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

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Calcium; Diglycerides; Hydroxyeicosatetraenoic Acids; Inositol Phosphates; Islets of Langerhans; Models, Biological; Phosphatidic Acids; Phospholipids; Prostaglandins

The 2 hemiketal (HK) eicosanoids HKD

Topics: Arachidonate 5-Lipoxygenase; Arachidonic Acid; Blood Platelets; Calcimycin; Cyclooxygenase 2; Dinoprostone; Humans; Hydroxyeicosatetraenoic Acids; Leukocytes; Prostaglandin D2

As a Chinese traditional herbal medicine, leaves of Platycladus orientalis (Linnaeus) Franco (LPO) are used to treat coughs, excessive mucus secretion, chronic bronchitis, bronchiectasis, and asthma, etc. The experiments were carried out to investigate their anti-inflammatory properties and mechanisms, which could support the Chinese traditional uses of treating inflammatory airway diseases.. The anti-inflammatory activities of the chloroform fraction (CHL) and pure compounds of LPO were evaluated for their abilities to inhibit pro-inflammatory enzymes in vitro, and production of tumor necrosis factor-α (TNF-α) and nitric oxide in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Furthermore, the arachidonic acid metabolites, stimulated by calcium ionophore A23187, were also determined by HPLC.. For the first time, the assays of eicosanoids in intact cells showed that the CHL, hinokiol, and acacetin had significant inhibitory effects on 5-hydroxy-eicosa-tetra-enoic acid (5-HETE) and leukotriene B(4) (LTB4) formations. And cell-free enzyme assays (5-lipoxygenase, leukotriene A(4)-hydrolase, cyclooxgenase-2) demonstrated the potent inhibitory effects of the CHL, hinokiol and acacetin on 5-lipoxygenase (5-LOX). Then, the inhibitions of the CHL, hinokiol on NO biosynthesis and the inhibitions of the CHL, 8(14),15-pimaradien-3β,18-diol, and hinokiol on TNF-α release were also confirmed in the RAW264.7 murine macrophages.. The data indicate that the inhibitory effects of the CHL and its components (hinokiol and acacetin) on 5-LOX contribute to the anti-inflammatory activity of LPO. Moreover, the CHL and its components also show beneficial effects on NO and TNF-α production. Consequently, these results provide a rationale for LPO's traditional applications in the treatment of inflammatory airway diseases.

Topics: Animals; Anti-Inflammatory Agents; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Calcimycin; Calcium Ionophores; Cell Line; Chloroform; Chromatography, High Pressure Liquid; Cupressaceae; Cyclooxygenase 2; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Epoxide Hydrolases; Fatty Acids, Unsaturated; Flavones; Hydroxyeicosatetraenoic Acids; Inflammation Mediators; Leukotriene B4; Lipoxygenase Inhibitors; Macrophages; Medicine, Chinese Traditional; Mice; Nitric Oxide; Plant Leaves; Plants, Medicinal; Rats; Rats, Sprague-Dawley; Solvents; Tumor Necrosis Factor-alpha

The arachidonic acid (AA) metabolic network produces key inflammatory mediators which have been considered as hallmark contributors in various inflammatory related diseases. Enzymes in this network, such as 5-lipoxygenase (5-LOX), cyclooxygenase (COX), leukotriene A(4) hydrolase (LTA4H) and prostaglandin E synthase (PGES), have been used as targets for anti-inflammatory drug discovery. Multi-target drugs and drug combinations have also been developed for this network. However, how the inhibitors alter the dynamics of metabolite production and which combinatorial target intervention solutions are better needs further exploration. We did a system based intervention analysis on the AA metabolic network. Using an LC-MS/MS method, we quantitatively studied the eicosanoid metabolites responses of AA metabolic network during stimulation of Sprague Dawley rat blood samples with the calcium ionophore. Our results indicate that inhibiting the upstream rather than the downstream target of 5-LOX pathway will simultaneously alter the AA metabolism to the COX pathway (and vice versa). Therefore, single-target inhibitors cannot control all the inflammatory mediators at the same time. We also suggest that in the case of multiple-target anti-inflammatory solutions, the combination of inhibitors of the downstream enzymes may have stronger inhibition efficiency and cause less side-effects compared to the other solutions. One therapeutic strategy, LTA4H/COX inhibition solution, was found promising for the intervention of inflammatory mediator biosynthesis and at the same time stimulating the production of anti-inflammatory agents.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Biosynthetic Pathways; Calcimycin; Chromatography, Liquid; Cyclooxygenase Inhibitors; Dinoprostone; Eicosanoids; Enzyme Inhibitors; Hydroxyeicosatetraenoic Acids; Intramolecular Oxidoreductases; Leukotriene B4; Lipoxygenase Inhibitors; Male; Mass Spectrometry; Metabolic Networks and Pathways; Models, Biological; Prostaglandin-E Synthases; Rats; Rats, Sprague-Dawley; Time Factors

Lipoxygenases (LOs) convert polyunsaturated fatty acids into lipid hydroperoxides. Homolytic decomposition of lipid hydroperoxides gives rise to endogenous genotoxins such as 4-oxo-2(E)-nonenal, which cause the formation of mutagenic DNA adducts. Chiral lipidomics analysis was employed to show that a 5-LO-derived lipid hydroperoxide was responsible for endogenous DNA-adduct formation. The study employed human lymphoblastoid CESS cells, which expressed both 5-LO and the required 5-LO-activating protein (FLAP). The major lipid peroxidation product was 5(S)-hydroperoxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid, which was analyzed as its reduction product, 5(S)-hydroxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid (5(S)-HETE)). Concentrations of 5(S)-HETE increased from 0.07 +/- 0.01 to 45.50 +/- 4.05 pmol/10(7) cells upon stimulation of the CESS cells with calcium ionophore A23187. There was a concomitant increase in the 4-oxo-2(E)-nonenal-derived DNA-adduct, heptanone-etheno-2'-deoxyguanosine (HepsilondGuo) from 2.41 +/- 0.35 to 6.31 +/- 0.73 adducts/10(7) normal bases. Biosynthesis of prostaglandins, 11(R)-hydroxy-5,8,12,14-(Z,Z,E,Z)-eicosatetraenoic acid, and 15(R,S)-hydroxy-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid revealed that there was cyclooxygenase (COX) activity in the CESS cells. Western blot analysis revealed that COX-1 was expressed by the cells, but there was no COX-2 or 15-LO-1. FLAP inhibitor reduced HepsilondGuo-adducts and 5(S)-HETE to basal levels. In contrast, aspirin, which had no effect on 5(S)-HETE, blocked the formation of prostaglandins, 15-HETE, and 11-HETE but did not inhibit HepsilondGuo-adduct formation. These data showed that 5-LO was the enzyme responsible for the generation of the HepsilondGuo DNA-adduct in CESS cells.

Topics: 5-Lipoxygenase-Activating Proteins; Arachidonate 5-Lipoxygenase; Aspirin; Calcimycin; Carrier Proteins; Cell Line; Cyclooxygenase 1; DNA Adducts; DNA Damage; Humans; Hydroxyeicosatetraenoic Acids; Ionophores; Leukotriene B4; Lipid Peroxidation; Lymphocytes; Membrane Proteins; Models, Biological; Prostaglandins

Recent studies have shown a correlation between 5-lipoxygenase (5-LO) pathway up-regulation and cardiovascular risk. Despite the existence of several assays for products of the 5-LO pathway, a reliable method for clinical determination of 5-LO activity remains to be established. In the present communication, we report conditions that allow measurement of 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B(4) (LTB(4)) in peripheral blood mononuclear cells (PBMCs) isolated from the blood of atherosclerosis patients before and after stimulation by the calcium ionophore, A23187. LTB(4), a potent mediator of inflammation-linked cardiovascular disease, was measured using an existing competitive enzyme immunoassay (EIA) kit after making specific methodological improvements that allowed PBMCs to be used in this format for the first time. LTB(4) was also measured by LC/MS/MS along with 5-HETE, a direct by-product of the action of 5-LO on arachidonic acid and a molecule for which no commercial EIA kit exists. The LC/MS/MS assay was validated over a range of 0.025-25ng/mL for LTB(4) and 0.1-25ng/mL for 5-HETE. The EIA method has a validated range covering 0.025-4ng/mL. When both assays were applied to analyze LTB(4) from stimulated PBMCs isolated from 25 subjects with various degrees of atherosclerosis, a high correlation was obtained (r=0.9426, Pearson's correlation coefficient). A high correlation was also observed between the levels of LTB(4) and 5-HETE measured by LC/MS/MS after ionophore stimulation (r=0.9159). Details are presented for optimized sample collection, processing, storage, and analysis in accordance with the logistical demands of clinical analysis.

Topics: Arachidonate 5-Lipoxygenase; Calcimycin; Chromatography, Liquid; Humans; Hydroxyeicosatetraenoic Acids; Immunoenzyme Techniques; Ionophores; Leukocytes, Mononuclear; Leukotriene B4; Reproducibility of Results; Tandem Mass Spectrometry; Temperature; Time Factors

Priming of human neutrophils with granulocyte-macrophage colony-stimulating factor (GM-CSF) followed by treatment with formyl-methionyl-leucyl-phenylalanine (fMLP) stimulates cells in a physiologically relevant manner with modest 5-lipoxygenase activation and formation of leukotrienes. However, pretreatment of neutrophils with thimerosal, an organomercury thiosalicylic acid derivative, led to a dramatic increase (>50-fold) in the production of leukotriene B(4) and 5-hydroxyeicosatetraenoic acid, significantly higher than that observed after stimulation with calcium ionophore A23187. Little or no effect was observed with thimerosal alone or in combination with either GM-CSF or fMLP. Elevation of [Ca(2+)](i) induced by thimerosal in neutrophils stimulated with GM-CSF/fMLP was similar but more sustained compared with samples where thimerosal was absent. However, [Ca(2+)](i) was significantly lower compared with calcium ionophore-treated cells, suggesting that a sustained calcium rise was necessary but not sufficient to explain the effects of this compound on the GM-CSF/fMLP-stimulated neutrophil. Thimerosal was found to directly inhibit neutrophil lysophospholipid:acyl-CoA acyltransferase activity at the doses that stimulate leukotriene production, and analysis of lysates from neutrophil preparations stimulated in the presence of thimerosal showed a marked increase in free arachidonic acid, supporting the inhibition of the reincorporation of this fatty acid into the membrane phospholipids as a mechanism of action for this compound. The dramatic increase in production of leukotrienes by neutrophils when a physiological stimulus such as GM-CSF/fMLP is employed in the presence of thimerosal suggests a critical regulatory role of arachidonate reacylation that limits leukotriene biosynthesis in concert with 5-lipoxygenase and cytosolic phospholipase A(2)alpha activation.

Topics: Arachidonate 5-Lipoxygenase; Arachidonic Acid; Calcimycin; Calcium; Cytosol; Dose-Response Relationship, Drug; Enzyme Activation; Gas Chromatography-Mass Spectrometry; Granulocyte-Macrophage Colony-Stimulating Factor; Group IV Phospholipases A2; Humans; Hydroxyeicosatetraenoic Acids; Ionophores; Leukotrienes; Mass Spectrometry; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Phospholipases A; Thimerosal; Time Factors

Polymorphonuclear leucocytes and blood platelets co-operate in several pathophysiological processes, and arachidonic acid (AA) metabolites produced in response to the activation of these cells are potent mediators of their functions. We studied the role of platelets in the formation of 5-lipoxygenase products from AA by autologous neutrophils, especially the chemotactic agent leucotriene (LT) B4. The formation of all products, namely 5-hydroxy-eicosatetraenoic acid (5-HETE), LTB4 and the other LTA4-derived metabolites, in response to the calcium ionophore A23187 was evaluated by high-performance liquid chromatography. All the 5-lipoxygenase products were significantly diminished by physiological concentrations of platelets. This inhibitory effect was lost when platelets were previously degranulated by thrombin in non-aggregating conditions. Peptides containing the Arg-Gly-Asp-Ser or His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val sequence, which prevent the adhesion of platelets to neutrophils via the fibrinogen released from platelet granules and the integrin glycoprotein IIb/IIIa, markedly decreased the inhibitory effect of non-degranulated platelets. The production of transcellular metabolites of AA such as LTC4, the dual 5- and 12-lipoxygenase product 5,12-diHETE and lipoxins could not account for the decreased formation of 5-HETE and LTA4-derived metabolites. It is concluded that platelets may inhibit the neutrophil 5-lipoxygenase activity at the integrin level and in turn may play a role in slowing down the production of LTB4 in the course of inflammation.

Topics: Arachidonate 5-Lipoxygenase; Arachidonic Acid; Blood Platelets; Calcimycin; Cells, Cultured; Chromatography, Gas; Humans; Hydroxyeicosatetraenoic Acids; Integrins; Ionophores; Leukotriene A4; Leukotriene B4; Leukotrienes; Neutrophils; Platelet Count

We recently reported the synthesis and anti-inflammatory properties of a novel long chain polyunsaturated fatty acid (PUFA) with an oxygen atom in the beta-position, beta-oxa-21:3 n-3 (Z,Z,Z)-(octadeca-9,12,15-trienyloxy) acetic acid). Our data, from studies aimed at elucidating the mechanism of its action, show that pretreatment of human neutrophils with the beta-oxa-PUFA substantially depresses the production of leukotriene B(4) (LTB(4)) in response to calcium ionophore, A23187, comparable to standard leukotriene inhibitors such as zileuton and nordihydroguaiaretic acid. Interestingly, the n-6 equivalent, beta-oxa 21:3 n-6, is also a strong inhibitor of LTB(4) production. In contrast, naturally occurring PUFA only slightly reduce, for eicosapentaenoic (20:5n-3) and docosahexaenoic (22:6n-3) acids, or increase, for arachidonic acid (20:4n-6), the formation of LTB(4). The parent beta-oxa-21:3n-3 molecule, rather than its derivatives (methyl ester, saturated, monohydroperoxy, or monohydroxy forms), is exclusively responsible for attenuation of LTB(4) formation. beta-Oxa-21:3n-3 inhibits the conversion of [(3)H]20:4n-6 to [(3)H]5-hydroxyeicosatetraenoic acid and [(3)H]LTB(4) by neutrophils in the presence of calcium ionophore and also suppresses the activity of purified 5-lipoxygenase, but not cyclooxygenase 1 and 2. Beta-oxa-21:3n-3 is taken up by neutrophils and incorporated into phospholipids and neutral lipids. In the presence of calcium ionophore, the leukocytes convert a marginal amount of beta-oxa-21:3n-3 to a 16-monohydroxy-beta-oxa-21:3n-3 derivative. After administration to rodents by gavage or i.p. injection, beta-oxa-21:3n-3 is found to be incorporated into the lipids of various tissues. Thus, beta-oxa-21:3n-3 has the potential to be used in the treatment of inflammatory diseases, which are mediated by products of the lipoxygenase pathway.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Arachidonate 5-Lipoxygenase; Calcimycin; Cholesterol Esters; Diglycerides; Enzyme Inhibitors; Fatty Acids, Unsaturated; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Lipoxygenase Inhibitors; Neutrophil Activation; Neutrophils; Phospholipids

5-Lipoxygenase (5-LO), which catalyzes the first two steps in leukotriene biosynthesis, is a target for pharmacological treatment of inflammatory disorders. Previous studies have shown that B-lymphocytes express 5-LO. Here we demonstrate that several stimuli of cell stress such as osmotic shock (sorbitol, NaCl), oxidative stress (hydrogen peroxide, diamide), chemical stress sodium arsenite, and inflammatory cytokines enhanced cellular 5-LO activity in a B cell line (BL41-E95-A), when added simultaneously with ionophore plus arachidonate. It is interesting that sorbitol alone was sufficient for 5-LO product formation in the presence of exogenous arachidonic acid. These stimuli also activated p38 mitogen-activated protein (MAP) kinase and downstream MAP kinase-activated protein kinases in BL41-E95-A cells, which could phosphorylate 5-LO or heat shock protein 27 in vitro. The p38 MAP kinase inhibitor SB203580 abolished stress-induced leukotriene synthesis in B cells, without inhibition of 5-LO catalytic activity in cell-free systems. Our results indicate that p38 MAP kinase activation by cell stress is required for efficient leukotriene synthesis in B-lymphocytes.

Topics: Arachidonate 5-Lipoxygenase; Arachidonic Acid; Arsenites; B-Lymphocytes; Calcimycin; Calcium; Cell Line; Cell-Free System; Enzyme Activation; Enzyme Inhibitors; Hydroxyeicosatetraenoic Acids; Hypertonic Solutions; Imidazoles; Interleukin-1; Intracellular Signaling Peptides and Proteins; Ionophores; Leukotrienes; Lipoxygenase Inhibitors; Mitogen-Activated Protein Kinases; Osmotic Pressure; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Kinases; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Protein Tyrosine Phosphatases; Pyridines; Sodium Compounds; Sorbitol; Subcellular Fractions; Thapsigargin; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Vanadates

The 5-lipoxygenase (5-LO) pathway in human CD34(+) hematopoietic progenitor cells, which were induced to differentiate into dendritic cells (DCs) by cytokines in vitro and in DCs of lymphoid tissues in situ, was examined. Extracts prepared from HPCs contained low levels of 5-LO or 5-LO-activating protein. Granulocyte-macrophage colony-stimulating factor (GM-CSF) plus tumor necrosis factor-alpha (TNF-alpha) promoted DC differentiation and induced a strong rise in 5-LO and FLAP expression. Fluorescence-activated cell sorter (FACS) analyses identified a major DC population coexpressing human leukocyte antigen (HLA)-DR/CD80 and monocytic or Langerhans cell markers. Transforming growth factor-beta1 (TGF-beta-1), added to support DC maturation, strongly promoted the appearance of CD1a(+)/Lag(+) Langerhans-type cells as well as mature CD83(+) DCs. TGF-beta-1 further increased 5-LO and FLAP expression, recruited additional cells into the 5-LO(+) DC population, and promoted production of 5-hydroxyeicosatetraenoic acid and leukotriene B(4) in response to calcium (Ca(++)) ionophore A23187. These in vitro findings were corroborated by 5-LO expression in distinct DC phenotypes in vivo. Scattered 5-LO and FLAP in situ hybridization signals were recorded in cells of paracortical T-lymphocyte-rich areas and germinal centers (GCs) of lymph nodes (LNs) and tonsil and in cells of mucosae overlying the Waldeyer tonsillar ring. 5-LO protein localized to both CD1a(+) immature DCs and to CD83(+) mature interdigitating DCs of T-lymphocyte-rich areas of LNs and tonsil. As DCs have the unique ability to initiate naive lymphocyte activation, our data support the hypothesis that leukotrienes act at proximal steps of adaptive immune responses. (Blood. 2000;96:3857-3865)

Topics: Antigens, CD34; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Calcimycin; Cell Differentiation; Cell Lineage; Chemotactic Factors; Cytokines; Dendritic Cells; Fetal Blood; Flow Cytometry; Hematopoietic Stem Cells; HLA-DR Antigens; Humans; Hydroxyeicosatetraenoic Acids; Immunohistochemistry; Immunophenotyping; Ionophores; Leukotriene B4; Lymph Nodes; Lymphoid Tissue; Macrophages; Palatine Tonsil

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

Arachidonic acid (AA) metabolism was assessed in cultured alveolar macrophages (AM) obtained from newborn (10 days old) and adult (2 months and 4 months old) rats. The AMs were stimulated with the calcium ionophore, A23187 (10 microM). The released radiolabelled AA metabolites were measured by thin layer chromatography. The results showed that among different aged rats, the synthesis of 5-lipoxygenase (5-LO) metabolites, LTB4, LTC4, LTD4 and 5-HETE were increased with age inspite of similar levels of [14C]AA release. In response to A23187, 5-LO metabolic capacity of 2 and 4 months old adult rat AMs were increased 21-fold and 34-fold, respectively, compared with 10 days old rat AMs. As the metabolic capacity increased, the release of prostaglandins and thromboxane B2 tended to decrease markedly. Newborn rats (10 days old) AM, at the initial developmental stage, did not produce a noticeable amount of 5-LO metabolites which, conceivably, contribute to high susceptibility of neonatal lung to infection.

Topics: Aging; Animals; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Calcimycin; Hydroxyeicosatetraenoic Acids; Leukotrienes; Macrophages, Alveolar; Prostaglandin-Endoperoxide Synthases; Prostaglandins; Rats; Rats, Inbred Strains; Thromboxane B2

The supposed 5-LO inhibitory activity of two N-omega-ethoxycarbonyl-4-quinolones was tested determining leukotriene B4 (LTB4) in RBL-1 cell cultures, pretreated with the two compounds of interest. LTB4, obtained by solid-phase extraction (SPE) from cell cultures supernatants, was determined by micellar electrokinetic chromatography (MEKC). The analysis was performed using an uncoated capillary, filled with borate buffer at pH 8.3, containing 12.5 mM SDS as micelles generator. Therefore, following the decreasing of LTB4 it was possible to verify the 5-LO inhibitory activity of two quinolone derivatives. To asses the suitability of the use of LTB4 as marker of the activity of the new compounds, the analysis was repeated using quercetin, a well known 5-LO inhibitor.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonate 5-Lipoxygenase; Biomarkers; Calcimycin; Chromatography, High Pressure Liquid; Chromatography, Micellar Electrokinetic Capillary; Culture Media, Conditioned; Electrophoresis, Capillary; Enzyme Activation; Evaluation Studies as Topic; Hydroxyeicosatetraenoic Acids; Leukemia, Basophilic, Acute; Leukotriene B4; Lipoxygenase Inhibitors; Prostaglandins B; Quercetin; Quinolones; Rats; Sodium Dodecyl Sulfate; Tumor Cells, Cultured

The overexpression of phospholipid hydroperoxide glutathione peroxidase (PHGPx) by RBL-2H3 cells was used as the basis for an investigation of the effects of PHGPx on the formation of leukotrienes. The rates of production of leukotriene C4 (LTC4) and leukotriene B4 (LTB4) in cells that overexpressed PHGPx were 8 times lower than those in a control line of cells. The reduction in rates of production of leukotrienes apparently resulted from the increase in the PHGPx activity since control rates of formation of leukotrienes could be achieved in PHGPx-overexpressing cells upon inhibition of PHGPx activity by diethyl malate. The conversion of radioactively labeled arachidonic acid to intermediates in the lipoxygenase pathway, such as 5-hydroxyeicosatetraenoic acid (5-HETE), LTC4, and LTB4, was strongly inhibited in PHGPx-overexpressing cells that had been prelabeled with [14C]arachidonic acid. PHGPx apparently inactivated the 5-lipoxygenase that catalyzed the conversion of arachidonic acid to 5-hydroperoxyeicosatetraenoic acid (5-HPETE) since 5-HPETE is a common precursor of 5-HETE, LTC4, and LTB4. The rates of formation of LTC4 and LTB4 in PHGPx-overexpressing cells returned to control rates upon the addition of a small amount of 12-HPETE. Flow cytometric analysis revealed that the rapid burst of formation of lipid hydroperoxides induced by A23187 was suppressed in PHGPx-overexpressing cells as compared with the control lines of cells. Subcellular fractionation analysis showed that the amount of PHGPx associated with nuclear fractions from PHGPx-overexpressing cells was 3.5 times higher than that from the control line of cells. These results indicate that PHGPx might be involved in inactivation of 5-lipoxygenase via reductions in levels of the fatty acid hydroperoxides that are required for the full activation of 5-lipoxygenase. Thus, in addition to its role as an antioxidant enzyme, PHGPx appears to have a novel function as a modulator of the production of leukotrienes.

