5-hydroxy-6-8-11-14-eicosatetraenoic-acid and 5-oxo-6-8-11-14-eicosatetraenoic-acid

Arachidonic acid can be oxygenated by a variety of different enzymes, including lipoxygenases, cyclooxygenases, and cytochrome P450s, and can be converted to a complex mixture of oxygenated products as a result of lipid peroxidation. The initial products in these reactions are hydroperoxyeicosatetraenoic acids (HpETEs) and hydroxyeicosatetraenoic acids (HETEs). Oxoeicosatetraenoic acids (oxo-ETEs) can be formed by the actions of various dehydrogenases on HETEs or by dehydration of HpETEs. Although a large number of different HETEs and oxo-ETEs have been identified, this review will focus principally on 5-oxo-ETE, 5S-HETE, 12S-HETE, and 15S-HETE. Other related arachidonic acid metabolites will also be discussed in less detail. 5-Oxo-ETE is synthesized by oxidation of the 5-lipoxygenase product 5S-HETE by the selective enzyme, 5-hydroxyeicosanoid dehydrogenase. It actions are mediated by the selective OXE receptor, which is highly expressed on eosinophils, suggesting that it may be important in eosinophilic diseases such as asthma. 5-Oxo-ETE also appears to stimulate tumor cell proliferation and may also be involved in cancer. Highly selective and potent OXE receptor antagonists have recently become available and could help to clarify its pathophysiological role. The 12-lipoxygenase product 12S-HETE acts by the GPR31 receptor and promotes tumor cell proliferation and metastasis and could therefore be a promising target in cancer therapy. It may also be involved as a proinflammatory mediator in diabetes. In contrast, 15S-HETE may have a protective effect in cancer. In addition to GPCRs, higher concentration of HETEs and oxo-ETEs can activate peroxisome proliferator-activated receptors (PPARs) and could potentially regulate a variety of processes by this mechanism. This article is part of a Special Issue entitled "Oxygenated metabolism of PUFA: analysis and biological relevance".

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Disease; Humans; Hydroxyeicosatetraenoic Acids; Oxidation-Reduction; Peroxisome Proliferator-Activated Receptors; Receptors, Eicosanoid; Receptors, G-Protein-Coupled; Signal Transduction

Arachidonic acid (AA) is converted to biologically active metabolites by different pathways, one of the most important of which is initiated by 5-lipoxygenase (5-LO). 5-Hydroxyeicosatetraenoic acid (5-HETE), although possessing only weak biological activity itself, is oxidized to 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE), a potent chemoattractant for eosinophils and neutrophils. Our main goal is to determine how the biosynthesis of 5-oxo-ETE is regulated and to determine its pathophysiological roles. To achieve this task, we designed and synthesized affinity chromatography ligands for the purification of 5-hydroxyeicosanoid dehydrogenase (5-HEDH), the enzyme responsible for the formation of 5-oxo-ETE.

Topics: Alcohol Oxidoreductases; Arachidonic Acids; Cell Line; Chromatography, Affinity; Humans; Hydroxyeicosatetraenoic Acids; Ligands; Neutrophils

The 5-lipoxygenase (5-LO) product 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE), which is a potent chemoattractant for myeloid cells, is known to promote the survival of prostate cancer cells. In the present study, we found that PC3 prostate cancer cells and cell lines derived from breast (MCF7) and lung (A-427) cancers contain 5-hydroxyeicosanoid dehydrogenase (5-HEDH) activity and have the ability to synthesize 5-oxo-ETE from its precursor 5S-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) when added as an exogenous substrate. H(2)O(2) strongly stimulated the synthesis of 5-oxo-ETE and induced dramatic increases in the levels of both glutathione disulfide and NADP(+). The effects of H(2)O(2) on 5-oxo-ETE and NADP(+) were blocked by N-ethylmaleimide (NEM), indicating that this effect was mediated by the glutathione reductase-dependent generation of NADP(+), the cofactor required by 5-HEDH. 5-Oxo-ETE synthesis was also stimulated by agents that have cytotoxic effects on tumor cells, including 4,7,10,13,16,19-docosahexaenoic acid, tamoxifen and MK-886. Because PC3 cells have only modest 5-LO activity compared with inflammatory cells, we investigated their ability to contribute to the transcellular biosynthesis of 5-oxo-ETE from neutrophil-derived 5-HETE. Stimulation of neutrophils with arachidonic acid and calcium ionophore in the presence of PC3 cells led to a large and selective increase in 5-oxo-ETE synthesis compared with controls in which PC3 cell 5-oxo-ETE synthesis was selectively blocked by pretreatment with NEM. The ability of prostate tumor cells to synthesize 5-oxo-ETE may contribute to tumor cell proliferation as well as the influx of inflammatory cells, which may further induce cell proliferation through the release of cytokines. 5-Oxo-ETE may be an attractive target in cancer therapy.

