Separation of lipoxins and leukotrienes from human granulocytes by high-performance liquid chromatography with a Radial-Pak cartridge after extraction with an octadecyl reversed-phase column

Steinhilber, Dieter; Herrmann, Thomas; Roth, H. J.

doi:10.1016/s0378-4347(00)82742-5

Cited by 63 publications

(41 citation statements)

References 18 publications

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“…After 10 min at 37°C, the reaction was stopped with 1 ml of methanol and 30 l of 1 N HCl, and 200 ng of prostaglandin B 1 and 500 l of PBS were added. Formed 5-LO metabolites were extracted and analyzed by HPLC as described (38). 5-LO product formation is expressed as nanograms of 5-LO products per 10 6 cells, which includes LTB 4 and its all-trans isomers, 5(S),12(S)-dihydroxy-6,10-trans-8,14-cis-eicosatetraenoic acid (5(S),12(S)-DiHETE), 5(S)-hydroxy-6-trans-8,11,14-cis-eicosatetraenoic acid (5-HETE), and 5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid (5-HPETE).…”

Section: Methodsmentioning

confidence: 99%

The C2-like β-Barrel Domain Mediates the Ca2+-dependent Resistance of 5-Lipoxygenase Activity Against Inhibition by Glutathione Peroxidase-1

Bürkert

Arnold

Hammarberg

et al. 2003

Journal of Biological Chemistry

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Recently, we reported that in crude enzyme preparations, a monocyte-derived soluble protein (M-DSP) renders 5-lipoxygenase (5-LO) activity Ca 2؉ -dependent. Here we provide evidence that this M-DSP is glutathione peroxidase (GPx)-1. Thus, the inhibitory effect of the M-DSP on 5-LO could be overcome by the GPx-1 inhibitor mercaptosuccinate and by the broad spectrum GPx inhibitor iodoacetate, as well as by addition of 13(S)-hydroperoxy-9Z,11E-octadecadienoic acid (13(S)-HPODE). Also, the chromatographic characteristics and the estimated molecular mass (80 -100 kDa) of the M-DSP fit to GPx-1 (87 kDa), and GPx-1, isolated from bovine erythrocytes, mimicked the effects of the M-DSP. Intriguingly, only a trace amount of thiol (10 M GSH) was required for reduction of 5-LO activity by GPx-1 or the M-DSP. Moreover, the requirement of Ca 2؉ allowing 5-LO product synthesis in various leukocytes correlated with the respective GPx-1 activities. Mutation of the Ca 2؉ binding sites within the C2-like domain of 5-LO resulted in strong reduction of 5-LO activity by M-DSP and GPx-1, also in the presence of Ca 2؉ . In summary, our data suggest that interaction of Ca 2؉ at the C2-like domain of 5-LO protects the enzyme against the effect of GPx-1. Apparently, in the presence of Ca 2؉ , a low lipid hydroperoxide level is sufficient for 5-LO activation. 5-Lipoxygenase (5-LO)1 catalyzes the initial steps in the biosynthesis of leukotrienes (LTs) and 5(S)-hydro(pero)xyeicosatetraenoic acid (5(S)-H(P)ETE) from arachidonic acid (AA) (for review, see Ref. 1). Due to the pivotal biological functions of 5-LO metabolites (2, 3), the activity of 5-LO is tightly regulated. In intact cells, Ca 2ϩ and phosphorylation seem to be primary signals that activate 5-LO. Moreover, the membranebound 5-LO-activating protein (FLAP) (4) and the redox state (5, 6) have a strong impact on cellular 5-LO product formation.In cell-free systems Ca 2ϩ , ATP, phospholipids (membranes), lipid hydroperoxides (LOOH), and leukocyte-derived proteins have been shown to enhance 5-LO catalysis (reviewed in Ref. 1). However, the degree of stimulation by each of these components depends on the assay conditions, i.e. the source of 5-LO (isolated, in crude homogenates or cellular fractions), presence of other cofactors, the concentration of AA, etc. LOOH are of importance for the initial conversion of the active site iron from the ferrous (resting) to the ferric (active) state (7, 8). Accordingly, glutathione peroxidases (GPx) that reduce LOOH inhibit 5-LO product synthesis in vitro and in intact cells (5, 9 -15), and conditions that are associated with an increased peroxidetone promote 5-LO product formation (6,16,17). Two Ca 2ϩ ions bind to the N-terminal C2-like domain of 5-LO with a K d of 6 M (18, 19). Half-maximal activation of purified 5-LO was determined at 1-2 M Ca 2ϩ , whereas 4 -10 M Ca 2ϩ causes maximal activation of the enzyme (20,21). In intact cells lower concentrations of Ca 2ϩ (200 -300 nM) seem to be sufficient for 5-LO activation (22,23). It was shown that C...

