一种新型神经肽Y Y5激动剂放射性配体的表征：[125I][cPP(1-7)，NPY(19-23)，Ala31，Aib32，Gln34]hPP

Dumont Yvan, Thakur Mira, Beck-Sickinger Annette, Fournier Alain, Quirion Rémi

Department of Psychiatry, Douglas Hospital Research Centre, McGill University, 6875 Boul. LaSalle, Montréal Verdun, Que., Canada H4H 1R3.

Neuropeptides. 2004 Aug;38(4):163-74. doi: 10.1016/j.npep.2004.04.007.

In order to optimally characterize a class of neuropeptide Y (NPY) receptors expressed in a tissue enriched with multiple subtypes (Y1, Y2, Y4 and Y5) and to establish its detailed distribution, it is critical to use highly selective and specific probes that possess very low non-specific binding. In that context, we recently reported on the development of [125I][hPP(1-17), Ala31, Aib32]NPY as Y5 receptor radioligand. However, the non-specific binding obtained with this radioligand was too high to allow for detailed receptor autoradiography studies [Br. J. Pharmacol. 139 (2003) 1360]. Iodinated [cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP may represent a better Y5 radioligand in that regard. Accordingly, [125I][cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP binding was investigated in rat brain membrane homogenates and its specificity and selectivity established in rat Y1, Y2, Y4 and Y5 transfected HEK293 cells. No specific binding was detected in HEK293 cells transfected with the rat Y1, Y2 or Y4 receptors, while saturable binding was observed in cells transfected with the rat Y5 receptor cDNA and in rat brain membrane homogenates (KD of 0.5-0.7 nM). Competition binding experiments performed in rat brain membrane homogenates demonstrated that specific [125I][cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP binding was competed with nanomolar affinities by Y5 agonists and antagonists such as [Leu31,Pro34]PYY, PYY(3-36), [cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP, [Ala31, Aib32]NPY, [hPP(1-17), Ala31, Aib32]NPY, CGP71683A and JCF109, but not by Y1 (BIBP3226 and BIBO3304), Y2 (BIIE0246) and Y4 (GR231118) ligands. Non-specific binding was also lower than that reported for [125I][hPP(1-17), Ala31, Aib32]NPY. Interestingly, detailed analysis of competition binding curves obtained with [Leu31, Pro34]PYY, hPP, PYY(3-36) and [cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP against specific [125I][cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP sites were best fitted to a two-site model. Additionally, receptor autoradiography studies revealed the presence of specific [125I][cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP binding sites in the lateral septum and area postrema while other brain regions contained much lower levels of specific binding. Taken together, these data suggest that [125I][cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP represents a useful tool to study the unique feature of the Y5 receptor subtype.

为了最佳地表征在富含多种亚型（Y1、Y2、Y4和Y5）的组织中表达的一类神经肽Y（NPY）受体，并确定其详细分布，使用具有非常低非特异性结合的高度选择性和特异性探针至关重要。在此背景下，我们最近报道了[125I][hPP(1 - 17)，Ala31，Aib32]NPY作为Y5受体放射性配体的开发。然而，这种放射性配体获得的非特异性结合过高，无法进行详细的受体放射自显影研究[《英国药理学期刊》139 (2003) 1360]。在这方面，碘化[cPP(1 - 7)，NPY(19 - 23)，Ala31，Aib32，Gln34]hPP可能是一种更好的Y5放射性配体。因此，研究了[125I][cPP(1 - 7)，NPY(19 - 23)，Ala31，Aib32，Gln34]hPP在大鼠脑膜匀浆中的结合情况，并在转染了大鼠Y1、Y2、Y4和Y5的HEK293细胞中确定了其特异性和选择性。在用大鼠Y1、Y2或Y4受体转染的HEK293细胞中未检测到特异性结合，而在用大鼠Y5受体cDNA转染的细胞和大鼠脑膜匀浆中观察到了饱和结合（KD为0.5 - 0.7 nM）。在大鼠脑膜匀浆中进行的竞争结合实验表明，特异性的[125I][cPP(1 - 7)，NPY(19 - 23)，Ala31，Aib32，Gln34]hPP结合可被Y5激动剂和拮抗剂如[Leu31，Pro34]PYY、PYY(3 - 36)、[cPP(1 - 7)，NPY(19 - 23)，Ala31，Aib32，Gln34]hPP、[Ala31，Aib32]NPY、[hPP(1 - 17)，Ala31，Aib32]NPY、CGP71683A和JCF109以纳摩尔亲和力竞争，但不能被Y1（BIBP3226和BIBO3304）、Y2（BIIE0246）和Y4（GR231118）配体竞争。非特异性结合也低于[125I][hPP(1 - 17)，Ala31，Aib32]NPY报道的水平。有趣的是，对[Leu31，Pro34]PYY、hPP、PYY(3 - 36)和[cPP(1 - 7)，NPY(19 - 23)，Ala31，Aib32，Gln34]hPP针对特异性[125I][cPP(1 - 7)，NPY(19 - 23)，Ala31，Aib32，Gln34]hPP位点获得的竞争结合曲线的详细分析最适合双位点模型。此外，受体放射自显影研究显示在外侧隔和最后区存在特异性的[125I][cPP(1 - 7)，NPY(19 - 23)，Ala31，Aib32，Gln34]hPP结合位点，而其他脑区的特异性结合水平要低得多。综上所述，这些数据表明[125I][cPP(1 - 7)，NPY(19 - 23)，Ala31，Aib32，Gln34]hPP是研究Y5受体亚型独特特征的有用工具。