Induction of inducible nitric oxide synthase increases the production of reactive oxygen species in RAW264.7 macrophages.
Induction of inducible nitric oxide synthase increases the production of reactive oxygen species in RAW264.7 macrophages.
复制标题
DOI：
10.1042/bsr20090048
复制
发表时间：
2010-08
影响因子：
4
通讯作者：
K. zhao;Zhenhao Huang;Hongling Lu;Juefei Zhou;T. Wei
中科院分区：
文献类型：
--
作者：
K. zhao;Zhenhao Huang;Hongling Lu;Juefei Zhou;T. Wei
巨噬细胞通过呼吸爆发产生大量活性氧。然而，iNOS [诱导型NOS（一氧化氮合酶）]激活对ROS产生的影响仍不清楚。在本研究中，活性氧在RAW264.7小鼠巨噬细胞的动力学产生监测化学发光。PMA在RAW264.7细胞中诱导强烈的化学发光，表明PKC（蛋白激酶C）相关的组装和NOX（NADPH氧化酶）的活化。检测iNOS诱导对ROS产生的影响。用LPS（脂多糖; 1 μ g/ml）诱导RAW264.7细胞中的iNOS表达导致PMA诱导的化学发光的显著增加，其可以被NOS底物L-精氨酸增强，并且可以被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PMA刺激的NOX的p47 phox亚基的磷酸化，并促进胞质p47 phox和p67 phox亚基的重新定位到膜。通过豆蔻酰化假底物抑制PKCzeta可显著降低LPS预处理细胞中PMA刺激的p47 phox磷酸化，表明PKCzeta参与iNOS依赖性组装和NOX激活。综上所述，本研究表明，诱导iNOS上调PMA诱导的NOX组装，并通过PKC ζ依赖性机制导致ROS的产生增加。
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.