engleski

The effect of short-term and long-term zolpidem treatment on recombinant alpha1beta2gamma2S subtype of GABA-A receptors

Introduction: GABA-A (gamma-aminobutyric acid-A) receptors are ligand-gated ion channels composed of various polypeptide subunits. They possess binding sites for several drugs, including benzodiazepines. The aim of this study was to explore the mechanisms that underlie adaptive changes in GABA-A receptors following their short-term and long-term exposure to hypnotic zolpidem, the non-benzodiazepine agonist of benzodiazepine binding sites. Such studies might be important as the prescription of zolpidem is increasing and prolonged zolpidem treatment, as recently shown, induces tolerance (Vlainić and Peričić, Neuropharmacology, 56:1124-30, 2009). Materials and methods: Human embryonic kidney (HEK 293) cells stably expressing recombinant alpha1beta2gamma2S GABA-A receptors, the most common type of GABA-A receptors found in the brain, were exposed to zolpidem (1 and 10 microM) for 2 h on one, two or three consecutive days or continuously for 48 h. Cell membranes were isolated 24 h after short-term or immediately after long-term treatment and radioligand binding studies were used to determine the parameters of [3H]flunitrazepam ([3H]FNZ) binding sites, as previously described (Peričić et al., Naunyn-Schmiedeberg’ s Arch. Pharmacol. 375: 177-87, 2007). Membrane preparations were incubated with increasing concentrations of non-radioactive flunitrazepam and a fixed concentration (1 nM) of [3H]FNZ) to determine the maximum number (Bmax) and dissociation constant (Kd) of benzodiazepine binding sites. In stimulation studies the ability of GABA to potentiate [3H]FNZ binding was used to assess the allosteric interactions between GABAA receptor binding sites. Results: A single (2 h) or repeated (2 or three days for 2 h daily) exposure of cells to zolpidem did not affect either the Bmax or the Kd value of [3H]FNZ binding sites. Addition of GABA enhanced [3H]FNZ binding to membranes obtained from control and zolpidem treated cells in a concentration-dependent manner, but there was no difference in either the potency (EC50) or efficacy (Emax) of GABA to potentiate [3H]FNZ binding. On the contrary, prolonged occupation (48 h) of benzodiazepine binding sites by zolpidem (10 microM) produced an up-regulation (~150%) of [3H]FNZ binding sites with no change in their affinity, as well as the functional uncoupling between GABA and benzodiazepine binding sites, as evidenced by a decreased ability of GABA to stimulate [3H]FNZ binding. Conclusions: The results suggest that in our model, characterized by the presence of a single well defined receptor subtype, the continuous, but not intermittent short-term exposure of cells to zolpidem (the latter being more similar to the therapy in humans), is able to induce adaptive changes in GABA-A receptors possibly related to development of tolerance and dependence. These changes are not substantially different from those obtained after prolonged exposure of cells to high doses of classical benzodiazepines (Švob Štrac et al., Brain Res. 1246:29-40, 2008).

GABA-A receptors ; zolpidem

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