Benzodiazepine binding to cultured human pituitary cells

Benzodiazepine receptors were investigated in a cell line of human pituitary cells (18-54,SF) grown in serum-free medium. Preparations of 18-54,SF whole cells and cell membranes were shown to possess saturable [3H]diazepam binding sites. Membrane sites were found to have a KD of 20 nM for diazepam while whole cells possessed a twofold higher value. The KD values determined from Rosenthal, Hill, and kinetic analyses were consistent for each preparation. Whole-cell binding of [3H]diazepam was observed to be more stable than binding to membranes at higher temperatures (37 degrees C) and when longer incubation times (60 min) were employed at 4 degrees C. The rank order potency of various benzodiazepines to inhibit [3H]diazepam binding to whole cells and membranes was Ro 5-4864, flunitrazepam, diazepam, and clonazepam. Representatives of other drug classes did not inhibit this benzodiazepine binding. When 18-54,SF cells were grown for 24 h with 100 nM diazepam and then extensively washed membranes prepared, the KD for diazepam increased to 38 nM whereas the Bmax was unchanged when compared with untreated controls. Overall, these findings indicate that pituitary cells possess a peripheral-type benzodiazepine receptor and that the whole cell receptor differs quantitatively when compared with the membrane receptor.