Dumont Yvan, Thakur Mira, Beck-Sickinger Annette, Fournier Alain, Quirion Rémi
Department of Psychiatry, Douglas Hospital Research Centre, McGill University, 6875 Boul LaSalle, Verdun (Montréal), QC, Canada H4H 1R3.
Br J Pharmacol. 2003 Aug;139(7):1360-8. doi: 10.1038/sj.bjp.0705376.
(1) The existence of multiple classes of neuropeptide Y (NPY) receptors (Y(1), Y(2), Y(4), Y(5) and y(6)) 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 Y(4) and Y(5) subtypes. (2) [hPP(1-17), Ala(31), Aib(32)]NPY, the first highly selective Y(5) agonist, was iodinated using the chloramine T method and purified by high-pressure liquid chromatography. (3) Binding performed in rat brain homogenates revealed that equilibrium was reached after 120 min (t(1/2)=21 min) and 60 min (t(1/2)=12 min) at 25 and 100 pM [(125)I][hPP(1-17), Ala(31), Aib(32)]NPY, respectively. (4) Isotherm saturation binding experiments demonstrated that [(125)I][hPP(1-17), Ala(31), Aib(32)]NPY binds to an apparent single population with high-affinity (K(D) of 1.2 and 1.7 nM) and low-capacity (B(max) of 14+/-3 fmol/100,000 cells and 20+/-5 fmol/mg protein) sites in Y(5) receptor HEK293-transfected cells and rat brain membrane homogenates, respectively. No specific [(125)I][hPP(1-17), Ala(31), Aib(32)]NPY binding sites could be detected in Y(1), Y(2) or Y(4) receptors transfected HEK293 cells, demonstrating the high selectivity of this ligand for the Y(5) subtype. (5) Competition binding experiments performed in rat brain membrane homogenates and Y(5)-receptor transfected HEK293 cells demonstrated that specific [(125)I][hPP(1-17), Ala(31), Aib(32)]NPY binding was competed with high affinity by Y(5) agonists and antagonists such as [Ala(31), Aib(32)]NPY, [hPP(1-17), Ala(31), Aib(32)]NPY, hPP, CGP71683A and JCF109, but not by Y(1) (BIBP3226), Y(2) (BIIE0246) and Y(1)/Y(4) (GR231118) preferential ligands. (6) Taken together, these data demonstrate that [(125)I][hPP(1-17), Ala(31), Aib(32)]NPY is the first highly selective Y(5) 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.
(1) 现已充分证实存在多种神经肽Y（NPY）受体类别（Y(1)、Y(2)、Y(4)、Y(5)和y(6)）。然而，研究这些不同受体亚型的主要困难之一是目前缺乏用于研究单一受体类别的高选择性探针。直到最近，对于Y(4)和Y(5)亚型尤其如此。(2) [hPP(1 - 17)，Ala(31)，Aib(32)]NPY，首个高选择性Y(5)激动剂，采用氯胺T法进行碘化，并通过高压液相色谱法纯化。(3) 在大鼠脑匀浆中进行的结合实验表明，在25 pM和100 pM的[(125)I][hPP(1 - 17)，Ala(31)，Aib(32)]NPY浓度下，分别在120分钟（t(1/2)=21分钟）和60分钟（t(1/2)=12分钟）后达到平衡。(4) 等温饱和结合实验表明，[(125)I][hPP(1 - 17)，Ala(31)，Aib(32)]NPY分别在Y(5)受体HEK293转染细胞和大鼠脑膜匀浆中以高亲和力（解离常数K(D)分别为1.2和1.7 nM）和低容量（最大结合量B(max)分别为14±3 fmol/100,000个细胞和20±5 fmol/mg蛋白质）与明显单一的群体结合。在Y(1)、Y(2)或Y(4)受体转染的HEK293细胞中未检测到特异性的[(125)I][hPP(1 - 17)，Ala(31)，Aib(32)]NPY结合位点，这表明该配体对Y(5)亚型具有高选择性。(5) 在大鼠脑膜匀浆和Y(5)受体转染的HEK293细胞中进行的竞争结合实验表明，特异性的[(125)I][hPP(1 - 17)，Ala(31)，Aib(32)]NPY结合可被Y(5)激动剂和拮抗剂如[Ala(31)，Aib(32)]NPY、[hPP(1 - 17)，Ala(31)，Aib(32)]NPY、hPP、CGP71683A和JCF109以高亲和力竞争，但不能被Y(1)（BIBP3226）、Y(2)（BIIE0246）和Y(1)/Y(4)（GR231118）优先配体竞争。(6) 综上所述，这些数据表明[(125)I][hPP(1 - 17)，Ala(31)，Aib(32)]NPY是首个研发出的高选择性Y(5)放射性配体。这种新探针对于进一步详细研究该受体亚型在脑和外周组织中的分子和药理学特性应证明是非常有用的。
