Kidney International, Vol. 50 (1996), pp. 499—505

Autoradiographic localization of vasopressin Via receptors in therat kidney using [3HJ-SR 49059

CLAUDINE SERRADEIL-LE GAL, DANIELLE RAUFASTE, ELENORE MARTY, CORINE GARCIA,JEAN-PIERRE MAFFRAND, and GERARD LE FUR

Sanofi Recherche, Exploratoiy Research Department, Toulouse, France

Autoradiographic localization of vasopressin 'ia receptors in the ratkidney using [3H]-SR 49059. Localization and characterization of bindingsites of the selective non-peptide vasopressin receptor Via ligand, [3Hj-SR49059, were investigated in the adult rat kidney by quantitative autora-diography using a fast-detecting radioluminographic phosphor-imagingplate system. [3H]-SR 49059, like the other Via ligands used, showed atotal absence of binding in the papilla, discrete and sparse labeling in thecortex and maximal binding in the outer part of the inner medulla. Thislabeling seemed to be mainly associated with medullary interstitial cellsand vascular elements of the vasa recta. Conversely, [3H1-AVP intenselylabeled the V2-enriched medullo-papillary portion of the kidney and, to alesser extent, the cortical structures. [3H]-SR 49059 binding, quantified inthe outer part of the inner medulla in rat kidney sections, was time-dependent, reversible, saturable and a single class of high affinity bindingsites (K = 1.48 0.16 nM) was identified. The relative potencies of thereference peptide and non-peptide compounds to inhibit [3H]-SR 49059binding confirm the V nature of the site and the stereospecificity of thisbinding. Thus, [3H]-SR 49059 allows the mapping and characterization ofthe Via receptor population present in the rat kidney. The stability and thehighly selective affinity of this non-peptide ligand for rat and human Viareceptors make it a suitable probe for the localization of Via receptors inorgans expressing heterogeneous populations of receptors.

The two neurohypophyseal hormones, vasopressin (AVP) andoxytocin (OT), elicit a variety of biological effects in mammalsthrough interaction with specific receptors (Via, Vib, V2 and OTreceptors), which have now been cloned and characterized in alarge number of tissues. The Via receptor subtype mainly exists invascular smooth muscle cells, platelets, the liver, adrenal, uterusand brain, whereas the Vib receptor has been initially identified inthe anterior pituitaly. The V2 receptor is present in great amountsin the kidney and mediates the well-known antidiuretic effect ofvasopressin. Finally, oxytocin receptors are essentially located inmammary glands, uterus and brain [1, 21.

However, most of the time, several target organs such as thebrain, kidney, and uterus express heterogeneous populations ofAVP/OT receptors [3—6]. Therefore, highly selective ligands foreach AVP/OT receptor subtype, with high stability and highspecific activity, constitute major tools for investigating the local-ization/mapping and the pharmacological functions of each entity.

Received for publication November 6, 1995and in revised form February 7, 1996Accepted for publication February 8, 1996

© 1996 by the International Society of Nephrology

A number of tritiated or iodinated Via peptide ligands havebeen described and used to characterize AVP Via receptors indifferent cell types by binding and autoradiographic analysis [2,7—111. We recently reported a potent, non-peptide, orally-activeAVP Via receptor antagonist, SR 49059, (Fig. 1) showing selectivehigh affinity for both animal and human Via receptors in vitro andin vivo [12]. Its tritiated derivative, [3H]-SR 49059, has beensynthetized and used in binding experiments as a suitable probefor labeling and characterizing AVP Via receptors in rat andhuman liver tissues [13].

The kidney predominantly expresses V2 receptors and, in smallamounts, Via and OT receptors, which are also able to controlrenal function [4, 14]. More recently, polymerase chain reactionstudies revealed that Vib receptor mRNA as well are present in anumber of tissues including the kidney [15, 161. Due to the highlyselective Via profile and the strong stability of [3Hj-SR 49059, thisfirst non-peptide ligand appears to be a valuable probe fordiscriminating between renal AVP/OT receptor subtypes and forgiving further insight into the localization of Via receptors presentin the discrete area of the kidney.

