Chronic exposure of cells expressing recombinant GABA(A) receptors to benzodiazepine antagonist flumazenil enhances the maximum number of benzodiazepine binding sites.

摘要

The aim of this study was to better understand the mechanisms that underlie adaptive changes in GABA(A) receptors following their prolonged exposure to drugs. Exposure (48 h) of human embryonic kidney (HEK) 293 cells stably expressing recombinant alpha(1)beta(2)gamma(2S) GABA(A) receptors to flumazenil (1 or 5 muM) in the presence of GABA (1 muM) enhanced the maximum number (B(max)) of [(3)H]flunitrazepam binding sites without affecting their affinity (K(d)). The flumazenil-induced enhancement in B(max) was not counteracted by diazepam (1 muM). GABA (1 nM-1 mM) enhanced [(3)H]flunitrazepam binding to membranes obtained from control and flumazenil-pretreated cells in a concentration-dependent manner. No significant differences were observed in either the potency (EC(50)) or efficacy (E(max)) of GABA to potentiate [(3)H]flunitrazepam binding. However, in flumazenil pretreated cells the basal [(3)H]flunitrazepam and [(3)H]TBOB binding were markedly enhanced. GABA produced almost complete inhibition of [(3)H]TBOB binding to membranes obtained from control and flumazenil treated cells. The potencies of GABA to inhibit this binding, as shown by a lack of significant changes in the IC(50) values, were not different between vehicle and drug treated cells. The results suggest that chronic exposure of HEK 293 cells stably expressing recombinant alpha(1)beta(2)gamma(2S) GABA(A) receptors to flumazenil (in the presence of GABA) up-regulates benzodiazepine and convulsant binding sites, but it does not affect the allosteric interactions between these sites and the GABA binding site. Further studies are needed to elucidate these phenomena.