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Section: Methodsmentioning

confidence: 99%

The C2-like β-Barrel Domain Mediates the Ca2+-dependent Resistance of 5-Lipoxygenase Activity Against Inhibition by Glutathione Peroxidase-1

Bürkert

Arnold

Hammarberg

et al. 2003

Journal of Biological Chemistry

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“…5-LO metabolites were extracted with 300 l of methanol. The extract was diluted with 120 l of water and 100 l of the diluted extract were analyzed by HPLC as described (Steinhilber et al, 1989).…”

Section: Methodsmentioning

confidence: 99%

Nonredox 5-Lipoxygenase Inhibitors Require Glutathione Peroxidase for Efficient Inhibition of 5-Lipoxygenase Activity

et al. 1998

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Nonredox type 5-lipoxygenase (5-LO) inhibitors, such as ZM 230487, its methyl analogue ZD 2138, or the Merck compound L-739,010, suppress cellular leukotriene synthesis of ionophore stimulated granulocytes with IC 50 values of about 50 nM. However, in cell homogenates or in preparations of purified enzyme, up to 150-fold higher concentrations are required for similar inhibition of 5-LO activity. This loss of 5-LO inhibition in cell homogenates was reversed by addition of glutathione or dithiothreitol, which increased the inhibitory potency of ZM 230487 or L-739,010 by about 100 to 150-fold so that 5-LO inhibition was comparable with that of intact cells. In the presence of thiols, addition of hydroperoxide [13(S)-HpODE], glutathioneperoxidase inhibition by iodacetate or selenium-deficiency lead to impaired 5-LO inhibition by ZM 230487 in cell homogenates. Moreover, addition of glutathione peroxidase was required for efficient inhibition of purified human 5-LO by ZM 230487. The data suggest that low hydroperoxide concentrations are important for efficient 5-LO inhibition by ZM 230487. The kinetic analysis revealed a noncompetitive inhibition of 5-LO by ZM 230487 at low hydroperoxide levels, whereas it acted as a competitive inhibitor with low affinity under nonreducing conditions in granulocyte homogenates. No such redox-dependent effects were observed with the 5-LO inhibitor BWA4C, the 5-LO activating protein-inhibitor MK-886 or the pentacyclic triterpene acetyl-11-keto-␤-boswellic acid. These data suggest that physiological conditions associated with oxidative stress and increased peroxide levels lead to impaired efficacy of nonredox type 5-LO inhibitors like ZM 230487 or L-739,010. This could explain the reported lack of activity of this class of 5-LO inhibitors in chronic inflammatory processes.