Topics: Alcohol Oxidoreductases; Arachidonate 5-Lipoxygenase; Arachidonic Acids; Breast Neoplasms; Chromatography, High Pressure Liquid; Docosahexaenoic Acids; Humans; Hydroxyeicosatetraenoic Acids; Lung Neoplasms; Male; Neutrophils; Oxidative Stress; Prostatic Neoplasms; Tumor Cells, Cultured

Neutrophils spontaneously undergo apoptosis, which is associated with increased oxidative stress. We found that there is a dramatic shift in the formation of 5-lipoxygenase products during this process. Freshly isolated neutrophils rapidly convert leukotriene B(4) (LTB(4)) and 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) to their biologically inactive omega-oxidation products. However, omega-oxidation is impaired in neutrophils cultured for 24 h, when only 25% of the cells are nonapoptotic, resulting in the persistence of LTB(4) and a dramatic shift in 5-HETE metabolism to the potent granulocyte chemoattractant 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE). The reduced omega-oxidation activity seems to be due to a reduction in LTB(4) 20-hydroxylase activity, whereas the increased 5-oxo-ETE formation is caused by a dramatic increase in the 5-hydroxyeicosanoid dehydrogenase cofactor NADP(+). NAD(+), but not NADPH, also increased, as did the GSSG/GSH ratio, indicative of oxidative stress. The changes in 5-HETE metabolism and pyridine nucleotides were inhibited by antiapoptotic agents (GM-CSF, forskolin) and antioxidants (diphenylene iodonium, catalase, deferoxamine), suggesting the involvement of H(2)O(2) and possibly other reactive oxygen species. These results suggest that in severe inflammation, aging neutrophils that have evaded rapid uptake by macrophages may produce increased amounts of the chemoattractants 5-oxo-ETE and LTB(4), resulting in delayed resolution or exacerbation of the inflammatory process.

Topics: Alcohol Oxidoreductases; Antioxidants; Apoptosis; Arachidonate 5-Lipoxygenase; Arachidonic Acids; Biomimetics; Cells, Cultured; Cellular Senescence; Chemotactic Factors; Colforsin; Cytochrome P-450 Enzyme System; Cytochrome P450 Family 4; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; NADP; Neutrophils; Oxidation-Reduction; Oxidative Stress; Pyridines; Superoxide Dismutase

There is increasing evidence that proinflammatory products of the 5-lipoxygenase pathway play an important role in cardiovascular disease. In the present study, we found that human endothelial cells rapidly oxidize the 5-lipoxygenase product 5S-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) to 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE), a potent chemoattractant for myeloid cells. 5-Oxo-ETE synthesis is strongly stimulated by oxidative stress. This effect is enhanced following inhibition of the pentose phosphate pathway with dehydroepiandrosterone and is mimicked by diamide, which oxidizes intracellular GSH to GSSG. Conversely, it is blocked by depletion of intracellular GSH/GSSG. The kinetics of H2O2-induced 5-oxo-ETE synthesis by endothelial cells correlate well with changes in the intracellular levels of GSSG and NADP+. These results suggest that exposure of the endothelium to oxidative stress and inflammation could result in the synthesis of 5-oxo-ETE, which could then induce the infiltration of inflammatory cells into the tissue.

Topics: Arachidonic Acids; Cells, Cultured; Endothelial Cells; Glutathione; Humans; Hydrogen Peroxide; Hydroxyeicosatetraenoic Acids; Oxidation-Reduction; Oxidative Stress; Pentose Phosphate Pathway

We have previously identified a Galpha(i/o)-protein-coupled receptor (TG1019/OXE) using 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-oxo-ETE) as its ligand. We investigated signal transduction from TG1019 following stimulation with 5-oxo-ETE and role of TG1019 in 5-oxo-ETE-induced chemotaxis, using Chinese hamster ovary cells expressing TG1019 (CHO/TG1019 cells). 5-Oxo-ETE induced intracellular calcium mobilization and rapid activation of MEK/ERK and PI3K/Akt pathways in CHO/TG1019 cells. CHO/TG1019 cells stimulated with 5-oxo-ETE and other eicosanoids exhibited chemotaxis with efficacies related to agonistic activity of each eicosanoid for TG1019. Pretreatment of the cells with pertussis toxin, a phospholipase C (PLC) inhibitor (U73122) or a PI3K inhibitor (LY294002), markedly suppressed 5-oxo-ETE-induced chemotaxis, whereas pretreatment with a MEK inhibitor (PD98059) had no significant effect on the chemotaxis. Our results show that TG1019 mediates 5-oxo-ETE-induced chemotaxis and that signals from TG1019 are transduced via Galpha(i/o) protein to PLC/calcium mobilization, MEK/ERK, and PI3K/Akt, among which PLC and PI3K would play important roles in the chemotaxis.