Topics: Animals; Antioxidants; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Calcimycin; Cell Line; Cell Separation; Chromatography, High Pressure Liquid; Flow Cytometry; Glutathione Peroxidase; Hydroxyeicosatetraenoic Acids; Ionophores; Leukotrienes; Phospholipid Hydroperoxide Glutathione Peroxidase; Proteins; Rats; Selenoproteins

We investigated the anti-inflammatory effect of YPE-01, a novel diarylheptanoid derivative in vitro and in vivo.. In the in vitro study, rat basophilic leukemia (RBL-1) cells were used. For the in vivo study, ICR and ddY mice (male, 7 weeks old) were used.. In the in vitro study, the supernatant of RBL-1 cells lysate was incubated with 50 microM arachidonic acid (AA) and 0.01-100 microM test drugs for 15 min. RBL-1 cells were preincubated with 0.01-100 microM test drugs for 10 min before incubation with 0.5 microM calcium ionophore A23187 for 10 min. In the in vivo study, YPE-01 (0.1-3 mg/ear) was applied to the ear of mice at the same time as a 12-O-tetradecanoylphorbol 13-acetate (TPA) application or 1 h before an AA application.. In the in vitro study, the amounts of 5-hydroxyeicosatetraenoic acid and leukotrienes were measured by high-performance liquid chromatography and an enzyme immunoassay, respectively. In the in vivo study, a circular tissue sample from the ear of the mice was weighed. Statistical analysis was done using Dunnett's test.. YPE-01 inhibited the 5-lipoxygenase activity (IC50, 0.28 microM) and the leukotriene B4 (IC50, 0.035 microM) and C4 (IC50, 0.046 microM) production by RBL-1 cells without any inhibition of cyclooxygenase activity in vitro. The topical application of YPE-01 significantly suppressed both the AA- and TPA-induced ear edemas in vivo.. YPE-01 is a selective 5-lipoxygenase inhibitor with a suppressive effect against dermal inflammation.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Calcimycin; Chemotactic Factors; Chromatography, High Pressure Liquid; Diarylheptanoids; Ear; Edema; Hydroxyeicosatetraenoic Acids; Ionophores; Ketones; Leukotriene B4; Lipoxygenase Inhibitors; Male; Mice; Mice, Inbred ICR; Phenols; Rats; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Upon an increasing cell density human neutrophils develop more cell-to-cell contacts in conjunction with an increase in the pHi. These changes are accompanied by decreased superoxide formation after adherence, and a decrease in the total amount of 5-lipoxygenase products after various stimuli. Among the various arachidonate metabolites, leukotriene formation remained almost constant but the yield in 5-HETE decreased. This drop in could account for the decrease in total 5-lipoxygenase products observed when the cell density increased. We conclude that cellular signalling can be affected by an increase of cell-cell interactions. Whether the increase in cellular pH is a cause or consequence of such contact inhibition has yet be answered.

Topics: Arachidonate 5-Lipoxygenase; Arachidonic Acid; Calcimycin; Calcium; Cell Adhesion; Cell Count; Cells, Cultured; Drug Synergism; Humans; Hydrogen-Ion Concentration; Hydroxyeicosatetraenoic Acids; Ionophores; Leukotriene B4; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Reactive Oxygen Species; Respiratory Burst; Signal Transduction; Tetradecanoylphorbol Acetate

The effects of two polyunsaturated fatty acids, 18:4n-3 and 16:4n-3 purified from the marine algae, Undaria pinnatifida and Ulva pertusa, on icosanoid production in MC/9 mouse mast cells were assessed. Both fatty acids suppressed the production of leukotriene B4 (LTB4), leukotriene C4 (LTC4), and 5-hydroxyeicosatetraenoic acid (5-HETE). The order of the suppressive activity for the two marine algae-derived fatty acids and three other common polyunsaturated fatty acids was as follows; 22:6n-3 = 18:4n-3 = 18:3n-3 > 20:5n-3 = 16:4n-3 for LTB4; 22:6n-3 = 18:4n-3 = 18:3n-3 > 16:4n-3 > 20:5n-3 (no suppression) for LTC4; 22:6n-3 = 18:4n-3 > 18:3n-3 > 20:5n-3 = 16:4n-3 for 5-HETE.

Topics: Animals; Calcimycin; Chromatography, High Pressure Liquid; Eukaryota; Fatty Acids, Unsaturated; Hydroxyeicosatetraenoic Acids; Ionophores; Leukotriene B4; Leukotriene C4; Mast Cells; Mice; Plants, Edible

The esterification of endogenously formed 5-hydroxyeicosatetraenoic acid (5-HETE) to cellular lipids in rat polymorphonuclear leukocytes (PMNL) was studied quantitatively by a fluorometric method using HPLC. Rapid and maximal production of free 5-HETE was observed after a 5-min stimulation of PMNL with A23187. The amount of free 5-HETE then declined rapidly, while that of 5-HETE esterified to phospholipids and triacylglycerol concomitantly increased in a time-dependent manner. Stimulation by A23187 yielded approximately 100 ng/10(7) cells esterified 5-HETE in 60 min, which corresponded to the decrease in the amount of free 5-HETE from 5 min to 60 min and indicated that free 5-HETE, which was formed endogenously, was metabolized predominantly by esterification to cellular lipids. The esterification profile of exogenous 5-HETE was different from that of endogenous 5-HETE. 5-[3H]HETE, which was added exogenously to the culture medium, was rapidly incorporated into PMNL and almost 80% of the total radioactivity was located in triacylglycerol. A quantitative study revealed that endogenous 5-HETE was esterified equally to phospholipids and triacylglycerol. Like PMNL, peritoneal macrophages treated with A23187 released significant amounts of 5-HETE. However, less 5-HETE was esterified to cellular lipids than in PMNL. Negligible amounts of 12-HETE, produced by activated peritoneal macrophages or activated platelets after a challenge with A23187, were esterified during the entire incubation. Exogenous 5-HETE was rapidly taken up by PMNL, but was incorporated into macrophages much more slowly than into PMNL. No uptake of 12-HETE into macrophages was observed. The rapid uptake of exogenous 5-HETE was strongly inhibited by the suppression of acylation of 5-HETE by triacsin C. These results suggest that esterification might be one of the factors that regulate the rate of incorporation of 5-HETE.

Topics: Animals; Blood Platelets; Calcimycin; Coenzyme A Ligases; Enzyme Inhibitors; Esterification; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Ionophores; Lipoxygenase Inhibitors; Macrophages, Peritoneal; Male; Masoprocol; Mice; Mice, Inbred ICR; Neutrophils; Phospholipids; Rabbits; Rats; Rats, Sprague-Dawley; Triazenes

The effect of tenidap on the metabolism of arachidonic acid via the 5-lipoxygenase (5-LO) pathway was investigated in vitro and in vivo.. In vitro (cells). Arachidonic acid (AA) stimulated rat basophilic leukemia, (RBL) cells; A23817 activated neutrophils (human rat, and rabbit), macrophages (rat), and blood (human). In vitro (enzyme activity). RBL-cell homogenate; purified human recombinant 5-LO. In vivo: Rat (Sprague-Dawley) models in which peritoneal leukotriene products were measured after challenge with zymosan (3 animals per group), A23187 (11 animals per group), and immune complexes (3-5 animals per group), respectively.. 5-Hydroxyeicosatetraenoic acid (5-HETE) and dihydroxyeicosatetraenoic acids (diHETEs, including LTB4) were measured as radiolabeled products (derived from [14C]-AA) or by absorbance at 235 or 280 nm, respectively, after separation by HPLC. Radiolabeled 5-HPETE was measured by a radio-TLC analyser after separation by thin layer chromatography (TLC). Deacylation of membrane bound [14C]-AA was determined by measuring radiolabel released into the extracellular medium. 5-LO translocation from cytosol to membrane was assessed by western analysis. Rat peritoneal fluid was assayed for PGE, 6-keto-PGF1 alpha, LTE4 or LTB4 content by EIA and for TXB2 by RIA.. Tenidap suppressed 5-LO mediated product production in cultured rat basophilic leukemia (RBL-1) cells from exogenously supplied AA, and in human and rat neutrophils, and rat peritoneal macrophages stimulated with A23187 (IC50, 5-15 microM). In addition, tenidap was less potent in inhibiting the release of radiolabeled AA from RBL-1 cells (IC50, 180 microM), suggesting that the decrease in 5-LO derived products could not be explained by an effect on cellular mobilization of AA (i.e., phospholipase). Tenidap blocked 5-hydroxyeicosatetraenoic acid (5-HETE) production by dissociated RBL-1 cell preparations (IC50, 7 microM), as well as by a 100000 x g supernatant of 5-LO/hydroperoxidase activity, suggesting a direct effect on the 5-LO enzyme itself. In addition, tenidap impaired 5-LO translocation from cytosol to its membrane-bound docking protein (FLAP) which occurs when human neutrophils are stimulated with calcium ionophore, indicating a second mechanism for inhibiting the 5-LO pathway. Surprisingly, tenidap did not block the binding of radiolabeled MK-0591, an indole ligand of FLAP, to neutrophil membranes. Although its ability to inhibit the cyclooxygenase pathway was readily observed in whole blood and in vivo, tenidap's 5-LO blockade could not be demonstrated by ionophore stimulated human blood, nor after oral dosing in rat models in which peritoneal leukotriene products were measured after challenge with three different stimuli. The presence of extracellular proteins greatly reduced the potency of tenidap as a 5-LO inhibitor in vitro, suggesting that protein binding is responsible for loss of activity in animal models.. Tenidap inhibits 5-lipoxygenase activity in vitro both directly and indirectly by interfering with its translocation from cytosol to the membrane compartment in neutrophils. A potential mechanism for the latter effect is discussed with reference to tenidap's ability to lower intracellular pH. Tenidap did not inhibit 5-LO pathway activity in three animal models.

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Anti-Inflammatory Agents, Non-Steroidal; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Calcimycin; Chemotactic Factors; Chromatography, High Pressure Liquid; Cyclooxygenase Inhibitors; Erythrocytes; Humans; Hydroxyeicosatetraenoic Acids; Immunoenzyme Techniques; Indoles; Ionophores; Leukemia, Basophilic, Acute; Leukotriene B4; Leukotriene E4; Lipoxygenase Inhibitors; Neutrophil Activation; Neutrophils; Oxindoles; Rabbits; Radioimmunoassay; Rats; Rats, Sprague-Dawley; Thromboxane B2; Zymosan

Arachidonic acid and 5-hydroxyeicosatetraenoic acid (5-HETE) are reported to induce reinitiation of meiosis in oocytes of the surf clam Spisula sachalinensis from the Sea of Japan (Varaksin et al., Comp. Biochem. Physiol. 101C, 627-630 (1992). As the Atlantic surf clam Spisula solidissima is a commonly used model for the study of meiosis reinitiation, we examined these cells for the possible occurrence of lipoxygenases and for the bioactivity of the products. Incubation of [14C]arachidonic acid with homogenates of S. solidissima oocytes led to the formation of two major metabolites: 5R-HETE, a novel lipoxygenase product, and 8R-HETE. The products were identified by HPLC, uv spectroscopy, and GC-MS. The corresponding hydroperoxy fatty acids, the primary lipoxygenase products, were isolated from incubations of ammonium sulfate fractionated oocyte cytosol. Arachidonic and eicosapentaenoic acids were identified as constituents of S. solidissima oocyte lipids and the free acids were equally good lipoxygenase substrates. We examined the activity of C18 and C20 polyunsaturated fatty acids and their lipoxygenase products on meiosis reinitiation in Spisula solidissima oocytes, using serotonin and ionophore A23187 as positive controls. The fatty acids and their derivatives were inactive. We conclude that in the surf clam, (as in starfish), there are responding and non-responding species in regard to the maturation-inducing activity of the oocyte lipoxygenase products, and that the lipoxygenase has another, as yet uncharacterized, function in oocyte physiology.

Topics: Animals; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Bivalvia; Calcimycin; Calcium Chloride; Cell Fractionation; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Eicosapentaenoic Acid; Enzyme Activation; Fatty Acids; Hydroxyeicosatetraenoic Acids; Molecular Conformation; Oocytes; Serotonin; Spectrophotometry

Serum-cultured rat W256 carcinosarcoma cells of the monocytoid origin undergo rapid apoptosis in response to the lipoxygenase inhibitor NDGA (nordihydroguaiaretic acid). Exogenous arachidonic acid (AA), in a time- and dose-dependent fashion, suppressed NDGA-induced W256 cell apoptosis as well as DNA fragmentation, with the maximal effect observed at approximately 25 microM. Mobilization of endogenous AA by calcium ionophore A23187 provided an even stronger and longer-lasting protection against NDGA-caused cell death. The A23187 effect on AA release as well as W256 cell death can be blocked by bromophenacyl bromide, thus suggesting involvement of phospholipase A2 activation. Serum withdrawal similarly caused W256 cells to undergo typical apoptosis, which was not rescued by several growth factors commonly found in serum. However, exogenous AA suppressed serum starvation-induced W256 cell apoptosis and significantly extended cell survival in a dose-dependent manner. Lipoxygenase products, 12(S)- and 15(S)-, but not 5(S)-hydroxyeicosatetraenoic acid (HETE), in a dose-dependent fashion, also prevented both NDGA- and serum-starvation-induced W256 cell apoptosis. AA appears to suppress W256 cell apoptosis via distinct signaling pathway(s) since it does not prevent cell death triggered by several other inducers. Examination of a panel of polyunsaturated fatty acids revealed that alpha-linolenic and linoleic acid can also suppress NDGA-induced W256 cell apoptosis. Our data suggest that AA and other polyunsaturated fatty acids and/or their metabolites may enhance tumor growth not only by promoting cell proliferation but also by suppressing apoptosis.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Acetophenones; Animals; Apoptosis; Arachidonic Acid; Calcimycin; Carcinosarcoma; Cell Line; Cell Survival; Cytokines; DNA Fragmentation; Fatty Acids, Nonesterified; Growth Substances; Hydroxyeicosatetraenoic Acids; Kinetics; Masoprocol; Rats; Tumor Cells, Cultured

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Adenocarcinoma; Calcimycin; Carcinoma, Small Cell; Carcinoma, Squamous Cell; Cell Communication; Cell Line; Coculture Techniques; Erythrocytes; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Lung Neoplasms; Models, Biological; Neutrophils; Phospholipases A; Phospholipases A2; Tumor Cells, Cultured

Changes in lipid composition were examined in peritoneal polymorphonuclear leukocytes (PMNL) from rats with streptozotocin-induced diabetes. The largest changes associated with the lipids in PMNL prepared from rats with insulin-dependent diabetes mellitus were found in the composition of fatty acids of phospholipids although no consistent changes were noted in the amounts of phospholipids in individual classes. The relative amount of arachidonic acid was significantly reduced and that of linoleic acid was significantly increased in PMNL from diabetic rats. The extent of A23187-stimulated release of arachidonic acid from CGP and IGP in PMNL from diabetic rats was much smaller than that from normal PMNL. Moreover, there were decreases in the production of free arachidonic acid and its metabolites, such as LTB4 and 5-HETE by A23187-stimulated PMNL from diabetic rats as compared to PMNL from normal rats. Aggregation of PMNL provoked by A23187 was significantly suppressed in PMNL from diabetic rats. However, addition of free arachidonic acid or LTB4 to normal PMNL and to those from diabetic rats resulted in aggregation to similar extents, a result that supports a role for enhanced availability of endogenous arachidonic acid in the induction of the aggregation of PMNL from diabetic rats. The present results suggest that diabetes-associated changes in arachidonic acid metabolism might play a critical role in the modulation of aggregation of PMNL.

Topics: Animals; Arachidonic Acid; Blood Glucose; Calcimycin; Cell Aggregation; Diabetes Mellitus, Experimental; Fatty Acids; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Kinetics; Leukotriene B4; Male; Neutrophils; Phospholipids; Rats; Rats, Sprague-Dawley; Reference Values

(15S)-Hydroxy-(5Z,8Z,11Z,13E)-eicosatetraenoic acid (15-HETE) suppresses in ionophore-A23187-stimulated human polymorphonuclear leucocytes (PMN) the conversion of exogenous arachidonic acid into leukotriene B(4) (LTB4) and (5S)-hydroxy-(6E,8Z,11Z,14Z)-eicosatetraenoic acid (5-HETE). However, contrary to earlier suggestions, 15-HETE is not a genuine 5-lipoxygenase inhibitor under these conditions, but rather suppresses the 5-lipoxygenation of arachidonic acid by switching-over of substrate utilization, as judged from a sizeable formation of labelled (5S,15S)-dihydroxy-(6E,8Z,11Z,13E)-eicosatetr aen oic acid (5,15-diHETE) from 15-[1(-14)C]HETE. Identical results were obtained with human recombinant 5-lipoxygenase. In PMN the formation of 5,15-diHETE is strongly stimulated by either hydroperoxypolyenoic fatty acids or arachidonic acid, suggesting a crucial role of the hydroperoxide tone of the cell. A comparison of a selection of hydroxypolyenoic fatty acids with respect to their capability of suppressing 5-lipoxygenation of arachidonic acid revealed that 15-mono-hydroxyeicosanoids throughout exhibit the highest inhibitory potencies, whereas the other HETEs, 5,15-diHETE as well as octadecanoids, are modest or poor inhibitors. The R and S enantiomers of 15-HETE do not differ from each other, excluding a receptor-like binding of the 15-hydroxy group.

Topics: Arachidonate 15-Lipoxygenase; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Calcimycin; Fatty Acids, Unsaturated; Humans; Hydroxyeicosatetraenoic Acids; Kinetics; Leukotriene B4; Lipoxygenase Inhibitors; Neutrophils; Recombinant Proteins; Structure-Activity Relationship; Substrate Specificity

In Ca2+ ionophore-activated HL-60 granulocytes the mitogen-activated protein kinase kinase-1 inhibitor, PD098059, blocked translocation of 5-lipoxygenase from the cytosol to the nuclear membrane and the corresponding enzyme activation. PD098059 inhibited 5-HETE formation with an IC50 = 9.4 microM in cells stimulated with A23187 alone, and with an IC50 = 12 microM in cells stimulated with A23187 plus 20 microM arachidonic acid. PD098059 inhibited translocation of 5-lipoxygenase in a concentration-dependent manner with an IC50 approximately 10 microM. At concentrations less than 100 microM PD098059 had no effect on purified recombinant 5-LO activity. Collectively, these data indicate that MAPKK-l participates in the molecular processes governing activation and translocation of 5-lipoxygenase from the cytosol to the nuclear membrane.

Topics: Arachidonate 5-Lipoxygenase; Arachidonic Acid; Base Sequence; Biological Transport; Calcimycin; Cell Differentiation; Cytosol; Enzyme Activation; Enzyme Inhibitors; Flavonoids; Granulocytes; HL-60 Cells; Humans; Hydroxyeicosatetraenoic Acids; Lipoxygenase Inhibitors; Mitogen-Activated Protein Kinase Kinases; Molecular Sequence Data; Nuclear Envelope; Protein Kinase Inhibitors; Recombinant Proteins

We recently demonstrated that the arachidonate metabolite 5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) is converted by a highly specific dehydrogenase in human neutrophils to 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE), which is a potent stimulator of these cells. The objective of this study was to determine whether 5-oxo-ETE is also formed by monocytes and lymphocytes. Human monocytes (74 +/- 2% pure) and lymphocytes (86 +/- 1% pure) were prepared by successive centrifugations of leukocytes over Ficoll-Paque and Percoll. Both cell types converted 5-HETE to a single major product, which was identified as 5-oxo-ETE. The formation of 5-oxo-ETE was stimulated about twofold by phorbol myristate acetate (PMA; 30 nM). Dehydrogenase activity in monocyte fractions did not appear to be due to platelet contamination, since depletion of platelets did not reduce enzyme activity. The dehydrogenase was localized in membrane fractions from monocytes and required NADP+ as a cofactor. It was specific for eicosanoids containing a 5S-hydroxyl group followed by a 6-trans double bond. We also investigated the formation of 5-oxo-ETE from endogenous arachidonic acid by monocytes. 5-Oxo-ETE, 5-HETE, and leukotriene B4 (LTB4) were present in comparable amounts after incubation of these cells with A23187. PMA (EC50 approximately 4 nM) stimulated the formation of 5-oxo-ETE and 5-HETE and, to a lesser extent, LTB4. Although monocytes released considerably less 5-HETE and LTB4 than neutrophils, they released comparable amounts of 5-oxo-ETE. Unlike neutrophils, monocytes did not convert any of these substances to detectable amounts of omega-oxidation products. Although lymphocytes were capable of converting 5-HETE to 5-oxo-ETE, they released little or no 5-lipoxygenase products in response to A23187. We conclude that monocytes have a high capacity to synthesize 5-oxo-ETE and that its formation is stimulated by activation of protein kinase C.

Topics: Alcohol Oxidoreductases; Calcimycin; Dose-Response Relationship, Drug; Humans; Hydroxyeicosatetraenoic Acids; Lymphocytes; Monocytes; Neutrophils; Tetradecanoylphorbol Acetate

The effects of tumor necrosis factor alpha (TNF-alpha) on arachidonic acid (AA) metabolism were investigated by prelabeling the human osteoblastic osteosarcoma cell line, G292, with [3H]AA. TNF-alpha differentially stimulates cyclooxygenase and lipoxygenase pathways of AA metabolism in a dose response manner in the cells. The highest concentration of TNF-alpha (10(-8)M) significantly increased the cyclooxygenase pathway, with prostaglandin E2 (PGE2) being a major product. However, at the lowest concentration (10(-10)M) of TNF-alpha, 15-hydroxyeicosatetraenoic acid (HETE) production was significantly increased, with no significant effects on the other identifiable products. When the concentration of TNF-alpha was increased to 10(-9) M leukotriene B4 (LTB4), 15-, 12-, and 5-HETE were significantly increased. The calcium ionophore A23187 (10(-6) M) significantly increased 15-HETE production, without significantly affecting cyclooxygenase metabolites. However, a combination of TNF-alpha (10(-8)M) and A23187 (10(-6)M) caused an inhibitory effect on each agent-induced PGE2 or 15-HETE production.

