MOLECULAR MECHANISMS OF NO INDUCED LUNG CELL INJURY

一氧化氮(NO)是肺内皮细胞的内源性产物 以及一种外源气体，已知在 动物和人类肺部的生理学和病理生理学。 尽管有调节功能，但一氧化氮具有细胞毒性，并导致S- 亚硝化和抑制酶活性，包括抑制 内皮细胞一氧化氮合酶(EcNOS)呈结构性表达。 NO还抑制氧化还原信号的mRNA和蛋白表达 动物体内硫氧还蛋白/硫氧还蛋白还原酶(T/tr)系统的研究 人肺内皮细胞。T/tr在监管中发挥着关键作用 对DNA合成和基因至关重要的氧化还原敏感酶 表情。因此，NO诱导的ecNOS和T/tr的抑制可能 与NO/cGMP和氧化还原信号功能受损有关 这些细胞。然而，NO诱导的分子机制 内皮细胞功能障碍仍然难以捉摸。要确定这些 机制，我们假设：1)NO与活性中心的相互作用 EcNOS的半胱氨酸与催化活性降低有因果关系 因此损害了NO/cGMP信号和2)NO诱导 T/tr表达减少与DNA合成受阻有关 和/或基因表达。为了检验这些假设，我们将i)确定 一氧化氮与ecNOS活性部位半胱氨酸的相互作用及其 利用细胞技术对内皮细胞功能的影响 生物学、生物化学和分子生物学，II)确定 NO诱导的DNA合成和基因表达减少 通过监测T/tr的活性而减少T/tr的表达 氧化还原敏感酶核糖核苷酸还原酶与DNA 氧化还原敏感型p50提交转录因子结合活性的研究 NFkB，以及III)确定内皮细胞是否补充了 含有硫醇的试剂或通过基因工程获得高表达T/tr 保护细胞免受NO诱导的细胞功能障碍。了解 NO诱导内皮细胞功能障碍的分子机制 导致开发出治疗患者的新策略 肺部疾病会产生过量的NO或患者 接受长期NO吸入治疗。

Nitric oxide (NO) is an endogenous product of lung endothelial cells as well as an exogenous gas known to play a critical role in the physiology and pathophysiology of the lung in animals and humans. Despite its regulatory function, NO is cytotoxic and causes S- nitrosylation and inhibition of enzyme activities including inhibition of the constitutively expressed endothelial cell NO synthase (ecNOS). NO also inhibits mRNA and protein expression of the redox signalling enzyme system thioredoxin/thioredoxin reductase (T/TR) in animal and human lung endothelial cells. T/TR plays a critical role in regulation of redox-sensitive enzymes essential for DNA synthesis and gene expression. As such, NO-induced inhibition of ecNOS and T/TR may be associated with impaired NO/cGMP and redox signalling functions of these cells. However, the molecular mechanisms of NO-induced endothelial cell dysfunction remain elusive. To identify these mechanisms, we hypothesize that: 1) NO's interaction with active site cysteines of ecNOS is causally linked with reduced catalytic activity and therefore impaired NO/cGMP signalling and 2) NO-induced reduction of T/TR expression is linked to inhibition of DNA synthesis and/or gene expression. To test these hypotheses, we will I) identify NO's interaction with active site cysteines of ecNOS and establish its implications for endothelial ell function by utilizing techniques of cell biology, biochemistry, and molecular biology, II) identify whether NO-induced reduction of DNA synthesis and gene expression are associated with reduced expression of T/TR by monitoring activity of redox-sensitive enzyme ribonucleotide reductase (RR) and DNA binding activity of redox-sensitive p50 submit of transcription factor NFKB, and III) determine whether endothelial cells supplemented with thiol-containing agents or genetically-engineered to hyperexpress T/TR are protected from NO-induced cell dysfunction. Understanding the molecular mechanisms of NO-induced endothelial cell dysfunction will result in the development of novel strategies for treatment of patients with pulmonary disorders that generate excessive NO or patients administered long term NO inhalation therapy.