Development and characterization of a highly selective neuropeptide Y Y5 receptor agonist radioligand: [125I][hPP1–17, Ala31, Aib32]NPY

摘要

The existence of multiple classes of neuropeptide Y (NPY) receptors (Y1, Y2, Y4, Y5 and y6) is now well established. However, one of the major difficulties in the study of these various receptor subtypes is the current lack of highly selective probes to investigate a single receptor class. Up to most recently, this was particularly true for the Y4 and Y5 subtypes.[hPP1–17, Ala31, Aib32]NPY, the first highly selective Y5 agonist, was iodinated using the chloramine T method and purified by high-pressure liquid chromatography.Binding performed in rat brain homogenates revealed that equilibrium was reached after 120 min (t1/2=21 min) and 60 min (t1/2=12 min) at 25 and 100 pM [125I][hPP1–17, Ala31, Aib32]NPY, respectively.Isotherm saturation binding experiments demonstrated that [125I][hPP1–17, Ala31, Aib32]NPY binds to an apparent single population with high-affinity (KD of 1.2 and 1.7 nM) and low-capacity (Bmax of 14±3 fmol/100,000 cells and 20±5 fmol/mg protein) sites in Y5 receptor HEK293-transfected cells and rat brain membrane homogenates, respectively. No specific [125I][hPP1–17, Ala31, Aib32]NPY binding sites could be detected in Y1, Y2 or Y4 receptors transfected HEK293 cells, demonstrating the high selectivity of this ligand for the Y5 subtype.Competition binding experiments performed in rat brain membrane homogenates and Y5-receptor transfected HEK293 cells demonstrated that specific [125I][hPP1–17, Ala31, Aib32]NPY binding was competed with high affinity by Y5 agonists and antagonists such as [Ala31, Aib32]NPY, [hPP1–17, Ala31, Aib32]NPY, hPP, CGP71683A and JCF109, but not by Y1 (BIBP3226), Y2 (BIIE0246) and Y1/Y4 (GR231118) preferential ligands.Taken together, these data demonstrate that [125I][hPP1–17, Ala31, Aib32]NPY is the first highly selective Y5 radioligand to be developed. This new probe should prove most useful for further detailed studies of the molecular and pharmacological properties of this receptor subtype in brain and peripheral tissues.