Nitric Oxide Modulation of CFTR Expression and Function

一氧化氮调节 CFTR 表达和功能

• 批准号: 6870706
• 负责人: Sadis Matalon
• 金额: $ 36.17万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-12-15 至 2008-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6870706
• 关键词: Xenopus oocyte; chloride channels; clinical research; confocal scanning microscopy; cyclic AMP; cystic fibrosis; free radical oxygen; gene expression; human tissue; immunocytochemistry; immunoprecipitation; inflammation; ion transport; laboratory mouse; mixed tissue /cell culture; nitric oxide; oxidative stress; phosphorylation; proteasome; protein structure function; respiratory epithelium; ubiquitin

DESCRIPTION (provided by applicant): The cystic fibrosis trans-membrane conductance regulator (CFTR), a 1480 amino acid protein, is a member of the traffic ATPase family (60) and functions as a cAMP-regulated CI channel. Based on our published results and preliminary data, we hypothesize that chronic exposure of mice and airway cells to agents which increase concentrations of reactive species (RONS), formed by the interaction of nitric oxide (NO) with partially reduced oxygen intermediates, results in oxidative modifications (oxidation, nitration and/or nitrosation) of key CFTR amino acids. These changes may: (1) decrease apical levels of CFTR by targeting it for ubiquitination and endoplasmic reticulum associated degradation by proteasomes and (2) impair Cl- secretion across the airway and alveolar epithelial cells following cAMP-stimulation by decreasing CFTR phosphorylation. These hypotheses will be tested both in vitro, by exposing Calu-3, primary human airway epithelial cells and mouse tracheocytes (MTE) to NO and RONS, as well as C57BL/6 mice to NO (1-10 ppm); nitrogen dioxide (NO2:1-10 ppm); intratracheal instillation of Mycoplasma pulmonis and measure the extent of oxidative modification and ubiquitination of CFTR as well as microscopic (single channel Cl currents) and macroscopic (whole cell Cl currents, nasal potential differences and alveolar fluid clearance) indices of its ability to act as a cAMP-activated Cl- channel. To identify specific amino acids modifications leading to loss of CFTR function, we will construct CFTR mutants by substituting each of the 40 CFTR tyrosines with alanine, express each cRNA in oocytes, and measure basal and cAMP-activated whole cell and single channel Cl- currents before and after exposure of oocyte to the RONS. Because of the well demonstrated vital importance of CFTR in both the hydration of airway fluid, as well as in cAMP-activated Na+ transport across the alveolar epithelium, the results of these studies may offer significant new insight into the pathophysiology of a number of pulmonary, non cystic fibrosis inflammatory diseases such as asthma, chronic obstructive lung disease and adult respiratory distress syndrome.

描述（由申请人提供）：囊性纤维化跨膜电导调节剂 (CFTR) 是一种 1480 个氨基酸的蛋白质，是交通 ATP 酶家族 (60) 的成员，并充当 cAMP 调节的 CI 通道。根据我们发表的结果和初步数据，我们假设小鼠和气道细胞长期暴露于增加活性物质（RONS）（由一氧化氮（NO）与部分还原的氧中间体相互作用形成）浓度的物质，导致关键CFTR氨基酸的氧化修饰（氧化、硝化和/或亚硝化）。这些变化可能：（1）通过蛋白酶体靶向泛素化和内质网相关降解来降低 CFTR 的顶端水平，（2）通过降低 CFTR 磷酸化，在 cAMP 刺激后损害气道和肺泡上皮细胞的 Cl- 分泌。这些假设将在体外进行测试，将 Calu-3、原代人气道上皮细胞和小鼠气管细胞 (MTE) 暴露于 NO 和 RONS，以及将 C57BL/6 小鼠暴露于 NO (1-10 ppm)；二氧化氮（NO2：1-10 ppm）；气管内滴注肺支原体，测量 CFTR 的氧化修饰和泛素化程度，以及其作为 cAMP 激活的 Cl 通道的微观（单通道 Cl 电流）和宏观（全细胞 Cl 电流、鼻电位差和肺泡液清除率）指标。为了识别导致 CFTR 功能丧失的特定氨基酸修饰，我们将通过用丙氨酸取代 40 个 CFTR 酪氨酸中的每一个来构建 CFTR 突变体，在卵母细胞中表达每种 cRNA，并测量卵母细胞暴露于 RONS 之前和之后的基础和 cAMP 激活的全细胞和单通道 Cl-电流。由于 CFTR 在气道液体水合以及 cAMP 激活的 Na+ 跨肺泡上皮转运中的重要性已得到充分证明，因此这些研究的结果可能为许多肺部非囊性纤维化炎症性疾病（例如哮喘、慢性阻塞性肺病和成人呼吸窘迫综合征）的病理生理学提供重要的新见解。