Biological characterization of rodent and human vasopressin V1b receptors using SSR-149415, a nonpeptide V1b receptor ligand

1 Exploratory Research Department, Sanofi-Aventis Recherche and Développement, Toulouse Cedex; 2 Isotope Chemistry and Metabolite Synthesis, Sanofi-Aventis Recherche and Développement, Chilly Mazarin Cedex; 3 Oncology Deparment, Sanofi-Aventis Recherche and Développement, Montpellier Cedex; 4 Institut Cochin, Département d'Endocrinologie Metabolisme et Cancer, Université Paris Descartes, CNRS (UMR 8104), Paris; and 5 Institut National de la Santé et de la Recherche Médicale, Unité 567, Paris, France Submitted 26 January 2007 ; accepted in final form 17 May 2007 [ 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 ) 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. vasopressin; V 1b receptor; rat; h