The purpose of the present work was to investigate the char-acteristics and the distribution of the binding sites of tritiated SR49059 in the adult rat kidney by quantitative autoradiography. Aclose comparison was established with the labeling obtained inthis organ by other peptide Via ligands such as [3H]-d(CH2)5Tyr(Me)AVP [8] and [1251]-linear AVP antagonist [7],and by the tritiated natural hormone, [3H]-AVP.

Moreover, to overcome the drawbacks related to the use oftritiated ligands, which provide the best spatial resolution butrequire very long exposure time, a phosphor-imaging plate systemwas used to allow fast detection, high sensitivity and accuratequantification of the radioactivity bound to histological sections,with a far wider dynamic range and greater dose-response linear-ity than traditional radiosensitive films [17].

Methods

Materials

The non-peptide molecules, SR 49059, [(2S) 1-[(2R 3S)-(5-chloro-3-(2-chlorophe-nyl)-1-(3,4-dimethoxybenzene-sulfonyl)-3-hydroxy-2,3-dihydro-1 H-indole-2-carbo-nyl]-pyrrolidine-2-carbox-amide; Fig. 11, its enantiomer (SR 49770), OPC-21268 [18] andOPC-31260 [19] were synthesized at Sanofi Recherche (Mont-pellier, France). All compounds were initially dissolved in DMSO

499

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500 Serradeil-Le Gal et al: [3H1-SR 49059 and autoradiography

Fig. 1. Structure of SR 49059. * indicates position of tritiation.

at a concentration of 102 M, then diluted in the appropriate testsolvent.

[3H]-SR 49059 (55 Ci/mmol) and [3H]-autoradiographic stan-dard microscales were obtained from Amersham (Les Ulis,France); [3H]-AVP (81 Ci/mmol) and [3H]-d(CH2)5Tyr(Me)AVP(47 Ci/mmol) were from New England Nuclear (Les Ulis,France); ['251]-linear AVP antagonist (phenylacetyl.D.Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2; 2,000 Ci/mmol) was synthe-sized by Dr. C, Barberis as described by Schmidt et al [7]. AVP,OT, d(CH2)5Tyr(Me)AVP, 1-desamino-8-D-arginine (DDAVP),(Phe2, Orn8)-Vasotocin (POVT) and [Deamino-Cys1 ,D-3-(Pyri-dyl)-A1a2,Arg8]-Vasopressine (d[D-3Pal]VP) were from SigmaChemical (L'Isle d'Abeau, France). All other chemicals werepurchased from Merck-Clevenot (Nogent sur Marne, France) orfrom Prolabo (France).

Autoradiography

Young developing male Sprague-Dawley rats (20 to 30 days old,100 g) were sacrified by decapitation and the organs (kidneys andliver) were rapidly removed and frozen in isopentane at —45°C.Serial sections (15 j.tm) were cut in a cryostat microtome andthaw-mounted into gelatin chrome-alum-gelatin coated glassslides (gelatin 1%, chrome-alum 0.05%). Sections were stored at—80°C until use. Slide-mounted sections (3 to 4 sections/slide),brought to room temperature, were preincubated for 15 minutesin the binding buffer (50 nM Tris-HC1, pH 7.4, 10 mrvt MgC12, 1 mMEGTA, 0.1% bovine serum albumin and bacitracin). Incubationwas carried out for one hour at room temperature in theincubating medium in the presence of either [3H]-SR 49059 (0.3to 18 nM for saturation or 1.8 to 2 nM for kinetic and competi-tion experiments) or other ligands {[3H]-AVP (2 nM),d(CH2)5Tyr(Me)AVP (1.8 nM) or {'251]-linear AVP antagonist(0.02 nM)}. Non-specific binding was determined in the presenceof 1 /.LM unlabeled AVP.

After incubation, the sections were washed three times for 10minutes each in ice-cold buffer, dipped briefly in distilled water,and dried under a stream of cold air.

