calcimycin and 5-15-dihydroxy-6-8-11-13-eicosatetraenoic-acid

(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

5-Lipoxygenase activation of human blood polymorphonuclear cells (PMN) from asthmatic patients (asthmatics) was studied to investigate whether differences may exist with healthy subjects (controls). The respective cell capacities to produce lipoxins (LXs), leukotrienes, and 5(S), 15(S)-dihydroxyeicosatetraenoic acid [5(S),15(S)-diHETE] were compared under in vitro stimulation by ionophore A23187, with or without exogenous 15(S)-hydroxyeicosatetraenoic acid [15(S)-diHETE]. Eicosanoids were analyzed by elution with an isocratic reverse-phase high performance liquid chromatography system, and their profiles, detected by simultaneous monitoring at 302, 280, and 246 nm, were evaluated on the basis of chromatographic behavior: UV spectral characteristics and coelution with synthetic standards. In the presence of exogenous 15(S)-HETE, human PMN were able to produce LXs and 5(S),15(S)-diHETE, PMN from asthmatics were able to produce 5(S), 5(S),15(S)-diHETE, and LXs from endogenous sources, whereas in the same experimental conditions, no detectable amounts of these compounds were released by PMN from controls. The levels of 5(S),15(S)-diHETE, and LXs biosynthesized from endogenous arachidonic acid were highly correlated. Two different LX patterns were observed involving two possible metabolic pathways: (a) via the intermediate 5,6-epoxytetraene alone for LXs generation from exogenous 15(S)-HETE; and (b) via 5,6- and/or 14,15-epoxytetraenes leading to the formation of an enzyme-bound delocalized carbocation for LXs generation from endogenous arachidonate, respectively. The enhanced 5-lipoxygenase activation of blood PMN from asthmatics and the metabolism of exogenous 15(S)-HETE may reflect a priming induced by various mediators released from environmental cells, and could be considered as a model of transcellular signalization between PMN and endothelial cells.

Topics: Arachidonate 5-Lipoxygenase; Arachidonic Acids; Asthma; Calcimycin; Chromatography, High Pressure Liquid; Female; Humans; Hydroxyeicosatetraenoic Acids; Male; Models, Biological; Neutrophil Activation; Substrate Specificity

The conversion of endogenous arachidonic acid (AA) by polymorphonuclear cells (PMN) from patients with rheumatoid arthritis (RA) was studied before (D0) and one day (D1) after antiinflammatory drug therapy. The biosynthesis of 5,15-diHETE and lipoxins (LXS), were investigated ex vivo, after PMN stimulation by ionophore A23187 without exogenous addition of 15-HETE. The eicosanoids were resolved by RP-HPLC and simultaneously detected at 246 and 302 nm respectively. Large amounts of 5,15-diHETE (50 to 400 ng/10(7) PMN) and significant levels of LXS (from 2 to 20 ng/10(7) PMN) were produced with individual differences between donors. Metabolite levels varied between patients but this work showed for the first time a linear relationship between the amounts of 5,15-diHETE and LXS. Moreover LXS production after treatment may be related to long-term clinical improvement of patients.

Topics: Adult; Anti-Inflammatory Agents; Arachidonic Acid; Arthritis, Rheumatoid; Calcimycin; Chromatography, High Pressure Liquid; Eicosapentaenoic Acid; Female; Humans; Hydroxyeicosatetraenoic Acids; Lipoxins; Male; Middle Aged; Neutrophils; Spectrophotometry, Ultraviolet

5-Lipoxygenase (5-LO) activation of human blood polymorphonuclear cells (PMN) from healthy subjects (HS) and from asthmatic patients (AP) was investigated comparing their respective capacities to produce lipoxins, 5,15-dihydroxyeicosatetraenoic acid (5,15-diHETE) and leukotrienes, under in vitro stimulation by ionophore A23187. PMN from AP were able to generate higher leukotriene levels from endogenous sources than PMN from HS. Moreover they produced 5,15-diHETE (from 50 to 280ng/10(7) cells) and lipoxins (from 1 to 30ng/10(7) cells), in a linear manner, whereas in the same experimental conditions no detectable amounts of these compounds appeared in PMN from HS. The enhanced 5-LO activation of blood PMN may reflect transcellular signalisation priming indicating that lipoxins and 5,15-diHETE could be much more specific inflammatory state biomarkers than leukotriene B4.

Topics: Arachidonic Acid; Asthma; Biomarkers; Calcimycin; Chromatography, High Pressure Liquid; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Neutrophils; Reference Values

1. The mechanism by which incubation of human peripheral blood neutrophils with exogenous arachidonic acid leads to 5-lipoxygenase product synthesis was investigated. 2. Incubation of neutrophils with arachidonic acid caused a concentration- and time-dependent synthesis of leukotriene B4, its omega-oxidation products, and 5-hydroxyeicosatetraenoic acid. 3. The threshold concentration of arachidonic acid required for this effect was equal to, or greater than 3.3 microM and the synthesis increased with up to 33 microM arachidonic acid, the highest concentration used. Synthesis induced by arachidonic acid increased with time for up to 15 min and the major products detected were the omega-oxidation products of leukotriene B4. 4. Pre-incubation of neutrophils with pertussis toxin inhibited the synthesis of 5-lipoxygenase products induced by arachidonic acid by 75% or more, but had no effect on either arachidonic acid-induced synthesis of the 15-lipoxygenase product, 15-hydroxyeicosatetraenoic acid, or activation of the 5-lipoxygenase induced by the calcium ionophore A23187. 5. Pre-incubation of neutrophils with granulocyte-macrophage colony-stimulating factor lead to enhanced leukotriene synthesis in response to arachidonic acid. 6. These results imply that exogenous arachidonic acid is not only used as a substrate, but also activates the 5-lipoxygenase. Possible mechanisms of action are discussed.

Topics: Arachidonate 5-Lipoxygenase; Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids; Blood Cells; Calcimycin; Chromatography, High Pressure Liquid; Colony-Stimulating Factors; Enzyme Activation; Granulocyte-Macrophage Colony-Stimulating Factor; Growth Substances; GTP-Binding Proteins; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Leukotrienes; Lipid Peroxides; Neutrophils; Pertussis Toxin; Virulence Factors, Bordetella