Enhancement of benzodiazepine binding sites following chronic treatment with flumazenil

The aim of this study was to improve our knowledge of the mechanisms leading to adaptive changes in γ-aminobutyric acid A (GABA A) receptors following chronic drug treatment. Exposure (48 h) of human embryonic kidney (HEK 293) cells stably expressing recombinant α 1β 2γ 2S GABA A receptors to the antagonist of benzodiazepine binding sites, flumazenil (5 μM), enhanced the maximum number ( B max) and the equilibrium dissociation constant ( K d) of [ 3H]flunitrazepam binding sites. The flumazenil-induced enhancement in B max was potentiated by GABA (50 μM) and reduced by the GABA A receptor antagonist, bicuculline (100 μM). Flumazenil-induced enhancement in K d was affected by neither of these treatments. GABA (50 μM) enhanced the density of [ 3H]flunitrazepam binding sites, and this enhancement was greater in the presence of diazepam (1 μM). The results suggest that chronic flumazenil treatment up-regulates in a bicuculline-sensitive manner benzodiazepine binding sites at stably expressed GABA A receptors.