Bicarbonate regulation of the pulmonary endothelial barrier

碳酸氢盐对肺内皮屏障的调节

• 批准号: 9187494
• 负责人: Sarah Louise Sayner
• 金额: $ 30.3万
• 依托单位: UNIVERSITY OF SOUTH ALABAMA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-12-01 至 2019-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9187494
• 关键词: Acidosis; Acute Lung Injury; Adenylate Cyclase; Adult Respiratory Distress Syndrome; Alveolar; Architecture; Arteries; Bicarbonates; Biological Assay; Blood; Blood capillaries; Cells; Chimera organism; Clinical; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cytosol; Data; Dependency; Detection; Development; Endothelial Cells; Endothelium; Environment; Environmental air flow; Experimental Models; Filtration; Fluorescence Resonance Energy Transfer; Forskolin; Fractionation; Gases; Generations; Goals; Image Analysis; Immunohistochemistry; Immunoprecipitation; In Situ; In Vitro; Infusion procedures; Liquid substance; Lung; Lung diseases; Measurement; Measures; Mechanical ventilation; Mediating; Membrane; Microtubules; Modeling; Morbidity - disease rate; Permeability; Phospho-Specific Antibodies; Phosphorylation; Phosphorylation Site; Plasma; Polymers; Prevention; Protein Isoforms; Proteins; Pseudomonas aeruginosa; Pulmonary Edema; Radioimmunoassay; Regulation; Research; Resistance; Respiration Disorders; Respiratory Acidosis; Signal Transduction; Signaling Molecule; Site-Directed Mutagenesis; Sodium; Techniques; Testing; Therapeutic Intervention; Tidal Volume; Tubulin; Video Microscopy; capillary; clinically significant; experimental study; extracellular; falls; interest; intraperitoneal; monolayer; mortality; novel; phosphoric diester hydrolase; prevent; protective effect; public health relevance; pulmonary artery endothelial cell; response; tau Proteins; tau phosphorylation; tau-1; vascular bed

DESCRIPTION (provided by applicant): Blood bicarbonate, which is in direct contact with the endothelium, can become elevated during respiratory diseases or following infusion (i.e to correct acidosis associated with lung protective mechanical ventilation strategies), yet its effect on the endotheliul are unknown. Na+-bicarbonate cotransporters (NBC) transport bicarbonate into cells. In the cytosol, bicarbonate can activate cytosolic adenylyl cyclase isoform 10, AC10, which generates cytosolic cAMP and disrupts the pulmonary endothelial barrier. The goal of this proposed research plan is to understand how the NBC is regulated and the mechanism of AC10 induced permeability in the setting of acute lung injury (ALI). It has been observed that while transmembrane AC generates juxtamembrane, barrier protective cAMP, the toxic bacterial soluble AC, ExoY, generates, barrier disruptive cAMP and increases permeability. Increased permeability contributes to the mortality and morbidity of Acute Lung Injury (ALI) and Acute Respiratory Distress Syndrome (ARDS). Thus, understanding how NBCs regulate AC10 activity and how endogenously generated cytosolic cAMP regulates pulmonary endothelial barrier integrity to exacerbate ALI and ARDS is of clinical interest. Specifically, in vitro, in situ and i vivo experimental models will be used to determine whether Na+-bicarbonate cotransporters and AC10 contribute to bicarbonate influx and generation of cycotsolic cAMP signals in pulmonary endothelial cells derived from either conduit arteries or the gas exchange segment of the vasculature, the septal capillaries (Specific Aim 1). We will determine whether the NBC, NBCn2, is inhibited by juxtamembrane cAMP signals (Specific Aim 2) and whether bicarbonate transport through NBCn2 to activate AC10 is required disrupt microtubule architecture and disrupt the endothelial barrier (Specific Aim 3). Techniques to be used include: immunohistochemistry and fractionation studies to determine NBC expression between different vascular beds; pH and sodium measurements to determine NBC activity; both cell-free and in-tact cell measurements of cAMP using cAMP turnover and radioimmunoassay in addition to spatial and temporal FRET-cAMP measurements using hyperspectral image analysis; immunoprecipitation assays and site directed mutagenesis to identify phosphorylation sites on NBCn2; Western analysis with phospho-specific antibody to detect changes in tau phosphorylation; detection by detection of free versus polymerized tubulin to identify changes in microtubule architecture; video microscopy, permeability and resistance measurements to detect changes in endothelial barrier integrity in vitro; and the isolated perfused lung model to measure the filtration coefficient (Kf), lung wet/dry ratio and alveolar fluid volume fraction in stu. Further, we will perform additional isolated lung studies following intraperitoneal LPS administration to induce ALI. These experimental technique and various experiment groups will be used to test our hypothesis.

描述（由申请人提供）：与内皮直接接触的血液碳酸氢盐在呼吸道疾病期间或输注后可以升高（即纠正与肺部保护性机械通气策略相关的酸中毒），但其对内皮的影响尚不清楚。 Na+ - 双碳酸盐共转运蛋白（NBC）将碳酸氢盐转运到细胞中。在细胞质中，碳酸氢盐可以激活胞质腺苷酸环化酶同工型10，AC10，从而产生胞质cAMP并破坏肺部内皮屏障。该拟议的研究计划的目的是了解如何调节NBC，并且在急性肺损伤（ALI）的情况下，AC10的机制引起了渗透性。已经观察到，尽管跨膜AC产生近去膜，屏障保护营，但有毒细菌可溶性AC，Exoy，产生，产生屏障破坏性营地并增加渗透性。渗透率提高有助于急性肺损伤（ALI）和急性呼吸遇险综合征（ARDS）的死亡率和发病率。因此，了解NBC如何调节AC10活性以及内源性产生的胞质cAMP如何调节肺内皮屏障完整性对加剧ALI和ARDS具有临床意义。具体而言，在体外，原位和I体内实验模型将用于确定Na+ - 贝克碳酸盐共转运蛋白和AC10是否有助于碳酸氢盐涌入和肺部内皮细胞中的环状cAMP信号产生。我们将确定NBC NBCN2是否受到近去膜营地信号的抑制（特定AIM 2）以及是否需要通过NBCN2运输碳酸氢盐，以激活AC10是否会破坏微管结构并破坏内皮屏障（特定目标3）。要使用的技术包括：免疫组织化学和分馏研究，以确定不同血管床之间的NBC表达； pH和钠测量值以确定NBC活性；除了使用高光谱图像分析外，还使用cAMP营业额和放射免疫测量的CAMP和放射免疫测量的CAMP和放射免疫测定法进行了cAMP的细胞测量；免疫沉淀测定和部位定向诱变，以鉴定NBCN2上的磷酸化位点；用磷酸特异性抗体的西方分析检测tau磷酸化的变化；通过检测自由与聚合小管蛋白的检测以识别微管结构的变化；视频显微镜，渗透性和抗性测量值，以检测体外内皮屏障完整性的变化；和孤立的灌注肺模型，以测量Stu中的过滤系数（KF），肺湿/干比和肺泡液体积分数。此外，腹膜内LPS给药后，我们将进行其他分离的肺部研究以诱导ALI。这些实验技术和各种实验组将用于检验我们的假设。