Flunitrazepam binding to intact and homogenized astrocytes and neurons in primary cultures.
[3H]Flunitrazepam binds to intact and homogenized mouse astrocytes and neurons in primary cultures. In intact cells, the binding is to a single, high-affinity, saturable population of benzodiazepine binding sites with a KD of 7 nM and Bmax of 6,033 fmol/mg protein in astrocytic cells and a KD of 5 nM and Bmax of 924 fmol/mg protein in neurons. After homogenization, the Bmax values decrease drastically in both cell types, but most in astrocytes. The temperature and time dependency are different for the two cell types, with a faster association and dissociation in astrocytes than in neurons and a greater temperature sensitivity in the astrocytes. Moreover, flunitrazepam binding sites on neuronal and astrocytic cells have different pharmacological profiles. In intact astrocytic cells, Ro 5-4864 (Ki = 4 nM) is the most potent displacing compound, followed by diazepam (Ki = 6 nM) and clonazepam (Ki = 600 nM). In intact neurons, the relative order of potency of these three compounds is different: diazepam (Ki = 7 nM) is the most potent, followed by clonazepam (Ki = 26 nM) and Ro 5-4864, which has little effect. After homogenization the potency of diazepam decreases. We conclude that both neuronal and astrocytic cells possess high-affinity [3H]flunitrazepam binding sites. The pharmacological profile and kinetic characteristics differ between the two cell types and are further altered by homogenization.
D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism*; D001570:Benzodiazepinones / Q000494:pharmacology; D002478:Cells, Cultured; D002998:Clonazepam / Q000494:pharmacology; D003975:Diazepam / Q000494:pharmacology; D005445:Flunitrazepam / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D011963:Receptors, GABA-A / Q000378:metabolism; D013696:Temperature
D001570:Benzodiazepinones; D011963:Receptors, GABA-A; C028513:4'-chlorodiazepam; D002998:Clonazepam; D005445:Flunitrazepam; D003975:Diazepam