Labeled sections were placed, together with autoradiographicstandards, on a phosphor-imaging plate (Fuji) for 4 to 10 days fortritiated and four hours for iodinated ligands. The exposedimaging plate was scanned and the emitted stored energy quan-tified as photo-stimulated luminescence (PSL) with a Bio-Image* Analyser (BAS 2000, Fuji) under specific reading conditions:gradation 1024; resolution 100; sensitivity 10,000; latitude 4. Hardcopies of the autoradiographic images were obtained using a highquality BAS HS printer (Fuji). The PSL were collected and* digitized to allow measurement of the relative radioactivity of thesection. The resulting 16 megabytes image exhibited a pixel size of100 sm and the value of each pixel is expressed between 0 and1023. Quantification of binding density was performed by calcu-lation of the mean gradation per mm2 (PSL/mm2). Prefabricated[3H]-labeled polymer strips (0.03 to 32.4 nCi/mg tissue equivalent,Amersham, microscale) were used to convert intensities intofmol/mg tissue equivalent [20].

Binding data analysisThe IC50 value was defined as the concentration of inhibitor

required to obtain 50% inhibition of the specific binding. Theinhibition constant (K1) values were calculated from IC50 valuesusing the Cheng and Prusoff equation [211. Data for equilibriumbinding (Kd, Bmax), competition experiments (IC50, H) andkinetic constants (KObS, K1) were analyzed using an iterativenon-linear regression program [22]. Results were expressed asmeans SD.

Results

Quantitative autoradiographic analysis by the imaging plate system

The autoradiographic technique provides quantitative databecause the film intensities observed are proportional to the timeof exposure and tissue content of radioactivity. In this study,phosphor-imaging plates for sensing radioactivity energy fromtritium or iodine were used instead of tradional X-ray films. Thisnewly-developed radioluminographic technique has a wide lineardynamic range as well as a high sensitivity ensuring a goodquantitative accuracy and great reduction in exposure-time (a fewdays instead of months in our case) [17, 23—251. On the otherhand, the resolution of imaging plates is, in general, less than thatof X-ray films due to the characteristics of the image reader. Asshown in Figure 2, using [3H]-labeled autoradiographic standardsas references, the imaging plate response is perfectly linearbetween 0.03 to 32.4 nCi/mg tissue equivalent and detection isproportionnal over exposure-time (r2> 0.99; not shown).

Autoradiographic distribution of AVPbinding sites in Sprague-

Dawley rat kidney sections

As expected, [3H]-AVP intensely labeled the medullo-papillaryportion of the kidney and, to a lesser extent, the cortical structurecorresponding to V2 and Via receptors, respectively, as previouslydescribed (Fig. 3A) [14, 26, 27].

Conversely, the non-peptide Via antagonist, [3H]-SR 49059(Fig. 3B) and the other selective peptide Via ligands used,[3Hj.d(CH2)5Tyr(Me)AVP (Fig. 3C) and ['251]-linear AVP antag-onist (Fig. 3D), showed an absence of binding in the papillary area

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Senudeil-Le Gal et al. [3H1-SR 49059 and autoradiography 501

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600

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400

200

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Fig. 2. Quantitative analysis of radioactive standards using the imaging platesystem. [3H]-autoradiographic standards (dose-ranging: 0.03 to 32.4nCi/mg tissue equivalent) were apposed 2 (LI), 5 (0), 8 (•) and 10 ()days to the phosphor-imaging plate. Inset. Dose-ranging from 0.03 to 6nCi/mg tissue equivalent. Density values of [3H]-labeled Amershamstandards (PSL/mm2) were analyzed at differents exposure times. Thecorrelation coefficient (r2) values are > 0.99 in all cases.

of the kidney, discrete labeling of the cortex and a dense andmassive labeling in the outer part of the inner medulla, probablyassociated to vascular structures in this area as shown previouslyby microscopic localization techniques [281. This binding spreadfrom the medulla to other structures in the adjacent part of theinner stripe of the outer medulla. In this latter region the labelinghas a thin striated apparence and probably corresponds to Viareceptors localized in vasa recta [28].

