5-L-hydroxy-6,8,11,14-eicosatetraenoate potentiates the human neutrophil degranulating action of platelet-activating factor (original) (raw)
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International journal of immunopharmacology, 2000
AS-35, (9-[4-acetyl-3-hydroxy-2-n-propylphenoxy) methyl]-3-(1H-tetrazol-5-yl)-4H-pyrido[1, 2-a] pyrimidin-4-one), was developed as a leukotriene (LT) receptor antagonist, which also inhibited IgE-mediated release of leukotrienes (LTs). We have investigated the action of AS-35 on the enzyme activities which are involved in the synthesis of LTC(4) and LTB(4) (LT-synthesizing enzymes); cytosolic phospholipase A(2) (cPLA(2)), 5-lipoxygenase (5-LO), leukotriene (LT)C(4) synthase and LTA(4) hydrolase. AS-35 dose-dependently inhibited IgE- and A23187-stimulated production of LTC(4) by up to 71.5-84.8% and that of LTB(4) by 48.3-49.2% at 2. 5x10(-5) M. The assays for cPLA(2)(-), 5-LO-, LTC(4) synthase- and LTA(4) hydrolase-activities revealed that the inhibition is attributable to suppression of cPLA(2), 5-LO and LTC(4) synthase but not LTA(4) hydrolase. We have also studied the action of AS-35 on the release of beta-hexosaminidase (beta-HEX) as a marker of preformed mediators. AS-35 had on...
Advances in prostaglandin, thromboxane, and leukotriene research, 1987
Leukotriene B4 (5S,12R-dihydroxy-6,14-cis, 8,10-trans-eicosatetraenoic acid, LTB4) is released from neutrophils exposed to calcium ionophores. To determine whether LTB4 might be produced by ligand-receptor interactions at the plasmalemma, we treated human neutrophils with serum-treated zymosan (STZ), heat-aggregated IgG and fMet-Leu-Phe (fMLP), agonists at the C3b, Fc and fMLP receptors respectively. STZ (10 mg/ml) provoked the formation of barely detectable amounts of LTB4 (0.74 ng/107 cells); no w-oxidized metabolites of LTB4 were found. Adding 10,M-arachidonate did not significantly increase production of LTB4 or its metabolites. Addition of 50 /LM-arachidonate (an amount which activates protein kinase C) before STZ caused a 40-fold increase in the quantity of LTB4 and its w-oxidation products. Neither phorbol myristate acetate (PMA, 200 ng/ml) nor linoleic acid (50 /SM), also activators of protein kinase C, augmented generation of LTB4 by cells stimulated with STZ. Neither fMLP (10-6 M) nor aggregated IgG (0.3 mg/ml) induced LTB4 formation (<0.01 ng/107 cells). Moreover, cells exposed to STZ, fMLP, or IgG did not form all-trans-LTB4 or 5-hydroxyeicosatetraenoic acid; their failure to make LTB4 was therefore due to inactivity ofneutrophil 5-lipoxygenase. However, adding 50 /sM-arachidonate to neutrophil suspensions before fMLP or IgG triggered LTB4 production, the majority of which was metabolized to its w-oxidized products (fMLP, 20.2 ng/ 107 cells; IgG, 17.1 ng/107 cells). The data show that neutrophils exposed to agonists at defined cell-surface receptors produce significant quantities of LTB4 only when treated with non-physiological concentrations of arachidonate.
