| Objective:New researches are dramatically altering our understanding of the molecular mechanisms of astrocytes underlying neuronal communication. Many researches indicate that astrocytes have a close relationship with epileptogenesis. The present study is aimed to examine the effects of chronic treatment of C6 glioma cells with sodium valproate (VAP) and withdrawal on amino acid transmitters release from C6 glioma cells and related changes at molecular level, and to explore the role of astrocytes involved in the VPA antiepileptic mechanism and the rebound effects caused by VPA withdrawal. Also, it is our purpose to provide the experimental information for more efficient and reliable means to prevent the rebound effects and for the development of new antiepileptic drugs as well. Methods:The experiments were performed on C6 glioma cells to establish the astrocyte model of chronic treatment with VPA at theraputic concentration (50mg/L). After chronic treatment of C6 cells with VPA and then immediate withdrawal of the drug, the parameters interested were detected at three levels, the amino acid transmitters release, mRNA expression and protein expression levels.The detected timepoints were O.Shours, 6hours, 12 hours, 24 hours and 48 hours after VPA withdrawal. High performance liquid chromatography (HPLC) was exploited to detect the release levels of Aspartate(Asp)> Glutamate(Glu) Asparatmine(Asn) Glutamine(Gln), Glycine(Gly) and r-Aminobutyric acid (GABA) from C6 glioma cells. The metabolic enzymes and transporters related to these amino acids released by C6 glioma cells were analyzed at mRNA level via RT-PCR assay. Meanwhile Western blot was used to determine the changes of GAD67 (Glutamate decarboxylase 67Kda) protein and GAD65 (Glutamate decarboxylase 65Kda) protein expressions. Results:VPA chronic treatment stimulated Asp, Glu and GABA release from C6glioma cells, and suppressed Gin release, but had no effect on Asn and Gly release. After withdrawal of VPA, the release of Glu, Asp and GABA exhibited rebound-like changes;RT-PCR assays showed that VPA chronic treatment upregulated GDH (Glutamate dehydrogenase), GAD67, GOTl(Glutamate oxaloacetate transaminase 1), GOT2 (Glutamate oxaloacetate transaminase 2), GABA-T(y-Aminobutyric acid transaminase), NAAT (Neutral amino acid transporter), GLT-1 (Glutamate transporter-1) and EAAC1 (Excitatory amino acid carrier 1) mRNA expressions, and downregulated GAT-3 (y-Aminobutyric acid transporter-3) mRNA expression. After VPA being withdrawed, GDH, GAD67, GOT1, GOT2, GABA-T, NAAT, GLT-1, EAAC1 and GAT-3 mRNA expressions displayed rebound-like changes;In our experiments, C6 glioma cells did not have any detectable GAD65 and GLAST (Glutamate/aspartate transporter) mRNA expressions, but showed clearly GLT-1 mRNA expression. Our observation that GLT-1 mRNA is expressed in C6 glioma cells is opposite to the current recognition that only the Glu transporter subtype EAAC1 but not GLT-1 and GLAST are expressed in the cells. This discrepancy needs further investigation.Chronic treatment of C6 cells with VPA obviously downregulated the mRNA expressions of PC (Pyruvate carboxylase) and GS (Glutamine synthetase). GS mRNA expression went up gradually but was still below the control level after withdrawal. In contrast, the change of PC mRNA expression showed a little difference from that of GS after withdrawal of VPA in that the mRNA expression of PC rose slightly at first and then went down to the level under control. This phenomenon might be attributable to a residual effect of this antiepileptic drug. The residual effect also could be seen in the changes of GOT2, GAT-3 and GABA-T mRNA expressions caused by withdrawal of VPA. Chronic treatment of the cells with VPA made an upregulation on Glutaminase (Glnase) mRNA expression, whereas the expression became declined after VPA withdrawal; SN1 Gln-T (System Nl glutamine transporter) mRNA expression was suppressed by VPA chronic treatment, but gradually increased following VPA withdrawal.Western blot analysis showe...
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