Division of Molecular Medicine, Ruder Bosković Institute, Zagreb, Croatia.
Naunyn Schmiedebergs Arch Pharmacol. 2010 Sep;382(3):201-12. doi: 10.1007/s00210-010-0539-0. Epub 2010 Jul 23.
Zolpidem, a widely used hypnotic drug which acts through benzodiazepine binding sites, is a positive allosteric modulator of gamma-aminobutyric acid (GABA) action with preferential affinity for GABA(A) receptors containing alpha1 subunit. The pharmacological profile of zolpidem is different from that of classical benzodiazepines. The aim of this study was to find out whether zolpidem treatment triggers adaptive changes in the recombinant alpha1 subunit-containing GABA(A) receptors other than those observed following treatment with classical benzodiazepine-diazepam. Radioligand binding studies showed that 2-day exposure of human embryonic kidney (HEK) 293 cells stably expressing recombinant alpha1beta2gamma2s GABA(A) receptors to zolpidem (10 muM) up-regulated the maximum number (B (max)) of [(3)H]flunitrazepam, [(3)H]muscimol, and [(3)H]t-butylbicycloorthobenzoate ([(3)H]TBOB) binding sites without changing their affinity (K (d)), suggesting an increase in total GABA(A) receptor number. Semi-quantitative RT-PCR analysis demonstrated increased levels of alpha1 subunit mRNA, while Western blot demonstrated up-regulated gamma2 subunit proteins, suggesting that zolpidem induced de novo synthesis of receptors proteins, at both the transcriptional and translational levels. GABA-induced potentiation of [(3)H]flunitrazepam binding to membranes obtained from zolpidem-treated cells was markedly reduced, indicating allosteric uncoupling between GABA and benzodiazepine binding sites. The number of benzodiazepine and convulsant binding sites as well as the functional coupling between GABA and benzodiazepine binding sites normalized in 24 h following discontinuation of zolpidem treatment. The results of our in vitro studies suggest that a 2-day exposure of recombinant alpha1 subunit-containing GABA(A) receptors stably transfected in HEK 293 cells to zolpidem induces adaptive changes in this selective GABA(A) receptor subtype, which are not substantially different from those obtained after prolonged exposure of cells to high concentrations of diazepam.
唑吡坦是一种广泛使用的催眠药物，通过苯二氮䓬结合位点发挥作用，是γ-氨基丁酸（GABA）作用的正变构调节剂，对含有α1亚基的 GABA(A)受体具有优先亲和力。唑吡坦的药理学特征不同于经典的苯二氮䓬类药物。本研究旨在探讨唑吡坦治疗是否会引发重组α1亚基 GABA(A)受体的适应性变化，而不仅仅是在细胞暴露于高浓度地西泮后观察到的变化。放射配体结合研究表明，在稳定表达重组α1β2γ2s GABA(A)受体的人胚肾（HEK）293 细胞中，2 天暴露于唑吡坦（10 μM）可上调[3H]氟硝西泮、[3H]muscimol 和[3H]叔丁基二环氧苯甲酸盐（[3H]TBOB）结合位点的最大数量（Bmax），而不改变其亲和力（Kd），表明总 GABA(A)受体数量增加。半定量 RT-PCR 分析表明α1 亚基 mRNA 水平升高，而 Western blot 分析表明γ2 亚基蛋白表达上调，表明唑吡坦诱导受体蛋白的从头合成，在转录和翻译水平上均有上调。与唑吡坦处理过的细胞的膜结合的 GABA 诱导的[3H]氟硝西泮结合的增强作用显著降低，表明 GABA 和苯二氮䓬结合位点之间的变构去耦。在停止唑吡坦治疗 24 小时后，苯二氮䓬和惊厥结合位点的数量以及 GABA 和苯二氮䓬结合位点的功能偶联均恢复正常。我们的体外研究结果表明，在 HEK 293 细胞中稳定转染重组α1 亚基 GABA(A)受体 2 天暴露于唑吡坦可诱导该选择性 GABA(A)受体亚型的适应性变化，与细胞长期暴露于高浓度地西泮后获得的变化基本不同。