Molecular Pharmacology, 2002
5-Lipoxygenase (5-LO) catalyzes the transformation of arachidonic acid to leukotrienes (LT). In stimulated human PMN, activation of 5-LO involves calcium, p38 MAP kinase (p38) phosphorylation, and translocation of 5-LO from the cytosol to nuclear membranes containing the 5-LO activating protein (FLAP). In this study, cAMP-elevating agents such as isoproterenol, prostaglandin E 2 , CGS-21680 (an adenosine A 2a receptor agonist), the type IV phosphodiesterase inhibitor RO 20-1724, the adenylate cyclase activator forskolin, and the Gsprotein activator cholera toxin all inhibited LT biosynthesis and 5-LO translocation to the nucleus in cytokine-primed human PMN stimulated with platelet-activating factor and in human PMN stimulated with the endomembrane Ca 2ϩ-ATPase blocker thapsigargin. Furthermore, monophosphorothioate analogs of cAMP, which activate protein kinase A (PKA), also inhibited LT biosynthesis and 5-LO translocation in stimulated cells. Treatment of PMN with CGS-21680 also prevented the phosphorylation of p38 by thapsigargin. Treatment of PMN with the PKA inhibitors H-89 and KT-5720 prevented the inhibitory effect of cAMP-elevating agents on LT biosynthesis, 5-LO translocation, and p38 phosphorylation, whereas the p38 inhibitor SB 203,580 dose-dependently inhibited arachidonic acidinduced LT biosynthesis. The 5-LO translocation was also inhibitable by the FLAP antagonist MK-0591 and correlated with LT biosynthesis in all experimental conditions tested. These results indicate that cAMP-mediated PKA activation in PMN results in the concomitant inhibition of 5-LO translocation and LT biosynthesis and support a role of p38 in the signaling pathway involved. This represents the first physiological downregulation mechanism of 5-LO translocation in human PMN. Leukotrienes (LT) are lipid mediators of inflammation that have been implicated in a number of pathological conditions including allergy, asthma, and other inflammatory diseases. The biosynthesis of LT involves the sequential release of arachidonic acid (AA) from cellular glycerolipids and its initial transformation by 5-lipoxygenase (5-LO), which catalyzes both the hydroperoxydation of AA at carbon 5 and a dehydrase reaction resulting in the formation of LTA 4. In human PMN, LTA 4 is further metabolized to the potent PMN activator and chemoattractant LTB 4 by the LTA 4 hydrolase. Pharmacological agents such as Ca 2ϩ ionophores or the Ca 2ϩ-ATPase blocker thapsigargin are routinely used as tools to study the regulation of AA metabolism in human PMN because they are potent inducers of LT biosynthesis. Physiological ligands such as platelet-activating factor (PAF) or N-formyl-methionyl-leucyl-phenylalanine (fMLP) also stimulate LT biosynthesis in human PMN, and this biosynthesis is strongly potentiated when cells are pre-exposed to priming agents such as TNF-␣, GM-CSF, or lipopolysaccharides (Roubin et al., 1987; DiPersio et al., 1988; McColl et al., These studies were supported by grants of the Canadian Institutes of Health Research (CIHR) and the Arthritis Society of Canada. N.F. is the recipient of a doctoral award from the CIHR. N.F. and M.E.S. contributed equally to this work.
Arch Dermatol Res, 1992
The purpose of the present study was to determine the effect of the n-6 fatty acid, dihomogammalinolenic acid (DGLA, 20 : 3, n-6) on arachidonic acid (AA) (C20:4) metabolism by human peripheral mononuclear leukocytes (HPML). After incubation of HPML with A23187 (5 ~tM) and DGLA, the cyclooxygenase (CO) and lipoxygenase (LO) products were separated and quantified by reversed-phase high-performance liquid chromatography (RP-HPLC) combined with radioimmunoassay. DGLA led to no change in PGE 2 formation, but at similar concentrations there was a dose-dependent decrease in LTB 4 formation (ICso = 45.0 ltM). The inhibition of LTB 4 formation by DGLA was associated with a dosedependent increase in its 15-LO metabolite 15-hydroxyeicosatrienoic acid (15-HETrE) and its CO metabolite prostaglandin E 1 (PGE1). Incubation of HPLM with 15-HETrE (0-1.5 pM) alone did not result in a change in PGE 2 formation, whereas 15-HETrE was a much more potent inhibitor of LTB 4 formation (ICso = 0.5 ltM) than DGLA. These results show that the addition of DGLA to HPML results in a selective inhibition of LTB 4 formation, presumably via its metabolite (15-HETrE).