Topics: Arachidonic Acid; Calcimycin; Dinoprostone; Dose-Response Relationship, Drug; Humans; Hydroxyeicosatetraenoic Acids; Indomethacin; Leukotriene B4; Lipoxygenase; Osteoblasts; Osteosarcoma; Prostaglandin-Endoperoxide Synthases; Tritium; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

We studied cellular interactions between human polymorphonuclear leukocytes (PMN) and lung cancer cell lines by investigating the influence of cancer cells on the production of leukotriene B4 (LTB4) and superoxide anion (O2-) by stimulated PMN. Of the nine cancer cell lines established from human lung cancers that we examined, H23 cells showed the highest LTA4 hydrolase activity. When PMN were stimulated by the calcium ionophore A23187 in the presence of H23 cells, the production of LTB4, 5(S)-hydroxyeicosatetraenoic acid (5-HETE), and 12(S)-hydroxyeicosatetraenoic acid (12-HETE) decreased in a dose-dependent manner. On the contrary, H23 did not inhibit O2- production by PMN. Two other cell lines (N417 and Q9) caused similar inhibition of LTB4 production by PMN. These three cancer cell lines alone did not generate any metabolites of the arachidonic acid (AA) lipoxygenase pathway or any O2- upon stimulation with A23187 alone. The addition of AA dose-dependently reversed the H23-induced inhibition of LTB4, 5-HETE, and 12-HETE production by PMN, suggesting inhibition at the phospholipase A2 (PLA2) level. Furthermore, addition of the cancer cell line Q9 inhibited 14C release from [14C]AA prelabeled PMN in a cell number-dependent manner in the buffer, with and without albumin. The supernatant of H23 cells also inhibited the production of LTB4 by PMN stimulated by A23187, as did the addition of H23 lysate or its 10(4) x g centrifugation supernatant. While neither the 10(5) x g supernatant (cytosol) nor the pellet (microsome) exhibited inhibitory activity, the combination of the separated cytosol and microsomal fractions restored the inhibitory activity. Furthermore, addition of the 10(4) x g supernatant of Q9 lysate to partially purified human cytosolic PLA2 inhibited PLA2 activity in a dose-dependent manner. Our results indicate that the lung cancer cell lines used in our study inhibit LTB4 production by human PMN through inhibition of phospholipase A2 activity, which may contribute to a predisposition to pulmonary infections in patients with lung cancer.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonic Acid; Calcimycin; Calcium; Coculture Techniques; Cyclooxygenase Inhibitors; Dinoprostone; Erythrocytes; Humans; Hydroxyeicosatetraenoic Acids; Indomethacin; Ionophores; Leukotriene B4; Lipoxygenase; Lung Neoplasms; Neutrophils; Phospholipases A; Phospholipases A2; Subcellular Fractions; Superoxides; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

14C arachidonic acid incorporation and 14C radioactivity release as well as prostaglandin (PG) and 5-hydroxyeicosatetraenoic acid (5-HETE) synthesis were measured in the pair of murine lymphoma L5178Y (LY) cell sublines differing in radiation sensitivity. Both LY sublines, LY-R (resistant) and LY-S (sensitive), incorporated exogenous arachidonic acid and released it from membrane phospholipids. Ca2+ ionophores (ionomycin and A23187) but not PMA stimulated the liberation of 14C arachidonic acid in LY cells. PMA did not potentiate the 14C arachidonic acid release both in the presence or in the absence of A23187; this observation suggests that protein kinase C activation is not essential for the regulation of arachidonic acid release by LY-R and LY-S cells. X-irradiation (5 Gy) did not change the uptake of 14C arachidonic acid into LY-R and LY-S cells and did not potentiate the release of its total radioactivity from the cells. PG synthesis was stimulated in irradiated LY-R cells but not in LY-S cells. The susceptibility of eicosanoid metabolism to A23187 and H2O2 was altered in irradiated LY-R cells. A23187 stimulated only PG and 5-HETE synthesis in irradiated LY-R cells. H2O2 did not stimulate the synthesis of PG from exogenous arachidonic acid in irradiated LY-R and LY-S cells and 5-HETE synthesis in LY-R cells. An implication of the increased PG synthesis in LY-R cells in the protection against radiation is discussed.

Topics: Animals; Arachidonic Acid; Calcimycin; Cell Line; Hydrogen Peroxide; Hydroxyeicosatetraenoic Acids; Ionomycin; Lymphoma; Mice; Phospholipids; Prostaglandins; Radiation Tolerance; Staurosporine; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; X-Rays

Dapsone (4,4'-diaminodiphenylsulfone) is effective in treating inflammatory skin diseases. Several lines of evidence suggest that the anti-inflammatory properties of this sulfone are partially due to modulation of functions of polymorphonuclear leukocytes (PMN). The goal of the present investigation is therefore to ascertain possible inhibitory effects of dapsone upon human 5-lipoxygenase (5-LOX) pathway. PMN of healthy donors were pretreated with dapsone in different concentrations (1.6-100 microM) for 5 min following by adding Ca ionophore A 23187 (10 microM) and subsequent incubation for 10 min. Thereupon the eicosanoids were assessed by reversed-phase high-performance liquid chromatography (RP-HPLC). Dapsone exhibited dose-dependent inhibitory activity showing 50% inhibition at 15 microM for leukotriene B4 (LTB4) with 5 x 10(6) PMN. The IC50 (half maximum inhibition concentration) of dapsone for 5-hydroxyeicosatetraenoic acid (5-HETE) and omega-OH-LTB4 amounted to similar values (5-HETE: 9 microM; omega-OH-LTB4: 11 microM). The inhibition of the conversion of arachidonic acid to several eicosanoids mainly suggests an effect on the 5-LOX enzyme. The comparison of inhibition values between intact PMN and a cell-free system (by sonification) indicates an additional effect of dapsone upon enzymes other than 5-LOX. Since the concentrations used are comparable with therapeutic conditions, dapsone may exert part of its anti-inflammatory effect by prevention of the generation of 5-LOX metabolites.

Topics: Calcimycin; Chromatography, High Pressure Liquid; Dapsone; Eicosanoids; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Ionophores; Leukotriene Antagonists; Leukotriene B4; Lipoxygenase; Lipoxygenase Inhibitors; Neutrophils

The effects of cyclosporin A (CSA) on arachidonic acid (AA) metabolism were investigated in intact rat basophilic leukemia-1 (RBL-1) cells and cell lysates. Calcium ionophore (A23187)-stimulated synthesis of cysteinyl leukotrienes (LTC4, LTD4, and LTE4), LTB4, and 5-hydroxyeicosatetraenoic acid (5-HETE) in intact cells in the absence or presence of CSA was measured by reversed-phase high-performance liquid chromatography (HPLC). CSA inhibited the production of cysteinyl LTs, LTB4, and 5-HETE in intact cells in a dose-dependent manner. The synthesis of cysteinyl LTs, LTB4, and 5-HETE was also measured after the incubation of cell lysates with free AA in the absence or presence of CSA. CSA did not inhibit synthesis of cysteinyl LTs, but rather stimulated production of LTB4 and 5-HETE in cell lysate. A23187-stimulated release of incorporated [3H]AA from intact cells was not inhibited by CSA. CSA did not inhibit the synthesis of cysteinyl LTs and LTB4 when cells incubated with LTA4 as the substrate. These results indicate that the inhibitory effects of CSA on the synthesis of LTs and 5-HETE in intact cells are attributable to a modulatory action on a step in the series of intracellular events that includes the activation of 5-lipoxygenase, which are initiated by Ca2+ influx and end in the release of metabolites from the cell membrane, rather than to a direct inhibitory action on enzymes in the LT biosynthetic pathway.

Topics: Animals; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Calcimycin; Chromatography, High Pressure Liquid; Cyclosporine; Hydroxyeicosatetraenoic Acids; Leukemia, Basophilic, Acute; Leukotriene B4; Leukotrienes; Phospholipases A; Rats; Tumor Cells, Cultured

Studies were made on the effects of stilbene derivatives isolated from medicinal plants on arachidonate metabolism and degranulation in human polymorphonuclear leukocytes (PMN-L). Resveratrol (3,4',5-trihydroxystilbene) isolated from the roots of Reynoutria japonica was found to inhibit the 5-lipoxygenase products 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE), 5,12-dihydroxy-6,8,10,14-eicosatetraenoic acid (5,12-diHETE) and leukotriene C4(LTC4); its concentrations for 50% inhibition (IC50) were 8.90 x 10(-6) M, 6.70 x 10(-6) M and 1.37 x 10(-6) M, respectively. The IC50 of 5-HETE, 5,12-diHETE and LTC4 formations of synthetic 3,3',4-trihydroxystilbene were 5.90 x 10(-6) M, 6.30 x 10(-7) M and 8.80 x 10(-7) M, respectively. Moreover, they inhibited the release of lysosomal enzyme such as lysozyme and beta-glucuronidase induced by calcium ionophore A 23187 from human PMN-L at 10(-3)-10(-4) M.

Topics: Arachidonic Acid; Autoradiography; Calcimycin; Cell Degranulation; Cyclic AMP; Fatty Acids, Unsaturated; Glucuronidase; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Leukotriene C4; Lysosomes; Muramidase; Neutrophils; Plant Roots; Plants, Medicinal; Resveratrol; Stilbenes; Structure-Activity Relationship

Products of arachidonic acid (AA) metabolism via the lipoxygenase pathways may have key roles in the maintenance of pregnancy and the onset of labor. We have determined whether calcium ionophores can modulate the rate of biosynthesis within the uterus of five important arachidonate lipoxygenase metabolites, i.e. leukotriene B4 (LTB4), LTC4, 5-hydroxyeicosatetraenoic acid (5-HETE), 12-HETE, and 15-HETE. Amnion, chorion, and decidual cells were isolated, grown to confluence and incubated with ionomycin. The production of LTB4, LTC4, 5-HETE, 12-HETE, and 15-HETE was determined using specific radioimmunoassays. Cell-specific, concentration-related stimulatory actions of ionomycin on 5-HETE, 12-HETE, 15-HETE, and LTC4 but not LTB4 production were found. A23187 had effects similar to ionomycin. Hence elevation of intracellular calcium levels can result in enhanced intrauterine production of arachidonate lipoxygenase metabolites that may affect pregnancy outcome.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Amnion; Arachidonic Acid; Calcimycin; Chorion; Decidua; Female; Humans; Hydroxyeicosatetraenoic Acids; Ionomycin; Leukotriene B4; Leukotriene C4; Lipoxygenase; Pregnancy; Uterus

The cutaneous antiinflammatory action of Dead-Sea brine is thought to be due to magnesium ions. To elucidate their mode of action, we studied the influence of isotonic solutions containing high concentrations of Mg2+ (up to 115mM) on the formation of 5-lipoxygenase-derived eicosanoids in human polymorphonuclear leukocytes. The cells were stimulated by either ionophore A23187 or the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine. We observed a pronounced inhibition of the formation of leukotriene B4 and 5-hydroxyeicosatetraenoic acid from either added [1-14C] or endogenously liberated arachidonic acid. In the latter case, the sum of arachidonic acid and its oxygenation products was also markedly diminished. The inhibitory effects of Mg2+ depended in a reciprocal manner on the concentration of Ca2+ in the incubation medium. An unspecific damage to cells as reason for the inhibitory effects was excluded. Human recombinant 5-lipoxygenase was also inhibited by Mg2+ in the same concentration range (IC50 16 mM). These data suggest that high concentrations of Mg2+ inhibit the eicosanoid metabolism both at the level of the liberation of arachidonic acid and by direct inhibition of the 5-lipoxygenase enzyme.

Topics: Arachidonate 5-Lipoxygenase; Arachidonic Acid; Calcimycin; Calcium; Chromatography, High Pressure Liquid; Eicosanoids; Fatty Acids; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Ionophores; Leukotriene B4; Lipoxygenase Inhibitors; Magnesium; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Recombinant Proteins

Ascites is a readily available source of human macrophages (M phi), which can be used to study M phi functions in vitro. We characterized the mediators of inflammation produced by human peritoneal M phi (hp-M phi) obtained from patients with portal hypertension and ascites. The production of the cytokines interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) was found to be lipopolysaccharide (LPS) concentration dependent (0-10 micrograms/ml) with a maximal production at 10 micrograms/ml and also dependent on the time of exposure to the stimulus (0-36 h). IL-1 beta, IL-6 and TNF-alpha production after LPS administration reached a plateau at 24 h. In vitro stimulation for 24 h with LPS does not influence the eicosanoid production from endogenous arachidonate. 13 min of exposure of the cells to the calcium ionophore A23187 gives a significant increase in eicosanoid production from both exogenous and endogenous arachidonate. The main eicosanoids produced are the 5-lipoxgenase products LTB4 and 5-hydroxyeicosatetraenoic acid (HETE). The increase in production of the other eicosanoids is not significant. The eicosanoid production depends on the stimulus concentration. The optimal A23187 concentration is 1 microM. Oxygen radical production was measured in the M phi by a flowcytometric method. The fluorescence intensity of phorbol 12-myristate 13-acetate stimulated and dihydro-rhodamine 123 loaded hp-M phi increases significantly after 15 min. We conclude that LPS stimulation of hp-M phi from liver disease results in similar production of IL-1 beta, IL-6 and TNF-alpha, but that the profile of the eicosanoid production of these M phi stimulated with LPS and A23187 differs from M phi of other origin and species.

Topics: Adult; Aged; Ascitic Fluid; Calcimycin; Cytokines; Eicosanoids; Female; Humans; Hydroxyeicosatetraenoic Acids; Interleukin-1; Interleukin-6; Leukotriene B4; Macrophages; Male; Middle Aged; Respiratory Burst; Tumor Necrosis Factor-alpha

Preincubation of human neutrophils with phorbol esters or soluble diglycerides enhances subsequent f-Met-Leu-Phe (fMLP)-stimulated arachidonate mobilization and leukotriene B4 (LTB4) synthesis. We have recently reported that 1,3-dioctanoylglycerol (1,3-diC8) is equipotent with 1,2-sn-dioctanoylglycerol (1,2-diC8) as priming agent, thus suggesting that the priming effects of diacylglycerols are protein kinase C (PKC) independent (Rosenthal et al., 1993, Biochim. Biophys. Acta 1177:79-86). In order to further investigate this question, the present study has directly compared the effects of oleoylacetylglycerol (OAG) and the PKC activator, phorbol 12-myristate 13-acetate (PMA), on agonist-stimulated lipid metabolism. The results indicate that both OAG and PMA dose dependently enhance f-Met-Leu-Phe (fMLP)-stimulated release of [3H]arachidonate. Optimal concentrations of OAG (5 microns) and PMA (10 nM) are equipotent in increasing fMLP-stimulated arachidonate mobilization as quantitated either with total radioactivity or by mass measurements of free arachidonate. By contrast OAG is sixfold more effective than PMA in enhancing synthesis of 5-lipoxygenase (5-LO) metabolites by mass and two to threefold more effective than PMA in enhancing synthesis of [3H]eicosanoids. Furthermore, OAG, but not PMA, enhances fMLP-stimulated synthesis of platelet-activating factor. By contrast, PMA directly stimulates [3H]arachidonate mobilization, while OAG (20 microM) does not; despite these differences, the combined effects of PMA + OAG on subsequent agonist-stimulated arachidonate release are not greater than those of PMA alone. In cells challenged with subthreshold concentrations (< 0.1 microM) of the calcium ionophore A23187, both OAG and PMA stimulate [3H]arachidonate release but not [3H]LTB4 synthesis. These findings suggest that OAG does not directly activate 5-LO, but instead couples arachidonate mobilization to leukotriene synthesis in a PKC-independent manner.

Topics: 1-Alkyl-2-acetylglycerophosphocholine Esterase; Arachidonic Acid; Biological Transport; Calcimycin; Diglycerides; Eicosanoids; Fatty Acids; Humans; Hydroxyeicosatetraenoic Acids; Indoles; Leukotriene Antagonists; Leukotriene B4; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Phospholipases A; Tetradecanoylphorbol Acetate

The hypothesis was tested that polymorphonuclear leukocytes (PMNL) from patients with gram-negative bacteremia are primed to produce leukotriene B4 (LTB4) or 5-hydroxyeicosatetraenoic acid (5-HETE), in response to concentrations of calcium ionophore A23187, which are substimulatory for control PMNL. PMNL from 11 bacteremic patients and 8 healthy subjects (11 samples) produced similar quantities of LTB4, omega-oxidation products of LTB4, and 5-HETE after incubation with 0.3 and 0.5 microM A23187 for 5 min. At the detection threshold of 0.3 microM A23187, LTB4 was present in PMNL preparations from 9 of 11 patients and 7 of 11 control samples and 5-HETE from the same 9 patients and from 6 controls. There was no correlation between LTB4 or 5-HETE and plasma levels of endotoxin. In this group of patients, priming of PMNL by gram-negative bacteremia did not lead to enhanced production of LTB4, its omega-oxidation products, or 5-HETE when PMNL were challenged with low concentrations of A23187.

Topics: Adult; Aged; Aged, 80 and over; Bacteremia; Calcimycin; Endotoxins; Female; Gram-Negative Bacterial Infections; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Male; Middle Aged; Neutrophils

Studies were undertaken to examine the effect of methotrexate (MTX) administered in vivo or added in vitro upon the production of the 5-lipoxygenase (5-LO) metabolites of arachidonic acid (AA) by rat neutrophils. Peptone-induced peritoneal exudate cells were stimulated by A23187 and the cell suspensions assayed for leukotriene B4 (LTB4), the all-trans isomers of LTB4 and 5-hydroxyeicosatetraenoic acid (5-HETE) using high-pressure liquid chromatography. MTX added in vitro to rat cells was weakly inhibitory; however, no inhibition of LTB4 production was seen following in vivo administration of MTX by oral, subcutaneous or intraperitoneal routes. On the basis of these findings, inhibition of 5-LO metabolism does not appear to explain the anti-inflammatory effects of MTX.

Topics: Administration, Oral; Animals; Arachidonic Acids; Calcimycin; Chromatography, High Pressure Liquid; Exudates and Transudates; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Injections, Intraperitoneal; Injections, Subcutaneous; Leukotriene B4; Lipoxygenase; Male; Methotrexate; Neutrophils; Rats; Rats, Wistar

The aim of the present work was to study interactions between the synthesis of platelet-activating factor (PAF) and leukotriene B4 (LTB4) in human polymorphonuclear leukocytes (PMNs) in vitro. PAF, at nanomolar concentrations, stimulated calcium ionophore A23187-activated PMNs to release LTB4 and 5-hydroxyeicosatetraenoic acid (5-HETE). This seems to be a receptor-mediated process as it was blocked by a PAF receptor antagonist WEB 2086 (IC50 6.6 +/- 3.9 microM). Moreover, LTB4 stimulated the formation of PAF in activated PMNs. WEB 2086 did not, however, alter PMN migration towards either LTB4 or the chemotactic peptide FMLP. This suggests that the enhancement of PAF synthesis in response to LTB4 is a concomitant event rather than a mediating process in LTB4-induced chemotactic movement of PMNs. These effects are implicated in the complex network of interactions between inflammatory mediators that results accumulation and activation of PMNs in the exacerbation of inflammatory processes.

Topics: Arachidonate 5-Lipoxygenase; Azepines; Calcimycin; Chemotaxis, Leukocyte; Drug Interactions; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Leukotriene B4; Neutrophils; Platelet Activating Factor; Platelet Membrane Glycoproteins; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Triazoles

The present study was carried out to further characterize the role of non-inflammatory cells in the inflammatory process. More specifically, we have investigated whether human epithelial cells can generate inflammatory lipid mediators via activation of the 5-lipoxygenase pathway. The cells were stimulated with the calcium ionophore A23187 (5 microM) for different periods of time, after which the production of eicosanoids was determined by gradient reverse-phase high performance liquid chromatography (RP-HPLC) and rapid spectral detection, permitting continuous ultraviolet spectroscopy. In both non-prelabeled cells and cells prelabeled with [1-14C]arachidonic acid, cell stimulation for 30 min or more resulted in the production of two important 5-lipoxygenase products: 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B4 (LTB4). Stimulation for 15 min or less, however, led solely to the formation of 5-HETE. The identities of 5-HETE and LTB4 were confirmed by HPLC retention times and UV spectra, as well as by gas chromatography-mass spectrometry for 5-HETE and radioimmunoassay for LTB4. It can therefore be concluded that human epithelial cells in general can produce important inflammatory mediators, which suggests that epithelial cells may play a more active role in the inflammatory process than is normally assumed.

Topics: Calcimycin; Cell Line; Chromatography, High Pressure Liquid; Epithelium; Gas Chromatography-Mass Spectrometry; Humans; Hydroxyeicosatetraenoic Acids; Intestinal Mucosa; Intestines; Leukotriene B4; Spectrophotometry, Ultraviolet

Cloricromene, an antithrombotic agent known to inhibit the release of arachidonic acid (AA) in stimulated human platelets, was tested for its effects on arachidonate release and metabolism in human polymorphonuclear leucocytes (PMNs). Cloricromene dose-dependently suppressed the release of leukotriene B4 (LTB4), as assessed by radioimmunoassay, from both isolated PMNs and human whole blood stimulated with the calcium ionophore A23187 or with serum-treated zymosan (STZ). The inhibitory effect was higher when the concentration of the stimulating agent was weaker. Cloricromene also inhibited dose-dependently the liberation of LTB4, LTC4, LTD4 and 5-hydroxy-6,8,11,14-eicosatraenoic acid as assessed by HPLC in the supernantant of A23187-stimulated PMNs. Finally, the drug was able to suppress the release of [3H]AA from purified human PMNs prelabeled with the radioactive fatty acid and stimulated with either A23187 or with STZ. The A23187-induced decrease in the radioactivity of phosphatidylinositol, the phospholipid class mainly involved in AA release in stimulated PMNs, was also inhibited by cloricromene. Cloricromene suppresses leukotriene formation in human PMNs by reducing AA release from membrane phospholipids, possibly through interference with phospholipase A2 activation; this activity may contribute to the leucocyte-inhibitory effects reported previously for cloricromene.

