Lipoxygenase Metabolism Of Arachidonic Acid Research Articles

Leukotriene receptors in GtoPdb v.2023.1

The leukotriene receptors (nomenclature as agreed by the NC-IUPHAR subcommittee on Leukotriene Receptors [35, 38]) are activated by the endogenous ligands leukotrienes (LT), synthesized from lipoxygenase metabolism of arachidonic acid. The human BLT1 receptor is the high affinity LTB4 receptor whereas the BLT2 receptor in addition to being a low-affinity LTB4 receptor also binds several other lipoxygenase-products, such as 12S-HETE, 12S-HPETE, 15S-HETE, and the thromboxane synthase product 12-hydroxyheptadecatrienoic acid. The BLT receptors mediate chemotaxis and immunomodulation in several leukocyte populations and are in addition expressed on non-myeloid cells, such as vascular smooth muscle and endothelial cells. In addition to BLT receptors, LTB4 has been reported to bind to the peroxisome proliferator activated receptor (PPAR) α [201] and the vanilloid TRPV1 ligand-gated nonselective cation channel [223]. The crystal structure of the BLT1 receptor was initially determined in complex with selective antagonists [141, 231] and has recently been extended to the cryo-electron microscopy structure of LTB4-bound human BLT1 receptor at 2.91 Å resolution [389]. The receptors for the cysteinyl-leukotrienes (i.e. LTC4, LTD4 and LTE4) are termed CysLT1 and CysLT2 and exhibit distinct expression patterns in human tissues, mediating for example smooth muscle cell contraction, regulation of vascular permeability, and leukocyte activation. Quite recently, the the crystal structures of both receptors have been solved, the CysLT1 in complex with zafirlukast and pranlukast [203] and the CysLT2 in complex with three dual CysLT1/CysLT2 antagonists [122]. There is also evidence in the literature for additional CysLT receptor subtypes, derived from functional in vitro studies, radioligand binding and in mice lacking both CysLT1 and CysLT2 receptors [38]. Cysteinyl-leukotrienes have also been suggested to signal through the P2Y12 receptor [99, 251, 280], GPR17 [60] and GPR99 [173].

CHARACTERISTICS AND THERAPEUTIC EFFECTS OF THE NOVEL FREE RADICAL SCAVENGER – EDARAVONE

Edaravone is the first free radical scavenger which approved clinically and has an ability to decrease the level of free radicals in cells. Edaravone is a strong antioxidant, which can protect different cells (e.g. endothelial cells) against damage by ROS by inhibiting the lipoxygenase metabolism of arachidonic acid, by trapping hydroxyl radicals, by increasing prostacyclin production, by inhibiting alloxan-induced lipid peroxidation, etc. Because of that, Edaravone is used in treatment of diseases which are associated with oxidative stress. Key words: edaravone, free radical, antioxidant, neuroprotective agent, oxidative stress

Leukotriene receptors (version 2020.3) in the IUPHAR/BPS Guide to Pharmacology Database

The leukotriene receptors (nomenclature as agreed by the NC-IUPHAR subcommittee on Leukotriene Receptors [34, 37]) are activated by the endogenous ligands leukotrienes (LT), synthesized from lipoxygenase metabolism of arachidonic acid. The human BLT1 receptor is the high affinity LTB4 receptor whereas the BLT2 receptor in addition to being a low-affinity LTB4 receptor also binds several other lipoxygenase-products, such as 12S-HETE, 12S-HPETE, 15S-HETE, and the thromboxane synthase product 12-hydroxyheptadecatrienoic acid. The BLT receptors mediate chemotaxis and immunomodulation in several leukocyte populations and are in addition expressed on non-myeloid cells, such as vascular smooth muscle and endothelial cells. In addition to BLT receptors, LTB4 has been reported to bind to the peroxisome proliferator activated receptor (PPAR) α [196] and the vanilloid TRPV1 ligand-gated nonselective cation channel [217]. The receptors for the cysteinyl-leukotrienes (i.e. LTC4, LTD4 and LTE4) are termed CysLT1 and CysLT2 and exhibit distinct expression patterns in human tissues, mediating for example smooth muscle cell contraction, regulation of vascular permeability, and leukocyte activation. There is also evidence in the literature for additional CysLT receptor subtypes, derived from functional in vitro studies, radioligand binding and in mice lacking both CysLT1 and CysLT2 receptors [37]. Cysteinyl-leukotrienes have also been suggested to signal through the P2Y12 receptor [96, 243, 272], GPR17 [57] and GPR99 [168].