Topics: Animals; Arachidonic Acids; Chemotaxis; CHO Cells; Cricetinae; Cricetulus; Dose-Response Relationship, Drug; Receptors, Eicosanoid; Receptors, G-Protein-Coupled; Recombinant Proteins

MDA-MB-231, MCF7, and SKOV3 cancer cells, but not HEK-293 cells, expressed mRNA for the leukocyte G protein-coupled 5-oxo-eicosatetraenoate (ETE) OXE receptor. 5-Oxo-ETE, 5-oxo-15-OH-ETE, and 5-HETE stimulated the cancer cell lines but not HEK-293 cells to mount pertussis toxin-sensitive proliferation responses. Their potencies in eliciting this response were similar to their known potencies in activating leukocytes and OXE receptor-transfected cells. However, high concentrations of 5-oxo-ETE and 5-oxo-15-OH-ETE, but not 5-HETE, arrested growth and caused apoptosis in all four cell lines; these responses were pertussis toxin-resistant. The same high concentrations of the oxo-ETEs but again not 5-HETE also activated peroxisome proliferator-activated receptor (PPAR)-gamma. Pharmacological studies indicated that this activation did not mediate their effects on proliferation. These results are the first to implicate the OXE receptor in malignant cell growth and to show that 5-oxo-ETEs activate cell death programs as well as PPARgamma independently of this receptor.

Topics: Anilides; Apoptosis; Arachidonic Acids; Binding Sites; Caspase 3; Caspases; Cell Line; Cell Line, Tumor; Cell Proliferation; Cell Survival; Gene Expression; Humans; Hydroxyeicosatetraenoic Acids; Mitosis; Peroxisome Proliferator-Activated Receptors; Pertussis Toxin; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; PPAR gamma; Prostaglandin D2; Protein Binding; Receptors, Eicosanoid; Transfection

The metabolism of arachidonic acid by the 5-lipoxygenase pathway not only leads to the formation of leukotrienes but also to the biologically active eicosanoid 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE). The synthesis of 5-oxo-ETE was investigated in the elicited peritoneal macrophage and the formation of 5-hydroxyeicosatetraenoic acid (5-HETE) as well as 5-oxo-ETE was quantitated using stable isotope dilution tandem mass spectrometry. The metabolism of 5-oxo-ETE in these same cells led to the formation of a series of novel less lipophilic metabolites oxidized near the methyl terminus that were structurally characterized using electrospray LC/MS and LC/MS/MS. Five novel metabolites of 5-oxo-ETE were identified including 5,18-diHETE, 5,19-diHETE, 5-oxo-19-HETrE, 5-oxo-18-HETrE, and 5,19-diHETrE. These metabolites corresponded to omega-1 and omega-2 oxidation of 5-oxo-ETE presumably formed by a specific cytochrome P450. There was no evidence for the formation of omega-oxidation (20-hydroxy metabolites), which are known products of metabolism of 5-oxo-ETE in other cell types. None of the metabolites were found to elevate intracellular calcium release, suggesting that this metabolic pathway may result in inactivation of 5-oxo-ETE. This is the first report of the biosynthesis of 5-oxo-ETE by tissue resident cell outside of the blood and the formation of novel omega-1 and omega-2 oxidation of this eicosanoid.

Topics: Animals; Arachidonic Acids; Biotransformation; Calcium; Catalysis; Cell Separation; Chromatography, High Pressure Liquid; Cytosol; Gas Chromatography-Mass Spectrometry; Hydrogen; Hydroxyeicosatetraenoic Acids; Macrophages; Mice; Mice, Inbred ICR; Oxidation-Reduction; Spectrometry, Mass, Electrospray Ionization; Spectrophotometry, Ultraviolet

