Radioligand binding assays

For radioligand assay, 8x10⁵cells were washed with HBSS and incubated with binding buffer (50 mM Tris-HCl pH 7.4, 5 mM MgCl₂, 1 mM EDTA) for 1h at 37°C. Saturation binding assays were performed with 2-[^125I]-MLT alone or with an excess of unlabelled MLT to evaluate non-specific binding. Similar experiments were done with the MT₂ receptor agonist [¹²⁵I]-{"type":"entrez-nucleotide","attrs":{"text":"S70254","term_id":"546525","term_text":"S70254"}}S70254 ± {"type":"entrez-nucleotide","attrs":{"text":"S70254","term_id":"546525","term_text":"S70254"}}S70254. Briefly, 0.08–1.5 nM radioligands alone or with 10 μM unlabelled ligand were incubated in binding buffer for 1 h at 37°C. The reaction was stopped by aspiration of the medium. Cells were incubated in a cold-acid buffer (50 mM glycine, 150 mM NaCl) for 3 min at 4°C and supernatants were stored to quantify the number of radioligands bound on cell surface. Then, cells were lysed in 1 M NaOH for 2 h at 37°C to measure intracellular ligand-receptor complexes. Radioactivity was measured with a Wizard gamma counter (Perkin Elmer) Data were analysed by using the PRISM software (GraphPad Software Inc., San Diego, CA, USA) and results were presented as the mean of duplicate measurements. The density of binding sites (B_max) and the dissociation constant of the radioligand (K_D) values were calculated according to the method of Scatchard (F test in GraphPad Prism, version 5).