Kidney International, Vol. 58 (2000), pp. 1613–1622

Binding properties of a selective tritiated vasopressin V2

receptor antagonist, [3H]-SR 121463

CLAUDINE SERRADEIL-LE GAL, DANIELLE RAUFASTE, ELEONORE 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

Binding properties of a selective tritiated vasopressin V2 recep- ron [1]. The V2 receptor belongs to the seven-transmem-tor antagonist, [3H]-SR 121463. brane, G-protein–coupled receptor family, and its intra-

Background. [3H]-SR 121463 is the first radiolabeled selec- cellular signaling pathway involves a Gs/adenylyl cy-tive nonpeptide vasopressin V2 receptor antagonist ligand thatclase-stimulating system that induces cAMP formation.has been reported to date. In the present work, we studied theThe renal V2 receptor has been cloned in different spe-binding properties of [3H]-SR 121463 for renal V2 receptors

from animal and human origins. cies, including rat, pig, bovine, and human, and shares aMethods. Binding studies were performed with [3H]-SR 121463 high sequence homology [2–5]. Despite this fact, marked

in Chinese hamster ovary (CHO) cells transfected with the species differences have been reported in the pharmacol-human V2 receptor and in various kidney preparations express-ogy of this receptor [6]. The V2 receptor is present ining the native V2 receptors (rat, rabbit, dog, pig, monkey, andgreatest amounts in the kidney, but recently, polymerasehuman). Autoradiographies were performed in rat and human

kidney sections. chain reaction studies revealed that V2 receptor mRNAResults. [3H]-SR 121463 binding to CHO cells stably trans- is also present in other tissues or cells such as the brain,

fected with the cloned human renal V2 receptor was specific, the liver, and the lung and in small cell lung cancerhighly stable, time dependent, saturable, and reversible. A single(SCLC) and corticotroph tumors [7–11]. Moreover, thepopulation of high-affinity binding sites was identified (Kd 5existence of functional extrarenal V2 receptors involved0.94 6 0.34 nmol/L, Bmax 5 9876 6 317 fmol/mg protein). Of

note, [3H]-SR 121463 revealed a higher number (about 40%) in clotting factor release (factor VIII and von Willebrandof V2 sites than [3H]-AVP in the same preparation. Displace- factor) has been clearly demonstrated; however, theirment of [3H]-SR 121463 binding by reference peptide and non- exact localization remains to be determined [12–14].peptide vasopressin/oxytocin compounds exhibited a typical

Highly selective AVP V2 receptor ligands with highAVP V2 profile. [3H]-SR 121463 also displayed a high affinityfor native V2 receptors in several kidney preparations from affinity and stability constitute major tools for investigat-rat, pig, dog, rabbit, bovine, monkey, and human. The autoradio- ing the localization/mapping and the pharmacologicalgraphic experiments using rat and human kidney sections functions of these receptors. From a therapeutic pointshowed intense labeling in the medullopapillary region and

of view, AVP V2 receptor-specific antagonists able tolower intensity in the cortex, consistent with a main localizationblock the action of AVP at the level of the renal collect-of V2 receptors on collecting tubules.

Conclusion. [3H]-SR 121463 is a useful ligand for the specific ing duct specifically promote water excretion. Suchlabeling of animal and human V2 receptors and could be a “aquaretic” agents could be of high interest for the treat-suitable probe for the search and in situ localization of V2 sites. ment of several water-retaining disorders such as syn-

drome of inappropriate antidiuretic hormone secretion(SIADH), hyponatremia, liver cirrhosis, certain stages

Vasopressin (AVP) plays a major role as an antidiure- of congestive heart failure, and hypertension [15, 16].tic hormone regulating water and solute excretion by the Extensive work in the design of peptide ligands withkidney through specific interaction with renal V2 recep- high affinity and specificity for animal and human AVP/tors present in the collecting duct of the mammalian neph- oxytocin (OT) receptors has been carried out [17, 18],

leading to the discovery of selective V1a [19–23] and OT[24] receptor ligands. Although a peptide-radioiodinatedKey words: kidney, antidiuretic hormone, arginine vasopressin, homeo-

stasis, water regulation, ligand. ligand for the AVP V2 receptor was recently reported,it displays high affinity for rat and human AVP V2 and V1aReceived for publication December 10, 1999receptors and, therefore, cannot easily be used to spe-and in revised form March 15, 2000

Accepted for publication May 12, 2000 cifically label V2 receptors [25]. Over the past decade,several orally active nonpeptide AVP receptor antago- 2000 by the International Society of Nephrology

