Urodilatin binds to and activates renal receptors for atrial natriuretic peptide

JP Valentin, LA Sechi, C Qui, M Schambelan and MH HumphreysUrodilatin binds to and activates renal receptors for atrial natriuretic peptide

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432

Urodilatin Binds to and Activates RenalReceptors for Atrial Natriuretic Peptide

Jean-Pierre Valentin, Leonardo A. Sechi, Changbin Qiu,Morris Schambelan, and Michael H. Humphreys

Urodilatin is a recently described member of the atrial natriuretic peptide family, thought possibly to besynthesized in the kidney. To determine if urodilatin binding sites are present in rat and human kidney, weevaluated the effect of urodilatin on iodine-125-Jabeled atrial natriuretic peptide (ANP) (100 pM) binding totissue sections using an in situ autoradiographic technique. 12iI-ANP binding occurred primarily in gloroeruliand medullary structures of both rat and human kidney. Increasing concentrations of urodilatin yielded amonophasic displacement of 125I-ANP binding with an IQ, of 42 nM, a value nearly identical to that achievedwith unlabeled ANP (12 nM). In additional experiments, rat glomeruli and inner medullary collecting ductcells were isolated and incubated in vitro with either ANP or urodilatin (10"" to 10"6 M) and cyclicguanosine-3',5'-monopaosphate accumulation measured by radioimmunoassay. Dose-response curves for thetwo peptides were superimposable in each tissue; at 10"* M, ANP generated 613±41 and urodilatin 603±55fmol cyclic guanosine monophosphate per 10 minutes per milligram protein in inner medullary collecting ductcells (p=NS). Thus, urodilatin is as effective as ANP in displacing I2JI-ANP binding to both rat and humanrenal tissue and in generating cyclic guanosine monophosphate in renal target cells in the rat, suggesting thatits physiological effects may occur through the same receptors and signaling pathways that mediate the actionsof ANP. (Hypertension 1993^1:432-438)

KEY WORDS • autoradiography • guanosine cyclic monophosphate • kidney glomerulus • kidneytubules, collecting • renal artery • natriuretic peptides, atrial

Three major types of receptor for atrial natriureticpeptide (ANP) have been identified in targettissues: ANP A-receptor and ANP B-receptor

contain a membrane-bound guanylate cyclase in theircytoplasmic domains that mediates the production ofthe intracellular second messenger cyclic guanosine-3',5'-monophosphate (cGMP). ANP has greatest affin-ity for the ANP A-receptor, whereas the naturallyoccurring ligand for ANP B-receptor may be the re-cently identified C-type natriuretic peptide (CNP).1

ANP C-receptor is a so-called clearance receptor, whichto date has no clearly established function but may bindand degrade natriuretic peptides.1-2 The C-receptor hasbeen identified because it binds biologically inactive

From the Divisions of Nephrology (J-P.V., C.Q., M.H.H.) andEndocrinology (L.A.S., M.S.), San Francisco General Hospitaland University of California San Francisco (California).

Presented, in part, at the Council for High Blood PressureResearch 45th Annual Fall Conference and Scientific Sessions,Chicago, 111., September 24-27,1991, and has appeared in abstractform {Hypertension 1991;18:393).

Supported by grants DK-31623 and HL-11046 from the Na-tional Institutes of Health, Bethesda, Md., and by grant-in-aid89-1124 from the American Heart Association. J-P.V. was therecipient of fellowship awards from ICI Pharma and the AmericanHeart Association, California Affiliate. LA.S. was the recipient ofawards from the Italian Society of Hypertension and the JuvenileDiabetes Foundation International.

Address for correspondence: Michael H. Humphreys, MD,Room 350, Building 100, San Francisco General Hospital, SanFrancisco, CA 94110.