It is important to note the intense labeling of the renal pelvicwall of the papillary surface which was a remnant of capsulafibrosa. All three Via ligands showed a similar distribution patterneven if we obtained a higher resolution with tritiated ligands thanwith the iodinated one by using the imaging plate system (Fig. 3).Moreover, the binding observed in these autoradiograms is judgedto be specific since it was eliminated by incubating in the presenceof 1 LM cold AVP (Fig. 3, inset).

Additional control experiments were performed with {3H]-SR49059 on rat liver sections under similar operating conditions (Fig.3B). As expected, intense labeling was observed in all the liver inagreement with the high level of expression of Via receptors inthis organ. Binding heterogeneity confirmed a distribution of Viareceptors to specialized hepatocytes and/or vascular epithelialcells as previously observed on autoradiograms in this tissue usinga selective radioiodinated Via ligand [11].

Characteristics of [3H]-SR 49059 binding to slide-mounted ratkidney sections

In these autoradiographic experiments performed in adultSprague-Dawley rat kidneys, we determined the properties of[3H]-SR 49059 binding by quantification analysis of [3H]-SR49059 labeling in the outer medulla region.

As shown in Figure 4, [3H]-SR 49059 binding to rat kidney

sections was time-dependent, reached an apparent equilibrium inabout 40 minutes, and demonstrated high stability (> 4 hours).Thus, an incubation time of 60 minutes was chosen for thefollowing saturation and competition experiments. An apparentequilibrium constant (KObS) of 0.125 0.026 minutes and ahalf-maximum association time (t½) of 6 2 minutes werecalculated from these time-course binding studies. Binding of[3H]-SR 49059 was reversible and the addition of unlabeled SR49059 (1 M) induced rapid and full dissociation of the peptide-receptor complex with an apparent dissociation constant (K_5)value of 0.059 0.012 min (Fig. 4). It is worth noting that underequilibrium conditions, non-specific binding represented 25 to30% of total binding.

Saturation experiments of [3H}-SR 49059 binding to slide-mounted rat kidney sections showed that the specific binding wassaturable (Fig. 5A). Scatchard analysis of data, resulting from thelabeling of [3H]-SR 49059 in the outer medulla, gave a linear plotconsistent with the presence of a single class of high affinitybinding sites with a Kd and a Bm value of 1.48 0.16 nM and 33

2 fmol/mg tissue equivalent, respectively (N = 3), A similaranalysis performed in the renal cortex yielded an equivalent Kdvalue (1.05 0.25 nM) but a much lower number of binding sites(Bm value of 4.9 0.5 fmol/mg tissue equivalent; not shown).

The binding of [3H]-SR 49059 to the outer medullary sites in ratkidney sections was further characterized by studying the relativepotencies of several peptide and non-peptide AVP/OT agonistsand antagonists (Table 1). The receptor identified by [3H]-SR49059 exhibited a full Via profile since d(CH2)5Tyr(Me)AVP, aselective V1 a antagonist, showed nanomolar affinity, whereas thespecific V2 agonist, DDAVP, the non-peptide AVP V2 antagonist,OPC-31260 [19], oxytocin and d[D-3Pal]VP, described as a selec-tive Vib agonist [29], were much less potent (IC50 values > 200nM).

As shown in Figure 6, SR 49059 dose-dependently inhibited the[3H]-SR 49059 binding to kidney slides with similar nanomolarpotency to that of d(CH2)5Tyr(Me)AVP. Conversely, the othernon-peptide Via compound, OPC-21268 exhibited a 100-foldlower affinity, in agreement with that previously reported for Viareceptors in rat liver membranes [18]. Furthermore, [3H]-SR49059 binding to renal Via receptors was highly stereospecificsince SR 49059's enantiomer, SR 49770, had 50-fold less affinitythan SR 49059 (Table 1; Fig. 6). These data are consistent withprevious results obtained with SR 49059 and SR 49770 in differentcell types expressing Via receptors such as Swiss 3T3 fibroblasts,human vascular smooth muscle cells, and rat and human liver [13,30, 311. In all cases, dose-response curves for these compoundsgave a linear Hill plot and pseudo Hill coefficients (nH) aboutunity indicating competitive antagonism with the ligand. More-over, K and Kd values for SR 49059, calculated from kinetic,saturation or competition experiments [21, 22] by analysis of thedifferent autoradiograms were highly similar (1, 1.48 and 1.25 ni,respectively).

