The pulmonary endothelium influences the redox status of blood borne redox active endogenous, pharmacological, toxicological and dietary compounds. It does so by acting as a large chemical reactor surface comprised of cell surface and intracellular redox enzymes situated between the venous and arterial circulations. The impact of the endothelium on the redox status of these compounds affects their bioactivity, including pro-or anti-oxidant effects, tissue permeation, and chemical reactivity. Quinones represent 1 important class of redox-active compounds with a range of physiological, pharmacological, and toxicological activities. The hypothesis underlying this proposal is that redox metabolism of quinones by pulmonary endothelial cells determines the quinone reduction products, and their bioactivity, within the lung and endothelial cells, the plasma, and downstream vessels and organ systems. The goal of the proposed research is to identify and quantify the factors, with regard to properties of both the cells and quinones, that determine the fate of a given quinone that comes in contact with the pulmonary endothelium.
The specific aims are to: 1. Identify and quantify the pulmonary endothelial quinone reductase and hydroquinone oxidase mediated reactions contributing to the fate of a series of quinones with varying physical and chemical properties: 2. Determine the dependence of the activities of quinone reductases contributing to the fate of a given quinone on the pulmonary endothelial redox status: 3. Determine the influence of adaptation of the pulmonary endothelium to hyperoxia on the redox processes contributing to quinone fate. To achieve these aims, the general approaches include: measuring the kinetics of intracellular and cell surface quinone reduction, hydroquinone oxidation and associations with cellular constituents using intact normoxic pulmonary arterial endothelial cells in culture and cells adapted to hyperoxia, as a model of oxidant stress. A panel of inhibitors and kinetic modeling will provide the means for functional and quantitative identification of the contributions of the quinone reductases and hydroquinone oxidases involved. Cell redox status will be manipulated experimentally to determine its role in the fate of the quinones that come in contact with the endothelial cells. Finally, the quinone reductases in the control and hyperoxia-adapted cells will be isolated and identified to further characterize their contributions to the net effect of both cell types to quinone fate. Since quinone reductases and hydroquinone oxidases are important in metabolism of a wide range of other redox active compounds, the results will have implications for endothelial processing of substances as well. The results will reveal pulmonary endothelial mechanisms involved in processing blood borne redox active substances, and their adaptation to oxidative stress, with implications for their pharmacological, physiological and toxicological activities throughout the body.
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