PERMEABILITY OF THE LUNG TO WATER SOLUBLE SOLUTES

拟议研究的长期目标是分析 内皮细胞和上皮细胞调节血管生成的机制 肺气-血屏障的渗透性。 完成 采用这两个模型系统。 1. 血浆无蛋白 氟碳交换输血大鼠; 2. 培养的上皮细胞。 生化和电生理数据与观察结果相关 用超微结构免疫细胞化学、形态计量学和冷冻法 骨折 具体目标包括利用碳氟化合物交换的研究 输注大鼠，以确定免疫球蛋白G（IgG），α 1- 酸性糖蛋白（α 1-AGP）、纤维蛋白原（FAN）、血浆纤维连接蛋白（FN） 和二丁酰cAMP，单独和组合，以增强 白蛋白在防止大分子转运增加和水肿， 发生在不含蛋白质的制剂中。 管理的本地化 将测定肺内皮上的测试蛋白 免疫细胞化学，并与形态测量相关的 铁蛋白运动，大分子运输的量度。 分布 ZO-1是一种TJ相关蛋白，免疫细胞化学定位于 正常和增强条件下的微血管内皮细胞 液体过滤 质粒pZipSVtsA 58，一种赋予 转染上皮细胞的温度敏感性生长潜力. 33 ℃，但允许在39 ℃表达分化功能，用于 使肺泡II型细胞永生化。 形成跨上皮细胞的克隆 选择电阻>100 Ω cm 2用于调节的研究， TJ的组成。 使用生物素化剂和抗肿瘤药物的新策略 异双官能交联剂用于鉴定和 表征将产生单克隆抗体的TJ蛋白。 膜脂质在TJ的调节和功能中的作用将被 在培养的上皮细胞中检测，当胆固醇 通过给予洛伐他汀而缺乏，达到稳态 跨上皮阻力比对照组高2至3倍。 拟议研究的结果将：i。 展示所发挥的关键作用 通过特定的血浆蛋白，除了白蛋白，在调节 内皮通透性 二. 鉴定TJ蛋白并提供试剂 利用分子生物学的工具来研究它们的结构， 三. 帮助解决有关膜脂质作用的争议 在TJ。 更好地理解这一重要结构的性质 将为肺水肿的治疗提供合理的依据。

The long term objective of the proposed research is to analyze the mechanisms by which endothelial and epithelial cells regulate the permeability properties of the pulmonary air-blood barrier. To accomplish this two model systems are employed. 1. The plasma protein-free fluorocarbon exchange transfused rat and 2. epithelial cells in culture. Biochemical and electrophysiological data are correlated with observations made by ultrastructural immunocytochemistry, morphometry and freeze fracture. The specific aims include studies utilizing the fluorocarbon exchange transfused rat, to determine the ability of immunoglobulin G (IgG), alpha1- acid glycoprotein (alpha1-AGP), fibrinogen (FAN), plasma fibronectin (FN) and dibutyryl cAMP, alone and in combination, to augment the action of albumin in preventing the increased macromolecular transport and edema that occurs in a protein-free preparation. The localization of administered test proteins on the pulmonary endothelium will be determined immunocytochemically, and correlated with morphometric measurements of ferritin movement, a measure of macromolecular transport. The distribution of ZO-1, a TJ associated protein, is mapped immunocytochemically in microvascular endothelial cells under conditions of normal and augmented fluid filtration. The plasmid pZipSVtsA58, a construct that confers temperature sensitive growth potential to transfected epithelial cells at 33oC, but allows expression of differentiated functions at 39oC, is used to immortalize alveolar type II cells. Clones that develop a transepithelial resistance >100 omega cm2 are selected for study of the regulation and composition of TJs. Novel strategies using biotinylating agents and a heterobifunctional crosslinking agent are employed to identify and characterize TJ protein(s) to which monoclonal antibodies will be raised. The role of membrane lipids in the regulation and function of TJs will be examined in cultured epithelial cells which, when made cholesterol deficient by administration of Lovastatin, develop steady state transepithelial resistances two to three-fold greater than controls. Results of the proposed research will: i. Demonstrate the key role played by specific plasma proteins, in addition to albumin, in regulating endothelial permeability. ii. Identify TJ proteins and provide reagents with which to study their structure using tools of molecular biology and iii. aid in resolving controversy concerning the role of membrane lipids in TJs. A better understanding of the nature of this important structure will provide a rational basis for the management of pulmonary edema.