Characteristics of flunitrazepam binding to intact primary cultured spinal cord neurons and its modulation by GABAergic drugs.
The interaction of [3H]flunitrazepam and its modulation by various drugs was studied in intact primary cultured spinal cord neurons. In the intact cells, the [3H]-flunitrazepam binding was rapid and saturable. The benzodiazepine binding sites exhibited high affinity and saturability, with an apparent KD of 6.1 +/- 1.6 nM and Bmax of 822 +/- 194 fmol/mg protein. The association and dissociation of [3H]flunitrazepam binding exhibited monoexponential kinetics. Specifically bound [3H]flunitrazepam was displaced in a concentration-dependent manner by benzodiazepines like flunitrazepam, clonazepam, diazepam, Ro 15-1788, and beta-carbolines like methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3'-carboxylate. Specific [3H]flunitrazepam binding to intact cells was enhanced in a concentration-dependent manner by gamma-aminobutyric acid (GABA) agonists and drugs which facilitate GABAergic transmission like etazolate, (+)-etomidate, and pentobarbital. The enhancing effect of GABA agonists was antagonized by bicuculline and picrotoxinin. These results suggest that the intact cultured spinal cord neurons exhibit the properties of benzodiazepine GABA receptor-ionophore complex. Since these cells can also be studied in parallel for characterizing GABA-induced 36Cl-influx, they provide an ideal in vitro assay preparation to study GABA synaptic pharmacology.
Mehta
A K
AK
Department of Pharmacology, University of Texas Health Science Center, San Antonio 78284-7764.
Ticku
M K
MK
D000818:Animals; D001569:Benzodiazepines / Q000378:metabolism; D001667:Binding, Competitive; D002243:Carbolines / Q000378:metabolism; D002478:Cells, Cultured; D004622:Embryo, Mammalian; D004974:Etazolate / Q000494:pharmacology; D005045:Etomidate / Q000494:pharmacology; D005445:Flunitrazepam / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D008810:Mice, Inbred C57BL; D009118:Muscimol / Q000494:pharmacology; D009474:Neurons / Q000378:metabolism*; D010424:Pentobarbital / Q000494:pharmacology; D011963:Receptors, GABA-A / Q000187:drug effects / Q000378:metabolism*; D013116:Spinal Cord / Q000378:metabolism*; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology / Q000502:physiology*
D002243:Carbolines; D011963:Receptors, GABA-A; D001569:Benzodiazepines; D009118:Muscimol; D005680:gamma-Aminobutyric Acid; D005445:Flunitrazepam; D010424:Pentobarbital; D004974:Etazolate; D005045:Etomidate