1613

Serradeil-Le Gal et al: V2 receptors and [ 3H]-SR 1214631614

nists have been reported [reviewed in 26]. One of them,SR 121463 (1-[4-(N-tert-butylcarbamoyl)-2-methoxyben-zene sulfonyl]-5-ethoxy-3-spiro-[4-(2-morpholinoethoxy-cyclohexane)] indoline-2-one, cis-isomer), has been shownto be a potent and selective AVP V2 receptor antagonist[27]. This highly stable molecule displays a high selectiveaffinity for V2 receptors from several species, includinghumans, and exhibits powerful intravenous and oralaquaretic effects [27]. Moreover, SR 121463 inhibits AVP-evoked cAMP formation in human kidney membranesand reverses extrarenal V2 receptor agonism of dDAVP-induced release of homeostasis factors in dogs [14].

Thus, SR 121463 represents an interesting probe forthe characterization and localization of V2 receptors inanimals and humans. This compound has been tritiatedand used in our current study for the characterization ofrenal V2 receptors. The present work reports the binding

Fig. 1. Chemical structure of [3H]-SR 121463. The * indicates the posi-properties of [3H]-SR 121463 to membranes from Chi-tion of tritium. (Reproduced from the Journal of Clinical Investigation

nese hamster ovary (CHO) cells stably transfected with [27], with permission from the American Society for Clinical Investigation.)the cloned human renal V2 receptor, mainly in terms ofkinetics, equilibrium-binding parameters, and specificity.We used a wide number of peptide and nonpeptide refer-

O-Me-Tyr2,Arg8]-AVP were obtained from Sigma Chemi-ence antagonists to characterize [3H]-SR 121463 bindingcal Co. (L’Isle d’Abeau, France). [Thr4,Gly7]-OT, [Phe2,further, and a close comparison was established withlle3,Orn8]-AVP, d(CH2)5,Tyr(Me)2,Thr4,Tyr-NH2-OVT,[pGlu4,binding results obtained using the natural tritiated hor-Cystine8]-AVP4–9, d(CH2)5Tyr(Me)AVP, [d(CH2)5,D-lle2,mone, [3H]-AVP. In addition, we used [3H]-SR 121463lle4,Arg8]-AVP, [d(CH2)5,D-Phe2,lle4,Ala9-NH2]-AVP werefor labeling native V2 receptors in different renal prepa-obtained from Peninsula Laboratories (Orsay, France). Des-rations from animal (that is, rat, rabbit, pig, dog, monkey,Gly-[Pha1,D-Tyr(Et)2-Lys6-Arg8]-AVP, SKF-105494 [o-ethyl-and bovine) and human origin. Finally, we performedD-Tyr1-Phe2-val3-Asn4-[1-(carboxymethyl)cyclohexyl]5-L-autoradiographic experiments with [3H]-SR 121463 onnorvalyl6-Arg7-D-ArgNH2

8, cyclic (5→1)] was a generousrat and human kidney sections for in situ mapping ofgift from Dr. L. Bankir and Dr. Kinter. SKF-101926AVP V2 receptors. Our results show that [3H]-SR 121463([d(CH2)5,D-Tyr(Et)2,Val4,Arg8, des-Gly9]-AVP) was ob-binds with high affinity to renal V2 receptors from varioustained from Bachem (Voisin Le Bretonneux, France).species, including humans, and is a suitable probe for[Des-Glycinamide9, d(CH2)5, O-Me-Tyr2,Thr4,Orn8]-vaso-the selective localization of AVP V2 receptors.tocin was obtained from Saxon/Interchim (Montlucon,France). Dulbecco’s modified Eagle medium (DMEM)

METHODS and phosphate-buffered saline (PBS) were from Boeh-Chemicals ringer Mannheim (Meylan, France). All other cell culture

reagents were from GIBCO (Life Technologies, GIBCOReference nonpeptide AVP receptor antagonists, SRBRL, Cergy Pontoise, France). Ethylenediaminetetraace-121463 (1-[4-(N-tert-butylcarbamoyl)-2-methoxybenzenetic acid (EDTA), Tris, and DMSO were obtained fromsulfonyl]-5-ethoxy-3-spiro-[4-(2-morpholinoethoxy)cyclo-Merck-Clevenot (Nogent sur Marne, France). All otherhexane]indol-2-one, monophosphate salt, molecular weightchemicals were from Prolabo (Paris, France).759.82-SR 121463B-) [27], SR 49059 [28], OPC-31260 [29],

OPC-21268 [30], YM-087 [31], and VPA-985 [32] were syn-Biological materialthesized at Sanofi-Synthelabo. The purity, measured by high-

