Characterization of a benzodiazepine receptor site with exceptionally high affinity for Ro 15-4513 in the rat CNS.

The binding of [3H]Ro 15-4513, [3H]flunitrazepam and [3H]flumazenil to rat CNS membranes was studied at 2 degrees C, 22 degrees C and 37 degrees C using ligand concentrations ranging from approximately 0.06 nM to 10 microM. Analysis of the binding saturation data suggested the existence of high-affinity sites (Kd < 10 nM) and low-affinity sites (Kd > 100 nM) for each ligand. When binding was performed using very low ligand concentrations a benzodiazepine site with an exceptionally high affinity for Ro 15-4513 (Kd approximately 0.1 nM) was evident in all major regions of the CNS except the cerebellum. This site was most prevalent in the hippocampus, medulla and spinal cord where it accounted for approximately 70% of the specific binding when [3H]Ro 15-4513 was approximately 0.06 nM. The selectivity of Ro 15-4513 for this site as compared to other high-affinity sites was 20- to 60-fold depending on the incubation temperature and CNS region. The affinity for the very high-affinity site was decreased approximately 3-fold as temperature was increased from 2 degrees C to 37 degrees C (Kd approximately 0.1 nM and approximately 0.3, respectively), which was similar to the effect of temperature on other high-affinity sites (Kd approximately 2.6 nM at 2 degrees C and approximately 8 nM at 37 degrees C). Flumazenil, flunitrazepam, and diazepam did not differentiate the very high-affinity [3H]Ro 15-4513 site from other BZ sites, but alpidem exhibited a low affinity for it (IC50 approximately 5 microM). GABA at 100 microM had little effect on the Kd value for the very high-affinity site (GABA shift: approximately 0.8 to 1.0), suggesting that Ro 15-4513 is a partial inverse agonist or an antagonist at this site. These findings provide further evidence for the pharmacologic diversity of BZ sites on different subtypes of GABAA receptors.