Ion Transport and Mucus Clearance in CF Airways

描述(申请人提供)：我们先前已经证明，正常(NL)呼吸道表面液体(ASL)的高度(体积)是受调节的，但潜在的机制尚不清楚，与疾病(例如囊性纤维化；CF)的相关性也存在争议。我们的长期目标是了解生理条件下NL和CF的ASL容量是如何自动调节的。这项研究背后的具体假设是，ATP和ADO作为切应力依赖的信号编码在ASL中，通过调节离子转运(Na+吸收和CI分泌)来自动调节NL和CFASL的体积。这一假设是基于一种新的系统获得的数据，该系统模拟了正常潮气呼吸期间活体肺所施加的剪切力。通过荧光共振能量转移(FRET)检测，剪切力诱导肌动蛋白细胞骨架沿施加剪切力的方向重新排列，增加了ATP释放到呼吸道表面液体中，并增加了5‘胞外核苷酸酶(负责在气道表面制造腺苷的胞外酶)与A2B腺苷受体之间的距离。这些研究为了解切应力如何在呼吸道上皮细胞中被感知和传递提供了第一步。我们还发现，剪切力激活了主要效应器：在正常气道上皮细胞中，依赖细胞外嘌呤核苷酸(ATP)和核苷(腺苷)的ASL自动调节途径增加，而囊性纤维化气道仅依赖于运动依赖的ATP途径来重新平衡异常的CF离子转运，并将ASL高度调整到适合粘液运输的水平。基于这些观察，本建议的具体目标是(1)识别ASL容量的传感器，(2)了解ASL容量调节是如何传递的，以及(3)使CF气道表面液体容量调节正常化。

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.