Tissue samples from human kidneys were collected inpressure liquid chromatography, thin layer chromatography,conformity with the national ethical rules and were ob-and elemental analysis, was more than 98%. They weretained at the time of nephrectomy from patients aged 40dissolved in dimethylsulfoxide (DMSO) at 1022 mol/L andto 60 years. Immediately after excision, tissues were dis-then diluted in the appropriate test solvent. [3H]-SR 121463sected, and reno-medullary samples were either frozen at(47.5 Ci/mmol; Fig. 1) and [3H]-AVP (30 to 70 Ci/mmol)2408C in isopentane and further stored at 2808C for auto-were purchased from New England Nuclear, Life Scienceradiographic experiments or crude plasma membranesProducts (Les Ulis, France). AVP, dDAVP (1-desamino-were prepared for binding studies. This project was ap-[D-Arg8]-AVP), [Lys8]-AVP, d(D-3Pal)AVP (deamino1

(D-3-(Pyridyl)Ala2, Arg(8)-AVP, OT, [deamino-Pen1, proved by the human subject ethical review committees

Serradeil-Le Gal et al: V2 receptors and [ 3H]-SR 121463 1615

of our institutions and was carried out in collaboration similar operating conditions using [3H]-AVP as a ligand(3 nmol/L). In this case, the nonspecific binding waswith Hopital Lapeyronie (Montpellier, France). Bovine

kidneys were from a local slaughterhouse; male New measured in the presence of 10 mmol/L unlabeled AVP.Data for equilibrium binding [apparent equilibriumSprague-Dawley rats (250 to 350 g) and male New

Zealand rabbits (2.5 to 3 kg) were purchased from Iffa- dissociation constant (Kd), maximum binding density(Bmax)], competition experiments [IC50, Hill coefficientCredo (Lyon, France). Bred mongrel dogs (16 to 22 kg)

and pigs were from les Souches and Cegan Ltd. (France), (nH)], and kinetic constants [observed association rateconstant (kobs), apparent dissociation rate constant (k21)respectively. Bred Cynomolgus monkeys, Macaca fascic-

ularis (3 to 5 kg), were supplied by CRP Ltd. (Mauritius, and apparent association rate constant k11 5 (kobs 2k21)/[L] with [L] equal to the radioligand concentration]France).were calculated using an interactive nonlinear regression

Cell culture and preparations of cell homogenates program [38]. The IC50 value was defined as the concen-tration of inhibitor required to obtain 50% inhibition ofCHO-DHFR2 cells (DXB11) were transfected with an

expression vector derived from plasmid 7055 containing the specific binding. Inhibition constant (Ki) values werecalculated from the IC50 values using the Cheng andthe cDNA encoding the human V2 receptor. Stably trans-

formed cell lines were isolated as described earlier [33, Prusoff equation [39]. For peptide compounds, an appar-ent IC50 value was determined considering the fraction34]. They were grown in 10 mmol/L HEPES, pH 7.4,

minimal essential medium (MEM) supplemented with of displaceable sites by these peptides.5% fetal calf serum and 8 g/L sodium bicarbonate and

Autoradiography300 mg/mL geneticin at 378C in a humidified atmospherecontaining 5% CO2. Wild-type CHO cells were routinely Human kidney samples dissected in the medullary

papillary region and whole rat kidney were used. Serialgrown in a similar culture medium. Cells, grown to con-fluence, were collected by scraping, and crude mem- sections (15 mm thick) from frozen rat and human kid-

neys were mounted onto gelatin chrome-alum slides,branes were prepared by homogenization of cells as pre-viously described [35]. Culture medium were removed rinsed to eliminate endogenous AVP, and incubated with

1.5 nmol/L [3H]-SR 121463 with (nonspecific binding) orevery other day, and cells were subcultured by treatmentwith 0.05% trypsin, 0.02% EDTA. without (total binding) 1 mmol/L unlabeled SR 121463 as

described previously [27]. After incubation, the sectionsMembrane preparations were washed three times for 10 minutes each in ice-cold

binding buffer, dipped briefly in distilled water, and driedReno-medullary membranes from rat, rabbit, dog, pig,bovine, monkey, and humans were prepared according under a stream of cold air. Rinsed labeled sections were

placed on a phosphor-imaging plate (Fuji, Tokyo, Japan)to Stassen et al [36] and were stored as aliquots in liquidnitrogen until use at a final concentration of about 10 mg for four days and further analyzed with a Bio-Image

Analyzer (BAS 5000; Fuji) as already described [40].protein/mL. The protein concentration was determinedby the method of Bradford using bovine serum albuminas a standard [37].

