The long term objectives of this proposal are to understand the mechanisms and regulation of albumin transport across the tracheal epithelium. Albumin is a major protein constituent of the airways lining fluid and has many important properties and functions. These include anti-oxidant and non-specific binding properties, participation in lipid and drug transport, and having profound reversible effects on the physical properties of airway mucus. Despite these important effects, it is generally held that albumin reaches, and leaves the airspaces by purely diffusive mechanisms. Recent studies however, suggest that albumin content in the airways may be highly regulated, and that this regulation involves active transport mechanisms. the alterations in airways albumin content seen in a wide range of airways diseases may reflect abnormalities in these transport processes. these studies will first evaluate albumin and dextran movement in both the serosa to mucosa direction (secretion), and in the opposite direction (absorption) in the intact in-vitro rabbit trachea. The effect of a range of agonists on bidirectional transport will be determined. these results will then be compared to bidirectional albumin and dextran movement across a confluent monolayer of rabbit tracheal epithelial cells in primary culture. The same agonists will be used, and the validity of the tissue culture system for the study of macro-molecular transport determined. Using this system, several mechanisms of albumin transport will be explored. The hypothesis that albumin transport involves specific albumin receptors with endocytotic vesicles and microtubular based transcytosis which may include fusion with lysosomes will be tested. The role of vesicular transport will be assessed by determining the temperature dependence of transport. The participation of the microtubular network determined by pre-treatment with the microtubule-disrupting agents nocodazole and colchicine. The specificity of the transport mechanisms and presence of albumin receptors will be assessed by studying the saturation kinetics of transport and the degree of specific albumin binding on the epithelial surface. Lastly, possible fusion with lysosomes will be sought by looking for degradation of albumin during transport with column chromatography.
NIH ExPorter
NIH ExPorter