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

Ashok K. Mehta, Richard P. Shank

Research output: Contribution to journalArticlepeer-review

25 Scopus citations

Abstract

The binding of [3H]Ro 15-4513, [3H]flunitrazepam and [3H[Fumazenil to rat CNS membranes was studied at 2°C, 22°C and 37°C using ligand concentrations ranging from ≈ 0.06 nM to 10 μM. Analysis of the binding saturation data suggested the existence of high-affinity sites (Kd < 10nM) and low-affinity sites (Kd > 100nM) 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 ≈ 0.1nM) 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 ≈ 70% of the specific binding when [3H]Ro 15-4513 was ≈ 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 ≈ 3-fold as temperature was increased from 2°C to 37°C (Kd ≈ 0.1nM and ≈ 0.3, respectively), which was similar to the effect of temperature on other high-affinity sites (Kd ≈ 2.6nM at 2°C and ≈ 8 nM at 37°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 ≈ 5 μM). GABA at 100 μM had little effect on theKd value for the very high-affinity site (GABA shift: ≈ 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.

Original languageEnglish (US)
Pages (from-to)289-297
Number of pages9
JournalBrain Research
Volume704
Issue number2
DOIs
StatePublished - Dec 18 1995
Externally publishedYes

Keywords

  • Benzodiazepine
  • Cerebellum
  • GABA receptor
  • Hippocampus
  • Medulla
  • Ro 15-4513
  • Spinal cord

ASJC Scopus subject areas

  • Neuroscience(all)
  • Molecular Biology
  • Clinical Neurology
  • Developmental Biology

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