Radioligand binding assays

Experimental procedures were performed according to the method of Abramovitz et al. [23]. [³H]PGE₂ (PerkinElmer, Waltham, MA, USA) binding assays for recombinant EP4 receptors were performed in 10 mM 2-(N-morpholino)ethanesulphonic acid-KOH buffer, pH 6, containing 10 mM MgCl₂ and 1 mM CaCl₂. Ten micrograms of protein from membrane fractions were incubated in a total volume of 0.1 ml with 1–1.5 nM [³H]PGE₂. To determine the total binding or non-specific binding, 1% dimethyl sulphoxide (DMSO) or 1 μM PGE₂ was added to the reaction mixture. Incubation was carried out in 96-well multi-well plates for 90 minutes at room temperature prior to separation of the bound and free radioligand by rapid filtration on glass-fibred filters (UniFilter-96 GF/B; PerkinElmer) pre-soaked in 0.3% polyethylenimine. Filters were washed with ice-cold buffer, pH 7.4 (50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES], NaCl 500 mM, BSA 0.1%), and dried for 30 minutes at 30 °C, then 0.2 ml of MicroScint-20 (PerkinElmer) was added. The residual [³H]PGE₂ binding was determined using a solid scintillation counter (TopCount; PerkinElmer) after at least 1 h of stabilisation. In saturation binding studies, isotherm-binding curves generated with six to eight different concentrations of [³H]PGE₂ (from 0.5 to 25 nM) were evaluated. In competition experiments, curves of compound under investigation ranged from 3 × 10⁻¹⁰ M to 10⁻⁶ M (eight concentration points). In both experimental protocols, all concentration points were performed in duplicate.