Characterization of Ligand Binding to the σ₁Receptor in a Human Tumor Cell Line (RPMI 8226) and Establishment of a Competitive Receptor Binding Assay

Abstract

The standard assay for the determination of σ(1) receptor affinities of novel compounds is a competitive binding assay using [(3)H]-(+)-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 σ(1) receptor. Membrane fragments of RPMI 8226 cells were prepared and characterized. A Western blot analysis confirmed the high density of σ(1) 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 [(3)H]-(+)-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 σ(1) binding sites per cell. The assay was validated by using six known σ(1) ligands and eight σ(1) 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 σ(1) receptors.

A 96-well filtration method for radioligand binding analysis of σ receptor ligands

“…This cell line was successfully used to analyze a wide range of compounds including 10 σ reference ligands [38]. Studies from the Wunsch group utilized cell lines with high endogenous expression levels of σ 1 or σ 2 (RPMI 8226 and RT-4 cells respectively) in conjunction with 96-well filtration; these studies were validated with σ reference ligands as well [39, 40]. The 96-well format has also been successfully employed for a number of studies using guinea pig brain for σ 1 and rat liver for σ 2 with filtration through filtermats [9] or filterplates [8, 10, 12], but our efforts to filter rat brain homogenates were unsuccessful with the filterplates we examined.…”

The Oligomeric States of the Purified Sigma-1 Receptor Are Stabilized by Ligands

“…Interestingly, these interactions were disrupted in the presence of ligands such as haloperidol and/or (+)-pentazocine, so one may speculate that a ligand-gated S1R oligomer/monomer equilibrium defines the availability of monomer S1R for interaction with client ion channels or G-protein-coupled receptors. An additional unusual feature of binding of the agonist, (+)-pentazocine, to S1R is the time (>90 min at 30 °C) needed to reach equilibrium (41, 74). It is possible that formation of stable interactions between oligomeric states of S1R in situ regulate the rate of (+)-pentazocine binding to S1R (perhaps also affected by interactions with accessory protein partners).…”

Bridging from Brain to Tumor Imaging: (S)-(−)- and (R)-(+)-[18F]Fluspidine for Investigation of Sigma-1 Receptors in Tumor-Bearing Mice

“…In this publication, we investigated the suitability of both enantiomers of [ 18 F]fluspidine to image Sig1R in different mouse tumor models. Current investigations [ 30 ] show strong expression of Sig1R and correlation with pathologic tumor tissue in human esophageal squamous cell carcinoma [ 36 ], prostate cancer [ 37 , 38 ], myeloma [ 39 ], melanoma [ 20 , 40 ], and glioma [ 41 ]. To assess the expression of the Sig1R protein in different human tumors, cell lines from epidermoid carcinoma, melanoma, and glioblastoma were investigated with Western blot in the current study.…”