RESULTS

Binding of [3H]-SR 121463 to human V2 receptorsAVP V2 binding assaysexpressed in CHO cellsChinese hamster ovary membranes expressing the hu-

man V2 receptors or reno-medullary preparations from Tissue-binding linearity. Preliminary experiments haveshown that [3H]-121463–specific binding to crude cellrat, rabbit, dog, pig, bovine, monkey, and humans were

incubated in a 50 mmol/L Tris-HCl buffer, pH 8.1, con- homogenates was linear with increasing protein concen-tration up to 30 mg/tube (that is, 150 mg/mL). Nonspecifictaining 2 mmol/L MgCl2, 1 mmol/L EDTA, 0.1% bovine

serum albumin (BSA), 0.1% bacitracin, and [3H]-SR binding determined in the presence of 1 mmol/L unla-beled SR 121463 was very low and ranged between 1121463 (0.03 to 10 nmol/L for saturation experiments or

2 nmol/L for kinetic and competition studies), and in- and 5% of the total binding (data not shown).Kinetics binding studies. As shown in Figure 2, [3H]-creasing amounts of the tested compound. The reaction

was started by the addition of membranes (30 mg/assay SR 121463–specific binding to V2 receptors expressed inCHO cell membranes was rapid (t1/2 5 5 6 3 min, N 5for CHO and 100 to 200 mg/assay for native renal mem-

branes) and incubation for 45 minutes at 258C (except 3) and time-dependent and reached equilibrium in about20 minutes at 258C (kobs 5 0.033 6 0010 min21). Thein association-dissociation studies) and was then stopped

by filtration through Whatman GF/B filters as previously apparent equilibrium binding level remained highly sta-ble and unchanged during the three-hour observationdescribed [27]. Nonspecific binding was determined in

the presence of 1 mmol/L SR 121463. Competition and period. Dissociation of the [3H]-SR 121463 receptor com-plex, initiated after 45 minutes of incubation by the addi-saturation-binding experiments were conducted under

Serradeil-Le Gal et al: V2 receptors and [ 3H]-SR 1214631616

from Scatchard plot and kinetics experiments. The selec-tive V2 receptor antagonist VPA-985 and the two non-peptide V2/V1a receptor antagonists OPC-31260 and YM-087 displaced bound [3H]-SR 121463 with nanomolar Ki

values (8.8 6 0.7 and 0.67 6 0.05 nmol/L, respectively)in agreement with data previously reported in variousanimal and human AVP V2 preparations [27, 32]. As ex-pected, the selective nonpeptide V1a receptor antagonistSR 49059 exhibited low potency in displacing [3H]-SR121463. Displacement curves obtained in each case gavelinear Hill plots and pseudo Hill coefficient (nH) of aboutone (data not shown).

It is important to note that with these nonpeptide li-gands, we observed total displacement of [3H]-SR 121463bound to membranes, whereas with peptide compounds

Fig. 2. Time–course of association (d) and dissociation (s) of [3H]- such as AVP, dDAVP (a V2 receptor agonist), SKF com-SR 121463 to membranes of Chinese hamster ovary (CHO) cells ex-pounds (V2 receptor antagonists), and d(CH2)5Tyr(Me)pressing the human renal arginine vasopressin (AVP) V2 receptors.

Incubations were carried out as described in the Methods section in AVP (a V1a receptor antagonist), even at high concentra-the presence of 2 nmol/L [3H]-SR 121463 for various periods of time. tions we observed only partial displacement of [3H]-SRThe arrow indicates time (45 min) at which unlabeled SR 121463

121463 bound to our preparation (Fig. 4B and Table 1).(1 mmol/L) was added to initiate the dissociation process. The resultsrepresent data from a typical experiment performed in duplicate and Computer analysis of the data obtained with peptiderepeated three times without noticeable modifications. compounds indicated that the binding was best fitted to

a two-site model: one site (about 60%) displaceable byAVP (and peptide analogues) and exhibiting the ex-pected V2 profile according to the rank order of affinitytion of 1 mmol/L unlabeled SR 121463 (Fig. 2), occurred

slowly with an apparent dissociation constant (k21) value found for the reference compounds studied (Table 1)and a second site (about 40%) totally insensitive to AVPof 0.0066 6 0.0012 min21. Two and a half hours after initi-

ation of the dissociation process, 30% of remaining spe- and to other agonist or antagonist peptide compounds(whatever their AVP/oxytocin profile) and only displace-cific binding to membranes was still observed indicating

slow dissociation. From kinetics experiments, an approx- able by nonpeptide AVP receptor antagonists accordingto a V2 rank order of potency. Interestingly, the proportionimate calculated Kd value of 0.4 nmol/L was obtained.

