endothelin-stimulated secretion peptides by rat atrial ... · endothelin-stimulated secretion...

460

Endothelin-Stimulated Secretion of NatriureticPeptides by Rat Atrial Myocytes Is Mediated by

Endothelin A ReceptorsG. Thibault, A.F. Doubell, R. Garcia, R. Lariviere, E.L. Schiffrin

Abstract Endothelin (ET), a potent vasoconstrictor pep-tide, is known to enhance the secretion of atrial natriureticfactor (ANF) by the heart. In the present study, we investi-gated the potency of ET isopeptides to stimulate ANF andbrain natriuretic peptide (BNP) secretion in primary culturesof neonatal atrial myocytes, and we characterized the receptormediating these effects. All ET isopeptides caused a twofoldincrease of ANF and BNP secretion with the following orderof potency: ET-1P:ET-2>sarafotoxin 6b>>ET-3. Secretion ofthe natriuretic peptides was blocked by BQ-123, an ETA-receptor antagonist, but was not affected by either IRL-1620or [Ala"3"""5]ET-1, two ETB-receptor agonists. ET receptorswere localized by autoradiography on the surface of atrialmyocytes, indicating that contaminating cells were not respon-sible for '251-ET-1 binding. Competition binding analyses werethen used to assess the ET-receptor subtype on atrial myocyte

membrane preparations. A high-affinity (100 pmol/L) bindingsite with high density (=1500 fmol/mg) was found to prefer-entially bind the ET isopeptides in the following order:ET-1.ET-2.sarafotoxin 6b>ET-3. Binding was totally dis-placed by BQ-123 but not by IRL-1620. The ET binding sitetherefore had the characteristics of an ETA-like receptor.Analysis by cross-linking and sodium dodecyl sulfate-poly-acrylamide gel electrophoresis showed that it possessed amolecular mass of ='50 kD. Northern blot analysis of bothETA- and ETB-receptor mRNAs allowed only the detection ofthe former, indicating that the ETB receptor may be expressedin very small amounts. These results demonstrate that ANFand BNP secretion by atrial myocytes is enhanced by ET viabinding to an ETA-like receptor. (Circ Res. 1994;74:460-470.)Key Words * endothelin * atrial natriuretic factor .

brain natriuretic peptide * endothelin-receptor subtypes

N atriuretic peptides found in the heart are nowwell recognized as hormones with potent ac-tions at the level of the kidneys, the vascula-

ture, and the adrenal glands, regulating salt and waterhomeostasis as well as blood pressure."2 Among themembers of this peptide family, atrial natriuretic factor(ANF) and brain natriuretic peptide (BNP) are synthe-sized by atrial myocytes of the rat heart and are storedin atrial secretory granules. We have shown that ANFand BNP are present in the same secretory granules ofthe rat,3 and some investigators have found BNP insecretory granules of pig atria but not evenly distributedacross the atrial wall.4 Others have reported that BNP isnot contained in all secretory granules of human atria.5These results may suggest that both peptides could besecreted concomitantly on stimulation.The factors that affect ANF and BNP secretion by

atrial myocytes are not fully known. The main in vivostimulus of release appears to be mechanical stretchingof the atrial wall. In a review by Ruskoaho et al,6 only afew agents have been documented to enhance ANFrelease. Among them, endothelin (ET) appears to beone of the most potent. The ETs comprise a family ofpeptides with high homology.7 Among them are ET-1,ET-2, and ET-3, which have been cloned from mamma-lian banks, and the sarafotoxins (S6a, S6b, and S6c),

Received May 11, 1993; accepted November 16, 1993.From the MRC Multidisciplinary Research Group on Hyper-

tension, Laboratoire de Biologie Cellulaire de l'Hypertension,Institut de recherches cliniques de Montreal and Faculte demedecine de l'Universite de Montreal (Canada).Correspondence to Dr G. Thibault, IRCM, 110 Pine Ave West,

Montreal, Quebec, Canada H2W 1R7.

which originate from venom of the asp Atractaspisengaddensis. These are all 21-amino-acid peptides withtwo disulfide-bond closed loops. ET-1, the principalpeptide of the ET family, is strongly expressed in thevascular endothelium but also in other tissues in vivo.ET-2 and ET-3 have a more restricted expression, suchas in the placenta or tumoral cells.7ETs have profound effects on cardiovascular regula-

tion through their influence on the vasculature andkidney or via the secretion of other hormones.5,8 At thelevel of the heart,9 ETs act as inotropic and chrono-tropic agents, vasoconstricting coronary arteries andpotentiating ANF release by myocytes. They affect ANFsecretion in vivo as well as in isolated systems such asthe perfused rat heart and neonatal atrial myocytes inculture.'0-14 In cardiac myocytes, ET activates varioussignal transduction pathways: inositol phosphate pro-duction, protein kinase C activation, or intracellularCa21 mobilization.'5-18 ET and ANF counteract eachother at different levels. ANF modulates in vivo therenal effects of ET'9 as well as its constrictor effect onaortic ring preparations.20 Conversely, ET can decreasethe cardiovascular and endocrine effects of ANF.21ANF inhibits the stimulated release of ET by mesangi-a122 and endothelial cells23'24 but enhances it in parathy-roid cells.25 ANF can block the mutagenesis induced byET.26 In view of these multiple interactions, it is there-fore important to understand by which mechanisms ETtriggers ANF secretion.

