Effects of GHB on GABAB binding in the presence and in the absence of valproate
| Crude Synaptosomal Membranes Prepared According to the Method of Bernasconiet al. (1992) | Crude Synaptosomal Membranes Prepared According to the Method of Hill and Bowery (1981) |
|---|---|
| Cerebellum | Cerebellum |
| Total binding: 5411 ± 217 cpm | Total binding: 6329 ± 256 cpm |
| Specific binding: 3390 cpm | Specific binding: 1827 cpm |
| Nonspecific binding: 2021 ± 111 cpm | Nonspecific binding: 4502 ± 318 cpm |
| GHB 100 μM: 1269 cpm displaced 37% of the specific binding | GHB 100 μM: 335 cpm displaced 18% of the specific binding |
| GHB 100 μM + valproate 5 mM: | GHB 100 μM + valproate 5 mM: |
| 0 cpm displaced | 0 cpm displaced |
| Cerebrum | Cerebrum |
| Total binding: 6922 ± 312 cpm | Total binding: 7212 ± 236 cpm |
| Specific binding: 3882 cpm | Specific binding: 2393 cpm |
| Nonspecific binding: 3040 ± 52 cpm | Nonspecific binding: 4848 ± 613 cpm |
| GHB 100 μM: 933 cpm displaced 24% of the specific binding | GHB 100 μM: 0 cpm displaced |
| GHB 100 μM + Valproate 5 mM: | GHB 100 μM + valproate 5 mM: |
| 0 cpm displaced | 0 cpm displaced |
Crude synaptosomal membranes were prepared according to Bernasconiet al. (1992) or Hill and Bowery (1981). Membranes were incubated in Tris-HCl 50 mM, CaCl2 2.5 mM, pH 7.4, containing 100 μM isoguvacine, [3H]GABA (25 nM, 74 Ci/mmol) and GHB 100 μM. In some experiments, valproate (5 mM) was added in order fully to inhibit GHB dehydrogenase. After a 15-min incubation at room temperature, bound [3H]GABA was separated from free [3H]GABA by rapid centrifugation at 40,000 × g for 30 min.