CELLULAR BASIS OF AIRWAY SECRETION CLEARANCE COUPLING

描述(申请人摘要)：气管上皮细胞发挥作用 维持足够的粘液纤毛在肺宿主防御中的重要作用 通行证。这个项目的长期目标是阐明细胞和 人呼吸道粘液纤毛调节的分子机制 通行证。两个主要的G蛋白偶联信号系统发挥着重要作用 在调节粘膜清除中：M受体(M3)被激活 ATP或UTP刺激的乙酰胆碱(ACh)和嘌呤能受体(P2Y)。 这一应用重点是ACh和ACh对纤毛运动的调节 并建议扩展以前的研究，使用最先进的、单一的 将细胞光学、生物物理和分子技术转化为人类细胞。我们有 发现P2Y和M3受体对纤毛具有双重的、相反的作用 心跳频率(CBF)，似乎是由 胞浆内钙浓度([Ca~(2+)]i)。这两种行为的结合 为纤毛细胞提供了非常精细的控制。 调节CBF反应，也可能与代谢活动相结合。在这 应用，我们将研究RGS蛋白，一个新描述的家族的作用 G蛋白信号的调节器以及它们影响的程度 [Ca~(2+)]i回应。我们将使用RGS基因导入细胞作为工具来 通过测量G蛋白之间的动力学耦合来操纵G蛋白信号 钙离子和脑血流量的变化，识别介导这种变化的信号分子 耦合，提出了钙离子调控脑血流量的定量模型。这个 具体目标有：i)人呼吸道钙信号转导与脑血流量的研究 上皮细胞生长在气液界面。II.)定义…的角色 RGS蛋白通过M3受体介导G蛋白偶联[Ca~(2+)]i信号。 三、)从分子水平上了解[Ca~(2+)]i与脑血流的偶联作用。 这个项目有很多创新的方面。我们将探索G蛋白偶联 用编码质粒转染呼吸道细胞的受体信号转导 RGS蛋白家族的成员。对于这些研究，我们将使用极化的， 我们可以在人体呼吸道上皮细胞中测量几种生理性的 单细胞和单纤毛同时反应(Ca~(2+)、CBF 水平。这一独特的能力将促进我们对钙离子处理的了解， 纤毛运动和G蛋白偶联信号。这样的研究不仅将 提高我们对呼吸道粘液纤毛清除的了解 更广泛地有助于我们对信号转导的理解。

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.