Until now, two ET receptors have been identified andcloned.27 These receptors have been classified as ETA(with higher affinity for ET-1 than ET-3) and ETB (withsimilar affinity for ET isopeptides). Both are ~430

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Thibault et al ANF and BNP Secretions Are Mediated by ETA Receptors 461

amino acids long, have a molecular mass of :50 kD, andshare high homology, particularly in the transmembranedomain, which is a potential G-protein binding site. ETreceptors are apparently linked to phospholipase C andthe inositol phosphate-diacylglycerol cascade as well asto phospholipase A2 and D. Pharmacologic tools havebeen developed to selectively activate or inhibit eachreceptor. Among these agents are BQ-123 and BQ-153,which are ETA-receptor antagonists,2829 and IRL-1620,BQ-3020, and [Ala"3'11"5]ET-1 (4Ala ET-1), which areETB-receptor agonists.30-32The present study was designed to evaluate the

impact of ET isopeptides on ANF and BNP secretion byatrial myocytes and to determine which ET-receptorsubtype mediates their actions. Primary cultures ofneonatal rat atrial myocytes were used to assess bothANF secretion and the receptor subtype. In addition,BQ-123, IRL-1620, and 4Ala ET-1 were used to char-acterize the ET-receptor subtype involved.

Materials and MethodsPeptidesThe ET isopeptides were purchased from Peninsula Labo-

ratories, Belmont, Calif, and Bachem California, Torrance,Calif. BQ-123 was obtained from Peptides International Inc,Louisville, Ky. IRL-1620 was a generous gift from Dr N.Kamei, CIBA-GEIGY (Japan) Limited, Takarazuka, Japan.Rat ANF-(99-126) was synthesized by Hukabel, Longueuil,Quebec, Canada, and rat BNP-32 and its antiserum, whichrecognizes equally rat BNP-32 and -45, were bought fromPeninsula Laboratories.

Atrial Myocyte CultureMyocyte cultures were prepared from 4-day-old neonatal

Sprague-Dawley rats (Charles River, St Constant, Quebec,Canada) as described by Shields and Glembotski.33 The ani-mals were first injected intraperitoneally with 50 puL (50 U)heparin sulfate (Hepalean, Organon Canada Ltd, Toronto,Ontario, Canada). After decapitation, they were dipped in70% ethanol, the chest was then opened, and the atria wereremoved with surgical forceps. Atria were collected from 3litters (40 to 50 animals) and washed in 10 mL Joklik mediumsupplemented with 250 U heparin sulfate to remove red bloodcells. They were digested at 37°C in 25 mL Joklik mediumcontaining 0.1% collagenase (CLS 2, Worthington Biochemi-cal Corp, Freehold, NJ) and 0.01% DNAse for 20 minutes withagitation (120 cycles per minute).

After incubation, the cells were dispersed by pipetting, andthe remaining tissue was digested for another 20 minutes in 15mL collagenase-DNAse solution. The dispersed cells weresuspended in 10 mL fetal calf serum, filtered through a nylonmesh, centrifuged, and resuspended in complete serum-freemedium (CSFM-1) plus 10% Nu-Serum IV (CollaborativeResearch Inc, Bedford, Mass).33 They were counted in ahematocytometer, diluted, and seeded in fibronectin-coatedculture dishes at a density of 1.25 x 105 cells per squarecentimeter. Fibronectin was purified from rat plasma bygelatin-Sepharose 4B (Pharmacia LKB Biotechnology, Upp-sala, Sweden) column34 and dialyzed against 0.02 mol/L so-dium phosphate, pH 7.4, and 0.15 mol/L NaCl. The culturedishes were coated for 2 hours at room temperature with asolution of 60 ,ug/mL fibronectin at a density of 7.5 gtg/cm2.The plated cells were cultured at 37°C in 5% C02/95% air.

The day after seeding, the medium was replaced with CSFM-233 supplemented with 10 ,ug/mL cytosine arabinofuronoside(Ara-C), which was removed from the medium after 3 days.The cells usually began beating in synchrony after 1 or 2 daysin culture. After 5 to 6 days in culture, cells in 96-well plateswere used for stimulation. The medium was changed for fresh

CSFM-2 containing the different agents. All the peptides weredissolved in water or 0.01 mol/L acetic acid and were dilutedfrom stock solution (10` mol/L) kept at -40°C. After 2 hoursat 37°C, the medium was collected and kept frozen untilassayed. Preliminary experiments have shown that secretion ofboth ANF and BNP were linear over a period of 8 hours.Addition of ET-1 to the medium also increases the amount ofANF and BNP in a linear fashion but at a higher level. In thepresent study, it is assumed that the maximal stimulatoryresponse for all the secretagogues occurred at a similar timepoint.

RadioimmunoassayRadioimmunoassays (RIAs) of ANF and BNP were per-

formed with specific antisera. The cross-reactivities of ANFantiserum on BNP and the converse were <0.01%. All meth-ods are fully described elsewhere.335 Five and 150 gL ofmedium were used without extraction for ANF and BNP RIA,respectively.

Atrial Membrane PreparationsAtrial membranes were prepared from both 4-day-old rats

and neonatal atrial myocyte cultures. Membranes from 4-day-old rats were obtained by homogenization and centrifugationof atria as described by Liu et al.36 In brief, atria werehomogenized in 0.05 mol/L NaHCO3, pH 7.4, and 0.1 mmol/Lphenylmethylsulfonyl fluoride (PMSF) with a Polytron (Brink-man, Rexdale, Ontario, Canada) (two times for 10 seconds)and centrifuged at 3000g for 10 minutes. The pellet wasrehomogenized, and both supernatants were pooled and cen-trifuged at 40 000g for 15 minutes. The pellet was washed in0.05 mol/L Tris-HCl, pH 7.4, recentrifuged at the same speed,and resuspended in the same buffer.

