Pericić Danka, Strac Dubravka Svob, Jembrek Maja Jazvinsćak, Vlainić Josipa
Division of Molecular Medicine, Ruder Bosković Institute, Laboratory for Molecular Neuropharmacology, P.O.Box 180, 10002 Zagreb, Croatia.
Naunyn Schmiedebergs Arch Pharmacol. 2007 May;375(3):177-87. doi: 10.1007/s00210-007-0152-z. Epub 2007 Mar 22.
Benzodiazepines are drugs known to produce tolerance and dependence and also to be abused and co-abused. The aim of this study was to further explore the mechanisms that underlie adaptive changes in GABA(A) receptors following prolonged exposure to these drugs. Human embryonic kidney (HEK 293) cells stably expressing recombinant alpha1beta2gamma2s GABA(A) receptors were exposed for 72 h to a high concentration of diazepam (50 microM) in the absence or presence of other drugs. Radioligand binding studies were used to determine the parameters of [(3)H]flunitrazepam and [(3)H]muscimol binding sites and allosteric interactions between these sites. Prolonged treatment with diazepam increased the maximum number (B (max)) of [(3)H]flunitrazepam and [(3)H]muscimol binding sites in the membranes, and of [(3)H]muscimol binding sites on the surface of HEK 293 cells. There was no change in the affinity (K (d)) of binding sites. The diazepam-induced increase in the B (max) value of [(3)H]flunitrazepam binding sites was reduced by two GABA(A) receptor antagonists, gabazine (1 and 10 microM) and picrotoxin (100 microM). In addition, it was reduced by cycloheximide (5 microg/ml), a protein synthesis inhibitor, and actinomycin D (7.5 microg/ml), an RNA synthesis inhibitor. Flumazenil (5 microM), the antagonist of benzodiazepine binding sites, also up-regulated [(3)H]flunitrazepam recognition sites. Simultaneous treatment with diazepam and flumazenil failed to produce an additive up-regulation. GABA (1 nM - 1 mM)-induced potentiation of [(3)H]flunitrazepam binding to membranes obtained from diazepam (50 microM)-pretreated cells was markedly reduced, suggesting functional uncoupling between GABA and benzodiazepine binding sites. The results suggest that diazepam up-regulated benzodiazepine binding sites on stably expressed GABA(A) receptors by stimulating their synthesis at both the transcriptional and translational levels. A comparable increase of [(3)H]muscimol binding sites expressed on the surface of intact HEK 293 cells suggests that internalisation of surface receptors presumably can not explain the uncoupling.
苯二氮䓬类药物已知会产生耐受性和依赖性，还会被滥用及合并滥用。本研究的目的是进一步探究长期接触这些药物后GABA(A)受体适应性变化的潜在机制。将稳定表达重组α1β2γ2s GABA(A)受体的人胚肾（HEK 293）细胞在不存在或存在其他药物的情况下，暴露于高浓度地西泮（50微摩尔）72小时。采用放射性配体结合研究来确定[(3)H]氟硝西泮和[(3)H]蝇蕈醇结合位点的参数以及这些位点之间的变构相互作用。用地西泮长期处理增加了膜中[(3)H]氟硝西泮和[(3)H]蝇蕈醇结合位点以及HEK 293细胞表面[(3)H]蝇蕈醇结合位点的最大数量（B(max)）。结合位点的亲和力（K(d)）没有变化。两种GABA(A)受体拮抗剂加巴喷丁（1和10微摩尔）和印防己毒素（100微摩尔）降低了地西泮诱导的[(3)H]氟硝西泮结合位点B(max)值的增加。此外，蛋白质合成抑制剂环己酰亚胺（5微克/毫升）和RNA合成抑制剂放线菌素D（7.5微克/毫升）也降低了该值。苯二氮䓬结合位点拮抗剂氟马西尼（5微摩尔）也上调了[(3)H]氟硝西泮识别位点。地西泮和氟马西尼同时处理未能产生累加上调作用。GABA（1纳摩尔 - 1毫摩尔）诱导的[(3)H]氟硝西泮与从地西泮（50微摩尔）预处理细胞获得的膜结合的增强作用明显降低，表明GABA与苯二氮䓬结合位点之间存在功能解偶联。结果表明，地西泮通过在转录和翻译水平刺激其合成，上调了稳定表达的GABA(A)受体上的苯二氮䓬结合位点。完整HEK 293细胞表面表达的[(3)H]蝇蕈醇结合位点有类似增加，这表明表面受体的内化可能无法解释这种解偶联。
