Alveolar epithelial B2-adrenergic receptors

描述（由申请方提供）：肺水肿液的清除通过肺泡上皮细胞的主动Na+转运进行。 广泛接受的是，肺泡上皮细胞的顶膜中的Na+和Cl-通道以及基底外侧方面中的Na，K-ATP酶的协调功能产生了导致流体离开肺泡的跨上皮渗透梯度。 在动物模型和人类中观察到，在某些类型的肺损伤中，主动Na+转运受损。大量实验数据表明，β 2-肾上腺素能激动剂可加速主动Na+转运并加速水肿消退。 这些数据提供了β 2-肾上腺素能受体激动剂可能用于治疗肺水肿的可能性。 我们实验计划的总体目标是提高我们对β 2-肾上腺素能受体（β 2AR）如何调节肺泡主动Na+转运的理解。 我们已经报道，没有β 1或β 2肾上腺素能受体的小鼠具有正常的总肺cAMP水平，但不能上调活性Na+转运以响应过量的肺泡液。 这一发现使我们认为β 2AR调节主动Na+转运需要的不仅仅是cAMP的产生。 最近已经注意到，α 2 AR与支架和衔接蛋白相互作用，所述支架和衔接蛋白与β 2 AR效应分子（例如PKA和CFTR）紧密接近，并且将其锚于亚膜细胞骨架。 这些蛋白质-蛋白质相互作用允许区室化信号传导和β 2AR功能的严格调节。在这种竞争性更新的准备中，我们进行了初步的免疫沉淀研究，表明肺泡上皮细胞（2ARS）与其他转运蛋白形成大分子复合物。 这一新的数据使我们假设：肺泡上皮细胞主动转运的β 2 AR调节通过与支架和/或衔接蛋白的高度调节的相互作用发生。 如果得到证实，这一假设将支持一个新的范例，即β 2 AR调节肺泡主动Na+转运不仅依赖于cAMP的产生，而且还在细胞膜上形成大分子调节复合物。 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β 2 AR-支架/衔接蛋白相互作用是否是必要的β 2 AR敏感的主动钠离子转运在体外肺泡上皮细胞的调节。 目的2：确定β 2 AR-支架相互作用是否是β 2 AR调节正常肺中主动Na+转运所必需的。 目的3：确定急性肺损伤是否改变小鼠肺中β 2 AR-支架/接头蛋白的相互作用。 在这种竞争性更新应用中的重点研究旨在确定肺泡β 2 AR与哪些支架和衔接蛋白相互作用，这些蛋白是调节主动Na+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，扩大我们的了解肺泡上皮细胞调节主动钠离子转运的机制。 希望我们的研究将确定损伤诱导的β 2 AR蛋白-蛋白相互作用的改变，这些改变将适合于治疗操作，以加速每年数百万急性肺水肿患者肺水肿的消退。

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.