The purpose of this project is to determine the capacity of rabbit alveolar macrophages to form eicosanoids and to determine the mechanism by which arachidonate (20:4) becomes available for conversion to eicosanoids. Recent evidence from our laboratory indicates that lyso(bis)phosphatidic acid (L(b)PA) and phosphatidylinositol (PI) have a large reservoir of 20:4 that is metabolically active and might be a major source of 20:4 for eicosanoid synthesis. A fundamental difference was found in this metabolism between normal and BCG-elicited macrophages. We have developed a model cell line of transformed macrophages, RAW 264.7, that is useful for probing certain metabolic pathways. To test a possible linkage between L(b)PA and PI metabolism and eicosanoid synthesis, we plan to do the following in normal and BCG-elicited macrophages: 1) Determine the eicosanoids synthesized under a variety of cell challenge conditions and relate those results with the determination of the radiolabeled phospholipids deacylated. 2) Determine the subcellular distribution of lipoxygenase, cyclooxygenase, phospholipases, and phospholipid donors for the 20:4. 3) Determine the pathway for L(b)PA from exogenous phosphatidylglycerol (PG), initially using RAW cells. These studies will include investigation of the stereoconversion of the glycerol backbone from 3-sn-glycerol to l-sn-glycerol and the incorporation of fatty acid into the precursor of L(b)PA via a transacylation involving Pl. Once the techniques for the study of L(h)PA synthesis are established, the alveolar macrophages will be studied in-depth. Further. we will determine if PG in lung surfactant is the source of PG for L(b)PA synthesis and therefore accounts for the high content of L(b)PA in normal alveolar macrophages and relates to the lower content of L(b)PA in BCG- elicited cells. Together these studies should provide new insight into the regulation of eicosanoid synthesis in alveolar macrophages, an important immunologic function of these cells in the lung. Further, the comparison of normal and BCG-elicited macrophages will help define a major difference between these cells as a consequence of immunologic challenge.
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