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“…methanol, 5-lipoxygenase metabolites were eluted with 300 pl methanol. The eluates were diluted with 120 p1 water, and 100 pl diluted extract was analyzed by HPLC as described [28].…”

Section: Methodsmentioning

confidence: 99%

Selenium‐Dependent Peroxidases Suppress 5‐Lipoxygenase Activity in B‐Lymphocytes and Immature Myeloid Cells

Werz

Steinhilber

1996

European Journal of Biochemistry

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Differentiation of HL-60 cells by dimethylsulfoxide induces 5-lipoxygenase protein expression, but only low cellular 5-lipoxygenase activity. Similarly, B-lymphocytes express 5-lipoxygenase protein and show activity in cell homogenates but not in intact cells. Here, we demonstrate that suppression of cellular 5-lipoxygenase activity in these cell lines is serum dependent and that the serum effect can be mimicked by selenium. Selenium-dependent inhibition of 5-lipoxygenase activity was also observed in the corresponding cell homogenates or 100 OOOXg supernatants when dithiothreitol or glutathione (GSH) was added. The properties of the endogenous selenium-dependent inhibitor, i.e. molecular mass, utilization of GSH and dithiothreitol as substrates, sensitivity to iodacetate, inhibition of 5-lipoxygenase activity in the presence of the GPx-1 inhibitor mercaptosuccinate, suggest that a selenoenzyme with properties of the phospholipid hydroperoxide glutathione peroxidase (GPx-4) is responsible for the 5-lipoxygenase inhibition in BL41-E95-A and immature HL-60 cells.Differentiation of HL-60 cells in the presence of 1,25-dihydroxyvitamin D, and transforming growth factor# (TGFP) upregulated cellular 5-lipox ygenase activity regardless of whether the cells were grown with or without serum or selenium. Also, 5-lipoxygenase activity in homogenates or lOOOOOXg supernatants of l ,25-dihydroxyvitamin DJTGFP differentiated HL-60 cells and of human granulocytes was not inhibited by dithiothreitol or GSH. Thus, after 1,25-dihydroxyvitamin DJTGFP differentiation, HL-60 cells resemble normal granulocytes with respect to the high 5-lipoxygenase activity in intact cells and to the dithiothreitol effects in broken cell preparations. Combination experiments with 100 OOOXg supernatants of BL41-E95-A cells and neutrophils revealed that the high 5-lipoxygenase activity of granulocytes is due to stability of the 5-lipoxygenase catalytic activity against selenium-dependent peroxidases, but not to low peroxidase activity. Our data suggest that the capability of mature myeloid cells to release large amounts of leukotrienes after stimulation is due to a peroxidase-insensitive 5-lipoxygenase catalytic activity.Keywords: 5-lipoxygenase; glutathione peroxidase; HL-60; cell differentiation; lymphocyte.Leukotrienes are mediators of inflammatory responses that can be formed in granulocytes, monocyteslmacrophages and mast cells after stimulation. 5-lipoxygenase catalyzes the first two steps of leukotriene biosynthesis from arachidonic acid [ l , 21. In neutrophils and HL-60 cells, 5-lipoxygenase is localized in the cytosol and translocates to the nuclear membrane after cell stimulation where it interacts with 5-lipoxygenase-activating protein [3-71. Recently, it has been shown that 5-lipoxygenase can associate with other proteins via interactions with SH3 domains and that tyrosine kinase activity has a prominent effect on cellular localization and activity of 5-lipoxygenase [8, 91. The capability of myeloid cells to release leukotrienes is upreg...

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Separation of lipoxins and leukotrienes from human granulocytes by high-performance liquid chromatography with a Radial-Pak cartridge after extraction with an octadecyl reversed-phase column

Cited by 63 publications

References 18 publications

The C2-like β-Barrel Domain Mediates the Ca2+-dependent Resistance of 5-Lipoxygenase Activity Against Inhibition by Glutathione Peroxidase-1

The C2-like β-Barrel Domain Mediates the Ca2+-dependent Resistance of 5-Lipoxygenase Activity Against Inhibition by Glutathione Peroxidase-1

Nonredox 5-Lipoxygenase Inhibitors Require Glutathione Peroxidase for Efficient Inhibition of 5-Lipoxygenase Activity

Selenium‐Dependent Peroxidases Suppress 5‐Lipoxygenase Activity in B‐Lymphocytes and Immature Myeloid Cells

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