Topics: Arachidonic Acid; Calcimycin; Chromonar; Dose-Response Relationship, Drug; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Membrane Lipids; Neutrophils; Phospholipids; SRS-A; Zymosan

The present studies were conducted to understand better the regulation of phospholipase A2 (PLA2)-dependent mobilization of lipid mediators by arachidonic acid (C20:4). After stimulation of human neutrophils, g.l.c./m.s. analysis of non-esterified fatty acids indicated that the quantity of C20:4 increased as a function of time after stimulation, from undetectable quantities to > 800 pmol/10(7) cells. In contrast with C20:4, the quantities of other free fatty acids such as oleic and linoleic were high in resting cells and did not change after stimulation. Some 15% of the C20:4 released from cellular lipids remained cell-associated. To examine the effect of C20:4 on its own release, neutrophils were exposed to [2H8]C20:4, to differentiate it by g.l.c./m.s. from naturally occurring C20:4. In A23187-stimulated neutrophils, low concentrations (5-10 microM) of [2H8]C20:4 added just before A23187 increased the quantity of C20:4 produced by the cell, whereas higher concentrations (30-50 microM) decreased the quantity of C20:4 released from phospholipids. As other measures of PLA2 activity, the effects of C20:4 on production of platelet-activity factor (PAF) and leukotriene B4 (LTB4) were assessed. C20:4 treatment just before stimulation of neutrophils blocked PAF and LTB4 production in a concentration-dependent manner (IC50 10-20 microM). The effect of C20:4 was not blocked by the cyclo-oxygenase inhibitor naproxine (10 microM), nor could it be mimicked by 1 microM LTB4, 5-hydroxyeicosa-6,8,11,14-tetraenoic acid (5HETE), 5-hydroperoxyeicosa-6,8,11,14-tetraenoic acid (5HPETE) or 15-hydroxyeicosa-5,8,11,13-tetraenoic acid (15HETE). The 5-lipoxygenase (5LO) inhibitor zileuton induced a concentration-dependent decrease in PAF, with a maximal effect of a 50% decrease at 10-50 microM. The decrease in PAF by the 5LO inhibitor could not be circumvented by addition of 1 microM 5HETE, 5HPETE and LTB4, and may be attributed to the capacity of zileuton to increase the quantity of C20:4 in A23187-treated neutrophils. The inhibitory effect of C20:4 (20-40 microM) on PAF production could be antagonized by the protein kinase C inhibitor staurosporine (30 nM), but not by inhibitors of protein kinase A, tyrosine kinase or calmodulin kinase II. Taken together, these data demonstrate that C20:4 is selectively released from membrane phospholipids of A23187-stimulated neutrophils, and this C20:4 may play an important role in regulating the mobilization of C20:4 by altering PLA2 activi

Topics: Arachidonic Acid; Calcimycin; Fatty Acids; Fatty Acids, Nonesterified; Gas Chromatography-Mass Spectrometry; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Leukotrienes; Neutrophils; Phospholipases A; Phospholipases A2; Platelet Activating Factor

It has been demonstrated that both auranofin and eicosapentaenoic acid (EPA) have anti-inflammatory properties and both inhibit neutrophil leukotriene B4 (LTB4) synthesis. In the present study, we examined interactions between auranofin and EPA with regard to inhibition of human neutrophil LTB4 synthesis. Auranofin inhibited A23187-stimulated LTB4 synthesis, but the dose required for inhibition of LTB4 was greater than that required for inhibition of other 5-lipoxygenase metabolites; namely, the all-trans isomers of LTB4 and 5-hydroxyeicosatetraenoic acid. These results were explained after a comparison of the rates of synthesis of these 5-lipoxygenase metabolites in the presence and absence of added arachidonic acid which led to the conclusion that leukotriene A hydrolase, the enzyme catalysing the formation of LTB4, was saturated with substrate and rate-limiting for LTB4 synthesis during A23187 stimulation. In combination, auranofin and EPA had a simple additive effect on inhibition of the 5-lipoxygenase pathway. Favorable drug/EPA combinations have the potential to provide a beneficial anti-inflammatory effect with lower levels of each component than are required when used individually.

Topics: Arachidonic Acid; Auranofin; Calcimycin; Dose-Response Relationship, Drug; Drug Interactions; Eicosapentaenoic Acid; Epoxide Hydrolases; Humans; Hydroxyeicosatetraenoic Acids; Isomerism; Leukotriene B4; Neutrophils

Among the cells which participate in amplification of the local inflammatory reaction in asthma, neutrophils (PMN) are pro-inflammatory cells that can generate inflammatory mediators and arachidonic acid derivatives in particular. In asthmatic patients (AP) with attacks, the capacity of blood PMN to produce 5-lipoxygenase metabolites was investigated and compared to the response in healthy subjects (HS). PMN from 6 AP and from 6 HS were stimulated by calcium ionophore A23187 and arachidonate 5-lipoxygenase metabolites were analyzed by reverse-phase HPLC. LTB4, 6-trans LTB4, omega OH-LTB4 and 5-HETE were identified. In AP, total LTB4 synthesis was enhanced as compared to synthesis with PMN in HS. But the total 5-HETE synthesis by PMN from AP was decreased. Thus, the inflammatory potential of PMN from AP was enhanced in comparison to HS. The anti-inflammatory effect of nedocromil sodium (NS) was studied in the 5-lipoxygenase metabolism of arachidonic acid. NS (10(-4) mol/l) inhibited total LTB4 synthesized by PMN in AP but not in HS. We conclude that NS affects leukotriene synthesis only in cells with enhanced inflammatory potential.

Topics: Adult; Anti-Inflammatory Agents, Non-Steroidal; Arachidonate 5-Lipoxygenase; Asthma; Calcimycin; Chromatography, High Pressure Liquid; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Leukotriene B4; Male; Middle Aged; Nedocromil; Neutrophils; Quinolones

Alveolar macrophages release greater amounts of leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) after A23187 stimulation than do blood monocytes. The mechanisms for this enhanced 5-lipoxygenase activity in alveolar macrophages are unknown. In these studies, we determined whether alveolar macrophages have greater amounts of the enzyme 5-lipoxygenase than do blood monocytes. We confirmed that alveolar macrophages released greater amounts of LTB4 after A23187 stimulation than did equivalent numbers of blood monocytes. In both the presence and absence of A23187, alveolar macrophages had greater amounts of immunoreactive 5-lipoxygenase, determined by Western analysis, on a per cell and a per protein basis than did blood monocytes. The amounts of 5-lipoxygenase enzyme in the cells roughly correlated with the amounts of LTB4 released by both types of cells. These observations suggest that A23187 stimulates alveolar macrophages to release greater amounts of LTB4 and 5-HETE than blood monocytes, in part, due to the greater amounts of 5-lipoxygenase.

Topics: Arachidonate 5-Lipoxygenase; Calcimycin; Cells, Cultured; Chromatography, High Pressure Liquid; Humans; Hydroxyeicosatetraenoic Acids; Immunoblotting; In Vitro Techniques; Leukotriene B4; Macrophages, Alveolar; Monocytes

The biosynthesis of leukotrienes is known to occur through a series of complex processes which, in part, can be influenced by cell-cell interactions. Several studies have suggested that arachidonic acid availability is a major limiting step for leukotriene biosynthesis and that its transfer between cells can represent a significant source of this precursor. Accordingly, effect of time and source of arachidonic acid on transcellular leukotriene synthesis was studied in mixed platelet/neutrophil populations challenged with the calcium ionophore A23187. A time-dependent contribution of platelet-derived as well as neutrophil-derived arachidonate was found in the selective formation of neutrophil 5-lipoxygenase metabolites. Utilization of platelet or neutrophil arachidonate was followed by incorporation of radiolabeled arachidonic acid into platelet or neutrophil phospholipids prior to stimulation. Specific activity of liberated arachidonic acid along with numerous 5-lipoxygenase products (including LTB4, 20-hydroxy-LTB4, 5-HETE and LTC4) was determined in order to follow mass and radiolabel. A large amount of platelet-derived arachidonic acid was released in the first 1.5 min, whereas 10 min platelet-derived arachidonate was much lower in amount but significantly higher in specific activity, suggesting different precursor pools. The platelet-derived arachidonate was heavily utilized by the neutrophils at the early time points for formation of 5-HETE and delta 6-trans-LTB4 isomers, but appeared to contribute only marginally to the constitutive metabolism of neutrophil arachidonate into LTB4. Results from these experiments suggest different pools of 5-lipoxygenase in the neutrophil and indicate a time and source dependent modulation of arachidonate metabolism in mixed cell interactions.

Topics: Arachidonate 5-Lipoxygenase; Arachidonic Acid; Blood Platelets; Calcimycin; Cells, Cultured; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Neutrophils; SRS-A; Stereoisomerism; Time Factors

Polymorphonuclear neutrophils (PMN) generate 5-HETE which can be retained within cells as free metabolites or esterified into cellular lipids. Since this metabolite has been shown to have certain inflammatory properties, we compared the generation and distribution profile of 5-HETE in A 23187-stimulated PMN from asthmatic patients (AP) and normal subjects (NS). 5-HETE was analyzed using RP-HPLC. After 5 min, total 5 HETE generation was similar in the two populations. However, esterified 5-HETE was significantly enhanced in AP (72 +/- 3% versus 47 +/- 2% of the total synthesis, p less than 0.005), whereas intracellular free 5-HETE was decreased (13 +/- 3% versus 37 +/- 4%, p less than 0.005) and similar low release was observed. Kinetic studies showed that PMN from AP esterified 5-HETE more rapidly and to a greater extent than PMN from NS. By contrast, more intracellular free 5-HETE was recovered in PMN from NS. Esterification seems to be the major pathway of 5-HETE metabolism in PMN from AP. Moreover, we showed that most of the 5-HETE added exogenously was esterified into cellular lipids. In these experimental conditions, PAF-induced migration of PMN was increased. The enhanced ability of PMN to migrate could be due to the increase of 5-HETE esterification process.

Topics: Adult; Asthma; Calcimycin; Cell Migration Inhibition; Chromatography, High Pressure Liquid; Esterification; Humans; Hydroxyeicosatetraenoic Acids; Middle Aged; Neutrophils; Platelet Activating Factor; Reference Values

Blood cells from the crab, Carcinus maenas, stimulated with calcium ionophore A23187, in the presence of exogenous fatty acid, produced cyclooxygenase, lipoxygenase and monooxygenase derivatives of eicosatetraenoic (20:4(n - 6)) and eicosapentaenoic (20:5(n - 3)) acids. Isolation, identification and quantification of these products was achieved using chiral and reverse phase-high performance liquid chromatography, gas-chromatography, radioimmunoassay and gas chromatography-mass spectrometry. The principle metabolites observed were 8-hydroxy fatty acids and 'E' series prostaglandins. Smaller amounts of thromboxane B2, 6-keto-prostaglandin F1 alpha and 5-, 9-, 11-, 12- and 15-hydroxy-eicosatetraenoic acids were also synthesised. Lipoxygenase, cyclooxygenase and cytochrome P-450 inhibitors were used to investigate the mode of product formation. Mixtures of hydroxy-fatty acid enantiomers were produced and the dominant chiral form varied with the position of the hydroxyl group. No leukotrienes or lipoxins were detected.

Topics: Animals; Arachidonic Acids; Blood Cells; Brachyura; Calcimycin; Chromatography, High Pressure Liquid; Cyclooxygenase Inhibitors; Eicosanoids; Enzyme Activation; Hemolymph; Hydroxyeicosatetraenoic Acids; Indomethacin; Masoprocol; Proadifen

The influence of exogenously supplied free arachidonic acid (AA) and eicosapentaenoic acid (EPA) on the 5-lipoxygenase metabolism in human neutrophils (PMN) was investigated. Simultaneous application of A23187 with incremental concentrations of free AA caused a dose-dependent augmentation of the ionophore-elicited eicosanoid generation [release of leukotriene B4 and its omega-oxidation products, nonenzymatic hydrolysis products of leukotriene A4, and 5-hydroxyeicosatetraeneoic acid (5-HETE)]. A23187 challenge in the presence of free EPA resulted in the dose-dependent appearance of corresponding n - 3-derived metabolites, parallelled by a decrease in 4-series leukotrienes and 5-HETE. The inflammatory ligands formyl-methionyl-leucyl-phenylalanine and platelet-activating factor evoked no substantial eicosanoid generation in the absence of exogenously supplied polyunsaturated fatty acids (PUFAs). Addition of free AA or EPA in parallel with the ligand challenge evoked exclusive and dose-dependent generation of the respective leukotrienes and 5-HETE or 5-hydroxyeicosapentaenoic acid. Total amounts of 5-lipoxygenase products elicited under these conditions approached those in ionophore-stimulated PMN, with platelet-activating factor challenge surpassing the formyl-methionyl-leucylphenylalanine-evoked effect by approximately 50%. Two thirds of the maximum effect was obtained in the presence of only 10 microM free PUFA. Use of labeled fatty acids suggested exclusive origin of the eicosanoids from the exogenously provided precursor PUFA. Critical dependence on timing was noted; maximum response occurred upon simultaneous application of PUFA and ligand, and only 5 min of delay between AA or EPA addition and ligand challenge sufficed to reduce the formation of respective metabolites to less than 20%. EPA competed with AA and was noted to be the preferred substrate for ligand-evoked eicosanoid synthesis. In contrast to the simultaneous addition of free PUFAs, preloading of PMN with AA or EPA for 60 min revealed only very moderate or even no influence on ionophore- or ligand-evoked eicosanoid synthesis. We conclude that inflammatory ligands induce marked stimulation of PMN eicosanoid synthesis, with critical dependence on the presence of free precursor PUFAs. Preference of EPA over AA is observed under these conditions.

Topics: Calcimycin; Chromatography, High Pressure Liquid; Fatty Acids, Nonesterified; Humans; Hydrolysis; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Leukotrienes; Ligands; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Oxidation-Reduction; Platelet Activating Factor

Leukotriene B4 (LTB4) and zymosan-activated plasma (ZAP) were each instilled into the lungs of steers to elicit alveolar neutrophils for subsequent functional analysis. Prior to instillation of either agent, bronchoalveolar lavage cell populations consisted of 95.8 +/- 0.4% macrophages (mean +/- SEM). Four hours after instillation of LTB4 or ZAP, the lavage cell populations consisted of 75.0 +/- 8.8% and 90.7 +/- 0.7% neutrophils, respectively. Alveolar neutrophils elicited with LTB4 and stimulated with the calcium ionophore A23187 released diminished amounts of LTB4 and increased amounts of 5-hydroxyeicosatetraenoic acid (5-HETE) as compared to circulating neutrophils. Release of superoxide anion was decreased for LTB4-elicited alveolar neutrophils as compared to circulating cells, while bacterial killing was unchanged. ZAP-elicited alveolar neutrophils released diminished amounts of LTB4 when stimulated with A23187 as compared to circulating neutrophils. There were no differences observed in 5-HETE levels between the two cell populations. In addition, release of superoxide anion was diminished among ZAP-elicited alveolar cells, while bacterial killing was unchanged. Incubation of circulating neutrophils with LTB4 did not influence the release of arachidonate metabolites, superoxide anion, or bacterial killing. However, incubation of circulating neutrophils with ZAP, followed by A23187 resulted in a reduction in the release of LTB4, as compared to control cells. Prior exposure to ZAP did not influence the release of superoxide anion or bacterial killing by the circulating neutrophils.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Bacteriolysis; Calcimycin; Cattle; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Male; Neutrophils; Pulmonary Alveoli; Superoxides; Zymosan

The N-substituted quinolylmethoxyphenylamines, ETH603, ETH615 and ETH647, inhibited the formation of LTB4 in rat peritoneal leukocytes, human peripheral polymorphonuclear leukocytes and canine whole blood. In rat and human cells, the compounds also inhibited the formation of 5-HETE and stimulated the synthesis of 15-HETE. In rat leukocytes, the compounds were 15-30 times more potent inhibitors of LTB4 synthesis than nordihydroguaiaretic acid, but in canine whole blood they were significantly less potent, possibly due to protein binding. However, after oral administration of the compounds to dogs a long-lasting inhibition of LTB4 production in peripheral blood was observed at serum concentrations much lower than those required in vitro. Furthermore, the compounds inhibited the LTB4-directed chemotaxis and the phagocytosis of C. albicans blastospores by canine polymorphonuclear leukocytes both in vitro and following oral administration. The calcium ionophore A23187-induced release of LTB4 in the peritoneal cavity of rats was also inhibited by systemic administration of the compounds. We therefore conclude that these novel quinolines are orally active 5-lipoxygenase inhibitors which may accumulate in inflammatory cells in vivo, leading to potent inhibition of leukotriene biosynthesis and cell function.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Chemotaxis, Leukocyte; Dogs; Free Radicals; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Leukotriene E4; Neutrophils; Phagocytosis; Quinolines; Rats; SRS-A

The production of 5-lipoxygenase products from arachidonic acid was investigated in polymorphonuclear leukocytes (PMNL) isolated from non-diabetic and alloxan-induced diabetic rabbits: (i) production of 5-hydroxyeicosatetraenoic acid, leukotriene B4, and the two 6-trans-leukotriene B4 isomers were significantly decreased in the PMNL of diabetic rabbits when compared to non-diabetic rabbits; (ii) production of LTB4 and 5-HETE from diabetic PMNL required the addition of Ca2+ and A23187 to a greater degree than control incubations; and (iii) the availability of substrate in the PMNL of diabetics was not a limiting factor for 5-lipoxygenase product formation. Alternative pathways of arachidonic acid metabolism were also evaluated: the recovery of exogenous leukotriene B4 and 5-hydroxyeicosatetraenoic acid were identical using PMNL from control and diabetic rabbits and peptido-leukotrienes were not detected by radioimmunoassay. The data suggest that the activity of 5-lipoxygenase and the production of 5-hydroperoxyeicosatetraenoic acid in the diabetic PMNL may be limiting factors since the formation of leukotriene B4, leukotriene B4 isomers, and 5-hydroxyeicosatetraenoic acid are depressed in PMNL of diabetic rabbits. Alternative pathways do not account for the conversion of arachidonic acid to other products nor are the elimination pathways for LTB4 and 5-HETE different. Decreased formation of 5-hydroxyeicosatetraenoic acid and leukotriene B4 could predispose diabetic subjects to infection due to a decrease in mediators leading to the local accumulation of PMNL in the inflammatory response.

Topics: Animals; Arachidonate 5-Lipoxygenase; Arachidonic Acids; Calcimycin; Calcium; Cell Separation; Chromatography, High Pressure Liquid; Diabetes Mellitus, Experimental; Dinoprostone; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Kinetics; Leukotriene B4; Male; Neutrophils; Rabbits; Reference Values; Thromboxane B2

Pyocyanin, a phenazine pigment produced by Pseudomonas aeruginosa, and its metabolite 1-hydroxyphenazine inhibited leukotriene B4 and 5-hydroxyeicosatetraenoic acid production by up to 70% in human neutrophils stimulated with the calcium ionophore A23187 (5 microM). This potential anti-inflammatory effect was dose dependent and occurred at low concentrations (10 to 50 microM) that did not inhibit neutrophil viability.

Topics: Calcimycin; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Lymphocyte Activation; Neutrophils; Phenazines; Pseudomonas aeruginosa

Changes in eicosanoid generation have been examined in stimulated human peripheral leukocytes incubated with plasma lipoprotein fractions. Leukocytes (2.5 X 10(7) cells/ml, 90% neutrophils) were incubated with physiological concentrations of low density lipoprotein (LDL), high density lipoprotein (HDL) and very low density lipoprotein (VLDL). No release of leukotriene B4 (LTB4) or platelet activating factor (PAF) was noted prior to cell stimulation with either calcium ionophore, opsonized zymosan or FMLP. After stimulation with ionophore, LDL led to a 40% enhancement of LTB4 release compared to control incubations while there was no effect on PAF production. HDL caused a small but not significant increase in LTB4 while VLDL had no effect on the release of LTB4. The formation of the other major lipoxygenase product 5-hydroxy-eicosatetraenoic acid (5-HETE) was decreased by 20% following LDL incubation and by more than 50% after VLDL incubation compared to controls. LTB4 release was also enhanced by 27% after incubation with LDL and stimulation with opsonized zymosan. LDL did not cause any increase in superoxide production by leukocytes stimulated with opsonized zymosan or PMA. PGE2 release was stimulated directly in cells incubated with lipoproteins, particularly LDL and VLDL. Oxidised LDL enhanced LTB4 production to an even greater extent than native LDL. The observed enhancement of LTB4 by LDL is not the result of LDL oxidation during incubation, the provision of arachidonic acid substrate by the lipoprotein nor the uptake of cholesterol by the cell. The effect is most likely associated with the binding of LDL to the cell membrane, since LTB4 enhancement was partially blocked by dextran sulphate.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Arachidonic Acid; Calcimycin; Dextran Sulfate; Dinoprostone; Fatty Acids; Humans; Hydroxyeicosatetraenoic Acids; Leukocytes; Leukotriene B4; Lipoproteins, LDL; Platelet Activating Factor

Topics: Acylation; Calcimycin; Cell Aggregation; Esterification; Humans; Hydroxyeicosatetraenoic Acids; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Phosphatidylinositols; Tetradecanoylphorbol Acetate

Topics: Arachidonate 15-Lipoxygenase; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Arachidonic Acids; Asthma; Bronchi; Calcimycin; Epithelium; Humans; Hydroxyeicosatetraenoic Acids; Lung

We had previously shown that inhibition of cyclooxygenase in vitro by indomethacin can cause increased formation of products of the 5-lipoxygenase pathway of arachidonic acid metabolism in leukocytes. To determine if this effect also occurred in vivo, we studied leukocyte arachidonic acid metabolism in 12 volunteers before and after ingestion of 150 mg indomethacin daily for 3 days. Blood was collected before treatment and 2 hours, 2 days, and 5 days after the final dose of indomethacin. Serum thromboxane B2, a measure of platelet cyclooxygenase activity, was profoundly suppressed 2 hours after the final dose of indomethacin but had recovered to control values at 2 days. Mixed leukocyte suspensions and purified neutrophil suspensions were prepared and stimulated with calcium ionophore A23187 and the resultant 5-lipoxygenase metabolites were quantified by HPLC. Two hours after the final dose of indomethacin, the stimulated levels of 5-hydroxyeicosatetraenoic acid, leukotriene B4, and leukotriene C4 were significantly increased to 247% +/- 68%, 135% +/- 14%, and 149% +/- 23% of pretreatment values, respectively. Two days after the final dose of indomethacin, 5-hydroxyeicosatetraenoic acid levels were still significantly elevated. By 5 days all parameters had returned to baseline. Similar effects were not observed in purified neutrophil suspensions, probably because of the loss of indomethacin from the cells during the multiple washing procedures used in their preparation. This is in accord with the reversible nature of the inhibitory effect of indomethacin on cyclooxygenase. We conclude that indomethacin at a commonly used dose increases the ability of circulating leukocytes to produce 5-lipoxygenase products.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Adult; Arachidonate 12-Lipoxygenase; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Arachidonic Acids; Calcimycin; Cell Membrane; Female; Glucuronidase; Humans; Hydroxyeicosatetraenoic Acids; Indomethacin; Leukocytes; Leukotriene B4; Male; Middle Aged; Neutrophils; Phospholipids; Prostaglandin-Endoperoxide Synthases; SRS-A; Superoxides; Thromboxane B2

Binding of LA350, a lymphoblastoid human B cell line, by phorbol myristate acetate (PMA) plus a calcium ionophore, either ionomycin or A23187, produced unique alterations in the release of arachidonic acid (AA) from cellular phospholipids. After equilibrium labeling of cells with radioactive fatty acids, [14C]AA demonstrated a selective enhanced release from the cells in response to the binding of PMA plus calcium ionophore as compared to the release of [14C]stearic acid (STE), [3H]oleic acid (OLE) and [3H]palmitic acid (PAL). The major phospholipid sources of the released [14C]AA were shown to be phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol. The participation of protein kinase C (PKC) in the enhanced synergistic release of [14C]AA was demonstrated by the inhibition of the release by the PKC inhibitor, staurosporine. Approximately 2-6% of the labeled AA liberated was converted to 5-hydroxyeicosatetraenoic acid by an endogenous 5-lipoxygenase. Therefore during cell activation the B cell is capable of liberating AA via a PKC-dependent mechanism, implicating AA and/or its metabolites in signal transduction.