Saturation-binding studies. Saturation experiments per- of “AVP-insensitive” sites (near 40%) was in good agree-ment with Bmax values obtained from Scatchard plots usingformed in the presence of increasing concentrations of

[3H]-SR 121463 showed that the specific binding was [3H]-SR 121463 and [3H]-AVP as ligands (5524 6 887 vs.9876 6 317 fmol/mg protein; Fig. 3).saturable (Fig. 3A). Scatchard analysis of these data gave

a linear plot consistent with the presence of a single Specificity of [3H]-SR 121463 binding. Despite thehighly selective profile previously reported for SR 121463class of high-affinity binding sites characterized by an

apparent Kd of 0.94 6 0.34 nmol/L and a maximal binding [27], one could speculate that the CHO cell line usedmight express other native binding sites recognized bycapacity (Bmax) of 9876 6 317 fmol/mg protein (that is,

approximately 500,000 sites/cell, N 5 3). Saturation bind- SR 121463 and unrelated to AVP receptors. This hypoth-esis can be discarded, since no specific binding could being experiments, performed under similar experimental

conditions, with the natural tritiated ligand [3H]-AVP detected in membranes of untransfected CHO wild-typecells when using [3H]-SR 121463 (data not shown). More-revealed a linear Scatchard plot with a nanomolar Kd

value (1.80 6 1.40 nmol/L)—similar to that of [3H]-SR over, additional binding experiments performed with[3H]-SR 121463 (2 nmol/L) in CHO or ltK2 cell lines121463—and a significantly lower number of binding

sites (about 40%, Bmax 5 5524 6 897 fmol/mg protein) stably expressing human V1a, V1b, or oxytocin receptorsdid not reveal any specific binding, in agreement withthan that obtained with [3H]-SR 121463 (Fig. 3B).

Competition-binding studies. The relative affinities of an extremely high specificity for the V2 receptor subtypeversus other AVP/oxytocin receptors. As expected, forseveral reference peptide or nonpeptide AVP/oxytocin

compounds were studied to characterize further the an antagonist radioligand able to recognize the differentstates of a G-protein–coupled receptor, binding of [3H]-binding of [3H]-SR 121463 (Table 1 and Fig. 4). As shown

in Figure 4A, [3H]-SR 121463 binding to the human V2 SR 121463 was insensitive to guanylnucleotides (100mmol/L GTP, GppNHp, GTPgs) and 140 mmol/L Na1receptor was inhibited in a dose-dependent manner by

unlabeled SR 121463, yielding a Ki value of 0.54 6 0.09 (data not shown), which are well-known conditions ableto promote the low-affinity state for the receptor.nmol/L (N 5 4), consistent with Kd values obtained both

Serradeil-Le Gal et al: V2 receptors and [ 3H]-SR 121463 1617

Fig. 3. Saturation of [3H]-SR 121463 (d) and[3H]-AVP (j) specific binding to membranesof CHO cells transfected with the human renalAVP V2 receptors. (A) Saturation isotherms.(B) Scatchard plots. Incubations were per-formed for 45 minutes at 258C in the presenceof 10 mg protein/mL and increasing concen-trations of [3H]-SR 121463 or [3H]-AVP asdescribed in the Methods section. Data aremeans calculated from a typical experimentperformed in duplicate and repeated threetimes without noticeable changes.

Table 1. Inhibition of [3H]-SR 121463 binding to CHO cells transfected with the human AVP V2 receptor by reference peptide andnonpeptide AVP/OT compounds

% Sites displacedKd nmol/L at 1025 mol/L

NonpeptideSR 121463 0.5460.09 100VPA-985 0.6160.21 100YM-087 0.6560.05 100OPC-31260 8.860.7 100SR 49059 232648 100OPC-21268 .10,000 3

PeptideAVP 1266 63dDAVP 51618 53[Lys8]-AVP 104610 67d(D-3Pal)AVP .10,000 14OT .1,000 21[Thr4,Gly7]-OT .10,000 6POVT .10,000 6d(CH2)5,Tyr(Me)2,Thr4,Tyr-NH2-OVT 990679 47[Des-glycinamide9,d(CH2)5

1,O-Me-Tyr2,Thr4,Orn9]-vasotocin .10,000 9[pGlu4,cystine8]-AVP 4-9 .10,000 3d(CH2)5Tyr(Me)AVP 160666 58[d(CH2)5,D-lle2,lle4,Arg8]-AVP 113621 65[d(CH2)5,D-lle2,lle4,Ala9-NH2]-AVP 2266 67des-Gly-[Pha1,D-Tyr(Et)2-Lys6-Arg8]-AVP 238642 62[deamino-Pen1,O-Me-Tyr2,Arg8]-AVP 546665 58SKF-105494 1.360.5 68SKF-101926 4.862.5 65

Inhibition constants (Ki) were calculated according to the Cheng and Prusoff equation [39] as described in Methods section. Values are means 6 SD (N 5 3 to 6).The % of site displaced at 1025 mol/L is indicated.

