Volume 58, Issue 4, Pages 1613-1622 (October 2000) 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, Jean Pierre Maffrand  Kidney International  Volume 58, Issue 4, Pages 1613-1622 (October 2000) DOI: 10.1046/j.1523-1755.2000.00322.x Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 1 Chemical structure of [3H]-SR 121463. The * indicates the position of tritium. (Reproduced from the Journal of Clinical Investigation27, with permission from the American Society for Clinical Investigation.) Kidney International 2000 58, 1613-1622DOI: (10.1046/j.1523-1755.2000.00322.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 2 Time–course of association (•) and dissociation (○) of [3H]-SR 121463 to membranes of Chinese hamster ovary (CHO) cells expressing the human renal arginine vasopressin (AVP) V2 receptors. Incubations were carried out as described in the Methods section in the presence of 2 nmol/L [3H]-SR 121463 for various periods of time. The arrow indicates time (45 min) at which unlabeled SR 121463 (1 μmol/L) was added to initiate the dissociation process. The results represent data from a typical experiment performed in duplicate and repeated three times without noticeable modifications. Kidney International 2000 58, 1613-1622DOI: (10.1046/j.1523-1755.2000.00322.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 3 Saturation of [3H]-SR 121463 (•) and [3H]-AVP (▪) specific binding to membranes of CHO cells transfected with the human renal AVP V2 receptors. (A) Saturation isotherms. (B) Scatchard plots. Incubations were performed for 45 minutes at 25°C in the presence of 10 μg protein/mL and increasing concentrations of [3H]-SR 121463 or [3H]-AVP as described in the Methods section. Data are means calculated from a typical experiment performed in duplicate and repeated three times without noticeable changes. Kidney International 2000 58, 1613-1622DOI: (10.1046/j.1523-1755.2000.00322.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 4 Inhibition of [3H]-SR 121463-specific binding to membranes of CHO cells expressing the human renal AVP V2 receptor by reference nonpeptide (A) and peptide (B) AVP/OT compounds. Incubations were carried out in the presence of 1.5 nmol/L [3H]-SR 121463 and increasing concentrations of the compound to be tested. Symbols in (A) for nonpeptide compounds: (○) SR 121463; (•) YM-087; (□) VPA 985; (▪) OPC-31260; (▾) SR 49059; (▿) OPC-21268. Symbols in (B) for peptide compounds: (▾) AVP; (○) dDAVP; (▪) d(CH2)5Tyr(Me)AVP; (•) SKF-101926; (□) d(CH2)5,D-lle2,lle4,Arg8]-AVP. Data are the means of a typical experiment performed in duplicate and repeated several times without noticeable changes. Kidney International 2000 58, 1613-1622DOI: (10.1046/j.1523-1755.2000.00322.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 5 Labeling of AVP V2 receptors with [3H]-SR 121463 in human (A and B) and rat (C and D) kidney. Serial sections from frozen human samples dissected in the medullopapillary region and whole rat kidney were used. Autoradiograms were obtained by incubating adjacent kidney sections with 1.5 nmol/L [3H]-SR 121463 with (B and D; nonspecific binding) and without (A, B; total binding) cold SR 121463 (1 μmol/L). Calibration bars correspond to 2 mm. Kidney International 2000 58, 1613-1622DOI: (10.1046/j.1523-1755.2000.00322.x) Copyright © 2000 International Society of Nephrology Terms and Conditions