Adenosine Regulation of Alveolar Fluid Homeostasis

描述（由申请人提供）：肺泡上皮细胞通过活跃的Na+转运从肺泡空气中清除肺水肿。这是直观的，主动运输增加上皮细胞的能量消耗时，细胞的能量储存可能受到损害。来自各种实验系统的数据表明，当能量储存减少时，细胞通过减少活性Na+运输来匹配能量消耗和能量供应。迄今为止，在肺泡上皮细胞中尚未发现这种反调控机制。腺苷是在细胞内ATP消耗和/或cAMP产生较高时由AMP代谢产生的。我们推测，肺损伤时cAMP（来自b受体信号传导）和AMP/ADP（来自Na、k - atp酶活性）水平的升高为肺泡中腺苷的产生提供了底物。我们最近注意到腺苷对离体大鼠肺的肺泡活性Na+转运具有浓度依赖的双向作用。具体来说，低浓度的腺苷（=10-8M）通过2a型腺苷受体（A2aR）增加肺泡活性Na+运输约100%，而高浓度（=10-6M）通过1型腺苷受体（A1R）减少。我们也在大鼠和小鼠远端肺组织和分离的大鼠和小鼠肺泡2型上皮细胞中发现了这些受体。这些新的观察结果首次描述了腺苷及其受体在肺泡上皮中的作用，以及第一个有条件地上下调节肺泡上皮活性Na+转运的自分泌/旁分泌机制。基于这些初步数据，我们假设：肺泡上皮腺苷受体参与正常和损伤肺中Na+活性运输的调节。已知的腺苷与cAMP产生和ATP消耗的相互关系以及我们的初步数据使我们提出了一种调节肺泡活性Na+运输的新范式。具体来说，我们认为在正常情况下，肺泡腺苷在细胞外空间的浓度较低，可能通过A2aR依赖途径作为肺泡活性Na+运输的积极调节剂。相反，在肺损伤期间，高ATP利用率和cAMP的产生导致细胞外腺苷浓度足以抑制腺苷酸环化酶并减少通过A1R依赖途径的主动转运。该模型表明腺苷及其受体参与了一个反馈回路，该反馈回路允许肺泡上皮细胞微调cAMP敏感的活性Na+转运，以响应ATP利用和/或cAMP产生的变化。为了验证我们的假设，我们提出了以下3个科学目标：目标1：表征肺泡上皮中的腺苷受体，并确定它们是否以及如何调节正常肺部肺泡活性Na+运输。目的2：确定腺苷受体调节肺泡活性Na+转运的机制。目的3：确定急性肺损伤时肺泡上皮腺苷受体信号传导是保护还是不适应。我们提出的重点研究整合了药理学操作、基因工程小鼠和生理模型的基因转移，以产生上皮腺苷受体功能的获得和丧失模型，这将使我们能够验证我们的假设，并确定腺苷受体在肺泡上皮中是否起到保护或不适应的作用。

DESCRIPTION (provided by applicant): Pulmonary edema is removed from the alveolar airspace by active Na+ transport by alveolar epithelial cells. It is intuitive that active transport increases epithelial cell energy consumption at times when cellular energy stores could be compromised. Data from a variety of experimental systems indicate that cells match energy consumption with energy supply by reducing active Na+ transport when energy stores are reduced. To date no such counter-regulatory mechanisms have been described in alveolar epithelial cells. Adenosine is produced from metabolism of AMP when intracellular ATP consumption and/or cAMP production are high. We speculate that increased levels of cAMP (from b-receptor signaling) and AMP/ADP (from Na,K-ATPase activity) in the setting of lung injury provide substrate for adenosine production in the alveolus. We recently noted that adenosine has concentration dependent, bidirectional effects on alveolar active Na+ transport in isolated rat lungs. Specifically, low concentrations of adenosine (=10-8M) increase alveolar active Na+ transport by ~100% via type 2a adenosine receptors (A2aR) whereas high concentrations (=10-6M) reduce it via type 1 adenosine receptors (A1R). We have also identified these receptors in distal rat and mouse lung tissue and isolated rat and mouse alveolar type 2 epithelial cells. These new observations are the first descriptions of a role for adenosine and its receptors in the alveolar epithelium and the first autocrine/paracrine mechanism that conditionally up- and down-regulates alveolar epithelial active Na+ transport. Based on this preliminary data, we hypothesize that: Alveolar epithelial adenosine receptors participate in the regulation of active Na+ transport in normal and injured lungs. The known inter-relationship of adenosine with cAMP production and ATP consumption and our preliminary data cause us to propose a new paradigm of regulation of alveolar active Na+ transport. Specifically, we believe that in normal lung alveolar adenosine concentrations in the extracellular space are low and serve as a positive modulator of alveolar active Na+ transport, probably via an A2aR dependent pathway. Conversely, during lung injury high ATP utilization and cAMP production lead to extracellular adenosine concentrations sufficient to inhibit adenylyl cyclase and reduce active transport via an A1R dependent pathway. This model suggests that adenosine and its receptors participate in a feedback loop that allows alveolar epithelial cells to fine tune cAMP sensitive active Na+ transport in response to changes in ATP utilization and/or cAMP production. To test our hypothesis we are proposing the following 3 scientific aims: Aim 1: Characterize adenosine receptors in the alveolar epithelium and determine if, and how, they regulate alveolar active Na+ transport in normal lungs. Aim 2: Determine the mechanism(s) by which adenosine receptors modulate alveolar active Na+ transport. Aim 3: Determine if alveolar epithelial adenosine receptor signaling is protective or maladaptive during acute lung injury. The focused studies we are proposing integrate pharmacologic manipulations, genetically engineered mice, and gene transfer with physiologic models to generate models of gain and loss of epithelial adenosine receptor function that will allow us to test our hypothesis and to determine if adenosine receptors serve protective or maladaptive roles in the alveolar epithelium.