Received June 18, 1992; accepted in revised form November 9,1992.

analogues of ANP with high affinity, whereas the gua-nylate cyclase receptors do not.3 Ligand binding studieswith radioactively labeled peptides have indicated thatA-receptors are concentrated in glomeruli, inner med-ullary collecting duct (IMCD), and vascular structures,whereas C-receptors are more concentrated in glomer-uli.2'3 Recently, a four-amino acid N-terminal extensionof ANP has been identified in renal tissue and urine.4-5

Termed urodilatin (URO) or renal natriuretic peptide,its function like that of ANP has not been fully deter-mined, although it has been advanced as a candidate tomediate renal tubular actions previously ascribed tocirculating ANP, including diurnal oscillations in uri-nary sodium excretion and the natriuretic response toan acute intravenous saline infusion.6'7 There is as yetlittle information on the binding properties of thispeptide or its ability to stimulate production of cGMPin responsive tissues.

The purpose of our study was therefore to character-ize, using the technique of autoradiography, the abilityof URO compared with CNP and unlabeled ANP todisplace 125I-ANP from its binding sites in renal tissue.In addition, we examined the effectiveness of URO instimulating production of cGMP by isolated glomeruliand IMCD cells studied in vitro. Our results are consis-tent with the interpretation that URO binds to the samebiologically active receptors as ANP in renal tissue.

MethodsMale Sprague-Dawley rats (Bantin Kingman, Fre-

mont, Calif.) weighing 280-350 g were housed in a



controlled environment with a 12-hour light/dark cycleand provided standard rat chow and water ad libitum.

Receptor Binding StudiesAnimals were killed by decapitation, and the kidneys

were quickly removed, decapsulated, snap frozen inliquid nitrogen, and stored at -80°C. Human renaltissue was obtained from the normal portion of twoneoplastic kidneys removed at nephrectomy and frozenin liquid nitrogen. The distribution of renal ANP recep-tors was assessed by a modification of an in situ receptorbinding assay.8 Briefly, tissue sections (10 uM) were cuton a cryostat at - 15°C, thaw mounted (two per slide) onpory-L-rysine-coated slides, dried in vacuo for 18 hoursat -4° to +4°C over silica gel, and stored in sealedBakelite boxes at -80°C. Before assay, the slides werebrought to room temperature. Endogenous ANP boundto receptors was removed by preincubating the sectionsfor 10 minutes at room temperature in 200 uh of abuffer (buffer A) containing 30 mM sodium phosphate(pH 7.2), 120 mM NaCl, 0.3% bacitracin, and 0.5%receptor grade bovine serum albumin. After preincuba-tion, the buffer was replaced with 200 jiL fresh buffer Acontaining 100 pM &I-ANP (2,200 /xCi/mmol, NewEngland Nuclear, Boston, Mass.), and the sections wereplaced in a humidified chamber and incubated at roomtemperature for 15 minutes. After incubation, the slideswere rinsed in an ice-cold buffer containing 30 mMsodium phosphate and 120 mM NaCl for 10 seconds,washed in the same buffer for 5 minutes, rinsed indeionized water for 10 seconds, and dried for 2 hours ina stream of cool air. For autoradiography, the slideswere exposed to LKB-Ultrofilm for 3-5 days at roomtemperature. Films were processed with Kodak D-19developer (Kodak, Rochester, N.Y.) and Kodak rapidfixer, and the amount of radioligand bound to the tissuesections was determined by counting the slides in agamma counter.

Nonspecific binding was determined on adjacent sec-tions under identical incubation conditions except forthe addition of 1 /iM unlabeled rat ANP-(l-28) (Penin-sula Laboratories, Belmont, Calif.). Clearance receptorswere identified by addition of 10 fiM CNP-(4-23)(Peninsula Laboratories). Emulsion autoradiographywas used to obtain more precise cellular localization ofthe ANP binding sites. After incubation with radioli-gand and washes as described above, serial sectionswere fixed in paraformaldehyde vapors for 2 hours at80°C, left in air for a minimum of 4 hours, dipped inphotographic emulsion (Kodak NTB2), and exposed for15-20 days at 4°C. The emulsion was developed usingKodak D-19 and Kodak fixer. To assist in the identifi-cation of structures associated with the grains, selectedsections were stained with hematoxylin-eosin.

