engleski

The effects of zolpidem treatmant and withdrawal on the in vitro expression of recombinant GABA-A receptors expressed in HEK 293 cells

Introduction:Imidazopyridine zolpidem is the most widely prescribed non-benzodiazepine hypnotic, which exerts its effects via benzodiazepine modulatory sites on GABA-A receptor. Zolpidem is a positive allostreic modulator of GABA-A receptors with preferential, although not exclusive, binding for alpha 1 subunit-containing receptors. The aim of this study was to explore the mechanisms that underlie adaptive changes in GABA-A receptors following exposure to zolpidem. Such studies might be important since the prescription of zolpidem is increasing substantially and prolonged zolpidem treatment, as recently shown, induces tolerance in mice (Vlainić and Peričić, Neuropharmacology, 56: 1124-1130, 2009). Materials and Methods: In order to evaluate the effects of prolonged zolpidem treatment on GABA-A receptors human embryonic kidney (HEK 293) cells stably expressing recombinant alpha1beta2gamma2s subtype of GABA-A receptors were exposed to zolpidem (10 microM, 48 h). Radioligand binding studies preformed under conditions previously described ( Peričić et al., Naunyn-Schmiedebergs Arch Pharmacol 375:177-187, 2007) were used to determine the parameters of (3H)flunitrazepam. (3H)muscimol and (3H)butylbicycloorthobenzoate (3H)TBOB) binding sites. Semi-quantitative RT-PCR and Western blot analysis were used to asses mRNA and protein levels. Results: Prolonged exposure of GABA-A receptors to Zolpidem up-regulated the maximum number (Bmax) of (3H)flunitrazepam, (3H)muscimol and (3H)TBOB) binding sites, without changing their affinity (Kd), suggesting an increase of total GABA-A receptor number. Prolonged occupation of benzodiazepine binding site by 10 microM zolpidem produced also the functional uncoupling between GABA and benzodiazepine binding sites, as evidenced by a decreased ability of GABA to stimulate (3H)flunitrazepam binding. The observed increase in gamma2 subunit proteins along with an increased mRNA for alpha1 subunit supports the hypothesis that zolpidem treatment stimulates de novo synthesis of GABA-A receptor proteins by acting at the transcriptional as well as translational level. Enhanced number of binding sites and gamma2 subunit proteins returned to control values 24 h after discontinuation of zolpidem treatment. THe observed functional uncoupling of binding sites normalized folowing zolpidem withdrawal as well. Conclusion: The results of our in vitro studies suggest that two-day exposure of recombinant alpha1 subunit-containing GABA-A receptors stably transfected in HEK 293 cells to zolpidem triggers adaptive changes of these selective GABA-A receptor subtype in a manner that is similar to that obtained with high concentrations of classical benzodiazepine-diazepam (Švob Štrac et al., Brain Res 1246:29-40, 2008).

zolpidem ; long-trem treatment ; withdrawal ; recombinant GABA-A receptor expression ; HEK 293 cells

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