Alveolar Type II cells secrete lung surfactant in the form of lamellar body contents into the aqueous alveolar subphase, where they re transformed into tubular myelin. Tubular myelin, and perhaps lamellar body contents too, provide material for adsorption into the alveolar air-water film itself. Good, but indirect evidence from surface balance studies indicate that this adsorption process is selective, in that dipalmitoylphosphatidylcholine (DPPC) is somehow sorted into the surface film, while most non-DPPC materials are sorted away from it surfactant components. Surfactant components reaching the surface film eventually journey back into the aqueous subphase by two mechanisms: (1) a rapid squeezeout of any resident DPPC-poor materials during initial film compression, and (2) a very slow collapse of DPPC-rich material over a period of hours. Alveolar stability is achieved by virtue of the prolonged presence of the DPPC-rich film which maintains a very low surface tension when compressed, and optimal function thus requires molecular sorting in the formation of such a film. Our objective is to provide direct biochemical evidence for this sorting process, and to find the roles played in it by individual surfactant proteins and lipids. We will test the general hypothesis that sorting actually occurs, by analyzing the lipid and protein contents of the adsorped surfactant film itself. To accomplish this we have produced a controlled foam of natural lung surfactant as a tractable model of the newly formed air-water surfactant film, which we can characterize chemically in a variety of ways. We have developed both osmium and gas-chromatographic methods for assaying saturated phospholipids (e.g., DPPC), and these will be supported by the use of phospholipase A2 (PLA2) and thin layer chromatography (TLC) for """"""""dissecting"""""""" the foams, to test for a hypothesized surface-associated compartment or compartments. We will also search with PLA2 and selected probes to see if """"""""presorting"""""""" might have already taken place within tubular myelin or secreted lamellar body structures. Using computer technologies designed for our captive bubble surfactometer (CBS) in the current grant period, we will do parallel surface activity studies of materials generating the foams. In addition,. We will subject the single bubbles of lung surfactants, lipids, and proteins studied to temperature ramps in the CBS. This latter procedures should test whether films of adsorped lung surfactants and adsorped DPPC give similar thermal transitions, thus further confirming their identity. The results of these studies should deepened our understanding of surfactant function, delineate the importance of surface sorting, and perhaps lead to improved design of therapeutic materials.
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