Naslov
Long-term diazepam treatment of HEK 293 cells stably expressing recombinant GABA-A receptors induces up-regulation of GABA-A receptor binding sites in parallel with their functional uncoupling.

Autori
Švob Štrac, Dubravka ; Jazvinšćak Jembrek, Maja ; Vlainić (Lazić), Josipa ; Peričić, Danka

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
FENS Forum Abstracts, Vol. 4 / - Ženeva, 2008, 011.23-011.23

Skup
6th Forum of European Neuroscience

Mjesto i datum
Ženeva, Švicarska, 12.07.2008. - 16.07.2008

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
diazepam; prolonged exposure; GABA-A receptors recombinant; radioligand binding. mRNA; Western blot

Sažetak
The aim of this study was to further explore the mechanisms leading to adaptive changes in GABA-A receptors following their prolonged exposure to benzodiazepines, drugs known to produce tolerance and dependence and also to be abused. Therefore, we studied the effects of chronic diazepam (an agonist of benzodiazepine binding sites) treatment on the recombinant alpha1 beta2 gamma2S GABA-A receptors, the most common type of GABA-A receptors found in the brain, stably expressed in human embryonic kidney (HEK) 293 cells. Aliquots of the cell membrane preparation or of the intact cell suspension, were used in binding studies with [3H]flunitrazepam, [3H]muscimol and t-[3H]butylbicycloorthobenzoate ([3H]TBOB), to determine the possible changes in binding parameters (Kd and Bmax) and in functional interactions between GABA-A receptor binding sites. Our results demonstrated that chronic (72 h) exposure of cells to diazepam (50 microM) enhanced in a gabazine (a competitive antagonist of GABA binding sites) sensitive manner the maximum number of binding sites for benzodiazepines, GABA and convulsants, suggesting the up-regulation of GABA-A receptors. The enhancement of cell surface [3H]muscimol binding sites suggested the increase of functionally relevant receptors. The results of semi-quantitative RT-PCR and Western blot analysis, showing elevated levels of alpha1 subunit mRNA and beta2 subunit proteins, respectively, indicated that prolonged diazepam treatment induced de novo receptor synthesis. The results of WST-1 proliferation assay demonstrated that the observed up-regulated receptor expression is not a consequence of stimulated growth of cells exposed to chronic diazepam. While the number of GABA-A receptors returned to control value 24 h following diazepam withdrawal, the effect on allosteric uncoupling between GABA and benzodiazepine binding sites persisted after the termination of drug treatment.

Izvorni jezik
Engleski

Znanstvena područja
Temeljne medicinske znanosti

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