The arachidonic acid metabolite, 5-oxo-eicosatetraenoic acid (5-oxo-ETE), is a potent chemotaxin for neutrophils and eosinophils. The aim of this study was to identify physiological conditions and stimulators of 5-oxo-ETE synthesis, because no such conditions have yet been identified.. Human neutrophils and monocyte-derived dendritic cells were prepared and 5-oxo-ETE synthesis analyzed using precolumn/reversed-phase HPLC under different conditions and with several physiological and unphysiological stimuli.. Incubation of neutrophils with 5-hydroxyeicosatetraenoic acid (5-HETE) resulted in the synthesis of about 3.4 nM 5-oxo-ETE per 10(6) cells in 1 ml under optimal conditions. The synthesis was enhanced about 8-fold with the unphysiological stimuli calcium ionophore A23187 and phorbol 12-myristate 13-acetate (PMA). No significant effect was observed with different physiological activators. Under optimal conditions, human dendritic cells produced about 50 nM 5-oxo-ETE per 10(6) cells in 1 ml. The synthesis could be increased with PMA and A23187 by about 50%. Again, no effect could be observed with physiological agents for dendritic cells such as complement fragment C5a, platelet activating factor, N-formyl peptides and interleukin-5.. These data identified dendritic cells as the only yet known physiological source of relevant amounts of 5-oxo-ETE. This suggests a regulatory function of dendritic cells in the induction of inflammatory neutrophil and eosinophil infiltration caused by 5-oxo-eicosatetraenoic acid.

Topics: Arachidonic Acids; Calcimycin; Chemotactic Factors; Chromatography, High Pressure Liquid; Dendritic Cells; Humans; Hydroxyeicosatetraenoic Acids; Ionophores; Neutrophils; Tetradecanoylphorbol Acetate

Neutrophil-derived 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a potent activator of neutrophils and eosinophils. In the present study we examined the biosynthesis and metabolism of this substance by platelets. Although platelets contain an abundant amount of 5-hydroxyeicosanoid dehydrogenase, the enzyme responsible for the formation of 5-oxo-ETE, they synthesize only very small amounts of this substance from exogenous 5-hydroxyeicosatetraenoic acid (5-HETE) unless endogenous NADPH is converted to NADP+ by addition of phenazine methosulfate. Similarly, relatively small amounts of 5-oxo-ETE were formed by A23187-stimulated mixtures of platelets and neutrophils, which instead formed substantial amounts of two 12-hydroxy metabolites of this substance, 5-oxo-12-HETE and 8-trans-5-oxo-12-HETE, which were identified by comparison with authentic chemically synthesized compounds. These metabolites were also formed from 5-oxo-ETE by platelets stimulated with thrombin or A23187. In contrast, unstimulated platelets converted 5-oxo-ETE principally to 5-HETE. Neither 5-oxo-12-HETE nor 8-trans-5-oxo-12-HETE had appreciable effects on neutrophil calcium levels or platelet aggregation at concentrations as high as 10 micromol/L, but both blocked 5-oxo-ETE-induced calcium mobilization in neutrophils with IC50 values of 0.5 and 2.5 micromol/L, respectively. We conclude that platelets can biologically inactivate 5-oxo-ETE. Unstimulated platelets convert 5-oxo-ETE to 5-HETE, with a 99% loss of biological potency, whereas stimulated platelets convert this substance to 12-hydroxy metabolites, which possess antagonist properties.

Topics: Alcohol Oxidoreductases; Arachidonic Acids; Blood Platelets; Calcimycin; Calcium Signaling; Eicosanoids; Humans; Hydroxyeicosatetraenoic Acids; Methylphenazonium Methosulfate; NADP; Neutrophils; Platelet Activation; Platelet Aggregation; Subcellular Fractions; Thrombin

Oxidation of red blood cell (RBC) ghost preparations initiated by tert-butyl hydroperoxide (tBuOOH) was employed to explore the formation of lipid products derived from endogenous phospholipids that specifically expressed biological activity toward the human polymorphonuclear leukocyte (PMN). Common measure of lipid peroxidation, thiobarbituric acid-reactive substances (TBARS) and the increased absorbance at 235 nm consistent with the formation of conjugated dienes, was observed following a 90-min incubation of RBC ghosts with tBuOOH. Saponification of phospholipids and separation of the resultant fatty acids by RP-HPLC permitted direct mass spectrometric analysis of oxidized fatty acids. Individual HPLC fractions were assayed for their ability to increase intracellular free calcium ion concentrations in human PMN to guide structural investigations. Two fractions were found to contain biologically active components, and tandem mass spectrometric analysis of the abundant ions observed in these fractions resulted in the characterization of several oxidized polyunsaturated fatty acids derived from arachidonic and linoleic acids. The major components in these fractions included 5-hydroxyeicosatetraenoic acid (5-HETE) and 5-hydroperoxyeicosatetraenoic acid (5-HpETE). The dose-dependent increases in intracellular calcium in the neutrophil using synthetic 5(rac)-HETE, 5(rac)-HpETE, and 5-oxo-ETE were found to have EC50's of 250, 6, and 3 nM, respectively. The quantity of 5-oxygenated arachidonate components present in oxidized RBC was consistent with the observed biological response elicited by fractions A and B. This study suggests that 5-HETE and 5-HpETE are abundant products of lipid peroxidation of cellular membranes and that these racemic products possess significant biological activity. Such compounds could play important roles as mediators of the cellular response to toxicologic stimuli that generate free radical species.