Leukotriene receptors (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

The leukotriene receptors (nomenclature as agreed by the NC-IUPHAR subcommittee on Leukotriene Receptors [31, 34]) are activated by the endogenous ligands leukotrienes (LT), synthesized from lipoxygenase metabolism of arachidonic acid. The human BLT1 receptor is the high affinity LTB4 receptor whereas the BLT2 receptor in addition to being a low-affinity LTB4 receptor also binds several other lipoxygenase-products, such as 12S-HETE, 12S-HPETE, 15S-HETE, and the thromboxane synthase product 12-hydroxyheptadecatrienoic acid. The BLT receptors mediate chemotaxis and immunomodulation in several leukocyte populations and are in addition expressed on non-myeloid cells, such as vascular smooth muscle and endothelial cells. In addition to BLT receptors, LTB4 has been reported to bind to the peroxisome proliferator activated receptor (PPAR) α [189] and the vanilloid TRPV1 ligand-gated nonselective cation channel [210]. The receptors for the cysteinyl-leukotrienes (i.e. LTC4, LTD4 and LTE4) are termed CysLT1 and CysLT2 and exhibit distinct expression patterns in human tissues, mediating for example smooth muscle cell contraction, regulation of vascular permeability, and leukocyte activation. There is also evidence in the literature for additional CysLT receptor subtypes, derived from functional in vitro studies, radioligand binding and in mice lacking both CysLT1 and CysLT2 receptors [34]. Cysteinyl-leukotrienes have also been suggested to signal through the P2Y12 receptor [91, 236, 265], GPR17 [53] and GPR99 [161].

Investigation of new combined cream Dermalipoin effect on the vascular wall permeability

Topicality. The problem of treating wounds, including burns, remains relevant in modern medicine. For today in the arsenal of doctors there is a wide choice of medicines for treatment of wound process, however none of them is universal, and their effects are far from optimal. This makes it necessary to search for and develop new drugs for pharmacotherapy of the wound process. Aim. To study the capillary-firming activity of new cream on the model of increased permeability of the vessels of the abdominal wall in rats caused by various phlogogens. Materials and methods. With the purpose of in-depth study of the effect of the cream on the exudative component of the inflammatory reaction, experiments were conducted to determine its effect on the permeability of the vessels of the abdominal wall, which was caused by the administration of solutions of histamine, formalin, carrageenan, egg protein. By the difference in time of staining of the papules, after the introduction of the dye, the conclusion was made about the effect of the cream on vascular permeability. Results and discussion. The presence of new vascular stabilizing action in the new combined cream has been established due to the stabilization of the cell membranes and suppression of the activity of the inflammatory mediators that are formed as a result of cyclo- and lipoxygenase metabolism of arachidonic acid and, thereby, reduce the permeability of the vascular wall, which is an important component of the pharmacodynamics of the investigational pharmacological preparation, which is proposed for inflammatory-destructive processes. Conclusions. The ability of the test cream to reduce vascular permeability when introducing various phlogogenic substances indicates a multicomponent anti-inflammatory effect, which is associated with the antioxidant and membrane-stabilizing properties of the active substances of the cream.

Abstract 1867: Application of oxylipin metabolomics to a celecoxib intervention in men at risk for colorectal neoplasia recurrence

