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.
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