Characterization of barbiturate-stimulated chloride efflux from rat brain synaptoneurosomes

Membrane chloride (Cl^-) permeability was studied in a novel subcellular brain preparation, the synaptoneurosome. Using a radioactive tracer exchange technique, Cl^- transport was determined by measuring ³⁶Cl^- efflux from rat cerebral cortical synaptoneurosomes. Barbiturates increased ³⁶Cl^- efflux in a dose-dependent manner with the following relative order of potency: 5-(1,3-dimethylbutyl)-5-ethyl barbituric acid ((-)-DMBB) > pentobarbital > secobarbital > (+)-DMBB > hexobarbital > amobarbital > mephobarbital. Phenobarbital and barbital were virtually inactive. A good correlation was observed between the potencies of these barbiturates in stimulating ³⁶Cl^- efflux and their anesthetic potencies in mice (r = 0.90, p < 0.01) and their abilities to enhance [³H]diazepam binding to brain membranes (r = 0.77, p < 0.05). The effect of pentobarbital in enhancing ³⁶Cl^- efflux was reversed by the γ-aminobutyric acid (GABA) antagonists picrotoxin and bicuculline. Picrotoxin and bicuculline both decreased ³⁶Cl^- efflux in the absence of pentobarbital, suggesting the presence of endogenous GABA. Incubation of synaptoneurosomes with 4,4'-di-isothiocyano- or dinitro-2,2'-disulfonic acid stilbene, inhibitors of anion transport, also decreased both basal and pentobarbital-induced ³⁶Cl^- efflux. Pentobarbital (500 μM) was most effective in inducing ³⁶Cl^- efflux in the cerebellum, hippocampus, and cortex (23.7, 23.6, and 22.5%, respectively), and was less effective in stimulating ³⁶Cl^- efflux in the striatum (15.1%) and pons-medulla (6.2%). The relative efficacy of pentobarbital in enhancing ³⁶Cl^- efflux among these various brain regions was highly correlated (r = 0.96, p = 0.01) with the relative densities of [³⁵S]-t-butylbicyclophosphorothionate-binding sites, a measure of GABA-gated Cl^- channel density. These data suggest that pharmacologically relevant Cl^- transport associated with the benzodiazepine/GABA/Cl^- ionophore receptor complex can be measured in a subcellular preparation from brain, the synaptoneurosome.