Submitted on January 26, 2007
Accepted on May 17, 2007
Biological characterization of rodent and human vasopressin V1b receptors using SSR149415, a nonpeptide V1b receptor ligand
Claudine Serradeil-Le Gal1*, Daniele Raufaste1, Sylvain Derick1, Jorg Blankenstein2, John Allen3, Brigitte Pouzet1, Marc Pascal1, Jean Wagnon4, and Maria Angeles Ventura5
1 Exploratory Research, Sanofi-aventis, Toulouse, France
2 Isotope Chemistry and Metabolite Synthesis, Sanofi-aventis, Chilly-Mazarin, France
3 Isotope Chemistry and Metabolite Synthesis, Sanofi-aventis, Chilly Mazarin, France
4 Oncology, Sanofi-aventis, Montpellier, France
5 Endocrinology, Institut Cochin, Paris, France; U.567, INSERM, Paris, France; UMR 8104, CNRS, Paris, France
* To whom correspondence should be addressed. E-mail: claudine.serradeil{at}sanofi-aventis.com.
[3H]-SSR149415 is the first tritiated nonpeptide vasopressin V1b receptor antagonist ligand. It was used for studying rodent (mouse, rat, hamster) and human V1b receptors from native or recombinant origin. Moreover, a close comparison between the human and the mouse V1bR was performed using SSR149415/[3H]-SSR149415 in binding and functional studies in vitro. [3H]-SSR149415 binding was time-dependent, reversible and saturable. Scatchard plot analysis gave a single class of high affinity binding sites with apparent equilibrium dissociation constant (Kd) around 1 nM and Bmax values from 7,000 to 300,000 sites/cell according to the cell line. In competition experiments, [3H]-SSR149415 binding was stereospecific and dose-dependently displaced by reference peptide and nonpeptide AVP/OT ligands following a V1b rank order of affinity: SSR149415=AVP>dCha>dPen>dPal>dDavp>SSR126768A>SR49059>SSR149424>OT>SR121463B. Species differences between human, rat, mouse and hamster V1b receptors were observed. Autoradiography studies with [3H]-SSR149415 on rat and human pituitary showed intense specific labelling confined to corticotroph cells and absence of labelling in the other tissues examined. SSR149415 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 V1b receptors. AVP (10-7 M) exposure of AtT20 cells expressing mouse or human EGFP-tagged V1b receptors induced their rapid internalization. Preincubation with 10-6 M SSR149415 counteracted the internalization process. Moreover, recycling of internalized receptors was observed upon 10-6 M SSR149415 treatment. Thus, SSR149415/[3H]-SSR149415 are unique tools for studying animal and human V1b receptors.