Cultured atrial myocytes in 10-cm plastic dishes werewashed twice with 0.02 mol/L NaHCO3, pH 7.4, and 0.1mmol/L PMSF and scraped in the same buffer (1 mL/60 cm2).The cells were homogenized in a Potter-Elvehjem homoge-nizer at 750 rpm with 10 strokes and centrifuged at 40 000g for15 minutes. The pellet was resuspended in 0.05 mol/L Tris-HCI, pH 7.4. All membrane preparations were kept at -40°Cuntil used. Preliminary experiments have shown that onefreezing and thawing cycle does not affect the binding prop-erties of the ET receptor. Proteins in the membrane prepara-tions were measured by the Bio-Rad protein assay (Bio-RadLaboratories, Hercules, Calif) with bovine serum albumin asstandard. Atrial myocyte cultures give 4 to 5 gg protein persquare centimeter.

Receptor-Binding AssayThe ET-receptor-binding properties of the membrane prep-

arations were characterized by rapid filtration on a glass filterwith iodinated ET-1 as tracer. ET-1 was radiolabeled by thelactoperoxidase method.37 The monoiodinated peptide wasthen purified by reverse-phase high-performance liquid chro-matography (HPLC) on a C18-Bondapak column with a linear0.1% trifluoroacetic-acetonitrile gradient. The specific activityof the labeled ET-1 (1500 Ci/mmol) was assessed by selfdisplacement.38 For saturation analysis, membrane proteins

10 tg) were incubated at room temperature for 90 minutes inthe presence of increasing concentrations of iodinated ET-1(20 000 to 800 000 cpm) in a total volume of 0.25 mL in 0.05Tris-HCI, pH 7.2, 50 U/mL aprotinin, 0.1% bacitracin, 5mmol/L MgCI2, and 0.5% bovine serum albumin. After 90minutes of incubation at room temperature, the tubes (induplicate) were filtered on a No. 34 glass filter (Schleicher &Schuell, Keene, NH) with a cell harvester (Brandel, Gaithers-burg, Md). The filters were finally washed three times with 0.05mol/L Tris, pH 7.2, and 0.15 mol/L NaCl. Before use, they weresoaked in 5% dry skim milk (Carnation) to decrease nonspecificbinding. Under these conditions, nonspecific binding due toabsorption to the filters is <0.3% of total radioactivity. Radio-



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activity on the filters was counted in a gamma counter (LKBWallac, Turku, Finland). Nonspecific binding to the membranes(<2%) was estimated in the presence of 10-6 mol/L ET-1. Forcompetition analysis, membrane proteins (:::z10 ,g, which gives10% to 15% binding) were incubated with 30 000 cpm of'5I-ET-1 in the presence of increasing concentrations of pep-tides (10-13 to 10-6 mol/L). All other parameters were identicalto those in the saturation experiments.

Preliminary studies showed that binding was linear up to 25,ug of proteins. Binding equilibrium was reached between 60and 90 minutes of incubation at room temperature. Analysis ofiodinated ET-1 by reverse-phase HPLC after incubation indi-cated that <5% of the tracer was degraded. All the data wereanalyzed by the EBDA and LIGAND computer software ofMcPherson39 (Biosoft, Cambridge, UK).

Cross-linking of ET ReceptorOne hundred micrograms of membranes was incubated in a

total volume of 100 ,uL in 0.05 mol/L NaPO4, pH 7.4, and 0.15mol/L NaCl (phosphate-buffered saline [PBS]) in the presenceof 300 000 cpm of 125I-ET-1 with or without 10-6 mol/L ET-1,10`6 mol/L BQ-123, or 10-f mol/L IRL-1620 for 90 minutes atroom temperature. The samples were then centrifuged at15 000 rpm for 10 minutes, and the supernatant was removed.The membrane pellet was resuspended in 50 ,uL PBS to whichdisuccinimidyl suberate (50 mmol/L in dimethyl sulfoxide) wasadded at a final concentration of 1 mmol/L. After 20 minutes,1.25 pL of 2 mol/L Tris was incorporated to quench thecross-linker, and 5 minutes later, the membranes were col-lected by centrifugation and dissolved in sodium dodecylsulfate (SDS) buffer for electrophoresis. Proteins were sepa-rated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on 12% acrylamide gel according to the methodologyof Laemmli.40 After electrophoresis, the gels were stained in0.05% Coomassie R-250, dried, and exposed to X-OmatKodak film (Eastman Kodak Co, Rochester, NY) for 10 to 20days.

AutoradiographyAtrial myocytes were seeded in two-well culture chamber

glass slides (Nunc Inc, Naperville, Ill). After 5 or 6 days inculture, the cells were incubated in 1 mL CSFM-2 containing1x106 cpm/mL '25I-ET-1 for 2 hours on ice. Incubation wasperformed at low temperature to avoid internalization anddegradation of the peptide. After incubation, the cells werewashed once with CSFM-2 and twice with PBS, fixed for 10minutes in 4% paraformaldehyde and 0.5% glutaraldehyde inPBS, washed once in PBS and once in 70% ethanol, andimmersed 10 minutes in acetone at -20°C. The slides werethen kept in 70% ethanol until they were immunostained.They were first incubated for 20 minutes in 1.5% normal goatserum (NGS) in PBS, washed, and then incubated overnight inANF antiserum diluted 1:400 in 1.5% NGS in PBS. Afterwashing the slides in PBS, the first antibody was revealed bythe Vectastain ABC kit (Vector Laboratories, Burlingame,Calif) according to the manufacturer's instructions. Controlexperiments to reveal nonspecific staining were performed bysaturation of the first antibody in the presence of excess (20,g) ANF-(99-126). To detect the bound radioactivity, theimmunostained slides were dipped in K-5 emulsion (IlfordScientific Product, Mabberley, UK) and exposed for 20 days.