Topics: Arachidonic Acid; Arachidonic Acids; B-Lymphocytes; Calcimycin; Cell Line; Humans; Hydroxyeicosatetraenoic Acids; Membrane Lipids; Phospholipids; Protein Kinase C; Stearic Acids; Tetradecanoylphorbol Acetate

In animal cells arachidonic acid is metabolized via the 5-, 12- and 15-lipoxygenase pathways. The kinetic mechanism of action of plant (soya) and animal (reticulocyte) 15-lipoxygenases is now well established. 5-Lipoxygenase possesses, in all probability, the most complex mechanism of activity regulation. At present several effectors of neutrophil 5-lipoxygenase, both cytosolic and membrane-bound ones, have been identified. The molecular and kinetic mechanisms of action of the enzyme are still open to question. A kinetic scheme of regulation of synthesis of arachidonic acid 5-lipoxygenase metabolites which does not exclude the presence of two binding sites on the enzyme molecule, is proposed. Within the framework of this kinetic scheme the enzyme activator complex may be the active form of the enzyme. There is evidence that the curve for the time dependence of 5-HETE accumulation in neutrophils stimulated by the Ca2+ ionophore A23187 has a maximum, while the corresponding curve for the LTB4 accumulation is a curve with saturation. It was shown that an increase in the concentration of exogenous arachidonate induces the synthesis of 5-HETE, whereas the concentration of LTB4 remains practically unchanged. The results of mathematical analysis of the above kinetic scheme and a comparison of experimental and calculated values suggest that the reaction effector, Ca2+, plays a crucial regulatory role in the observed kinetic dependencies reflecting the formation of two sequential products of 5-lipoxygenase oxidation of arachidonate. In this way Ca2+ strongly influences the first step of the reaction, i.e., 5-HETE formation; its effect on the second reaction step (5-HETE conversion into LTA4) is far less apparent.

Topics: Algorithms; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Arachidonic Acids; Calcimycin; Humans; Hydroxyeicosatetraenoic Acids; Kinetics; Leukotriene B4; Mathematics; Models, Biological; Neutrophils

Azelastine produced a concentration-dependent inhibition of calcium ionophore A23187 (0.2 microM) stimulated generation of 5-HETE, leukotriene B4, and leukotriene C4 in rat mixed peritoneal cells, yielding IC50 values of 35.5, 47.4, and 31.7 microM, respectively. Nordihydroguaiaretic acid (a potent 5-lipoxygenase inhibitor) also exerted a strong and concentration-dependent inhibition of 5-HETE and leukotriene B4 and C4 formation with IC50 values of 0.15, 0.09, and 0.1 microM, respectively. The inhibition of the formation of the products of the lipoxygenase pathway of arachidonic acid metabolism by azelastine may contribute to its overall antiallergic, antiasthmatic, and pulmonary anti-inflammatory activities.

Topics: Animals; Calcimycin; Depression, Chemical; Histamine H1 Antagonists; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Peritoneal Cavity; Phthalazines; Radioimmunoassay; Rats; SRS-A

Ligand binding to B lymphocytes via membrane Ig initiates a cascade of events beginning with the hydrolysis of phosphatidylinositol 4,5-bisphosphate into diacylglycerol and inositol 1,4,5-trisphosphate. Subsequent to the activation of protein kinase C and the induction of a rise in intracellular calcium by diacylglycerol and inositol 1,4,5-trisphosphate, there is gene transcription and eventually cellular activation. By mimicking the initial event of B cell activation with phorbol ester and calcium ionophore one can begin to identify the many mediators used in signaling between the membrane and the nucleus. We have examined the effect of calcium on arachidonic acid (AA) metabolism in several EBV-transformed human B cell lines. The cells were labelled with [3H]AA and stimulated with the calcium ionophore A23187. Analysis of the supernatant by reversed-phase HPLC demonstrated a dose-dependent release of an AA metabolite that coeluted with authentic 5-hydroxyeicosatetraenoic acid (5-HETE). In addition, the AA metabolite coeluted with standard 5-HETE under straight-phase chromatography. Further analysis by RIA confirmed the identification of 5-HETE and revealed an additional metabolite, 5-HETE lactone (5-HL). 5-HL is the intramolecular ester of 5-HETE generated in the presence of acid. We were unable to convert [3H] 5-HETE into 5-HL during sample preparation unless cells were present, suggesting that the 5-HL, is of cellular origin. These results suggest that the AA metabolites 5-HETE and its intramolecular ester 5-HL may play a role in B cell activation because they are produced subsequent to a rise in intracellular Ca2+, an event that occurs during cross-linking of membrane Ig.

Topics: Arachidonate 5-Lipoxygenase; Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids; B-Lymphocytes; Calcimycin; Cell Line; Cell Transformation, Viral; Chromatography, High Pressure Liquid; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Signal Transduction; T-Lymphocytes

Coupling of the dopamine (DA) receptor to the pathway of arachidonate (AA) through the islet-activating protein (IAP)-sensitive GTP-binding (G) protein was examined in primary cultures of anterior pituitary cells. The inhibitions by 1 microM DA of the stimulations of prolactin (PRL) release by 100 nM thyrotropin-releasing hormone and by 10 microM calcium ionophore A23187 were blocked by 100 ng/ml IAP. DA at 1 microM did not inhibit the release of [3H]-AA induced by 100 mU/ml phospholipase A2 (PLA2), but it inhibited the release of PRL induced by 100 mU/ml PLA2. This inhibition was blocked by 100 ng/ml IAP. IAP also blocked the inhibitions by DA of the releases of PRL by 5 microM AA and by 5 microM 5-hydroxyeicosatetraenoic acid. These results suggest that the DA receptor on lactotrophs is coupled to the pathway following the release of AA (lipoxygenase) through the IAP-sensitive G protein.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Cells, Cultured; Dopamine; Female; GTP-Binding Proteins; Hydroxyeicosatetraenoic Acids; Pertussis Toxin; Phospholipases A; Phospholipases A2; Pituitary Gland, Anterior; Prolactin; Radioimmunoassay; Rats; Rats, Inbred Strains; Receptors, Dopamine; Thyrotropin-Releasing Hormone; Tritium; Virulence Factors, Bordetella

The effect of auranofin on the 5-lipoxygenase pathway was studied in human neutrophils stimulated with either fMLP or A23187 (with or without arachidonic acid). The synthesis of leukotriene B4 (LTB4), 5-HETE and the all-trans isomers of LTB4 was measured by HPLC. At low concentrations (0.5-2.0 microM), auranofin stimulated LTB4 synthesis, but inhibited it at higher concentrations (100% inhibition at less than 10 microM). In contrast auranofin caused dose-dependent inhibition of the synthesis of 5-HETE and the all-trans isomers of LTB4. Similar observations were made with each agonist. The stimulation of LTB4 synthesis and inhibition of the trans isomer production suggests that auranofin at low concentrations stimulates LTA hydrolase--the enzyme that converts LTA4 to LTB4, whereas the inhibition of synthesis of all lipoxygenase products at higher auranofin concentrations, suggests inhibition of 5-lipoxygenase/LTA synthase.

Topics: Arachidonate 5-Lipoxygenase; Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids; Auranofin; Calcimycin; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Epoxide Hydrolases; Humans; Hydroxyeicosatetraenoic Acids; Isomerism; Leukotriene B4; Lipoxygenase Inhibitors; Models, Biological; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils

The in vitro release of arachidonic acid (AA) metabolites from caprine alveolar macrophages (CAM) stimulated with the calcium ionophore A23187 or opsonized zymosan was examined. Leukotriene B4 [5(S),12(R)-6,14-cis-8,10-trans-dihydroxyeicosatetraenoic acid] was the major AA metabolite elicited with either agonist; smaller amounts of 12- and 5-mono-hydroxyeicosatetraenoic acid (HETE), and 12-hydroxyheptadecatrienoic acid (HHT) were also detected. Zymosan stimulation also caused the release of very small quantities of prostaglandins E2 and F2 alpha, and thromboxane B2. Our report is the first to describe arachidonic acid metabolism in CAM.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Bronchoalveolar Lavage Fluid; Calcimycin; Fatty Acids, Unsaturated; Goats; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Lipid Metabolism; Macrophages; Zymosan

We studied the effects of a single, oral dose of methotrexate (MTX) on arachidonic acid metabolism in neutrophils from 6 patients with rheumatoid arthritis, which were obtained 1 day before and 1 day after their usual weekly MTX dose. The 6 patients had received a mean weekly MTX dose of 9.6 mg (range 5-15) for a mean of 61.7 months (range 58-64), and none received concomitant corticosteroids. Total generation of leukotriene B4 (LTB4) in neutrophils stimulated ex vivo with 10 microM calcium ionophore A23187 for 20 minutes was significantly suppressed, by a mean of 53%, after the MTX dose compared with the predose levels (mean +/- SEM 13.0 +/- 1.4 ng/10(6) cells versus 6.0 +/- 0.9 ng/10(6) cells; P = 0.0019), reflecting a comparable suppression of both released and cell-retained LTB4. A 49% decrease in omega-oxidation products of LTB4 demonstrates that decreased LTB4 synthesis, rather than increased degradation, is responsible for the decrease in LTB4 generation. The absence of a significant change in either 3H-labeled arachidonic acid release or platelet-activating factor generation indicates that the observed decrease in LTB4 synthesis was apparently not caused by diminished phospholipase A2 activity. A 28% decrease in the total formation of the 5-lipoxygenase products 5-hydroxyeicosatetraenoic acid and the 6-trans-LTB4 diastereoisomers, and a 48% suppression of production of LTB4 plus its omega-oxidation metabolites after the MTX dose suggest inhibition of 5-lipoxygenase activity and possible suppression of leukotriene A4 epoxide hydrolase activity.

Topics: Aged; Arachidonic Acid; Arachidonic Acids; Arthritis, Rheumatoid; Calcimycin; Chemotaxis, Leukocyte; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Methotrexate; Middle Aged; Neutrophils; Phospholipases; Platelet Activating Factor

Continuous i.v. infusion of a nonlethal dose of Escherichia coli endotoxin induced an early (3-h) accumulation of neutrophils in the rat liver followed by a later (30-h) greater extravasation of mononuclear phagocytes (MNP). These inflammatory cells, recovered together by centrifugal elutriation, were analyzed for their potential capacity to metabolize [1-14C]-AA. Ca2+ ionophore A23187 (5 microM) stimulated the release of [1-14C]-AA from PC and PI both in cells from saline- and ET-infused rats, the latter showing a higher capacity to further metabolize AA to eicosanoids. LTB4 and 5-HETE were the major metabolites accumulated in cells from rats infused with ET for 3 h, while PGD2 played the main role in cells from saline-infused rats. This could reflect [1-14C]-AA metabolism by PMNP and Kupffer cells, respectively. By 30 h of ET-infusion, a shift from PGD2 to PGE2 release was observed. These results suggest that eicosanoids released by nonparenchymal cells (i.e., Kupffer and endothelial cells) and PMNP in the liver of ET-infused rats may alter the normal intercellular information flow between parenchymal and nonparenchymal cells, contributing to the severe impairment in liver function and metabolism during endotoxicosis and sepsis.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Dinoprostone; Eicosanoids; Endotoxins; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Liver; Male; Neutrophils; Rats; Rats, Inbred Strains

Hydroxyeicosatetraenoic acids (HETEs) have previously been reported to stimulate the relatively inactive 15-lipoxygenase in A23187-activated human neutrophils to metabolize exogenously added arachidonic acid to 15-HETE. Several aspects of this HETE-induced activation process were examined. Pretreatment of intact PMNs with 3-20 microM 15-HETE, A23187 and the 5-lipoxygenase inhibitor NDGA (or the dual cyclooxygenase/lipoxygenase inhibitor BW755C) followed by [14C]arachidonic acid addition resulted in an unexpected synergistic activation of the cryptic 15-lipoxygenase activity. The ability of several HETE derivatives or analogues to stimulate the inactive 15-lipoxygenase was also investigated. The presence of the hydroxyl group but not its position was essential since 5-, 12- and 15-HETE were approximately equipotent and about ten times more effective than arachidonic acid. 5-HETE was more potent than 5-HETE methyl ester which suggested that a free carboxyl group was important. Both 5-HPETE and 5.15-diHETE were found to be less potent than 5-HETE. The C18 hydroxy fatty acid analogues 9- and 13-HODE were the least effective activators of the PMN 15-lipoxygenase activity that were tested.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonate 15-Lipoxygenase; Arachidonate 5-Lipoxygenase; Calcimycin; Humans; Hydroxyeicosatetraenoic Acids; Neutrophils

Azelastine, a newly synthesized antiallergic agent, strikingly inhibited the production of leukotriene B4 and C4 (LTB4 and LTC4) in murine peritoneal cells which had been stimulated by calcium ionophore A23187. The 50% inhibitory concentrations (IC50) of the agent were approximately 1.0 x 10(-5) M. In addition, azelastine significantly inhibited also 5-lipoxygenase activity in peritoneal cells with an IC50 of 1.0 x 10(-5) M, but not on LTC4 synthetase, LTA4 hydrolase or phospholipase A2 activity. Furthermore, azelastine showed little effect on either 12-lipoxygenase activity or thromboxane synthesis in human platelets. These results suggest that at least the drug's antiallergic effects can be attributed to its inhibiting action of 5-lipoxygenase in regard to arachidonate metabolism.

Topics: Animals; Blood Platelets; Calcimycin; Dose-Response Relationship, Drug; Epoxide Hydrolases; Glutathione Transferase; Hydroxyeicosatetraenoic Acids; Ketotifen; Leukotrienes; Lipoxygenase; Mice; ortho-Aminobenzoates; Peritoneal Cavity; Phospholipases A; Phospholipases A2; Phthalazines; Piperazines; Pyridazines; Thromboxanes

Platelet-activating factor (PAF) is a potent neutrophil agonist operating through specific receptors located on the cell surface. Binding of PAF to its receptor may also stimulate further PAF synthesis, thus providing a means of amplifying the PAF signal for the cell of origin and/or other responsive cells. In this report we demonstrate that 1-O-alkyl-2-N-methylcarbamyl-sn-glycero-3-phosphocholine (C-PAF), a nonmetabolizable bioactive analog of PAF, stimulates human neutrophils to synthesize PAF, as detected by [3H]acetate incorporation into PAF. This approach allowed us to conclude that [3H]acetate-labeled PAF was formed from endogenous precursor rather than mere turnover of the stimulatory dose of PAF. PAF's ability to initiate further PAF synthesis was confirmed by measuring the PAF-stimulated conversion of 1-O-[3H]alkyl-2-acylglycerophosphocholine to 1-O-[3H]alkyl-2-acetylglycerophosphocholine by prelabeled human neutrophils and by determining the molecular species of 1-O-alkyl-2-[3H]acetylglycerophosphocholine produced by cells stimulated with a single molecular species of PAF (C15:0). Degradation of exogenously added [3H]PAF was not inhibited by C-PAF/5-hydroxyeicosatetraenoic acid treatment. Thus, inhibition of PAF degradation was ruled out as the mechanism accounting for the appearance of labeled PAF in the stimulated cells. Synthesis of PAF in response to C-PAF was not dependent on cytochalasin B pretreatment but was dramatically potentiated by 5-hydroxyeicosatetraenoic acid, which alone was without effect. Additionally, we have demonstrated that another major arachidonate metabolite of neutrophils, leukotriene B4, stimulates PAF production. Thus, at least three products of activated neutrophils, including PAF itself, can promote PAF synthesis by these cells. This positive feedback effect may amplify autacoid production and the final cellular response.

Topics: 1-Alkyl-2-acetylglycerophosphocholine Esterase; Acetates; Arachidonic Acid; Arachidonic Acids; Calcimycin; Humans; Hydrolysis; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Neutrophils; Phospholipases A; Platelet Activating Factor

Alveolar macrophages (AM) are the principal resident phagocytes in the human lung, and play a major role in local defence against environmental agents. It is now known that during asthma these cells take part in the amplification of the inflammatory mechanism. It has been demonstrated in vitro that they can be activated to generate leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE), mediators with potent pharmacological properties. These two arachidonic metabolites were identified and quantified by reversed phase high performance liquid chromatography (HPLC) performed in cell suspensions, and in cell free supernatants. AM from asthmatics, after stimulation by the calcium ionophore A23187 or opsonized zymosan, released significantly (p less than 0.05) more LTB4 than those from healthy subjects. The increase in LTB4 release could be evidence for in vivo activation. On the other hand, the levels of 5-HETE in the AM from asthmatics were significantly (p less than 0.03) higher than those in cells from healthy subjects. This intracellular increase could be correlated with a greater migratory ability of these inflammatory macrophages, as observed for eosinophils. The clinical efficacy of nedocromil sodium may be partly related to the decreases in LTB4 releasability and intracellular 5-HETE levels observed only in AM from asthmatic patients.

Topics: Adolescent; Adult; Asthma; Bronchoalveolar Lavage Fluid; Calcimycin; Cells, Cultured; Chromatography, High Pressure Liquid; Female; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Macrophages; Male; Middle Aged; Nedocromil; Pulmonary Alveoli; Quinolones; Zymosan

Lipoxygenase (LO) products generated by human PMN were examined utilizing a gradient-HPLC and rapid spectral detector which permitted continuous UV-spectral monitoring of leukotrienes, lipoxins and related oxygenated products of arachidonic acid. When exposed to the ionophore A23187, PMN generated LTB4 and its omega-oxidation products as well as LXA4, LXB4, and 7-cis-11-trans-LXA4 from endogenous sources. Addition of 15-HETE changed the profile of products generated by activated PMN and led to a time- and dose-dependent increase in lipoxins and related compounds while the production of LTB4 and its omega-oxidation products was inhibited. Results of time-course and radiolabel studies revealed that 15-HETE is rapidly transformed within 15 s to 5,15-DHETE and conjugated tetraene-containing products, and that the inhibition of leukotriene formation followed a similar time-course. In contrast, PMN did not generate either lipoxins or related products from 5-[3H]HETE, nor did 5-HETE block leukotriene formation. Stimulated PMN generated 5,15-DHETE from exogenous 5-HETE, while in the absence of ionophore, 5-HETE was transformed to 5,20-HETE. These results indicate that PMN can generate lipoxins and related products from endogenous sources and that 15-HETE and 5-HETE are transformed by different routes.

Topics: Calcimycin; Chromatography, High Pressure Liquid; Gas Chromatography-Mass Spectrometry; Humans; Hydroxyeicosatetraenoic Acids; Kinetics; Leukotriene B4; Leukotrienes; Lipoxins; Neutrophils; Spectrophotometry, Ultraviolet

Neutrophils treated with optimal amounts of tumor promoters that activate protein kinase C (e.g. mezerein) release large quantities of superoxide (O2-) and exhibit an intense phosphorylation of two proteins with molecular masses of approximately 47 and 49 kDa. These cells can also be stimulated synergistically to release a comparable amount of O2-. This involves treatment with a suboptimal amount of a tumor promoter and an agent capable of elevating cellular Ca2+. Neutrophils treated in the former fashion exhibit a redistribution of the activity of protein kinase C from a soluble to a particulate fraction that is stable in the presence of Ca2+ chelators, whereas cells stimulated synergistically do not do so to an appreciable extent (Badwey, J. A., Robinson, J. M., Horn, W., Soberman, R. J., Karnovsky, M. J., and Karnovsky, M. L. (1988) J. Biol. Chem. 263, 2779-2786). In this paper, we report that neutrophils stimulated synergistically do exhibit a significant incorporation of 32P into the 47-kDa protein, but with little labeling of the 49-kDa species. This labeling of the 47-kDa protein was greater than the sum of those observed with each agent added separately but was less than that observed in cells stimulated with optimal amounts of tumor promoters alone. An inhibitor of protein kinase C (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine) blocked O2- release and the phosphorylation of the 47-kDa protein under all conditions of stimulation mentioned, whereas an inhibitor of cyclic nucleotide-dependent kinases had no effect on these phenomena. Thus, labeling of the 47-kDa protein can occur in the absence of a "tight" translocation of protein kinase C to membrane and was always observed during synergy. The data support a role for protein kinase C and the 47-kDa phosphoprotein in the synergistic stimulation of neutrophils.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Calcimycin; Diterpenes; Drug Synergism; Guinea Pigs; Hydroxyeicosatetraenoic Acids; Isoquinolines; Molecular Weight; Neutrophils; Phosphoproteins; Phosphorus Radioisotopes; Phosphorylation; Piperazines; Sulfonamides; Superoxides; Terpenes

We studied the effect of phorbol myristate acetate (PMA) on endogenous leukotriene B4 (LTB4) metabolism of calcium ionophore A23187-stimulated human neutrophils. Preincubation of normal neutrophils with PMA significantly suppressed the recovery of endogenous LTB4 induced by A23187. PMA did not suppress the recovery of LTB4 produced by neutrophils from patients with chronic granulomatous disease (CGD), which is known to be defective in NADPH oxidase activation to produce reactive oxygen species (ROS). PMA inhibited the formation of omega-oxidation products of LTB4, but enhanced arachidonic acid release in normal and CGD neutrophils. Furthermore, 5-lipoxygenase activity of 10,000 x g supernatants from normal neutrophils pretreated with PMA was equivalent to that of the controls. Decrease in LTB4 recovery was not attributed to the suppression of the intracellular Ca2+ increase. Thus, it is suggested that reactive oxygen species (ROS) produced by PMA may directly affect endogenous LTB4 and convert it into metabolite(s) distinct from omega-oxidation products.

