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系统疾病期间或输注后可能会升高（即纠正与肺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+-碳酸氢盐协同转运蛋白 (NBC) 将碳酸氢盐转运至细胞内。在细胞质中，碳酸氢盐可以激活细胞质腺苷酸环化酶亚型 10 (AC10)，产生细胞质 cAMP 并破坏肺内皮屏障。该研究计划的目标是了解 NBC 在急性肺损伤 (ALI) 情况下的调节方式以及 AC10 诱导通透性的机制。据观察，虽然跨膜 AC 产生近膜屏障保护性 cAMP，但有毒细菌可溶性 AC ExoY 会产生屏障破坏性 cAMP 并增加通透性。通透性增加会导致急性肺损伤 (ALI) 和急性呼吸窘迫综合征 (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从而加剧 ALI 和 ARDS 具有临床意义。具体来说，体外、原位和体内实验模型将用于确定 Na+-碳酸氢盐协同转运蛋白和 AC10 是否有助于碳酸氢盐流入以及源自导管动脉或肺动脉气体交换段的肺内皮细胞中胞质 cAMP 信号的产生。脉管系统，间隔毛细血管（具体目标 1）。我们将确定 NBC、NBCn2 是否被近膜 cAMP 信号抑制（具体目标 2），以及是否需要通过 NBCn2 转运碳酸氢盐来激活 AC10，从而破坏微管结构并破坏内皮屏障（具体目标 3）。使用的技术包括：免疫组织化学和分级研究，以确定不同血管床之间的 NBC 表达； pH 和钠测量以确定 NBC 活性；除了使用高光谱图像分析进行空间和时间 FRET-cAMP 测量外，还使用 ​​cAMP 周转和放射免疫测定对 cAMP 进行无细胞和完整细胞测量；免疫沉淀测定和定点诱变以确定 NBCn2 上的磷酸化位点；使用磷酸化特异性抗体进行 Western 分析，检测 tau 磷酸化的变化；通过检测游离微管蛋白与聚合微管蛋白来识别微管结构的变化；视频显微镜、渗透性和阻力测量，以检测体外内皮屏障完整性的变化；并建立离体灌注肺模型，测量stu中的滤过系数（Kf）、肺湿/干比和肺泡液体积分数。此外，我们将在腹膜内 LPS 诱导 ALI 后进行额外的离体肺研究。这些实验技术和各种实验组将用于检验我们的假设。