calcimycin and arachidonic-acid-5-hydroperoxide

Propofol is a clinically important intravenous anesthetic. We previously reported that it directly inhibited 5-lipoxygenase (5-LOX), a key enzyme for leukotriene biosynthesis. Because the hydroxyl group in propofol (propofol 1-hydroxyl) is critical for its anesthetic effect, we examined if its presence would be inevitable for 5-lipoxygenase recognition. Fropofol is developed by substituting the hydroxy group in propofol with fluorine. We found that propofol 1-hydroxyl was important for 5-lipoxygenase recognition, but it was not absolutely necessary. Azi-fropofol bound to 5-LOX at one of the two propofol binding sites of 5-LOX (pocket around Phe-187), suggesting that propofol 1-hydroxyl is important for 5-LOX inhibition at the other propofol binding site (pocket around Val-431). Interestingly, 5-hydroperoxyeicosatetraenoic acid (5-HpETE) production was significantly increased by stimulation with calcium ionophore A23187 in HEK293 cells expressing 5-LOX, suggesting that the fropofol binding site is important for the conversion from 5-HpETE to leukotriene A

Topics: Anesthetics, Intravenous; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Binding Sites; Calcimycin; Calcium Ionophores; HEK293 Cells; Humans; Leukotriene B4; Leukotrienes; Lipoxygenase Inhibitors; Molecular Docking Simulation; Mutagenesis; Propofol; Protein Conformation; Structure-Activity Relationship

A simple and sensitive method, applicable to quantification of 5-hydroperoxyeicosatetraenoic acid (5-HPETE) produced in cells has been developed using high-performance liquid chromatography on a silica gel column with chemiluminescence detection. 5-HPETE was clearly separated from other positional isomers of HPETEs and hydroxyeicosatetraenoic acids with hexane-isopropanol-acetic acid (97:3:0.01, v/v) as the mobile phase. The lower limit of detection was about 100 pg. 5-HPETE produced in 10(7) cells of RBL-2H3 cells stimulated with A23187 was determined as 480+/-30 pg. In the present study, 5-HPETE, which occurs naturally, was detected and quantitated for the first time in intact cells.

Topics: Animals; Calcimycin; Calibration; Chromatography, High Pressure Liquid; Leukemia, Basophilic, Acute; Leukotriene B4; Leukotrienes; Luminescent Measurements; Malates; Rats; 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

Leukotriene biosynthesis inhibitors have potential as new therapies for asthma and inflammatory diseases. The recently disclosed thiopyrano[2,3,4-cd]indole class of 5-lipoxygenase (5-LO) inhibitors has been investigated with particular emphasis on the side chain bearing the acidic functionality. The SAR studies have shown that the inclusion of a heteroatom (O or S) in conjunction with an alpha-ethyl substituted acid leads to inhibitors of improved potency. The most potent inhibitor prepared contains a 2-ethoxybutanoic acid side chain. This compound, 14d (2-[2-[1-(4-chlorobenzyl)-4-methyl-6-[(5-phenylpyridin-2-yl)methox y]- 4,5-dihydro-1H-thiopyrano[2,3,4-cd]indol-2-yl]ethoxy]-butanoic acid, L-699,333), inhibits 5-HPETE production by human 5-LO and LTB4 biosynthesis by human PMN leukocytes and human whole blood (IC50s of 22 nM, 7 nM and 3.8 microM, respectively). The racemic acid 14d has been shown to be functionally active in a rat pleurisy model (inhibition of LTB4, ED50 = 0.65 mg/kg, 6 h pretreatment) and in the hyperreactive rat model of antigen-induced dyspnea (50% inhibition at 2 and 4 h pretreatment; 0.5 mg/kg po). In addition, 14d shows excellent functional activity against antigen-induced bronchoconstriction in the conscious squirrel monkey [89% inhibition of the increase in RL and 68% inhibition in the decrease in Cdyn (0.1 mg/kg, n = 3)] and in the conscious sheep models of asthma (iv infusion at 2.5 micrograms/kg/min). Acid 14d is highly selective as an inhibitor of 5-LO activity when compared to the inhibition of human 15-LO, porcine 12-LO and ram seminal vesicle cyclooxygenase (IC50 > 5 microM) or competition in a FLAP binding assay (IC50 > 10 microM). Resolution of 14d affords 14g, the most potent diastereomer, which inhibits the 5-HPETE production of human 5-LO and LTB4 biosynthesis of human PMN leukocytes and human whole blood with IC50s of 8 nM, 4 nM, and 1 microM respectively. The in vitro and in vivo profile of 14d is comparable to that of MK-0591, which has showed biochemical efficacy in inhibiting ex vivo LTB4 biosynthesis and urinary LTE4 excretion in clinical trials.

