Radioligand binding

Competition experiments were performed with [³H]{"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 and [³H]NECA in a final volume of 400 μl. The vial contained 10 μl of dissolved test compound in DMSO, 90 μl of Tris buffer (50 mM, pH 7.4), 100 μl of MgCl₂ solution (final concentration: 10 mM), 100 μl of radioligand solution (final radioligand concentration: [³H]{"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680, 5 nM; [³H]NECA, 3 nM), and 100 μl of membrane preparation (70–150 μg/vial), which was previously incubated with 2 U of adenosine deaminase per milligram of protein for 20 min. Total binding was measured in the absence of test compound, while non-specific binding was determined in the presence of 50 μM NECA in experiments with [³H]{"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680, and 100 μM of N⁶-(R)-(2-phenylisopropyl)adenosine (R-PIA) in experiments with [³H]NECA. After incubation ([³H]{"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680, 1 h; [³H]NECA, 3 h), the assay mixture was filtered through GF/B glass fiber filters using a Brandel harvester (Brandel, Gaithersburg, USA). The filters were washed three times with 300 μl ice-cold Tris buffer (50 mM, pH 7.4), transferred into scintillation vials and incubated for 6 h with 2.5 ml of scintillation cocktail LUMASAFE. The vials were then counted in a liquid scintillation counter (Tricarb 2900TR, Perkin Elmer, Rodgau, Germany). Independent experiments were performed in duplicates. In the homologous competitive binding, a single concentration of radiolabeled ligand and various concentrations of the identical unlabeled ligand were used. For the analysis, it is assumed that the system is at equilibrium and that both hot and cold ligands bind with identical affinities. Thus, the K_D and K_i values are identical. With the Cheng and Prussoff equation, the K_i value is calculated from the IC₅₀ resulting in K_d = K_i = IC₅₀—[radioligand].