engleski

Prolonged exposure of HEK-293 cells stably expressing recombinant GABAA receptors to diazepam induces allosteric uncoupling and up-regulation of benzodiazepine binding sites.

The aim of this study was to improve our understanding of the mechanisms underlying adaptive changes in GABAA receptor following their prolonged exposure to benzodiazepines, drugs known to produce tolerance and dependence. Therefore, we studied the effects of prolonged diazepam (an agonist of benzodiazepine binding sites) exposure on the recombinant alpha1 beta2 gamma2S GABA-A receptors, the most common type of GABA-A receptors found in the brain. Human embryonic kidney, HEK 293 cells stably expressing alpha1 beta2 gamma2S subtype of GABA-A receptors were exposed for 72 h to diazepam (50 microM), or to diazepam in combination with flumazenil (5 microM), actinomycin (7.5 microg/ml), cycloheximide (5 microg/ml) or picrotoxin (100 microM). Aliquots of cell membrane preparations (~75 microg protein/ml) obtained from control and drug treated cells were used in saturation binding studies with [3H]flunitrazepam (0.3-100 nM). In stimulation studies different concentration of GABA (1 nM-1 mM) were incubated with [3H]flunitrazepam (1nM). Our results demonstrated that exposure of cells for 72 h to 50 microM diazepam enhanced in a picrotoxin (a noncompetitive antagonist of GABA binding sites) sensitive manner the maximum number (Bmax) of [3H]flunitrazepam binding sites without affecting their affinity (Kd). The diazepam-induced increase in Bmax was reduced by actinomycin, a RNA synthesis inhibitor and by cycloheximide, a protein synthesis inhibitor, indicating that this effect appears to be due to increased synthesis at both transcriptional and translational level. In diazepam-pretreated cells GABA-induced potentiation of [3H]flunitrazepam binding was reduced, suggesting functional uncoupling of GABA and benzodiazepine binding sites. This effect was counteracted by chronic flumazenil, which per se produced an up-regulation but not allosteric uncoupling.

recombinant GABA A receptors; HEK 293 cells; diazepam; chronic treatment

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