Biological characterization of rodent and human vasopressin V sub(1b) receptors using SSR-149415, a nonpeptide V sub(1b) receptor ligand

[ super(3)H]SSR-149415 is the first tritiated nonpeptide vasopressin V sub(1b) receptor (V sub(1b)R) antagonist ligand. It was used for studying rodent (mouse, rat, hamster) and human V sub(1b)R from native or recombinant origin. Moreover, a close comparison between the human and the mouse V sub(1b)R was performed using SSR-149415/[ super(3)H]SSR-149415 in binding and functional studies in vitro. [ super(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 sub(d)) similar to 1 nM and maximum binding density (B sub(max)) values from 7,000 to 300,000 sites/cell according to the cell line. In competition experiments, [ super(3)H]SSR-149415 binding was stereospecific and dose-dependently displaced by reference peptide and nonpeptide arginine vasopressin (AVP)/OT ligands following a V sub(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 sub(1b)R were observed. Autoradiography studies with [ super(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 super(2+) increase (EC sub(50) from 1.83 to 3.05 nM) in recombinant cell lines expressing either the mouse or the human V sub(1b)R. AVP (10 super(-7) M) exposure of AtT20 cells expressing mouse or human EGFP-tagged V sub(1b)R induced their rapid internalization. Preincubation with 10 super(-6) M SSR-149415 counteracted the internalization process. Moreover, recycling of internalized receptors was observed upon 10 super(-6) M SSR-149415 treatment. Thus SSR-149415/[ super(3)H]SSR-149415 are unique tools for studying animal and human V sub(1b)R.