Bivalent Argininamide-Type NPY Y 1 Receptor Antagonists
Scheme 6. Synthesis of the potential bivalent tritium ligands 3.42 and 3.43 as well as the potential bivalent PET ligand 3.44
3.3.4 Pharmacology: Cell Culture, Fura-2 Assay and Competition Binding Assay
Cell culture. HEL and SK-N-MC cells were cultured as described elsewhere.31, 32 HEL cells were subcultured by 1:6-dilution with fresh culture medium 24 h prior to the Fura Ca2+-assay.
MCF-7-Y1 cellsa were maintained in MEM (Sigma, Deisenhofen, Germany), supplemented with 5 % FCS (Biochrom AG, Berlin, Germany).
aThis cell line was subcloned from MCF-7 cells in passage 157th and shows a higher Y1R expression (by a factor 2 -3 higher than that of the MCF-7 cells (ATCC number HTB 22)).
Chapter 3 74
Fura-2 assay on HEL cells. The Fura assay was performed with HEL cells as previously described28 using a Perkin-Elmer LS50 B spectrofluorimeter (Perkin Elmer, Überlingen, Germany).
Radioligand competition binding assay. The procedure performed in the radioligand binding assay was deduced from a previously described protocol31 and [3H]-UR-MK114 (2.8b, cf.
chapter 2) was used instead [3H]-propionyl-pNPY. Radioligand concentration was set to 1.5 nM for SK-N-MC cells (KD = 1.2 nM) and 2 nM for MCF-7 cells (KD = 2.9 nM). Cells were seeded in 24-well plates 2 or 3 days prior to the experiment. MCF-7-Y1a cells were seeded in culture medium with 1 % -Estradiol in order to up-regulate the Y1 receptor. On the day of the experiment confluency of the cells was at least 70 %. The culture medium was sucked off, cells were washed once with buffer (500 µL) and covered with binding buffer (200 µL for SK-N-MC, 400 µL for MCF-7 cells). Binding buffer (25/50 µL) and binding buffer (25/50 µL) with radioligand (ten fold concentrated) was added for total binding. For non-specific binding and competition of test compounds with radioligand binding buffer (25/50 µL) with the competing agent (pNPY or test substance, ten fold concentrated) and binding buffer (25/50 µL) with radioligand (ten fold concentrated) was added. During incubation at room temperature (22 – 25 °C) the plates were gently shaken. After incubation (20 min for SK-N-MC, 30 min for MCF-7-Y1 cells) the binding buffer was removed, the cells were washed twice with buffer (500 µL, 4 °C, ≤ 30 s) and covered with lysis solution (200 µL). The plates were gently shaken for at least 30 min. The lysis solution was transferred into 6-mL scintillation vials filled with scintillator (3 mL) and the dishes were washed once with lysis solution (100 µL). Assays were performed in triplicate or duplicate.
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