Abstract
A comparative study of real-time kinetics of respiratory burst, monitored by H2O2-dependent chemiluminescence, and phospholipase D (PLD)-mediated phosphatidylcholine breakdown has been undertaken on human neutrophils stimulated by N-formylmethionyl-leucylphenylalanine in the absence of cytochalasin B. The fungal metabolite 17-hydroxywortmannin (HWT), an inhibitor of NADPH oxidase activation, decreases phosphatidic acid (PA) production by 30% at a concentration of 1 nM. Higher concentrations (10 nM-1 microM) inhibit PA formation maximally by 50% as compared with control. In all cases, the inhibition is delayed by 20-30 s after addition of the agonist. Thus the full PA generation is actually the result of an early (HWT-insensitive) and a late (HWT-sensitive) phosphatidylcholine breakdown. However, under all conditions, alkylacylglycerol remains at the basal level. PLD activity is dependent on Ca2+ influx, but is fully inhibited in cells depleted of Ca2+ with EGTA and Quin 2. The effect of HWT on the respiratory burst was investigated by measuring the kinetics of H2O2-induced chemiluminescence. This method allows to distinguish various phases of superoxide ion production: a lag, an increase in H2O2 formation (early phase), the duration of H2O2 production (late phase) and the termination of the oxidative burst. The lag remains constant for all HWT concentrations. A concentration of 10 nM-HWT, which fully inhibits the HWT-sensitive part of PA production, decreases superoxide ion production with a delay of about 20 s after addition of the agonist. Higher HWT concentrations, which have no additional effect on PLD inhibition, equally affect an early and a late phase of the burst. Thus high doses of HWT have a site of action which decreases the whole burst but does not affect the PLD any more. Therefore HWT and Ca2+ provide evidence for a two-step process for PLD activation. Only the delayed PA generation is functionally linked to a late phase of the oxidative burst.