Synergistic stimulation of neutrophils. Possible involvement of 5-hydroxy-6,8,11,14-eicosatetraenoate in superoxide...

Neutrophils stimulated with optimal amounts of tumor-promoters that activate protein kinase C (e.g. mezerein, phorbol 12,13-dibutyrate) are known to release large quantities of superoxide: approximately 40-50 nmol O2-/min/10(7) cells. Previous studies have shown that treatment of neutrophils with the calcium ionophore A23187, or with 5-hydroxy-6,8,11,14-eicosatetraenonate (5-HETE), dramatically increased the ability of these cells to release O2- in response to suboptimal concentrations of the stimulants mentioned. In this manuscript, we provide data relevant to the basis of this augmentation of O2- release. The synergy with ionophore A23187 exhibited a partial requirement for extracellular Ca2+, whereas that with 5-HETE exhibited a near absolute requirement for that cation. Neutrophils stimulated with optimal amounts of tumor-promoters are known to exhibit a redistribution of protein kinase C activity from the soluble to a particulate fraction. A redistribution of kinase activity was not observed in cells stimulated synergistically. On the other hand, ionophore A23187 and 5-HETE increased the binding of a suboptimal amount of [3H] phorbol 12,13-dibutyrate to intact neutrophils by approximately 25 and 50%, respectively. Inhibitors of protein kinase C (i.e. sphingosine, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine) substantially blocked O-2 release from neutrophils stimulated either synergistically or with optimal levels of tumor-promoters. These data suggest a role for 5-HETE in modulating O-2 release by neutrophils and are discussed in relation to models of the interactions of protein kinase C with membranes.