Topics: Animals; Bronchoconstriction; Calcimycin; Chromatography, High Pressure Liquid; Humans; Indoles; Leukotriene B4; Leukotrienes; Lipoxygenase Inhibitors; Male; Molecular Structure; Pyridines; Rats; Rats, Sprague-Dawley; Saimiri; Sheep; Stereoisomerism; Structure-Activity Relationship; Swine

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

Prostaglandin E2 (PGE2), a potent mediator of inflammation released in large amounts by endotoxin-stimulated alveolar macrophages (AM), has been shown to inhibit leukotriene B4 (LTB4) release by activated neutrophils. We investigated the hypothesis that LTB4 synthesis by AM can be regulated by PGE2 and performed experiments to determine the biochemical site of regulation. AM obtained from Sprague-Dawley rats were preincubated with PGE2 before stimulation with the calcium ionophore A23187. LTB4, platelet-activating factor (PAF), lysoPAF, (AA), and 5-hydroxyeicosatetraenoic acid were isolated from AM cells and supernatants, then quantified by gas chromatography/electron capture negative ion mass spectrometry. Stimulated AM released 30.50 +/- 4.52 ng LTB4/10(6) cells and were inhibited by PGE2 in a dose dependent manner. PGE2 (1 microM) inhibited LTB4 synthesis by 37% and decreased release of both 5-hydroxyeicosatetraenoic acid and arachidonic acid by stimulated AM, but did not alter synthesis of PAF or lysoPAF. These mass measurements suggest that PGE2 does not affect the activity of phospholipase A2, PAF acetyltransferase, leukotriene A4 hydrolase. We conclude that PGE2 attenuates LTB4 production in alveolar macrophages by altering the activity of lipases other than phospholipase A2. PGE2-mediated inhibition of LTB4 synthesis by AM may regulate the initiation of lung inflammation.

Topics: Animals; Arachidonic Acid; Calcimycin; Dinoprostone; Leukotriene B4; Leukotrienes; Macrophages, Alveolar; Phospholipases A; Phospholipases A2; Platelet Activating Factor; Rats

Ionophore activation of the human polymorphonuclear neutrophil results in eicosanoid synthesis and the accumulation of inactive 5-lipoxygenase in a membrane compartment. We report here that inhibition of self-inactivation of 5-lipoxygenase in ionophore-treated neutrophils with the reversible inhibitor zileuton, results in the accumulation of active 5-lipoxygenase in the membrane fraction. In zileuton plus ionophore-treated cells, 77% of the specific activity of the cytosolic enzyme from resting cells was diverted to the membrane fraction compared to 22% of the activity translocated when ionophore alone was used to activate the neutrophils. Accumulation of active membrane-associated 5-lipoxygenase was inhibited and reversed by the 5-lipoxygenase translocation inhibitor MK-886. The membrane-associated 5-lipoxygenase was two times more efficient in the production of leukotriene A4 from arachidonate-derived 5-hydroperoxyeicosatetraenoic acid than the cytosolic enzyme. Unlike the cytosolic enzyme, membrane-associated 5-lipoxygenase could metabolize 12(S)- and 15(S)-hydroxyeicosatetraenoic acid to 5(S),12(S)- and 5(S),15(S)-dihydroxyeicosatetraenoic acid, respectively. The ability to metabolize hydroxy fatty acids was dependent upon 5-lipoxygenase-activating protein association, but was lost if 5-lipoxygenase was eluted from the membrane by MK-886. These studies reveal for the first time that significant quantities of active 5-lipoxygenase can be detected in the membrane fraction of activated neutrophils and show that membrane association can alter the substrate specificity of 5-lipoxygenase which is further evidence for the role of the membrane-associated enzyme in the synthesis of 5-lipoxygenase metabolites.

