Gay J C, Raddassi K, Truett A P, Murray J J
Department of Pediatrics, Vanderbilt University Medical Center, Nashville, TN 37232-2574, USA.
Biochim Biophys Acta. 1997 Aug 29;1336(2):243-53. doi: 10.1016/s0304-4165(97)00034-2.
Phosphorylation of components of the neutrophil NADPH oxidase plays a critical role in activation and maintenance of superoxide anion (O2-) generation. To investigate the role of dephosphorylation by phosphatases in regulating O2- production, human neutrophils were treated with calyculin A, a potent inhibitor of protein phosphatases 1 and 2A, prior to stimulation. Calyculin A alone did not stimulate O2- production. However, neutrophils exposed to 50 nM calyculin A and the chemotactic peptide formyl-met-leu-phe (FMLP, 100 nM) displayed markedly enhanced O2- production in comparison to cells stimulated with FMLP alone (28.63 +/- 7.00 versus 8.69 +/- 3.69 nmol O2-/1.5 x 10(6) neutrophils/5 min, respectively, n = 18, p < 0.001), with an increased duration of O2- production. In contrast, phosphatase-inhibition decreased oxidative responsiveness to phorbol myristate acetate (PMA, > or = 16 nM). We next examined the effect of calyculin A on products of the phosphatidylcholine-specific phospholipase D (PLD) pathway by assaying the mass levels of phosphatidic acid (PA), choline and diacylglycerol (DAG). Calyculin A increased both PA and choline production to 224 +/- 28% and 315 +/- 61% of FMLP-stimulated controls, respectively (p < 0.01, n = 7) without significantly increasing DAG. Also, membrane protein kinase C activity increased more than 10-fold in FMLP-stimulated cells exposed to calyculin A but decreased in cells stimulated with PMA following calyculin A pre-treatment. These results suggest that phosphatases exert variable and stimulus-dependent effects on pathways leading to O2- production. Further, it appears that phospholipase D activity and PA generation represent important steps in the pathway for NADPH activation triggered by FMLP.
中性粒细胞NADPH氧化酶成分的磷酸化在超氧阴离子（O2-）生成的激活和维持中起关键作用。为了研究磷酸酶去磷酸化在调节O2-产生中的作用，在刺激前，用蛋白磷酸酶1和2A的强效抑制剂花萼海绵诱癌素A处理人中性粒细胞。单独的花萼海绵诱癌素A不会刺激O2-的产生。然而，与仅用趋化肽甲酰-蛋-亮-苯丙氨酸（FMLP，100 nM）刺激的细胞相比，暴露于50 nM花萼海绵诱癌素A和趋化肽FMLP的中性粒细胞显示出O2-产生明显增强（分别为28.63±7.00和8.69±3.69 nmol O2-/1.5×10(6)个中性粒细胞/5分钟，n = 18，p < 0.001），且O2-产生的持续时间增加。相反，磷酸酶抑制降低了对佛波酯肉豆蔻酸酯乙酸酯（PMA，≥16 nM）的氧化反应性。接下来，我们通过检测磷脂酸（PA）、胆碱和二酰基甘油（DAG）的质量水平，研究了花萼海绵诱癌素A对磷脂酰胆碱特异性磷脂酶D（PLD）途径产物的影响。花萼海绵诱癌素A分别将PA和胆碱的产生增加到FMLP刺激对照组的224±28%和315±61%（p < 0.01，n = 7），而没有显著增加DAG。此外，在暴露于花萼海绵诱癌素A的FMLP刺激细胞中，膜蛋白激酶C活性增加了10倍以上，但在花萼海绵诱癌素A预处理后用PMA刺激的细胞中活性降低。这些结果表明，磷酸酶对导致O2-产生的途径发挥可变的、依赖刺激的作用。此外，似乎磷脂酶D活性和PA生成是FMLP触发的NADPH激活途径中的重要步骤。
