MACROMOLECULAR TRANSPORT ACROSS LUNG ALVEOLAR EPITHELIUM

拟议研究的具体目标是调查 大分子跨膜转运的特性和调控 肺泡上皮屏障 我们最近对单向14 C- 肺泡上皮白蛋白通量显示出明显的不对称性， 放射性示踪剂流，其中流出肺泡腔的流量约为4 比进入肺泡腔的流量大10倍。 的幅度 标记的白蛋白通量也大于小得多的 多糖、菊粉。 这些观察使我们假设， 存在专门的大分子（例如，白蛋白）转运 肺泡上皮细胞中的机制。 我们将调查可能的 大分子跨膜转运的机制和途径 肺泡上皮屏障利用两个分离的模型， 组织，即大鼠肺II型肺细胞单层在原发性 培养和完整的两栖动物肺。 大鼠II型肺细胞单层 生长在多孔表面和完整的牛蛙肺制备为平 张将安装在尤辛室，并将研究其 大分子转运特性 单向放射性示踪剂 蛋白质和葡聚糖的通量测量，以及表观 渗透性能，将执行。 特定传输 研究的机制包括肺泡上皮细胞的蛋白水解， 示踪剂和大分子的囊泡运输。 测试 大分子通过囊泡转运的假说 途径，我们将研究吸附和内化的动力学 大分子的肺泡上皮细胞，以及 囊泡转运抑制剂的作用。 形态学观察 的大分子运输途径将进行利用 超微结构标记（例如，胶体金吸附大分子） 用电子显微镜技术进行内吞作用。 的影响 电荷密度将通过使用中性、阳离子和 阴离子大分子。 偶联的可能刺激作用 还将测定具有聚阳离子的蛋白质。 非特异性效应 大分子运输对小溶质和水通量将是 研究以进一步确定大量内吞途径在 肺泡上皮 预计这些研究将提供 大分子的特性和调控信息 运输通过肺泡上皮屏障，并将有助于 阐明哺乳动物肺泡上皮细胞的转运特性， 肺泡上皮屏障在预防，形成， 和体内肺泡肺水肿的消退。

The specific objectives of the proposed studies are to investigate the characteristics and regulation of macromolecular transport across the alveolar epithelial barrier. Our recent studies on unidirectional 14 C- albumin fluxes across the alveolar epithelium show marked asymmetry of radiotracer flow, in which flux out of the alveolar space is about four times greater than flux into the alveolar space. The magnitudes of labeled albumin fluxes are also greater than that for the much smaller polysaccharide, inulin. These observations led us to postulate that there is a specialized macromolecular (e.g., albumin) transport mechanism in alveolar epithelium. We will investigate possible mechanisms and pathways for the translocation of macromolecules across the alveolar epithelial barrier utilizing two isolated models of the tissue, namely, rat lung type II pneumocyte monolayers in primary culture and intact amphibian lungs. Rat type II pneumocyte monolayers grown on porous surfaces and intact bullfrog lungs prepared as flat sheets will be mounted in Ussing chambers and will be studied for their macromolecular transport characteristics. Unidirectional radiotracer flux measurements of proteins and dextrans, and analysis of apparent permeability properties, will be performed. Specific transport mechanisms to be studied include alveolar epithelial proteolysis of tracers and vesicular transport of macromolecules. To test the hypothesis that macromolecules are translocated via vesicular transport pathways, we will study the kinetics of adsorption and internalization of macromolecules by the alveolar epithelial cells, as well as the effects of vesicular transport inhibitors. Morphological investigation of the macromolecular transport pathways will be performed utilizing ultrastructural markers (e.g., colloidal gold-absorbed macromolecules) for endocytosis with electron microscopic techniques. The influence of charge density will be investigated by utilizing neutral, cationic, and anionic macromolecules. Possible stimulatory effects of conjugation of proteins with polycations will also be determined. Nonspecific effects of macromolecular transport on small solute and water fluxes will be studied to further determine the role of bulk endocytotic pathways in the alveolar epithelium. It is expected that these studies will provide information on the characteristics and regulation of macromolecular transport across the alveolar epithelial barrier, and will help elucidate mammalian alveolar epithelial transport properties and the role of the alveolar epithelial barrier in the prevention, formation, and resolution of alveolar pulmonary edema in vivo.