and Specific Aims.) Epithelial tissues (in skin, gut, and lung) bear the critical responsibility of defining a boundary between host and environment. This task includes protection against injury by acting as a barrier and modulating immune and inflammatory responses to environmental agents. These same cellular functions are often regulated at a molecular level by the products of phospholipid- arachidonic acid metabolism. Thus, the determination of biochemical mechanisms for epithelial cell biosynthesis and degradation of phospholipid- arachidonate metabolites is a critical step towards understanding the biologic function of epithelial tissues in general (and pulmonary airway epithelium in particular). The long-term goal of the present investigations is to develop the hypothesis that pulmonary airway epithelial pathways for phospholipid- fatty acid metabolism are critical modulators of airway function and that critical components of these pathways pose suitable pharmacologic targets for intervention in airway disease. The proposed studies aim at specifically dissecting airway epithelial pathways for PGH synthase-catalyzed generation of prostaglandins and the lipoxygenase-catalyzed peroxidation of membrane fatty acids. The overall strategy is to define specific molecular probes for each of these enzymatic pathways in order to lay the groundwork for determining mechanisms for their regulation in intact tissue in health and disease. The primary focus of the work is the human airway epithelial cell isolated from tracheal or bronchial mucosa, although there are also plans to develop models using transformed human cells and ovine airway epithelial cells that express similar enzymatic activities. The application proposes to use analytical lipid chemistry, immunochemistry, and molecular biology in studies of airway epithelial cells and epithelial tissue to examine two hypothesis. The first hypothesis is that the PGH synthase/PGE isomerase pathway regulates prostaglandin formation under physiologic conditions whereas the PGH synthase/PGF synthetase/PGD isomerase pathway regulates prostaglandin formation during inflammation. The second hypothesis is that arachidonate 12- lipoxygenase (and the homologous 15-lipoxygenase) control the degree of membrane fatty acid peroxidation in airway epithelial cells as a mechanism underlying epithelial cell damage during inflammation.
The Specific Aims are to: 1) determine the mechanisms controlling the expression and regulation of enzymatic activity for airway epithelial PGH synthase-1 and PGH synthase-2, and compare the relative importance of each PGH synthase versus phospholipase and PG isomerase activities for controlling prostaglandin formation; and 2) determine the mechanisms controlling the expression and regulation of enzymatic activity for airway epithelial 12- and 15-lipoxygenases, and determine their capacity for peroxidation of membrane fatty acids.
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