The biological activities of P-450 products of arachidonic acid metabolism are often mediated by the cyclooxygenase enzyme. This could occur through at least two separate mechanisms. The P-450 products could release biologically active cyclooxygenase products (perhaps through a receptor mediated process) and these could then be responsible for the observed activity. Alternatively, the P-450 metabolites themselves could undergo cyclooxygenase-mediated transcellular metabolism to provide compounds with different biological activities. There is evidence from our own laboratory and from several other laboratories that this latter mechanism may be the most dominant for both epoxygenase and omega/omega-1 oxidation products of arachidonic acid metabolism. There is evidence that metabolism of some of the P-450 metabolites can also be carried out by lipoxygenase enzymes. The major goals of Project 3 are: to further refine our understanding of transcellular metabolism of P-450 arachidonic acid metabolites by cyclooxygenase and lipoxygenase, to determine whether glucuronidation of the metabolites in normal physiological processes and to ascertain whether they are involved in pathophysiological processes. Structural characterization of urinary metabolites will be carried out primarily by gas chromatography/electron ionization mass spectrometry. Quantitative studies will involve the development of analytical methodology based on stable isotope dilution gas chromatography/electron capture negative ion chemical ionization mass spectrometry. The transcellular metabolites will be analyzed in urine from animal models and from human subjects to provide a measure of the renal biosynthesis of these compounds. the contribution of these metabolites to the overall eicosanoid excretion rate will then be assessed. In vitro model systems to examine the generation of transcellular metabolites will be developed and these model systems will be used to delineate the mechanism by which the individual transcellular metabolites are formed. We will also focus on possible participation of transcellular metabolites in signal transduction mechanisms. In particular, the possibility that some of the transcellular metabolites are incorporated into specific phospholipid pools will be explored. This could lead to the generation of diacylglycerols with enhanced activity in phosphoinositide signalling pathways.
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