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.
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