engleski

Prolonged exposure of recombinant GABA-A receptors to diazepam induces differential effects on functional coupling and expression of benzodiazepine binding sites

Variety of drugs, including clinically relevant benzodiazepines, exerts most of their pharmacological effects via GABA-A receptors, the major fast inhibitory neurotransmitter receptors in the mammalian brain. The aim of this study was to further explore the mechanisms that underlie 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 investigated the effects of chronic diazepam treatment on the recombinant alpha1beta2gamma2S 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 preparations were used in binding studies to compare the effects of different diazepam concentrations on the number and affinity of (3H)flunitrazepam binding sites and on their allosteric interactions with GABA recognition sites. The results demonstrated that long-term exposure of cells to a high concentration of diazepam (50 microM) enhanced the maximum number (Bmax) of (3H)flunitrazepam biding sites, without changing their affinity (Kd). This effect appears to be the consequence of chronic diazepam administration, since short-term diazepam treatment (30 min.) has not affected the number of benzodiazepine binding sites. In contrast, a lower concentration of diazepam (1 microM) failed to modify the parameters (Bmax and Kd) of (3H)flunitrazepam binding. However, diazepam applied in both concentrations (1 microM and 50 microM) produced functional uncoupling between GABA and benzodiazepine binding sites. The increased number of benzodiazepine binding sites as well as allosteric uncoupling, observed after long-term treatment with 50 microM diazepam, were reduced by gabazine and bicuculline (competitive antagonists of GABA binding sites), respectively, suggesting a role of GABA recognition sites in these effects. Nevertheless, co-treatment with GABA has not potentiated the enhancement of (3H)flunitrazepam binding sites induced by 50 microM diazepam. On the other hand, the combination of 1 microM diazepam with GABA had an additive effect on the maximum number of benzodiazepine biding sites. The results suggested up-regulation of benzodiazepine binding sites after prolonged treatment with diazepam, applied in a concentration that might be found in drug abusers. On the other hand, the observed decrease in allosteric coupling between benzodiazepine and GABA binding sites has been already found with significantly lower doses of diazepam. In addition to benzodiazepine binding sites, GABA recognition sites seem to be important for the observed effects of diazepam. The present study suggests that at least two separable processes could be occuring during the long-term diazepam treatment. We assume that these results might be useful for understanding the effects of chronic high dose benzodiazepine use on the nervous system.

recombinant GABA-A receptors; diazepam; chronic treatment; expression; functional expression

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