MECHANISMS OF ALVEOLAR EPITHELIAL ALBUMIN TRANSPORT

我们的具体目标是调查的机制和调节 肺泡上皮白蛋白转运 白蛋白（最丰富的蛋白质 血清中）已显示存在于肺泡衬里液中， 据报道，肺泡液中的白蛋白浓度升高， 受伤的肺 体内大分子浓度的稳态控制 肺泡液可能涉及专门的肺泡上皮运输 流程. 来自我们对白蛋白转运的初步研究的数据， 肺泡上皮细胞与以下中心一致 假设：I）（完整）白蛋白通过 肺泡上皮细胞主要通过受体介导的胞吞作用发生 需要特异性白蛋白受体（约60 kDa）的途径 在肺细胞质膜上，和2） 白蛋白可通过特定的实验刺激来调节。 为了验证这些核心假设，我们建议研究以下四个问题 具体目标： 1)阐明快速净吸收的具体机制 白蛋白穿过肺泡上皮。 这些研究将包括 研究温度和内吞抑制剂对 单向白蛋白通量，动力学特性， 结合/内化/转胞吞，表征（例如，不对称 白蛋白受体的细胞表面表达），以及形态学 转胞吞途径的研究。 2)描述肺泡上皮白蛋白转运的调控因子。 这些研究将包括诸如肺泡液pH和 表皮生长因子 3)氧化损伤对肺泡上皮细胞损伤机制的研究 白蛋白转运 4)确定完整的哺乳动物肺具有白蛋白 转运类似于肺泡上皮细胞单层中发现的转运， 原代培养 我们将利用大鼠肺泡上皮细胞单层培养的组织 培养物处理的聚碳酸酯过滤器（特定目的1-3）和分离的 灌注的大鼠肺（特定目标4）。 以确定是否存在， 肺泡上皮细胞白蛋白受体的差异表达 表面，我们将使用方法的组合（例如，光亲和 白蛋白的探针、免疫印迹和脉冲追踪/免疫沉淀 受体）。 肺泡灌洗液的机制和途径的表征 白蛋白的上皮转运将代表 了解肺泡液的性质，肺泡 上皮在其调节，其改变的机制， 健康和疾病。

Our specific objectives are to investigate the mechanisms and regulation of alveolar epithelial albumin transport. Albumin (the most abundant protein in serum) has been shown to be present in alveolar lining fluid, and albumin concentration in alveolar fluid is reported to be elevated in injured lungs. Homeostatic control of macromolecule concentrations in alveolar fluid likely involves specialized alveolar epithelial transport processes. Data from our preliminary studies on albumin transport across the alveolar epithelium are consistent with the following central hypotheses: I) asymmetric and rapid fluxes of (intact) albumin across the alveolar epithelium take place primarily via receptor-mediated transcytotic pathways which require specific albumin receptors (of approximately 60kDa) on pneumocyte plasma membranes, and 2) net transepithelial absorption of albumin is regulable by specific experimental stimuli. To test these central hypotheses, we propose to study the following four Specific Aims: 1) Elucidate the specific mechanisms underlying rapid, net absorption of albumin across the alveolar epithelium. These studies will include investigation of effects of temperature and endocytosis inhibitors on unidirectional albumin fluxes, kinetic properties of binding/internalization/transcytosis, characterization (e.g., asymmetric cell surface expression) of albumin receptors, and morphological investigations of transcytotic pathways. 2) Delineate regulatory factors in alveolar epithelial albumin transport. These studies will include effects of factors such as alveolar fluid pH and epidermal growth factor. 3) Study effects of oxidant injury on mechanisms of alveolar epithelial albumin transport. 4) Determine that intact mammalian lungs possess mechanisms for albumin transport similar to those found in alveolar epithelial cell monolayers in primary culture. We will utilize rat alveolar epithelial cell monolayers cultured on tissue culture-treated polycarbonate filters (Specific Aims 1-3) and isolated perfused rat lungs (Specific Aim 4). To ascertain the presence and differential expression of albumin receptors on alveolar epithelial cell surfaces, we will use a combination of approaches (e.g., photoaffinity probes, immunoblot, and pulse-chase/immunoprecipitation of albumin receptors). Characterization of the mechanisms and pathways for alveolar epithelial transport of albumin would represent significant progress toward understanding the properties of alveolar fluid, the role of alveolar epithelium in its regulation, and the mechanisms of its alteration in health and disease.