l-663536 and 15-hydroxy-5-8-11-13-eicosatetraenoic-acid

Inflammation is a multi-staged process whose expansive phase is thought to be driven by acutely released arachidonic acid (AA) and its metabolites. Inhibition of cyclooxygenase (COX), lipoxygenase (LOX), or soluble epoxide hydrolase (sEH) is known to be anti-inflammatory. Inhibition of sEH stabilizes the cytochrome P450 (CYP450) products epoxyeicosatrienoic acids (EETs). Here we used a non-selective COX inhibitor aspirin, a 5-lipoxygenase activation protein (FLAP) inhibitor MK886, and a sEH inhibitor t-AUCB to selectively modulate the branches of AA metabolism in a lipopolysaccharide (LPS)-challenged murine model. We used metabolomic profiling to simultaneously monitor representative AA metabolites of each branch. In addition to the significant crosstalk among branches of the AA cascade during selective modulation of COX, LOX, or sEH, we demonstrated that co-administration of t-AUCB enhanced the anti-inflammatory effects of aspirin or MK886, which was evidenced by the observations that co-administration resulted in favorable eicosanoid profiles and better control of LPS-mediated hypotension as well as hepatic protein expression of COX-2 and 5-LOX. Targeted disruption of the sEH gene displayed a parallel profile to that produced by t-AUCB. These observations demonstrate a significant level of crosstalk among the three major branches of the AA cascade and that they are not simply parallel pathways. These data illustrate that inhibition of sEH by both pharmacological intervention and gene knockout enhances the anti-inflammatory effects of aspirin and MK886, suggesting the possibility of modulating multiple branches to achieve better therapeutic effects.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Aspirin; Benzoates; Blood Pressure; Dose-Response Relationship, Drug; Enzyme Activation; Epoxide Hydrolases; Gene Expression Regulation, Enzymologic; Hydroxyeicosatetraenoic Acids; Indoles; Inflammation; Lipopolysaccharides; Lipoxygenase Inhibitors; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Prostaglandin-Endoperoxide Synthases; Urea

Topics: Bronchi; Bronchoconstriction; Humans; Hydroxyeicosatetraenoic Acids; Indoles; Leukotriene Antagonists; Leukotrienes; Monocytes; Muscle, Smooth; Propionates; Quinolines

5-Lipoxygenase-activating protein is required for cellular leukotriene synthesis and is the target of the leukotriene biosynthesis inhibitors MK-886 (3-[1-(p-chlorophenyl)-5-isopropyl-3-tert-butylthio-1H- indol-2-yl]-2,2-dimethylpropanoic acid) and MK-591 (3-[1-(4-chlorobenzyl)-3-(t-butylthio)-5-(quinolin-2-ylmethoxy)-indol-2-yl] - 2,2-dimethylpropanoic acid). Recent studies demonstrate that 5-lipoxygenase-activating protein binds arachidonic acid and stimulates the utilization of this substrate by 5-lipoxygenase. The present study utilizes a radioligand binding assay to assess the affinity of 5-lipoxygenase-activating protein for arachidonic acid and the specificity of the fatty acid binding site on 5-lipoxygenase-activating protein. Our findings demonstrate that the presence of a free carboxyl group on fatty acids or leukotriene biosynthesis inhibitors which interact with 5-lipoxygenase-activating protein is not required for specific binding to the protein. However, the degree of saturation significantly affects the affinity of fatty acids for 5-lipoxygenase-activating protein.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 5-Lipoxygenase-Activating Proteins; Arachidonic Acid; Binding Sites; Carrier Proteins; Humans; Hydroxyeicosatetraenoic Acids; Indoles; Leukocytes; Leukotrienes; Membrane Proteins; Quinolines; Radioligand Assay