CELLULAR BASIS OF AIRWAY SECRETION CLEARANCE COUPLING

描述（申请人摘要）：气管上皮细胞发挥着重要作用， 通过维持足够的粘膜纤毛在肺宿主防御中起重要作用 间隙该项目的长期目标是阐明细胞和 调节人气道粘膜纤毛的分子机制 间隙两个主要的G蛋白偶联信号系统发挥重要作用 在调节粘膜清除：毒蕈碱受体（M3）刺激 乙酰胆碱（ACh）和嘌呤能受体（P2 Y）。 本申请集中于ACh对纤毛运动的调节， ATP/UTP，并建议使用最先进的单 细胞光学、生物物理和分子技术应用于人类细胞。我们有 发现P2 Y和M3受体对睫状体具有双重、相反的作用， 搏动频率（CBF），似乎是由 胞浆Ca ~（2+）浓度（[Ca ~（2+）]i）。这两个动作的结合 有助于使纤毛细胞对细胞的生长 调节CBF反应，并且还可以与代谢活性偶联。在这 应用，我们将研究RGS蛋白，一个新描述的家庭的作用 G蛋白信号的调节剂，以及它们影响的程度 [Ca2+]i答复。我们将使用RGS转染细胞作为工具， 通过测量G蛋白和G蛋白之间的动力学耦合来操纵G蛋白信号传导， Ca 2+和CBF的变化，确定介导这种变化的信号分子， 偶联，并提出了我们的定量模型的钙调节CBF。的 具体目标是：（一）表征人气道中的Ca 2+信号和CBF 上皮细胞在气液界面生长。二.）定义的角色 RGS蛋白通过M3受体介导G蛋白偶联的[Ca 2 +]i信号传导。 三.）在分子水平上了解[Ca ~（2+）]i与CBF之间的耦合。 这个项目有许多创新的方面。我们将探索G蛋白偶联 使用编码以下的质粒转染气道细胞的受体信号传导 RGS蛋白家族成员。对于这些研究，我们将使用偏振， 我们可以在人类呼吸道上皮细胞中测量几种生理 同时在单个细胞和单个纤毛上的Ca 2+，CBF反应 水平这种独特的能力将促进我们对Ca 2+处理的了解， 纤毛运动和G蛋白偶联信号传导。这些研究不仅 提高我们对气道中粘膜纤毛清除的理解， 有助于我们更广泛地理解信号转导。

DESCRIPTION (Applicant's Abstract): The cells of the tracheal epithelium play a vital role in pulmonary host defense by maintaining adequate mucociliary clearance. The long-term goal of this project is to elucidate the cellular and molecular mechanisms responsible for regulation of human airway mucociliary clearance. Two major G-protein-coupled signaling systems play important roles in regulating mucociiary clearance: muscarinic receptors (M3) stimulated by acetylcholine (ACh) and purinergic receptors (P2Y) stimulated by ATP or UTP. This application focuses on the regulation of ciliary motility by ACh and ATP/UTP and proposes to extend previous studies using state-of-the-art, single cell optical, biophysical, and molecular techniques to human cells. We have found that both P2Y and M3 receptors have dual, opposing actions on ciliary beating frequency (CBF), actions that seem to be mediated by changes in cytoplasmic Ca2+ concentration ([Ca2+]i). The combination of these two actions serves to give the ciliated cell an extraordinary degree of fine control in regulating CBF responses and may also couple to metabolic activity. In this application, we will examine the role of RGS proteins, a newly described family of regulators of G-protein signaling, and the extent to which they influence [Ca2+]i responses. We will use RGS transfections into cells as tools to manipulate G-protein signaling by measuring the kinetic coupling between changes in Ca2+ and CBF, identifying signaling molecules that mediate such coupling, and advancing our quantitative model of Ca2+ regulation of CBF. The specific aims are: I.) To characterize Ca2+ signaling and CBF in human airway epithelial cells grown at the air-liquid interface. II.) To define the role of RGS proteins in mediating G-protein coupled [Ca2+]i signaling by M3 receptors. III.) To understand coupling between [Ca2+]i and CBF at the molecular level. This project has numerous innovative aspects. We will explore G-protein coupled receptor signaling using transfection of airway cells with plasmids encoding members of the RGS protein family. For these studies, we will use polarized, human airway epithelial cells in which we can measure several physiological responses (Ca2+, CBF) simultaneously at the single cell and single cilium level. This unique capability will advance our knowledge of Ca2+ handling, ciliary motility, and G-protein coupled signaling. Such studies will not only improve our understanding of mucociliary clearance in the airway but also contribute more broadly to our understanding of signal transduction.