Nitric Oxide Modulation of CFTR Expression and Function

描述(由申请人提供)：囊性纤维化跨膜电导调节器(CFTR)是一种1480个氨基酸的蛋白质，是流量ATPase家族(60)的成员，具有cAMP调节的CI通道的功能。根据我们已发表的结果和初步数据，我们假设，小鼠和呼吸道细胞长期暴露于由一氧化氮(NO)与部分还原的氧中间体相互作用形成的活性物种(RON)浓度增加的试剂中，会导致关键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)通过蛋白酶体泛素化和内质网相关的降解来降低CFTR的顶端水平；(2)通过减少cAMP刺激后呼吸道和肺泡上皮细胞的氯离子分泌，减少CFTR的磷酸化。这些假说将通过使CALU-3、原代人呼吸道上皮细胞和小鼠气管细胞(MTE)暴露于NO和RONS以及C57BL/6小鼠暴露于NO(1-10ppm)、二氧化氮(NO2：1-10ppm)、气管内滴注肺支原体和测量CFTR氧化修饰和泛素化的程度以及微观(单通道氯电流)和宏观(全细胞氯电流、鼻电位差和肺泡液清除)指标来检验其作为cAMP激活的氯通道的能力。为了确定导致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酪氨酸构建CFTR突变体，在卵母细胞中表达每个cRNA，并测量卵母细胞暴露于RONS前后的基础和cAMP激活的全细胞和单通道氯电流。由于CFTR在呼吸道液体水化以及cAMP激活的钠跨肺泡上皮运输中具有重要作用，这些研究结果可能为一些肺、非囊性纤维性炎症性疾病，如哮喘、慢性阻塞性肺疾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。

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.