Mucociliary Function in Chronic Bronchitis

描述(申请人提供)：在慢性支气管炎中，粘液纤毛清除功能障碍，特别是在疾病恶化期间。慢性支气管炎的恶化通常与活性氧簇(ROS)的产生增加和呼吸道酸化有关。本项目将研究ROS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粘液纤毛运输系统的关键成分：细胞外ATP和透明质酸片段。我们认为，与连接蛋白相关但不同于连接蛋白的蛋白，形成通向细胞外的通道(称为连接蛋白)，并至少部分地负责在呼吸道上皮细胞顶端ATP的释放。我们假设，在疾病恶化的早期，ROS的产生不仅增加了细胞内钙离子浓度([Ca~(2+)]i)以刺激纤毛搏动频率(CBF)和通过膜联蛋白释放心尖ATP，而且还降解了心尖透明质酸，后者反过来刺激CBF并增加气道表面液体(ASL)的量。随后的细胞内酸化可能是由细胞因子介导的呼吸道表达的NADPH氧化酶(DUOX)活性上调所介导的，通过抑制纤毛活性和pannins而导致粘液纤毛功能障碍。这一假设将通过三个具体目标进行检验。具体目标1将测试这一假设，即外源ROS激活包括初始[Ca~(2+)]i增加的信号级联以刺激CBF，尽管由于DUOX产生H而导致轻微和暂时的细胞内酸化；此外，顶端透明质酸直接降解激活RHMM和RON以刺激CBF并可能增加ASL容量。具体目的2将验证Pannins负责将ATP释放到呼吸道上皮细胞顶面的假设，在那里，ATP在调节包括CBF和ASL体积在内的粘液纤毛功能中发挥重要作用。具体目标3将测试这一假说，即持续的细胞内酸化通过直接作用于纤毛搏动以及通过阻止通过膜上膜蛋白释放ATP来抑制粘液纤毛清除。拟议的实验结果，使用新的和最先进的方法，将为调节慢性支气管炎的粘液纤毛清除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.