Pericić Danka, Lazić Josipa, Strac Dubravka Svob
Ruder Bosković Institute, Laboratory for Molecular Neuropharmacology, Division of Molecular Medicine, P.O.B. 180, 10002 Zagreb, Croatia.
Biomed Pharmacother. 2005 Aug;59(7):408-14. doi: 10.1016/j.biopha.2005.02.003.
GABA(A) receptors mediate most of the fast inhibitory neurotransmission in the brain. Prolonged occupancy of these receptors by ligands leads to regulatory changes often resulting in reduction of receptor function. The mechanism of these changes is still unknown. In this study, stably transfected human embryonic kidney (HEK) 293 cells were used as a model to study the effects of prolonged flumazenil (antagonist of benzodiazepine binding sites at GABA(A) receptors) exposure on the recombinant alpha(1)beta(2)gamma(2S) GABA(A) receptors, the most common type of GABA(A) receptors found in the brain. Exposure (48 h) of HEK 293 cells stably expressing recombinant alpha(1)beta(2)gamma(2S) GABA(A) receptors to flumazenil (1 or 5 microM) in the presence of GABA (1 microM), enhanced the maximum number (B(max)) without affecting the affinity (K(d)) of [(3)H]TBOB labeled binding sites for convulsants. Diazepam (1 nM-1 mM) in the presence of GABA (1 microM) modulated [(3)H]TBOB binding to control and flumazenil pretreated cells according to a two-site model. No significant differences between the groups were observed in either the potency or efficacy of diazepam to modulate [(3)H]TBOB binding, as evidenced by a lack of significant changes between their IC(50) and I(max) values. The results suggest that chronic exposure of HEK 293 cells stably expressing recombinant alpha(1)beta(2)gamma(2S) GABA(A) receptors to flumazenil up-regulates the binding sites for convulsants, but it does not appear to affect the functional coupling between these sites and benzodiazepine binding sites. Along with our recent data, these results suggest that chronic treatment with flumazenil enhances the number of GABA(A) receptors.
GABA(A)受体介导大脑中大部分快速抑制性神经传递。配体对这些受体的长期占据会导致调节性变化，常导致受体功能降低。这些变化的机制尚不清楚。在本研究中，稳定转染的人胚肾（HEK）293细胞被用作模型，以研究长期暴露于氟马西尼（GABA(A)受体上苯二氮䓬结合位点的拮抗剂）对重组α(1)β(2)γ(2S) GABA(A)受体的影响，这是大脑中最常见的GABA(A)受体类型。在存在GABA（1μM）的情况下，将稳定表达重组α(1)β(2)γ(2S) GABA(A)受体的HEK 293细胞暴露于氟马西尼（1或5μM）48小时，可增加最大结合量（B(max)），而不影响[(3)H]TBOB标记的惊厥剂结合位点的亲和力（K(d)）。在存在GABA（1μM）的情况下，地西泮（1 nM至-1 mM）根据双位点模型调节[(3)H]TBOB与对照细胞和氟马西尼预处理细胞的结合。地西泮调节[(3)H]TBOB结合的效力或效能在各组之间未观察到显著差异，这由它们的IC(50)和I(max)值之间缺乏显著变化所证明。结果表明，将稳定表达重组α(1)β(2)γ(2S) GABA(A)受体的HEK 293细胞长期暴露于氟马西尼会上调惊厥剂的结合位点，但似乎并不影响这些位点与苯二氮䓬结合位点之间的功能偶联。连同我们最近的数据，这些结果表明，氟马西尼的长期治疗可增加GABA(A)受体的数量。
