double daggerCancer Institute and Hospital, Chinese Academy of Medical Sciences, 17 Panjiayuan Nanli, Beijing 100021, People's Republic of China.
Biosci Rep. 2010 Mar 12;30(4):233-41. doi: 10.1042/BSR20090048.
Macrophages produce a large volume of ROS (reactive oxygen species) through respiratory burst. However, the influence of iNOS [inducible NOS (nitric oxide synthase)] activation on ROS production remains unclear. In the present study, the kinetic generation of ROS in RAW264.7 murine macrophages was monitored by chemiluminescence. PMA induces a robust chemiluminescence in RAW264.7 cells, suggesting PKC (protein kinase C)-related assembly and activation of NOX (NADPH oxidase). The effects of iNOS induction on ROS production were examined. Induction of iNOS expression in RAW264.7 cells with LPS (lipopolysaccharide; 1 microg/ml) causes a significant increase in PMA-induced chemiluminescence, which could be enhanced by the NOS substrate, L-arginine, and could be abolished by the NOS inhibitor, L-NNA (NG-nitro-L-arginine). Further experiments reveal that induction of iNOS expression enhances the PMA-stimulated phosphorylation of the p47phox subunit of NOX, and promotes the relocalization of cytosolic p47phox and p67phox subunits to the membrane. Inhibition of PKCzeta by its myristoylated pseudosubstrate significantly decreased the PMA-stimulated phosphorylation of the p47phox in LPS-pretreated cells, suggesting that PKCzeta is involved in the iNOS-dependent assembly and activation of NOX. Taken together, the present study suggests that the induction of iNOS upregulates the PMA-induced assembly of NOX and leads to the enhanced production of ROS via a PKCzeta-dependent mechanism.
巨噬细胞通过呼吸爆发产生大量的 ROS（活性氧）。然而，iNOS[诱导型 NOS（一氧化氮合酶）]激活对 ROS 产生的影响尚不清楚。在本研究中，通过化学发光监测 RAW264.7 鼠巨噬细胞中 ROS 的动力学产生。PMA 诱导 RAW264.7 细胞产生强烈的化学发光，表明 PKC（蛋白激酶 C）相关的 NOX（NADPH 氧化酶）组装和激活。检查了 iNOS 诱导对 ROS 产生的影响。用 LPS（1μg/ml）诱导 RAW264.7 细胞中 iNOS 的表达导致 PMA 诱导的化学发光显着增加，这可以通过 NOS 底物 L-精氨酸增强，并且可以通过 NOS 抑制剂 L-NNA（NG-硝基-L-精氨酸）消除。进一步的实验表明，诱导 iNOS 表达增强了 PMA 刺激的 NOX 的 p47phox 亚基的磷酸化，并促进了胞质 p47phox 和 p67phox 亚基向膜的重新定位。PKCzeta 的豆蔻酰化伪底物对其的抑制通过其显著降低 LPS 预处理细胞中 PMA 刺激的 p47phox 的磷酸化，表明 PKCzeta 参与了 iNOS 依赖性的 NOX 组装和激活。总之，本研究表明，iNOS 的诱导上调了 PMA 诱导的 NOX 组装，并通过 PKCzeta 依赖的机制导致 ROS 的产生增强。
