Binding of [3H]Ro 5-4864 in primary cultures of astrocytes

Intact primary cultures of astrocytes display benzodiazepine receptors, which can be labeled with [3H]Ro 5-4864. Binding of [3H]Ro 5-4864 is specific, saturable and temperature dependent, being maximal at 0 degrees C. Scatchard analyses show a single population of high affinity binding sites with a Kd value of 6.7 nM and a Bmax value of congruent to 12,000 fmol/mg protein. The binding reaches equilibrium at congruent to 100 min, with k+1 of 0.0078 nM-1 X min-1 and is rapidly reversible with k-1 of 0.057 min-1. [3H]Ro 5-4864 binding is not modulated by GABA. Certain benzodiazepines (flunitrazepam, diazepam, Ro 7-3351) and dipyridamole displace this binding with IC50 values in the nanomolar range, whereas other benzodiazepines (alprazolam, clonazepam, chlordiazepoxide) as well as carbamazepine, phenytoin and phenobarbital have IC50 values in the micromolar range. These characteristics resemble those of Ro 5-4864 binding to brain membrane preparations reported by other authors and thus indicate that the cultured astrocytes are good models of their in vivo counterparts.