5-hydroxy-6-8-11-14-eicosatetraenoic-acid and Leukemia--Basophilic--Acute

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 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

Synthetic routes were developed to access a variety of novel 1-aryl-2H,4H-tetrahydro-1,2,4-triazin-3-one analogs which were evaluated as 5-lipoxygenase (5-LO) inhibitors. The parent structure, 1-phenylperhydro-1,2,4-triazin-3-one (4), was found to be a selective inhibitor of 5-LO in broken cell, intact cell, and human blood assays with IC50 values of 5-21 microM. In a rat anaphylaxis model, 4 blocked leukotriene formation with an ED50 = 7 mg/kg when administered orally. Compound 4 exhibited selectivity for inhibition of 5-LO with little activity against related enzymes: 12-LO from human platelets, 15-LO from soybean, and cyclooxygenase (COX) from sheep seminal vesicle. In pilot subacute toxicity testing, 4 did not produce methemoglobinemia in rats (400 mg/kg po daily for 9 days) or in dogs (200 mg/kg po daily for 28 days). These results indicated that the triazinone structure provided a 5-LO inhibitor template devoid of the toxicity problems observed in the related phenidone (1) and pyridazinone (3) classes of 5-LO inhibitors. The parent compound 4 is a selective, orally bioavailable 5-LO inhibitor which can serve as a useful reference standard for in vivo pharmacological studies involving leukotriene-mediated phenonmena.

Topics: Animals; Arachidonate 12-Lipoxygenase; Arachidonate 5-Lipoxygenase; Blood Platelets; Dogs; Glycine max; Humans; Hydroxyeicosatetraenoic Acids; Leukemia, Basophilic, Acute; Leukotriene B4; Lipoxygenase Inhibitors; Macaca fascicularis; Male; Methemoglobin; Molecular Structure; Rats; Seminal Vesicles; Sheep; Structure-Activity Relationship; Triazines; Tumor Cells, Cultured

We investigated the inhibitory action of FK506 (0.0005-5 micrograms/ml) on the metabolism of arachidonate 5-lipoxygenase in rat basophilic leukemia-1 cells. Cells were stimulated with A23187 (10(-5) mol/l) for 15 min in the absence or presence of various concentrations of FK506. Arachidonate 5-lipoxygenase metabolites, peptide leukotrienes (LTs), leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) were measured by high-performance liquid chromatography. FK506 inhibited A23187-stimulated production of peptide LTs, LTB4 and 5-HETE in intact cells by up to 77, 73 and 60%, respectively. Phospholipase A2 activity, measured by the release of 3H-arachidonic acid (AA), was not significantly inhibited by FK506. The synthesis of peptide LTs and LTB4 was not inhibited by FK506 when leukotriene A4-free acid was added to the culture medium. The synthesis of peptide LTs, LTB4 and 5-HETE was not affected by FK506 in a cell lysate study using AA as the substrate. These results indicate that FK506 inhibits the production of peptide LTs, LTB4 and 5-HETE by inhibiting 5-lipoxygenase activity in intact cells. The inhibition is not a direct action on 5-lipoxygenase but results from the activating processes of this enzyme.

Topics: Animals; Hydroxyeicosatetraenoic Acids; Leukemia, Basophilic, Acute; Leukotrienes; Lipoxygenase Inhibitors; Phospholipases A; Phospholipases A2; Rats; Tacrolimus; Tumor Cells, Cultured

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

Rat basophilic leukemia cells exhibit 12-lipoxygenase activity only upon cell disruption. 12-Lipoxygenase may also possess 15-lipoxygenase activity, as is indicated by the formation of low amounts of 15(S)-HETE, in addition to the predominant product 12(S)-HETE, upon incubation of partially purified 12-lipoxygenase with arachidonic acid. With 5(S)-HPETE as substrate not only 5(S), 12(S)-diHETE and 5(S), 15(S)-diHETE are formed, but also LTA4, as was indicated by the presence of LTA4-derived LTB4-isomers. 12-Lipoxygenase from rat basophilic leukemia cells has many features in common with 12-lipoxygenase from bovine leukocytes. As was suggested for the latter enzyme, 12-lipoxygenase from rat basophilic leukemia cells may represent the remaining LTA4-synthase activity of 5-lipoxygenase, of which the 5-dioxygenase activity has disappeared upon cell disruption. Such a possible shift from 5-lipoxygenase activity to 12-lipoxygenase activity could not simply be induced by interaction of cytosolic 5-lipoxygenase with a membrane fraction after cell disruption, but may involve release of membrane-associated 5-lipoxygenase upon disruption of activated rat basophilic leukemia cells.

