title: gamma-Aminobutyric acid- and benzodiazepine-induced modulation of [35S]-t-butylbicyclophosphorothionate binding to cerebellar granule cells.
abstract: t-Butylbicyclophosphorothionate (TBPS) is a bicyclophosphate derivative with potent picrotoxin-like convulsant activity that binds with high affinity and specificity to a Cl- channel-modulatory site of the gamma-aminobutyric acid (GABA)/benzodiazepine receptor complex. Using intact cerebellar granule cells maintained in primary culture, we have studied the modifications induced by GABA and diazepam on the ion channel-modulatory binding site labeled by [35S]TBPS. At 25 degrees C, and in a modified Locke solution, the [35S]TBPS specific binding, determined by displacing the radioligand with an excess (10(-4) M) of picrotoxin, was approximately 70% of the total radioactivity bound to the cells. [35S]TBPS specific binding was saturable with a Kd of approximately 100 nM, a Bmax of approximately 440 fmol/mg of protein, and a Hill coefficient of 1.18. Neither cerebellar astrocytes maintained in culture for 2 weeks nor a neuroblastoma cell line (NB-2A) exhibited any specific [35S]TBPS binding. Muscimol (0.3 to 5 microM) enhanced and bicuculline (0.1 to 5 microM) inhibited [35S]TBPS specific binding to intact cerebellar granule cells. The effect of muscimol and bicuculline on [35S]TBPS binding was noncompetitive. Muscimol (0.1 to 5 microM) reversed bicuculline inhibition in a dose-dependent fashion but failed to reverse picrotoxin-induced inhibition. [35S]TBPS binding was also modulated by benzodiazepine receptor ligands. The binding was increased by diazepam and decreased by 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylic acid methylester. Muscimol (0.05 microM) failed to reverse bicuculline inhibition in the absence of diazepam, but it became effective in the presence of 0.1 to 1 microM diazepam.(ABSTRACT TRUNCATED AT 250 WORDS)
mesh_terms: D000818:Animals; D001569:Benzodiazepines / Q000502:physiology*; D001640:Bicuculline / Q000494:pharmacology; D001643:Bridged Bicyclo Compounds / Q000378:metabolism*; D019086:Bridged Bicyclo Compounds, Heterocyclic; D001952:Bridged-Ring Compounds / Q000378:metabolism*; D002243:Carbolines / Q000494:pharmacology; D002531:Cerebellum / Q000166:cytology / Q000378:metabolism*; D003975:Diazepam / Q000494:pharmacology; D006098:Granulocytes / Q000378:metabolism*; D009118:Muscimol / Q000494:pharmacology; D010852:Picrotoxin / Q000494:pharmacology; D051381:Rats; D013462:Sulfur Radioisotopes; D013696:Temperature; D005680:gamma-Aminobutyric Acid / Q000502:physiology*
chemical_list: D001569:Benzodiazepines; D001640:Bicuculline; D001643:Bridged Bicyclo Compounds; D019086:Bridged Bicyclo Compounds, Heterocyclic; D001952:Bridged-Ring Compounds; D002243:Carbolines; D003975:Diazepam; D009118:Muscimol; D010852:Picrotoxin; D013462:Sulfur Radioisotopes; D005680:gamma-Aminobutyric Acid; C034818:methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate; C037476:tert-butylbicyclophosphorothionate