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n‐[3H]Butyl‐β‐Carboline‐3‐Carboxylate, a Putative Endogenous Ligand, Binds Preferentially to Subtype 1 of Central Benzodiazepine Receptors

Abstract: Synthetic n‐butyl β‐carboline‐3‐carboxylate, an endogenous central benzodiazepine receptor inhibitor found in brain, was tritium‐labeled from the butenyl ester. Binding of this [3H]β‐carboline was concentrated particularly in the synaptosomal membrane fraction of the cerebral cortex; this fraction showed a single type of high‐affinity site (KD= 2.7 ± 0.1 nM) with a Bmax of 1.16 ± 0.08 pmol/mg of protein. The number of sites labeled was about half of that obtained with [3H]flunitrazepam binding (Bmax= 2.36 ± 0.06 pmol/mg of protein). On the other hand, in the cerebellum, both ligands bound to practically the same number of sites. When [3H]flunitrazepam binding was done in the presence of 10−11‐10−5M butyl β‐carboline, the differences between the two brain regions were more apparent. In cerebellar membranes the data fitted a straight line in the Eadie‐Hofstee plot; this finding and a Hill number near unity suggest a single type of binding site. In the cortical membranes the data of binding fitted a concave curve, and the Hill number was 0.6. These are characteristics of two types of binding sites with different affinities (KDi= 0.6–1.5 nM and KD2= 12–18 nM). The differentiation of a high‐ and low‐affinity site in the cerebral cortex was corroborated by experiments in which [3H]butyl β‐carboline binding was displaced by the triazolopyridazine CL 218,872. These results demonstrate that in the cerebral cortex there are two subtypes of sites (1 and 2) of central benzodiazepine receptors and that CL 218,872 binds preferentially to subtype 1. γ‐Aminobutyric acid (GABA) stimulated the binding of [3H]flunitrazepam, whereas the effect on the binding of [3H]butyl β‐carboline was negative. This finding, together with some pharmacological experiments in mice, suggests that n‐butyl β‐carboline‐3‐carboxylate acts as an inverse agonist on the central benzodiazepine receptor, inhibiting GABAergic neurotransmission.