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

1. In the present study, we demonstrate that leukotriene B4 (LTB4) has the ability to activate the human neutrophil 5-lipoxygenase (5-LO). 2. Stimulation of neutrophils with 30 nM 14,15-dideuterio-LTB4 (D2-LTB4) failed to induce the synthesis of LTB4 from endogenous arachidonic acid (AA), but stimulated the formation of LTB4 from 3.3 microM exogenous AA, as determined by GC-MS analysis. 3. The stimulatory effect of LTB4 on 5-LO activity was further examined with an alternative substrate; LTB4 time- and dose-dependently stimulated the 5-LO-mediated conversion of exogenous 15(S)-hydroperoxy-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoate (15-HpETE) into 5(S),15(S)-dihydroxy-6,8,11,13,-(E,Z,Z,E)-eicosatetraenoate (5,15-DiHETE), with a threshold effect at 300 pM. 4. The ability of LTB4 to activate the 5-LO showed structural specificity, since LTB4 was found to be 100 times more potent than omega-hydroxy-LTB4, and 300 times more potent than its delta 6-trans-12-epi-isomer. 5. The LTB4-induced 5-LO activation was effectively inhibited by MK-886 (an inhibitor of 5-LO translocation), by pertussis toxin, and by the LTB4 receptor antagonist, LY-223982. 6. These results demonstrate that the binding of LTB4 to its cell-surface receptor results in 5-LO activation in a process mediated by pertussis toxin-sensitive guanine nucleotide-binding proteins. Our data also suggest that the underlying mechanism involves a translocation of the 5-LO to the membrane. These findings raise the possibility that LTB4 produced by phagocytes may positively feedback on its own synthesis.

Topics: Arachidonate 5-Lipoxygenase; Arachidonic Acid; Benzophenones; Enzyme Activation; Humans; Hydroxyeicosatetraenoic Acids; Indoles; Leukotriene B4; Leukotrienes; Lipid Peroxides; Neutrophils; Pertussis Toxin; Virulence Factors, Bordetella

Preincubation with granulocyte-macrophage colony-stimulating factor (GM-CSF) increased the synthesis of leukotriene B4 and its omega-oxidation products by neutrophils in response to 10(-7) M platelet-activating factor (PAF) by more than 10-fold compared to untreated cells. Pretreatment with GM-CSF also enabled the detection of these products in response to lower concentrations of PAF (greater than or equal to 10(-9) M), under which conditions synthesis of 5-lipoxygenase products was not observed in non-GM-CSF-treated cells. While PAF induced the release of arachidonic acid from neutrophils, GM-CSF alone did not stimulate the release of detectable amounts of the fatty acid. However, the levels of free arachidonic acid in response to PAF were enhanced in neutrophils pretreated with GM-CSF. Similarly, GM-CSF alone did not directly activate the 5-lipoxygenase as determined by the 5-lipoxygenase-mediated transformation of exogenous 15-hydroperoxyeicosatetraenoic acid into 5,15-dihydroxyeicosatetraenoic acid (5,15-diHETE). However, stimulation of neutrophils with PAF resulted in the transformation of the 15-hydroperoxy acid into the 5,15-diHETE, and furthermore, preincubation of neutrophils with GM-CSF resulted in enhanced formation of 5,15-diHETE in response to PAF. Taken together, these data indicate that GM-CSF does not enhance leukotriene synthesis in neutrophils in response to PAF by either directly activating the 5-lipoxygenase or stimulating the liberation of endogenous arachidonic acid, but rather by augmenting the effect of PAF on these two responses.

Topics: Arachidonate 5-Lipoxygenase; Arachidonic Acid; Drug Synergism; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Leukotriene B4; Leukotrienes; Lipid Peroxides; Neutrophils; Platelet Activating Factor

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