Abstract
The radiochemical synthesis and pharmacological properties are described of [3H]RY 80 (ethyl-8-acetylene-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a][1,4]benzodiazepine-3-carboxylate, [ethyl-3H]). This compound is one of a series of 8-substituted imidazobenzodiazepines that exhibits both high affinity and selectivity for γ-aminobutyric acid (GABA)Areceptors containing alpha-5 subunits. Saturable, high-affinity (Kd ∼0.7 nM) binding of [3H]RY 80 was observed in hippocampal membranes. The maximum number (Bmax) of [3H]RY 80 binding sites was ∼18% of that obtained with [3H]flunitrazepam, a radioligand that labels all “diazepam-sensitive” GABAA receptors. This value is consistent with previous estimates (10–20%) of the proportion of rat hippocampal GABAA receptors containingalpha-5 subunits determined by immunoprecipitation with selective antibodies and competition experiments using analpha-5-selective ligand. In recombinant GABAA receptors composed of alpha-5beta-3 gamma-2 subunits, theKd of [3H]RY 80 (∼0.5 nM) was consistent with the value obtained in hippocampus, whereas theBmax value was not significantly different from that obtained with [3H]flunitrazepam. The potencies of several benzodiazepine site ligands to inhibit [3H]RY 80 binding to hippocampal membranes were in agreement with the values obtained in recombinant (alpha-5 beta-3gamma-2) GABAA receptors. [3H]RY 80 was used both in a “GABA shift” assay to correctly predict the in vivo actions of a novel,alpha-5-selective ligand and to characterize a population of GABAA receptors containingalpha-5 subunits in neonatal rat cortex. These findings demonstrate that [3H]RY 80 can be used as a radioligand to examine the properties of GABAA receptors containingalpha-5 subunits.
Footnotes
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Send reprint requests to: Dr. P. Skolnick, Chief, Laboratory of Neuroscience, NIDDK/NIH, Building 8/111, Bethesda, MD 20892-0008. E-mail: dpopa{at}helix.nih.gov
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↵1 Portions of this work were presented at the 35th Annual Meeting of the American College of Neuropsychpharmacology, December 9–13, 1996, San Juan, PR.
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↵2 This work was supported in part by a predoctoral fellowship from the American Society for Pharmacology and Experimental Therapeutics (C.M.C.) and NIMH Grant MH-46851 (J.M.C.). R.J.S. is a PRAT Fellow, NIGMS.
- Abbreviations:
- GABA
- γ-aminobutyric acid
- DMCM
- methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate
- HEK
- human embryonic kidney
- Received March 19, 1997.
- Accepted July 29, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
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