Discussion

The present study demonstrates that [3H]-SR 49059, which isthe first tritium-labeled non-peptide vasopressin receptor Vialigand yet described, is a suitable probe for investigating thedistribution of Via receptors in complex tissues such as the kidneyexpressing a low density of Via sites together with mixed popula-tions of AVP/OT receptor subtypes.

0 5 10 15 20 25 30 35

nd/mg tissue equivalent

C rH)-d(cH2)5Tyr(Me)Avp D [1251]-Iinear AVP antagonist

A rH]-AVP

2.5 mm 2.5 mm

Graduation bar:

Iower'"T4higher intensity of labeling

Liver

B rHl-sR 49059

Kidney

A (3H]-AVP

Graduation bar:

lower higher intensity of labeling

I I :12.5 mm 2.5 mm

B (3H]-SR 49059

Kidney Liver

C [3H]-d(CH2)5Tyr(Me)AVP D (1l]-linear AVP antagonist

Fig. 3. Localization of vasopressin binding sites in rat kidney and liver tissue sections. [3H1-AVP (A), [3H1-SR 49059 (B), [3H1-d(CH2)5Tyr(Me)AVP (C),and ['251J-linear AVP antagonist (D) labeling in Sprague-Dawley rat autoradiograms. Inset. Non-specific binding obtained for each ligand by incubatingin the presence of 1 zM unlabeled AVP. Graduation color bar: lower higher intensity of labeling.

Serradeil-Le Gal et a!: [3HJ-SR 49059 and autoradiography 503

SR 49059 1 JIM

I

0 30 60 90 120 150 180

Time, minutes

Fig. 4. Time-course of association (•) and dissociation (0) of [3H1-SR49059 to slide-mounted rat kidney sections. Incubations were carried out asdescribed in the text in the presence of [3H1-SR 49059 (1.9 nM) for variousperiods of time. Arrow indicated time (60 mm) at which cold SR 49059 (1

was added to initiate the dissociation process. Results represent datafrom a typical experiment performed in triplicate.

In spite of these difficulties, the complete characterization ofthe site labeled by [3H]-SR 49059 was achieved in slide-mountedadult rat kidney sections by receptor autoradiography using, forthe first time, a phosphor-imaging plate detecting system. In thepresent case, this method was of high interest in order to benefitfrom the best spatial resolution provided by tritiated ligands andto avoid the very long exposure-times with traditional photo-graphic emulsions. Therefore, using the selective AVP Via ligand,[3H]-SR 49059, together with highly sensitive and faster autora-diographic phosphor-screens (10 to 100 times more than X-rayfilms), it took on average a four day-exposure, instead of months,in order to obtain the full mapping of AVP Via receptors indiscrete areas of rat kidneys. The wide dynamic range and thelinear response obtained with extremely strong signals on radio-luminographic plates allowed accurate quantification, even insections with both low and intense labeling (that is, saturationexperiments on tissue sections, whole body autoradiography [23—251). This expanding methodology has been recently successfullyapplied to detecting the complex distribution of neuropeptidereceptors such as cholecystokinin binding sites in rat brain [32] orendothelin receptors in human microvessels [331.

