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 以及一种已知在 动物和人类肺的生理学和病理生理学。 尽管具有调节功能，但NO具有细胞毒性并导致S- 亚硝基化和酶活性的抑制，包括 组成型表达的内皮细胞一氧化氮合酶（ecNOS）。 NO还抑制氧化还原信号的mRNA和蛋白质表达 酶系统硫氧还蛋白/硫氧还蛋白还原酶（T/TR）在动物和 人肺内皮细胞 T/TR在调节中起着关键作用 对DNA合成和基因表达至关重要的氧化还原敏感酶 表情 因此，NO诱导的ecNOS和T/TR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是难以捉摸的。 识别这些 机制，我们推测：1）NO与活性位点的相互作用 ecNOS的半胱氨酸与降低的催化活性有因果关系 因此受损的NO/cGMP信号和2）NO诱导的 T/TR表达的减少与DNA合成的抑制有关 和/或基因表达。 为了验证这些假设，我们将（I）确定 NO与ecNOS活性位点半胱氨酸的相互作用， 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）确定是否 NO诱导的DNA合成和基因表达的减少， 与T/TR表达减少相关， 氧化还原敏感酶核糖核苷酸还原酶（RR）和DNA 氧化还原敏感性p50转录因子的结合活性 和III）确定补充有NF κ B的内皮细胞是否 含巯基的试剂或经基因工程改造以超表达T/TR 免受NO诱导的细胞功能障碍。 了解 NO诱导的内皮细胞功能障碍的分子机制将 导致开发用于治疗患者的新策略 产生过量NO的肺部疾病或患者 给予长期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.