of the superoxide anion quenching enyzme, manganese superoxide dismutase (MnSOD), perhaps contributing to progression of disease. In this study, it is confirmed through immunoanalysis that nitrated proteins are produced during EIU, and furthermore, that MnSOD is a target of nitration during the inflammatory response. In addition, through microsequencing analyses, nitrated albumin--apparent in both control and EIU eyes--was identified. Positive immunostaining of nitrated proteins was seen in the ciliary epithelium, inflammatory cells, and protein exudate of eyes from rats injected with endotoxin. Incubation of nitrotyrosine immunoprecipitates from the iris and ciliary body (ICB) with a polyclonal antibody against MnSOD revealed that nitrated MnSOD was present only in the ICB of EIU rats. When the total activity of the enzyme was examined, it was observed that despite the presence of nitrated MnSOD, activity was increased relative to control. Analysis of MnSOD mRNA and protein from the ICB of both groups demonstrated an increase in mRNA expression and consequently a three- to five-fold increase in MnSOD protein in EIU rats as compared to control rats. Further examination of MnSOD protein expression through immunohistochemistry noted enhanced immunostaining in the ciliary epithelium of eyes of EIU rats. Additional investigation of a 70 kDa band apparent in nitrotyrosine immunoprecipitates from the ICB of control and EIU rats revealed that the plasma protein albumin is nitrated as well. This protein is present as a result of the breakdown of the blood-aqueous barrier during inflammation. In summary, two endogenous nitration targets, albumin and MnSOD, were identified. Nitrated MnSOD appears to be specifically targeted to the ICB during inflammation, underscoring the importance of the interface in EIU. Furthermore, the expression and activity of the enzyme is increased in the ICB during EIU, perhaps regulating reactive nitrogen species produced within the cells. This study implicates ONOO(-) in the pathogenesis of EIU and imparts the putative role MnSOD plays in disease resolution.
活性氮物质，特别是过氧亚硝酸根（ONOO(-)），被认为在内毒素诱导的葡萄膜炎（EIU）发病机制中起重要作用。在其他模型系统中已表明，ONOO(-)介导的酪氨酸硝化作用会抑制超氧阴离子淬灭酶锰超氧化物歧化酶（MnSOD）的活性，这可能促使疾病进展。在本研究中，通过免疫分析证实EIU过程中会产生硝化蛋白，而且在炎症反应期间MnSOD是硝化作用的靶点。此外，通过微量测序分析，在对照眼和EIU眼中均发现了硝化白蛋白。在内毒素注射大鼠的眼内，睫状体上皮、炎性细胞和蛋白渗出物中可见硝化蛋白的阳性免疫染色。用抗MnSOD多克隆抗体孵育虹膜和睫状体（ICB）的硝基酪氨酸免疫沉淀物，结果显示硝化MnSOD仅存在于EIU大鼠的ICB中。当检测该酶的总活性时，发现尽管存在硝化MnSOD，但与对照相比活性仍有所增加。对两组ICB中的MnSOD mRNA和蛋白进行分析，结果表明与对照大鼠相比，EIU大鼠的mRNA表达增加，因此MnSOD蛋白增加了三到五倍。通过免疫组织化学进一步检测MnSOD蛋白表达，发现EIU大鼠眼内睫状体上皮的免疫染色增强。对对照大鼠和EIU大鼠ICB的硝基酪氨酸免疫沉淀物中出现的一条70 kDa条带进行的额外研究表明，血浆蛋白白蛋白也被硝化。这种蛋白是炎症期间血-房水屏障破坏的结果。总之，确定了两个内源性硝化靶点，即白蛋白和MnSOD。硝化MnSOD在炎症期间似乎特异性定位于ICB，突出了该界面在EIU中的重要性。此外，在EIU期间ICB中该酶的表达和活性增加，这可能调节细胞内产生的活性氮物质。本研究表明ONOO(-)参与EIU的发病机制，并揭示了MnSOD在疾病消退中的假定作用。
