Characteristics of GABA_Breceptor binding sites on rat whole brain synaptic membranes

Abstract

Saturable binding of (±)‐[ 3 H]‐baclofen and [ 3 H]‐γ‐aminobutyric acid ([ 3 H]‐GABA) to rat brain crude synaptic membranes has been examined by means of a centrifugation assay. The binding of [ 3 H]‐baclofen could be detected in fresh or previously frozen tissue and was dependent on the presence of physiological concentrations of Ca 2+ or Mg 2+ 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 [ 3 H]‐baclofen formed 20.3 ± 6.9% of total bound ligand. This could be displaced by GABA (IC 50 = 0.04 μ m ), (–)‐baclofen (0.04 μ m ) and to a much lesser extent by (+)‐baclofen (33 μ m ). Isoguvacine, piperidine‐4‐sulphonic acid and bicuculline methobromide were inactive (up to 100 μ m ) and muscimol was only weakly active (IC 50 = 12.3 μ m ). Saturable binding of [ 3 H]‐GABA increased on adding CaCl 2 or MgSO 4 (up to 2.5 M m and 5.0 M m respectively) to the Tris‐HCl incubation solution. This binding (GABA B site binding) was additional to the bicuculline‐sensitive binding of GABA (GABA A site binding) and could be completely displaced by (–)‐baclofen (IC 50 = 0.13 μ m ). Increasing the Ca 2+ concentration (0 to 2.5 M m ) increased the binding capacity of the membranes without changing their affinity for the ligand. The binding of [ 3 H]‐GABA to GABA B 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 [ 3 H]‐GABA from GABA B sites correlated well with that for [ 3 H]‐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 [ 3 H]‐baclofen or [ 3 H]‐GABA are both ligands for the same bicuculline‐insensitive, divalent cation‐dependent binding sites in the rat brain.