Effect of diazepam on the low affinity GABA binding sites at different developmental stages of the chick optic lobe

Abstract

The developmental profile of GABA receptor sites was studied under experimental conditions where the low affinity binding site is mainly measured. The in vitro effects of nanomolar concentrations of diazepam upon low affinity GABA binding sites during development were also examined. The addition of 50 nM diazepam enhanced specific [3H]GABA binding at all stages of development, this stimulation being greater at early stages (10th–14th embryonic day). Scatchard analysis performed at the 14th embryonic day revealed only the binding sites of low affinity with a Kd of 520 Nm and a maximal number of binding sites (Bmax) of 7.60 pmol/mg protein. At hatching two populations of GABA binding sites were present: one with high affinity (Kd = 10.47 nM and Bmax = 0.22 pmol/mg protein) and the other with low affinity (Kd = 686.51 nM and Bmax = 13.28 pmol/mg protein). The addition of 50 nM diazepam increased the affinity of the low affinity GABA binding sites in both stages of development, while that of the high affinity ones remained unchanged. The apparent Kds of [H3]GABA binding at the 14th embryonic day and at hatching were 120.50 and 452.06 nM, respectively, while the densities of receptor sites were unchanged. Our results show that diazepam increases low affinity GABA binding in an age-dependent manner, this effect being higher in tissue obtained from embryonic chicken optic lobe as compared to the adult one. The binding data obtained in this study suggest a tighter coupling of the GABA-benzodiazepine complex at early stages of development.