PERMEABILITY OF THE LUNG TO WATER SOLUBLE SOLUTES

描述(申请人摘要)：这项建议的长期目标是 确定紧密连接(TJ)蛋白与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 地址。1.occludin、claudin-1和ZO-1在调节中的作用是什么 TJ的屏障功能是什么？这是使用独特的可感应的、双重的 转染上皮细胞克隆以改变这些基因的相对表达 蛋白质；然后检测对TJ通透性的影响。2.如何具体 Claudins的结构域决定TJ屏障功能？为了检验这一点， 特定的氨基酸序列从细胞质和胞外被删除 Claudin-1或-4的结构域。上皮细胞的TJ通透性 对表达这些修饰蛋白的克隆进行了评估。3.深奥 胆固醇(CH)外流对洗涤剂不溶物相关TJ功能的影响 CH/糖脂筏，CH与TJ蛋白密切相关吗？一本小说 使用放射性标记的、可光激活的CH类似物来确定CH是否 与闭塞蛋白和/或克拉丁蛋白结合。在比例上的差异 整体TJ蛋白与高、低TJ筏的脂质组成 上皮细胞的耐药株将为其作用提供新的线索 在TJ渗透率中。激活脂质2M信使产生增加 CH外流过程中的磷脂酶提示它们是 TJ屏障功能。这些磷脂酶将被鉴定和表征。 4.肺TJ通透性在多大程度上受 它们的蛋白质组成呢？免疫金标记包涵蛋白和6-氨基丁酸 肺相关的Claudins被用来将它们的位置映射到特定的上皮细胞 肺内皮细胞TJ。采用脂多糖诱导的急性炎症模型。 探索从呼吸道分离的TJ蛋白和相关脂筏是如何 上皮细胞，在炎性细胞的迁移过程中被修改。建议数 研究将为TJ生物学提供新的见解，并允许开发新的 调节治疗药物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.