cAMP Phosphodiesterase and Lung Endothelial Cell Permeability

cAMP 磷酸二酯酶和肺内皮细胞通透性

基本信息

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
项目类别：
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&apos ,5&apos -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

项目摘要

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，内皮屏障在响应生物体的需求而维持气体交换方面发挥着关键作用。当液体在间质中积聚并最终在远端中时，肺内皮屏障的破坏导致肺水肿。在培养的肺微血管内皮细胞 (PMVEC) 和体内动物模型中，这会导致气体交换严重受损，并最终导致死亡。由质膜定位的腺苷酸环化酶产生的 (cAMP) 具有屏障保护作用，相反，由细菌毒素、ExoY 或可溶性腺苷酸环化酶在细胞质中产生的 cAMP 会破坏肺动脉内皮细胞 (PAEC) 的屏障。这种渗漏的机制尚未确定，但近膜 cAMP 水平的增加会阻止这种渗漏。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 水平的增加被认为具有屏障保护作用。 M) 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 的稳定敲低具有屏障保护作用。这些和本文描述的其他观察结果使我们得出以下工作假设：高亲和力 PDE7 降低 cAMP 水平并增加肺动脉内皮细胞的屏障通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、离体肺 (离体肺) 和体内内皮屏障功能的影响。 AIMS 概述了整合细胞信号传导和系统生理学方法的计划，以确定 PDE7 在调节内皮屏障功能中的生理作用。具体目标 1. 确定 PDE7 活性是否降低。培养的大鼠肺动脉内皮细胞中的近膜 cAMP 水平具体目标 2. 确定 PDE7 活性的抑制是否在体外、离体和体内对肺动脉内皮具有屏障保护作用。公众健康相关性：肺动脉内皮通透性增加会导致气道顺应性降低、呼吸做功增加、呼吸窘迫患者的常见并发症。目前尚无针对预防肺动脉内皮通透性增加的策略。特定蛋白质（7 型磷酸二酯酶）的药理和分子抑制剂是否可以防止这些动脉内皮通透性增加，从而使呼吸变得更容易。