GABA-agonists induce the formation of low-affinity GABA-receptors on cultured cerebellar granule cells via preexisting high affinity GABA receptors.
The kinetics of specific GABA-binding to membranes isolated from cerebellar granule cells, cultured for 12 days from dissociated cerebella of 7-day-old rats was studied using [3H]GABA as the ligand. The granule cells were cultured in the presence of the specific GABA receptor agonist 4, 5, 6, 7-tetrahydroisoxazolo [5,4-c]pyridin-3-ol (THIP, 150 microM) or THIP plus the antagonist bicuculline methobromide (150 microM of each) or in the absence of the agonist or antagonist. Membranes isolated from granule cells cultured in a medium without the GABA agonist revealed a single binding site for GABA with a binding constant (KD) of 7.9 +/- 0.4 nM and a Bmax of 3.42 +/- 0.08 pmol X mg-1 protein. Membranes from cells cultured in the presence of THIP had two binding sites for GABA with KD-values of 6.8 +/- 0.9 nM and 476 +/- 311 nM, respectively. The corresponding Bmax values were 4.41 +/- 0.42 pmol X mg-1 and 5.81 +/- 1.20 pmol X mg-1. The effect of culturing the cells in THIP was antagonized by the simultaneous presence of bicuculline in the culture media, i.e. no significant low-affinity binding for GABA was found on the membranes from granule cells cultured in both THIP and bicuculline. The KD value (14.3 +/- 1.4 nM) for the high affinity binding site was, however, slightly increased compared to the non-treated cells. These findings suggest that the ability of THIP to induce formation of low-affinity GABA receptors is mediated by preexisting high-affinity GABA-receptors on the granule cells.
D000818:Animals; D001640:Bicuculline / Q000494:pharmacology; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism; D007555:Isoxazoles / Q000494:pharmacology*; D007700:Kinetics; D010080:Oxazoles / Q000494:pharmacology*; D051381:Rats; D011919:Rats, Inbred Strains; D011963:Receptors, GABA-A / Q000032:analysis / Q000187:drug effects*
D007555:Isoxazoles; D010080:Oxazoles; D011963:Receptors, GABA-A; C015542:gaboxadol; D001640:Bicuculline