CHARACTERIZATION OF [H-3] IMIDAZENIL BINDING TO RAT-BRAIN MEMBRANES

1 The binding of [H-3]-imidazenil, an imidazobenzodiazepine carboxamid e, to rat cerebellar membranes was characterized at different temperat ures. 2 Specific binding was linear with tissue concentrations and rea ched maximum after 90, 30 and 5 min incubation at 0, 21 and 37 degrees C, respectively. The binding was of high affinity; specific and satur able; non linear regression and Scatchard analysis of the data was com patible with the presence of a single population of receptor sites wit h B-max of 0.74+/-0.020, 0.90+/-0.011 and 1.0+/-0.036 pmol mg(-1) prot ein at 0, 21 and 27 degrees C, respectively. Binding affmity decreased with increasing temperature: K-d were 0.29+/-0.051 nM (0 degrees C), 1.0+/-0.080 nM (21 degrees C) and 2.4+/-0.38 nM (37 degrees C). 3 At a ll tested temperatures, [H-3]-imidazenil binding was reversible and th e K-d calculated from the dissociation and association rate constants approximated the equilibrium K-d. 4 In the presence of gamma-aminobuty ric acid (GABA), K-d increased 4 fold at 0 degrees C, whereas B-max in creased, albeit slightly, at all temperatures. 5 Benzodiazepines (BZDs ), imidazopyridines and methyl-beta-carboline-3-carboxylate (beta CCM) were effective inhibitors of [H-3]-imidazenil binding. Conversely, GA BA(A) antagonists, barbiturates, picrotoxin and peripheral BZD recepto r ligands were devoid of any activity. 6 Comparing [H-3]-imidazenil to [H-3]-flumazenil binding in various brain greas, similar densities of recognition sites as well as like regional differences in the distrib ution of binding sites for both radioligands were observed (cortex = s triatum>cerebelium>spinal cord). 7 The present results indicate that [ H-3]-imidazenil specifically binds to the BZD sites of GABA(A) recepto rs. Furthermore, the effects of GABA and temperature differentiate imi dazenil from classical BZDs. It is suggested that the characteristics of imidazenii binding may be relevant to thein vive pharmacology of th e drug.