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Modification of γ‐Aminobutyric Acid A Receptor Binding and Function by N ‐Ethoxycarbonyl‐2‐Ethoxy‐1,2‐Dihydroquinoline In Vitro and In Vivo: Effects of Aging
Author(s) -
Miller Lawrence G.,
Lumpkin Monica,
Galpern Wendy R.,
Greenblatt David J.,
Shader Richard I.
Publication year - 1991
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1991.tb11417.x
Subject(s) - in vivo , in vitro , alkoxy group , chemistry , function (biology) , aminobutyric acid , receptor , pharmacology , biochemistry , biology , microbiology and biotechnology , organic chemistry , genetics , alkyl
The irreversible protein‐modifying reagent N ‐ethoxycarbonyl‐2‐ethoxy‐1,2‐dihydroquinoline (EEDQ) was used to investigate binding site characteristics on the γ‐aminobutyric acid A (GABA A ) receptor complex. In vitro, preincubation with EEDQ led to a concentration‐dependent decrease in receptor number for benzodiazepine, t ‐butylbicyclophosphorothionate (TBPS), and GABA binding sites in cerebral cortex. The effect was maximal at the highest concentration of EEDQ used (10 −4 M ) and was greatest for the benzodiazepine site. Pretreatment of membranes with the benzodiazepine antagonist Ro 15‐1788, 1 or 10 μ M , or the agonist lorazepam, 10 μ M , largely prevented the effects of EEDQ. Scatchard analysis indicated no effect of EEDQ, 10 −4 M , on apparent affinity, but a decrease in receptor density for each site. Administration of EEDQ to mice, 12.5 mg/kg i.p., led to a substantial (55‐65%) decrease in number of benzodiazepine binding sites in cortex after 4 h. Slightly smaller changes were observed for TBPS and GABA binding. No changes were observed in apparent affinity at any site. Prior administration of Ro 15‐1788, 5 mg/kg, prevented the effect of EEDQ on benzodiazepine binding. Density of benzodiazepine binding sites gradually recovered over time, and receptor density returned to control values by 96 h after EEDQ injection. Number of binding sites in cortex for TBPS and GABA also increased over time after EEDQ. Benzodiazepine sites in cerebellum were decreased proportionally to cortex after EEDQ, and increased over a similar time course. Function of the GABA A receptor in chloride uptake in cortex was markedly reduced (65%) by EEDQ. Administration of EEDQ to senescent mice (20 months) led to a similar decrease in benzodiazepine sites compared to young mice, but receptor density returned more slowly to control levels (calculated receptor t 1/2 : young, 25.3 h; senescent, 75.1 h). EEDQ modifies all three major sites on the GABA A receptor, with the greatest effect at the benzodiazepine site. This compound may be useful in assessing benzodiazepine receptor half‐life in vivo.