Change in Bmax and Kd for [3H]flunitrazepam observed in the course washing rat brain tissue with distilled water

A method for the removal of an endogenous inhibitor(s) of benzodiazepine receptor binding (extraction in distilled water at 20 degrees) was used to evaluate the possible influence of this inhibitor(s) on the affinity of the benzodiazepine receptor in vivo. The results indicate that the presence of an inhibitor(s) in the concentration which exists in the rat brain leads to a 30-fold increase in the Kd value as compared with the results obtained in vitro. The endogenous inhibitor(s) appeared to be a thermostable, proteinase K-resistant substance(s) with Mr between 500 and 10,000 daltons. Repeated washings of the brain tissue with distilled water were accompanied by a decrease in the Bmax for [3H]flunitrazepam. The [3H]flunitrazepam binding sites with Kd = 1.7 nM and Bmax = 61 fmoles/mg of protein were found in the supernatant collected after the second and third washings in distilled water. The presence of diazepam and the benzodiazepine antagonist Ro 15-1788 prevented in a dose-dependent manner the decrease in the Bmax value for [3H]flunitrazepam during the tissue washing with distilled water.