ETA- and ETB-Receptor ProbesRat ETA-receptor cDNA generously donated by Dr Herbert

Y. Lin, Whitehead Institute for Biochemical Research, Cam-bridge, Mass, was used as a cDNA probe.41 The rat ETB-receptorprobe was prepared by polymerase chain reaction (PCR) ampli-fication with first-strand cDNA from the rat lung. First-strandcDNA was synthesized by incubating 5 ,ug of total RNA in a50-,Lp reaction mixture containing 50 mmol/L Tris-HCl (pH8.3), 50 mmol/L KCI, 10 mmol/L MgCl2, 10 mmol/L dithiothrei-

tol, 1 mmol/L dNTPs, 0.5 mmol/L spermidine, 1 ,ug oligo(dT)12-18(Pharmacia LKB Biotechnology), and 10 U/,g RNA avianmyeloblastosis virus reverse transcriptase (Promega Corp, Mad-ison, Wis) for 1 hour at 42°C. The tubes were then boiled for 5minutes, and bovine pancreas ribonuclease (Pharmacia LKBBiotechnology) was added to give a final concentration of 1,g/mL and incubated for 30 minutes at 37C. Five microliters offirst-strand cDNA was subjected to PCR amplification in a 50-pLreaction mixture containing 10 mmol/L Tris-HCl (pH 8.3), 1.5mmol/L MgCl2, 50 mmol/LKCI, 0.1% Triton X-100, 200 pmol/LdNTPs, 100 pmol of each primer, and 2 U Thermus aquaticusDNA polymerase (Promega). PCR amplification was performedin an Easy Cycler (Ericomp, San Diego, Calif) programmed asfollows: 5 minutes of initial denaturation at 95°C, then 30 cycleswith 1 minute of denaturation at 95°C, 1 minute of annealing at50°C, and 1 minute of elongation at 72°C with final elongation for10 minutes at 72°C. A 494-bp ETB-receptor PCR product wasobtained using a 5' forward primer (5'-CTTAATCCCTTTCA-GAAAACAGC-3) and a 3' reverse primer (5'-AAGCAG-GATTGCTTCTCCTCTAA-3'). located at nucleotides 766 to788 and 1237 to 1259 of the coding sequence, respectively.42 ThisPCR product was then cloned into pGEM-7zf(+) plasmid(Promega). The sequence was determined by the Sanger methodof dideoxy-mediated chain termination43 and was identical to thecloned rat ETB receptor.42

Radiolabeled ETA- and ETB-receptor cDNA probes wereprepared by random-prime labeling,37 using [32P]dCTP (3000Ci/mmol, Amersham Corp, Arlington Heights, Ill) and theKlenow fragment of DNA polymerase (Promega). 18S ribo-somal RNA was analyzed with a specific oligonucleotide probe(5'-CTTCCTCTAGATAGTCAAGTTCGACCGTCT-3 )44labeled with T4 polynucleotide kinase (Pharmacia LKB Bio-technology) and [32P]ATP (3000 Ci/mmol, Amersham). The32P-labeled probes were purified by chromatography usingNACS cartridges (GIBCO-BRL, Burlington, Ontario, Canada).

Northern Blot AnalysisTotal RNA was extracted from cultured neonatal atrial

myocytes by the guanidium isothiocyanate-phenol-chloroformmethod.45 The samples were resuspended in diethylpyrocar-bonate-treated H20, and optical density was determined at260 and 280 nm. Typically, a ratio of 1.6 to 1.75 was obtained.Total RNA samples (20 ,ug) were ethanol-precipitated, resus-pended in 20 uL denaturation solution containing 1x runningbuffer (20 mmol/L MOPS [pH 7.0], 6 mmol/L sodium acetate,and 1 mmol/L EDTA), 6% formaldehyde, and 50% forma-mide, and then heated for 15 minutes at 65°C before loading.RNA samples were run on 1.0% agarose gel containing 1 xrunning buffer and 6% formaldehyde. The samples weretransferred from the gel to a Duralon-UV nylon membrane(Stratagene, La Jolla, Calif) by capillary action using 3 mol/LNaCl and 0.3 mol/L sodium citrate (20x standard saline citrate[SSC]). After blotting, the filters were completely dried, andRNA was fixed to the membranes by UV irradiation with UVStratalinker (Stratagene). To verify the evenness of loading ineach lane and to identify the location of the 18S and 28Sribosomal RNA species, the filters were stained with 0.02%methylene blue in 0.3 mol/L sodium acetate (pH 5.5), followedby rapid destaining in diethylpyrocarbonate-treated H20. Af-ter recording each of the ribosomal RNA positions, themembranes were destained in 0.1xSSC and 1% SDS for 15minutes. They were prehybridized for 2 hours at 42°C in 50mmol/L sodium phosphate buffer (pH 7.0) containing 5x SSC,1% SDS, SxDenhardt's solution (SOxDenhardt's solutioncontains 1% Ficoll, 1% polyvinylpyrolidone, and 1% bovineserum albumin), 50% formamide, and 100 ug/mL sonicatedsalmon sperm DNA. Hybridization began with the addition ofthe 32P-labeled probe. It was carried out for 18 to 20 hours at42°C. The membranes were washed three times in 12.5mmol/L NaCl and 0.1% SDS at 42°C for 20 minutes. Theywere patted dry with paper towels, enveloped in plastic wrap,



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Peptide concentration (log M)FIG 1. Graph showing stimulation by endothelin (Et) isopep-tides of atrial natriuretic factor (ANF) secretion by atrial myo-cytes. S6b indicates sarafotoxin 6b. Since basal ANF secretionvaried from culture to culture from 600 to 1500 fmol per well for2 hours, the results are expressed as percentage of changes inANF secretion. These results are mean±SEM of four to six cellpreparations with six to eight wells per preparation. Secretions ofeach peptide were compared with the lowest concentration byone-way ANOVA and the a posteriori Bonferroni t test. *P<.05.

and exposed to Kodak XAR-5 film with intensifying screens at-70°C for 5 to 7 days.

