Expression of Inducible
Nitric Oxide Synthase Gene in Neuronal Cells Mediated by Retrovirus Vector
ZANG Meng-Wei, SHEN Qi, WANG Qing and LIU Jing-Sheng*
( Department of Pharmacology, National Laboratory of Medical Molecular
Biology, Institute of Basic Medical Sciences,Chinese Academy of
Medical Sciences & Peking Union Medical College, Beijing 100005, China
)
PENG Xue-Xian
( Institute of Microbiology, the Chinese Academy of Sciences, Beijing
100080, China )
Abstract The role of an inducible
nitric oxide synthase(iNOS)expression in the mechanisms of opioid tolerance and
dependence was investigated. A recombinant retroviral expression vector
containing a cDNA fragment of iNOS was transfected into the
neuroblastoma×glioma NG108-15 cells by lipofectamine gene transferring
technique. G418-resistant clones were selected and were named NG-LNCXiNOS
cells. Using Southern blot, PCR amplification for Neo gene, RT-PCR and
Western blot analysis, NG-LNCXiNOS cells were confirmed to have an integral exogenous
iNOS gene which was being transcribed and translated into protein.
NADPH-diaphorase(NADPH-d)histochemical staining and immunohistochemical
staining with iNOS-specific antibody demonstrated that high-level expression of
iNOS protein was present in the cytoplasm of NG-LNCXiNOS cells. The catalytic
activity and NO－2content in supernatant medium
were obviously enhanced in iNOS gene-transfected cells. The results
show that the biochemical and pharmacological properties of the recombinant
enzyme were similar to those of native enzyme. The recombinant enzyme activity
was completely dependent on NADPH and failed to be stimulated by the addition
of calcium and calmodulin. Chelating agents failed to decrease its activity.
NOS inhibitors could markedly reduce NO－2production at a
concentration-dependent manner. The expression of iNOS gene was involved in the
up-regulation of NO-cGMP signal transduction cascade. Therefore, an iNOS
gene-modified neuronal cell line was successfully established, offering an
excellent model system for seeking and screening new drugs to treat opioid
tolerance and dependence.
Key Words Nitric oxide synthase； retrovirus； cyclic GMP； neuroblastoma； cell line
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