engleski

Up-regulation of stably expressed recombinant GABA A receptors by chronic flumazenil treatment

g-Aminobutyirc acid (GABA), the major inhibitory neurotransmitter in the brain, fulfils most of its physiological actions via GABAA receptors. GABAA receptors are ligand-gated ion-channels composed of various polypeptide subunits. The functional and pharmacological properties of these receptors depend on their subunit composition. GABAA receptors possess binding sites for a variety of drugs, including clinically relevant benzodiazepines, barbiturates, general anesthetics and neurosteroides, which are known to elicit at least some of their pharmacological effects via GABAA receptors. Chronic occupation of benzodiazepine binding sites by agonists leads to regulatory changes generally resulting in down-regulation of receptor levels and function. Changes produced following prolonged treatment with antagonists are less clear. Therefore, in this study, stably transfected HEK 293 cells were used as a model to study the effects of prolonged flumazenil (antagonist of benzodiazepine binding sites) exposure on the recombinant a1b2g2s GABAA receptors. Materials and Methods: Stably transfected human embryonic kidney (HEK) 293 cells expressing the a1b2g2s subtype of GABAA receptors were exposed for 48 h to flumazenil (1 and 5 mM), or to flumazenil (5 mM) in combination with GABA (50 mM), bicuculline (100 mM), diazepam (1 mM) or b-carboline-3-carboxylic acid methyl ester (b-CCM, 1 mM). Aliquots of membrane preparations (~ 100 mg protein) obtained from control and drug treated cells were used in saturation binding studies with [3H]flunitrazepam (0.2 - 16 nM) under conditions (4°C, 90 min) previously described (Peričić et al. Eur. J. Pharmacol., 482: 117-125, 2003). Results: Exposure of HEK 293 cells stably expressing recombinant a1b2g2s GABAA receptors to flumazenil (5 mM), even in the absence of GABA, enhanced the maximum number (Bmax) and the equilibrium dissociation constant (Kd) of [3H]flunitrazepam binding sites. Flumazenil-induced increase in the Bmax value was further enhanced by GABA, diminished by bicuculline (the competitive antagonist of GABA binding sites), and not affected by diazepam, the agonist, and b-CCM, an inverse agonist of benzodiazepine binding sites. While GABA by itself also up-regulated, diazepam in the absence of GABA failed to affect the number of benzodiazepine binding sites. Conclusions: The results suggest that chronic exposure of HEK 293 cells expressing recombinant a1b2g2s GABAA receptors, to benzodiazepine antagonist flumazenil up-regulates, in a diazepam and b-CCM insensitive manner, benzodiazepine binding sites and decreases their affinity. They further suggest the involvement of GABA binding site in this effect of chronic flumazenil treatment. Along with our data showing an enhancement of binding sites for GABA and convulsants, these results suggest that prolonged exposure to flumazenil up-regulates the number of GABAA receptors.

recombinant GABA A receptors; HEK 293 cells; benzodiazepine receptor ligands; chronic treatment

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