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WATER AND ELECTROLYTE HOMEOSTASIS

Biological characterization of rodent and human vasopressin V_1b receptors using SSR-149415, a nonpeptide V_1b receptor ligand

¹Exploratory Research Department, Sanofi-Aventis Recherche and Développement, Toulouse Cedex; ²Isotope Chemistry and Metabolite Synthesis, Sanofi-Aventis Recherche and Développement, Chilly Mazarin Cedex; ³Oncology Deparment, Sanofi-Aventis Recherche and Développement, Montpellier Cedex; ⁴Institut Cochin, Département d'Endocrinologie Metabolisme et Cancer, Université Paris Descartes, CNRS (UMR 8104), Paris; and ⁵Institut National de la Santé et de la Recherche Médicale, Unité 567, Paris, France

Submitted 26 January 2007 ; accepted in final form 17 May 2007

[³H]SSR-149415 is the first tritiated nonpeptide vasopressin V_1b receptor (V_1bR) antagonist ligand. It was used for studying rodent (mouse, rat, hamster) and human V_1bR from native or recombinant origin. Moreover, a close comparison between the human and the mouse V_1bR was performed using SSR-149415/[³H]SSR-149415 in binding and functional studies in vitro. [³H]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) 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, [³H]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_1bR were observed. Autoradiography studies with [³H]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 Ca²⁺ increase (EC₅₀ from 1.83 to 3.05 nM) in recombinant cell lines expressing either the mouse or the human V_1bR. AVP (10^–7 M) exposure of AtT20 cells expressing mouse or human EGFP-tagged V_1bR 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/[³H]SSR-149415 are unique tools for studying animal and human V_1bR.

vasopressin; V_1b receptor; rat; hamster; mouse; human