Expression of σ receptors of human urinary bladder tumor cells (RT-4 cells) and development of a competitive receptor binding assay for the determination of ligand affinity to human σ2 receptors

A selective competitive binding assay for the determination of the affinity of compounds to the human σ2 receptor using 96-well multiplates and a solid state scintillator was developed. In the assay system, [3H]ditolylguanidine (DTG) was used as radioligand and membrane homogenates from human RT-4 cells physiologically expressing σ2 receptors served as receptor material. In order to block the interaction of the unselective radioligand [3H]DTG with σ1 receptors, all experiments were performed in the presence of the σ1 selective ligand (+)-pentazocine. The density of σ2 receptors of the cells was analyzed by a saturation experiment with [3H]DTG. The radioligand [3H]DTG was bound to a single, saturable site on human σ2 receptors, resulting in a Bmax value of 2108 ± 162 fmol/mg protein and Kd-value of 8.3 ± 2.0 nM. The expression of competing σ1 receptors was evaluated by performing a saturation experiment using the σ1 selective radioligand [3H](+)-pentazocine, which resulted in a Bmax value of 279 ± 40 fmol/mg protein and Kd value of 13.4 ± 1.6 nM. For validation of the σ2 binding assay, the Ki-values of four σ2 ligands (ditolylguanidine, haloperidol, rimczole and BMY-14802) were determined with RT-4 cell membrane preparations. The Ki values obtained from these experiments are in good accordance with the Ki-values obtained with rat liver membrane preparations as receptor material and with Ki values given in the literature.