Benzodiazepines have high-affinity binding sites and induce melanogenesis in B16/C3 melanoma cells.

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Abstract
Two markers of differentiation, tyrosinase (monophenol, dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) activity and melanin synthesis, are induced by diazepam in B16/C3 mouse melanoma cells. High-affinity binding sites were demonstrated for [3H]diazepam in these cells by radioreceptor assay, and binding to the cell surface by fluorescence microscopy was visualized with a benzodiazepine analog conjugated to a fluorescein-labeled protein. There are differences between the binding characteristics in intact cells and in membrane fractions prepared from the cells. Scatchard analysis of the binding data from membrane fractions gave a linear plot (Kd = 9.1 .times. 10-8 M). With intact cells, a curvilinear Scatchard plot was obtained. This was resolved into 2 components defining binding sites with affinity constants of 1.7 .times. 10-9 M and 4.6 .times. 10-7 M. [3H]Diazepam binding in intact cells is more complex than in isolated membranes. Several related benzodiazepines, including flunitrazepam, Ro-5-4864, nitrazepam, oxazepam, lorazepam, Ro-5-3072, chlordiazepoxide, and clonazepam induced melanogenesis. When these compounds were tested for their ability to inhibit [3H]diazepam binding, flunitrazepam, diazepam, and Ro-5-4864 [7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2H-1,4-benzodiazepine-2-one] were the most effective inhibitors. The 3 compounds were the most potent in inducing melanogenesis. The benzodiazepines may modulate cell differentiation. The presence of high-affinity binding sites in this homogeneous, easily grown cell line may provide a useful model for studies on the mechanism of action of these compounds.