Gélas P, Von Tscharner V, Record M, Baggiolini M, Chap H
INSERM U 326, Hôpital Purpan, Toulouse, France.
Biochem J. 1992 Oct 1;287 ( Pt 1)(Pt 1):67-72. doi: 10.1042/bj2870067.
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.
在不存在细胞松弛素B的情况下，对由N-甲酰甲硫氨酰-亮氨酰-苯丙氨酸刺激的人中性粒细胞进行了一项比较研究，该研究通过依赖H2O2的化学发光监测呼吸爆发的实时动力学，并研究磷脂酶D（PLD）介导的磷脂酰胆碱分解。真菌代谢产物17-羟基渥曼青霉素（HWT）是一种NADPH氧化酶激活抑制剂，在浓度为1 nM时可使磷脂酸（PA）生成减少30%。与对照相比，更高浓度（10 nM - 1 μM）最大程度抑制PA形成达50%。在所有情况下，加入激动剂后抑制作用延迟20 - 30秒。因此，完整的PA生成实际上是早期（对HWT不敏感）和晚期（对HWT敏感）磷脂酰胆碱分解的结果。然而，在所有条件下，烷基酰基甘油保持在基础水平。PLD活性依赖于Ca2+内流，但在用EGTA和喹胺酸2耗尽Ca2+的细胞中被完全抑制。通过测量H2O2诱导的化学发光动力学研究了HWT对呼吸爆发的影响。该方法能够区分超氧离子产生的各个阶段：一个延迟期、H2O2形成增加（早期阶段）、H2O2产生持续时间（晚期阶段）以及氧化爆发的终止。对于所有HWT浓度，延迟期保持恒定。浓度为10 nM的HWT完全抑制PA产生的HWT敏感部分，在加入激动剂后约20秒延迟降低超氧离子产生。更高的HWT浓度对PLD抑制没有额外影响，同样影响爆发的早期和晚期阶段。因此，高剂量的HWT有一个作用位点，可降低整个爆发但不再影响PLD。所以HWT和Ca2+为PLD激活的两步过程提供了证据。只有延迟的PA生成在功能上与氧化爆发的晚期阶段相关联。
