The effects of zolpidem treatment and withdrawal on the in vitro expression of recombinant α1β2γ2s GABAA receptors expressed in HEK 293 cells

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 GABAA receptors containing α1 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 α1 subunit-containing GABAA 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 α1β2γ2s GABAA receptors to zolpidem (10 μM) up-regulated the maximum number (Bmax) of [3H]flunitrazepam, [3H]muscimol, and [3H]t-butylbicycloorthobenzoate ([3H]TBOB) binding sites without changing their affinity (Kd), suggesting an increase in total GABAA receptor number. Semi-quantitative RT-PCR analysis demonstrated increased levels of α1 subunit mRNA, while Western blot demonstrated up-regulated γ2 subunit proteins, suggesting that zolpidem induced de novo synthesis of receptors proteins, at both the transcriptional and translational levels. GABA-induced potentiation of [3H]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 α1 subunit-containing GABAA receptors stably transfected in HEK 293 cells to zolpidem induces adaptive changes in this selective GABAA receptor subtype, which are not substantially different from those obtained after prolonged exposure of cells to high concentrations of diazepam.