Alveolar epithelial B2-adrenergic receptors

描述(申请人提供)：肺水肿液的清除是通过肺泡上皮细胞主动的钠转运来实现的。人们普遍认为，肺泡上皮细胞顶膜上的Na和CI-通道与肺泡上皮细胞基底外侧的Na，K-ATPase的协同作用产生了跨跨皮渗透梯度，导致液体流出肺泡。已经在动物模型和人类中观察到，在某些类型的肺损伤中，主动钠转运受到损害。大量实验数据表明，β2-肾上腺素能激动剂可加速Na的主动转运，加速水肿消退。这些数据提供了一种可能性，即β2-肾上腺素能激动剂可能用于治疗肺水肿。我们实验计划的总体目标是提高我们对β2肾上腺素能受体(β2AR)如何调节肺泡活性钠转运的理解。 我们已经报道，没有Beta1或Beta2肾上腺素能受体的小鼠肺总cAMP水平正常，但无法上调活性钠转运，以响应过多的肺泡液。这一发现使我们认为，β2AR调节活性钠转运需要的不仅仅是cAMP的产生。最近人们注意到，a2AR与与β2AR效应分子如PKA和CFTR密切相关的支架和接头蛋白相互作用，并将其锚定在亚膜细胞骨架上。这些蛋白质-蛋白质的相互作用允许信号的区隔和对Beta2AR功能的严格调节。在为这种竞争性更新做准备时，我们进行了初步的免疫沉淀研究，表明肺泡上皮(2ARs)与其他运输蛋白形成大分子复合体。这一新的数据使我们假设：β2AR对肺泡上皮主动运输的调节是通过与支架和/或适配器蛋白的高度调控的相互作用而发生的。 如果得到证实，这一假说将支持一种新的范式，即β2AR对肺泡活性钠运输的调节不仅依赖于cAMP的产生，还依赖于细胞膜上大分子调节复合体的形成。为了解决这一假设，我们制定了以下三个具体目标：目标1：确定β2AR-支架/适配器蛋白相互作用是否在体外调节肺泡上皮细胞对β2AR敏感的活性钠转运是必要的。目的2：确定β2AR-支架相互作用是否对β2AR调节正常肺内活性钠转运是必需的。目的3：确定急性肺损伤是否改变了小鼠肺组织中β2AR-支架/适配器蛋白的相互作用。在这一竞争性更新应用中，重点研究的是确定哪些支架和适配器蛋白与肺泡β2AR相互作用，哪些是调节活性钠转运所必需的，以及这些蛋白是否受到急性肺损伤的影响。这些研究提供了一个机会来扩大我们对肺泡上皮调节活性钠转运的机制的理解。希望我们的研究将确定损伤诱导的Beta2AR蛋白-蛋白相互作用的变化，这些变化将适用于治疗操作，目的是加速每年数百万急性肺水肿患者的肺水肿缓解。

DESCRIPTION (provided by applicant): The clearance of pulmonary edema fluid occurs by means of active Na+ transport by alveolar epithelial cells. It is widely accepted that the coordinated function of Na+ and CI- channels in the apical membrane and Na,K-ATPase in the basolateral aspect of alveolar epithelial cells creates a transepithelial osmotic gradient that causes fluid to exit the alveolus. It has been observed in animal models and humans that in some types of lung injury active Na+ transport is impaired. Substantial experimental data suggests that beta2-adrenergic agonists accelerate active Na+ transport and speed edema resolution. These data offer the possibility that beta2-adrenergic agonists may be useful for the treatment of pulmonary edema. The overall goal of our experimental program is to improve our understanding of how beta2-adrenergic receptors (beta2AR) regulate alveolar active Na+ transport. We have reported that mice with no beta1 or beta2 adrenergic receptors have normal total lung cAMP levels but are unable to upregulate active Na+ transport in response to excess alveolar fluid. This finding led us to consider that beta2AR regulation of active Na+ transport requires more than cAMP production. It has recently been noted that the a2AR interacts with scaffold and adaptor proteins that are in close proximity to beta2AR effector molecules such as PKA and CFTR and, anchor it to the sub-membrane cytoskeleton. These protein-protein interactions allow for compartmentalized signaling and tight regulation of beta2AR function. In preparation for this competitive renewal we conducted preliminary immunoprecipitation studies that suggest that alveolar epithelial (2ARS form macromolecular complexes with other transport proteins. This new data led us to hypothesize that: beta2AR regulation of alveolar epithelial active transport occurs via highly regulated interactions with scaffold and/or adaptor proteins. If confirmed, this hypothesis would support a new paradigm where beta2AR regulation of alveolar active Na+ transport is dependent not only on cAMP generation but also formation of a macromolecular regulatory complex at the cell membrane. To address this hypothesis we have formulated the following three specific aims: Aim 1: Determine if beta2AR-scaffold/adaptor protein interactions are necessary for regulation of beta2AR sensitive active Na+ transport in alveolar epithelial cells in vitro. Aim 2: Ascertain if beta2AR -scaffold interactions are necessary for beta2AR regulation of active Na+ transport in normal lungs. Aim 3: Determine if acute lung injury alters beta2AR -scaffold/adaptor protein interactions in mouse lung. The focused studies in this competitive renewal application are structured to define which scaffold and adaptor proteins alveolar beta2AR interacts with, which are required for regulation of active Na+ transport, and if these proteins are affected by acute lung injury. These studies offer an opportunity to expand our understanding of the mechanisms by which the alveolar epithelium regulates active Na+ transport. It is hoped that our studies will identify injury-induced alterations in beta2AR protein-protein interactions that would be amenable to therapeutic manipulation for purposes of speeding resolution of pulmonary edema in the millions of patients with acute pulmonary edema each year.