Effect Of Abrupt Withdrawal Of Phenytoin On Release Of Transmitter Amino Acids And MRNA Expressions Of Related Enzymes And Transporter Proteins

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 Human Astrocytes(HA) with Phenytoin Sodium and withdrawal on amino acid transmitters release from the cells and related changes at molecular level, and to explore the role of astrocytes involved in the Phenytoin Sodium antiepileptic mechanism and the rebound effects caused by 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 HA to establish the astrocyte model of chronic treatment with Phenytoin Sodium at therapeutic concentration (20μg/ml). After chronic treatment of HA cells with Phenytoin Sodium and then immediate withdrawal of the drug, the parameters of interest were obtained at three levels, the amino acid transmitters release, mRNA expression and protein expression level. The sample time points were 0,12,24,36,48 and 60hours after drug withdrawal. High performance liquid chromatography (HPLC) was exploited to detect the release amount of Asp, Glu, Asn, Gin, Gly and GABA from HA cells. The metabolic enzymes and transporters related to these amino acids released by cells were analyzed at mRNA level via real-time quantitative PCR assay. Meanwhile western blot was used to determine the changes of GLUL protein.Results:1. Phenytoin Sodium chronic treatment stimulated Gly and GABA release from HA and suppressed Glu release, but had no effect on Asn, Asp and Gin release. After withdrawal, the release of Glu, Gly and GABA exhibited rebound-like changes.2. Real-time quantitative PCR assay showed that Phenytoin Sodium chronic treatment upregulated GAD-1, GAD-2, GLAST and GLT-1 mRNA expression and down regulated GCSH, ABAT and GAT--3 mRNA expression. After withdrawal, all of these mRNA expressions displayed rebound-like changes. In our experiments, HA cells did not have any detectable GLUD1, GOT1, GOT2, GPT, GLUL, PC, GLS, ASRGL1, SLC1A6, SLC6A12, SLC6A15, SLC6A19 mRNA expression changes. Western blot analysis showed that phenytoin sodium chronic treatment made no effects on GLUL protein which was coincident with the previous results.Conclusion:Phenytoin sodium chronic treatment can increase Gly and GABA release and decrease Glu release from HA cells. Glu, Gly and GABA release exhibited rebound-like changes after withdrawal and the mRNA expressions of related metabolic enzymes and transporters demonstrated the same changes. These results may have some relevance to the rebound effect of clinical withdrawal of phenytoin sodium. It is supposed that astrocytes might play an important role in the phenytoin sodium antiepileptic action and the rebound effect caused by withdrawal.