engleski

The differential effects of chronically administered GABA-A receptor ligands on recombinant GABA-A receptor expression

The main inhibitory neurotransmitter in the mammalian brain, gamma-aminobutyric acid (GABA), fulfils most of its physiological actions via GABA-A receptors. GABA-A receptors possess binding sites for a variety of different drugs, including clinically relevant benzodiazepines, barbiturates, general anesthetics and neurosteroides. Occupancy of these receptors by different drugs leads to regulatory changes often affecting receptor expression and function. Therefore, the aim of this study was to better understand the mechanisms that underlie adaptive changes in GABA-A receptors following their prolonged exposure to different GABA-A receptor ligands. Human embryonic kidney (HEK) 293 cells stably expressing alpha1beta2gamma2S recombinant GABA-A receptors were chronically exposed to GABA, muscimol (an agonist of GABA binding sites), diazepam (an agonist of benzodiazepine binding sites), gabazine and bicuculline (competitive antagonists of GABA binding sites), picrotoxin (a non competitive GABA-A receptor antagonist), as well as to flumazenil (an antagonist of benzodiazepine binding sites). Aliquots of cell membrane preparations obtained from control and drug treated cells were used in saturation binding studies to compare the effects of different drugs on the number and affinity of [3H]flunitrazepam. To measure the viability and growth of HEK 293 cells in response to long-term diazepam or flumazenil exposure, WST-1 cell proliferation assay was used. Furthermore, as the pharmacology of GABA-A receptors at the benzodiazepine binding sites is primary determined by the isoforms of alpha subunit, the steady-state level of mRNA encoding alpha1 receptor subunit was determined by semiquantitative RT-PCR analysis in diazepam- and flumazenil-treated cells. Prolonged exposure of HEK 293 cells expressing recombinant alpha1beta2gamma2S GABA-A receptors to GABA and its agonist muscimol, but not to gabazine, bicucculline and picrotoxin, the GABA antagonists, enhanced the maximum number of [3H]flunitrazepam binding sites. However, chronic treatment with benzodiazepine antagonist flumazenil also up-regulated the number of benzodiazepine binding sites. On the other hand, although a lower concentration of diazepam (1 microM) failed to modify the parameters of [3H]flunitrazepam binding, the combination of diazepam with GABA had an additive effect on the maximum number of benzodiazepine binding sites. Nevertheless, long-term exposure of cells to a high concentration of diazepam (50 microM) produced by itself an increase in the number of benzodiazepine biding sites. WST-1 proliferation assay demonstrated that increased GABA-A receptor expression following high dose diazepam, as well as flumazenil treatment, was not a consequence of stimulated growth of cells exposed to these drugs. Furthermore, semi-quantitative RT-PCR analysis showing elevated levels of alpha1 subunit mRNA suggested that prolonged high dose diazepam as well as flumazenil administration, both induced de novo receptor synthesis. Regardless of the mechanism involved, this study suggests that chronic treatment with the benzodiazepine antagonist, and not only with agonists of benzodiazepine binding sites, is able to induce adaptive changes in GABA-A receptors in an in vitro system.

recombinant GABA-A receptor expression; GABAergic drugs; long-term treatment; HEK 293 cells

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