Considering the Via receptor distribution in (20 to 30 days-old)rat kidneys, [3HJ-SR 49059 and other peptide Via ligands used{[3H]-d(CH2)5Tyr(Me)AVP and ['251]-linear AVP antagonist} [7,8] provided a similar labeling pattern expressed in three mainregions: a large and dense band in the outer part of the innermedulla, radiating streaks in the inner part of the outer stripe ofthe medulla and sparse and low labeling of the cortex. Whateverthe Via ligand used, no radioactivity was detected in the papilla.Conversely, [3H]-AVP intensely labeled this latter V2 receptor-enriched medullo-papillary region correlating with the AVP re-ceptor distribution reported by others [14, 26, 27].

Full characterization of [3H]-SR 49059 binding sites, by quan-tification analysis in the intensely labeled outer part of the inner

medulla region, confirmed the Via nature of the site identified.Binding of [3H1-SR 49059 to rat kidney sections was time-dependent, reversible, saturable and exhibited similar propertiesto those already described for liver Via receptors in rat andhuman tissues [13]: a single high affinity binding site (Kd = 1.48

0.16 nM), high binding stability, rapid dissociation of the receptor-ligand complex (t½ = 5 mm) and stereospecificity. The relativepotencies of reference peptide and non-peptide compounds ininhibiting [3H]-SR 49059 binding showed that the binding siteidentified by [3H]-SR 49059 exhibited a fully Via profile: AVPd(CH2)5Tyr(Me)AVP SR 49059 << oxytocin or DDAVP ord[D-3Pal]VP or OPC-31260. Moreover, the high selectivity profilepreviously described for SR 49059 [121 was of particular interest inautoradiographic studies in tissues such as kidneys expressingboth AVP/OT sites (Via, V2, UT and also Vib [15, 16]) and a widevariety of receptors for peptide or non-peptide ligands such asendothelin, neurotensin, beta-adrenergic, neurokinin, angioten-sin, benzodiazepin receptors.

As regards the AVP Via receptor localization in rat kidney,although the autoradiographic technique used did not allowlocalization at the cellular level, it seems reasonable to assumethat [3H]-SR 49059 binding sites are associated with medullaryinterstitial cells and vascular elements of inner medulla vasa rectaand vascular bundles of outer stripe of medulla accounting for theradiating streaks seen in the autoradiograms. Taken overall, thispattern is consistent with the ontogenic expression of mRNAencoding the Via receptors as reported by Ostrowski et a! in adultSprague-Dawley rats using in situ hybridization histochemistry, atechnique allowing microscopic localization [28, 34]. They evi-denced the V a receptor mRNA predominantly in vascular ele-ments adjacent to and coursing alongside collecting ducts whereasthe basic pattern of V2 mRNA expression remained in medullo-papillary, cortical collecting ducts and moderately in the thickascending limbs of the ioop of Henle in the outer medulla.Moreover, this latter study emphazises that Via receptor (and V2)expression depends on the stage of development and more Viareceptor transcripts are detected in the developing kidney (21 to60 days-old rats, as in the present study) than in the mature organ(60 days-old). Besides the localization to vascular structures, anassociation of Via receptors to the functional nephron segmentscannot be excluded. The reverse transcription-polymerase chainreaction method, allowing quantification of Via and V2 mRNA invarious segments of rat Sprague-Dawley nephrons isolated bymicrodissection, demonstrated consistent amounts of Via mRNAin the cortical collecting ducts and to a lesser extent in the outermedullary collecting duct [35]. Functional data also underline theexpression of efficient Via receptors in these segments andphysiological studies showed that specific AVP Via receptorantagonists inhibited AVP-induced PGE2 synthesis by isolatedmedullary collecting duct cells [36] and totally blocked AVP-evoked Ca2 transients in the cortical and outer medullaryportions of the collecting ducts, consistent with a role of Viareceptors in the renal function [37]. A number of in vivo studiessupport the renal role of Via receptors both at a vascular level inregulating the renal medullary hemodynamics and the tubularfunction. The direct tubular effect of Via receptor stimulationappears to favor diuresis and/or natriuresis. Moreover, by use ofvideomicroscopy, laser-Doppler fiowmetry techniques and spe-cific Via anologues such as POVT, it is clearly demonstrated thatVia receptor stimulation in the renal medulla reduces renal

