Cell Biology – Immunology – Pathology
Kidney International (2000) 58, 1613–1622; doi:10.1046/j.1523-1755.2000.00322.x
Binding properties of a selective tritiated vasopressin V2 receptor antagonist, [3H]-SR 121463
Claudine Serradeil-Le Gal, Danielle Raufaste, Eléonore Double-Cazanave, Gilles Guillon, Corinne Garcia, Marc Pascal and Jean Pierre Maffrand
Exploratory Research Department, Sanofi-Synthelabo Recherche, Toulouse, and INSERM U-469, CCIPE, Montpellier, France
Correspondence: Dr Claudine Serradeil-Le Gal, Exploratory Research Department, Sanofi-Synthelabo Recherche, 195, route d'Espagne, 31036 Toulouse Cedex, France. E-mail: claudine.serradeil@sanofi-synthelabo.com
Received 10 December 1999; Revised 15 March 2000; Accepted 12 May 2000.
Abstract
Binding properties of a selective tritiated vasopressin V2 receptor antagonist, [3H]-SR 121463.
Background
[3H]-SR 121463 is the first radiolabeled selective nonpeptide vasopressin V2 receptor antagonist ligand that has been reported to date. In the present work, we studied the binding properties of [3H]-SR 121463 for renal V2 receptors from animal and human origins.
Methods
Binding studies were performed with [3H]-SR 121463 in Chinese hamster ovary (CHO) cells transfected with the human V2 receptor and in various kidney preparations expressing the native V2 receptors (rat, rabbit, dog, pig, monkey, and human). Autoradiographies were performed in rat and human kidney sections.
Results
[3H]-SR 121463 binding to CHO cells stably transfected with the cloned human renal V2 receptor was specific, highly stable, time dependent, saturable, and reversible. A single population of high-affinity binding sites was identified (Kd = 0.94 
0.34 nmol/L, Bmax = 9876 317 fmol/mg protein). Of note, [3H]-SR 121463 revealed a higher number (about 40%) of V2 sites than [3H]-AVP in the same preparation. Displacement of [3H]-SR 121463 binding by reference peptide and nonpeptide vasopressin/oxytocin compounds exhibited a typical AVP V2 profile. [3H]-SR 121463 also displayed a high affinity for native V2 receptors in several kidney preparations from rat, pig, dog, rabbit, bovine, monkey, and human. The autoradiographic experiments using rat and human kidney sections showed intense labeling in the medullopapillary region and lower intensity in the cortex, consistent with a main localization of V2 receptors on collecting tubules.
Conclusion
[3H]-SR 121463 is a useful ligand for the specific labeling of animal and human V2 receptors and could be a suitable probe for the search and in situ localization of V2 sites.
Keywords:
kidney, antidiuretic hormone, arginine vasopressin, homeostasis, water regulation, ligand
Abbreviations:
AVP, arginine vasopressin; Bmax, maximum binding density; BSA, bovine serum albumin; CHO, Chinese hamster ovary; dDAVP, 1-desamino-[D-Arg8]-vasopressin; DMEM, Dulbecco's modified Eagle's medium; DMSO, dimethyl sulfoxide; EDTA, ethylediaminetetraacetic acid; k+1, association rate constant; k-1, dissociation rate constant; Kd, dissociation constant; Ki, inhibition constant; kobs, observed rate constant; MEM, minimal essential medium; nH, Hill coefficient; OT, oxytocin; SCLC, small cell lung cancer; SIADH, syndrome of inappropriate antidiuretic hormone secretion; SR 121463, 1-[4-(N-tert-butylcarbamoyl)-2-meth-oxybenzene sulfonyl]-5-ethoxy-3-spiro-[4-(2-morpholinoethoxy-cyclohexane)] indoline-2-one, cis-isomer
