Flunitrazepam Binding to Intact and Homogenized Astrocytes and Neurons in Primary Cultures

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Abstract
[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 76 nM and Bmax [maximum binding capacity] 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 2 cell types, with a faster association and dissociation in astrocytes than in neurons and a greater temperature sensitivity in the astrocytes. Flunitrazepam binding sites on neuronal and astrocytic cells have different pharmacological profiles. In intact astrocytic cells, Ro 5-4864 [7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2H-1,4-benzodiazepine-2-one] (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 3 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. Both neuronal and astrocytic cells possess high-affinity [3H]flunitrazepam binding sites. The pharmacological profile and kinetic characteristics differ between the 2 cell types and are further altered by homogenization.