Characteristics of GABAB receptor binding sites on rat whole brain synaptic membranes. 1983.

Abstract

Saturable binding of (±)‐[3H]‐baclofen and [3H]‐γ‐aminobutyric acid ([3H]‐GABA) to rat brain crude synaptic membranes has been examined by means of a centrifugation assay. 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. 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 μm), (–)‐baclofen (0.04 μm) and to a much lesser extent by (+)‐baclofen (33 μm). Isoguvacine, piperidine‐4‐sulphonic acid and bicuculline methobromide were inactive (up to 100 μm) and muscimol was only weakly active (IC50 = 12.3 μm). 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 μm). Increasing the Ca2+ concentration (0 to 2.5 Mm) increased the binding capacity of the membranes without changing their affinity for the ligand. 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%. 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. 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.