Topics: Arachidonic Acids; Calcium; Chromatography, High Pressure Liquid; Erythrocyte Membrane; Fatty Acids; Humans; Hydroxyeicosatetraenoic Acids; Leukotrienes; Lipid Peroxidation; Lipids; Mass Spectrometry; Neutrophil Activation; Neutrophils; Oxidation-Reduction

Arachidonic acid (5,8,11,14-eicosatetraenoic acid), a member of the omega-6 poly-unsaturated fatty acids, was found to be an effective stimulator of human prostate cancer cell growth in vitro at micromolar concentrations. Selective blockade of the different metabolic pathways of arachidonic acid (e.g. ibuprofen for cyclooxygenase, SKF-525A for cytochrome P-450, baicalein and BHPP for 12-lipoxygenase, AA861 and MK886 for 5-lipoxygenase, etc.) revealed that the growth stimulatory effect of arachidonic acid is inhibited by the 5-lipoxygenase specific inhibitors, AA861 and MK886, but not by others. Addition of the eicosatetraenoid products of 5-lipoxygenase (5-HETEs) showed stimulation of prostate cancer cell growth similar to that of arachidonic acid, whereas the leukotrienes were ineffective. Moreover, the 5-series of eicosatetraenoids could reverse the growth inhibitory effect of MK886. Finally, prostate cancer cells fed with arachidonic acid showed a dramatic increase in the production of 5-HETEs which is effectively blocked by MK886. These experimental observations suggest that arachidonic acid needs to be metabolized through the 5-lipoxygenase pathway to produce 5-HETE series of eicosatetraenoids for its growth stimulatory effects on human prostate cancer cells.

Topics: Arachidonate 5-Lipoxygenase; Arachidonic Acid; Arachidonic Acids; Benzoquinones; Cell Division; Cyclooxygenase Inhibitors; Eicosanoids; Enzyme Inhibitors; Flavanones; Flavonoids; Humans; Hydroxyeicosatetraenoic Acids; Ibuprofen; Indoles; Lipoxygenase Inhibitors; Male; Masoprocol; Proadifen; Prostatic Neoplasms; Tumor Cells, Cultured

The newly described products of 5-hydroxyeicosanoid dehydrogenase, 5-oxo-6,8,11,14-eicosatetraenoic acid (ETE) and 5-oxo-15(OH)ETE, induced directional migration and actin polymerization of human monocytes in vitro. At peak concentrations, the two eicosanoids had a chemotactic activity of about 40% of that observed in the presence of an optimal concentration of FMLP and twice the activity elicited by the related eicosanoid 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE). 15-Oxo-ETE showed a very low but detectable chemotactic activity. All of these chemotactic responses were blocked by Bordetella pertussis toxin, but were resistant to LY255283, a leukotriene B4 (LTB4) receptor antagonist. 5-Oxo-ETEs and 5-HETE induced homologous desensitization of chemotactic response, but did not cross-desensitize to other chemotactic agonists (e.g., monocyte chemotactic protein (MCP)-1 and LTB4). 5-Oxo-ETEs increased in a synergistic fashion the monocyte migration to MCP-1 and MCP-3. In the same range of concentrations, 5-oxo-ETE increased MCP-1-induced release of arachidonic acid from labeled monocytes. No synergistic interaction was observed when FMLP was used as chemoattractant. Thus, this study identifies monocytes as cells responsive to 5-oxo-ETEs and shows that monocyte activation by 5-oxo-ETEs occurs through an LTB4 receptor-independent mechanism that associates with pertussis toxin-sensitive G proteins. The synergistic interaction between 5-oxo-ETEs and C-C chemokines, two families of mediators both synthesized by phagocytic cells, may be relevant in vivo for the regulation of monocyte accumulation at sites of allergic and inflammatory reactions.

Topics: Actins; Arachidonic Acids; Chemokine CCL2; Chemokine CCL7; Chemotactic Factors; Chemotaxis, Leukocyte; Cytokines; Drug Synergism; Humans; Hydroxyeicosatetraenoic Acids; Macrophage Activation; Monocyte Chemoattractant Proteins; Monocytes

