Sequestration of gamma-aminobutyric acidA receptors on clathrin-coated vesicles during chronic benzodiazepine administration in vivo
Tehrani, M.H.; Barnes, E.M.
Journal of Pharmacology and Experimental Therapeutics 283(1): 384-390
1997
ISSN/ISBN: 0022-3565 PMID: 9336347 Document Number: 471339
Chronic administration of benzodiazepine agonists produces behavioral tolerance. For induction of tolerance, the use-dependent down-regulation of gamma-aminobutyric acid-A (GABA-A)/ benzodiazepine receptors is a potential cellular mechanism. We previously identified GABA-A receptors on clathrin-coated vesicles from rat brain, suggesting that surface receptors can be internalized via endocytosis. To examine a role for coated vesicles in GABA-A receptor down-regulation in vivo, fractions were obtained from mouse brain microsomes through density centrifugation and treatment with 0.1% Triton X-100. This coated vesicle preparation was enriched in clathrin subunits and clathrin light-chain kinase and had twice the level of (3H)flunitrazepam binding as did vesicles not exposed to Triton. Adult mice were treated with lorazepam (2 mg/kg/day) for 7 days via osmotic minipump, achieving a serum level of 103 +- 8.9 ng/ml. The level of flunitrazepam bound to coated vesicles was increased by 83 +- 13% in the lorazepam-treated mice compared with vehicle-treated controls. The B-max value for (3H)flunitrazepam binding to synaptic membranes from lorazepam-treated animals was 33 +- 4% lower than that of controls. The amount of GABA-A receptor alpha-1 subunits, as quantified by Western blotting, followed a similar pattern. Relative to controls, immunoreactivity for alpha-1 subunits in coated vesicles from lorazepam-treated mice was increased by 60.0 +- 10.3%, whereas that in synaptic membranes declined by 12 +- 6%. These results indicate that lorazepam-dependent GABA-A receptor sequestration occurs in mouse brain. Furthermore, it is suggested that this sequestration may play a role in GABA-A receptor down-regulation in vivo.