Combadière C, el Benna J, Pedruzzi E, Hakim J, Périanin A
Laboratoire d'Hématologie, INSERM U 294, Hôspital Bichat, Paris, France.
Blood. 1993 Nov 1;82(9):2890-8.
Stimulation of polymorphonuclear neutrophils (PMN) by phorbol esters or formyl peptides (fMLP) generates large quantities of superoxide anion, the so-called respiratory burst (RB), a phenomenon associated with intense phosphorylation of a 47-kD protein (p47 phox). Staurosporine, a potent protein kinase C (PKC) antagonist, inhibits both responses when PMN are stimulated by phorbol myristate acetate (PMA), suggesting a positive role of PKC. In this study, we reassessed these PMN responses in fMLP-stimulated cells and found that staurosporine had opposite effects depending on the duration of PMN treatment with staurosporine. Short PMN incubation (0.5 to 3 minutes) with 25 to 100 nmol/L staurosporine inhibited the fMLP-induced RB, whereas longer treatment (15 to 20 minutes) enhanced it by up to about 200% relative to controls. In contrast, the PMA-mediated RB was depressed by staurosporine in a time-dependent manner. A primed fMLP-induced RB was also observed after long (15 minutes) PMN treatment with 5 to 100 mumol/L H-7, whereas shorter treatment (5 minutes) resulted in a small decrease in RB. By contrast, the tyrosine kinase inhibitor genistein (2 to 80 mumol/L) depressed fMLP-induced RB whatever the duration of PMN treatment. Analysis of 32P-phosphorylated proteins in fMLP-stimulated cells showed that short PMN treatment (< 8 minutes) with staurosporine abolished the phosphorylation of the 47-kD protein, which was identified as p47 phox, whereas long treatment partially restored p47 phox phosphorylation up to approximately 50% of the control value. In PMA-stimulated PMN, phosphorylation was reduced in a time-dependent manner. Furthermore, the staurosporine-primed RB and the staurosporine-induced recovery of phosphorylation were inhibited by sphingosine but not by genistein. Thus, in addition to its known depressive effect, staurosporine markedly potentiated fMLP-stimulated RB as a function of the duration of PMN treatment. The restoration of p47 phox phosphorylation suggests that staurosporine may alter the interactions between different protein kinases, producing marked time-dependent changes in signalling pathways. These data emphasize the care that should be taken in interpreting data obtained using this kinase inhibitor that may, however, be helpful analyzing in signalling pathways.
佛波酯或甲酰肽（fMLP）刺激多形核中性粒细胞（PMN）会产生大量超氧阴离子，即所谓的呼吸爆发（RB），这一现象与一种47-kD蛋白（p47 phox）的强烈磷酸化有关。星形孢菌素是一种有效的蛋白激酶C（PKC）拮抗剂，当PMN受到佛波醇肉豆蔻酸酯乙酸酯（PMA）刺激时，它会抑制这两种反应，提示PKC具有正向作用。在本研究中，我们重新评估了fMLP刺激细胞中的这些PMN反应，发现星形孢菌素的作用取决于PMN用星形孢菌素处理的持续时间。PMN用25至100 nmol/L星形孢菌素短时间孵育（0.5至3分钟）可抑制fMLP诱导的RB，而较长时间处理（15至20分钟）则使其相对于对照增强约200%。相反，星形孢菌素以时间依赖性方式抑制PMA介导的RB。在用5至100 μmol/L H-7对PMN进行长时间（15分钟）处理后，也观察到预激发的fMLP诱导的RB，而较短时间处理（5分钟）则导致RB略有下降。相比之下，无论PMN处理持续时间如何，酪氨酸激酶抑制剂染料木黄酮（2至80 μmol/L）都会抑制fMLP诱导的RB。对fMLP刺激细胞中32P磷酸化蛋白的分析表明，PMN用星形孢菌素短时间处理（<8分钟）可消除47-kD蛋白的磷酸化，该蛋白被鉴定为p47 phox，而长时间处理可使p47 phox磷酸化部分恢复至对照值的约50%。在PMA刺激的PMN中，磷酸化以时间依赖性方式降低。此外，鞘氨醇可抑制星形孢菌素预激发的RB和星形孢菌素诱导的磷酸化恢复，但染料木黄酮则不能。因此，除了其已知的抑制作用外，星形孢菌素还可根据PMN处理的持续时间显著增强fMLP刺激的RB。p47 phox磷酸化的恢复表明，星形孢菌素可能会改变不同蛋白激酶之间的相互作用，从而在信号通路中产生明显的时间依赖性变化。这些数据强调了在解释使用这种激酶抑制剂获得的数据时应谨慎，不过它可能有助于分析信号通路。
