Identification, characterization, and developmental regulation of embryonic benzodiazepine binding sites

Journal Article · Mon Jun 01 00:00:00 EDT 1987 · J. Neurosci.; (United States)

OSTI ID:6244037

Borden, L A; Gibbs, T T; Farb, D H

We report the identification and characterization of 2 classes of benzodiazepine binding sites in the embryonic chick CNS. Binding was examined by competition and saturation binding experiments, using as radioligands /sup 3/H-flunitrazepam, a classical benzodiazepine anxiolytic, and /sup 3/H-Ro5-4864, a convulsant benzodiazepine. The results demonstrate that high-affinity (KD = 2.3 nM) /sup 3/H-flunitrazepam binding sites (site-A) are present by embryonic day 5 (Hamburger and Hamilton stage 27) and increase throughout development (Bmax = 0.3 and 1.3 pmol/mg protein in 7 and 20 d brain membranes, respectively). When 7 or 20 d brain membranes are photoaffinity-labeled with /sup 3/H-flunitrazepam and ultraviolet light, the radioactivity migrates as 2 bands on SDS-PAGE, consistent with Mrs of 48,000 and 51,000. GABA potentiates /sup 3/H-flunitrazepam binding at both 7 and 20 d of development, indicating that site-A is coupled to receptors for GABA early in development. Importantly, we have also identified a novel site (site-B) that binds classical benzodiazepine agonists with low affinity (micromolar) but displays high affinity for Ro5-4864 (KD = 41 nM). Site-B displays characteristics expected for a functional receptor, including stereospecificity and sensitivity to inactivation by heat and protease treatment. Saturation binding studies employing /sup 3/H-Ro5-4864 indicate that the levels of site-B are similar in 7 and 20 d brain (ca. 2.5 pmol/mg protein). The function of site-B is not known, but its preponderance in 7 d brain, relative to site-A, suggests that it might be important during early embryonic development.

• Research Organization: State Univ. of New York, Brooklyn
• OSTI ID: 6244037
• Journal Information: J. Neurosci.; (United States), Vol. 7:6
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
RECEPTORS
CHEMICAL COMPOSITION
TRANQUILIZERS
BIOCHEMICAL REACTION KINETICS
AFFINITY
BRAIN
CELL MEMBRANES
CHICKENS
ELECTROPHORESIS
EMBRYOS
LIGANDS
ONTOGENESIS
TRACER TECHNIQUES
TRITIUM COMPOUNDS
ANIMALS
BIRDS
BODY
CELL CONSTITUENTS
CENTRAL NERVOUS SYSTEM
CENTRAL NERVOUS SYSTEM AGENTS
DRUGS
FOWL
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
MEMBRANE PROTEINS
MEMBRANES
NERVOUS SYSTEM
ORGANIC COMPOUNDS
ORGANS
PROTEINS
PSYCHOTROPIC DRUGS
REACTION KINETICS
VERTEBRATES
550201* - Biochemistry- Tracer Techniques