The goal of this program is to evaluate control of lung surfactant phosphatidylcholine synthesis with special emphasis on the role of choline as a rate-limiting substrate. Using nutritional choline deficiency as a probe, we have shown that in contrast to a large decrease in liver phosphatidylcholine, there is only a slight decrease in the pool of lung phosphatidylcholine. The apparent compensatory mechanisms that maintain lung phosphatidylcholine concentrations are increased activity of the CTP: cholinephosphate cytidyltransferase, an increased rate of surfactant turnover, and increased epithelial transport of choline. The focus of this application will be on the mechanisms for transport of choline by granular pneumocytes. Our preliminary data have indicated that granular pneumocytes accumulate choline against a concentration gradient by an energy dependent, sodium independent mechanism. This uptake mechanism has characteristics of the low affinity active transport system described but poorly characterized in other tissues. Choline uptake will be studied in granular pneumocytes isolated from rat lungs and maintained in primary culture on collagen coated filters. Isolated perfused rat lungs will also be studied for correlation with an intact system.
The first aim will be to fully describe the choline transport system in granular pneumocytes with measurement of time course for accumulation, kinetic constants based on concentration, sodium and energy dependence, hemicholinium-3 and fluorocholine as inhibitors of transport, choline binding to cell constituents, and efflux of choline from prelabelled cells.
The second aim will established the structural specificity for the choline transport system using a variety of probes to determine the requirement for the quaternary ammonium and the alkyl OH groups, the spatial requirements for the head group, and the alkyl chain length.
The third aim will utilize membrane vesicles prepared from isolated cells in order to define mechanisms for transport and to separate the transport step from subsequent metabolic transformations. The fourth am will investigate alterations of transport in granular pneumocytes from choline deficient rats and in (de) differentiated granular pneumocytes. The proposed studies will provide new information on mechanisms through which these cells control the availability of choline for synthesis of the major constituent of lung surfactant.