Fig. 4. Inhibition of [3H]-SR 121463-specificbinding to membranes of CHO cells express-ing the human renal AVP V2 receptor by refer-ence nonpeptide (A) and peptide (B) AVP/OTcompounds. Incubations were carried out inthe presence of 1.5 nmol/L [3H]-SR 121463 andincreasing concentrations of the compound tobe tested. Symbols in (A) for nonpeptide com-pounds: (s) SR 121463; (d) YM-087; (h)VPA 985; (j) OPC-31260; (.) SR 49059;(,) OPC-21268. Symbols in (B) for peptidecompounds: (.) AVP; (s) dDAVP; (j)d(CH2)5Tyr(Me)AVP; (d) SKF-101926; (h)d(CH2)5,D-lle2,lle4,Arg8]-AVP. Data are themeans of a typical experiment performed induplicate and repeated several times withoutnoticeable changes.

Serradeil-Le Gal et al: V2 receptors and [ 3H]-SR 1214631618

Table 2. Equilibrium binding parameters (Kd, Bmax) of likely corresponding to cortical collecting tubules from[3H]-SR 121463 and [3H]-AVP for V2 receptors in various

long-loop nephrons. This is consistent with the knowncrude animal and human kidney preparationsrenal V2 receptor localization in the rat kidney [25]. Bind-

[3H]-SR 121463 [3H]-AVP ing in the medulla was more easily displaced by cold SRKi Bmax Ki Bmax 121463 than in the cortical region (Fig. 5D).

nmol/L fmol/mg protein nmol/L fmol/mg protein

Rat 1.5160.70 220636 0.6360.25 93650Rabbit 0.9060.15 113622 0.85633 123642 DISCUSSIONPig 0.4660.12 161631 0.7360.20 216616

The present study demonstrates that [3H]-SR 121463Dog 0.6560.16 151629 1.1160.65 109612Monkey 0.7760.42 3196153 1.2060.20 129618 displays high and specific affinity for AVP V2 receptorsBovine 0.8360.32 10386217 0.5660.14 6706324 from animal and human origin and constitutes a suitableHuman 0.5460.08 223619 1.3560.46 151657

probe for investigating these receptors, especially in com-plex organs expressing mixed populations of AVP/OTreceptor subtypes.

Receptors for AVP and OT are widely distributed andBinding of [3H]-SR 121463 to V2 receptors fromhave been identified with various functions. However,various animal and human native kidney preparationsmany pharmacological experiments and binding/auto-

Saturation binding experiments performed with [3H]- radiographic studies have been hampered or limited inSR 121463 in membranes prepared from the medulla their interpretation by the lack of potent and highlyand the papillary area of the kidney from various species selective ligands for V1a, V1b, V2, and OT receptors. Selec-(rat, rabbit, dog, bovine, monkey, and humans) demon- tive peptide and nonpeptide, tritiated or iodinated, V1astrated an excellent affinity of [3H]-SR 121463 for V2 or OT ligands are currently available [17, 24], but up toreceptors (Kd value in the nanomolar range; Table 2). now, the design of peptide or nonpeptide radiolabeledWhatever the species, [3H]-SR 121463 bound to a single ligands with high affinity and specificity for animal andclass of high-affinity binding sites and displayed a higher human AVP V2 receptors has been unsuccessful. Species(or at least comparable) affinity than the natural hor- differences and a lack of selectivity are the major draw-mone, [3H]-AVP, for these native V2 receptors. Interest- backs of the potent peptide AVP V2 compounds reportedingly, we found similar binding affinity of [3H]-SR 121463 so far [6, 25, 41, 42]. Although attempts have been madein CHO cells stably transfected with the human V2 recep- to label V2 receptors with [3H]-dDAVP [43], a well-tor and in crude human kidney preparations (Kd values known peptide V2 agonist, recent data demonstratedobtained from saturation experiments and Scatchard high affinity of this compound for human V1b receptorsanalysis, 0.94 6 0.34 vs. 0.54 6 0.08 nmol/L, respectively). as well [44]. Another potent and selective V2 receptorIt is also important to note a marked heterogeneity in antagonist ligand, [3H]-desGly-NH2

