Purification and Characterization of Naturally Occurring Benzodiazepine Receptor Ligands in Rat and Human Brain

Chemicals that are active at the benzodiazepine receptor (endozepines) are naturally present in the CNS. These substances are present in tissue from humans and animals and in plants and fungi. Using selective extraction protocols, HPLC purification, receptor binding displacement studies, and selective anti‐benzodiazepine antibodies, we have identified six or seven peaks of endozepines in rat and human brain. All material could competitively displace [ 3 H]flunitrazepam binding to cerebellar benzodiazepine binding sites. Two peaks also competitively displaced Ro 5‐4864 binding to the mitochondrial benzodiazepine binding site. Total amounts of brain endozepines were estimated to be present in potentially physiological concentrations, based on their ability to displace [ 3 H]flunitrazepam binding. Although endozepine peaks 1 and 2 had HPLC retention profiles similar to those of nordiazepam and diazepam, respectively, gas chromatography‐mass spectrometry as well as high‐performance TLC revealed biologically insignificant amounts of diazepam (< 0.02 pg/g) and nordiazepam (<0.02 pg/g) in the purified material. Electrophysiologically, some purified endozepines positively modulated γ‐aminobutyric acid (GABA) action on Cl − conductance, monitored in patch‐clamped cultured cortical neurons or in mammalian cells transfected with cDNA encoding various GABA A receptor subunits. These studies demonstrate that mammalian brains contain endozepines that could serve as potent endogenous positive allosteric modulators of GABA A receptors.