Mucociliary Function in Chronic Bronchitis

描述（由申请人提供）：在慢性支气管炎中，粘膜纤毛清除功能失调，特别是在疾病加重期间。慢性支气管炎的恶化通常与活性氧（ROS）的产生增加和气道酸化有关。该项目将研究活性氧和细胞内酸化如何调节和/或失调粘液纤毛功能。至少有两种内源性和旁分泌介质控制着纤毛粘膜运输系统的关键成分：细胞外ATP和透明质酸片段。我们认为，pannexins是一种与连接蛋白相关但又不同于连接蛋白的蛋白质，可形成通往细胞外部的通道（称为pannexons），至少在一定程度上负责气道上皮中顶端ATP的释放。我们假设，在疾病恶化的早期，ROS的产生不仅增加细胞内钙浓度（[Ca2+]i）以刺激纤毛搏动频率（CBF）和通过pannexons释放的根尖ATP，而且还降解根尖透明质酸，从而刺激CBF并增加气道表面液体（ASL）体积。随后的细胞内酸化，可能是由细胞因子介导的气道中表达的NADPH氧化酶活性上调（Duox）介导的，通过抑制纤毛活性和泛内联蛋白导致纤毛粘膜功能障碍。这一假设将通过三个具体目标进行检验。特异性目标1将验证外源性ROS激活信号级联的假设，包括初始的[Ca2+]i增加以刺激CBF，尽管由于Duox产生H+导致细胞内轻微和暂时的酸化；此外，直接根尖透明质酸降解激活RHAMM和RON刺激CBF并可能增加ASL体积。特异性Aim 2将验证pannexins负责向气道上皮细胞顶表面释放ATP的假设，其中ATP在调节包括CBF和ASL体积在内的纤毛粘膜功能中起重要作用。特异性目的3将验证持续的细胞内酸化通过直接作用于纤毛搏动以及通过泛子阻止ATP释放来抑制纤毛粘液清除的假设。实验结果，采用新颖的和最先进的方法，将为慢性支气管炎粘膜纤毛清除的调节提供新的和重要的机制见解。

DESCRIPTION (provided by applicant): In chronic bronchitis, mucociliary clearance is dysfunctional, especially during disease exacerbation. Chronic bronchitis exacerbations are commonly associated with increased production of reactive oxygen species (ROS) and airway acidification. This project will examine how ROS and intracellular acidification may regulate and/or dysregulate mucociliary function. At least two endogenous and paracrine mediators control key components of the mucociliary transport system: extracellular ATP and hyaluronan fragments. We propose that pannexins, proteins related to but different from connexins, form channels to the outside of cells (called pannexons) and are responsible, at least in part, for the release of apical ATP in the airway epithelium. We hypothesize that early in disease exacerbation, the production of ROS not only increases intracellular calcium concentrations ([Ca2+]i) to stimulate ciliary beat frequency (CBF) and apical ATP release through pannexons but also degrades apical hyaluronan which in turn stimulates CBF and increases airway surface liquid (ASL) volume. Subsequent intracellular acidification, possibly mediated by cytokine-mediated upregulation of an NADPH oxidase activity expressed in the airway (Duox), leads to mucociliary dysfunction by inhibiting ciliary activity and pannexins. This hypothesis will be tested with three specific aims. Specific Aim 1 will test the hypothesis that exogenous ROS activate a signaling cascade that includes an initial [Ca2+]i increase to stimulate CBF, despite a mild and temporary intracellular acidification due to H+ production by Duox; in addition, direct apical hyaluronan degradation activates RHAMM and RON to stimulate CBF and possibly increase ASL volume. Specific Aim 2 will test the hypothesis that pannexins are responsible for releasing ATP to the apical surface of airway epithelial cells where ATP plays an important role in regulating mucociliary functions including CBF and ASL volume. Specific Aim 3 will test the hypothesis that persistent intracellular acidification inhibits mucociliary clearance by a direct action on ciliary beating as well as by preventing ATP release through pannexons. The results of the proposed experiments, using novel and state-of-the-art methods, will provide new and important mechanistic insights into the regulation of mucociliary clearance in chronic bronchitis.