PERMEABILITY OF THE LUNG TO WATER SOLUBLE SOLUTES

描述（申请人摘要）：本提案的长期目标是 定义紧密连接（TJ）蛋白和因子之间的相互作用 调节它们在肺部的活动。TJ生物学的四个核心问题是 处理。1.封闭蛋白、封闭蛋白-1和ZO-1在调节 TJ的屏障功能这是评估使用独特的诱导，双重 转染的上皮细胞克隆，以改变这些 蛋白质;然后检查对TJ渗透性的影响。2.如何具体 封闭蛋白的结构域决定TJ屏障功能？为了检验这一点， 从细胞质和细胞外缺失特定的氨基酸序列 紧密连接蛋白-1或-4的结构域。上皮细胞TJ通透性特性 评价表达这些修饰蛋白的克隆。3.如何深刻 胆固醇（CH）流出对与洗涤剂不溶物相关TJ功能的影响 CH/糖脂筏，CH与TJ蛋白密切相关吗？一种新型 使用放射性标记的光活化CH类似物来确定CH是否是 与闭合蛋白和/或密蛋白结合。比例差异 完整的TJ蛋白和TJ筏的脂质组成，在高和低， 上皮细胞的耐药菌株将为它们的作用提供新的线索 在TJ渗透性方面。通过激活脂质2 M信使的产生增加 在CH流出过程中的磷脂酶表明，它们是重要的调节剂， TJ屏障功能。这些磷脂酶将被鉴定和表征。 4.肺TJ的渗透性在多大程度上取决于 它们的蛋白质组成occludin和six的免疫金标记 肺相关的claudins用于将它们的位置映射到特定的上皮细胞。 肺的内皮TJ。使用LPS诱导的急性炎症模型 探索从气道中分离的TJ蛋白和相关的脂筏 上皮细胞在炎性细胞迁移过程中被修饰。拟议 研究将为TJ生物学提供新的见解，并允许开发新的 调节治疗剂通过肺上皮细胞的策略 和/或防止过敏原穿透TJ屏障。

DESCRIPTION (Applicant's Abstract): The long term goal of this proposal is to define the interactions between tight junction (TJ) proteins and the factors regulating their activity in the lung. Four central questions in TJ biology are addressed. 1. What is the role of occludin, claudin-1 and ZO-1 in regulating the barrier function of TJs? This is assessed using unique inducible, doubly transfected epithelial cell clones to vary the relative expression of these proteins; the effect on TJ permeability is then examined. 2. How do specific domains of the claudins determine TJ barrier function? To examine this, specific amino acid sequences are deleted from cytoplasmic and extra-cellular domains of claudin-1 or -4. The TJ permeability properties of epithelial cell clones expressing these modified proteins are evaluated. 3. How is the profound effect of cholesterol (CH) efflux on TJ function related to detergent insoluble CH/glycolipid rafts and is CH closely associated with TJ proteins? A novel radiolabeled, photoactivatable CH analog is used to determine whether CH is bound to occludin and/or the claudins. Differences in the proportions of integral TJ proteins and the lipid composition of the TJ rafts in high and low resistance strains of epithelial cells will provide new clues as to their role in TJ permeability. Increased lipid 2M messenger generation by activation of phospholipases during CH efflux suggest that they are important regulators of TJ barrier function. These phospholipases will be identified and characterized. 4. To what extent are the permeability properties of pulmonary TJs dictated by their protein composition? Immunogold labeling of occludin and six lung-associated claudins is used to map their location to specific epitheliall endothelial TJs of the lung. An LPS-induced model of acute inflammation is used to explore how TJ proteins and the associated lipid rafts, isolated from airway epithelium, are modified during migration of inflammatory cells. The proposed studies will provide new insights into TJ biology and allow development of new strategies to regulate passage of therapeutic agents across lung epithelia and/or to prevent the penetration of allergens through the TJ barrier.