Regulation Of Inducible Nitric Oxide Synthase Gene Expression On Receptor & Signal Transduction Mechanism Of Opioid Tolerance And Dependence

In this report, regulation of inducible nitric oxide synthase (iNOS) gene expression in the mechanism of opioid tolerance and dependence has been investigated using molecular biological techniques and molecular pharmacological methods. First, a retroviral vector and an eukaryotic expression vector containing the full-length cDNA for iNOS were constructed respectively. The neuroblastomaxglioma NG108-15 cells were transfected with the resulting two recombinant plasmids containing iNOS gene, thus stable iNOS-gene expressing cell lines were successfully obtained. Then, a cell model of opioid tolerance and dependence was established by long-term exposure of iNOS gene-transfected cells to opioid agonists. The relationships between the iNOS-gene functional expression and the 5-opioid receptor-coupled AC-cAMP system and Ca²⁺ system, and NO-cGMP signal system were studied. In addition, we also further explored the neurochemical mechanism of the drugs interfering NMDA-NO-cGMP pathway in treating opioid tolerance and dependence. Our studies not only have great theoretical values to elucidate the iNOS gene expression and regulation, but also provide strong experimental evidence for the interactions between opioid receptor- and NMDA receptor-mediated signal transduction systems in development of opioid tolerance and dependence, offering novel research strategies and drug-acting targets for seeking new drugs against opioid tolerance and dependence.1. Stable Expression of Recombinant iNOS Gene Mediated by Retroviral Vector in NG108-15 cellsUsing recombinant DNA techniques, the full-length cDNA encoding murine macrophage iNOS was isolated from pKSiNOS plasmid and subcloned into intermediate vector pSP72, adjusting the restriction enzyme sites in both 5'- and 3'-flanking end of inserted cDNA. The retroviral vector pLNCXiNOS, which contains cytomegalovirus promoter, iNOS coding region and neomycin resistance (neo) gene as a selectable marker, was further constructed. The authenticity of inserting size and orientation of iNOS sequence was verified by restriction mapping analysis and PCR amplification with iNOS gene-specific primers. Retroviral expression vector carrying cDNA fragment of iNOS was constructed. NG108-15 cells was transfected with iNOS cDNA by LipofectAMINE gene transferring technique and G418 resistant clones were selected and named as NG-pLNCXiNOS cells. Southern blot, PCR amplification, RT-PCR and Western blot analysis testified the integrity and expression of the foreign iNOS gene in NG-pLNCXiNOS cells. NADPH-diaphorase (NADPH-d) histochemical staining and immunohistochemical staining with iNOS specific antibody displayed the iNOS gene functional expression in cytoplasm of NG-pLNCXiNOS cells. The cytosolic iNOS catalytic activity in NG-pLNCXiNOS cells was increased by 2- to 4- fold with assessing the conversion of ³H-Arg to ³H-Cit. Such high activity could remain stable for more than 6 months of continuous cultivation. Activity was concentration-dependently reduced by NOS inhibitor such as N^G-nitro-L-Arginine (L-NNA) and aminoguadine (AG). Therefore, a neural cell line with stable expression of the recombinant iNOS gene is successfully established for the first time.