Ion Transport and Mucus Clearance in CF Airways

描述（由申请人提供）：我们之前已经证明正常（NL）气道表面液体（ASL）的高度（体积）是受调节的，但其潜在机制尚不清楚，并且与疾病（如囊性纤维化；CF）的相关性存在争议。我们的长期目标是了解生理条件下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编码的剪切应力依赖信号，通过调节离子运输（Na+吸收和ci分泌）来自动调节NL和CF ASL体积。这一假设是基于一种新型系统获得的数据，该系统模拟了正常潮汐呼吸过程中肺在体内传递的剪切应力。通过荧光共振能量转移（FRET）测量，剪切应力诱导肌动蛋白细胞骨架在施加剪切的方向上重新排列，增加ATP释放到气道表面液体中，并增加5'外核苷酶（负责在气道表面制造腺苷的外酶）与A2b腺苷受体之间的距离。这些研究为理解剪切应力如何在气道上皮中被感知和传导提供了第一步。我们还发现主要效应物被剪切应力激活：正常气道上皮细胞外嘌呤核苷酸（ATP）和核苷（腺苷）依赖的ASL自我调节途径增加，而囊性纤维化气道仅依赖运动依赖的ATP途径来重新平衡异常的CF离子运输并调整ASL高度至足以运输粘液的水平。基于这些观察结果，本提案的具体目的是(1)识别ASL体积传感器，(2)了解ASL体积调节是如何转导的，(3)规范CF气道表面液体体积调节。

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.