title: 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 σ(2) receptors.
abstract: A selective competitive binding assay for the determination of the affinity of compounds to the human σ(2) receptor using 96-well multiplates and a solid state scintillator was developed. In the assay system, [(3)H]ditolylguanidine (DTG) was used as radioligand and membrane homogenates from human RT-4 cells physiologically expressing σ(2) receptors served as receptor material. In order to block the interaction of the unselective radioligand [(3)H]DTG with σ(1) receptors, all experiments were performed in the presence of the σ(1) selective ligand (+)-pentazocine. The density of σ(2) receptors of the cells was analyzed by a saturation experiment with [(3)H]DTG. The radioligand [(3)H]DTG was bound to a single, saturable site on human σ(2) receptors, resulting in a B(max) value of 2108±162fmol/mg protein and K(d)-value of 8.3±2.0nM. The expression of competing σ(1) receptors was evaluated by performing a saturation experiment using the σ(1) selective radioligand [(3)H](+)-pentazocine, which resulted in a B(max) value of 279±40fmol/mg protein and K(d) value of 13.4±1.6nM. For validation of the σ(2) binding assay, the K(i)-values of four σ(2) ligands (ditolylguanidine, haloperidol, rimczole and BMY-14802) were determined with RT-4 cell membrane preparations. The K(i) values obtained from these experiments are in good accordance with the K(i)-values obtained with rat liver membrane preparations as receptor material and with K(i) values given in the literature.
authors.affiliation: Institut für Pharmazeutische und Medizinische Chemie der Universität Münster, Hittorfstraße 58-62, D-48149 Münster, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Schepmann
authors.forename: Kirstin
authors.initials: K
authors.lastname: Lehmkuhl
authors.forename: Stefanie
authors.initials: S
authors.lastname: Brune
authors.forename: Bernhard
authors.initials: B
authors.lastname: Wünsch
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D002623:Chemistry Techniques, Analytical; D004305:Dose-Response Relationship, Drug; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008099:Liver / Q000378:metabolism; D008956:Models, Chemical; D010423:Pentazocine / Q000494:pharmacology; D051381:Rats; D017480:Receptors, sigma / Q000737:chemistry; D013696:Temperature; D001743:Urinary Bladder / Q000187:drug effects / Q000378:metabolism*; D001749:Urinary Bladder Neoplasms / Q000188:drug therapy / Q000378:metabolism*
chemical_list: D008024:Ligands; D017480:Receptors, sigma; D010423:Pentazocine