Abstract Background. In randomized controlled trials, non-steroidal anti-inflammatory agents (NSAIDs) have been shown to prevent the development of colorectal adenoma (CRA) by 15-40% depending on the agent, dose and duration of exposure. Toxicity concerns limit the acceptability of these agents for general use in the population, particularly for COX2 selective agents such as celecoxib. COX2 metabolizes arachidonic acid (AA) to the pro-inflammatory prostaglandins (PGs); an established pathway in CRA development. AA is also metabolized by cytochrome P450 (CYP450) and lipoxygenase (LOX) enzymes to produce other biologically active metabolites called oxylipins that mediate pain, hypertension, and other physiologic functions. Methods. In 2001, we initiated a randomized phase III clinical trial to evaluate the chemopreventive effect of the COX2 inhibitor celecoxib in individuals at risk for CRA. In a subset of participants (N=126 men and N=59 women) who received celecoxib or placebo for a minimum of 12 months, we performed plasma oxylipin profiling analysis using HPLC coupled to a 4000 QTRAP mass spectrometer (MS). Fasting plasma samples for this ancillary study were collected at the one-year clinic visit and immediately stored at -80C at collection. These samples have never undergone prior freeze-thaw. Once thawed, triphenylphosphine (TPP) and butylated hydroxytoluene (BHT) (0.2% w/w) were added to preserve the oxylipins. Plasma samples (250 µL) were subjected to solid phase extraction before analysis as described previously (Liu et al 2009). Results. Using HPLC-MS, we were able to separate and quantify 89 oxylipins (CV's <30%). On average, the levels of the COX2 metabolite and primary target of the intervention, prostaglandin E2 (PGE2), were non-significantly lower (23%) in the celecoxib treated individuals when compared to placebo (P=0.20). The 5-LOX metabolite, 5-HETE (inducer of pulmonary vasoconstriction and edema) was higher in the celecoxib group (P=0.033) whereas CYP450 metabolites: 8,9-DiHETrE (P=0.018), 11,12-DiHETrE (P=0.053), 16,17-DiHDPE (P=0.059) and 10,11-DiHDPE (P=0.040) - associated with hypertension - were higher in the celecoxib treated individuals when compared to placebo. Conclusions. Upregulation of COX2 (and subsequent production of pro-inflammatory PGE2) is a well-established mechanism in the pathogenesis of colorectal cancer, and thus an attractive chemoprevention target. Our results suggest that CYP450 and LOX metabolites increase in celecoxib treated subjects, when compared to placebo, while achieving only modest inhibition of COX2 dependent PGE2. These findings suggest that selective perturbation of the AA metabolic pathway may induce the P450 and LOX metabolism of AA resulting in increased levels of potentially harmful oxylipins. Preliminary analyses of the relationship between oxylipins, CRA and toxicity outcomes in this sub-study by treatment status will be presented. Citation Format: Jessica Anne Miller, Jun Yang, Bruce Hammock, Peter Lance, Erin Ashbeck, Patricia A. Thompson. Application of oxylipin metabolomics to a celecoxib intervention in men at risk for colorectal neoplasia recurrence. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1867. doi:10.1158/1538-7445.AM2014-1867

Prevention of hypertension in DOCA-salt rats by an inhibitor of soluble expoxide hydrolase

Cyclooxygenase and lipoxygenase metabolism of arachidonic acid produces compounds important in cardiovascular control. Further, arachidonic acid can be metabolised by cytochrome p450 to produce epoxyeicosatrienoic acids (EETs). These derivatives are inactivated by soluble epoxide hydrolase (sEH). The potential role of these EETs in hypertension and cardiac remodelling has been determined using the selective sEH inhibitor, N-adamantyl-N'-dodecylurea (ADU), in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Experiments were performed on male Wistar rats following uninephrectomy alone (UNX rats) or uninephrectomy with administration of DOCA (25 mg every fourth day subcutaneously) and 1% NaCl in drinking water (DOCA-salt rats). ADU (10 mg/kg/d subcutaneously) was administered for 2 wk starting 2 wk after surgery. Cardiovascular structure and function were determined using organ wet weights, histological analysis of collagen and inflammation, isolated heart and thoracic aortic ring preparations, and electrophysiological measurements. DOCA-salt hypertensive rats developed hypertension, hypertrophy, perivascular and interstitial fibrosis, endothelial dysfunction, and prolongation of the cardiac action potential duration within 4 wk. Administration of ADU prevented the further increase in systolic blood pressure and left-ventricular wet weight and normalized endothelial function. ADU treatment did not change inflammatory cell infiltration, collagen deposition, or cardiac action potential duration. EETs may be involved in the development of hypertension and endothelial dysfunction in DOCA-salt rats, but not in excessive collagen deposition or electrophysiological abnormalities.

