engleski

Long-term exposure to flumazenil enhances the maximum number of benzodiazepine binding sites on the recombinant GABA-A receptors stably expressed in HEK 293 cells.

The aim of our study was to explain the possible mechanisms that are involved in adaptive changes of GABA-A receptors following prolonged treatment with flumazenil, a selective antagonist of benzodiazepine binding sites on GABA-A receptors. The effects of long-term treatment (48 h) was studied on human embryonic kidney (HEK) 293 cells that stably express the alpha1 beta2 gamma2s subtype of GABA-A receptor, the predominant subtype of GABA-A receptors in mammalian brain. Radioligand binding studies with [3H] -flunitrazepam revealed that chronic exposure of HEK 293 cells to flumazenil increased the maximum number (Bmax) of benzodiazepine binding sites (Pericic el al., Eur. J. Pharmacol 507:7-13, 2005). This increase was reduced with cycloheximide (5 microg/ml), an inhibitor of protein synthesis, and with actinomycin D (7.5 microg/ml), an inhibitor of RNA synthesis. To further investigate flumazenil-induced increase in the number of benzodiazepine binding sites, the possible change in steady-state level of mRNA encoding alpha1 receptor subunit was studied by semiquantitative RT-PCR, using beta-actin as the internal standard. In flumazenil-treated cells the level of alpha1 mRNA was enhanced, although not as much as the maximum number of benzodiazepine binding sites. This result, taken together with the results obtained with cycloheximide and actinomycin, suggests that chronic flumazenil-induced up-regulation of benzodiazepine binding sites is presumably due to de novo synthesis of receptor proteins.

recombinant GABA A receptors; HEK 293 cells; chronic treatment; flumazenil; actinomycin; cycloheximide; mRNA

nije evidentirano