The long range goals of the project are a mechanistic understanding of inflammation of the pulmonary airway and the development of biochemical strategies for use against airway inflammatory diseases such as bronchitis and asthma. The investigations are aimed at the role of the airway epithelial cell as an interface between inhaled agents and underlying cells and, specifically, at the epithelial capacity for fatty acid oxygenation and lipid mediator release. Recent discoveries of airway epithelial stores of fatty acid substrates, the capacity to release these subtrates through the action of phospholipases, and the expression of activity for selective lipoxygenation at a high level make epithelial oxygenation pathways excellent candidates to serve as a mechanism for regulating inflammation. The proposed studies aim at dissecting the pathways leading to oxygenation of fatty acids, especially arachidonic acid, in isolated epithelial cells. The primary focus of the work is the human airway epithelial cell, but the studies will be supplemented when necessary by work on transformed human cells, epithelial cells from other species, and leukocytes with similar oxygenation pathways.
Specific aims are to: Identify the types of lipoxygenase, cyclooxygenase, and monooxygenase pathways for fatty acid metabolism in airway epithelial cells using recently developed methods for cell culture and analytical lipid chemistry. Defining enzymatic pathways and their products will lay the groundwork for eventual studies of product function. Determine the mechanisms for endogenous generation of epithelial oxygenation products using the same techniques. Knowledge of the uptake of fatty acid substrate, the types of phospholipases responsible for substrate release, and the conditions for activation or expression of phospholipase activity is needed as a basis for anti-inflammatory strategies. Purify and characterize the arachidonate 15-lipoxygenase which often predominates in human epithelial cells using conventional, immunological, and molecular biological methods for protein purification. The availability of purified enzyme will facilitate studies of enzymatic regulation and the development of selective inhibitors.
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