Characteristics of GABAB receptor binding sites on rat whole brain synaptic membranes.

1 Saturable binding of (+/-)-[3H]-baclofen and [3H]-gamma- aminobutyric acid ([3H]-GABA) to rat brain crude synaptic membranes has been examined by means of a centrifugation assay. 2 The binding of [3H]-baclofen could be detected in fresh or previously frozen tissue and was dependent on the presence of physiological concentrations of Ca2+ or Mg2+ although a lower affinity Na+ -dependent component could also be observed. Both components probably reflect binding to receptor recognition sites. 3 The saturable portion of bound [3H]-baclofen formed 20.3 +/- 6.9% of total bound ligand. This could be displaced by GABA (IC50 = 0.04 microM), (-)-baclofen (0.04 microM) and to a much lesser extent by (+)-baclofen (33 microM). Isoguvacine, piperidine-4-sulphonic acid and bicuculline methobromide were inactive (up to 100 microM) and muscimol was only weakly active (IC50 = 12.3 microM). 4 Saturable binding of [3H]-GABA increased on adding CaCl2 or MgSO4 (up to 2.5 mM and 5.0 mM respectively) to the Tris-HCl incubation solution. This binding (GABAB site binding) was additional to the bicuculline-sensitive binding of GABA (GABAA site binding) and could be completely displaced by (-)-baclofen (IC50 = 0.13 microM). 5 Increasing the Ca2+ concentration (0 to 2.5 mM) increased the binding capacity of the membranes without changing their affinity for the ligand. 6 The binding of [3H]-GABA to GABAB sites could be demonstrated in fresh as well as previously frozen membranes with a doubling of the affinity being produced by freezing. Further incubation with the non-ionic detergent Triton-X-100 (0.05% v/v) reduced the binding capacity by 50%. 7 The pharmacological profile of displacers of [3H]-GABA from GABAB sites correlated well with that for [3H]-baclofen displacement. A correlation with data previously obtained in isolated preparations of rat atria and mouse vas deferens was also apparent. 8 It is concluded that [3H]-baclofen or [3H]-GABA are both ligands for the same bicuculline-insensitive, divalent cation-dependent binding sites in the rat brain.