Statistical AnalysisAll the data are presented as the mean+SEM. Statistical

analyses of ANF and BNP secretion were performed byone-way ANOVA followed by an appropriate a posteriori testas described in the legends of figures. A value of Pc.05 wasconsidered significant. Correlations between two variableswere analyzed by the Pearson product moment test. Thestatistical software SIGMASTAT (Jandel Scientific, San Rafael,Calif) was used in all cases.

ResultsSince ANF secretion is reported to be enhanced by

ET, we examined the potency of different ET isopep-tides on this parameter in a cardiac myocyte culturesystem. Neonatal atrial myocytes were incubated for 2hours in the presence of increasing concentrations ofETs. ANF and BNP were measured in the media byspecific RIAs. Fig 1 shows the dose-response curve ofANF secretion in the presence of ET-1, ET-2, ET-3,and S6b. Basal secretion of ANF from one preparationto the other varied from 600 to 1500 fmol per well for 2hours (920±100 fmol per well for 2 hours, n=9 cellpreparations), and results were expressed as percentageof changes in ANF secretion. All four peptides doubledANF secretion but with different potencies. ET-1 wasthe most potent, with an EC50 of 46 pmol/L. Thepotency of ET-2 (54 pmol/L) was comparable to that ofET-1. In both cases, responses doubled at 10-9 mol/L(203±3.7% for ET-1 and 200±3.9% for ET-2). S6b andET-3 were less potent, with EC50s of 385 pmol/L and14.1 nmol/L, respectively, and maximal responses at10`8 and 10` mol/L, respectively (202±5.0% and207±8.0%, respectively). The order of potency of theET isopeptides was therefore as follows: ET-1=ET-2>S6b>>ET-3. High doses of ET isopeptides also

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FIG 2. A, Bar graph shows inhibition of endothelin (ET)-1-stimulated atrial natriuretic factor (ANF) secretion by increasingconcentrations of ETA-receptor antagonist BQ-123. The resultsare mean+SEM of ANF secretion of three cell preparations withsix wells per preparation. The results were compared by one-way ANOVA and the a posteriori Student-Newman-Keuls test.*P<.05 vs control (CONT). B, Bar graph shows representativeET-isopeptide-stimulated ANF secretion with or without 10-6mol/L BQ-123. S6b indicates sarafotoxin 6b. The results aremean+SEM of six to eight wells compared by one-way ANOVAand the a posteriori Student-Newman-Keuls test. *P<.05 vsCONT. #P<.05 vs the respective stimulated secretion withoutBQ-1 23.

showed a slight, although not significant when com-

pared with the maximal stimulation, inhibition of ANFsecretion. A similar situation was observed by Lew andBaertschi.46 In addition to chronotropic and inotropiceffects, ET has been reported to have cytoxic effects.47ET at high doses may therefore profoundly alter themetabolism of myocytes and consequently its secretoryfunction.The availability of an ETA-receptor antagonist al-

lowed us to test whether this compound could blockET-stimulated ANF secretion. BQ-123 in increasingdoses significantly blocked ET-stimulated ANF secre-tion (Fig 2A). A similar pattern of inhibition was seenwhen ANF secretion was stimulated by ET-2, S6b, orET-3 (Fig 2B).

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FIG 3. Bar graphs showing the effect of endothelin (ET) B-re-ceptor agonists (IRL-1620 and [Ala1 3 11'151ET-1 [4Ala ET-1]) onbasal and ET-1-stimulated natriuretic peptide secretion. BNPindicates brain natriuretic peptide; BQ-1 23, ETA-receptor antag-onist. A, Effect of IRL-1 620 is shown. The results are mean±SEMof 42 to 72 wells. B, Effect of 4Ala ET-1 is shown. The results aremean±SEM of eight wells. All the data were analyzed byone-way ANOVA and the a posteriori Bonferroni t test. *P< .05 vsbasal secretion.

We also investigated the potential stimulation ofANF secretion by IRL-1620, an ETB-receptor agonist(Fig 3A). Alone or in combination with ET-1, it had noeffect on ANF secretion. Since BNP is present in ratatria and is stored in atrial secretory granules,3 we alsoexamined simultaneous ANF and BNP secretion byatrial myocytes. As illustrated in Fig 3B, BNP secretion,measured by a specific RIA, always represented = 1% ofbasal or stimulated ANF secretion. This figure alsodepicts the effect of 4Ala ET-1, another ETB agonist,which did not modify basal or stimulated ANF and BNPsecretion. As with ANF, BQ-123 blocked BNP secre-tion. Fig 4 illustrates the relation between secretions ofANF and BNP by atrial myocytes. Significant correla-tions were obtained for these two parameters, indicatingthat, under basal or ET-stimulated secretion, BNP isproportional to ANF and probably originates from thesame source, the atrial secretory granules.The order of potency of the ET isopeptides, the

inhibition of secretion of the natriuretic peptides byBQ-123, and the lack of response to IRL-1620 and 4Ala

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ANF (fmol/well/2h)FIG 4. Regression lines between atrial natriuretic factor (ANF)and brain natriuretic peptide (BNP) secretion in two different cellpreparations (A and *). Concentrations of ANF in the medium foreach experiment (basal and endothelin stimulated) were plottedvs those of BNP. Correlations were analyzed by the Pearsonproduct moment test.

