Studies on the activation of human neutrophil 5-lipoxygenase induced by natural agonists and Ca2+ ionophore A23187.
By using exogenous substrates, activation of human neutrophil 5-lipoxygenase can be investigated independently of the release of endogenous arachidonic acid. We have developed a sensitive assay to measure 5-LO activation which takes advantage of the 5-LO-mediated conversion of 15S-hydroperoxy-5,8,11,13(Z,Z,Z,E)-eicosatetraenoic acid (15-HpETE) into 5S,15S-dihydroxy-6,8,11,13(E,Z,Z,E)-eicosatetraenoic acid (5,15-DiHETE). When resting neutrophils were incubated with low micromolar concentrations of 15-HpETE, a minor dose- and time-dependent formation of 5,15-DiHETE was observed. In contrast, co-addition of 15-HpETE with Ca2+ ionophore A23187 or with the neutrophil agonists platelet-activating factor (PAF), fMetLeuPhe or complement component C5a resulted in a sizeable concentration-dependent synthesis of 5,15-DiHETE, while lyso-PAF and phorbol myristate acetate were without effect on 5,15-DiHETE formation from 15-HpETE. This stimulation of 5,15-DiHETE synthesis by A23187 or by natural agonists was effectively inhibited by MK-886, a compound that has recently been reported to inhibit the A23187-induced translocation of 5-LO to membrane structures. Furthermore, natural-agonist-induced activation of the 5-LO-mediated transformation of 15-HpETE was inhibited by pertussis toxin, indicating the involvement of a GTP-binding protein in the 5-LO activation process.
McDonald
P P
PP
Centre de recherche en Inflammation, Immunologie et Rhumatologie, l'Université Laval, Sainte-Foy, Québec, Canada.
McColl
S R
SR
Naccache
P H
PH
Borgeat
P
P
D001094:Arachidonate 5-Lipoxygenase / Q000378:metabolism*; D000001:Calcimycin / Q000494:pharmacology; D002384:Catalysis; D002772:Cholera Toxin / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D015777:Eicosanoids / Q000494:pharmacology; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007700:Kinetics; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology*; D037342:Pertussis Toxin; D013379:Substrate Specificity; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D002722:Chlorobenzenes
D001094:Arachidonate 5-Lipoxygenase; D000001:Calcimycin; D002772:Cholera Toxin; D015777:Eicosanoids; D019204:GTP-Binding Proteins; D007211:Indoles; D037342:Pertussis Toxin; D010566:Virulence Factors, Bordetella; C060893:MK-886; D002722:Chlorobenzenes