This proposal describes the investigation of the regulation and function of the critical enzymes and products (prostaglandins, thromboxane, leukotrienes) of eicosanoid metabolism under physiological, pharmacological and pathological conditions. We will employ: (a) cDNA probes and selective antibodies for cyclooxygenase, LTA4 hydrolase, 5-lipoxygenase and thromboxane synthetase; (b) immunoassays for prostaglandin (PG) E2, thromboxane B2 6-keto-PGF1alpha, leukotriene cyclooxygenase, thromboxane synthetase, 5-lipoxygenase, and phospholipase A2 to investigate the qualitative, quantitative and temporal role of these enzymes and their products in the regulation, differentiation, and pharmacological and pathological modification of fibroblasts and mononuclear cells. Cellular regulation will be studied by analyses of transcription, translation, expression and turnover of the eicosanoid enzymes relative to metabolite biosynthesis. We plan to study changes in arachidonate metabolism which will serve as a phenotypic marker and/or modulator of cellular maturation and differentiation by comparing the profile of enzymes and metabolite changes in differentiating mononuclear cells (i.e., bone marrow cells, blood monocytes, comparative tissue macrophage populations (peritoneal, alveolar, splenic and hepatic) that are resident (unstimulated) or activated (in vivo stimulation with endotoxin, ConA, bacteria) or inhibited (glucocorticoids and the dexamethasone-induced protein). In experiments analyzing the molecular mechanisms of fibroblast cyclooxygenase, we have already uncovered suggestive evidence for two forms of the enzyme (during differentiation or stimulation) and have discovered a unique and potent ability of glucocorticoids to selectively inhibit (post-transcriptionally) the enzyme. In summary, the comprehensive application of the biochemical and pharmacological reagents we have assembled for studies in isolated cells and in vivo, utilizing experimental systems that can be selectively manipulated, should identify the steps in eicosanoid metabolism that are functionally critical and therefore amenable to pharmacological and therapeutic manipulation.
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