ET-1 suggest that the ETA-receptor subtype is involvedin the stimulatory action.To verify the presence of the ETA receptor in atrial

myocyte cultures, membranes were prepared from cul-tured cells as well as from the atria of 4-day-old animals.The latter preparations were used to verify that therewas no major change in ET-receptor properties whenthe myocytes were put in culture. Atrial myocyte mem-brane preparations were also used to assess the bindingproperties of iodinated ET-1 by means of saturationcurves (Fig 5). Specific binding was saturated with 800pmol/L of 125I-ET-1. Scatchard analysis of the dataindicated the presence of a single class of binding siteswith an affinity in the picomolar range. Three differentmembrane preparations were used to estimate thebinding parameters: the apparent dissociation constantwas 204±45 pmol/L, and the maximum density ofbinding sites (Bma) was 1280±380 fmol/mg protein.

Both atrial tissue and atrial myocyte membrane prep-arations were then examined in competition experi-ments with 125I-ET-1 as the tracer in conjunction witheither ET-1, ET-2, ET-3, S6b, BQ-123, or IRL-1620(Fig 6). The results of analysis of the curves are given inthe Table. ET-1, ET-2, and S6b displaced 125I-ET-1 withvery high and comparable affinity, and a high-affinitybinding site was detected. ET-3 also displaced the tracerbut with somewhat lower potency. With both S6b andET-3, a second site was detected. BQ-123, the ETAantagonist, totally displaced iodinated ET-1 on theatrial myocyte preparations, but two sites of differentaffinities were found. Finally, IRL-1620 displaced= 10% of the tracer on myocyte preparations, indicatingthe possible presence of a very low density of ETBreceptors. In atrial tissue preparations (Fig 6A), aslightly different picture was seen: BQ-123 displaced80% of the tracer, and IRL-1620 displaced =15%,suggesting the presence of a low proportion of ETBreceptors. These results thus demonstrated only minor

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0 2 4 6

ET-1 (bound.fmol/0.25m1)

0

0 50 100 150 200

125I-ET-1 added (fmol)FIG 5. Representative saturation curves of binding of 1251-endothelin (ET)-1 to membrane preparation of cultured atrial myocytes. Inset:Scatchard curve.

differences between atrial tissue and cultured atrialmyocytes.There was a significant linear relation (Fig 7) between

the log of the EC50 (from results presented in Fig 1) andthe Kds of ET-1, ET-2, S6b, and ET-3 (Table), suggest-ing that this high-affinity binding site was directly re-lated to ANF secretion. Logarithmic transformation ofthe EC50 may reflect the different experimental condi-

'-

0

10

mm,-0

"I

CU-4

100

80 -

60 -

40-

20-

0-

A

,IRL- 1620

ET-1-

ET-2

BQ- 123

-1

0 -12 -11 -10 -9 -8 -7 -6 -5

Peptide (log M)

100

tions (37°C versus room temperature, membranes ver-sus cells, ionic states, etc) used for each system.Primary cultures of atrial myocytes are often contam-

inated by other cell types. To establish whether the ETreceptor was present only in atrial myocytes and not incontaminating cells, in vitro autoradiography was per-formed concomitantly with immunostaining (Fig 8). Byimmunostaining with ANF antiserum, >95% of the cells

0 -12 -11 -10 -9 -8 -7 -6 -5

Peptide (log M)

0C0

mr-4

E-

-4

FIG 6. Representative competition curves of binding of 1251-endothelin (ET)-1 to membrane preparations of 4-day-old rat atrial tissues(A) and cultured atrial myocytes (B). B/B, indicates the bound-to-free ratio; S6b, sarafotoxin 6b; IRL-1620, ETB-receptor agonist; andBQ-123, ETA-receptor antagonist.

25000

3 20000

c.)

0 15000

0~01.1

1 10000

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Binding Parameters of Endothelin Isopeptides on Membrane Preparations of Cultured Neonatal Atrial Myocytes andAtrial Tissues of 4-Day-Old Rats

Atrial Tissues Cultured Atrial Myocytes

High Affinity Low Affinity High Affinity Low AffinityET-1 71.4±3.1 pmol/L ... 68.4+2.9 pmol/L ...

(1690+190) (1796+50)ET-2 123±12.4 pmol/L ... 92.6±9.5 pmol/L

(1461 ±218) (1725+171)S6b 187±43.4 pmol/L 43.4+24.8 nmol/L 109±44.6 pmol/L 12.2±13.6 nmol/L

(1282±69) (128±52) (1666±68) (310±121)ET-3 401 ±157 pmol/L 45.4±23.9 nmol/L 220±39.6 pmol/L 14.4±5.1 nmol/L

(1182+214) (280±19) (1148±167) (763±42)BQ-123 1.25±0.14 nmol/L 19.9±4.1 j.bmol/L 0.75±0.23 nmol/L 22.6±31.5 nmol/L

(1180±80) (283±55) (1591 ±347) (389±284)ET indicates endothelin; S6b, sarafotoxin 6b; and BQ-123, ETA-receptor antagonist. Values are mean+SEM of three binding

experiments with different preparations. Data in parentheses are densities of the binding sites in femtomoles per milligram.Displacement by ETB-receptor agonist IRL-1620 was <15%. Affinities and densities when IRL-1620 was used could not be

mathematically evaluated adequately.

were positive. Staining was more intense at the periph-ery of the nucleus, where storage granules are mainlylocated.48 Preincubation of the primary ANF antibodywith ANF-(99-126) completely abolished the reaction(Fig 8B). The location of the ET receptor was visualizedby incubating the cells with 'PI-ET-1 and coating themwith a photographic emulsion. The ET receptor willappear as black silver grains at the surface of cells,which may be positively stained. Only cells that stainedpositively for ANF demonstrated the presence of silvergrains, which were distributed randomly all over thesurface (Fig 8A). Cells that did not stain possessed onlya few grains corresponding to background (Fig 8A and8E, arrowheads). The silver grains were totally dis-placed by 10-6 mol/L ET-1 (Fig 8C) and 10-6 mol/LBQ-123 (Fig 8D) but not by 10`6 mol/L IRL-1620 (Fig

10000

5000

Z 10001-

o 500

PQ

100

50

log(EC50)=0.934 + 0.008Kd

r=0.993p<o.oI

0

I .