The time course of binding was assessed by determin-ing the total amount of radioligand bound after 2.5-45minutes of incubation. The reversibility of binding wasassessed by the addition, after 15 minutes of incubation,of either unlabeled rat ANP (1 uM) or URO (1 fiM)(Peninsula Laboratories). To determine the extent towhich radioligand binding was inhibited by unlabeledpeptides, adjacent sections were incubated with 100 pM125I-ANP in the presence of increasing concentrations(10 pM-10 uM) of rat ANP or URO. The concentrationof unlabeled peptides that resulted in 50% inhibition

Valentin et al Urodilatin and Renal Receptors

A ^ B

433

FIGURE 1. In situ autoradiographic localization of at rialnatriuretic peptide (ANP) receptor subtypes in rat kidney.Panel A: Binding of 125I-ANP was distributed primarily inglomeruli and in the inner medulla. Panel B: Incubation withI (JLM unlabeled ANP markedly inhibited radioligand binding.Panel C: Incubation with 10 \LM C-type natriuretic peptideinhibited radioligand binding to glomeruli to a lesser degree.Panel D: Incubation with 1 fxM unlabeled urodilatin inhibitedradioligand binding throughout the kidney to an extent similarto that seen with unlabeled ANP.

of radioligand binding was calculated using theLIGAND program.9

Because binding studies of ANP may be influenced byendogenous hormone bound to its receptors,10 we at-tempted to establish that our preincubation with bufferA was adequate to remove already bound peptide bycomparing the binding of 100 pM 125I-ANP to adjacentkidney slices (n=6) that were preincubated for 10minutes with buffer A or an acidic wash formed byadding 0.5% acetic acid to buffer A. Total counts perminute (corrected for background) with buffer A were3,532±437 compared with 4,254±362 with the acidwash (p=NS). These results suggest that preboundligand did not materially influence our binding resultsand are in accord with the more extensive studies ofBrown et al.11

Preparation of Glomeruli and Inner MedullaryCollecting Duct Cells

Rats were anesthetized with pentobarbital (50 mg/kgi.p.). Glomeruli were isolated as follows12: the kidneys



434 Hypertension Vol 21, No 4 AprU 1993



Valentin et al Urodilatin and Renal Receptors 435

FIGURE 2. Emulsion autoradiography of atrial natriureticpeptide (ANP) receptors in rat kidney. Sections were incu-bated with 123I-ANP as described in "Methods." Panel A:Silver grains in a major branch of the renal artery (a). PanelB: Silver grains are present predominantly overlying twoglomeruli (g). Original magnification x200.

were perfused with cold isotonic heparinized salineuntil blanched (50-60 mL saline in 2 minutes). Therenal cortices were dissected and minced to a pastelikeconsistency. The homogenate was passed successivelythrough a 106 -um sieve that excluded tubules and bloodvessels and a 75 -fim sieve that retained the glomeruliand allowed cells and small debris to pass through.Glomeruli were suspended in ice-cold 20 mM tris(hy-droxymethyl)aminomethane hydrochloride (tris HC1)buffer, pH 7.4, containing (mM) NaCl 125, KC1 10,sodium acetate 10, and glucose 5 (buffer B) and werecentrifuged at 120g for 2 minutes. The supernatant wasdiscarded, and the pellet was resuspended in the samebuffer and recentrifuged. By light microscopic examina-tion, samples of the final pellet consisted of nearly pureisolated decapsulated glomeruli with less than 5% tu-bular contamination. No afferent or efferent arterioleswere observed.