Edaravone (3-Methyl-1-Phenyl-2-Pyrazolin-5-one), A Novel Free Radical Scavenger, for Treatment of Cardiovascular Diseases

Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one), a strong novel free radical scavenger, is used for treatment of patients with acute brain infarction. Edaravone has preventive effects on myocardial injury following ischemia and reperfusion in patients with acute myocardial infarction. Antioxidant actions of edaravone include enhancement of prostacyclin production, inhibition of lipoxygenase metabolism of arachidonic acid by trapping hydroxyl radicals, inhibition of alloxan-induced lipid peroxidation, and quenching of active oxygen, leading to protection of various cells, such as endothelial cells, against damage by reactive oxygen species (ROS). Recently, we have shown that edaravone improves endothelial function through a decrease in ROS in smokers. From a clinical perspective, it is important to select an appropriate drug that is effective in improving endothelial function in patients with cardiovascular diseases. The novel free radical scavenger edaravone may represent a new therapeutic intervention for endothelial dysfunction in the setting of atherosclerosis, chronic heart failure, diabetes mellitus, or hypertension. This review focuses on clinical findings and on putative mechanisms underlying the beneficial effects of the antioxidative agent edaravone on the artherosclerotic process in patients with cardiovascular diseases.

Leukotriene B 4 signaling through NF-κB-dependent BLT 1 receptors on vascular smooth muscle cells in atherosclerosis and intimal hyperplasia

Leukotriene B(4) (LTB(4)), a potent leukocyte chemoattractant derived from the 5-lipoxygenase metabolism of arachidonic acid, exerts its action by means of specific cell surface receptors, denoted BLT(1) and BLT(2). In this study, BLT(1) receptor proteins were detected in human carotid artery atherosclerotic plaques, colocalizing with markers for macrophages, endothelial cells, and vascular smooth muscle cells (SMC). Challenge of human coronary artery SMC with either LTB(4) or U75302, a partial agonist that is selective for the BLT(1) receptor, induced an approximately 4-fold increase of whole-cell currents by using the patch-clamp technique, indicating that these cells express functional BLT(1) receptors. LTB(4) induced migration and proliferation of SMC in vitro, and treatment with the BLT receptor antagonist BIIL 284 (10 mg/kg, once daily) for 14 days after carotid artery balloon injury in vivo inhibited intimal hyperplasia in rats. In the latter model, SMC derived from the intima exhibited increased levels of BLT(1) receptor mRNA compared with medial SMC. BLT receptor up-regulation in the intima in vivo, as well as that induced by IL-1beta in vitro, were prevented by transfection with a dominant-negative form of Ikappa kinase beta carried by adenovirus, indicating that BLT(1) receptor expression depends on NF-kappaBeta. These results show that LTB(4) activates functional BLT(1) receptors on vascular SMC, inducing chemotaxis and proliferation, and that BLT(1) receptors were up-regulated through an Ikappa kinase beta/NF-kappaB-dependent pathway. Inhibition of LTB(4)/BLT(1) signaling during the response to vascular injury reduced intimal hyperplasia, suggesting this pathway as a possible target for therapy.

15(S)-Lipoxygenase-2 Mediates Arachidonic Acid-stimulated Adhesion of Human Breast Carcinoma Cells through the Activation of TAK1, MKK6, and p38 MAPK

