Dual action of pentobarbitone on GABA binding: role of binding site integrity.

The effects of pentobarbitone on the binding of gamma-aminobutyric acid (GABA) to crude synaptosomal rat brain membranes were studied. In extensively washed P2 membranes, pentobarbitone had a biphasic action: at concentrations ranging between 12.5 and 500 microM, pentobarbitone enhanced GABA binding in a concentration-dependent manner; at concentrations greater than 500 microM, this enhancement was progressively reversed towards control levels of GABA binding. The effect of pentobarbitone seen at higher concentrations may reflect a GABA-mimetic action, since similar concentrations enhanced diazepam binding to washed P2 membranes, an effect antagonized by bicuculline methochloride and picrotoxinin. When washed P2 membranes were incubated in 0.5% Triton X-100 (30 min at 37 degrees C), the enhancement of GABA binding by low concentrations of pentobarbitone was abolished, while at higher concentrations GABA binding was progressively inhibited, suggesting that the GABA-mimetic action is retained. When washed P2 membranes were subjected to high-frequency homogenization, the biphasic dose-response relationship for pentobarbitone was markedly shifted to the right. The choice of membrane preparation appears to be a critical factor in examining drug-receptor interactions in vitro, at least for those involving GABA and the barbiturates.