IMCD cells were isolated as follows13: the kidneyswere perfused with 50-60 mL of Joklik's minimalessential medium (Applied Scientific, San Francisco,Calif.) followed by 5 mL of a solution of Joklik'smedium containing 0.2% of collagenase (Type II, SigmaChemical Co., St. Louis, Mo.). The inner medullas wereexcised, finely minced, and incubated in the samesolution for 90 minutes at 37°C. The resulting suspen-sion of inner medullary cells was layered on a 16%Ficoll (Sigma) solution in nonbicarbonate Ringer buffer(buffer C) and centrifuged at 2,300g for 40 minutes.Cells were subsequently washed through buffer C andthen a solution of buffer C containing 7.5% albumin toremove any traces of contaminating collagenase. Sam-ples of IMCD cells were then embedded in pory/BED812 and examined by transmission electron microscopyusing a JEM JEOL 100 SC microscope; photographswere magnified x 6,900. Cell preparations were homo-geneous and exhibited the characteristic ultrastructuralappearance of IMCD cells.13

Determination of Cyclic GMP Generation byGlomeruli and Inner Medullary Collecting Duct Cells

Freshly isolated glomeruli were resuspended in bufferB containing 1 mM CaCl2 and 1 mg/mL bacitracin, andIMCD cells in buffer C containing bacitracin, 7.5 mMglucose, and no pyruvate or acetate and preincubated inroom air for 10 minutes at 37°C in a shaking water bath;in one set of experiments using isolated glomeruli, theprotease inhibitors aprotinin (500 KIU/mL), pepstatinA (20 Mg/mL), phenylmethylsulfonyl fluoride (200 u.g/mL), and SQ28.603 (1 mg/mL) were added to theincubation medium. Incubations were carried out for 10minutes at 37°C in the presence of increasing doses(10-" to 10"6 M) of ANP or URO, dissolved in theincubation medium in volumes of 10 uL. Incubationstook place in the absence of any phosphodiesteraseinhibitor. Incubations were terminated by adding 750(JLL of ice-cold trichloroacetic acid (TCA) (final concen-

tration 6.6%) and cooling to 4°C. The precipitatedprotein was sedimented by centrifugation at 4,500 rpmfor 15 minutes at 4°C, and the pellets were dissolved inIN NaOH and assayed for protein content by themethod of Lowry et al14 using bovine serum albumin asstandard. The supernatant was extracted five times withfour volumes of water-saturated ethyl ether to removethe TCA before being evaporated to dryness under astream of air; it was stored at -70°C until assayed forcGMP content. For the cGMP assay, samples weredissolved in 50 mM sodium acetate buffer, pH 6.2,mixed thoroughly, and 100 /JLL aliquots acetylated ac-cording to the manufacturer's instructions (New En-gland Nuclear). Average results of triplicate determina-tions are expressed as femtomoles cGMP generated per10 minutes per milligram protein.

Data Presentation and Statistical AnalysisData are presented as mean±SEM. Comparisons

between groups were done by the use of Student's t testfor paired or unpaired variables, and one-way analysisof variance with repeated measures when multiplecomparisons were carried out. A value of p<0.05 wasused to assign statistical significance.

ResultsReceptor Binding Studies

Binding of 125I-ANP was found in both cortical andmedullary regions of rat kidney (Figure 1). In thecortex, the radioligand binding was localized primarilyin glomeruli (Figure 2). In the outer medulla, boundradioligand was found primarily in longitudinal bands inthe inner stripe, corresponding to medullary vascularbundles.8 Minimal radioligand binding was found in theinterbundle areas, whereas binding to papillae wasmoderate. An additional area of specific radioligandbinding was observed in major branches of the renalartery (Figure 2) and in the muscular layer of theintrarenal pelvis. Nonspecific binding was less than 10%(Figure 1). Incubation with CNP markedly reducedbinding of the radioligand to glomeruli (Figure 1).Incubation with URO also inhibited binding of theradioligand (Figure 1) to an extent similar to that seenwith unlabeled rat ANP.