9-d(CH2)5 [D-Ileu2,the number of V2 sites found in the different studied Ileu4]-AVP, exhibited marked species difference and wasspecies (Bmax values ranged from 113 6 22 fmol/mg pro- a suitable ligand only for labeling rat V2 receptors [45]. Intein in the rabbit to 1038 6 217 fmol/mg protein in bovine comparison, the nonpeptide AVP V2 receptor antagonistrenal membranes). Notably, compared with [3H]-AVP [3H]-SR 121463 binds with high affinity to V2 receptors(a V1 and V2 ligand), [3H]-SR 121463 identified a similar in native kidney membrane preparations from rat, rabbit,(rabbit, pig, and dog) or even higher (rat, monkey, and dog, pig, bovine, monkey, and human origin, with Kd

humans) number of binding sites according to the origin values similar to, or even higher than, those found for theof the preparation (Table 2). natural hormone [3H]-AVP. The difference in receptor

number (Bmax values) observed between these variousAutoradiography experiments with [3H]-SR 121463 kidney preparations (obtained under similar operatingin rat and human kidney sections conditions) showed marked species heterogeneity in re-

Figure 5 shows autoradiograms obtained from human nal V2 receptor density as already described for the liverand rat kidney sections incubated with [3H]-SR 121463 V1a receptors [46].in the presence or absence of unlabeled SR 121463. A The present study provides a detailed characterizationdense labeling was observed in the medullopapillary por- of this new nonpeptide ligand by studying the bindingtion dissected from the human kidney, with lower label- properties of [3H]-SR 121463 to membranes from CHOing in the outer medulla/cortical extremity showing a cells stably transfected with the cloned human renal AVPtubular pattern (Fig. 5A). In rat whole kidney sections, V2 receptor. [3H]-SR 121463 binding was specific, highlyspecific binding was mainly seen in the inner/outer me- stable, time-dependent, saturable, and reversible, withdulla and papilla corresponding to the main localization minimal nonspecific binding measured in the presenceof collecting ducts. Much less intense specific binding of 1 mmol/L unlabeled SR 121463 (less than 5%). Associ-

ation occurred rapidly (t1/2 about 5 min) to reach a re-was also detected in the cortical portion of the kidney

Serradeil-Le Gal et al: V2 receptors and [ 3H]-SR 121463 1619

Fig. 5. Labeling of AVP V2 receptors with[3H]-SR 121463 in human (A and B) and rat(C and D) kidney. Serial sections from frozenhuman samples dissected in the medullopapil-lary region and whole rat kidney were used.Autoradiograms were obtained by incubatingadjacent kidney sections with 1.5 nmol/L [3H]-SR 121463 with (B and D; nonspecific binding)and without (A, B; total binding) cold SR121463 (1 mmol/L). Calibration bars corre-spond to 2 mm.

markably stable steady state. The dissociation process nonpeptide compounds tested indicated that the homog-enous population of sites labeled by [3H]-SR 121463 ex-of the receptor-ligand complex, using 1 mmol/L unlabeled

SR 121463, was slow and still partial after three hours, hibited the expected profile for human AVP V2 receptors(Table 2), in good agreement with published values usingdemonstrating a tight association of [3H]-SR 121463 to

its binding sites. Both in CHO cells transfected with the [3H]-AVP as a ligand [47]: SR 121463 . SKF com-pounds . AVP . OPC-31260 . dDAVP . SR 49059 ≈human V2 receptors and in renomedullary membranes

from native human kidney, which constitutively ex- d(CH2)5Tyr(Me)AVP . OT and OT analogues .d(D-3Pal)AVP. A striking finding was that in CHO cellpresses AVP V2 receptors, [3H]-SR 121463 bound to a

single class of high-affinity binding sites (Kd 5 0.94 6 homogenates, [3H]-SR 121463 bound to a population ofAVP V2 sites that could not be displaced by AVP and0.34 nmol/L, Bmax 5 9876 6 317 fmol/mg protein—

approximately 500,000 sites/cell—and Kd 5 0.54 6 0.01 by other AVP V2-related peptides (that is, dDAVP, SKFcompounds). Similarly, saturation binding experimentsnmol/L, Bmax 5 223 6 19 fmol/mg protein, respectively).

The rank order of affinity of the reference peptide and performed simultaneously with [3H]-AVP and [3H]-SR

Serradeil-Le Gal et al: V2 receptors and [ 3H]-SR 1214631620

121463 in the same CHO preparation revealed a differ- 121463 may be a valuable tool for the localization ofextrarenal V2 receptors. Recent polymerase chain reac-ence in receptor density. A first hypothesis that [3H]-SR

121463 bound to sites unrelated to AVP V2 receptors tion studies have shown the presence of small amountsof V2 mRNA in various animal and human tissues suchwas discarded since no specific binding could be detected

in untransfected CHO. An alternative explanation that as brain and lung tissues or cells [7, 11], and several invivo data suggest the presence of V2 or V2-like receptors[3H]-SR 121463 binds to a population of V2 receptors

with reduced affinity for agonists, as previously observed located in the vascular system, probably in endothelialcells, responsible for vasodilation via a nitric oxide re-in various systems [48, 49], was rejected since neither

peptide agonists nor antagonists displaced this [3H]-SR lease pathway [55, 56]. In addition, the involvement ofAVP V2 extrarenal sites in clotting factor release has121463 AVP-resistant binding. It appears likely that the

