Radioligand Binding Assay: The affinity of a test compound for the human dopamine receptor subtype hDRD2 was determined by radioligand binding assays in CHO-K1 cells stably transfected with hDRD2 receptor, as follows. The cDNA coding sequence of hDRD2 (GenBank accession No. X51362) was subcloned into the mammalian expression vector pcDNA3.1/GS (Life Technologies). Clonal cell lines stably expressing hDRD2 were obtained by transfection into CHO-K1 cells and subsequently selected with culture medium containing 0.3 mg/mL of zeocin (Life Technologies).Membranes for in vitro receptor binding assays were obtained by the following procedures. CHO-K1 cells expressing hDRD2 were homogenized in ice-cold buffer with 10 mM Tris-HCl, 5 mM EDTA, 3 mM EGTA, 1 mM phenylmethylsuphonyl fluoride, pH 7.6 using Polytron PT10-35GT (Kinematica) at 18,000 rpm for 30 seconds and centrifuged at 500xg for 10 minutes. The supernatant containing the plasma membranes was centrifuged at 100,000xg for 30 minutes and the pellet was resuspended.
Radioligand Binding Assay: Membranes for in vitro receptor binding assays were obtained by the following procedures. CHO-K1 cells expressing one of the somatostatin receptors were homogenized in ice-cold buffer with 10 mM Tris-HCl, 5 mM EDTA, 3 mM EGTA, 1 mM phenylmethylsuphonyl fluoride, pH 7.6, using Polytron PT10-35GT (Kinematica) at 18,000 rpm for 30 seconds and centrifuged at 500xg for 10 minutes. The supernatant containing the plasma membranes was centrifuged at 100,000xg for 30 minutes and the pellet was resuspended in buffer containing 20 mM glycine-glycine, 1 mM MgCl2, 250 mM sucrose, pH 7.2, for storage at -80 C.For the SSTR1, 2 and 5 assays, membranes and various concentrations of test compounds were incubated in 96-well plates for 60 minutes at 25 C. with 0.05 nM [125I-Tyr11]-SRIF-14 (for hSSTR1; PerkinElmer Life Science), 0.05 nM [125I-Tyr]-seglitide (for hSSTR2; PerkinElmer Life Science) or 0.05 nM [125I-Tyr]-[DPhe-cyclo(Cys-Tyr-DTrp-Lys-Val-Cys)-Thr-NH2] (for hSSTR5.
Radioligand Binding Assay: Membranes for in vitro receptor binding assays were obtained by the following procedures. CHO-K1 cells expressing one of the somatostatin receptors were homogenized in ice-cold buffer with 10 mM Tris-HCl, 5 mM EDTA, 3 mM EGTA, 1 mM phenylmethylsuphonyl fluoride, pH 7.6, using Polytron PT10-35GT (Kinematica) at 18,000 rpm for 30 seconds and centrifuged at 500xg for 10 minutes. The supernatant containing the plasma membranes was centrifuged at 100,000xg for 30 minutes and the pellet was resuspended in buffer containing 20 mM glycine-glycine, 1 mM MgCl2, 250 mM sucrose, pH 7.2, for storage at -80 C.For the SSTR1, 2 and 5 assays, membranes and various concentrations of test compounds were incubated in 96-well plates for 60 minutes at 25 C. with 0.05 nM [125I-Tyr11]-SRIF-14 (for hSSTR1; PerkinElmer Life Science), 0.05 nM [125I-Tyr]-seglitide (for hSSTR2; PerkinElmer Life Science) or 0.05 nM [125I-Tyr]-[DPhe-cyclo(Cys-Tyr-DTrp-Lys-Val-Cys)-Thr-NH2] (for hSSTR5.
Radioligand Binding Assay: Membranes for in vitro receptor binding assays were obtained by the following procedures. CHO-K1 cells expressing one of the somatostatin receptors were homogenized in ice-cold buffer with 10 mM Tris-HCl, 5 mM EDTA, 3 mM EGTA, 1 mM phenylmethylsuphonyl fluoride, pH 7.6, using Polytron PT10-35GT (Kinematica) at 18,000 rpm for 30 seconds and centrifuged at 500xg for 10 minutes. The supernatant containing the plasma membranes was centrifuged at 100,000xg for 30 minutes and the pellet was resuspended in buffer containing 20 mM glycine-glycine, 1 mM MgCl2, 250 mM sucrose, pH 7.2, for storage at -80 C.For the SSTR1, 2 and 5 assays, membranes and various concentrations of test compounds were incubated in 96-well plates for 60 minutes at 25 C. with 0.05 nM [125I-Tyr11]-SRIF-14 (for hSSTR1; PerkinElmer Life Science), 0.05 nM [125I-Tyr]-seglitide (for hSSTR2; PerkinElmer Life Science) or 0.05 nM [125I-Tyr]-[DPhe-cyclo(Cys-Tyr-DTrp-Lys-Val-Cys)-Thr-NH2] (for hSSTR5.
Radioligand Binding Assay: Membranes for in vitro receptor binding assays were obtained by the following procedures. CHO-K1 cells expressing one of the somatostatin receptors were homogenized in ice-cold buffer with 10 mM Tris-HCl, 5 mM EDTA, 3 mM EGTA, 1 mM phenylmethylsuphonyl fluoride, pH 7.6, using Polytron PT10-35GT (Kinematica) at 18,000 rpm for 30 seconds and centrifuged at 500xg for 10 minutes. The supernatant containing the plasma membranes was centrifuged at 100,000xg for 30 minutes and the pellet was resuspended in buffer containing 20 mM glycine-glycine, 1 mM MgCl2, 250 mM sucrose, pH 7.2, for storage at -80 C.For the SSTR1, 2 and 5 assays, membranes and various concentrations of test compounds were incubated in 96-well plates for 60 minutes at 25 C. with 0.05 nM [125I-Tyr11]-SRIF-14 (for hSSTR1; PerkinElmer Life Science), 0.05 nM [125I-Tyr]-seglitide (for hSSTR2; PerkinElmer Life Science) or 0.05 nM [125I-Tyr]-[DPhe-cyclo(Cys-Tyr-DTrp-Lys-Val-Cys)-Thr-NH2] (for hSSTR5.
Radioligand Binding Assay: Membranes for in vitro receptor binding assays were obtained by the following procedures. CHO-K1 cells expressing one of the somatostatin receptors were homogenized in ice-cold buffer with 10 mM Tris-HCl, 5 mM EDTA, 3 mM EGTA, 1 mM phenylmethylsuphonyl fluoride, pH 7.6, using Polytron PT10-35GT (Kinematica) at 18,000 rpm for 30 seconds and centrifuged at 500xg for 10 minutes. The supernatant containing the plasma membranes was centrifuged at 100,000xg for 30 minutes and the pellet was resuspended in buffer containing 20 mM glycine-glycine, 1 mM MgCl2, 250 mM sucrose, pH 7.2, for storage at -80 C.For the SSTR1, 2 and 5 assays, membranes and various concentrations of test compounds were incubated in 96-well plates for 60 minutes at 25 C. with 0.05 nM [125I-Tyr11]-SRIF-14 (for hSSTR1; PerkinElmer Life Science), 0.05 nM [125I-Tyr]-seglitide (for hSSTR2; PerkinElmer Life Science) or 0.05 nM [125I-Tyr]-[DPhe-cyclo(Cys-Tyr-DTrp-Lys-Val-Cys)-Thr-NH2] (for hSSTR5.