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(EZ,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.