Addition of submicromolar concentrations of arachidonic acid (AA) to human neutrophils induced a 2-fold increase in the activity of a cytosolic phospholipase A2 (PLA2) when measured using sonicated vesicles of 1-stearoyl-2-[14C]arachidonoylphosphatidylcholine as substrate. A similar increase in cytosolic PLA2 activity was induced by stimulation of neutrophils with leukotriene B4 (LTB4), 5-oxoeicosatetraenoic acid, or 5-hydroxyeicosatetraenoic acid (5-HETE). LTB4 was the most potent of the agonists, showing maximal effect at 1 nM. Inhibition of 5-lipoxygenase with either eicosatetraynoic acid or zileuton prevented the AA-induced increase in PLA2 activity but had no effect on the response induced by LTB4. Furthermore, pretreatment of neutrophils with a LTB4-receptor antagonist, LY 255283, blocked the AA- and LTB4-induced activation of PLA2 but did not influence the action of 5-HETE. Treatment of neutrophils with pancreatic PLA2 also induced an increase in the activity of the cytosolic PLA2; this response was inhibited by both eicosatetraynoic acid or LY 255283. The increases in PLA2 activity in response to stimulation correlated with a shift in electrophoretic mobility of the 85-kDa PLA2, as determined by Western blot analysis, suggesting that phosphorylation of the 85-kDa PLA2 likely underlies its increase in catalytic activity. Although stimulation of neutrophils with individual lipoxygenase metabolites did not induce significant mobilization of endogenous AA, they greatly enhanced the N-formylmethionyl-leucyl-phenylalanine-induced mobilization of AA as determined by mass spectrometry analysis. Our findings support a positive-feedback model in which stimulus-induced release of AA or exocytosis of secretory PLA2 modulate the activity of the cytosolic 85-kDa PLA2 by initiating the formation of LTB4. The nascent LTB4 is then released to act on the LTB4 receptor and thereby promote further activation of the 85-kDa PLA2. Since 5-HETE and LTB4 are known to prime the synthesis of platelet-activating factor, the findings suggest that 85-kDa PLA2 plays a role in platelet-activating factor synthesis.

Topics: 5,8,11,14-Eicosatetraynoic Acid; alpha-Linolenic Acid; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Arachidonic Acids; Cytosol; Enzyme Activation; Exocytosis; Humans; Hydroxyeicosatetraenoic Acids; Kinetics; Leukotriene B4; Molecular Weight; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Phospholipases A; Phospholipases A2; Tetrazoles

Human neutrophils and monocytes contain a highly specific dehydrogenase which converts 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) to 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE). We have previously shown that 5-oxo-ETE is a potent stimulus of neutrophil calcium levels and migration and have now investigated its effects on human eosinophils. 5-Oxo-ETE is a potent stimulus of eosinophil migration, with significant effects being detected at concentrations as low as 1 nM and a maximal response at 1 microM. The responses elicited by 5-oxo-ETE were about two to three times greater than those to platelet-activating factor (PAF) and 5-oxo-15-hydroxy-6,8,11,13-eicosatetraenoic acid (5-oxo-15-hydroxy-ETE) at all concentrations tested between 10 nM and 1 microM. Leukotrienes B4 and D4 also significantly stimulated eosinophil migration, but the maximal responses to these agonists were only about 4% of the maximal response to 5-oxo-ETE. A low concentration of 5-oxo-ETE (1 nM) potentiated eosinophil migration in response to PAF. Eosinophils were capable of converting 5-HETE to 5-oxo-ETE, and this reaction was enhanced by phorbol myristate acetate. Stimulation of eosinophils with A23187 in the presence of low concentrations of arachidonic acid and phorbol 12-myristate 13-acetate led to the formation of 5-oxo-ETE and 5-oxo-15-hydroxy-ETE, but the amounts were considerably less than those of other eicosanoids such as leukotriene C4, cysteine-containing lipoxins, and 5,15-dihydroxy-6E,8Z,11Z,13E-eicosatetraenoic acid. In summary, of all the lipid mediators tested, 5-oxo-ETE was the most effective in stimulating migration of human eosinophils. Although eosinophils are capable of synthesizing 5-oxo-eicosanoids, the amounts detected were relatively small, and other leukocytes such as neutrophils, monocytes, or macrophages may be more important sites for the synthesis of this compound.

Topics: Arachidonic Acid; Arachidonic Acids; Cell Movement; Chemotaxis, Leukocyte; Eosinophils; Glucuronidase; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Platelet Activating Factor; Tetradecanoylphorbol Acetate

