Elshourbagy Nabil A, Douglas Stephen A, Shabon Usman, Harrison Stephen, Duddy Graham, Sechler Jan L, Ao Zhaohui, Maleeff Beverly E, Naselsky Diane, Disa Jyoti, Aiyar Nambi V
Department of Expression Genomics, GlaxoSmithKline Pharmaceuticals, King of Prussia, Pennsylvania, PA 19406, USA.
Br J Pharmacol. 2002 May;136(1):9-22. doi: 10.1038/sj.bjp.0704671.
Urotensin-II (U-II) and its receptor (UT) represent novel therapeutic targets for management of a variety of cardiovascular diseases. To test such hypothesis, it will be necessary to develop experimental animal models for the manipulation of U-II/UT receptor system. The goal of this study was to clone mouse and primate preproU-II and UT for pharmacological profiling. Monkey and mouse preproU-II genes were identified to encode 123 and 125 amino acids. Monkey and mouse UT receptors were 389, and 386 amino acids, respectively. Genomic organization of mouse genes showed that the preproU-II has four exons, while the UT receptor has one exon. Although initially viewed by many exclusively as cardiovascular targets, the present study demonstrates expression of mouse and monkey U-II/UT receptor mRNA in extra-vascular tissue including lung, pancreas, skeletal muscle, kidney and liver. Ligand binding studies showed that [125I]h U-II bound to a single sites to the cloned receptors in a saturable/high affinity manner (Kd 654+/-154 and 214+/-65 pM and Bmax of 1011+/-125 and 497+/-68 fmol mg-1 for mouse and monkey UT receptors, respectively). Competition binding analysis demonstrated equipotent, high affinity binding of numerous mammalian, amphibian and piscine U-II isopeptides to these receptors (Ki=0.8 - 3 nM). Fluorescein isothiocyanate (FITC) labelled U-II, bound specifically to HEK-293 cells expressing mouse or monkey UT receptor, confirming cell surface expression of recombinant UT receptor. Exposure of these cells to human U-II resulted in an increase in intracellular [Ca2+] concentrations (EC50 3.2+/-0.8 and 1.1+/-0.3 nM for mouse and monkey UT receptors, respectively) and inositol phosphate (Ip) formation (EC50 7.2+/-1.8 and 0.9+/-0.2 nM for mouse and monkey UT receptors, respectively) consistent with the primary signalling pathway for UT receptor involving phospholipase C activation.
尾加压素 II（U-II）及其受体（UT）是多种心血管疾病治疗的新靶点。为验证这一假设，有必要建立用于操控 U-II/UT 受体系统的实验动物模型。本研究的目的是克隆小鼠和灵长类动物的前体 U-II 和 UT，以进行药理学分析。已鉴定出猴和小鼠的前体 U-II 基因分别编码 123 和 125 个氨基酸。猴和小鼠的 UT 受体分别为 389 和 386 个氨基酸。小鼠基因的基因组结构显示，前体 U-II 有四个外显子，而 UT 受体有一个外显子。尽管许多人最初仅将其视为心血管靶点，但本研究表明，小鼠和猴的 U-II/UT 受体 mRNA 在包括肺、胰腺、骨骼肌、肾脏和肝脏在内的血管外组织中表达。配体结合研究表明，[125I]h U-II 以饱和/高亲和力的方式与克隆的受体结合于单一位点（小鼠和猴 UT 受体的解离常数 Kd 分别为 654±154 和 214±65 pM，最大结合量 Bmax 分别为 1011±125 和 497±68 fmol mg-1）。竞争结合分析表明，多种哺乳动物、两栖动物和鱼类的 U-II 异肽对这些受体具有等效、高亲和力的结合（抑制常数 Ki = 0.8 - 3 nM）。异硫氰酸荧光素（FITC）标记的 U-II 特异性结合表达小鼠或猴 UT 受体的 HEK-293 细胞，证实重组 UT 受体在细胞表面表达。将这些细胞暴露于人类 U-II 会导致细胞内[Ca2+]浓度升高（小鼠和猴 UT 受体的半数有效浓度 EC50 分别为 3.2±0.8 和 1.1±0.3 nM）以及肌醇磷酸（Ip）生成增加（小鼠和猴 UT 受体的 EC50 分别为 7.2±1.8 和 0.9±0.2 nM），这与 UT 受体涉及磷脂酶 C 激活的主要信号通路一致。
