Biological characterization of rodent and human vasopressin V1b receptors using SSR-149415, a nonpeptide V1b receptor ligand.
[(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.
Serradeil-Le Gal
Claudine
C
Sanofi-Aventis Recherche and Développement, Exploratory Research Department, 195, route d'Espagne, BP 1169, 31036 Toulouse Cedex, France. claudine.serradeil@sanofi-aventis.com
Raufaste
Danièle
D
Derick
Sylvain
S
Blankenstein
Jörg
J
Allen
John
J
Pouzet
Brigitte
B
Pascal
Marc
M
Wagnon
Jean
J
Ventura
Maria Angeles
MA
D000818:Animals; D001345:Autoradiography / Q000379:methods*; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D006224:Cricetinae; D018076:DNA, Complementary; D004705:Endocytosis; D006801:Humans; D007211:Indoles / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D007295:Inositol Phosphates / Q000378:metabolism; D008024:Ligands; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D011759:Pyrrolidines / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011869:Radioligand Assay / Q000379:methods*; D017483:Receptors, Vasopressin / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D014316:Tritium
C456029:1-(5-chloro-1-((2,4-dimethoxyphenyl)sulfonyl)-3-(2-methoxyphenyl)-2-oxo-2,3-dihydro-1H-indol-3-yl)-4-hydroxy-N,N-dimethyl-2-pyrrolidinecarboxamide; D018076:DNA, Complementary; D007211:Indoles; D007295:Inositol Phosphates; D008024:Ligands; D008565:Membrane Proteins; D011759:Pyrrolidines; D017483:Receptors, Vasopressin; D011994:Recombinant Proteins; D014316:Tritium; D002118:Calcium