Eicosatetraenoates (ETEs) with 5-oxo residues are known to induce human neutrophil (PMN) Ca2+ transients and chemotaxis. We find that 5-oxoETE, 5-oxo-8-trans-ETE, 5-oxo-15-hydroxy-ETE, 5-hydroxy-ETE, 5-hydroxy-15-oxoETE, 5,15-dioxoETE, and 5,15-dihydroxy-ETE have respective relative potencies of 10, 5, 3, 1, 0.2, 0.1, and 0.02 in: a) causing PMN to mobilize Ca2+, aggregate, and release small amounts of granule enzymes and b) promoting large degranulation and oxidative burst responses in PMN co-challenged with platelet-activating factor, tumor necrosis factor-alpha, or ATP. Contrastingly, 12(R)-hydroxy-ETE, 12(S)-hydroxy-ETE, and 12-oxoETE induced PMN Ca2+ transients and aggregation [respective potencies (5-hydroxy-ETE = 1) of 0.1, 0.01, and 0.003] but did not effect degranulation, and 15-hydroxy-ETE, 15-oxoETE, and 15-oxo-11-trans-ETE were inactive in all assays. Finally, 5-oxo/hydroxy-ETEs desensitized PMN to themselves but not to 12-oxo/hydroxy-ETEs or leukotriene (LT)B4; 12-oxo/hydroxy-ETEs and LTB4 desensitized PMN to themselves and each other but not to 5-oxo/hydroxy-ETEs; 15-oxo/hydroxy-ETEs did not desensitize PMN; and a LTB4 receptor antagonist blocked responses to LTB4 and 12-oxo/hydroxy-ETEs but not to 5-oxo/hydroxy-ETEs. Thus, 5-oxo/hydroxy-ETEs act by a common, LTB4 receptor-independent mechanism that recognizes 5- but not 12- or 15-oxo/hydroxy-ETEs and prefers oxo over hydroxy residues at C5 whereas 12-oxo/hydroxy-ETEs act via a LTB4 receptor mechanism that recognizes 12- but not 5- or 15-oxo/hydroxy-ETEs and prefers hydroxy over oxo residues at C12.

Topics: Arachidonic Acids; Isomerism

We have shown previously that human neutrophil microsomes contain a highly specific dehydrogenase which, in the presence of NADP+, converts 5S-hydroxy-6,8,11,14-eicosatetraenoic acid (5S-HETE) to its 5-oxo metabolite, 5-oxo-ETE, a potent agonist of these cells. However, intact neutrophils convert 5S-HETE principally to its omega-oxidation product, 5,20-diHETE, and to only small amounts of 5-oxo-ETE. Phorbol myristate acetate (PMA) dramatically shifts the metabolism of 5S-HETE by intact cells so that 5-oxo-ETE is the major metabolite. The objective of this investigation was to determine the mechanism for the stimulatory effect of PMA on 5-oxo-ETE formation. The possibility that oxidants released in response to PMA nonenzymatically oxidized 5S-HETE was ruled out, since PMA did not appreciably stimulate the formation of 5-oxo-ETE from 5R-HETE. On the other hand, inhibition of NADPH oxidase either by diphenylene iodonium or by mild heating nearly completely prevented the stimulatory effect of PMA on the formation of 5-oxo-ETE. The possibility that this effect was mediated by superoxide seems unlikely, since it was still observed, although somewhat attenuated, in the presence of superoxide dismutase. Moreover, superoxide generated by another mechanism (xanthine/xanthine oxidase) did not appreciably affect the formation of 5-oxo-ETE by neutrophils. However, phenazine methosulfate, which can nonenzymatically convert NADPH to NADP+, mimicked the effect of PMA on 5-oxo-ETE formation by intact neutrophils. It is concluded that PMA acts by activating NADPH oxidase, resulting in conversion of NADPH to NADP+, which enhances the formation of 5-oxo-ETE and reduces the formation of 5,20-diHETE. Serum-treated zymosan has an effect on the metabolism of 5S-HETE similar to that of PMA in that it also stimulates the formation of 5-oxo-ETE and inhibits that of 5,20-diHETE.

Topics: Arachidonic Acids; Azides; Dose-Response Relationship, Drug; Enzyme Activation; Humans; Hydroxyeicosatetraenoic Acids; Methionine; Microsomes; Models, Biological; NADH, NADPH Oxidoreductases; NADP; NADPH Oxidases; Neutrophils; Oxidation-Reduction; Prostaglandins B; Superoxides; Tetradecanoylphorbol Acetate; Zymosan

5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a newly discovered chemotactic agent for human polymorphonuclear leukocytes (PMNL) which has potent stimulatory effects on cytosolic calcium levels in these cells. Although we have shown that it is synthesized from 5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) by a highly specific microsomal dehydrogenase, little is known about the synthesis of this substance by intact PMNL. In the present study we found that in contrast to PMNL microsomes, intact, unstimulated PMNL produced relatively small amounts of 5-oxo-ETE from 5-HETE, but instead converted 5-HETE primarily to its omega-oxidation product, 5,20-diHETE. However, preincubation of PMNL with phorbol myristate acetate (PMA; EC50, ca. 4 nM) dramatically increased the ratio of 5-oxo-ETE to 5,20-diHETE from 0.07 in its absence to 1.85 in the presence of 100 nM PMA. Both effects were completely reversed by staurosporine, indicated that they were mediated by a protein kinase. PMA also stimulated the formation of 5-oxo-ETE, 5-HETE, and leukotriene B4 (LTB4) from exogenous arachidonic acid. The greatest enhancement was observed for 5-oxo-ETE, which, under all conditions, was produced in greater quantities than LTB4. PMA stimulated the formation of 5-oxo-ETE by PMNL stimulated with either A23187 or zymosan. A23187-stimulated PMNL initially produced more LTB4 than 5-oxo-ETE, but at longer time points, 5-oxo-ETE predominated. These results demonstrate that PMA-activated human PMNL can synthesize substantial amounts of 5-oxo-ETE and raise the possibility that this substance may be an important inflammatory mediator.