Figure 3 shows the time course of binding of radioli-gand to sections of rat kidney. Total binding increasedrapidly, reaching a plateau after 15 minutes of incuba-tion. Both unlabeled rat ANP and URO progressivelydisplaced the labeled ligand by 31% and 33% of thecontrol binding, respectively. Increasing concentrationsof both rat ANP and URO resulted in a monophasicdisplacement of radioligand binding, with an IQo of 7.2and 4.2 nM, respectively (Figure 4).

The pattern of radioligand binding to human kidneytissue is illustrated in Figure 5. As in the rat kidney,binding was present primarily in glomeruli. This bindingwas markedly inhibited by unlabeled ANP and URO,but only slightly by CNP (Figure 5).

Cyclic GMP Generation in Glomeruli and InnerMedullary Collecting Duct Cells

Figure 6 illustrates the stimulation of cGMP genera-tion in glomeruli and IMCD cells in response to increas-ing concentrations of ANP and URO. ANP induced a



436 Hypertension Vol 21, No 4 April 1993

15 4530

minutesFIGURE 3. Line graph shows time course of binding anddisplacement oflOOpM 125I-atrial natriuretic peptide (ANP).Total binding increased progressively during the first 15minutes, reaching a plateau thereafter. Addition of bothunlabeled ANP and urodilatin (URO) at this point progres-sively displaced the radioligand, reaching values of 31% and33% of the control value, respectively. Data are mean±SEMof sections obtained from four rats.

dose-dependent increase in cGMP generation in bothglomeruli and IMCD cells. At 10"6 M, the values wereroughly 10-fold and sixfold higher, respectively, forglomeruli and IMCD cells than basal values. Superim-posable dose-response curves were obtained in bothglomeruli and IMCD cells with URO. The presence ofprotease inhibitors in the incubation medium did notalter these results: at 10"6 M ANP, cGMP accumulationin glomeruli in the absence of the inhibitors was 638 ±81versus 628 ±103 fmol/mg per 10 minutes in their pres-ence. With 10"6 M URO, the corresponding resultswere 599±105 versus 608±129 fmol/mg per 10 minutes(«=6 for each).

DiscussionOur results indicate that URO is as effective as

unlabeled ANP in displacing 125I-ANP from bindingsites in both rat and human kidney. The virtually

• • ANPO---O URO

-11 -10 -9 -8 -7 - 6 - 5

log Peptide, MFIGURE 4. Competition of unlabeled atrial natriuretic pep-tide (ANP) and urodilatin (URO) for ml-ANP to longitudinalsections of rat kidney. Addition of increasing concentrationsof either unlabeled peptide resulted in a monophasic displace-ment of specific radioligand binding, with an ICJO of 7.2 and4.2 nM, respectively. Data are mean±SEM of sections ob-tained from five rats.

superimposable displacement curves and the nearlyidentical IQo values obtained with URO and ANP offervery strong evidence that each peptide has equal affinityfor the same receptor or receptors. This is not anunexpected result in view of the identical structure ofthe two peptides save for the N-terminal extension offour amino acids possessed by URO. Addition andsubstitution studies of the binding of ANP to theA-receptor have identified several factors necessary foroptimum binding. These include integrity of the ringstructure and the three C-terminal amino acids; exten-sion at the N-terminus has relatively little effect onbinding.23 Since each of these key structural propertiesis shared by both ANP and URO, it is not surprisingthat URO should be effective in displacing 12SI-ANPfrom these receptors. The fact that the displacementcurve and the IQo are nearly identical provides evi-dence that the N-terminal extension of URO neitherimpedes nor enhances its ability to displace 125I-ANPcompared with unlabeled ANP. Our data also indicatethat URO interacts with the C-receptor in glomeruli ina manner closely resembling that of ANP (Figure 1).

