Chronic exposure of cells expressing recombinant GABAA receptors to benzodiazepine antagonist flumazenil enhances the maximum number of benzodiazepine binding sites.
The aim of this study was to better understand the mechanisms that underlie adaptive changes in GABAA receptors following their prolonged exposure to drugs. Exposure (48 h) of human embryonic kidney (HEK) 293 cells stably expressing recombinant alpha1beta2gamma2S GABAA receptors to flumazenil (1 or 5 microM) in the presence of GABA (1 microM) enhanced the maximum number (Bmax) of [3H]flunitrazepam binding sites without affecting their affinity (Kd). The flumazenil-induced enhancement in Bmax was not counteracted by diazepam (1 microM). GABA (1 nM-1 mM) enhanced [3H]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 (EC50) or efficacy (Emax) of GABA to potentiate [3H]flunitrazepam binding. However, in flumazenil pretreated cells the basal [3H]flunitrazepam and [3H]TBOB binding were markedly enhanced. GABA produced almost complete inhibition of [3H]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 IC50 values, were not different between vehicle and drug treated cells. The results suggest that chronic exposure of HEK 293 cells stably expressing recombinant alpha1beta2gamma2S GABAA 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.
Pericić
Danka
D
Ruder Bosković Institute, Laboratory for Molecular Neuropharmacology, Division of Molecular Medicine, POB 180, 10002 Zagreb, Croatia. pericic@irb.hr
Lazić
Josipa
J
Jembrek
Maja Jazvinsćak
MJ
Strac
Dubravka Svob
DS
Rajcan
Ivana
I
D001665:Binding Sites / Q000187:drug effects; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D003975:Diazepam / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004338:Drug Combinations; D005442:Flumazenil / Q000494:pharmacology*; D005445:Flunitrazepam / Q000378:metabolism; D018757:GABA Modulators / Q000494:pharmacology*; D006801:Humans; D007668:Kidney / Q000187:drug effects* / Q000378:metabolism; D008024:Ligands; D011963:Receptors, GABA-A / Q000378:metabolism*; D011994:Recombinant Proteins; D014316:Tritium; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
D019086:Bridged Bicyclo Compounds, Heterocyclic; D004338:Drug Combinations; D018757:GABA Modulators; D008024:Ligands; D011963:Receptors, GABA-A; D011994:Recombinant Proteins; D014316:Tritium; D005442:Flumazenil; D005680:gamma-Aminobutyric Acid; D005445:Flunitrazepam; C046308:tert-butylbicyclo-2-benzoate; D003975:Diazepam