Effects Of Morphine On Levels Of Nitric Oxide, Glutamate And Glutamine And Expression Of The Related Metabolic Key Enzymes In Rat C6 Glioma Cell

Opioids and drug abuse and addiction and popularity has become an important social problem in today's world, but so far, their tolerance and dependence on the occurrence and development of the withdrawal mechanisms are not completely clear now.The purpose of this study is through the action of morphine in C6 glioma cells drug-dependent cell lines, research extracellular NO, Gln and Glu content changes and metabolic cycle of Gln-Glu key enzyme of the GS and GDH changes of C6 cells with a view to the study opiates add new content.Neurons, glial cells and cerebral vascular endothelial cell can produce NO, as a atypical neurotransmitter, NO plays an important role in the central nervous system. NO has bidirectional capability on the CNS. Low concentrations of NO act as the messenger, while the high concentration of NO has cytotoxicity.In the central nervous system, glutamate (glutamate, Glu) is considered to be a distribution of a wide range of excitatory amino acids (excitatory amino acids, EAAs). Recent studies have confirmed that, Glu occupies an important position in drug dependence and withdrawal process, and morphine addiction and withdrawal symptoms has been plagued with problems of the world. Therefore, the study of Glu in the morphine addiction and withdrawal symptoms on the mechanism is necessary. Addiction morphine tolerance in the excitatory neurotransmitter, glutamate changes can cause in the level of excitement. Scholars believe that through the role ofμopioid receptor, the exogenous opioids regulating the release of excitatory amino acids, which affect dopamine reward system, we speculate morphine addiction, physical dependence and forced to find drug-related behavior reference to the mechanism.1. Objective and MethodsThis study using rat C6 glioma cell to set up the model of morphine dependence, the morphology and cell cycle and apoptosis of C6 rat glioma cell were observed and studied, the extracellular NO, Glu and glutamine contents changes and key enzyme glutamine synthetase (GS) and glutamate dehydrogenase (GDH) changes of glutamate and glutamine cycle, with a view to add new content of the mechanism of.the withdrawal of naloxone and morphine dependence and tolerance.In this experiment, whit the use of morphine at the same time giveμreceptor antagonist naloxone intervention into solvent, it were divided into control group (group C), morphine group (M, at the concentration of 27μM), morphine and naloxone group (N + M, after naloxone effect at the concentration of 1μM , add morphine at the concentration of 27μM) and naloxone group (N, at the concentration of 1μM). Each group administration once every 24 h, after the third administration cultured for more 24h, take the cell cultured supernatant frozen in -70℃or extract protein and total RNA.The First Chapter research morphine and naloxone on rat C6 glioma cell morphology and cell cycle and cell apoptosis by AO staining and flow cytometry method. Investigate NO, Gln and Glu content changes of C6 rat glioma cell culture supernatant by NO, Gln and Glu Detection Kit. Chapter II research morphine on rat C6 glioma cells in the inducible nitric oxide synthase (iNOS) expression by Western blot method. RT-PCR method were used to study morphine on C6 rat glioma cells in the glutamate dehydrogenase (GDH) and glutamine synthetase (GS) m-RNA expression of the impact.2. Results(1)Effects of morphine on morphology and cell cycle and apoptosis and contents of nitric oxide, glutamate and glutamine The Experimental results of the first chapter show that: In the effects of morphine and naloxone, respectively, as well as commonly, there was no obvious decrease in the number of cells and the cytoplasm shrinkage phenomenon on AO staining. FCM also observed no significant reduction in the number of cells and apoptosis peaks induced by morphine and naloxone. Morphine administered alone can reduce extracellular NO content significantly, morphine and naloxone administered together did not reduced the extracellular concentration of NO significantly; Morphine or naloxone alone can increase the extracellular Glu content significantly, and application of morphine and naloxone together can make a significant reduction on morphine induced the increase of extracellular Glu content.(2) Effects of morphine on expression of iNOS and glutamate , glutamine gene expression of the related metabolic key enzymes in rat C6 glioma cell Chapter II molecular level Experimental results show that: Morphine may inhibitμreceptor expression of C6 glioma cells and reduce the expression of iNOS protein. Morphine or naloxone administered alone can significantly decrease the GS mRNA transcription activity, and compared with the M group, the GS mRNA transcription activity of N + M Group were significantly increased. The GDH mRNA transcription activity were significantly increased in M group and N + M group.3. ConclusionAbove all results indicate that: morphine and naloxone can affecting extracellular glutamate and nitric oxide release and uptake of C6 glioma cells by the regulation of iNOS protein expression, GS and GDH gene transcriptional activity to adjust morphine tolerance and dependence.Above results suggest that: morphine and naloxone possiblely act through the surfaceμreceptor of C6 glioma cell, affecting the release of nitric oxide and glutamate, involved in morphine tolerance and dependence. Morphine may regulate the key enzyme GS and GDH of metabolic cycle of Gln-Glu to regulate the concentration of extracellular Glu and Gln, and non-specific opioid receptor antagonist naloxone can be antagonism the effects of morphine partly , suggesting that morphine regulate the GS and GDH gene transcriptional activity and extracellular Gln and Glu contents changes by the binding with opioid receptor. On morphine regulate the GS and GDH gene transcriptional activity of C6 glioma cells through opioid receptor and the changes of extracellular Glu and Gln contents to be involved in such as the role of morphine dependent and tolerance mechanism needs to be further confirmed by experiments.