The same line of reasoning applies to the functionalconsequences of ligand binding to the A-receptor. Intarget tissues, the A-receptor contains a guanylate cy-clase in its cytoplasmic domain so that receptor occu-pancy activates the enzyme and results in production ofcGMP. It consequently follows that URO should beequally potent in stimulating cGMP production as ANP,given that it exhibits equivalent potency in displacingmI-ANP from its receptors. This was indeed the case inboth isolated glomeruli and IMCD cells studied in vitro:the dose-response relation of cGMP produced withincreasing concentrations of added peptide was againvirtually identical between URO and ANP in bothtissues. These results were not altered by the presenceof four different peptidase inhibitors in the incubationmedium, suggesting that degradation of the peptides didnot occur to any large extent. Previous data regardingthe cellular effects of URO are scanty. Heim andassociates15 showed that URO was equipotent to ANPin stimulating particulate guanylate cyclase from bovineadrenal cortical membranes, and the two peptides pro-duced equivalent decreases in medullary sodium reab-sorption when given intravenously.16 This would suggestidentical activity in medullary target sites, consistentwith our in vitro observations. If URO is indeed actingthrough the same renal receptors as ANP, then onewould predict that URO could not further stimulatecGMP accumulation above that seen with a maximalconcentration of ANP. Such an experiment will be animportant test of our hypothesis.

The site of synthesis and action and physiologicalfunctions of URO are unknown at present. Peptideisolation techniques have indicated that URO can berecovered from kidney tissue,3 and proANP is releasedfrom renal epithelial cells grown in culture.17 35S-Methionine incorporation studies demonstrate thatthe prohormone can be synthesized in renal cells,17

and a recent preliminary report demonstrates tran-scripts of the ANP gene in renal tissue using reversetranscription and the polymerase chain reaction.18

URO has been linked to sodium excretion only byvirtue of the coordinacy of their respective excretionrates during diurnal variations and after the intrave-



Valentin et al Urodilatin and Renal Receptors 437

B

FIGURE 5. In situ autoradiographiclocalization of atrial natriwetic peptide(ANP) receptor subtypes in human kid-ney. Adjacent sections (10 /j,m) wereincubated with 125I-ANP. Panel A: Li-gand binding occurred primarily in glo-meruli. Panel B: Incubation with 1 \iMunlabeledANP markedly inhibited bind-ing of the radioligand. Panel C: Incuba-tion with 10 \i.M C-type ANP partiallyinhibited binding of the radioligand toglomeruli. Panel D: Incubation with 1fiM unlabeled urodilatin inhibited radi-oligand binding to an extent similar tothat seen with unlabeled ANP. No me-dulla was obtained for examination.

nous infusion of a saline load.3619 The signal (orsignals) governing the fluctuations in its excretion,presumably reflecting changes in synthesis and secre-tion, are unknown. An entirely plausible possibility

OLOUERUU IMCOCctlj

cGMP In vitro

600

500

100

200

— -11-10-9 -8 -7 - • — -11-10-9 -8 -7 -6log Peptide, M

• •ANf O--OUR0

FIGURE 6. Line graphs show effect of increasing concentra-tions of atrial natriuretic peptide (ANP) and urodilatin(URO) on generation of cyclic GMP (cGMP) by isolatedintact glomeruli (left panel) and by inner medullary collectingduct (IMCD) cells (right panel). Dose-response curves forANP and URO were superimposable in both glomeruli andIMCD cells. Data are mean±SEM of results from three andfour glomerular preparations for ANP and URO, respectively,and four preparations of IMCD cells for each peptide. Valuesat lO'6 M for each peptide were significantly greater thanvehicle alone or results with 10'" to 10~9 M in each tissue(one-way analysis of variance), and results with URO did notdiffer from those with ANP at any concentration.

that is not addressed by our studies is that URO mayact through a different set of receptors that recognizethe N-terminal extension of the peptide. Receptorbinding studies that use 125I-URO as the ligand will benecessary to test for this possibility.

