Expression of inducible nitric oxide synthase gene in neuronal cells mediated by retrovirus vector

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 x 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 NO2- content 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 NO2- production 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.