100 200 300 400 500 800

Kd (pM)FIG 7. Regression line between the EC5o of endothelin (ET)isopeptides on atrial natriuretic factor secretion measured fromthe stimulation dose-response curves of Fig 1 and the Kd ofhigh-affinity sites for ET-1, ET-2, sarafotoxin 6b, and ET-3 takenfrom the Table. Correlation was analyzed by the Pearson prod-uct moment test.

8E), confirming the results obtained by the displace-ment curves with the membrane preparations.The molecular characteristics of the ET receptor

were investigated by cross-linking and electrophoresis.'251-ET-1 was cross-linked with dissuccinimidyl suberateto the ET receptor and then analyzed by SDS-PAGEand autoradiography. Fig 9 illustrates that the cross-linked ET receptor had a molecular mass of ;50 kD.The labeled specific band was absent when the mem-brane was incubated with 10C6 mol/L ET-1 and BQ-123but not with IRL-1620, confirming that the labeledreceptor was of the ETA subtype.

Finally, to determine whether ETA- and ETB-receptorgenes were expressed in atrial myocytes, we performedNorthern blot analysis on total RNA prepared fromcultured cells using specific 32P-labeled cDNA probes(Fig 10). Two mRNA bands of 4 and 5 kb were detectedas expected for the ETA subtype. With a similar amountof total RNA, the ETB-receptor mRNA band of 4.5 kbcould not be detected.

DiscussionIn addition to the mechanical stretching of the atrial

wall, many agents have been shown to consistently en-hance in vivo the secretion of the natriuretic peptides byatrial myocytes. Among them, the effect of ETs in vivo orwith perfused heart are well documented.6,10-13,49,50 How-ever, in these experiments, it is difficult to dissociatestretch-induced secretion and its direct effects on atrialmyocytes.

Neonatal atrial myocytes in culture are a good modelfor the evaluation of secretagogue independent of anyhemodynamic modifications. In these cultured cells,calcium ionophores, a-adrenergic agents, and ET havebeen shown to be secretagogues of ANF. Indeed, thesecells respond well to the presence of ET-1 with atwofold to fivefold ANF secretion increase.14 Most ofthe investigations on ANF release have been conductedwith ET-1 and rarely or incompletely with other isopep-tides. In one study, Pitkanen et aP15 found that 2.7



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.1 a y'V

.

,,'¢"S

FIG 8. Autoradiography and immunostaining ofcultured atrial myocytes. After preincubation with1251-endothelin (ET)-1 (A), 10-6 mol/L ET-1 (C),10 6 mol/L ETA-receptor antagonist BO-123 (D),and 10-6 mol/L ETB-receptor agonist IRL-1620 (E),myocytes were fixed and immunostained with atrialnatriuretic factor (ANF) antiserum and the avidin-biotin complex second antibody. Preincubation ofANF antiserum with excess ANF (99-126) com-pletely blocked the positive reaction (B). Note thepresence of silver grains in panels A and E andtheir absence in panels C and D. Arrowheadsindicate the position of contaminating cells. Origi-nal magnification x400.

nmol/L S6b showed comparable potency with the samedose of ET-1 to release ANF from perfused rat heart.The cultured atrial myocyte system has also been used

to assess intracellular mechanisms involved in the ETresponse. 15- However although many reports havebeen published on the characterization of ET receptorson cultured ventricular myocytes2,53 as well as on thepresence of ET receptors and their expression in adultatrial tissue,7 754-57 no data are yet available on culturedneonatal atrial myocytes and their relation with natri-uretic peptide secretion.One of the major problems encountered with cul-

tured cells is that the phenotype of the original tissuecan be lost in culture. This problem was eliminated inthe present study by comparing the binding propertiesof the ET receptor on atrial myocytes with those of theatrial tissue of 4-day-old rats from which the cultureoriginated. As shown in the Table, there were no majordifferences in the affinities of the ET isopeptides be-tween both membrane preparations, indicating that thephenotype in cultured cells was preserved. Moreover,receptor density was roughly equal, although the mem-brane preparations were obtained by different proto-

cols. A minor difference was seen: atrial tissue pos-sessed a slightly larger amount of ETB receptorcompared with cultured atrial myocytes. This differencecan be attributed to the fact that atrial tissue prepara-tions contain membranes not only from myocytes butalso from other cell types, such as endothelial cellsoriginating from the coronary circulation, which are

known to contain the ETB receptor.58Another important problem is purity of the culture.

The addition of Ara-C, an antimitotic agent, to theculture medium greatly improved myocyte purity,59and typically, the culture contained <5% of contami-nating cells on the basis of anti-ANF immunostaining.The possibility that these contaminating cells possessthe ET receptor and therefore affect the overallbinding parameters was assessed by autoradiography.Cells that did not stain with ANF antiserum did notbind iodinated ET-1.The ET receptor subtype on atrial myocyte mem-

brane preparation was characterized by competitionanalysis with '25I-ET-1 as tracer and ET isopeptides as

well as the ETB-receptor agonist IRL-1620 and theETA-receptor antagonist BQ- 123. The competition

c 4

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e

04*



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a b c d e f gkDo 80-5 7*

49.5-

32.5-t

27.5-

18.5-

FIG 9. Autoradiography of cross-linked endothelin (ET) recep-tors with 1251-ET-1 in the presence of disuccinimidyl suberate.Lanes are as follows: a, total binding; b, 10 9 mol/L ET-1; c, 10-6mol/L ET-1; d, 5 x 1 o-8 mol/L ETA-receptor antagonist BQ-123; e,10-6 mol/L BQ-123; f, 1 0-6 mol/L ETB-receptor agonist IRL-1 620;and g, 10 5 mol/L IRL-1620.