AcknowledgmentsMaria Narvaez provided secretarial assistance. SQ28,603

was a kind gift from Dr. James R. Powell, Squibb Institute forMedical Research, Princeton, NJ.

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Matsuo H, Goeddel DV: Selective activation of the B natriureticpeptide receptor by C-type natriuretic peptide (CNP). Science1991^252:120-123

2. Brenner BM, Ballennann BJ, Gunning ME, Zeidel ML: Diversebiological actions of atrial natnuretic peptide. Physiol Rev 1990;70:665-699

3. Maack T, Suzuki M, Almeida FA, Nussenzveig D, ScarboroughRM, McEnroe GA, Lewicki JA: Physiological role of silent recep-tors of atrial natriuretic factor. Science 1987;238:675-678

4. Schulz-Knappe P, Forssraann K, Herbst F, Hock D, Pipkorn R,Forssmann WG: Isolation and structural analysis of "urodilatin," anew peptide of the cardiodilatin-(ANP)-family, extracted fromhuman urine. Klin Wochenschr 1988;66:752-759

5. Feller SM, Gagelmann M, Forssmann WG: Urodilatin: A newlydescribed member of the ANP family. Trends Pharmacol Sci 1989;16337-41

6. Goetz D, Drummer C, Zhu JL, Leadley R, Fiedler F, Gerzer R:Evidence that urodilatin, rather than ANP, regulates renal sodiumexcretion. J Am Soc Nephrot 1990;l:867-874

7. Drummer C, Fiedler F, Konig A, Gerzer R: Urodilatin, a kidney-derived natriuretic factor, is excreted with a circadian rhythm andis stimulated by saline infusion in man. J Am Soc Nephrol 1991;1:1109-1113



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8. Chai SY, Sexton PM, Allen AM, Figdor R, Mendelsohn FAO: Invitro autoradiographic localization of ANP receptors in rat kidneyand adrenal gland. Am J Physioi 1986;250:F753-F757

9. Munson PJ, Rodbard D: UGAND: A versatile computerizedapproach for characterization of ligand binding systems. Anal Bio-chem 1980;107:220-239

10. Martin ER, Lewkki JA, Scarborough RJ, Ballerman BJ: Expres-sion and regulation of ANP receptor subtypes in rat renal glomer-uli and papillae. Am J Physioi 1989;257:F649-F657

11. Brown J, Salas SP, Singleton A, Polak JM, Dollery CT: Autora-diographic localization of atrial natriuretic peptide receptor sub-types in rat kidney. Am J Physioi 1990;259:F26-F39

12. Don BR, Blake S, Hutchison FN, Kaysen GA, Schambelan M:Dietary protein modulates glomerular eicosanoid production inthe rat. Am J Physioi 1989;256:F711-F718

13. Zeidl ML, Sirva P, Brenner BM, Seifter SU cGMP mediates effectsof atrial peptides on medullary collecting duct cells. Am J Physioi1987;252:F551-F559

14. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein mea-surement with the Folin phenol reagent. / Biol Chan 1951;193:265-272

15. Heim J-M, Kiefersauer S, Fuller H-J, Gerzer R: Urodilatin and/3-ANF: Binding properties and activation of paniculate guanylatecyclase. Biochem Biophys Res Commun 1989;163:37-41

16. Schulz-Knappe R, Honrath V, Forssman WG, Sonnenberg H:Endogenous natriuretic peptides: Effect on collecting duct func-tion in rat kidney. Am J Physioi 1990;259:F415-F418

17. Ritter D, Needleman P, Greenwald JE: Synthesis and secretion ofan atriopeptin-like protein in rat kidney cell culture. / Clin Invest1991;87:208-212

18. Golomb E, Friedman E, Abassi ZA, Trachewsky D, Reiser HR:Renal transcription of the gene encoding for atrial natriureticpeptide. (abstract) Program and Abstracts, The Endocrine Society1992;74J34

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Urodilatin binds to and activates renal receptors for atrial natriuretic peptide