Topics: Arachidonate 5-Lipoxygenase; Arachidonic Acid; Arachidonic Acids; Calcimycin; Calcium; Granulomatous Disease, Chronic; Humans; Hydroxyeicosatetraenoic Acids; Kinetics; Leukotriene B4; Male; NADH, NADPH Oxidoreductases; NADPH Oxidases; Neutrophils; SRS-A; Superoxides; Tetradecanoylphorbol Acetate

Neutrophils stimulated with optimal amounts of tumor-promoters that activate protein kinase C (e.g. mezerein, phorbol 12,13-dibutyrate) are known to release large quantities of superoxide: approximately 40-50 nmol O2-/min/10(7) cells. Previous studies have shown that treatment of neutrophils with the calcium ionophore A23187, or with 5-hydroxy-6,8,11,14-eicosatetraenonate (5-HETE), dramatically increased the ability of these cells to release O2- in response to suboptimal concentrations of the stimulants mentioned. In this manuscript, we provide data relevant to the basis of this augmentation of O2- release. The synergy with ionophore A23187 exhibited a partial requirement for extracellular Ca2+, whereas that with 5-HETE exhibited a near absolute requirement for that cation. Neutrophils stimulated with optimal amounts of tumor-promoters are known to exhibit a redistribution of protein kinase C activity from the soluble to a particulate fraction. A redistribution of kinase activity was not observed in cells stimulated synergistically. On the other hand, ionophore A23187 and 5-HETE increased the binding of a suboptimal amount of [3H] phorbol 12,13-dibutyrate to intact neutrophils by approximately 25 and 50%, respectively. Inhibitors of protein kinase C (i.e. sphingosine, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine) substantially blocked O-2 release from neutrophils stimulated either synergistically or with optimal levels of tumor-promoters. These data suggest a role for 5-HETE in modulating O-2 release by neutrophils and are discussed in relation to models of the interactions of protein kinase C with membranes.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Calcimycin; Cations, Divalent; Drug Synergism; Guinea Pigs; Hydroxyeicosatetraenoic Acids; Isoquinolines; Neutrophils; Phorbol 12,13-Dibutyrate; Phorbol Esters; Piperazines; Protein Kinase C; Sphingosine; Sulfonamides; Superoxides

CGS 8515 inhibited 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B4 synthesis in guinea pig leukocytes (IC50 = 0.1 microM). The compound did not appreciably affect cyclooxygenase (sheep seminal vesicles), 12-lipoxygenase (human platelets), 15-lipoxygenase (human leukocytes) and thromboxane synthetase (human platelets) at concentrations up to 100 microM. CGS 8515 inhibited A23187-induced formation of leukotriene products in whole blood (IC50 values of 0.8 and 4 microM, respectively, for human and rat) and in isolated rat lung (IC50 less than 1 microM) in vitro. The selectivity of the compound as a 5-lipoxygenase inhibitor was confirmed in rat whole blood by the 20-70-fold separation of inhibitory effects on the formation of leukotriene from prostaglandin products. Ex vivo and in vivo studies with rats showed that CGS 8515, at an oral dose of 2-50 mg/kg, significantly inhibited A23187-induced production of leukotrienes in whole blood and in the lung. The effect persisted for at least 6 h in the ex vivo whole blood model. CGS 8515, at oral doses as low as 5 mg/kg, significantly suppressed exudate volume and leukocyte migration in the carrageenan-induced pleurisy and sponge models in the rat. Inhibitory effects of the compound on inflammatory responses and leukotriene production in leukocytes and target organs are important parameters suggestive of its therapeutic potential in asthma, psoriasis and inflammatory conditions.

Topics: Animals; Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids; Benzoquinones; Biotransformation; Blood Platelets; Calcimycin; Dexamethasone; Guinea Pigs; Humans; Hydroxyeicosatetraenoic Acids; Indomethacin; Leukocytes; Leukotriene B4; Lipoxygenase Inhibitors; Male; Naphthoquinones; ortho-Aminobenzoates; Pleurisy; Quinones; Rats; Rats, Inbred Strains; Sheep

The formation of 5-lipoxygenase products (5-hydroxyeicosatetraenoic acid [5-HETE], leukotriene B4 [LTB4], and leukotriene C4 [LTC4]) by polymorphonuclear and mononuclear leukocytes isolated from peripheral blood of patients with rheumatoid arthritis was evaluated and compared with the data obtained from a group of control subjects. Although the levels of arachidonic acid metabolites via 5-lipoxygenase pathway by stimulated polymorphonuclear cells were comparable between patients and controls, mononuclear leukocytes from patients synthesized, when stimulated, significantly greater amounts of 5-HETE, LTB4, and LTC4 than did cells isolated from normal subjects. In addition, the release of superoxide anion, stimulated by either a particulate or a soluble stimulus, was increased in mononuclear cells from patients. The enhanced capacity of peripheral mononuclear leukocytes isolated from patients with rheumatoid arthritis to generate proinflammatory metabolites of arachidonic acid and oxygenated species with bactericidal and tissue-damaging properties may contribute to the pathogenesis of this complex disease.

Topics: Arthritis, Rheumatoid; Calcimycin; Cell Aggregation; Humans; Hydroxyeicosatetraenoic Acids; Leukocytes, Mononuclear; Leukotriene B4; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Phagocytosis; SRS-A; Superoxides

Both 1,2-diacyl- and 1-O-alkyl-2-acylglycerols are formed during stimulation of human neutrophils (PMN), and both can prime respiratory burst responses for stimulation by the chemotactic peptide, N-formyl-Met-Leu-Phe (fMLP); however, mechanisms of priming are unknown. Arachidonic acid (AA) release through phospholipase A2 activation and metabolism by 5-lipoxygenase are important activities of PMN during inflammation and could be involved in the process of primed stimulation. Therefore, we have examined the ability of diacyl- and alkylacylglycerols to act as priming agents for AA release and metabolism in human neutrophils. After prelabeling PMN phospholipids with [3H]AA, priming was tested by incubating human PMN with the diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG), or its alkylacyl analog, 1-O-delta 9-octadecenyl-2-acetylglycerol (EAG) before stimulating with fMLP. fMLP (1 microM), OAG (20 microM), or EAG (20 microM) individually caused little or no release of labeled AA. However, after priming PMN with the same concentrations of either OAG or EAG, stimulation with 1 microM fMLP caused rapid (peak after 1 min) release of 6-8% of [3H]AA from cellular phospholipids; total release was similar with either diglyceride. Priming cells with OAG also enhanced conversion of released AA to leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) upon subsequent fMLP stimulation, but AA metabolites were not increased in EAG-primed PMN. If fMLP was replaced with the calcium ionophore A23187 (which directly causes release of AA and production of LTB4 and 5-HETE), priming by both diglycerides again enhanced release of [3H]AA, but only OAG priming increased lipoxygenase activity. Indeed, EAG pretreatment markedly reduced LTB4 and 5-HETE production. Thus, both diglycerides prime release of AA from membrane phospholipids but have opposite actions on the subsequent metabolism of AA.

Topics: Arachidonic Acid; Arachidonic Acids; Calcimycin; Cytochalasin B; Diglycerides; Humans; Hydroxyeicosatetraenoic Acids; Kinetics; Leukotriene B4; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Phospholipases; Phospholipases A; Phospholipases A2

The generation of LTB4 by peripheral blood neutrophils (PMN) isolated before and for as long as 6 h after exercise-induced asthma (EIA) has been analyzed. Three and 6 h after the development of EIA, PMN isolated from 10 asthmatic subjects and stimulated in vitro by 2 x 10(8) and 4 x 10(8) zymosan particles per 2 x 10(6) PMN demonstrated a 12- and 4-fold enhancement, respectively, in the production of immunoreactive LTB4 as compared with PMN isolated before exercise. At 6 h after EIA, there was a redistribution of generated LTB4 such that 30 to 40% of LTB4 produced by zymosan-activated PMN was released extracellularly as compared with 10% before exercise. There was no significant enhancement in the generation of LTB4 by unstimulated PMN at any time point after exercise. Resolution by reverse-phase high performance liquid chromatography (HPLC) of products from [3H]arachidonic-acid-labeled and zymosan-activated PMN demonstrated that, in addition to LTB4, there was enhanced metabolism to 6-trans-LTB4, omega-oxidation metabolites of LTB4 and 5-HETE. Stimulation of PMN with 10 microM A23187 revealed a 2-, 6-, and 5-fold enhancement in the production of LTB4, 6-trans-LTB4, and 5-HETE, respectively, at 6 h after EIA, as measured by integrated ultraviolet absorbance after HPLC. There was no significant enhancement in LTB4 generation by PMN in 6 asthmatic subjects after methacholine-induced bronchospasm, and after exercise in 6 subjects who did not develop asthma. The augmentation of PMN LTB4 generation in EIA correlated with the extent of the early decrease in SGaw.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adolescent; Adult; Airway Resistance; Arachidonate 5-Lipoxygenase; Asthma; Asthma, Exercise-Induced; Bronchial Provocation Tests; Calcimycin; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Female; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Male; Methacholine Chloride; Methacholine Compounds; Middle Aged; Neutrophils; Stereoisomerism; Zymosan

Cultured porcine aortic endothelial cells were conditioned through two passages to mimic euglycemic and hyperglycemic conditions (5.2 mM, normal glucose; 15.6 mM, elevated glucose). After incubation with 1 microM [14C]arachidonic acid for 24 h, the cells were stimulated with 1 microM A23187 for times up to 30 min. Uptake of [14C]arachidonic acid and its distribution among cell lipids were unaffected by the increased glucose concentration. The release of eicosanoids from labeled cells and unlabeled cells was measured by reverse-phase HPLC and by RIA, respectively. Compared with cells stimulated in the presence of normal glucose concentrations, cells stimulated in the presence of elevated glucose released 62.6% less free [14C]arachidonic acid, but released 129% more 14C-labeled 15-hydroxyeicosatetraenoic acid (HETE). Increased release of 15-HETE in the presence of elevated glucose in response to A23187, bradykinin, and thrombin was confirmed by RIA. A similar increase in 5-HETE release was observed by RIA after A23187 treatment. The release of both radiolabeled and unlabeled prostanoids was equal at both glucose concentrations. The data indicate that glucose may play an important role in the regulation of release and metabolism of arachidonic acid after agonist stimulation. In the presence of elevated glucose concentrations, such as those associated with diabetes mellitus, the extent and pattern of eicosanoid release from endothelial cells is markedly altered.

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Aorta; Arachidonic Acid; Arachidonic Acids; Bradykinin; Calcimycin; Eicosanoic Acids; Endothelium, Vascular; Glucose; Hydroxyeicosatetraenoic Acids; Swine; Thrombin

The phorbol ester, phorbol 12-myristate 13-acetate enhanced leukotriene B4 production stimulated by formyl-methionyl-leucyl-phenylalanine and arachidonic acid and reduced the production of the all-trans isomers of LTB4 by human neutrophils. Production of 5-hydroxyeicosatetraenoic acid was unaffected. These observations are consistent with a stimulatory effect of phorbol ester on LTA hydrolase, the enzyme which catalyses the conversion of LTA4 to LTB4. We demonstrate that a protein of the same molecular weight as LTA hydrolase is phosphorylated upon stimulation of neutrophils with PMA. These data suggest that the activity of LTA hydrolase may be regulated by protein kinase C-dependent phosphorylation.

Topics: Arachidonic Acid; Arachidonic Acids; Calcimycin; Drug Interactions; Epoxide Hydrolases; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Phosphorylation; Tetradecanoylphorbol Acetate

The influence of the glutathione status of human polymorphonuclear leukocytes (PMN) on 5-lipoxygenase activity was studied by treating cells with increasing concentrations of 1-chloro-2,4-dinitrobenzene (Dnp-Cl) or azodicarboxylic acid bis(dimethylamide) (Diamide). Subsequent incubation with arachidonate resulted in an up to tenfold-stimulated formation of 5-hydroxyeicosatetraenoic acid, leukotriene B4, leukotriene B4 isomers and omega-hydroxyleukotriene B4. Higher concentrations of the GSH reagents were inhibitory. At maximal stimulation by Dnp-Cl, 5-hydroperoxyeicosatetraenoic acid started to be built up at the expense of 5-HETE at glutathione levels which were diminished by about 50% compared to resting cells. No increase in cytosolic Ca2+ could be measured under these conditions by the fura-2 method. In PMN homogenates Dnp-Cl and Diamide were without effect and even caused inhibition when 5-lipoxygenase was stimulated by Ca2+ and ATP. 15-Lipoxygenase was either unchanged in the case of Diamide, or even increased after pretreatment with Dnp-Cl. The results allow us to conclude that 5-lipoxygenase activity in intact PMN is regulated not only by Ca2+ but in a complex manner also by the glutathione redox status. Conditions of oxidative stress increase the activity which may reflect the in vivo situation under phagocytosis and oxidative burst.

Topics: Adult; Arachidonate 5-Lipoxygenase; Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids; Calcimycin; Calcium; Chromatography, High Pressure Liquid; Diamide; Dinitrochlorobenzene; Glutathione; Humans; Hydroxyeicosatetraenoic Acids; Kinetics; Leukotriene B4; Leukotrienes; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils

The effects of an inhalation anesthetic, halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) on the formation of 5-lipoxygenase metabolites such as leukotriene B4, 5(S)-hydroxyeicosatetraenoic acid (5-HETE), 6-trans-isomers of leukotriene B4 and leukotriene C4 were studied in human leukocytes stimulated with calcium ionophore A23187. Halothane inhibited the formation of all these metabolites dose dependently and the formation was restored by removal of the drug. The anesthetic also reversibly inhibited the release of [3H]arachidonic acid from neutrophils with a half-inhibition concentration of less than 0.19 mM. The formation of 5-lipoxygenase metabolites was not inhibited by the anesthetic when leukocytes were stimulated with the ionophore in the presence of exogenous arachidonic acid. These observations indicate that the inhibitory effect of halothane on the formation of 5-lipoxygenase metabolites in leukocytes is mainly due to the inhibition of arachidonic acid release.

Topics: Arachidonate 5-Lipoxygenase; Arachidonic Acid; Arachidonic Acids; Calcimycin; Halothane; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Kinetics; Leukocytes; Leukotriene B4; Neutrophils; SRS-A

12-Hydroxyheptadecatrienoic acid (HHT), a UV chromophore, has been used to assess cyclooxygenase (CO) pathway metabolism of arachidonic acid (AA) by rat peritoneal cells. Simultaneous monitoring at 235 and 280 nm after HPLC permits the measurement of both CO and 5-lipoxygenase (5-LO) pathway fluxes in a single system.

Topics: Animals; Arachidonate 5-Lipoxygenase; Arachidonic Acids; Ascitic Fluid; Calcimycin; Chromatography, High Pressure Liquid; Cyclooxygenase Inhibitors; Eosinophils; Fatty Acids, Unsaturated; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Lipoxygenase Inhibitors; Macrophages; Male; Mast Cells; Prostaglandin Antagonists; Prostaglandin-Endoperoxide Synthases; Rats; Rats, Inbred Strains; Reference Standards; Spectrophotometry, Ultraviolet

Effects of chronic ethanol ingestion (3 weeks) on the capacity of peritoneal macrophages, lung and heart tissues, to metabolize endogenous arachidonic acid (AA) in rats, and the in vitro effect of ethanol on 3'-5' cyclic adenosine monophosphate (cAMP) levels in rat platelets were studied. Peritoneal resident macrophages were stimulated by calcium ionophore (A23187) and levels of 5-hydroxyeicosatetraenoic acid (5-HETE), leukotriene B4 (LTB4) and PGE2 were measured by radioimmunoassay. There were no differences in levels of the eicosanoids synthesized by macrophages between the ethanol treated and the control group. There were also no differences found in levels of the eicosanoids synthesized by heart or lung homogenate between the two groups. These results suggest that chronic ethanol ingestion does not alter the capacity to synthesize the eicosanoids from the endogenous precursor in tissues studied here. Preincubation of ethanol with platelet rich plasma resulted in a dose dependent increase in cAMP levels. The well documented inhibitory effects of ethanol in vitro on aggregation and AA metabolism in platelets may be due to the enhanced cAMP levels.

Topics: Alcohol Drinking; Animals; Arachidonic Acid; Arachidonic Acids; Blood Platelets; Calcimycin; Cyclic AMP; Dinoprostone; Ethanol; Heart; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Lung; Macrophages; Male; Myocardium; Prostaglandins E; Rats; Rats, Inbred Strains

A reliable system for evaluating 5-lipoxygenase (5-LO) pathway inhibitors employing human whole blood stimulated by the calcium ionophore, A-23187, and yeast cell walls (YCW) is described. In developing this system, we have shown that leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) can be recovered quantitatively from whole blood, and can be measured with accuracy and a precision (standard deviation) of +/- 12%. Apparent differences in LTB4/5-HETE levels between donors can be minimized by normalizing the LTB4/5-HETE production to neutrophil number. Variability in LTB4/5-HETE production among different donors was reduced by increasing the ionophore concentration. The kinetics of ionophore stimulated product production display a 1-4 min lag which is dependent on ionophore concentration. The lag is removed by pretreatment of blood with 5 micrograms/ml cytochalasin B. Likewise, the kinetics of product formation after stimulation with yeast cell walls demonstrated a lag period, which could be shortened by prior opsonization of the YCW. The amount of LTB4 metabolism to 20-OH-LTB4 and 20-COOH-LTB4 in this system is approximately 20%. Phenidone, nordihydroguaiaretic acid, and nafazatrom, known inhibitors of the 5-LO pathway, display half-maximal inhibition points of 0.4, 1.5, and 9 micrograms/ml, respectively. In summary, we believe that this assay offers a guide for predicting systemic levels of drug needed to be achieved for effective inhibition of cellular LTB4/5-HETE synthesis/release in humans.

Topics: Arachidonate 5-Lipoxygenase; Arachidonate Lipoxygenases; Arachidonic Acids; Calcimycin; Chromatography, High Pressure Liquid; Cytochalasin B; Egtazic Acid; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Kinetics; Leukotriene A4; Leukotriene B4; Lipoxygenase Inhibitors; Radioimmunoassay; Spectrophotometry, Ultraviolet; Yeast, Dried

We infused exogenous arachidonic acid (AA) into salt-perfused isolated dog lungs. This led to elevations in adenosine 3',5'-cyclic monophosphate (cAMP) which were from conversion of the AA to cyclooxygenase products. The maximal levels of cAMP occurred at far less than maximal levels of cyclooxygenase products. Next, we infused A 23187 to release endogenous pulmonary AA. This led to elevations in cAMP that were from conversion of this endogenous AA to cyclooxygenase products. The level of these products was far less than maximal levels from exogenous AA. However, maximal levels of cAMP from conversion of endogenous AA were similar to maximal levels of cAMP from conversion of exogenous AA. We conclude that maximal levels of pulmonary cAMP from endogenous or exogenous AA are from conversion of the AA to far less than maximal levels of pulmonary cyclooxygenase products. This indicates that levels of cAMP rather than levels of cyclooxygenase products are a potential rate-limiting step in cAMP-linked pulmonary actions of such products from pulmonary conversion of endogenous or exogenous AA.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Cyclic AMP; Dogs; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Kinetics; Lung; Prostaglandin-Endoperoxide Synthases; Prostaglandins; Thromboxane B2

Substitution of dietary fatty acids has potential for altering the inflammatory response. The purpose of the present study was to define the metabolites of arachidonic acid (AA) and eicosapentaenoic acid (EPA) secreted by bovine peripheral blood neutrophils and platelets. High performance liquid chromatography was used to characterize cyclooxygenase and lipoxygenase metabolites secreted in response to the calcium ionophore A23187. Cells were prelabelled with 3H-AA or 3H-EPA prior to challenge with the calcium ionophore. Bovine neutrophils secreted leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) as the major metabolites of AA, as well as the corresponding leukotriene B5 (LTB5) and 5-hydroxyeicosapentaenoic acid (5-HEPE) metabolites of EPA. Peptidoleukotrienes derived from 3H-AA or 3H-EPA were not detected under these conditions. The major tritiated metabolites secreted from bovine platelets were: thromboxane A2, measured as the stable metabolite thromboxane B2 (TXB2); hydroxyheptadecatrienoic acid (HHT) and 12-HETE derived from 3H-AA; and the omega-3 analogs TXB3 and 12-HEPE, derived from 3H-EPA. Preferred substrate specificities existed amongst the AA- and EPA-derived metabolites for the intermediary enzymes involved in the arachidonic acid cascade. These findings support the hypothesis that substitution of membrane-bound AA by EPA has potential for modulation of the host inflammatory response following cellular phospholipid mobilization.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Blood Platelets; Calcimycin; Cattle; Eicosapentaenoic Acid; Gas Chromatography-Mass Spectrometry; Hydroxyeicosatetraenoic Acids; Leukotriene A4; Leukotriene B4; Male; Neutrophils; Tritium

Confluent cultures of porcine aortic endothelial cells were prelabeled with 1 microM [14C]arachidonic acid complexed to 1 microM bovine serum albumin. After washing, the cells were stimulated with 1 microM A23187 for time intervals between 30 s and 30 min. Cellular lipids were extracted and separated into major lipid classes and phospholipid subclasses. The external medium was analyzed for released radioactive eicosanoids. The time-course of total release of 14C radioactivity demonstrated a biphasic nature of A23187-induced changes in endothelial cell lipids. Early, from 30 s to 5 min, substantial losses of [14C]arachidonic acid from diacylphosphatidylethanolamine and phosphatidylinositol, as well as an abrupt increase in diacylphosphatidylcholine-associated radioactivity were observed. These initial changes coincided with the release of 14C-labeled cyclooxygenase products. Later changes (5-30 min) included a sustained progressive loss of 14C radioactivity from alkenyl (alk-1-enyl) acylphosphatidylethanolamine and diacylphosphatidylcholine. These later changes coincided with the elaboration of 14C-labeled lipoxygenase products. Although unequivocal assignments cannot be made, the data suggest that specific pools of arachidonic acid provide precursors for individual classes of eicosanoids.