Topics: Arachidonate 5-Lipoxygenase; Arachidonic Acid; Calcimycin; Cell Compartmentation; Cell Membrane; Cytosol; Humans; Hydroxyeicosatetraenoic Acids; Hydroxyurea; In Vitro Techniques; Indoles; Leukotrienes; Neutrophils; Substrate Specificity

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

Human polymorphonuclear leukocytes (PMN) stimulated with the Ca-ionophore A23187 or opsonized zymosan not only release marker enzymes of specific granules but secrete 5-lipoxygenase activity as well. In the presence of BSA cells incubated with [14C]AA were able to synthetize 5-HPETE but failed to produce 5-HETE, LTB4, and its omega-oxidation metabolites. Subcellular fractionation studies by differential and isopycnic equilibrium density centrifugation demonstrated main lipoxygenase activity in particulate fractions consisting of specific granules, but not in cytosolic fractions. These results suggest the association of 5-lipoxygenase with specific granules. 5-lipoxygenase released from the cells upon appropriate stimulation reached its peak activity after 10 min and was then rapidly inactivated. It appears that the intermediate 5-HPETE may be generated extracellularly but has to re-enter the intracellular space for further metabolization.

Topics: Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids; Calcimycin; Carbon Radioisotopes; Cytoplasmic Granules; Humans; Leukotrienes; Lipoxygenase; Neutrophils; Serum Albumin, Bovine

Prostaglandin (PG) D2 is the major cyclooxygenase metabolite of arachidonic acid released after immunologic stimulation of mast cells. In this report, we demonstrate that this PG is unlike other PGs previously investigated in that it enhances human basophil histamine release at concentrations of 1 to 100 nM. Enhancement is seen when antigen, the phorbol diester, 12-O-tetradecanoylphorbol-13-acetate and ionophore A23187 are used to initiate release and, in preparations of basophils, purified to 51 to 66% of total cell number as well as in preparations of mixed leukocytes. This enhancement is a late or "second stage" phenomenon as defined by the two stages of histamine release and is additive with the enhancement produced by D2O, but not with the enhancement produced by indomethacin or 5-hydroperoxyeicosatetraenoic acid. PGD2 also reverses the inhibition of release produced by drugs and hormones such as dimaprit and PGE2 that activate adenylate cyclase to increase cellular cyclic AMP levels. These data suggest that PGD2 may play an important role in allergic and immunologic reactions and suggest a mechanism by which mast cells and basophils can interact during these reactions.

Topics: Arachidonic Acids; Basophils; Calcimycin; Dimaprit; Dinoprostone; Histamine Release; Humans; Indomethacin; Leukotrienes; Prostaglandin D2; Prostaglandins D; Prostaglandins E; Receptors, Histamine H2; Stimulation, Chemical; Tetradecanoylphorbol Acetate; Thiourea

Topics: Allergens; Antibodies, Anti-Idiotypic; Antigens, Plant; Arachidonic Acids; Basophils; Calcimycin; Dimaprit; Dinoprostone; Histamine Release; Humans; Immunoglobulin E; Leukotrienes; Lipoxygenase; Plant Proteins; Pollen; Prostaglandins E; Tetradecanoylphorbol Acetate; Thiourea

Topics: Arachidonic Acid; Arachidonic Acids; Calcimycin; Cell Line; Chemotaxis, Leukocyte; Granulocytes; Humans; Leukemia, Myeloid; Leukotrienes; Oligopeptides