engleski

Regulation of stably expressed GABA A receptors by chronic exposure to benzodiazepine receptor ligands

Aims: The aim of this study was to better understand the mechanisms that underlie adaptive changes in GABAA receptors following their prolonged exposure to drugs. Methods: Cells of the human embryonic kidney (HEK) 293 cell line expressing the a1b2g2S subtype of GABAA receptor were exposed for 48 h to diazepam or flumazenil (agonist and antagonist of benzodiazepine binding sites), or the combination thereof. Aliquots of membrane preparations (~100 mg protein) obtained from control and drug treated cells were used in saturation binding studies with [3H]flunitrazepam in the presence or absence of GABA (100 microM), under conditions (4°C, 90 min) previously described (Pericic et al., Eur. J. Pharmacol., 2003). Results and Conclusions: Exposure of HEK 293 cells stably expressing recombinant a1b2g2S GABAA receptors to flumazenil (5 microM), but not to diazepam (1 microM), enhanced the maximum number and the equilibrium dissociation constant of [3H]flunitrazepam binding sites. After addition of GABA (100 microM) the potentiation of [3H]flunitrazepam binding (GABA shift) was decreased in flumazenil, in diazepam (1 microM), as well as in flumazenil (1 or 5 microM) + diazepam pretreated HEK 293 cells. The exposure to diazepam also could not abolish either the flumazenil induced decrease in the affinity or increase in the maximum number of benzodiazepine binding sites. The results suggest that chronic flumazenil (5 microM) treatment of HEK 293 cells expressing recombinant a1b2g2S GABAA receptors up-regulates benzodiazepine binding sites and decreases their affinity. They further demonstrate that in the absence of GABA the prolonged occupation of benzodiazepine binding sites either by an agonist or the antagonist produces functional uncoupling between GABA and benzodiazepine binding sites, as evidenced by a decreased ability of GABA to potentiate [3H]flunitrazepam binding.

Recombinant GABA A receptors; HEK 293 cells; Benzodiazepine receptor ligands; Chronic treatment

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