Autocrine regulation of ciliary beat frequency

纤毛搏动在人体呼吸道的宿主防御中起着至关重要的作用，但我们对控制纤毛搏动频率的内源信号，或者信号如何从细胞表面受体传导到细胞内的效应蛋白知之甚少。我们假设，物理应激诱导腔核苷酸释放来激活自分泌和旁分泌途径，这些途径调节粘液纤毛清除的成分，包括纤毛跳动。通过P2Y2受体作用的核苷酸和通过A2B受体作用的腺苷刺激纤毛搏动频率，但涉及的机制尚不清楚。我们将使用功能和生化分析来研究核苷酸/核苷对呼吸道纤毛功能调节的极性。我们将确定睫状膜上是否存在G蛋白偶联受体，局部产生cAMP(A2B受体)和Ca2+/IP3(P2Y2受体)，或者这些受体是否存在于顶膜微绒毛上，需要第二信使扩散到睫状轴。我们还将研究IP3受体的分布，以确定通过激活P2Y2受体而产生的IP3是否作用于细胞体、纤毛或两个隔室中的钙储存。P2Y2受体存在于顶端和基底外侧表面，但激动剂只有在腔表面应用时才会增加纤毛。我们假设线粒体施加了一个扩散屏障，对呼吸道细胞中钙信号的区隔至关重要，并将在上皮片和分离的细胞中检验这一假设。我们还将研究A2B受体途径的最终效应者蛋白激酶A的区隔作用，因为我们的结果表明，蛋白激酶A通过与一种新的A-激酶锚定蛋白结合而锚定在纤毛中，并且锚定的蛋白激酶优先磷酸化单个纤毛靶标。我们将研究锚定的PKA的功能，这是一种新型的纤毛锚定蛋白，也是分离的纤毛中磷酸化的主要蛋白。最后，我们将确定当呼吸道上皮细胞暴露在呼吸道上皮细胞通常所见的物理应力下时，我们确定的这些通路是否被激活，包括流动、振动和剪切。我们的研究将阐明参与调节人体呼吸道纤毛功能的机制，以响应管腔刺激，并可能导致新的治疗策略，以提高呼吸道疾病的清除。

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.