The dietary cis-polyunsaturated fatty acid, arachidonic acid, stimulates adhesion of metastatic human breast carcinoma cells (MDA-MB-435) to the extracellular matrix, but the molecular mechanisms by which fatty acids modify the behavior of these cells are unclear. Exposure to arachidonic acid activates multiple signaling pathways. Activation of p38 mitogen-activated protein kinase (p38 MAPK) is required for increased cell adhesion to type IV collagen, and this activation is sensitive to inhibitors of lipoxygenases, suggesting a requirement for arachidonic acid metabolism. The goals of the current study were to identify the one or more key metabolites of arachidonic acid that are responsible for activation of p38 MAPK and to elucidate the upstream kinases that lead to p38 MAPK activation. High performance liquid chromatographic analysis revealed that MDA-MB-435 cells metabolize exogenous arachidonic acid predominantly to 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE). Immunoblot analysis with antibodies specific to 15(S)-lipoxygenase-1 (LOX-1) and 15(S)-lipoxygenase-2 (LOX-2) demonstrated the expression of 15-LOX-2, but not 15-LOX-1, in these tumor cells. A LOX inhibitor, nordihydroguaiaretic acid, attenuated production of 15(S)-HETE and inhibited the phosphorylation of p38 MAPK following exposure to arachidonic acid. In contrast, overexpression of LOX-2 sensitized the cells to the addition of arachidonic acid, leading to increased activation of p38 MAPK. Addition of exogenous 15(S)-HETE to MDA-MB-435 cells stimulated cell adhesion to type IV collagen and activated the p38 MAPK pathway, including the upstream kinases transforming growth factor-beta1-activated protein kinase-1 (TAK1) and MAPK kinase 6. Transfection of these cells with a dominant negative form of TAK1 blocked arachidonic acid-stimulated p38 MAPK phosphorylation. These data demonstrate that 15(S)-LOX-2 generation of 15(S)-HETE activates specific growth factor receptor-related signaling pathways, thereby initiating signal transduction events leading to increased cell adhesion to the extracellular matrix.

The role of leukotrienes in allergic rhinitis

To review the role of cysteinyl leukotrienes (cysLTs) in allergic rhinitis and the scientific rationale for therapy with leukotriene receptor antagonists (LTRAs). Relevant basic science and clinical articles were identified by a search of the PubMed database for articles published from 1984 to 2004 using the following keywords: allergic rhinitis; nose; immune response; allergen challenge; leukotrienes C, D, and E; cysteinyl leukotriene; cysteinyl leukotriene receptor; cytokine; leukocyte; montelukast; zafirlukast; and pranlukast. The authors' expert opinion was used to select studies for inclusion in this review. CysLTs are synthesized via 5-lipoxygenase metabolism of arachidonic acid by mast cells and basophils during the early-phase response to antigen and by eosinophils and macrophages during the late phase. The cysLT levels in nasal secretions are elevated after short-term allergen instillation and in allergy season in patients with allergic rhinitis. These lipid mediators act locally and systemically by interacting with receptors, particularly the cysLT1 receptor, on target cells. Evidence derived from topical application of cysLTs in the nose and from the effects of LTRAs indicates that cysLTs contribute to nasal mucous secretion, congestion, and inflammation. CysLTs promote allergic inflammation by enhancing immune responses and the production, adhesion, migration, and survival of inflammatory cells such as eosinophils. They also increase the generation of an array of other proinflammatory mediators, such as cytokines, which in turn increase the production of and receptors for cysLTs. Clinical trials have demonstrated that LTRAs have significant but modest efficacy as single agents but additive efficacy when used with other classes of agents. CysLTs fulfill the criteria for relevant mediators of allergic rhinitis via their diverse effects on immune, inflammatory, and local structural components of disease. By blocking the cysLT1 receptor responsible for most of these effects, LTRAs represent a useful approach to treatment of this important and prevalent disorder.

Stabilization of Leukotriene A4 by Epithelial Fatty Acid-binding Protein in the Rat Basophilic Leukemia Cell

Leukotriene A(4) (LTA(4)) is a chemically unstable triene epoxide product of 5-lipoxygenase metabolism of arachidonic acid. Despite this chemical reactivity and its synthesis at the perinuclear membrane, LTA(4) is enzymatically converted into the cysteinyl leukotrienes and leukotriene B(4). Furthermore, LTA(4) participates in transcellular biosynthesis and is thus transferred between cells as an intact molecule. A cytosolic fatty acid-binding protein present in the rat basophilic leukemia cells was identified using mass spectrometry. This protein was determined to be the stabilizing factor present in the cell cytosol responsible for increasing the effective chemical half-life of LTA(4). Rat epithelial fatty acid-binding protein (E-FABP) was isolated using partial protein purification and immunoprecipitation. In-gel digestion with trypsin followed by peptide fingerprint analysis using matrix-assisted laser desorption ionization mass spectrometry and sequencing the major tryptic peptide obtained from liquid chromatography/mass spectrometry/mass spectrometry analysis identified E-FABP in the active fraction. Semi-quantitative Western blot analysis indicated that E-FABP in the cytosolic fraction of RBL-1 cells was present at approximately 1-3 pmol/10(6) cells. E-FABP (9 microm) was tested for its ability to stabilize LTA(4), and at 37 degrees C E-FABP was able to increase the half-life of LTA(4) from the previously reported half-life less than 3 s to a half-life of approximately 7 min. These results present a novel function for the well studied fatty acid-binding protein as a participant in leukotriene biosynthesis that permits LTA(4) to be available for further enzymatic processing in various cellular regions.