Topics: 6-Ketoprostaglandin F1 alpha; Acylation; Animals; Aorta, Thoracic; Arachidonic Acid; Arachidonic Acids; Calcimycin; Cells, Cultured; Endothelium, Vascular; Ethers; Fatty Acids, Unsaturated; Hydroxyeicosatetraenoic Acids; Kinetics; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylinositols; Phospholipids; Swine

CI-922 (3,7-dimethoxy-4-phenyl-N-1H-tetrazol-5-yl-4H-furo[3,2-b]indole-2- carboxamide, 1,2-ethanediamine, 2:1), an antiallergy compound, was tested for its effects on arachidonic acid metabolism, and its potency was compared to that of proxicromil, BW-755C, indomethacin, and nordihydroguaiaretic acid (NDGA). CI-922 was both a relatively potent and selective inhibitor of 5-HETE and LTB4 formation in human leukocytes, being equipotent to BW-755C and about four-fold more potent than proxicromil. However, CI-922 was rather weak in inhibiting the formation of PGE2 in bovine seminal vesicles. The parallel inhibition of 5-HETE and LTB4 formation by CI-922 suggests that either direct or indirect inhibition of the 5-lipoxygenase pathway may explain, at least in part, its antiallergy properties.

Topics: 4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyrazol-3-amine; Adult; Animals; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Arachidonic Acids; Azoles; Calcimycin; Cattle; Histamine Release; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Indoles; Indomethacin; Leukocytes; Male; Masoprocol; Pyrazoles; Seminal Vesicles; Tetrazoles

Amoxanox has potent antiallergic activity because it inhibits the release of chemical mediators such as histamine and leukotrienes. We studied the in vitro effect of amoxanox on arachidonic acid metabolism, including the lipoxygenase and cyclooxygenase pathways. Amoxanox inhibited calcium ionophore A23187-induced formation of 5-HETE, LTB4, SRS-A (LTC4, LTD4 and LTE4), and 12-HETE in rat peritoneal resident monocytes. These results indicate that amoxanox inhibits 5- and 12-lipoxygenases. The compound, however, did not affect the formation of TXB2 or 6-keto-PGF1 alpha in guinea pig lung fragments and PGE2 or PGF2 alpha in bovine seminal vesicles, suggesting that it did not inhibit cyclooxygenase. These results show that the antiallergic action of amoxanox is associated, at least in part, with the reduction of leukotrienes due to the inhibition of lipoxygenases.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 5,8,11,14-Eicosatetraynoic Acid; Aminopyridines; Animals; Asthma; Calcimycin; Cattle; Guinea Pigs; Histamine H1 Antagonists; Hydroxyeicosatetraenoic Acids; Hypersensitivity; In Vitro Techniques; Leukotriene B4; Lipoxygenase Inhibitors; Lung; Male; Monocytes; Prostaglandins; Rats; Rats, Inbred Strains; SRS-A; Thromboxane B2

A cone and plate viscometer and Coulter Counter were used to study platelet modulation of polymorphonuclear leukocyte (PMNL) aggregation caused by controlled shear stress. As an index of aggregation, the large-particle percentage (LPP) was calculated. This represents the ratio of aggregated cell count to total cell count. PMNL suspensions in buffer (1.0 X 10(7) cells per milliliter, final concentration) did not show any aggregate formation at shear stresses below 150 dynes/cm2 for one minute exposure time (LPP less than 3%). However, there was PMNL aggregation in mixed PMNL and platelet-rich plasma suspensions in this shear stress range. Supernatant plasma from sheared platelets initiated PMNL aggregation at moderate shear stress (150 dynes/cm2 for one minute; LPP, 20.3% +/- 2.5%). In contrast, platelet release factors, such as adenosine diphosphate (2 mumol/L) and serotonin (2 mumol/L) did not cause PMNL aggregation (LPP, 2.9% +/- 1.2% and 3.3% +/- 0.8%, respectively). The use of a cyclo-oxygenase inhibitor (acetylsalicylic acid, 50 mumol/L) did not suppress the aggregation of PMNLs after shear (LPP, 20.1% +/- 2.4%). However, preincubation with nordihydroguaiaretic acid (10 mumol/L), an inhibitor of C-5 and C-12 lipoxygenase, and 6,9-deepoxy-6,9-(phenylimino)-6,8-prostaglandin I1 (U-60257, 10 mumol/L), an inhibitor of C-5 lipoxygenase in human leukocytes, suppressed this aggregation (LPP, 9.1% +/- 2.5% and 10.4% +/- 3.2%, respectively). Also, the formation of lipoxygenase products (5-HETE, 12-HETE, 15-HETE, and LTB4) activated by shear stress was documented by reversed phase-high-performance liquid chromatography (RP-HPLC). These data support the possibility of a cooperation between platelets and leukocytes in shear-induced PMNL aggregation that is dependent on C-12 or C-5 lipoxygenase activity, or both.

Topics: Arachidonic Acid; Arachidonic Acids; Blood Platelets; Calcimycin; Cell Aggregation; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Lipoxygenase; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils

The production of lipoxygenase metabolites of arachidonic acid was studied in bovine alveolar macrophages (BAM). Unstimulated macrophages produced small amounts of LTB4 (0.2 +/- 0.2 ng/10(6) BAM) but monohydroxyeicosatetraenoic acids (5-, 12-, and 15-HETE) usually were not detectable. Both exogenous arachidonic acid and the calcium ionophore A23187 induced production of LTB4, 5-, 12-, and 15-HETE, of which 60-80% was 5-HETE. Combined challenge of BAM with both exogenous arachidonic acid and A23187 was more effective in the production of these metabolites than with either stimulus alone. The generation of the peptidoleukotrienes LTC4, LTD4, and LTE4 by BAM could not be detected under these in vitro conditions. Our results demonstrate that bovine alveolar macrophages produce similar lipoxygenase metabolites of arachidonic acid in response to A23187, as do human alveolar macrophages stimulated with the same agonist.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Cattle; Glutathione; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Lipoxygenase; Macrophages; Male; Pulmonary Alveoli; SRS-A

Caseinate elicited suspension of guinea pig peritoneal PMNs synthesized LTB4, 6t-LTB4, 12-epi-6t-LTB4 and 5HETE after incubations with A23187 and arachidonic acid. Concentrations of LTB4 peaked in 3 minutes and were then rapidly depleted. 6t-LTB4 and 12-epi-6t-LTB4 also peaked in concentrations in 3 min but were depleted slower than LTB4. NaCN inhibited the depletion of LTB4 in a dose dependent fashion without dramatically affecting biosynthesis.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Cyanides; Guinea Pigs; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Kinetics; Leukotriene B4; Neutrophils; Sodium Cyanide; Stereoisomerism

Topics: Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids; Calcimycin; Chromatography, High Pressure Liquid; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Leukotriene B4; Lipoxygenase; Neutrophils; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet; Time Factors

Chronic inflammation has long been associated with carcinogenesis. Phorbol esters which are potent promoters of tumors in mouse skin are also potent inflammatory agents in skin and cause inflammatory cells to release large quantities of reactive oxygen intermediates and oxidized lipid products. SENCAR mice have been bred for their sensitivity to the promotion of tumors by phorbol esters and C57BL/6 mice have been shown to be resistant. We quantified the release of H2O2 and metabolites of arachidonic acid by macrophages obtained from SENCAR and C57BL/6 mice, following exposure to phorbol esters and other stimulants. The basal level for secretion of H2O2 in resident peritoneal macrophages was negligible in cells from both strains. Conversely, inflammatory macrophages from SENCAR mice, elicited by the injection of sterile casein, secreted 4 times more H2O2 than the corresponding cells from C57BL/6 mice. Furthermore, cells from SENCAR mice required less than one-third the amount of phorbol ester to obtain 50% of the maximal response than that required by cells from C57BL/6 mice. This difference was less when zymosan was used as a stimulant. Both resident and inflammatory macrophages from SENCAR mice released more metabolites of arachidonic acid than cells from C57BL/6 mice when exposed to phorbol esters, but macrophages from C57BL/6 mice released more metabolites when stimulated with zymosan. Few differences in the pattern of released metabolites were noted between the strains of mice. There were large differences in the relative amounts of individual metabolites released when different stimulants were used. The enhanced response to phorbol esters of chronic inflammatory cells from SENCAR mice correlates with the enhanced sensitivity to the promotion of tumors by phorbol esters in these animals.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Caseins; Hydrogen Peroxide; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Macrophages; Mice; Mice, Inbred Strains; Phorbol Esters; Prostaglandins; SRS-A; Zymosan

The effect of anthralin and its oxidation products danthrone and anthralin-dimer on the production of 5-lipoxygenase products (5-HETE, leukotriene B4, omega-oxidized LTB4) by Ca-ionophore A 23187-stimulated human neutrophils has been studied in vitro. Anthralin exhibited dose-dependent inhibitory activity showing 50% inhibition at 7 microM with 10(7) neutrophils. Inhibitory effects strongly depended upon cell densities and maximal inhibition occurred at low cell concentrations, whereas inhibitory rates of anthralin were low at high cell densities. Inhibition of leukotriene production persisted after washing of anthralin-treated neutrophils. Also, with increasing amounts of arachidonic acid as substrate only slight changes of inhibitory activity were detected, indicating a noncompetitive way of action. In addition to the inhibition of leukotriene-production, the formation of omega-OH-LTB4 from LTB4 as well as omega-COOH-LTB4 from omega-OH-LTB4 was inhibited with IC50 (half maximum inhibition concentration) near 4.4 microM and 2.2 microM, respectively. In contrast to anthralin, both metabolites--danthrone as well as anthralin-dimer--did not show any effect on leukotriene production and omega-oxidation even at high concentrations (up to 70 microM and 44 microM, respectively).

Topics: Anthralin; Anthraquinones; Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids; Calcimycin; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Lipoxygenase Inhibitors; Neutrophils; Oxidation-Reduction

Phorbol myristate acetate augmented the release of 3H-AA and the synthesis of leukotriene B4 and 5-hydroxyeicosatetraenoic acid by human polymorphonuclear leukocytes stimulated by A23187. PMA alone had no effect. Enhancement of the response to A23187 was not seen when the inactive phorbol ester 4-alpha phorbol didecanoate was added with A23187. These data are consistent with the hypothesis that activation of protein kinase C enhances AA release and metabolism in stimulated polymorphonuclear leukocytes.

Topics: Arachidonic Acid; Arachidonic Acids; Calcimycin; Drug Synergism; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Neutrophils; Tetradecanoylphorbol Acetate; Time Factors

To determine whether chronic glucocorticoid excess influences the metabolism of arachidonic acid to prostaglandins (PGs) in the renal cortex, the authors investigated the effects of dexamethasone treatment (2.5 mg/kg/week) on the metabolism of arachidonic acid by renal cortex homogenates and microsomes and by isolated glomeruli, and on the release of immunoreactive prostanoids from isolated glomeruli incubated for 30 min in buffered salt solution at 37 degrees C. Glomeruli from dexamethasone-treated rats released, during basal incubation conditions, about twice (P less than .01) as much PGE2 and PGF2 alpha as did glomeruli from vehicle-treated rats. During incubation with arachidonic acid (33 microM) or calcium ionophore, A23187 (2.0 micrograms/ml), the release of PGE2 and PGF2 alpha from glomeruli of rats receiving dexamethasone also exceeded (P less than .01) the release from glomeruli of control rats. The rate of conversion of [1-14C]arachidonic acid to PGE2 and PGF2 alpha and to less polar metabolites having the chromatographic mobility of 5-hydroxyeicosatetraenoic acid and 12-hydroxyeicosatetraenoic acid, by isolated glomeruli and by renal cortex homogenates and microsomes from dexamethasone-treated rats, was higher (P less than .01) than the conversion by glomeruli and renal cortex homogenates and microsomes from control rats. The metabolism of arachidonic acid to the nonpolar metabolite(s) was not inhibited by indomethacin (10 microM), suggesting that it is not catalyzed by cyclooxygenase. The authors conclude that chronic dexamethasone treatment increases the release of glomerular PGE2 and PGF2 alpha and the metabolic transformation of arachidonic acid by glomeruli and by renal cortex homogenates and microsomes via both cyclooxygenase and noncyclooxygenase pathways.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Dexamethasone; Dinoprost; Dinoprostone; Hydroxyeicosatetraenoic Acids; Indomethacin; Kidney Cortex; Kidney Glomerulus; Male; Microsomes; Prostaglandins; Prostaglandins E; Prostaglandins F; Rats; Rats, Inbred Strains

Fatty acids are labeled with 18O in the carboxyl group during ester hydrolysis in H2(18)O. We utilized this principle to develop a novel mass spectrometric method for study of the turnover of arachidonic acid in intact cell systems. Analysis of 18O incorporations was by negative ion chemical ionization GC-MS. The use of deuterium-labeled exogenous substrates allowed the metabolic fate of exogenous and intrinsic compounds to be distinguished. Thus, it was shown that the preferred route of 5-lipoxygenase metabolism of exogenous arachidonic acid in ionophore-stimulated human neutrophils is via direct reaction with the enzyme and not via incorporation into cellular lipids. The re-uptake of 5-HETE was also studied. For investigation of intracellular reactions which involve ester hydrolysis, the 18O labeling method is unique in providing direct evidence of the intermediate metabolic pathway of endogenous and exogenous products associated with the arachidonic acid cascade.

Topics: Arachidonic Acid; Arachidonic Acids; Calcimycin; Deuterium; Esters; Gas Chromatography-Mass Spectrometry; Humans; Hydrolysis; Hydroxyeicosatetraenoic Acids; Lipoxygenase; Neutrophils

Human neutrophils incorporated 5-hydroxy-E,Z,Z,Z-6,8,11,14-eicosatetraenoic acid (5-HETE) into cellular triglyceride and phospholipid. They also metabolized 5-HETE into a novel, extracellularly released derivative, 5,20-dihydroxy-E,Z,Z,Z-6,8,11,14-eicosatetraenoic acid (5,20-diHETE). 5,20-diHETE formation predominated at higher substrate concentrations and longer incubation intervals. In the absence of added 5-HETE, 1 X 10(8) neutrophils stimulated with 20 microM ionophore A23187 produced up to 243 ng of 5,20-diHETE, indicating that both endogenously formed and exogenously added substrate could be oxidized at carbon 20. 5,20-diHETE was about 10- to 100-fold weaker than 5-HETE in enhancing human neutrophil degranulation responses to platelet-activating factor. omega-Oxidation appears to be a general enzymatic mechanism for inactivation of arachidonic acid metabolites.

Topics: Calcimycin; Glucuronidase; Humans; Hydroxyeicosatetraenoic Acids; Muramidase; Neutrophils; Oxidation-Reduction; Phospholipids; Platelet Activating Factor; Spectrum Analysis; Triglycerides

A cloned murine mast cell line designated MC9 expresses a 5-lipoxygenase activity when stimulated with the ionophore A23187. Upon addition of 0.5 microM ionophore, MC9 cells produce 270 +/- 43 pmoles 5-HETE, 74 +/- 40 pmoles 5,12 diHETEs and 65 +/- 31 pmoles LTC4/10(6) cells from 37 microM exogenously added [1-14C]arachidonic acid in two minutes. 5-HETE and 5,12-diHETES, including LTB4 were identified by GC/MS whereas LTC4 was confirmed by HPLC mobility, bio-assay, RIA and enzymatic transformation. The principal cyclooxygenase products were PGD2 and TxB2 (8.5 +/- 2.4 and 5.4 +/- 1.2 pmoles/10(6) cells respectively). Prostanoids were identified by comigration with authentic standards on two-dimensional thin layer chromatograms. Production of arachidonic acid lipoxygenase metabolites stimulated with ionophore proved relatively insensitive to removal of extracellular Ca+2 and chelation by EGTA. In addition, MC9 5-lipoxygenase required only low micromolar amounts of exogenous arachidonic acid for maximal activity. Whereas production of arachidonic acid metabolites lasted only two to five minutes, histamine release stimulated with ionophore was not initiated until 5 minutes (12 +/- 3% cellular histamine) and continued for 30 minutes (37 +/- 7% cellular histamine). Although these cells metabolize arachidonic acid differently from the classic peritoneal-derived mast cell, they resemble subpopulations found in certain tissues (such as mucosa) and should be useful in understanding the biochemistry of mast cell mediator release.

Topics: Animals; Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids; Calcimycin; Clone Cells; Dose-Response Relationship, Drug; Histamine Release; Hydroxyeicosatetraenoic Acids; Lipoxygenase; Mast Cells; Mice; SRS-A

The inhibitory effect of the drug BW755C on the 5-lipoxygenase pathway was analyzed for bone marrow-derived murine mast cells, termed E-mast cells. The drug prevented the formation of 5-HETE from exogenous [14C]arachidonic acid when IgE-sensitized cells were challenged by the antigen. BW755C also prevented formation of leukotriene C4 in a dose-dependent fashion when IgE-sensitized mast cells, preincubated with the drug, were activated with either the specific antigen or the ionophore. Leukotriene C4 inhibition occurred with a minimal drug preincubation period of 1 min before the cells were subjected to antigen-dependent activation. BW755C did not affect the degranulation response of these cells. Thus in an intact cell system BW755C prevents 5-lipoxygenation of arachidonic acid. Furthermore, this study reveals that even with transmembrane activation of E-mast cells through their IgE-Fc receptors, granule secretion is not dependent upon corresponding metabolites from the 5-lipoxygenase pathway.

Topics: 4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyrazol-3-amine; Animals; beta-N-Acetylhexosaminidases; Bone Marrow Cells; Calcimycin; Cytoplasmic Granules; Dose-Response Relationship, Drug; Hexosaminidases; Histamine Release; Hydroxyeicosatetraenoic Acids; Immunoglobulin E; Lipoxygenase Inhibitors; Male; Mast Cells; Mice; Mice, Inbred BALB C; Pyrazoles; SRS-A

Inhibition of prostaglandin formation from [14C]arachidonic acid by rat peritoneal leucocytes occurred with nonsteroidal anti-inflammatory drugs, their order of potency being indomethacin greater than piroxicam greater than naproxen greater than ibuprofen greater than isoxicam. At the lowest concentration tested (1 microgram ml-1), indomethacin markedly increased the accumulation of lipoxygenase products in the cell incubates. Naproxen, ibuprofen or piroxicam 1 or 10 micrograms ml-1 resulted in smaller increases of lipoxygenase products, and there was only a small rise with these concentrations of isoxicam.

Topics: Animals; Anti-Inflammatory Agents; Arachidonic Acid; Arachidonic Acids; Calcimycin; Hydroxyeicosatetraenoic Acids; Ibuprofen; In Vitro Techniques; Indomethacin; Leukocytes; Male; Naproxen; Piroxicam; Prostaglandins; Rats; Rats, Inbred Strains; Thiazines

Stimulus-activated polymorphonuclear neutrophils (PMN) produce leukotriene B4 (LTB4), 5-hydroxyeicosatetraenoate (5-HETE), and platelet-activating factor (PAF). Each of these lipids promotes PMN degranulation; in combination they have additive and potentiating effects that result in prominent degranulation responses at relatively low concentrations. Thus, the combined interactions of LTB4, 5-HETE, and PAF may mediate responses in PMN activated by other stimuli. This possibility was examined by measuring the responses of PMN made insensitive to one or more of these lipids. Cells were pretreated with LTB4, 5-HETE, and/or PAF for 8 min; exposed for 2 min to cytochalasin B (which is required for lipid-induced degranulation); and then challenged. PMN challenged with only buffer released minimal amounts of granule-bound enzymes. Furthermore, the lipid-pretreated cells were hyporesponsive to challenge with 1) various combinations of these same lipids or 2) ionophore A23187. The relative potencies of the lipids in producing hyporesponsiveness to themselves or A23187 were: 5-HETE less than PAF less than or equal to LTB4 less than PAF + LTB4 less than PAF + LTB4 + 5-HETE. For both types of challenge, reduced responsiveness occurred in cells pretreated with greater than 0.1 nM LTB4 and/or greater than 0.2 nM PAF, persisted in cells washed after lipid pretreatment, and did not develop in cells pretreated with various combinations of bioinactive structural analogues of the lipids. Thus, PAF, LTB4, and 5-HETE interacted to desensitize PMN, and the degranulating actions of A23187 required cells that were fully responsive to each of the three lipids. This supports the concept that the lipids act together in mediating certain of the ionophore's effects. However, lipid-desensitized PMN degranulated fully when challenged with C5a, a formylated oligopeptide, or phorbol myristate acetate. Degranulation responses, therefore, may proceed through various pathways, only some of which involve the lipid products studied here.

Topics: Animals; Calcimycin; Complement C5; Complement C5a; Cytoplasmic Granules; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Lipids; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Platelet Activating Factor; Rabbits

Arachidonic acid metabolism via the lipoxygenase pathway was examined in HL-60 cells before and after N,N-dimethylformamide induced differentiation along granulocytic lines. Untreated HL-60 cells produced small amounts of the 5-lipoxygenase products, 5-hydroxy-eicosatetraenoic acid and leukotriene B4 upon stimulation with calcium ionophore A23187. N,N-dimethylformamide treatment, caused a 10 to 20 fold increase in the amount of ionophore A23187-induced 5-lipoxygenase metabolites. An additional, and as yet unidentified arachidonic acid metabolite was routinely observed during reverse-phase high pressure liquid chromatography analyses of lipoxygenase products. Sensitivity to inhibition by less than 10(-7)M indomethacin coupled with other characteristics of its production, strongly suggest the compound is a cyclooxygenase product. The unusual UV absorbance and chromatographic elution pattern, however, suggest that it is not a typical prostaglandin, thromboxane or prostacyclin product.

Topics: Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids; Calcimycin; Cell Line; Chromatography, High Pressure Liquid; Humans; Hydroxyeicosatetraenoic Acids; Leukemia, Myeloid, Acute; Leukotriene B4; Lipoxygenase; Prostaglandin-Endoperoxide Synthases

The effects of alcohols on the formation of leukotrienes, 5-HETE and prostaglandin D2 in mastocytoma cells and human neutrophils were studied. In murine mastocytoma cells, alcohols appear to have at least two different effects on the production of these arachidonic acid metabolites. At low levels of cellular arachidonic acid achieved after stimulation with calcium ionophore A23187 or addition of low levels of exogenous arachidonic acid, alcohols appear to have a general inhibitory effect on the production of lipoxygenase metabolites. In the presence of higher concentrations of cellular arachidonic acid, ethanol and methanol stimulated the production of lipoxygenase metabolites, but had no large stimulatory effect on the cyclo-oxygenase metabolite, prostaglandin D2. Under these conditions, n-propanol and t-butanol have inhibitory effects on leukotriene production. Human neutrophils are less sensitive to ethanol than mastocytoma cells, but stimulatory effects were still found at high ethanol concentrations (220-430 mM).

