Article date: 1990/2/1
PubMed ID: 2303862
Journal name: The Journal of neuroscience : the official journal of the Society for Neuroscience (ISSN: 0270-6474)
35S-t-butylbicyclophosphorothionate (TBPS) binding to slide-mounted rat brain sections was characterized for subsequent autoradiographic analysis. Cortical brain mash slices, preincubated with EDTA to remove endogenous GABA, were used for biochemical characterization. Steady state for 35S-TBPS binding was reached by 3 hr of incubation at 22 degrees C. The association rate constant (K1) and dissociation rate constant (K2) were 0.377 min-1 microM-1 and 0.011 min-1, respectively. Dissociation was monophasic and slow (t1/2 = 80 min). The kinetically derived KD was 29.4 nM. Scatchard analysis indicated a single population of binding sites with a KD of 21.0 +/- 2.2 nM and a Bmax of 1.59 +/- 0.13 pmol/mg protein. Both picrotoxin and muscimol inhibited 35S-TBPS binding completely with IC50s of 251 +/- 13 nM and 203 +/- 41 nM and nHs of 0.98 and 1.4, respectively. The distribution of 35S-TBPS binding sites in the rat brain resembles that of other ligands that bind to GABAA receptor complex with some regionally specific differences. Regions with a high degree of 35S-TBPS binding included the inferior colliculus, medial septal nucleus, central and paracentral nuclei of the thalamus, olfactory tubercle, zona incerta, dentate gyrus, and substantia nigra. 35S-TBPS preferentially bound to the molecular vs granular layer of the cerebellum. Omission of the preincubation markedly but variably decreased 35S-TBPS binding. The greatest regional decreases occurred in areas with a high degree of GABA synthesis. In addition, 35S-TBPS binding was inhibited to different degrees in the cell layers of the cerebellum. The addition of 1 microM GABA to the incubation medium of preincubated slices also produced variable decreases in 35S-TBPS binding to cerebellar layers. These findings support previous studies that demonstrate GABAA receptor heterogeneity. Our study confirms the suitability of 35S-TBPS for use as a ligand in autoradiography and demonstrates that the distribution of 35S-TBPS binding sites is significantly influenced by the preincubation-incubation conditions used.
Author List: Edgar P P, Schwartz R D
Publication Types: Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.; Research Support, U.S. Gov't, P.H.S.
Substances mentioned in the article: Bridged Bicyclo Compounds; Bridged Bicyclo Compounds, Heterocyclic; Bridged-Ring Compounds; Sulfur Radioisotopes; gamma-Aminobutyric Acid; tert-butylbicyclophosphorothionate;
Mesh terms: Animals; Autoradiography; Binding Sites; Brain/metabolism; Bridged Bicyclo Compounds/metabolism; Bridged Bicyclo Compounds, Heterocyclic; Bridged-Ring Compounds/metabolism; Male; Rats; Rats, Inbred Strains; Sulfur Radioisotopes; Tissue Distribution; gamma-Aminobutyric Acid/pharmacology;