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Enhanced binding of the cage convulsant [3H]TBOB following chronic exposure of HEK 293 cells expressing recombinant alpha1 beta2 gamma2S GABA A receptors to flumazenil

The aim of this study was to assess whether prolonged exposure to benzodiazepine antagonist flumazenil, previously shown to up-regulate benzodiazepine binding sites on stably expressed recombinant a1b2g2S GABAA receptors (Pericic et al., submitted), affects the functional state of GABAA receptors. Human embryonic kidney (HEK) 293 cells stably expressing the a1b2g2S subtype of GABAA receptor were exposed for 48 h to 1 microM GABA in the absence (control) or presence of flumazenil (1 or 5 microM). Aliquots of membrane preparations (~ 100 microg protein) obtained from control and drug treated cells were used to study the potency and efficacy of added GABA and diazepam to inhibit the binding of the cage convulsant t-[3H]butylbicycloorthobenzoate ([3H]TBOB, 8 nM) under conditions (25°C, 90 min) previously described (Pericic et al., Eur. J. Pharmacol., 1998). In all membrane preparations GABA inhibited the binding of [3H]TBOB in a concentration-dependent manner. The inhibitory potencies of GABA (IC50 ~ 2 microM) were not different between the preparations of control and flumazenil pretreated cells. In the presence of 1microM GABA, diazepam inhibited the binding of [3H]TBOB according to a two-site model. There were no significant differences between the groups in either the potency or the efficacy of diazepam to inhibit [3H]TBOB binding. However, in membranes obtained from flumazenil (1 or 5 microM) treated cells the basal [3H]TBOB binding was markedly enhanced. These results along with our previous data suggest that prolonged exposure of cells expressing recombinant a1b2g2S GABAA receptors to flumazenil up-regulates convulsant binding sites, but in the presence of GABA it does not affect the functional state of GABAA receptors, as evidenced by an unchanged ability of GABA and diazepam to modulate the binding of [3H]TBOB.

Recombinant GABA A receptors; HEK 293 cells; Flumazenil; Chronic treatment

DOI: 10.1111/j.1472-8206.2004.00260.x