cAMP Phosphodiesterase and Lung Endothelial Cell Permeability

cAMP 磷酸二酯酶和肺内皮细胞通透性

• 批准号: 7924691
• 负责人: THOMAS C RICH
• 金额: $ 42.8万
• 依托单位: UNIVERSITY OF SOUTH ALABAMA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7924691
• 关键词: Adenylate Cyclase; Adrenergic Receptor; Affinity; Agonist; Animal Model; Arteries; Bacterial Toxins; Biochemical; Blood Circulation; Blood Vessels; Breathing; Cell membrane; Cells; Cessation of life; Corrosion Casting; Cyclic AMP; Cytosol; Data; Dinoprostone; Distal; Down-Regulation; Dyes; Endothelial Cells; Endothelium; Energy Transfer; Ensure; Epoprostenol Receptors; Evans blue stain; Extravascular Lung Water; Filtration; Gases; Genetic; Hydrolysis; Imaging Techniques; Impairment; In Vitro; Inflammation Mediators; Isoproterenol; Kinetics; Liquid substance; Location; Lung; Measurement; Measures; Mechanics; Membrane; Microscopy; Molecular; Monitor; Organism; Patients; Permeability; Physiological; Play; Prostaglandins; Prostaglandins I; Proteins; Pulmonary Edema; Pulmonary artery structure; Rattus; Respiratory distress; Role; Shapes; Signal Transduction; Specificity; System; Techniques; Testing; Thapsigargin; Thrombin; Work; Work of Breathing; adenosine cyclic-3' ,5' -monophosphate binding proteins; cellular imaging; in vivo; indexing; inhibitor/antagonist; interstitial; lung injury; monolayer; overexpression; phosphoric diester hydrolase; prevent; public health relevance; receptor; research study; response; sensor

DESCRIPTION (provided by applicant): In the lung, the endothelial barrier plays a critical role in maintaining gas exchange in response to the organism's demands. Disruption of the pulmonary endothelial barrier results in pulmonary edema as fluid accumulates in the interstitium and ultimately in the distal airspaces. This leads to severe impairment in gas exchange and eventually, death. In both cultured pulmonary microvascular endothelial cells (PMVECs) and in vivo animal models, cyclic AMP (cAMP) generated by plasma membrane-localized adenylyl cyclase is barrier protective. In contrast, cAMP generated in the cytosol by a bacterial toxin, ExoY, or a soluble adenylyl cyclase is barrier disruptive. Pulmonary arterial endothelial cells (PAECs) form barriers with higher constitutive permeability than PMVECs. The mechanisms underlying this leakiness have not been identified. It is known that increases in the near- membrane cAMP levels prevent thrombin- and thapsigargin-induced endothelial barrier disruption in pulmonary arteries. As such, increases in near-membrane cAMP levels are considered barrier protective. PAECs express both the high affinity PDE7 (Km ~0.1 ¿M) and low affinity PDE4 (Km ~3 ¿M), with similar hydrolysis rates. We have demonstrated that stable knockdown of PDE7A in PAECs is barrier protective. These and other observations described herein led us to the following working hypothesis: The activity of the high affinity PDE7 lowers cAMP levels and increases barrier permeability of pulmonary arterial endothelial cells. To test this hypothesis we will use a variety of biochemical and cellular imaging techniques to dissect the roles of PDE7 activity in regulating cAMP signals in cultured PAECs in vitro. We will then use both pharmacological and genetic approaches to examine the effects of altering PDE7 activity on endothelial barrier function in vitro, ex vivo (in the isolated lung), and in vivo. The following SPECIFIC AIMS outline a plan to integrate cellular signaling and systems physiological approaches in order to establish the physiological roles of PDE7 in regulating endothelial barrier function. SPECIFIC AIM 1. Determine whether PDE7 activity lowers near-membrane cAMP levels in cultured rat pulmonary arterial endothelial cells. SPECIFIC AIM 2. Determine whether inhibition of PDE7 activity is barrier protective in the pulmonary arterial endothelium in vitro, ex vivo, and in vivo. PUBLIC HEALTH RELEVANCE: Increased endothelial permeability in pulmonary arteries results in decreased airway compliance, increased work of breathing, common complications of patients with respiratory distress. There are currently no strategies directed toward preventing increases in endothelial permeability in pulmonary arteries. Here we propose to determine whether pharmacological and molecular inhibitors of a specific protein, phosphodiesterase type 7, prevent increases in endothelial permeability in these arteries, and hence, make breathing easier.

描述（由适用提供）：在肺中，内皮屏障在响应有机体需求的情况下维持气体交换中起着至关重要的作用。肺部内皮屏障的破坏会导致肺水肿，因为液体在间隙中积聚并最终在远端空间中。这导致天然气交换的严重损害，最终导致死亡。在培养的肺微血管内皮细胞（PMVEC）和体内动物模型中，由质膜 - 膜 - 位置化腺苷环酶产生的环状AMP（CAMP）均受到屏障的保护。相反，细菌毒素，Exoy或固体腺苷酸环化酶在细胞质中产生的cAMP是屏障破坏性的。肺部伪像的内皮细胞（PAECS）形成比PMVEC更高的结构渗透性的屏障。尚未确定这种泄漏的机制。众所周知，近膜cAMP水平的增加可预防肺动脉动脉中凝血酶和thapigargin引起的内皮屏障破坏。因此，近膜营地的增加被认为是障碍保护性。 PAECS同时表达高亲和力PDE7（km〜0.1€）和低亲和力PDE4（km〜3€m），具有相似的水解速率。我们已经证明，PAEC中PDE7A的稳定敲低受到屏障的保护。本文所述的这些和其他观察结果使我们提出了以下工作假设：高亲和力PDE7的活性降低了营地水平，并增加了肺动脉内皮细胞的屏障渗透性。为了检验这一假设，我们将使用各种生化和细胞成像技术来剖析PDE7活性在调节cAMP信号中的作用。然后，我们将使用药理学和遗传学方法来检查改变PDE7活性对体外，离体（分离的肺）和体内内皮屏障功能的影响。以下特定目的概述了整合细胞信号传导和系统物理方法的计划，以确定PDE7在调节内皮屏障功能中的生理作用。具体目的1。确定PDE7活性在培养的大鼠肺动脉内皮细胞中是否降低了近膜cAMP水平。具体目的2。确定抑制PDE7活性是否在体外，体内和体内受到屏障保护。 公共卫生相关性：肺动脉动脉内皮渗透性的增加导致气道依从性降低，呼吸工作增加，呼吸窘迫患者的常见并发症。目前，尚无策略用于防止肺动脉内皮内皮渗透性增加。在这里，我们建议确定特定蛋白质的药物和分子抑制剂，即7型磷酸二酯酶，防止这些动脉中内皮渗透性的增加，从而使呼吸更容易。