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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL031338-07
Application #
3342437
Study Section
Special Emphasis Panel (SSS (B))
Project Start
1984-02-01
Project End
1993-07-31
Budget Start
1990-08-01
Budget End
1991-07-31
Support Year
7
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Wake Forest University Health Sciences
Department
Type
Schools of Medicine
DUNS #
041418799
City
Winston-Salem
State
NC
Country
United States
Zip Code
27106
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Thuren, T; Weisgraber, K H; Sisson, P et al. (1992) Role of apolipoprotein E in hepatic lipase catalyzed hydrolysis of phospholipid in high-density lipoproteins. Biochemistry 31:2332-8
Waite, M; Osthoff, G; Sisson, P et al. (1992) Action of lysosomal phospholipase A1 on bis(monoacylglycerol)phosphate. Biochim Biophys Acta 1128:281-4
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Thornburg, T; Miller, C; Thuren, T et al. (1991) Glycerol reorientation during the conversion of phosphatidylglycerol to bis(monoacylglycerol)phosphate in macrophage-like RAW 264.7 cells. J Biol Chem 266:6834-40
Thuren, T; Sisson, P; Waite, M (1990) Hydrolysis of lipid mixtures by rat hepatic lipase. Biochim Biophys Acta 1046:178-84
Waite, M; King, L; Thornburg, T et al. (1990) Metabolism of phosphatidylglycerol and bis(monoacylglycero)-phosphate in macrophage subcellular fractions. J Biol Chem 265:21720-6
Kucera, G L; Miller, C; Sisson, P J et al. (1988) Hydrolysis of thioester analogs by rat liver phospholipase A1. J Biol Chem 263:12964-9

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