[3H]-SR 121463 binding sites not displaced by AVP (and been clearly evidenced using the selective AVP V2 recep-tor antagonist SR 121463 [14].other related V2 peptides) result from the sequestration

of a fraction of vesiculated V2 receptors in “right side In conclusion, this study reports the binding propertiesof [3H]-SR 121463, the first radiolabeled selective non-in” vesicles that are available to small hydrophobic non-

peptide ligands, but not to more hydrophilic peptide peptide V2 receptor antagonist, described so far. Thepotent affinity of this radioligand for both animal andanalogues. According to this hypothesis, more lipophylic

ligands, with a higher or lower affinity for V2 receptors, human AVP V2 receptors as well as its selectivity andstability suggests that it is ideally suited for labeling V2totally displaced the bound [3H]-SR 121463. Thus, SR

49059, a V1a receptor antagonist exhibiting a moderate receptors in tissues expressing heterogenous populationsof AVP/OT receptors. This compound is also of interestaffinity for V2 receptors, was able to displace 100%

of [3H]-SR 121463 bound at high concentrations (1025 for further investigation of the role of AVP and V2 recep-tors in physiological and pathological processes.mol/L), consistent with its V2 affinity (Ki 5 232 nmol/L).

In every case, the Hill coefficient about one is consistentwith a unique population of sites in our preparation. In ACKNOWLEDGMENTSsupport of this hypothesis, a recent study demonstrated The authors thank Drs. J. Wagnon, G. Garcia, and G. Valette fromthat treatment with nonpeptide antagonists SR 121463 Sanofi-Synthelabo for the synthesis of nonpeptide SR compounds and

references. We are grateful to Dr. R. Pruss and A.J. Patacchini forand VPA-985, but not with cell impermeable peptidehelpful comments on the manuscript, and to M. Laborde for her skillfulantagonists such as d(CH2)5

1,D-Tyr(Et)2, Val4,Arg8, des- secretarial assistance in preparing this work. We are thankful to Dr.Gly9-AVP (so-called SKF-101926), increased cell surface Bankir and Dr. Kinter for providing us with the SKF-105494 compound.

We would like to acknowledge Dr. Machard and the team of Newexpression of mutant V2 receptors causing nephrogenicEngland Nuclear Life Sciences Products for the synthesis and the giftdiabetes insipidus [50]. These data suggest that by bind- of [3H]-SR 121463.

ing intracellularly, nonpeptide antagonists facilitate fold-Reprint requests to Dr. Claudine Serradeil-Le Gal, Exploratory Re-ing and translocation of mutant V2 receptors to the

search Department, Sanofi-Synthelabo Recherche, 195, route d’Espagne,plasma membrane. Despite high binding affinity for hu-31036 Toulouse Cedex, France.

man V2 receptors, SKF-101926, also used in the present E-mail: claudine.serradeil@sanofi-synthelabo.comstudy, was unable to totally displace [3H]-SR 121463binding sites in our preparation, whereas nonpeptide

APPENDIXcompounds such as SR 121463 and VPA 985, able toAbbreviations used in this article are: AVP, arginine vasopressin;permeate membranes, did displace it. Other explanations

Bmax, maximum binding density; BSA, bovine serum albumin; CHO,involving different conformations of the V2 receptor, Chinese hamster ovary; dDAVP, 1-desamino-[D-Arg8]-vasopressin;discriminated by peptide ligands, cannot be excluded. DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethyl sulf-

oxide; EDTA, ethylediaminetetraacetic acid; k11, association rate con-In addition to its use as a selective ligand to measurestant; k21, dissociation rate constant; Kd, dissociation constant; Ki, inhibi-V2 receptors, [3H]-SR 121463 can be used to localize V2 tion constant; kobs, observed rate constant; MEM, minimal essential

receptors in situ in animal and human tissue. The pattern medium; nH, Hill coefficient; OT, oxytocin; SCLC, small cell lung cancer;SIADH, syndrome of inappropriate antidiuretic hormone secretion; SRof [3H]-SR 121463 labeling in the rat kidney was consis-121463, 1-[4-(N-tert-butylcarbamoyl)-2-meth-oxybenzene sulfonyl]-5-tent with previous autoradiographic data [51], and with ethoxy-3-spiro-[4-(2-morpholinoethoxy-cyclohexane)] indoline-2-one,

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