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: 7737658
• 负责人: THOMAS C RICH
• 金额: $ 41.87万
• 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 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7737658
• 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（由申请人提供）：在肺中，内皮屏障在维持气体交换以响应生物体的需求方面起着关键作用。肺内皮屏障的破坏导致肺水肿，因为液体积聚在肺动脉中，并最终积聚在远端空气间隙中。这会导致气体交换严重受损，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，由质膜定位的腺苷酸环化酶产生的环腺苷酸（cAMP）具有屏障保护作用。相比之下，由细菌毒素ExoY或可溶性腺苷酸环化酶在胞质溶胶中产生的cAMP是屏障破坏性的。肺动脉内皮细胞（PAEC）形成的屏障具有比PMVEC更高的组成性通透性。造成这种泄漏的机制尚未查明。众所周知，近膜cAMP水平的增加可以防止凝血酶和毒胡萝卜素诱导的肺动脉内皮屏障破坏。因此，近膜cAMP水平的增加被认为是屏障保护。PAEC表达高亲和力PDE 7（Km ~0.1 <$M）和低亲和力PDE 4（Km ~3 <$M），具有相似的水解速率。我们已经证明PAEC中PDE 7A的稳定敲低是屏障保护性的。本文所述的这些和其他观察结果使我们得出以下工作假设：高亲和力PDE 7的活性降低cAMP水平并增加肺动脉内皮细胞的屏障通透性。为了验证这一假设，我们将使用各种生物化学和细胞成像技术来剖析PDE 7活性在体外培养的PAEC中调节cAMP信号的作用。然后，我们将使用药理学和遗传学方法来检查改变PDE 7活性对体外、离体（在离体肺中）和体内内皮屏障功能的影响。以下具体目标概述了整合细胞信号传导和系统生理方法的计划，以确定PDE 7在调节内皮屏障功能中的生理作用。具体目标1.确定PDE 7活性是否降低培养的大鼠肺动脉内皮细胞近膜cAMP水平。具体目标2.确定PDE 7活性的抑制是否在体外、离体和体内肺动脉内皮中具有屏障保护作用。 公共卫生关系：肺动脉内皮通透性增加导致气道顺应性降低，呼吸作功增加，是呼吸窘迫患者常见的并发症。目前还没有针对预防肺动脉内皮通透性增加的策略。在这里，我们建议确定特定蛋白质（磷酸二酯酶7型）的药理学和分子抑制剂是否可以防止这些动脉中内皮渗透性的增加，从而使呼吸更轻松。