Labelling of diazepam-sensitive and -insensitive benzodiazepine receptors with [ 3H]tert-butyl-8-chloro-5,6-dihydro-5-methyl-6-oxo-4H-imidazol[1,5-a][1,4]benzodiazepine 3-carboxylate (ZG-63)

Abstract

A diazepam-insensitive subtype of benzodiazepine receptor has been identified in the cerebella of several species, including man. t-Butyl-8-chloro-5,6-dihydro-5-methyl-6-oxo-4 H-imidazo[1,5- a][1,4]benzodiazepine 3-carboxylate (ZG-63) was recently described as a selective, high affinity ligand at diazepam-insensitive benzodiazepine receptors. This compound was tritiated, and its properties as a radioligand evaluated in rat brain membranes. Consistent with the high affinity and selectivity described for the non-radioactive form of this compound, saturation analyses of [ 3H]ZG-63 binding to cerebellar diazepam-insensitive and other, diazepam-sensitive benzodiazepine receptors revealed K d values of 2.6 ± 0.2 nM and 10.6 ± 1.4 nM, respectively. The density ( B max) of cerebellar diazepam-insensitive receptors labelled with [ 3H]ZG-63 was not significantly different from values obtained with the prototypical diazepam-insensitive receptor ligand [ 3H]Ro 15-4513, representing ∼30% of total cerebellar benzodiazepine receptors. [ 3H]ZG-63 also labelled cortical diazepam-sensitive benzodiazepine receptors, with B max values that were not significantly different from those obtained with [ 3H]flunitrazepam. Diazepam-insensitive benzodiazepine receptors in rat cerebral cortex could be detected with [ 3H]ZG-63, but the densities of these sites are a very minor component ( $ ̌ = 5% ) of total benzodiazepine receptors. In the presence of GABA, [ 3H]ZG-63 behaved as a ‘γ-aminobutyric acid (GABA) -positive’, ‘GABA-negative’, and ‘GABA-neutral’ ligand at cortical diazepam-sensitive receptors, cerebellar diazepam-sensitive receptors, and cerebellar diazepam-insensitive benzodiazepine receptors, respectively. This profile differs from the prototype diazepam-insensitive receptor ligand, [ 3H]Ro 15-4513. Competition studies demonstrated a very high correlation ( r 2 = 0.98; P < 0.002) between the potencies of a series of benzodiazepine receptor ligands to inhibit [ 3H]ZG-63 and [ 3H]Ro 15-4513 binding to cerebellar diazepam-insensitive receptors. The high affinity and selectivity of [ 3H]ZG-63 for diazepam-insensitive receptors (diazepam-insensitive/diazepam-sensitive ratio of ∼ 0.25) together with a GABA-shift profile which differs from Ro 15-4513 suggests that this compound may be useful in elucidating the function(s) of this benzodiazepine receptor subtype.