Comparison of the Kinetics of [³H]Diazepam and [³H]Flunitrazepam Binding to Cortical Synaptosomal Membranes

Abstract

[3H]Diazepam and [3H]flunitrazepam ([3H]FNP) binding to washed and frozen synaptosomal membranes from rat cerebral cortex were compared. In Tris-citrate buffer, gamma-aminobutyric acid (GABA) and NaCl both increased [3H]diazepam binding more than [3H]FNP binding. GABA and pentobarbital both enhanced this effect of NaCl. Because of the extremely rapid dissociation of [3H]diazepam in the absence of NaCl and GABA, the Bmax (maximal binding capacity) was smaller by the filtration assay than by the centrifugation assay. [3H]FNP, which dissociates more slowly, had the same Bmax in both assays. [3H]Diazepam association had two components, and was faster than [3H]FNP association. [3H]Diazepam dissociation, which also had two components, was faster than that of [3H]FNP, and also had a greater fraction of rapidly dissociating species. [3H]FNP dissociation was similar when initiated by diazepam, flunitrazepam, clonazepam, or Ro15-1788, which is a benzodiazepine antagonist. [3H]Diazepam dissociation with Ro15-1788, flunitrazepam, or clonazepam was slower than with diazepam. GABA and NaCl, but not pentobarbital, increased the percentage of slowly dissociating species. This effect of NaCl was potentiated by GABA and pentobarbital. The results support the cyclic model of benzodiazepine receptors existing in two interconvertible conformations, and suggest that, distinct from their binding affinity, some ligands (like flunitrazepam) are better than others (like diazepam) in inducing the conversion of the receptor to the higher-affinity state.

Characterization of Na+‐Independent GABA and Flunitrazepam Binding Sites in Preparations of Synaptic Membranes and Postsynaptic Densities Isolated from Canine Cerebral Cortex and Cerebellum

“…However, it is clear that the PSD preparations isolated both from cerebral cortex and from cerebellum have single-affinity GABA-binding sites, a finding in agreement with that of Horng and Wong (1979) that Triton X-100-treated membranes from rat cerebral cortex and cerebellum have approximately equal GABA-binding capacities. In both cerebral cortex and cerebellar synaptic membranes there is only one apparent binding site with a K , value of 4-5 nM, a value similar to that found earlier with rat cerebral cortex synaptic membranes (Chiu and Rosenberg, 1982), with slightly lower values being reported by Asano et al (1983) using bovine cerebral and cerebellar synaptic membranes. We also confirmed the earlier results (Asano et al, 1983) that cerebral cortex membrane preparations have more binding sites than do cerebellar membrane preparations.…”

Attention Span, Anxiety and Benzodiazepine Receptors

“…1980), there are reports that concentrations of GABA greater than 10"5 M, in the presence or absence of NaCl. increase the Bmax of 13H~diazepam (Chiu and Rosenberg, 1979;Rosenberg, 1982) and[3Hlflunitrazepam binding (Squires, 1981). Thus, the small but significant increase in Bmax values might represent cooperativity of CABA and NaCI in facilitating [ 3 Hjflunitrazepam binding.…”

Το Συστημα Gaba-Βενζοδιαζεπινες: Μελετη Της Μεμβρανικης Και Της Πυρηνικης Δεσμευσης Στον Εγκεφαλο

“…Η φαρμακολογική διάκριση των κεντρικών υποδοχέων σε δυο τύπους εξηγείται με την ύπαρξη δυο διαμορφώσεων του υποδοχέα με διαφορετική σύνδεση με τη θέση δέσμευσης του GABA και το ιονοφόρο του Cl", όπως αυτό δείχνεται από θερμοδυναμικές μελέτες του υποδοχέα και την εμφάνιση συνέργειας σε πειράματα δέσμευσης (Gee and Yamamura 1982, Chiù and Rosenberg 1982, 1983a,b, Martin and Doble 1983, Gee et al, 1984. GABA-τροποποιητίνης (Guidotti et al, 1982;Hamon & Soubrie, 1983).…”