engleski

Allosteric uncoupling and up-regulation of benzodiazepine and GABA recognition sites following chronic diazepam treatment of HEK 293 cells stably transfected with alpha1 beta2 gamma2s subunits of GABA-A receptors

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 alpha1 beta2 gamma2s 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 [3H]flunitrazepam and [3H]muscimol binding sites and allosteric interactions between these sites. Prolonged treatment with diazepam increased the maximum number (Bmax) of [3H]flunitrazepam and [3H]muscimol binding sites in the membranes, and of [3H]muscimol binding sites on the surface of HEK 293 cells. There was no change in the affinity (Kd) of binding sites. The diazepam-induced increase in the Bmax value of [3H]flunitrazepam binding sites was reduced by two GABA-A receptor antagonists, gabazine (1 and 10 microM) and picrotoxin (100 microM). Further, 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 [3H]flunitrazepam recognition sites. Simultaneous treatment with diazepam and flumazenil failed to produce an additive up-regulation. GABA (1 nM - 1 mM) induced potentiation of [3H]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 [3H]muscimol binding sites expressed on the surface of intact HEK 293 cells suggests that internalisation of surface receptors presumably can not explain the uncoupling.

GABAA receptor recombinant; HEK 293 cells; chronic diazepam; Gabazine; Picrotoxin; inhibitors of RNA and protein synthesis; [3H]flunitrazepam; [3H]muscimol binding

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