COX‐LOX INHIBITION: CURRENT EVIDENCE FOR AN EMERGING NEW THERAPY

SUMMARYSafe and effective drug treatment is an important objective of all doctors. In the treatment of arthritis, non‐steroidal anti‐inflammatory drugs offer effective treatment but safety is significantly limited, largely due to gastrointestinal toxicity. Attention has recently focused on exploiting increased knowledge of metabolism of arachidonic acid to allow the development of safer anti‐inflammatory drugs. Dual inhibitors of cyclo‐oxygenase and lipoxygenase are planned. These drugs may inhibit formation of both prostaglandins and leukotrienes. This review outlines the salient features of cyclo‐oxygenase and lipoxygenase metabolism of arachidonic acid. The role of the eicosanoids in mediating inflammation and gastrointestinal integrity is delineated. Evidence is presented regarding action of licofelone, one COX/LOX inhibitor that is currently in advanced stages of clinical trials. This review examines the hypothesis that licofelone is an effective anti‐inflammatory agent that does not cause peptic damage.

Lipoxygenase metabolism of arachidonic acid in ischemic preconditioning and PKC-induced protection in heart.

We tested the hypothesis that activation of the 12-lipoxygenase (12-LO) pathway of arachidonic acid metabolism contributes to the protective effect of protein kinase C (PKC) activation and ischemic preconditioning (PC), and we report, in perfused rat heart, that both PC and the PKC activator 1,2-dioctanoyl-sn-glycerol (DOG) confer a similar protective effect and stimulate a comparable accumulation of 12-LO metabolites. The 12-LO product, 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], was increased in DOG-treated (22.8 +/- 4.4 ng/g wet wt) and PC hearts (26.8 +/- 5.5 ng/g wet wt) compared with control (13.8 +/- 2.1 ng/g wet wt, P < 0. 05), and this increase was blocked by 12-LO or PKC inhibitors. Both DOG pretreatment and PC improved recovery of left ventricular developed pressure (LVDP) nearly twofold after 20 min of ischemia; this improvement was blocked by 12-LO inhibitors and was mimicked by infusion of 12-hydroperoxyeicosatetraenoic acid [12(S)-HpETE; 67 +/- 6% recovery of LVDP vs. 35 +/- 3% for untreated hearts]. Also, the protection afforded by 12(S)-HpETE, as well as by PC, was attenuated by the K+-channel blocker 5-hydroxydecanoate, suggesting that the downstream mechanisms of 12(S)-HpETE-mediated protection are similar to PC. Furthermore, PC stimulates 12-LO metabolism in perfused rabbit heart, and 12-LO inhibition blocks PC-induced cardioprotection. Thus the data suggest that 12-LO metabolism plays an important role in cardioprotection.

Discovery of a second 15S-lipoxygenase in humans.

The lipoxygenase metabolism of arachidonic acid occurs in specific blood cell types and epithelial tissues and is activated in inflammation and tissue injury. In the course of studying lipoxygenase expression in human skin, we detected and characterized a previously unrecognized enzyme that at least partly accounts for the 15S-lipoxygenase metabolism of arachidonic acid in certain epithelial tissues. The cDNA was cloned from human hair roots, and expression of the mRNA was detected also in prostate, lung, and cornea; an additional 16 human tissues, including peripheral blood leukocytes, were negative for the mRNA. The cDNA encodes a protein of 676 amino acids with a calculated molecular mass of 76 kDa. The amino acid sequence has approximately 40% identity to the known human 5S-, 12S-, and 15S-lipoxygenases. When expressed in HEK 293 cells, the newly discovered enzyme converts arachidonic acid exclusively to 15S-hydroperoxyeicosatetraenoic acid, while linoleic acid is less well metabolized. These features contrast with the previously reported 15S-lipoxygenase, which oxygenates arachidonic acid mainly at C-15, but also partly at C-12, and for which linoleic acid is an excellent substrate. The different catalytic activities and tissue distribution suggest a distinct function for the new enzyme compared with the previously reported human 15S-lipoxygenase.

