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.
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