Differential effects of diazepam treatment and withdrawal on recombinant GABA_A receptor expression and functional coupling

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• 作者：Dubravka &#x160 ; vob &#x160 ; trac ; Josipa Vlainić ; Maja Jazvin&#x161 ; ć ; ak Jembrek ; Danka Perič ; ić
• 关键词：Diazepam treatment ; Withdrawal ; Recombinant GABA_A receptor ; HEK 293 cell ; Ligand binding ; mRNA and protein expression
• 刊名：Brain Research
• 出版年：2008
• 出版时间：30 December 2008
• 年：2008
• 卷：1246
• 期：Complete
• 页码：29-40
• 全文大小：574 K

文摘

Prolonged exposure to benzodiazepines, drugs known to produce tolerance and dependence and also to be abused, leads to adaptive changes in GABA_A receptors. To further explore the mechanisms responsible for these phenomena, we studied the effects of prolonged diazepam treatment on the recombinant α₁β₂γ_2S GABA_A receptors, stably expressed in human embryonic kidney (HEK) 293 cells. The results demonstrating that long-term (48 and 72 h) exposure of cells to a high concentration of diazepam (50 μM) enhanced the maximum number (B_max) of [³H]flunitrazepam, [³H]muscimol and [³H]t-butylbicycloorthobenzoate ([³H]TBOB) binding sites, without changing their affinity (K_d), suggested the up-regulation of GABA_A receptors. As demonstrated by cell counting and WST-1 proliferation assay, the observed increase in receptor expression was not a consequence of stimulated growth of cells exposed to diazepam. Semi-quantitative RT-PCR and Western blot analysis, showing elevated levels of α₁ subunit mRNA as well as β₂ and γ₂ subunit proteins, respectively, suggested that prolonged high dose diazepam treatment induced de novo receptor synthesis by acting at both transcriptional and translational levels. The finding that the number of GABA_A receptor binding sites returned to control value 24 h following diazepam withdrawal, makes this process less likely to account for the development of benzodiazepine tolerance and dependence. On the other hand, the results demonstrating that observed functional uncoupling between GABA and benzodiazepine binding sites persisted after the termination of diazepam treatment supported the hypothesis of its possible role in these phenomena.