Release of Leukotriene A4Versus Leukotriene B4 from Human Polymorphonuclear Leukocytes

The reactive intermediate formed by 5-lipoxygenase metabolism of arachidonic acid, leukotriene A4, is known to be released from cells and subsequently taken up by other cells for biochemical processing. The objective of this study was to determine the relative amount of leukotriene A4 synthesized by human polymorphonuclear leukocytes (PMNL) that is available for transcellular biosynthetic processes. This was accomplished by diluting cell suspensions and measuring the relative amounts of enzymatic versus nonenzymatic leukotriene A4-derived metabolites after challenge with the Ca2+ ionophore A23187. Nonenzymatic leukotriene A4-derived metabolites were used as a quantitative index of the amount of leukotriene A4 released into the extracellular milieu. The results obtained demonstrated that in human PMNL, the relative amounts of nonenzymatic versus enzymatic leukotriene A4-derived metabolites increased with decreasing cell concentrations. After a 20-fold dilution of PMNL in cell preparations, a doubling in the amount of nonenzymatic leukotriene A4-derived metabolites was observed following challenge (from 53.9 +/- 1.3 to 110.4 +/- 8.9 pmol/10(6) PMNL, p < 0.01). Reduction of possible cell-cell interactions by dilution suggested that over 50% of leukotriene A4 synthesized is released from the PMNL. These data provide evidence that, in human PMNL preparations, transfer of leukotriene A4 to neighboring PMNL is taking place, resulting in additional formation of leukotriene B4 and its omega-oxidized metabolites 20-hydroxy- and 20-carboxy-leukotriene B4. Neutrophil reuptake of extracellular leukotriene A4 leads to an underestimation of the fraction of leukotriene A4 that is in fact available for transcellular metabolism when tight cell-cell interactions occur, such as during PMNL adhesion to the microvascular endothelium and diapedesis.

Ca 2+] Modulates the ratio between cycloxygenase and lipoxygenase metabolism of arachidonic acid in homogenates of hippocampal astroglial cultures

While studying the enzymatic processing of arachidonic acid (AA) to eicosanoids in homogenates of hippocampal astrocytes, we observed that all the HPLC peaks corresponding to AA metabolites displayed significantly different levels depending on the presence or not of free Ca 2+ in the incubation medium. A specific pattern was noticed, i.e. lipoxygenase (LOX) derivatives, in particular 12-hydroxyeicosatetraenoic acid (12-HETE), showed higher levels in medium containing 1 mM Ca 2+, while cycloxygenase (COX) products including prostaglandins (PG) F 2α, E 2 and D 2 12-hydroxyheptadecatrienoic acid (12-HHT), were higher in Ca 2+-free medium. COX metabolism exceeded LOX metabolism by threefold in Ca 2+-free medium, while it was only 60% of it in 1 mM Ca 2+. The total amount of AA processed under the two conditions was identical. These data suggest that free [Ca 2+] influences the pattern of AA metabolites formed in hippocampal astrocytes, with possible important implications in view of the distinct roles played by COX and LOX eicosanoids in synaptic transmission and neurotoxicity in this area.

Inhibition of Radiation-Enhanced Expression of Integrin and Metastatic Potential in B16 Melanoma Cells by a Lipoxygenase Inhibitor

Low-dose gamma radiation stimulates expression of phenotypic characteristics in B16 melanoma cells which regulate metastatic potential. A transient increase in the expression of an integrin receptor (alpha IIb beta 3) was observed after exposure of B16 melanoma cells to 0.25 to 2.0 Gy of gamma radiation. This increased receptor expression resulted in enhanced adhesion of tumor cells to fibronectin in vitro and increased experimentally induced metastasis in vivo. In this report, we determined a role for the 12-lipoxygenase metabolite, 12-HETE, in radiation-enhanced metastasis. A significant increase in biosynthesis of 12-HETE in B16 melanoma cells was detected < 5 min after exposure to 0.5 Gy gamma radiation. We then determined that radiation-enhanced expression of alpha IIb beta 3 integrin and adhesion of B16 melanoma cells to fibronectin in vitro and metastasis in vivo were reduced by treatment of the cells with the lipoxygenase inhibitor NDGA prior to irradiation. These findings suggest that low-dose radiation, at levels comparable to those used in fractionated or hyper-fractionated radiotherapy, increases the metastatic potential of surviving tumor cells via a rapid and transient alteration in lipoxygenase metabolism of arachidonic acid and surface expression of an integrin receptor.

