MECHANISMS OF ALVEOLAR EPITHELIAL ALBUMIN TRANSPORT

我们的具体目标是研究 肺泡上皮白蛋白转运。白蛋白(最丰富的蛋白质 在血清中)已被证明存在于肺泡衬里液体中，并且 据报道，肺泡液中的白蛋白浓度在 肺部受伤。大分子浓度的动态平衡控制 肺泡液可能涉及特殊的肺泡上皮运输 流程。白蛋白跨膜转运的初步研究数据 肺泡上皮与下列中央区一致 假设：i)不对称和快速的(完整的)白蛋白跨 肺泡上皮细胞主要通过受体介导的跨细胞因子发生 需要特定白蛋白受体(约60 kDa)的途径 在肺细胞质膜上，2)跨上皮净吸收 白蛋白可通过特定的实验刺激进行调节。 为了检验这些中心假设，我们建议研究以下四个方面 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 温度和内吞抑制剂对血管内皮细胞生长的影响 单向白蛋白通量，动力学特性 结合/内化/跨细胞反应，特征(例如，不对称 白蛋白受体的细胞表面表达)，以及形态 跨细胞途径的研究。 2)阐明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损伤机制的研究 白蛋白运输。 4)确定完整的哺乳动物肺具有白蛋白的机制 与肺泡上皮细胞单层的转运相似 原生文化。 我们将利用在组织上培养的大鼠肺泡上皮细胞单层 培养处理的聚碳酸酯过滤器(特定目标1-3)和隔离 大鼠肺灌流(靶标4)。以确定是否存在和 白蛋白受体在肺泡上皮细胞上的差异表达 表面，我们将使用多种方法的组合(例如，光亲和 白蛋白的探针、免疫印迹和脉冲追逐/免疫沉淀 受体)。肺泡发育机制和途径的研究进展 白蛋白的上皮转运将代表着以下重大进展 了解肺泡液的性质，肺泡液的作用 上皮细胞在其调控中的作用及其改变机制 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.