NO TOXICITY: REDOX REGULATION OF LUNG CELL THIOREDOXIN

外源性一氧化氮（NO）已越来越多地用于吸入治疗新生儿，儿童和成人患者的各种肺部疾病。体内外研究表明，外源性NO抑制血管内皮细胞基础NO释放。这种NO诱导的负反馈调节与在转录后水平抑制组成型表达的内皮NO合酶（eNOS）有关，涉及eNOS中活性位点巯基的S-亚硝基化和分子内二硫键形成。eNOS活性的丧失是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）可逆的，其基因表达通过Rel家族转录因子NF κ B介导。还已知NO抑制Thx/ThxR和NF κ B亚基蛋白的mRNA和蛋白表达以及肺内皮细胞和完整肺中的NF κ B/DNA结合活性。因为NFkB调节多个基因的表达，包括其自身亚基和Thx/ThxR的基因，并且因为Thx/ThxR水平对于调节氧化还原敏感性过程和内皮依赖性肺功能至关重要，所以理解NO诱导的Thx/ThxR表达的调节是重要的。我们的初步数据使我们假设，NO诱导的上游氧化还原敏感性效应激酶，蛋白激酶C亚型的抑制，在转录信号传导中的PKC-ζ和λ激酶以及在翻译信号传导途径中的p70核糖体S6激酶（p70 S6 k），作为NF κ B亚基蛋白和Thx/ThxR合成减少的分子转换机制，Thx/ThxR基因表达可恢复NO介导的内皮细胞功能障碍。为了验证这个假设，我们将：1）鉴定Thx/ThxR在NO诱导的PKC亚型和p70 S6 k表达和活性调节中的作用，2）鉴定参与NO诱导的NF κ B相关蛋白和Thx/ThxR蛋白合成抑制的转录和翻译信号传导途径，3）证实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NO诱导的氧化还原调节和内皮细胞功能障碍的分子机制，将导致开发新的治疗方法，用于产生过量NO的肺部疾病患者或给予长期NO吸入治疗的患者。

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.