NO TOXICITY: REDOX REGULATION OF LUNG CELL THIOREDOXIN

外源性一氧化氮（NO）已越来越多地用于新生儿、儿童和各种肺部疾病的成人患者的吸入治疗。体外和体内研究表明，外源性NO抑制血管内皮基底NO释放。这种一氧化氮诱导的负反馈调节与在转录后水平上抑制组成性表达的内皮一氧化氮合成酶（eNOS）有关，包括活性位点硫醇的s -亚硝基化和eNOS分子内二硫化物的形成。eNOS活性的丧失可通过氧化还原调节蛋白硫氧还蛋白/硫氧还蛋白还原酶（Thx/ThxR）逆转，其基因表达是通过Rel家族转录因子NFkB介导的。在肺内皮细胞和完整肺中，NO还可以抑制Thx/ThxR和NFkB亚基蛋白的mRNA和蛋白表达，以及NFkB/DNA结合活性。由于NFkB调节多种基因的表达，包括其自身亚基基因和Thx/ThxR基因，并且由于Thx/ThxR水平对氧化还原敏感过程和内皮依赖性肺功能的调节至关重要，因此了解no诱导的Thx/ThxR表达调控非常重要。我们的初步数据使我们假设NO诱导的上游氧化还原敏感效应激酶、转录信号通路中的蛋白激酶C亚型（PKC-zeta和lambda）和翻译信号通路中的p70核糖体S6激酶（p70S6k）的抑制是NFkB亚基蛋白和Thx/ThxR合成减少的分子开关机制，并且Thx/ThxR基因表达可以恢复NO介导的内皮细胞功能障碍。为了验证这一假设，我们将：1)确定Thx/ThxR在NO诱导的PKC亚型和p70S6k表达和活性调节中的作用；2)确定NO诱导的NFkB相关蛋白和Thx/ThxR蛋白合成抑制中的转录和翻译信号通路；3)验证Thx/ThxR基因转染可恢复离体肺动脉内皮依赖性NO/cGMP介导的血管调节功能。了解一氧化氮诱导氧化还原调节和内皮细胞功能障碍的分子机制，将有助于开发新的治疗方法，用于治疗产生过量一氧化氮的肺部疾病患者或长期吸入一氧化氮的患者。

Exogenous nitric oxide (NO) has been increasingly used in inhalation therapy in newborns, children, and adult patients with diverse pulmonary disorders. In vitro and in vivo studies show that exogenous NO inhibits basal NO release from vascular endothelium. This NO-induced negative- feedback regulation is associated with inhibition of constitutively expressed endothelial NO synthase (eNOS) at a post-transcriptional level involving S-nitrosylation of active site thiol and intramolecular disulfide formation in eNOS. The loss of eNOS activity is reversible by the redox regulatory proteins thioredoxin/thioredoxin - reductase (Thx/ThxR) whose gene expression is mediated through the Rel family transcription factor NFkB. NO is also known to inhibit mRNA and protein expression of Thx/ThxR and of NFkB subunit proteins as well as NFkB/DNA binding activity in lung endothelial cells and in intact lung. Because NFkB regulates expression of multiple genes including genes of its own subunits and of Thx/ThxR, and because Thx/ThxR levels are critical for regulation of redox-sensitive processes and endothelium-dependent lung function, understanding NO-induced regulation of Thx/ThxR expression is important. Our preliminary data lead us to hypothesize that NO- induced inhibition of the upstream redox-sensitive effector kinase, protein kinase C isoforms (PKC-zeta and lambda) in transcriptional signaling, and p70 ribosomal S6 kinase (p70S6k) in translational signaling pathways, serves as a molecular switching mechanism for reduced synthesis of NFkB subunit proteins and of Thx/ThxR, and that Thx/ThxR gene expression can restore NO-mediated endothelial cell dysfunction. To test this hypothesis, we will: 1) identify the role of Thx/ThxR in NO-induced modulations of PKC isoforms and p70S6k expression and activities, 2) identify the transcriptional and translational signaling pathways involved in NO-induced inhibition of NFkB- associated proteins and of Thx/ThxR protein synthesis, and 3) verify that Thx/ThxR gene transfection restores endothelium-dependent NO/cGMP- mediated vasoregulatory function in isolated pulmonary artery. Understanding the molecular mechanisms of NO-induced redox modulation and endothelial cell dysfunction will result in the development of novel treatments for patients with pulmonary disorders that generate excessive NO or patients administered long term NO inhalation therapy.