Serradeil-Le Gal Claudine, Raufaste Danièle, Derick Sylvain, Blankenstein Jörg, Allen John, Pouzet Brigitte, Pascal Marc, Wagnon Jean, Ventura Maria Angeles
Sanofi-Aventis Recherche and Développement, Exploratory Research Department, 195, route d'Espagne, BP 1169, 31036 Toulouse Cedex, France.
Am J Physiol Regul Integr Comp Physiol. 2007 Aug;293(2):R938-49. doi: 10.1152/ajpregu.00062.2007. Epub 2007 May 23.
[(3)H]SSR-149415 is the first tritiated nonpeptide vasopressin V(1b) receptor (V(1b)R) antagonist ligand. It was used for studying rodent (mouse, rat, hamster) and human V(1b)R from native or recombinant origin. Moreover, a close comparison between the human and the mouse V(1b)R was performed using SSR-149415/[(3)H]SSR-149415 in binding and functional studies in vitro. [(3)H]SSR-149415 binding was time-dependent, reversible, and saturable. Scatchard plot analysis gave a single class of high-affinity binding sites with apparent equilibrium dissociation constant (K(d)) approximately 1 nM and maximum binding density (B(max)) values from 7,000 to 300,000 sites/cell according to the cell line. In competition experiments, [(3)H]SSR-149415 binding was stereospecific and dose-dependently displaced by reference peptide and nonpeptide arginine vasopressin (AVP)/OT ligands following a V(1b) rank order of affinity: SSR-149415 = AVP > dCha > dPen > dPal > dDavp > SSR-126768A > SR-49059 > SSR-149424 > OT > SR-121463B. Species differences between human, rat, mouse, and hamster V(1b)R were observed. Autoradiography studies with [(3)H]SSR-149415 on rat and human pituitary showed intense specific labeling confined to corticotroph cells and absence of labeling in the other tissues examined. SSR-149415 potently and stereospecifically antagonized the AVP-induced inositol phosphate production and intracellular Ca(2+) increase (EC(50) from 1.83 to 3.05 nM) in recombinant cell lines expressing either the mouse or the human V(1b)R. AVP (10(-7) M) exposure of AtT20 cells expressing mouse or human EGFP-tagged V(1b)R induced their rapid internalization. Preincubation with 10(-6) M SSR-149415 counteracted the internalization process. Moreover, recycling of internalized receptors was observed upon 10(-6) M SSR-149415 treatment. Thus SSR-149415/[(3)H]SSR-149415 are unique tools for studying animal and human V(1b)R.
[³H]SSR - 149415是首个氚标记的非肽类血管加压素V(1b)受体（V(1b)R）拮抗剂配体。它被用于研究来自天然或重组来源的啮齿动物（小鼠、大鼠、仓鼠）及人类的V(1b)R。此外，在体外结合和功能研究中，使用SSR - 149415/[³H]SSR - 149415对人类和小鼠的V(1b)R进行了细致比较。[³H]SSR - 149415的结合具有时间依赖性、可逆性和饱和性。Scatchard图分析显示存在一类单一的高亲和力结合位点，其表观平衡解离常数（K(d)）约为1 nM，根据细胞系不同，最大结合密度（B(max)）值在7000至300000个位点/细胞之间。在竞争实验中，[³H]SSR - 149415的结合具有立体特异性，并且按照V(1b)亲和力排序，参考肽和非肽类精氨酸血管加压素（AVP）/催产素（OT）配体可剂量依赖性地取代[³H]SSR - 149415的结合：SSR - 149415 = AVP > dCha > dPen > dPal > dDavp > SSR - 126768A > SR - 49059 > SSR - 149424 > OT > SR - 121463B。观察到人类、大鼠、小鼠和仓鼠V(1b)R之间存在物种差异。用[³H]SSR - 149415对大鼠和人类垂体进行放射自显影研究显示，强烈的特异性标记局限于促肾上腺皮质激素细胞，在所检查的其他组织中无标记。SSR - 149415在表达小鼠或人类V(1b)R的重组细胞系中，能有效且立体特异性地拮抗AVP诱导的肌醇磷酸生成和细胞内Ca²⁺增加（半数有效浓度（EC(50)）为1.83至3.05 nM）。暴露于10⁻⁷ M的AVP会诱导表达小鼠或人类EGFP标记的V(1b)R的AtT20细胞快速内化。用10⁻⁶ M SSR - 149415预孵育可抵消内化过程。此外，在10⁻⁶ M SSR - 149415处理后观察到内化受体的再循环。因此，SSR - 149415/[³H]SSR - 149415是研究动物和人类V(1b)R的独特工具。