American Journal of Respiratory and Critical Care Medicine, 1999
Persistent neutrophilia is a feature of chronic obstructive pulmonary disease (COPD). Leukotriene synthesis inhibitors and cysteinyl leukotriene receptor antagonists have shown efficacy in the treatment of asthma. Antagonism of leukotriene (LT)B 4 receptors is being considered as a mode of treating COPD. We examined the capacity for inhibition of leukotriene synthesis and LTB 4 receptor antagonism to reduce survival of neutrophils from patients with COPD and those from normal subjects. The basal apoptosis level of these cells was 55.4 Ϯ 2.4% (mean Ϯ SEM) of total cells. Separate exposure to lipopolysaccharide (LPS), granulocyte-macrophage colony-stimulating factor (GM-CSF), dexamethasone (DEX), and LTB 4 increased neutrophil survival (p Ͻ 0.001). The LTB 4 receptor antagonist SB201146 abolished LPS-induced survival in a concentration-dependent manner (10 pmol to 0.1 M), with an IC 50 of 1.9 nM. Combined exposure to SB201146 and to the cysteinyl leukotriene antagonist SKF104353 did not have a greater effect on survival than did exposure to SB201146 alone. Inhibition of 5-lipoxygenase (5-LO) with BWA4C and of 5-LO-activating protein (FLAP) with MK886 abolished GM-CSF-and DEX-induced neutrophil survival. BWA4C and MK886 abolished GM-CSFinduced neotrophil survival in a concentration-dependent manner (1 nM to 10 M), with IC 50 values of 182.0 nM and 63.1 nM, respectively. These findings demonstrate reversal of LPS-, GM-CSF-, and DEX-induced neutrophil survival by LTB 4 receptor antagonism and inhibitors of 5-LO and FLAP. They also suggest a potential additional antiinflammatory mode of action of these compounds through reduction of cell survival. Lee E,
Archives For Dermatological Research, 1992
The purpose of the present study was to determine the effect of the n-6 fatty acid, dihomogammalinolenic acid (DGLA, 20 : 3, n-6) on arachidonic acid (AA) (C20:4) metabolism by human peripheral mononuclear leukocytes (HPML). After incubation of HPML with A23187 (5 ~tM) and DGLA, the cyclooxygenase (CO) and lipoxygenase (LO) products were separated and quantified by reversed-phase high-performance liquid chromatography (RP-HPLC) combined with radioimmunoassay. DGLA led to no change in PGE 2 formation, but at similar concentrations there was a dose-dependent decrease in LTB 4 formation (ICso = 45.0 ltM). The inhibition of LTB 4 formation by DGLA was associated with a dosedependent increase in its 15-LO metabolite 15-hydroxyeicosatrienoic acid (15-HETrE) and its CO metabolite prostaglandin E 1 (PGE1). Incubation of HPLM with 15-HETrE (0-1.5 pM) alone did not result in a change in PGE 2 formation, whereas 15-HETrE was a much more potent inhibitor of LTB 4 formation (ICso = 0.5 ltM) than DGLA. These results show that the addition of DGLA to HPML results in a selective inhibition of LTB 4 formation, presumably via its metabolite (15-HETrE).
Prostaglandins, Leukotrienes and Medicine, 1985
Human platelets metabolize arachidonic acid via cyclooxygenase (E.C. 1.14.99.1) to thromboxane A and the 12-lipoxygenase to 12-hydroxyeicosatetraenoic a&d (12-HETE). Aspirin inhibits cyclooxygenase while the neutrophil product 15-Hydroxyeicosatetraenoic acid (15-HETE) is a selective inhibitor of platelet 12-lipoxygenase. The unexpected observation was made that the platelet 12-lipoxygenase of individuals who had ingested aspirin showed up to a twenty-fold increase in sensitivity to inhibition by 15-HETE. This observation was confirmed in platelets treated with aspirin js vitrp. Aspirin pretreatment consistently resulted in a decrease in the I for 15-HETE from an average of 21.5 ti! 5.2 + 1.5 uM, ind%ating a probable interaction between the to only cyclooxygenase and lipoxygenase pathways. 3NTRODUCTION Mammalian platelets, upon activation of platelet phospholipases, release arachidonic acid (AA) from membrane phospholipids. The released AA is the substrate for the enzymes Fatty Acid Cyclooxygenase and 12-Lipoxygenase yielding a number of products including thromboxane A (TXA2), a potent vasoconstrictor and proaggregatory agent (IS, and 12-HETE, a powerful chemotactic agent for vascular smooth muscle cells (2).