Topics: Alcohols; Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Cell Line; Cell-Free System; Chromatography, High Pressure Liquid; Ethanol; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Lipoxygenase; Mast-Cell Sarcoma; Methanol; Mice; Neutrophils; Prostaglandin D2; Prostaglandins D; Time Factors

The cellular and extracellular distribution of leukotriene B4 (LTB4) generated in human neutrophilic polymorphonuclear leukocytes (PMN) stimulated with unopsonized zymosan has been compared with that generated in PMN activated by the calcium ionophore. The amounts of extracellular and intracellular LTB4 were quantitated by radioimmunoassay. The authenticity of the immunoreactive LTB4 was confirmed by the elution of a single immunoreactive peak after reverse phase-high performance liquid chromatography (RP-HPLC) at the retention time of synthetic LTB4, by the identical elution time of a peak of radiolabeled product derived from [3H]arachidonic acid-labeled PMN with the immunoreactive product, and by the comparable chemotactic activity on a weight basis of immunoreactive LTB4 and synthetic LTB4 standard. Under optimal conditions of stimulation by unopsonized zymosan, more than 78% of the generated immunoreactive LTB4 remained intracellular, whereas with optimal activation by the ionophore, less than 8.6% of immunoreactive LTB4 was retained. Resolution by RP-HPLC of the products from the supernatants and cell extracts of [3H]arachidonic acid-labeled PMN stimulated with unopsonized zymosan and those stimulated with calcium ionophore allowed identification and measurement of 5-hydroxyeicosatetraenoic acid (5-HETE), 6-trans-LTB4, LTB4, and omega oxidation products of LTB4 by radioactivity. With zymosan stimulation of PMN, 5-HETE and the 6-trans-LTB4 diastereoisomers were not released, LTB4 was partially released, and the omega oxidation products of LTB4 were preferentially extracellular in distribution. In contrast, with ionophore stimulation, only 5-HETE had any duration of intracellular residence being equally distributed intra- and extracellularly throughout the 30-min period of observation; 6-trans-LTB4, LTB4, and the omega oxidation products of LTB4 were retained at less than 19%. The respective distributions of 5-HETE after zymosan and ionophore stimulation were not altered by the introduction of albumin to the reaction mixtures to prevent reacylation, or by hydrolysis of the cell extract to uncover any product that had been reacylated. The finding that stimulation of PMN with unopsonized zymosan results in the cellular retention of 5-lipoxygenase products suggests that release of these metabolites may be an event that is regulated separately from their generation.

Topics: Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids; Calcimycin; Humans; Hydroxyeicosatetraenoic Acids; Kinetics; Leukotriene B4; Lipoxygenase; Neutrophils; Opsonin Proteins; Phagocytosis; Zymosan

Rat isolated intestine incubated in Krebs solution converted exogenous [14C]-arachidonic acid into products that chromatographed with prostaglandins, leukotriene B4 and 5-hydroxy-eicosatetraenoic acid. Accumulation of these products was increased by the laxative ricinoleic acid (0.34 mM) or the calcium ionophore A23187 (7.6 microM). In the presence of the calcium antagonists TMB-8 (0.43 microM) or verapamil (0.2 microM) the mean effects of ricinoleic acid or the calcium ionophore were smaller. Stimulation of arachidonic acid metabolism by ricinoleic acid therefore seems likely to involve a calcium-dependent mechanism.

Topics: Animals; Arachidonic Acids; Calcimycin; Calcium; Colon; Fatty Acids, Unsaturated; Gallic Acid; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Leukotriene B4; Male; Prostaglandins; Rats; Ricinoleic Acids; Verapamil

We investigated the effects of monohydroxyeicosatetraenoic acids (monoHETEs) on lipoxygenase- and cyclooxygenase-catalyzed reactions in glycogen-elicited rat PMNs challenged with A23187 and exogenous [14C]arachidonic acid. A23187 (10 microM) stimulated a 10-, 4-, 1.7- and 1.8-fold increase in the synthesis of radiolabeled 5-HETE, LTB4, TxB2, and PGE2 by rat PMNs. Addition of 5-HETE, 5 lactone-HETE, 12-HETE, and 15-HETE led to a dose-related reduction in [14C]5-HETE and [14C]LTB4 synthesis by these cells. These monoHETEs also inhibited [14C]TxB2 synthesis, but only 5-HETE and 5 lactone-HETE inhibited the synthesis of [14C]PGE2. Both 12-HETE and 15-HETE failed to reduce the formation of [14C]PGE2. These results suggest that monoHETEs differ significantly in their effects on arachidonic acid metabolism in rat PMNs and may play a role in modulating the synthesis of both lipoxygenase and cyclooxygenase products.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acids; Calcimycin; Carbon Radioisotopes; Dinoprostone; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Neutrophils; Prostaglandins E; Rats; Thromboxane A2

The mechanism of action of gonadotropin-releasing hormone (GnRH) upon pituitary luteinizing hormone (LH) secretion has not yet been elucidated, but recent evidence has suggested that arachidonic acid or its metabolites are involved in GnRH action. In cultured rat pituitary cells, arachidonic acid and 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) elicited concentration-dependent release of LH with EC50 of about 12 microM. Other lipoxygenase derivatives including 11-, 12- and 15-HETE, had no consistent effect on LH release, and leukotrienes (B4 and C4) exerted only minor stimulatory actions on LH release. The lipoxygenase inhibitors nordihydroguaiaretic acid (NDGA), 5,8,11,14-eicosatetraynoic acid (ETYA), and 3-amino-1-(3-trifluoromethyl phenyl)-2-pyrazoline hydrochloride (BW 755C) caused dose-dependent inhibition of GnRH-induced LH release, with IC50 values of 5, 8.5, and 175 microM, respectively. In contrast, the cyclooxygenase inhibitor, indomethacin, had a biphasic action on GnRH-stimulated LH release, with potentiation of GnRH action at low doses (up to 25 microM) and no effect at higher concentrations. These findings are consistent with the potential role of a 5-lipoxygenase product of arachidonic acid in the mechanism of action of GnRH on pituitary gonadotropin release.

Topics: 4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyrazol-3-amine; 5,8,11,14-Eicosatetraynoic Acid; Animals; Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids; Calcimycin; Catechols; Cells, Cultured; Gonadotropin-Releasing Hormone; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Lipoxygenase; Luteinizing Hormone; Masoprocol; Pituitary Gland; Pyrazoles; Rats; SRS-A

When human neutrophils, previously labeled in their phospholipids with [14C]arachidonate, were stimulated with the Ca2+-ionophore, A23187, plus Ca2+ in the presence of [3H]acetate, these cells released [14C]arachidonate from membrane phospholipids, produced 5-hydroxy-6,8,11,14-[14C]eicosatetraenoic acid (5-HETE) and 14C-labeled 5S,12R-dihydroxy-6-cis,8,10-trans, 14-cis-eicosatetraenoic acid ([14C]leukotriene B4), and incorporated [3H]acetate into platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine). Ionophore A23187-induced formation of these radiolabeled products was greatly augmented by submicromolar concentrations of exogenous 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE), 5-HETE, and leukotriene B4. In the absence of ionophore A23187, these arachidonic acid metabolites were virtually ineffective. Nordihydroguaiaretic acid (NDGA) and several other lipoxygenase/cyclooxygenase inhibitors (butylated hydroxyanisole, 3-amino-1-(3-trifluoromethylphenyl)-2-pyrazoline and 1-phenyl-2-pyrazolidinone) caused parallel inhibition of [14C]arachidonate release and [3H]PAF formation in a dose-dependent manner. Specific cyclooxygenase inhibitors, such as indomethacin and naproxen, did not inhibit but rather slightly augmented the formation of these products. Furthermore, addition of 5-HPETE, 5-HETE, or leukotriene B4 (but not 8-HETE or 15-HETE) to neutrophils caused substantial relief of NDGA inhibition of [3H]PAF formation and [14C]arachidonate release. As opposed to [3H]acetate incorporation into PAF, [3H]lyso-PAF incorporation into PAF by activated neutrophils was little affected by NDGA. In addition, NDGA had no effect on lyso-PAF:acetyl-CoA acetyltransferase as measured in neutrophil homogenate preparations. It is concluded that in activated human neutrophils 5-lipoxygenase products can modulate PAF formation by enhancing the expression of phospholipase A2.

Topics: Acetates; Acetic Acid; Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids; Calcimycin; Catechols; Humans; Hydroxyeicosatetraenoic Acids; Lipoxygenase; Masoprocol; Membrane Lipids; Neutrophils; Phospholipases; Phospholipases A; Phospholipases A2; Platelet Activating Factor; Trifluoperazine

Arachidonic acid metabolism in ionophore A23187-activated human polymorphonuclear leukocytes (PMNs) proceeds predominantly via the 5-lipoxygenase pathway in comparison to metabolism by the 15-lipoxygenase route. Products of both lipoxygenase pathways appear to be involved in the mediation of inflammatory reactions. Pretreatment of polymorphonuclear leukocytes with micromolar amounts of the platelet-derived 12-lipoxygenase product 12-hydroxy-5,8,10,14- eicosatetraenoic acid (12-HETE) prior to the addition of A23187 and [14C]arachidonic acid resulted in the unexpected dose-dependent stimulation of the 15-lipoxygenase pathway, as evidenced by the formation of [14C]15-HETE. A concomitant inhibition of the 5-lipoxygenase pathway was also observed. The structural identity of 15-HETE was confirmed by retention times on straight-phase and reverse-phase high pressure liquid chromatography in comparison with an authentic standard, radioimmunoassay, and chemical derivatization. When other isomeric HETEs were tested, the order of stimulatory potencies was 15-HETE greater than 12-HETE greater than 5-HETE. When arachidonic acid metabolism via the 5-lipoxygenase route was inhibited by nordihydroguaiaretic acid, previously ineffective concentrations of exogenous 12-HETE were now able to stimulate the polymorphonuclear leukocyte 15-lipoxygenase. Thus, blockade of the 5-lipoxygenase pathway appeared to be a prerequisite for the activation of the 15-lipoxygenase. The HETE-induced activation of the 15-lipoxygenase occurred within 1-2 min, was a reversible process, and was enhanced in the presence of A23187. In nine donors tested, up to 14-fold stimulation of [14C]15-HETE production was observed. Our results indicate that endogenous HETEs can have a dual role in the post-phospholipase regulation of arachidonic acid metabolism since they can act as physiological stimulators of the 15-lipoxygenase as well as inhibitors of the 5-lipoxygenase.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids; Calcimycin; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Humans; Hydroxyeicosatetraenoic Acids; Lipoxygenase; Models, Chemical; Neutrophils; Time Factors

Suspensions of human blood leukocytes and platelets were stimulated with the ionophore A23187 in the presence of increasing concentrations of arachidonic acid. Nine metabolites of the 5-, 12- and 15-lipoxygenase and the cyclooxygenase pathways were analyzed by high performance liquid chromatography in order to study the rate-limiting steps and other characteristics of their biosynthesis. The data indicate that the LTA4 hydrolase and the LTA4 glutathione transferase activities are respectively the limiting factors in the synthesis of LTB4 and LTC4. At high substrate concentrations (5 X 10(-5)M and above), the 5-lipoxygenase activity was inhibited whereas the synthesis of the 15-HETE (15-lipoxygenase product) increased linearly up to the highest concentration tested (3 X 10(-4)M). The data also indicate that contrary to 5-HETE and LTs, 15-HETE is not formed upon stimulation with the ionophore, but only following incubation with exogenous arachidonic acid (20 microM and above), pointing out major differences in the synthesis of 5- and 15-lipoxygenase products.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonate Lipoxygenases; Arachidonic Acids; Blood Platelets; Calcimycin; Humans; Hydroxyeicosatetraenoic Acids; Leukocytes; Leukotriene B4; Lipoxygenase; SRS-A

We studied interactions of human platelets and neutrophils with particular reference to the arachidonic acid pathway. Suspensions of [3H]-arachidonate-labeled platelets and unlabeled neutrophils were stimulated with ionophore A23187. We detected several radioactive arachidonate metabolites, which are not produced by platelets alone. This included [3H]-labeled leukotriene B4 (LTB4), dihydroxyeicosatetraenoic acid (DHETE) and 5-hydroxyeicosatetraenoic acid (5-HETE). When the platelet product, [3H]12-HETE, was added to ionophore-stimulated neutrophils, DHETE was formed. In addition, when [3H]5-HETE, a neutrophil arachidonate product, was added to stimulated platelets, DHETE was the major metabolite. We, therefore, suggest that upon stimulation, the platelet derived arachidonate can serve as precursor for the neutrophil-derived eicosanoids LTB4 and 5-HETE and that the platelet-derived product 12-HETE can be metabolized to DHETE by stimulated human neutrophils. It would appear that the platelet and neutrophil lipoxygenase pathways take part in cell-cell interactions - an observation which suggests a role for the neutrophils which are present in hemostatic plugs, thrombi and inflammatory processes.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids; Blood Platelets; Calcimycin; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Lipoxygenase; Neutrophils

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonate Lipoxygenases; Arachidonic Acids; Calcimycin; Humans; Hydroxyeicosatetraenoic Acids; Leukocytes; Leukotrienes; Lipoxygenase; SRS-A

The syntheses of 7,7-dimethyl- (1), 10,10-dimethyl- (2), and 5,6-benzoarachidonic acid (3), potential substrate analogue inhibitors of leukotriene biosynthesis, are described. Two of these compounds (1 and 2) apparently stimulated, while 3 inhibited, the activity of lipoxygenase from intact human polymorphonuclear leukocytes in vitro when stimulated with Ca2+ and calcium ionophore A23187 in the presence of BSA and arachidonic acid.

Topics: Arachidonic Acids; Calcimycin; Calcium; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Neutrophils; SRS-A

Platelet-activating factor (AAGPC) and two of its structural analogues degranulated human neutrophils with respective potencies that were increased up to 100 to 1000-fold by 16 nM to 5 microM of 5-L-hydroxyeicosatetraenoate (5-L-HETE). 5-rac-HETE had similar actions but 8-rac-HETE was without effect. Furthermore, 5-L-HETE did not influence the degranulating actions of C5a, A23187 or a formalated oligopeptide chemotactic factor and none of the HETEs, by themselves, caused degranulation. Thus, 5-L-HETE and AAGPC selectively interact to induce degranulation. Since these products rapidly form in stimulated PMNs, they may serve as potentiator and agonist, respectively, to transduce biological signals into cell function.

Topics: Arachidonic Acids; Calcimycin; Complement C5; Complement C5a; Drug Synergism; Glucuronidase; Humans; Hydroxyeicosatetraenoic Acids; Muramidase; N-Formylmethionine; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Oligopeptides; Platelet Activating Factor; Time Factors

HeLa cells incubated with 12-O-tetradecanoylphorbol-13-acetate (TPA), and rat basophilic leukemia (RBL-1) cells incubated with calcium ionophore, showed increased levels of the protease plasminogen activator. These treatments have previously been shown to stimulate the cellular metabolism of arachidonic acid. The induction of plasminogen activator in both cell types was inhibited in a dose-dependent manner by 5,8,11,14-eicosatetraynoic acid and nordihydroguaiaretic acid, two compounds known to inhibit arachidonate metabolism via lipoxygenases. In contrast, indomethacin, which selectively inhibits arachidonate metabolism via cyclooxygenase, was inactive. The levels of four enzyme markers in HeLa cells were unchanged by treatment with TPA plus the lipoxygenase inhibitors, indicating that the inhibitors did not exert their effects on plasminogen activator via general cell toxicity. HeLa cells preincubated with [3H]arachidonate and subsequently challenged with TPA produced small amounts of material with the chromatographic mobilities and resistance to indomethacin expected of hydroxylated fatty acids derived via lipoxygenase. RBL-1 cells have been shown previously to produce leukotrienes and other lipoxygenase metabolites when treated with calcium ionophore. Plasminogen activator in HeLa cells was stimulated by up to 2.5-fold by incubation with 0.5-2 micrograms/ml 5-hydroxyeicosatetraenoic acid. Our results suggest that the induction of plasminogen activator in HeLa and RBL-1 cells is not mediated by prostaglandins or thromboxanes, but may be mediated or modulated by arachidonate metabolites derived via a lipoxygenase pathway.

Topics: 5,8,11,14-Eicosatetraynoic Acid; Animals; Anti-Bacterial Agents; Arachidonic Acid; Arachidonic Acids; Calcimycin; Catechols; Cells, Cultured; Enzyme Induction; Fatty Acids; HeLa Cells; Humans; Hydroxyeicosatetraenoic Acids; Leukemia, Experimental; Lipoxygenase Inhibitors; Masoprocol; Phorbols; Plasminogen Activators; Rats; Tetradecanoylphorbol Acetate

The effect of the calcium ionophore A23187 on the release and metabolism of [3H]arachidonic acid was examined in normal polymorphonuclear leukocytes and those obtained from patients with chronic granulomatous disease. The ionophore A23187 which stimulates oxidative metabolism in normal polymorphonuclear leukocytes was ineffective in increasing oxidative metabolism (chemiluminescence) in polymorphonuclear leukocytes from patients with chronic granulomatous disease. However, the ionophore A23187 stimulated the release of [3H]arachidonic acid from chronic granulomatous disease neutrophil phospholipids and stimulated its metabolism into hydroxyeicosatetraenoic acids and leukotrienes.

Topics: Arachidonic Acid; Arachidonic Acids; Calcimycin; Granulomatous Disease, Chronic; Humans; Hydroxyeicosatetraenoic Acids; Luminescent Measurements; Neutrophils; Phospholipids

Neutrophils metabolize arachidonic acid through the liposygenase pathway to 5-hydroxy-6,8,11,14-eicosatetrenoic acid (5-HETE) and 5,12-dihydroxy-6,8,10,14-eicosatraenoic acid (5,12 diHETE). 5-HETE and 5,12diHETE are potent chemotactic agents and are thought to have important roles in the inflammatory response. In this study we demonstrate the sulfasalazine, at concentrations found in the stool of patients being treated for ulcerative colitis, blocks the synthesis of both 5-HETE and 5,12 diHETE by human neutrophils. A sulfasalazine metabolite, 5-aminosalicylate, also blocks the synthesis of 5,12 diHETE.

Topics: Aminosalicylic Acids; Arachidonic Acid; Arachidonic Acids; Calcimycin; Dose-Response Relationship, Drug; Humans; Hydroxyeicosatetraenoic Acids; Indomethacin; Leukotriene B4; Mesalamine; Neutrophils; Sulfasalazine

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonic Acids; Calcimycin; Chromatography, High Pressure Liquid; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Lipopolysaccharides; Lipoxygenase Inhibitors; Neisseria meningitidis; Neutrophils

Incubation of peripheral blood leukocytes with arachidonic acid (and ionophore A23187) led to the formation of leukotriene B4, delta 6-trans-leukotriene B4, delta 6-trans-12-epi-leukotriene B4, 5-hydroxy-icosatetraenoic acid, 12-hydroxy-icosatetraenoic acid and of 5S,12S-dihydroxy-6,8,10,14-(E,Z,E,Z)-icosatetraenoic acid (5S,12S-DiHETE). Incubation of leukocytes with leukotriene A4 resulted in the formation of leukotriene B4 and of its two delta 6-trans-isomers but not of the 5S, 12S-DiHETE. 18O2 labeling experiments have shown that the hydroxyl groups at C5 and C12 in the 5S,12S-DiHETE are derived from molecular oxygen. The tetraacetylenic analog of arachidonic acid was found to be potent inhibitor of the formation of the 5S,12S-DiHETE whereas it potentiated the synthesis of the 5-hydroxy acid and of leukotriene B4. Addition of the 12-hydroxy-icosatetraenoic acid to leukocytes, or of the 5-hydroxy-icosatetraenoic acid to a suspension of platelets caused the formation of the 5S,12S-DiHETE. It is concluded that the 5S,12S-DiHETE is not derived from leukotriene A4 but is a product of the successive reactions of arachidonic acid with two lipoxygenases of different positional specificities.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 5,8,11,14-Eicosatetraynoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Chromatography, High Pressure Liquid; Gas Chromatography-Mass Spectrometry; Hydroxyeicosatetraenoic Acids; Leukocytes; Leukotriene B4; Swine

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonic Acid; Arachidonic Acids; Biological Transport; Calcimycin; Complement C5; Deoxy Sugars; Deoxyglucose; Humans; Hydroxyeicosatetraenoic Acids; N-Formylmethionine; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Oligopeptides

Rat neutrophils isolated from three-hour carrageenan pleural exudates actively metabolize arachidonic acid into three major metabolites, HHT, 11-HETE and 15-HETE. However, in the presence of the calcium ionophore, A23187, or the non-ionic detergent, BRIJ 56, these cells also produce 5-HETE and LTB. The production of these lipoxygenase products is calcium dependent. While non-steroidal anti-inflammatory drugs do not affect 5-HETE or LTB production, BW 755C and ETYA inhibit formation of these metabolites from exogenously added arachidonic acid.

Topics: Animals; Arachidonic Acids; Calcimycin; Carrageenan; Cetomacrogol; Exudates and Transudates; Fatty Acids, Unsaturated; Hydroxy Acids; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Lipoxygenase; Male; Neutrophils; Pleura; Rats

Lipoxygenase product formation by rabbit PMNs on exposure to the divalent ionophore A23187 was dependent upon pH, metabolic energy and substrate availability. Formation of 5-HETE and 5,12-diHETE (the major arachidonate oxygenation products) was inhibited by calcium depletion, by the metabolic inhibitors iodoacetate and 2-deoxyglucose, and by the substrate inhibitor TYA. The reaction had a pH optimum of 7.0-7.5. Colchicine, cytochalasin B and isoprenaline had no significant effects on HETE formation.

Topics: Animals; Arachidonic Acids; Calcimycin; Colchicine; Cytochalasin B; Hydroxyeicosatetraenoic Acids; Isoproterenol; Leukotriene B4; Lipoxygenase; Neutrophils; Rabbits; Time Factors

[14C]Arachidonic acid incubated with human neutrophils was esterified into phospholipids and triglycerides. Stimulation of these labeled neutrophils with ionophore A23187 (2 microM) results in release of [14C]arachidonate from phospholipid and its metabolism to prostaglandin E2 and 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE), a lipoxygenase product. The released arachidonate is also metabolized to a polar lipid of unknown composition here disignated compound A. 5-HETE was found to be released into the medium and then taken up again by the cells. To determine its metabolic fate, [14C]5-HETE was prepared biosynthetically, purified, and incubated with stimulated, unlabeled neutrophils. Most of the radioactivity entered the cells and was esterified into phospholipids and triglycerides. The radiolabeled complex lipids were saponified, and the released fatty acids cochromatographed with authentic 5-HETE. The esterification of 5-HETE, a hydroxylated fatty acid, into membrane phospholipids may be an example of a more generalized mechanism for altering membrane characteristics.

Topics: Arachidonic Acids; Calcimycin; Carbon Radioisotopes; Cells, Cultured; Esterification; Humans; Hydroxy Acids; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Isotope Labeling; Neutrophils; Phospholipids; Prostaglandins E; Stimulation, Chemical; Triglycerides