Increased benzodiazepine receptor number elicited in vitro by a novel purine, EMD 28422

EMD 28422 (N6-[2-(4-chlorophenyl)-bicyclo-2.2.2.octyl-(3)]-adenosine) was demonstrated to increase the number of binding sites for [3H]diazepam (Bmax) in vitro without an accompanying increase in receptor affinity (KD). The increase in receptor number was observed in both crude synaptosomal preparations (P2) and thrice-washed membrane preparations with and without the addition of 50 microM GABA. Furthermore, this effect appeared to be independent of the concentration of chloride ion, since the increases in Bmax were observed in both Tris-HCl and Tris-maleate buffer. The effects of EMD 28422 were stereospecifically antagonized by the GABA antagonist bicuculline, despite the lack of effect of EMD 28422 on [3H]muscimol binding at concentrations which markedly increased benzodiazepine receptor number. Neither EMD 39011 nor adenosine, the two parent moieties of EMD 28422, increased [3H]diazepam binding at concentrations of up to 1 mM. The increases in benzodiazepine receptor number observed with EMD 28422 in vitro suggests that this compound induces a conformational change in the benzodiazepine receptor which may cause the dissociation of an endogenous noncompetitive inhibitor of [3H]diazepam binding from the membrane, thus 'unmasking' binding sites. The stereospecific antagonism of this effect by bicuculline and the apparent inability of GABA to alter the action of EMD 28422 suggests the presence of a novel type or different functional state of GABA receptor which may play a permissive role in the rapid modulation of benzodiazepine receptor number in vitro.