Autocrine regulation of ciliary beat frequency

纤毛跳动频率的自分泌调节

• 批准号: 6576228
• 负责人: C. William Davis
• 金额: $ 7.38万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6576228
• 关键词: A kinase anchoring protein; autocrine; calcium flux; cellular polarity; cilium /flagellum motility; fluid flow; human tissue; laboratory mouse; mitochondria; phosphorylation; physiologic stressor; protein kinase A; purinergic receptor; receptor binding; receptor coupling; respiratory epithelium; vibration

Ciliary beating plays a critical role in host defense in the human airway, but we know little about the endogenous signals that control ciliary beat frequency, or how the signals are transduced from cell surface receptors to effector proteins in the cell. We hypothesize that physical stresses induce luminal nucleotide release to activate autocrine and paracrine pathways that modulate components of mucociliary clearance., including ciliary beating. Nucleotides, acting via P2Y2 receptors, and adenosine, acting via A2B receptors, stimulate ciliary beat frequency but the mechanisms involved are poorly understood. We will use functional and biochemical assays to study the polarity of nucleotide/nucleoside regulation of ciliary function in airway. We will determine whether G protein coupled receptors are present on ciliary membranes generate cAMP (A2B receptor) and Ca2+/IP3 (P2Y2 receptor) locally, or whether the receptors are present on apical membrane microvilli, requiring the diffusion of second messengers into the ciliary shaft. We will also study the distribution of IP3 receptors to determine whether IP3 generate by activation of P2Y2 receptors acts on Ca2+ stores in the cell body, in the ciliary, or in both compartments. P2Y2 receptors are present on apical and basolateral surfaces, but agonist increases ciliary only when applied to the luminal surface. We hypothesize that mitochondria impose a diffusion barrier critical for the compartmentalization of Ca++ signaling in airway cells, and will test this hypothesis in epithelial sheets and in isolated cells. We will also study the compartmentalization of protein kinase A, the final effector of the A2B receptor pathway, because our results indicate that protein kinase A is anchored in the cilia by association with a novel A-kinase anchoring protein, and that the anchored kinase preferentially phosphorylates a single ciliary target. We will study the functions of anchored PKA, the novel ciliary anchoring protein, and the major protein phosphorylated in isolated cilia. Finally, we will determine whether the pathways we identify are activated when airway epithelial cells are exposed to physical stresses typically seen by the respiratory epithelial, including flow, vibration, and shear. Together our studies will elucidate will elucidate mechanisms involved in modulating cilia function in human airway in response to luminal stimuli and may lead to novel therapeutic strategies to enhance clearance in airway disease.

纤毛跳动在人类气道的宿主防御中发挥着关键作用，但我们对控制纤毛跳动频率的内源信号或信号如何从细胞表面受体转导到细胞中的效应蛋白知之甚少。我们假设物理压力诱导管腔核苷酸释放，以激活自分泌和旁分泌途径，调节粘膜纤毛清除的成分，包括纤毛跳动。通过 P2Y2 受体发挥作用的核苷酸和通过 A2B 受体发挥作用的腺苷可刺激纤毛跳动频率，但所涉及的机制尚不清楚。我们将使用功能和生化测定来研究气道中纤毛功能的核苷酸/核苷调节的极性。我们将确定G蛋白偶联受体是否存在于睫状膜上，局部产生cAMP（A2B受体）和Ca2+/IP3（P2Y2受体），或者受体是否存在于顶膜微绒毛上，需要第二信使扩散到睫状干中。我们还将研究 IP3 受体的分布，以确定 P2Y2 受体激活产生的 IP3 是否作用于细胞体、睫状体或两个区室中的 Ca2+ 储存。 P2Y2 受体存在于顶端和基底外侧表面，但激动剂仅在应用于管腔表面时才会增加睫状体。我们假设线粒体施加了一个对于气道细胞中 Ca++ 信号传导至关重要的扩散屏障，并将在上皮层和分离的细胞中检验这一假设。我们还将研究蛋白激酶 A（A2B 受体途径的最终效应器）的区室化，因为我们的结果表明蛋白激酶 A 通过与新型 A 激酶锚定蛋白结合而锚定在纤毛中，并且锚定的激酶优先磷酸化单个纤毛靶标。我们将研究锚定 PKA、新型纤毛锚定蛋白以及分离纤毛中主要磷酸化蛋白的功能。最后，我们将确定当气道上皮细胞暴露于呼吸道上皮细胞通常看到的物理压力（包括流动、振动和剪切）时，我们确定的通路是否被激活。我们的研究将共同​​阐明参与调节人类气道纤毛功能以响应管腔刺激的机制，并可能导致新的治疗策略来增强气道疾病的清除。