Characterization of Flunitrazepam and Beta-Carboline High Affinity Binding in Bovine Pineal Gland

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Abstract
High affinity binding of 3H-flunitrazepam (FNZP) to crude membrane preparations of bovine pineal membranes was examined by a rapid filtration procedure through Whatman GFB paper. At 0 °C binding reached equilibrium in about 20 min. Scatchard analysis of data at equilibrium revealed a single population of binding sites with dissociation constant (Kd) = 3.14 ± 0.45 nM and binding site concentration (Bmax) = 55.6 ± 5.6 fmol/mg protein. Kinetic analysis of the association and dissociation curves indicated a kinetic Kd = 1.13 nM, in reasonable agreement to that obtained at equilibrium. When various benzodiazepine (BZP) analogues were tested for their ability to inhibit 3H-FNZP binding, the following Ki (nM) were obtained:clonazepam (0.22), Ro 15–1788 (0.48), FNZP (0.95), Ro 5–4864 (>10,000). When the β-carboline derivative 3H-ethyl-β-carboline-3-carboxylate ester (EβCEE) was used as a radioligand, Kd at equilibrium (0.98 nM), kinetic Kd (1.69 nM) and affinity order for analogues were in close agreement to those found for FNZP binding; however, Bmax was about 60% that observed for 3H-FNZP binding. Addition of GABA or pentobarbital (100 μM) to pineal membranes increased 3H-FNZP binding by 55 and 72%. These results suggest the existence of a mixed population of type 1 and type 2 central BZP receptor subclass in bovine pineal gland.

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