Characterization of Ligand Binding to the σ 1 Receptor in a Human Tumor Cell Line (RPMI 8226) and Establishment of a Competitive Receptor Binding Assay

Abstract

The standard assay for the determination of σ₁ receptor affinities of novel compounds is a competitive binding assay using [³H]-(+)-pentazocine as radioligand and membrane preparations from guinea pig brain. Herein, a novel competitive binding assay was developed employing the hematopoietic cell line of human multiple myeloma (RPMI 8226), which expresses a large amount of the human σ₁ receptor. Membrane fragments of RPMI 8226 cells were prepared and characterized. A Western blot analysis confirmed the high density of σ₁ receptors in this cell line. Assay conditions were carefully optimized leading to an incubation period of 120 min, an incubation temperature of 37°C, and receptor material for each well was prepared from 300,000 cells. It was shown that a large excess (10 μM) of (+)-pentazocine, haloperidol, and di-o-tolylguanidine provided the same results during determination of the nonspecific binding. Saturation experiments with the radioligand [³H]-(+)-pentazocine led to a K _d -value of 36±0.3 nM and a B _max -value of 477±7 fmol/mg protein. These data resulted in approximately 122,000 σ₁ binding sites per cell. The assay was validated by using six known σ₁ ligands and eight σ₁ ligands prepared in our lab. The K _i -values determined with RPMI 8226–derived receptor material are in good accordance with the K _i -values obtained with guinea pig brain membrane preparations. Compared with guinea pig brain preparations, the RPMI 8226–derived receptor material represents a better standardized receptor material with a high density of human σ₁ receptors.