Binding Assay: cDNA sequence encoding mouse NPY Y5 receptor (Biochem. Biophys. Acta 1328: 83-89, 1997) was cloned in the expression vector (pME18S, Takebe et al. Mol. Cell. Biol. 8, 466-472). The obtained expression vector was transfected into CHO cells as a host according to the instruction manual using Lipofectamine reagent (Trademark, Gibco BRL Co., Ltd.). The cells that stably express NPY Y5 receptor were obtained.The membrane samples prepared from the CHO cells expressing NPY Y5 receptor, the compound of the invention and 30,000 cpm 125I peptide YY (60 pM of final concentration: Amersham) were incubated in the assay buffer (20 mM HEPES-Hanks buffer containing 0.1% bovine serum albumin, pH 7.4) at 25° C. for 2 hours. The membrane samples were then filtered from the mixture through a glassfilter (GF/C) presoaked with 1% polyethyleneimine. After washing with 50 mM Tris-HCl buffer (pH 7.4), radioactivity retained on the filters was determined using gamma counter.
Binding Assay: cDNA sequence encoding a mouse NPY Y5 receptor (Biochim. Biophys. Acta 1328:83-89, 1997) was cloned in a vector (pME18S, Takebe et al. Mol. Cell. Biol. 8, 8957). The obtained expression vector was transfected into CHO cells as a host by using Lipofect AMINE reagent (Trademark, Gico BRL Co., Ltd.) according to the instruction manual. The cells that stably express NPY Y5 receptor were obtained.The membranes prepared from the CHO cells expressing NPY Y5 receptor, the compound of this invention and 30,000 cpm [125I] peptide YY (60 pM of final concentration: Healthcare) were incubated in the assay buffer (20 mM HEPES-Hanks buffer containing 0.1% bovine serum albumin, pH 7.4) at 25° C. for 2 hours, and then the membrane was filtered from the mixture through a glass filter (GF/C) presoaked with 1% polyethyleneimine. After washing with 50 mM Tris-HCl buffer (pH 7.4), radioactivity retained on the filters was quantified with a gamma counter.
Allosteric antagonist activity at NPY5R in mouse descending colon mucosa assessed as reduction in rPP-induced ion transport at 30 uM by electrophysiology