Ion Transport and Mucus Clearance in CF Airways

描述（由申请人提供）：我们先前已经表明，正常（NL）气道表面液体（ASL）的高度（体积）是受调节的，但潜在的机制是未知的，与疾病的相关性（例如，囊性纤维化; CF）存在争议。我们的长期目标是了解在生理条件下NL和CF气道上皮中ASL体积是如何自动调节的。提出的研究背后的具体假设是ATP和ADO作为ASL中编码的剪切应力依赖性信号，通过调节离子转运（Na+吸收和CI分泌）自动调节NL和CF ASL体积。这一假设是基于一个新的系统，模拟正常的潮气呼吸过程中，在体内的肺所赋予的剪切应力获得的数据。剪切应力诱导肌动蛋白细胞骨架在所施加的剪切方向上重新排列，增加ATP释放到气道表面液体中，并增加5'外核苷酸酶（负责在气道表面上产生腺苷的外酶）与A2 b腺苷受体之间的距离，如通过荧光共振能量转移（FRET）所测量的。这些研究提供了第一步，了解如何剪切应力可以感觉到和气道上皮细胞转导。我们还发现，剪切应力激活的主要效应：细胞外嘌呤核苷酸（ATP）和核苷（腺苷）的ASL自动调节依赖性途径在正常的气道上皮细胞增加，而囊性纤维化气道完全依赖于运动依赖性ATP途径，以重新平衡异常CF离子转运，并调整ASL的高度，足以粘液运输的水平。基于这些观察结果，本提案的具体目的是（1）识别ASL容量的传感器，（2）了解ASL容量调节如何被转换，以及（3）使CF气道表面液体容量调节标准化。

DESCRIPTION (provided by applicant): We have previously shown that the height (volume) of normal (NL) airway surface liquid (ASL) is regulated, but the underlying mechanisms are unknown and the relevance to disease (e.g., cystic fibrosis; CF) controversial. Our long-term goal is to understand how ASL volume is autoregulated in NL and CF airway epithelia under physiological conditions. The specific hypothesis behind the proposed research is that ATP and ADO act as shear stress-dependent signals encoded in the ASL to autoregulate NL and CF ASL volume by modulating ion transport (Na+absorption and CI-secretion). This hypothesis is based on data obtained with a novel system that mimics the shear stress imparted by the lung in vivo during normal tidal breathing. Shear stress induced a realignment of the actin cytoskeleton in the direction of the applied shear, increased ATP release into the airway surface liquid and increased the distance between the 5' ectonucleotidase (the ecto-enzyme responsible for making adenosine on airway surfaces) and the A2b adenosine receptor, as measured by fluorescence resonance energy transfer (FRET). These studies provide a first step towards understanding how shear stress may be sensed and transduced in airway epithelia. We also found that principal effectors were activated by shear stress: extracellular purine nucleotide (ATP)- and nucleoside (adenosine)-dependent pathways for ASL autoregulation were increased in normal airway epithelia while cystic fibrosis airways relied solely on a motion-dependent ATP pathway to rebalance abnormal CF ion transport and adjust ASL height to levels adequate for mucus transport. Based on these observations, the specific aims of this proposal are (1) to identify sensors of ASL volume, (2) to understand how ASL volume regulation is transduced and (3) to normalize CF airway surface liquid volume regulation.