15-keto-5-8-11-13-eicosatetraenoic-acid and 15-hydroxy-5-8-11-13-eicosatetraenoic-acid

Mast cells play a key role in the pathophysiology of asthma. These cells exert their effector functions by releasing a variety of proinflammatory and immunoregulatory compounds. Mast cells infiltrate the bronchial epithelium and smooth muscle to a higher degree in patients with asthma compared to control subjects. 15-Lipoxygenase type-1 (15-LO-1) is a prooxidant enzyme which is expressed in asthmatic lungs leading to formation of pro- and anti-inflammatory mediators. Here we report that interleukin-4 (IL-4) induced the expression of 15-LO-1 in human cord blood derived mast cells (CBMC) as demonstrated by RT-PCR, western blot and immunocytochemistry. The major metabolite of arachidonic acid formed via the 15-LO pathway in IL-4 treated CBMC was identified as 15-ketoeicosatetraenoic acid (15-KETE, also named 15-oxo-ETE) with smaller amounts of 15-hydroxyeicosatetraenoic acid (15-HETE) as identified by HPLC and mass spectrometry (MS/MS). Furthermore, immunohistochemical stainings demonstrated the expression of 15-LO-1 in mast cells in lung and skin in vivo. Osmotic activation of CBMC with mannitol resulted in activation of the 15-LO-1 pathway. In conclusion, the expression of 15-LO-1 and release of 15-LO-1 derived products by mast cells may contribute to the role of these cells in asthma and other inflammatory diseases.

Topics: Arachidonate 15-Lipoxygenase; Arachidonic Acids; Asthma; Fetal Blood; Humans; Hydroxyeicosatetraenoic Acids; Interleukin-4; Isoenzymes; Leukotrienes; Lipid Peroxides; Lung; Mannitol; Mast Cells; Skin; Tryptases

Reaction of manganese-reconstituted prostaglandin endoperoxide synthase (Mn-PGHS) with 15-hydroperoxyeicosatetraenoic acid (15-HPETE) generates two products in nearly equal amounts: 15-hydroxyeicosatetraenoic acid (15-HETE) and 15-ketoeicosatetraenoic acid (15-KETE) [Kulmacz et al. (1994) Biochemistry 33, 5428-5439]. Their proposed mechanism to explain 15-KETE formation, namely oxidation of 15-HETE by the peroxidase activity of MnPGHS, was tested and found not to occur. Instead, 15-KETE may arise by one-electron reduction of 15-HPETE followed by oxidation of an intermediate alkoxyl radical. The mechanism of hydroperoxide reduction by Mn-PGHS was investigated using 10-hydroperoxyoctadeca-8,12-dienoic acid (10-OOH-18:2), a diagnostic probe of hydroperoxide reduction pathways. Reaction of Mn-PGHS with 10-OOH-18:2 generated the two-electron reduced product, 10-hydroxyoctadeca-8,12-dienoic acid (10-OH-18:2), as well as the one-electron reduction products, 10-oxooctadeca-8,12 dienoic acid (10-oxo-18:2) and 10-oxodec-8-enoic acid (10-oxo-10:1) in relative yields of 82, 10, and 7%, respectively. The identity of the one-electron reduction products was confirmed by electrospray ionization mass spectrometry. The detection of 10-oxo-10:1 provides strong evidence for the production of an alkoxyl radical during 10-OOH-18:2 reduction by Mn-PGHS. Like 15-HPETE, reaction of Mn-PGHS with 13-hydroperoxyoctadeca-8,12-dienoic acid (13-OOH-18:2) generated two products in equal amounts: 13-hydroxyoctadeca-8,12-dienoic acid (13-OH-18:2) and the keto fatty acid 13-oxooctadeca-8,12-dienoic acid (13-oxo-18:2). Comparison of the three hydroperoxides demonstrates that 15-HPETE is a much better substrate for Mn-PGHS than 10-OOH-18:2 or 13-OOH-18:2 with 10-fold greater turnovers. The results show that Mn-PGHS catalyzes both one- and two-electron hydroperoxide reduction and that the pathway of alkoxyl radical decomposition is influenced by the protein component of Mn-PGHS and the structure of the alkoxyl radical intermediate.

Topics: Animals; Arachidonic Acids; Electrons; Free Radicals; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Leukotrienes; Lipid Peroxides; Male; Manganese; Molecular Structure; Oxidation-Reduction; Prostaglandin-Endoperoxide Synthases; Sheep; Substrate Specificity

The structural requirements for inhibition of RBL-1 (rat basophilic leukemia) 5-lipoxygenase by 15-hydroxyeicosa-5,8,11,13-tetraenoic acid (15-HETE, 1) were studied by systematic chemical modifications of the molecule at the hydroxyl and carboxyl groups, the double bonds, and the carboxylate and omega side chains. The most potent inhibitors were analogues that contained a 5,8-cis,cis-diene system and acted as alternate substrates for the enzyme. However, several analogues in which the 5,8-diene had been reduced were also found to inhibit the enzyme. Inhibition of 5-lipoxygenase by 15-hydroxyeicosa-11,13-dienoic acid (15-HEDE) analogues was optimal in compounds that generally contained a free carboxyl group, a carboxylate side chain of nine carbons, an omega side chain of five or six carbons, a cis,trans- or trans,cis-11,13-diene or 11,13-diyne system, and a 15-hydroxyl group. Conversion of 15-HEDE to its 16-membered lactone reduced but did not eliminate 5-lipoxygenase inhibitory activity. In contrast, a 3- to 10-fold enhancement of activity occurred when 5,15-diHETE (58) or 5-HETE (56) were cyclized to their respective delta-lactones. Molecular modeling of 15-HEDE analogues, modified in the C11-C15 region, showed that inactive analogues protrude into regions in space not occupied by active analogues. These structural studies indicate that multiple regions are important for 5-lipoxygenase inhibition by both 15-HETE and 15-HEDE analogues and that no single region plays a predominant role in inhibition.

Topics: Animals; Arachidonate Lipoxygenases; Basophils; Hydroxyeicosatetraenoic Acids; Indicators and Reagents; Leukemia, Experimental; Lipoxygenase Inhibitors; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Conformation; Rats; Spectrophotometry, Infrared; Structure-Activity Relationship