Topics: Arachidonic Acid; Arachidonic Acids; Chemotactic Factors; Chemotaxis, Leukocyte; Humans; Hydroxyeicosatetraenoic Acids; Neutrophils; Protein Kinase C; Tetradecanoylphorbol Acetate

We recently identified a novel pathway for the metabolism of 5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) by human neutrophils, resulting in oxidation of the 5-hydroxyl group to give 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) (Powell, W. S., Gravelle, F., and Gravel, S. (1992) J. Biol. Chem. 267, 19233-19241). This pathway is quite specific for 5-HETE and other eicosanoids containing a 5(S)-hydroxyl group followed by a 6-trans double bond. In the present study we have shown that 5-oxo-ETE is very potent in raising cytosolic calcium levels in human neutrophils. This effect was reproducibly observed at concentrations as low as 0.3 nM, and the EC50 was found to be 2 nM. The mechanism of action of 5-oxo-ETE on neutrophils appeared to be distinct from that of leukotriene B4 (LTB4), since it was not blocked by the LTB4 antagonist LY255283 at a concentration which completely prevented the response to LTB4. As would be expected for a receptor-mediated mechanism, the response to 5-oxo-ETE was subject to homologous desensitization and was completely abolished by prior treatment of neutrophils with 5-oxo-ETE (100 nM) but was not affected by pretreatment of these cells with the same concentration of LTB4. 5-Oxo-15(S)-hydroxy-6,8,11,13- eicosatetraenoic acid (5-oxo-15-hydroxy-ETE), formed from 5(S),15(S)-dihydroxy-6,8,11,13- eicosatetraenoic acid (5,15-di-HETE) by the pathway responsible for the formation of 5-oxo-ETE, also raised cytosolic calcium levels in human neutrophils, with an EC50 of about 15 nM. 5-HETE, the precursor of 5-oxo-ETE, also had this effect but was about 100 times less potent than the latter substance. Desensitization experiments indicated that both 5-oxo-15-hydroxy-ETE and 5-HETE act by a mechanism similar to that of 5-oxo-ETE, but different from that of LTB4. In addition to their effects on calcium levels, both 5-oxo-ETE and 5-oxo-15-hydroxy-ETE had chemotactic effects on human neutrophils. Related eicosanoids, including 15-oxo-5,8,11,13-eicosatetraenoic acid, 5,15-diHETE, and 5(S)-hydroxy-15-oxo-6,8,11,13-eicosatetraenoic acid were much less potent, as both chemotactic and calcium-mobilizing agents. These results suggest that neutrophils possess a specific recognition mechanism for 5-oxo-ETE, which may be an important regulator of the activity of neutrophils, especially if they become desensitized to LTB4.

Topics: Arachidonic Acids; Calcium; Cytosol; Eicosanoids; Humans; In Vitro Techniques; Kinetics; Leukotriene B4; Neutrophils; Receptors, Immunologic; Receptors, Leukotriene B4; Tetrazoles; Time Factors

5-Oxo-eicosatetraenoate (5-oxoETE), a newly defined arachidonate metabolite, resembled 5-hydroxyeicosatetraenoate (5-HETE) in stimulating neutrophils to mobilize Ca2+ an in promoting PMN degranulation responses to other agents. It was, however, 10-fold stronger than 5-HETE and, like leukotriene (LT) B4, had intrinsic PMN degranulating effects. Nonetheless, 5-oxoETE and 5-HETE desensitized PMN to themselves or each other but not to LTB4; LTB4 desensitized to itself but not to 5-oxoETE or 5-HETE; and an antagonist blocked LTB4 but not 5-oxoETE or 5-HETE. 5-OxoETE and 5-HETE thus induce diverse PMN responses using a shared, down-regulatable, and receptor-like mechanism that does not involve LTB4 receptors; 5-oxoETE is the preferred natural agonist for this mechanism.

Topics: Arachidonic Acids; Calcium; Cell Degranulation; Cells, Cultured; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Leukotriene B4; Neutrophils; Tetrazoles