Expression of inducible nitric oxide synthase potentiates the respiratory burst in RAW264.7 macrophages

【作者】 陆红玲； 赵凯； 卫涛涛；

【Author】 Hong-ling Lu~2,Kai Zhao~1,Tao-tao Wei~1 1 National Laboratory of Biomacromolecules,Institute of Biophysics,Chinese Academy of Sciences,15 Datun Road,Beijing 100101,China 2 Department of Biochemistry,Zunyi Medical College,201 Dalian Road, Zunyi 563003,China

【机构】 遵义医学院生化教研室； 中国科学院生物物理研究所生物大分子国家重点实验室；

【摘要】 <正>Macrophages produce a large volume of reactive oxygen species（ROS） through respiratory burst. However,the influence of inducible nitric oxide synthase（iNOS） activation on ROS production remains unclear.In the present investigation,the kinetic generation of ROS in RAW264.7 murine macrophages was monitored by chemiluminescence.Phorbol 12-myristate 13-acetate（PMA） induces a robust chemiluminescence in RAW264.7 cells,suggesting protein kinase C（PKC）-related assembly and activation of nicotinamide adenine dinucleotide phosphate（NADPH） oxidase（NOX）.The effects of iNOS induction on ROS production were examined.Induction of iNOS expression in RAW264.7 cells with lipopolysaccharide（LPS; 1μg/ml） causes a significant increase in PMA-induced chemiluminescence,which could be enhanced by the NOS substrate,L-arginine （L-Arg）,and could be abolished by the NOS inhibitor,L-N^G-nitroarginine（L-NNA）.Further experiments reveal that induction of iNOS expression enhances the PMA-stimulated phosphorylation of the p47^phox subunit of NOX, and promotes the relocalization of cytosolic p47^phox and p67^phox subunits to the membrane.Inhibition of PKCζ,by its myristoylated pseudosubstrate significantly decreased the PMA-stimulated phosphorylation of the p47^phox in LPS-pretreated cells,suggesting that PKCζis involved in the iNOS-dependent assembly and activation of NOX. Taken together,the present investigation suggests that the induction of iNOS up-regulates the PMA-induced assembly of NOX and leads to the enhanced production of ROS via a PKCζ-dependent mechanism.更多还原

【Abstract】 Macrophages produce a large volume of reactive oxygen species（ROS） through respiratory burst. However,the influence of inducible nitric oxide synthase（iNOS） activation on ROS production remains unclear.In the present investigation,the kinetic generation of ROS in RAW264.7 murine macrophages was monitored by chemiluminescence.Phorbol 12-myristate 13-acetate（PMA） induces a robust chemiluminescence in RAW264.7 cells,suggesting protein kinase C（PKC）-related assembly and activation of nicotinamide adenine dinucleotide phosphate（NADPH） oxidase（NOX）.The effects of iNOS induction on ROS production were examined.Induction of iNOS expression in RAW264.7 cells with lipopolysaccharide（LPS; 1μg/ml） causes a significant increase in PMA-induced chemiluminescence,which could be enhanced by the NOS substrate,L-arginine （L-Arg）,and could be abolished by the NOS inhibitor,L-N^G-nitroarginine（L-NNA）.Further experiments reveal that induction of iNOS expression enhances the PMA-stimulated phosphorylation of the p47^phox subunit of NOX, and promotes the relocalization of cytosolic p47^phox and p67^phox subunits to the membrane.Inhibition of PKCζ,by its myristoylated pseudosubstrate significantly decreased the PMA-stimulated phosphorylation of the p47^phox in LPS-pretreated cells,suggesting that PKCζis involved in the iNOS-dependent assembly and activation of NOX. Taken together,the present investigation suggests that the induction of iNOS up-regulates the PMA-induced assembly of NOX and leads to the enhanced production of ROS via a PKCζ-dependent mechanism.更多还原