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Table 1. Binding affinity of several peptide and non-peptide AVP/OTagonists and antagonists in [3H]-SR 49059 binding to Sprague-Dawley

rat kidney sections

Compounds IC50 nM

PeptideAVP 3.50 0.11POVT 19.53 3.12d(CH2)5Tyr(Me)AVP 2.07 0.60d[D-3 Pall VP 433 205

> 1000DDAVP (24% inhibition of binding at 1 jsM)OT 243±85

NonpeptideSR 49059 2.97 0.90SR 49770 165 88OPC-21268 435 168OPC-31260 2360 1053Values are means SD obtamed from 3 to 6 determinations.

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Fig. 6. Inhibition of specific /3H]-SR 49059 binding to slide-mounted ratkidney sections by AVP (LI), d(CH2)5Tyr(Me)AVP (V), SR 49059 (U), SR49770 (Y), OPC-21268 (•) and OPC-31260 (0). Incubations were carriedout with 1.8 nM [3Hl-SR 49059 and increasing concentrations of the testcompound for 60 minutes at room temperature. Data were means from 3to 6 determinations.

characterization of Via receptors even in discrete area of renalstructures. The low level of non-specific binding together withhigh specific-activity, stability and potent affinity for both animaland human Via receptors [121 ensure that this non-peptide ligandis of interest for labeling Via receptors in a variety of complexorgans expressing heterogeneous populations of AVP/OT recep-tors.

Acknowledgments

The authors wish to thank Dr. C. Barberis from INSERM U.401,CCIPE, Montpellier (France), for preparing the [1251l-linear AVP antag-onist ligand and Dr. J. Wagnon and Dr. G. Garcia from SANOFI,Montpellier, for synthesis of SR and OPC compounds. We extend ourthanks to Dr. J. Simiand and her team for the use of the Bio-ImageAnalyser (BAS 2000, Fuji) and their technical assistance. We also thankA.J. Patacchini for comments on the manuscript and M. Laborde for hersecretarial assistance.

Reprint requests to Dr. Claudine Serradeil-Le Gal, Sanofi Recherche,Exploratoiy Research Department, 195 Route d'Espagne, 31036 ToulouseGedex, France.

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Fig. 5. Saturation of [3H]-SR 49059 specificbinding to slide-mounted rat kidney sections. (A)Saturation isotherm performed in the presenceof various concentrations of [3H]-SR 49059 (0.3to 18 nM) as described in Methods. (B)Scatchard plot. Data are means calculated froma typical experiment performed in triplicate.

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medullary blood flow and decreases red blood cells velocity indescending and ascending vasa recta. In addition, chronic stimu-lation of Via receptors could even mediate the development ofhypertension in chronically instrumented rats [38].

Since renal glomerular mesangial cells are known to possessV10 receptors, the low labeling observed with [3H]-SR 49059(sixfold less than in the outer part of the inner medulla accordingto our quantification) and with other Via ligands in the cortexseems an intriguing observation. In addition, the selective peptideV0 ligand d(CH2)5Tyr(Me)AVP has shown high potency ininhibiting both [3H]-AVP binding and AVP-induced [Ca2]1 incultured rat mesangial cells in vitro [39]. However, autoradio-graphic and hybridation in situ experiments confirmed very smallamounts of V10 receptor labeling over glomeruli in the adult ratkidney [14, 34]. Different phenotypic states between in vitroproliferating mesangial cells and in vivo contractile cells couldexplain this discrepancy. Likewise, the presence of a highernumber of mRNA V, transcripts in developing glomerular cellsthan in cells at the adult stages has been described and isconsistent with the role played by V0 receptors in mediatinggrowth effect [34].

In conclusion, this study describes the localization and quanti-fication of V10 receptors in adult rat kidney sections using [3H1-SR49059 and a fast phosphor-imaging plate radioluminographictechnique. The present report points out the interest of [3H]-SR49059 as a suitable probe for specific detection and accurate

Serradeil-Le Gal et a!: [3H]-SR 49059 and autoradiography 505

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