The specific objectives of the proposed studies are to investigate the characteristics and regulation of macromolecular transport across the normal and abnormal pulmonary epithelial barrier. We will study two preparations of isolated alveolar epithelium, namely, hollow amphibian lungs and rat lung type II cell monolayers. These preparations will be utilized to study macromolecular transport properties and determine pathways and mechanisms for macromolecule translocation across the alveolar epithelium. Bullfrog lungs will be studied as flat sheets mounted in Ussing-type flux chambers or as suspended sacs, and monolayers of mammalian lung type II cells will be grown on porous surfaces and subsequently studied for their macromolecular transport characteristics. Unidirectional radiotracer flux measurements and analysis of apparent permeability properties will be performed. Specific pathways of macromolecular transport will be investigated by studying a number of potential mechanisms in detail. We will investigate possible fragmentation of proteins due to digestion by the alveolar epithelium, effects of transepithelial electrical gradients, saturation, competition (inhibition) and stimulation of macromolecular fluxes. In order to test the hypothesis that macramolecules are transported via vesicular pathways, we will study the kinetics of adsorption and internalization of macromolecules, as well as the effects of vesicular transport inhibitors. The influence of charge density will be investigated utilizing cationic, neutral, and anionic macromolecules. Possible stimulatory effects of conjugation of proteins (with polycations) will also be determined. Non-specific effects of macromolecular transport on small solute and water fluxes will be studied to further determine the role of a bulk endocytotic pathway in the alveolar epithelium. We will investigate the effects of metabolic inhibitors, temperature, pH, lung injury, and exogenous and endogenous substances on macromolecular transport properties of the alveolar epithelium. It is expected that our 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 in pathophysiological conditions and their roles in the prevention, formation, and resolution of alveolar pulmonary edema in vivo.
