'High-affinity' peripheral-type benzodiazepine-binding sites.

Abstract

1 ,4-Benzodiazepines are widely prescribed as minor tranquillizers or anxiolytics, as sedative-hypnotics and anticonvulsants. Some or possibly all of these effects may be explained by these compounds potentiating the action of the inhibitory neurotransmitter, y-aminobutyrate (GABA) (for reviews, see Turner & Whittle, 1983; Haefely et al., 1985). Thus some clinically active benzodiazepines potentiate neuronal postsynaptic GABA-receptor mediated chloride ion influx leading to membrane hyperpolarization. This receptor has been purified and shown to be an oligomeric complex of two protein subunits with binding sites for GABA and benzodiazepines (for reviews, see Turner & Whittle, 1983; Haefely et al., 1985). High-affinity benzodiazepine-binding sites were first reported by Braestrup & Squires (l977), who showed highaffinity diazepam-binding sites in brain and peripheral tissues. Central and peripheral binding sites could be distinguished from each other by (a) both types having high affinity for diazepam, (b) central sites having high affinity for clonazepam but low affinity for Ro-5-4864 (4’chlorodiazepam), (c) peripheral sites having low affinity for clonazepam and high affinity for Ro-5-4864. These peripheral-type sites were subsequently found in brain (Richards et al., 1982; Shoemaker et al., 1983) and have been proposed to be glial on the basis of neuronal cell lesioning studies (Schoemaker et a[., 1982). Several functions have been proposed for these peripheral/ non-neuronal benzodiazepine high-affinity binding sites, for example, in prostaglandin D, formation (Majewska & Chuang, 1985) in Ca2+ channel function (Doble et al., 1985) and in regulation of cell proliferation (Wang et al., 1984). We have previously reported (O’Beirne & Williams, 1984) that whereas we could measure high levels of peripheraltype benzodiazepine high-affinity binding to platelets from rodents, we were unable to detect this binding to platelets from non-rodents, and that this ‘lack’ of binding was probably not due to either states of activation nor presence of inhibitors or lack of activators. Whereas high-affinity binding sites of the peripheral-type could be shown using [3H]Ro-5-4864 or [3H]flunitrazepam, when membranes from rat kidney, liver and brain were, assayed, no specific binding of the peripheral-type could be shown to membranes from ox kidney, liver or brain nor human post-mortem membranes from kidney, liver or cerebral cortex. We conclude that in these tissues in these species peripheral-type binding is absent, immeasurably low or of low affinity. In rat brain, subcellular fractionation by differential centrifugation (Laduron et al., 1983) showed the nuclear fraction to have greatest enrichment of ‘peripheral-type’ binding sites. When brain fractions were resolved further using sucrose isopycnic density gradient centrifugation, for the ‘N’ fraction the binding sites were found enriched at an