binding curves revealed high-affinity binding sites (100pmol/L) for ET-1, ET-2, and S6b with high density(1500 fmol/mg). IRL-1620 displaced 10% of iodinatedET-1. As reported by Takai et al,31 the selectivity of thisdrug is 105 times higher for the ETB receptor than forthe ETA receptor in porcine tissues. Therefore, theETB-receptor subtype seems to be present in very lowdensity in our myocyte membrane preparations. Incontrast, BQ-123, the ETA antagonist with a 103 timesgreater selectivity for the ETA subtype,29 completelydisplaced '25I-ET-1, indicating a predominance of thisETA receptor. Displacement curves with ET-3, S6b, andBQ-123 should therefore behave according to a one-sitemodel, corresponding to the ETA-receptor subtype.However, analysis of the data (Hofstee transformation)suggested the presence of a second lower-affinity bind-ing site (20 nmol/L, Table), which represented -25% of

total receptors. Indeed, Panek et a155 have alreadyobserved that the ET binding site in adult rat atria doesnot behave exactly as an ETA receptor. Furthermore,this site is clearly distinct from the putative ETC-receptor subtype postulated by Emori et al,60 whichpreferentially binds ET-3. In a recent study, Ishikawa eta156 compared ET binding sites in neonatal rat atria andventricles by saturation curves with either 125 I-ET-1 or

ETA-R ETB-R

L A L A

28S-

18S- 4* *

FIG 10. Northern blot analysis of endothelin A (ETA)- andendothelin B (ETB)-receptor mRNAs. ET-receptor subtypes wereanalyzed in cultured atrial myocytes (A) with 20 gg of total RNAand in rat lung extracts (L) with 10 gtg of total RNA. The lungextracts were used as a positive control to verify the quality of thereagents and methods. The amount of RNA present on the blotswas assessed by hybridization with an 18S ribosomal RNA32P-labeled oligonucleotide probe.

25I-ET-3. Although one site was observed with 125-ET-1, two sites were seen with '25I-ET-3. In the absenceof any experiments with specific receptor drugs, theauthor could only establish the presence of two siteswithout appropriate identification. On the basis of theorder of affinity (ET-1.ET-2.S6b>ET-3) of the ETisopeptides, of displacement with an ETA antagonist,and of ETA-receptor mRNA expression, it can be con-cluded that the high-affinity binding site observed onatrial myocytes is most probably an ETA-like receptor.However, the lack of displacement with IRL-1620(:15%) and the proportion of low-affinity binding sites(:25%) suggest that some heterogeneity still exists thatcannot be adequately classified with the pharmacologictools presently available.The molecular mass of the ET receptor (-50 kD),

measured by cross-linking of iodinated ET-1 and SDS-PAGE, is well within the range of molecular massalready reported (from 30 to 70 kD)27 and in factcorresponds to the mass of ET receptors calculatedfrom their amino acid numbers. This technique, whichalso showed that the ET receptor does not appear to bedegraded in small, less active fragments, does not allowdiscrimination between ET-receptor subtypes becauseboth possess a similar mass.

All the results also supported the concept that theETB-receptor subtype, if present in our cultured atrialmyocyte preparation, is expressed in very low amountsthat cannot be easily detected by available pharmaco-logic tools or by Northern blot analysis.ANE secretion by atrial myocytes was enhanced by

the ET isopeptides, and the order of their potency wasET-NlET-2>S6b>>ET-3. Moreover, the ETA antago-nist BQ-123 inhibited ANF secretion, whereas IRL-1620 and 4Ala ET-1, two ETB agonists, did not alterbasal or ET-stimulated secretion. Therefore, we canassume that ANF secretion is mediated by activation ofan ETA-like receptor. Indeed, a linear logarithmic rela-tion was established between the potency of the ETisopeptides to enhance ANF secretion and their corre-sponding Kds on the high-affinity binding site. BNPsecretion followed a pattern similar to ANF, indicatingconcomitant excretion. These findings confirm and fur-ther extend our previous results3 as well as otherreports61,62 on BNP secretion.ETs are synthesized and released mainly but not

exclusively by the vascular endothelium.58 They arebelieved to act as paracrine hormones on surroundingcells, principally vascular smooth muscle cells. Theheart contains and releases immunoreactive ET.6364 Inthe myocardium, endothelial cells of the capillaries andof the endocardium are in close juxtaposition to myo-cytes. It is therefore plausible that ETs have a direct andshort-term action with inotropic and chronotropic re-sults and modulate ANF and BNP secretion. Over alonger term, ETs enhance the expression of cardiacgenes, such as myosin light chain, a-actin, ANF, andBNP. l65-67 Moreover, the elevation of ET expression ina pathological situation such as congestive heart fail-ure68 may be of prime importance in the control ofsecretion of the natriuretic peptides by the heart.

In conclusion, the present study demonstrates thatthe stimulation of natriuretic peptide secretion by ET ismediated through binding to the ETA receptor on the



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surface of atrial myocytes and further substantiates thepotential role of ET in cardiovascular regulation.

AcknowledgmentsThis study was supported by grants from the Medical

Research Council of Canada to the Multidisciplinary Re-search Group on Hypertension and from the Heart and StrokeFoundation of Canada. The authors thank Chantal Arguin andPavol Sventek for their invaluable technical assistance as wellas Vivianne Jodoin for her excellent secretarial assistance.

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G Thibault, A F Doubell, R Garcia, R Larivière and E L Schiffrinby endothelin A receptors.

Endothelin-stimulated secretion of natriuretic peptides by rat atrial myocytes is mediated

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