This proposal seeks to determine how 1-0-alkyl-glycerolipids (platelet-activating factor (PAF), others) mediate cell function to produce bronchospasm and anaphylaxis. It postulates that PAF uniformly acts through receptors. To test this, radiolabeled, synthetic analogues will be used. These analogues stimulate cells by a mechanism exactly like PAF but are resistent to the widespread enzymes degrading the natural compound. The radiolabels will be used to demonstrate, enumerate, and further characterize PAF receptors in human lung and neutrophils; identify morphologically lung cells with these receptors; and track receptor movements in neutrophils. The studies employ classical receptor-binding techniques plus autoradiography combined with light-electron microscopy. In associated studies, further mechanisms of PAF-induced human lung contraction will be explored. Release of endogenous mediators (e.g., leukotrienes, thromboxanes, histamine, etc.) by these PAF-challenged tissues will be measured and the influences of selective mediator inhibitors on the contractile response will be assessed in order to link specific mediators to function. Finally, other 1-0-alkyl-glycerolipids will be evaluated. Many studies give us the view that stimulated neutrophils, platelets, and alveolar macrophages metabolize their stores of 1-0-acyl-phospholipids (e.g., phosphatidylinositol, etc.) into products (e.g., phosphatidic acids, etc.) that promote cellular responses and anaphylaxis. These studies do not explain three recent findings: these same cells contain appreciable amounts of 1-0-alkyl-glycerolipids; certain 1-0-alkyl-glycerolipids (e.g., 1-0-alkyl-phosphatidates) are greater than 30 times more bioactive than their 1-0-acyl analogues; and the glycerolipids of these cells can be functionally segregated into two separately mobilizable pools. This proposal explains these findings by suggesting that 1-0-alkyl-glycerolipid turnover is also involved in mediating cell function. Accordingly, it will use newly-developed lipid-separating and radiolabeling techniques to trace the turnover and de novo synthesis of both types of glycerolipids in the above cell types; and, it will compare the bioactions of the product glycerolipids in assays of platelet/neutrophil aggregation and degranulation, lung contraction, PAF receptor binding, and anaphylaxis in animals. To do this, it uses organic synthesis, bioassays, various chromatographic techniques (thin layer, high-performance, and gas-liquid) and mass spectroscopy.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL027799-07
Application #
3339327
Study Section
Pathology A Study Section (PTHA)
Project Start
1981-08-01
Project End
1989-07-31
Budget Start
1987-08-01
Budget End
1988-07-31
Support Year
7
Fiscal Year
1987
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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O'Flaherty, J T; Kuroki, M; Nixon, A B et al. (1996) 5-Oxo-eicosatetraenoate is a broadly active, eosinophil-selective stimulus for human granulocytes. J Immunol 157:336-42
Sozzani, S; Zhou, D; Locati, M et al. (1996) Stimulating properties of 5-oxo-eicosanoids for human monocytes: synergism with monocyte chemotactic protein-1 and -3. J Immunol 157:4664-71
O'Flaherty, J T; Tessner, T; Greene, D et al. (1994) Comparison of 1-O-alkyl-, 1-O-alk-1'-enyl-, and 1-O-acyl-2-acetyl-sn-glycero-3-phosphoethanolamines and -3-phosphocholines as agonists of the platelet-activating factor family. Biochim Biophys Acta 1210:209-16
O'Flaherty, J T; Rossi, A G (1993) 5-hydroxyicosatetraenoate stimulates neutrophils by a stereospecific, G protein-linked mechanism. J Biol Chem 268:14708-14
O'Flaherty, J T; Cordes, J; Redman, J et al. (1993) 5-Oxo-eicosatetraenoate, a potent human neutrophil stimulus. Biochem Biophys Res Commun 192:129-34
O'Flaherty, J T; Jacobson, D P; Redman, J F (1992) Regulation of platelet-activating-factor receptors and the desensitization response in polymorphonuclear neutrophils. Biochem J 288 ( Pt 1):241-8
Sozzani, S; Agwu, D E; McCall, C E et al. (1992) Propranolol, a phosphatidate phosphohydrolase inhibitor, also inhibits protein kinase C. J Biol Chem 267:20481-8
O'Flaherty, J T; Rossi, A G; Redman, J F et al. (1991) Tumor necrosis factor-alpha regulates expression of receptors for formyl-methionyl-leucyl-phenylalanine, leukotriene B4, and platelet-activating factor. Dissociation from priming in human polymorphonuclear neutrophils. J Immunol 147:3842-7
Rossi, A G; O'Flaherty, J T (1991) Bioactions of 5-hydroxyicosatetraenoate and its interaction with platelet-activating factor. Lipids 26:1184-8

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