Isoprostanes (IsoPs) are novel bioactive prostaglandin (PG)-like compounds formed in vivo from the free radical-initiated peroxidation of arachidonic acid. Quantification of IsoPs has greatly expanded our knowledge of the role of oxidant stress in human diseases. We have characterized IsoPs with F-type and E/D-type prostane rings. We recently discovered that highly reactive cyclopentenone (A2/J2) IsoPs, which adduct proteins, DNA, and thiols, are formed from the dehydration of Ez/D2-IsoPs. We also determined that IsoP-like compounds, termed neuroprostanes (NPs) are generated in vivo from docosahexanenoic acid (DHA). Our hypothesis is that IsoPs and NPs contribute importantly to the pathophysiological sequelae of oxidant stress. Studies will delineate mechanisms by which IsoPs and NPs are formed and metabolized and examine pathways regulating their bioactivity. We will determine the extent to which F-ring PGs are formed in vivo via the IsoP pathway. We will test the hypothesis that compounds identical to cyclooxygenase-derived PGF2a, and its enantiomer, are generated in significant amounts by a kinetically-driven mechanism involving the non-enzymatic generation of racemic PGH2. We have previously determined that racemic PGE2 and PGD2 are formed via the IsoP pathway by epimerization of E/D-ring IsoPs but PGF2a is not generated in this manner. The extent to which these two mechanisms contribute to PG formation in vivo will be determined. We will also examine whether the enantiomer of PGF2alpha is metabolized similarly to PGF2a and compare the interactions of PGF2a and its enantiomer with the PGF2alpha (FP) receptor. Studies will elucidate mechanisms responsible for the formation of NPs and related oxidation products of DHA in vivo. The peroxidation of DHA is complex because of its degree of unsaturation. Based on studies utilizing novel mass spectrometric methods, we hypothesize that DHA oxidation leads not only to endoperoxides but to products containing cyclic peroxides and dioxolane-endoperoxides. We will test this hypothesis. We will also examine the biochemical pharmacology of cyclopentenone NPs, the most abundant NPs generated in vivo. We have found that IsoPs are formed in vivo esterified to glycerol (glyceryl-IsoPs) that are presumably derived from 2-arachidonoylglycerol (2-AG), a ligand for cannabinoid receptors. One glyceryl-IsoP, 2-glyceryl-15-F2t-IsoP, interacts with the thromboxane receptor, suggesting that oxygenated products of 2-AG can regulate physiological consequences of oxidative injury in a receptor-mediated manner. Studies will examine mechanisms related to the formation, metabolism and biological actions of glyceryMsoPs in vivo.
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