CHARACTERIZATION OF A BENZODIAZEPINE RECEPTOR-SITE WITH EXCEPTIONALLYHIGH-AFFINITY FOR RO-15-4513 IN THE RAT CNS

The binding of [H-3]Ro 15-4513, [H-3]flunitrazepam and [H-3]flumazenil to rat CNS membranes was studied at 2 degrees C, 22 degrees C and 37 degrees C using ligand concentrations ranging from approximate to 0.06 nM to 10 mu M. Analysis of the binding saturation data suggested the existence of high-affinity sites (K-d < 10 nM) and low-affinity sites (K-d > 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 (K-d approximate to 0.1 nM) was evident i n all major regions of the CNS except the cerebellum. This site was mo st prevalent in the hippocampus, medulla and spinal cord where it acco unted for approximate to 70% of the specific binding when [H-3]Ro 15-4 513 was approximate to 0.06 nM. The selectivity of Ro 15-4513 for this site as compared to other high-affinity sites was 20- to 60-fold depe nding on the incubation temperature and CNS region. The affinity for t he very high-affinity site was decreased approximate to 3-fold as temp erature was increased from 2 degrees C to 37 degrees C (K-d approximat e to 0.1 nM and approximate to 0.3, respectively), which was similar t o the effect of temperature on other high-affinity sites (K-d approxim ate to 2.6 nM at 2 degrees C and approximate to 8 nM at 37 degrees C). Flumazenil, flunitrazepam, and diazepam did not differentiate the ver y high-affinity [H-3]Ro 15-4513 site from other BZ sites, but alpidem exhibited a low affinity for it (IC50 approximate to 5 mu M). GABA at 100 mu M had little effect on the K-d value for the very high-affinity site (GABA shift: approximate to 0.8 to 1.0), suggesting that Ro 15-4 513 is a partial inverse agonist or an antagonist at this site. These findings provide further evidence for the pharmacologic diversity of B Z sites on different subtypes of GABA(A) receptors.