Reduced 5-lipoxygenase metabolism of arachidonic acid in macrophages rrom 1,25-dihydroxyvitamin D 3-deficient rats

The peripheral blood monocyte (PBM) migrates into tissues and differentiates into mature tissue macrophages. Previous investigations from our laboratory have demonstrated that PBM have reduced 5-lipoxygenase (5-LO) metabolism of arachidonic acid (AA) and 5-LO activating protein (FLAP) expression as compared to differentiated alveolar macrophages (AM). Moreover, PBM differentiated with 1,25-dihydroxyvitamin D 3 (1,25-(OH) 2D 3) displayed increased leukotriene synthesis and a parallel increase in FLAP expression. In the present study, we sought to examine the physiological role of 1,25-(OH) 2D 3 in the regulation of eicosanoid metabolism in terminally differentiated alveolar and peritoneal macrophages (PM), utilizing a well characterized rat model of vitamin D 3-deficiency. AM from vitamin D 3-deficient rats demonstrated reduced 5-LO metabolism of AA and a parallel reduction in FLAP expression compared to control rats. Similarly, PM from vitamin D 3-deficient rats demonstrated reduced 5-LO metabolism of AA. The effect of vitamin D 3 was specific for the 5-LO pathway, not affecting total release of AA or its metabolism via 12-lipoxygenase or cyclooxoygenase (COX) pathways in macrophages. Furthermore, it did not affect COX protein expression in macrophages or type II alveolar epithelial cells. In control animals, 1,25-(OH) 2D 3 concentrations were greater in bronchoalveolar lavage fluid (2.,6-fold) and peritoneal lavage fluid (1.6-fold) than in serum, which may account for the greater FLAP expression in AM and PM than in PBM. These observations suggest that 1,25-(OH) 2D 3 plays a physiological role in upregulating the 5-LO pathway in tissue macrophages in vivo.

Eicosanoids in the Phytophthora infestans-potato interaction: lipoxygenase metabolism of arachidonic acid and biological activities of selected lipoxygenase products

Potato tuber and leaf discs were treated with the fungal elicitor arachidonic acid. Hydroperoxy-eicosatetraenoic acids (HpETEs), produced by reaction of lipoxygenase with arachidonic acid, were then extracted from the tissues and analysed. 5-, 8-, 11-, 12-, and 15-HpETE were formed rapidly by both leaf and tuber tissues. 5-HpETE was the predominant lipoxygenase product in treated tuber discs, whereas 12- and 15-HpETE were the principal products produced by leaf discs. HpETEs were formed in tuber discs within 10 min after addition of the elicitor and increased during the first 5 h after treatment. The quantity of HpETEs, however, was inversely related to the amount of arachidonic acid initially applied to the discs and to the sesquiterpene phytoalexin levels induced. Treatments that enhance ( β -glucans from Phytophthora infestans ) or inhibit (abscisic acid) elicitor activity generally did not have a corresponding effect on the levels of HpETEs formed in tuber tissues, suggesting that these treatments primarily affect the response at sites different from the initial lipoxygenase reaction with arachidonic acid. Eicosatetraynoic acid, an acetylenic analogue of arachidonic acid and irreversible inhibitor of lipoxygenases, suppressed the elicitor-induced hypersensitive response in tuber discs and inhibited formation of HpETEs from arachidonic acid by microsomes from tuber discs. A collection of purified lipoxygenase products of arachidonic acid were tested for elicitor activity in potato and fungitoxicity to P. infestans and P. capsici . None of these possessed elicitor activity but several HpETEs and hydroxy-acids were inhibitory to cystospore germination. The results are discussed in relation to the proposed role of lipoxygenase and lipoxygenase products of arachidonic acid in hypersensitivity expression and disease resistance in potato.