Topics: Animals; Arachidonate 12-Lipoxygenase; Arachidonate 5-Lipoxygenase; Cell Fractionation; Chromatography, High Pressure Liquid; Cytosol; Hydroxyeicosatetraenoic Acids; Kinetics; Leukemia, Basophilic, Acute; Rats; Tumor Cells, Cultured

Apart from the generation of potent inflammatory mediators, the effects of fatty acid oxygenase activation, per se, on the host cell have not been well-delineated. Fatty acid oxygenases were activated in rat basophilic leukemia cells (RBL-1) by incubating them for 2-4 hr with 33-300 microM of arachidonic acid (AA) or linoleic acid (LA). As a control, the cells were incubated with one of two analogs of these fatty acids which are not oxygenase substrates: eicosatetraynoic acid or linoelaidic acid. Effects of oxygenase activation on cell viability were monitored by an assay for mitochondrial function. Cytotoxicity occurred in incubations with exogenous AA or LA in direct proportion to the substrate concentration but was not found in the control incubations or in incubations with the principal monohydroxylated AA products, 5-, 15-, and 12-HETE. Nordihydroguaiaretic acid (80 microM) and alpha-tocopherol (100 microM) significantly decreased the cell death observed during incubations with AA or LA. It is concluded that extensive oxygenase activation can result in cell death from intermediates produced proximal to the stable monohydroxylated derivatives.

Topics: Animals; Arachidonic Acid; Cell Survival; Enzyme Activation; Hydroxyeicosatetraenoic Acids; Leukemia, Basophilic, Acute; Linoleic Acid; Linoleic Acids; Lipoxygenase; Masoprocol; Rats; Tumor Cells, Cultured; Vitamin E

The principal monohydroxyeicosatetraenoic acids (HETEs), 5-, 12-, and 15-HETE, which can be produced by rat basophilic leukemia (RBL-1) cells, are also esterified by these cells. Exogenously added 5-, 12-, and 15-HETE were rapidly incorporated as esters in RBL cells, reaching plateau levels within 25 min. In incubations in culture medium with protein added, all three HETEs were essentially completely metabolized within 24 h. 5-HETE was esterified more rapidly and to a greater extent than 12-HETE or 15-HETE when these were incubated together with RBL cells, indicating some degree of selectivity in the esterification pathways. When arachidonic acid (AA) was incubated in increasing concentrations with constant concentrations of 15-HETE and RBL cells, the free 15-HETE concentration increased and esterified 15-HETE concentration decreased markedly at AA: 15-HETE molar ratios above 9. 15-HETE esterification in RBL cells was also markedly inhibited by the polyunsaturated fatty acids, eicosatetraynoic and eicosapentanoic acids, but not by oleic or linoleic acids. In separate experiments with unlabeled and radiolabeled substrates, the extent of incorporation of esterified HETE in RBL cells decreased at higher concentrations of 15-HETE and AA, which showed that the pathway was saturable. The shapes of the curves for these fatty acid inhibitors suggest a concentration-dependent two-compartment pathway of esterification. These data indicate that the HETEs and other 20 carbon fatty acid substrates probably compete for activity of a specific arachidonyl-CoA synthetase, which is the first and rate-limiting step for esterification of arachidonic acid by many human cells. Esterified 15-HETE was found to be predominantly in the phosphatidylethanolamine fraction of RBL cell lipids.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Coenzyme A Ligases; Esterification; Fatty Acids; Hydroxyeicosatetraenoic Acids; Leukemia, Basophilic, Acute; Phospholipids; Rats; Tumor Cells, Cultured

The arachidonate lipoxygenase from rat basophilic leukemia cells (RBL-1) is widely utilized as a model to dissect the primary enzymatic reactions leading to leukotriene formation. The purpose of the present study was to optimize the specific activity of 5-lipoxygenase prepared from a high speed supernatant of RBL-1 cell homogenates. Activation of 5-lipoxygenase was observed in the presence of micromolar levels of calcium. A synergistic enhancement of 5-lipoxygenase was observed upon addition of equally low levels of ATP; maximal activation was induced by 5 microM CaCl2 plus 5 microM ATP. Addition of a microsomal-membrane preparation and NADPH further augmented 5-HETE biosynthesis. High concentrations (330 microM) of NADPH reversed the microsomal-induced stimulation of RBL-1 5-lipoxygenase, resulting in enzyme inhibition.

Topics: Adenosine Triphosphate; Animals; Arachidonate 5-Lipoxygenase; Arachidonate Lipoxygenases; Calcium Chloride; Cell Line; Homeostasis; Hydroxyeicosatetraenoic Acids; Kinetics; Leukemia, Basophilic, Acute; NADP; Rats

Topics: Animals; Arachidonate 5-Lipoxygenase; Butyrates; Butyric Acid; Hydroxyeicosatetraenoic Acids; Leukemia, Basophilic, Acute; Prostaglandin D2; Prostaglandin-Endoperoxide Synthases; Rats; Tumor Cells, Cultured