2232

Cardiorenal and Neurohumoral Effects ofEndogenous Atrial Natriuretic Peptide in DogsWith Severe Congestive Heart Failure Using a

Specific Antagonist for GuanylateCyclase-Coupled Receptors

Atsuyuki Wada, MD; Takayoshi Tsutamoto, MD;Yuzuru Matsuda, PhD; Masahiko Kinoshita, MD

Background To elucidate the extent of the compensatoryrole of endogenous atrial natriuretic peptide (ANP) in severecongestive heart failure (CHF), we examined the changes inhemodynamics and neuroendocrine and renal functions afterincremental administration of an ANP antagonist, HS-142-1(HS), in dogs with CHF.Methods and Results We assessed the effects of HS on the

suppression of plasma and urinary cGMP levels as a marker ofendogenous ANP activity in dogs without CHF. Bolus injec-tions of 0.3 and 1.0 mg/kg HS reduced plasma cGMP levels to77% and 60% and urinary cGMP excretion to 78% and 61% ofthe relevant control levels, respectively. Then the study wasperformed in dogs with CHF induced by chronic rapid ven-tricular pacing, and the plasma ANP level was sixfold higherthan that in the controls. Hemodynamic, hormonal, and renalvariables were determined both before and after subsequentincremental administration (0.3, 1.0, and 3.0 mg/kg every 30

minutes) of HS. HS lowered the plasma and urinary cGMPlevels dose dependently to 32% and 37% of the control levels,respectively. Mean arterial, pulmonary capillary wedge, andright atrial pressures and cardiac output did not changesignificantly. However, plasma renin activity, aldosteronelevel, and norepinephrine level increased rapidly to 226%,179%, and 252% of the control values, respectively. Urine flowrate and urinary sodium excretion were significantly inhibited,with no concomitant change in glomerular filtration rate orrenal plasma flow.

Conclusions These findings suggest that endogenous ANPcontributes to the suppression of the activation of the renin-aldosterone system and sympathetic nervous activity and bodyfluid retention but that the vasodilative action of this peptide isattenuated in advanced CHF. (Circulation. 1994;89:.2232-2240.)Key Words * atrial natriuretic factor * guanosine -

heart failure, congestive

P revious studies have shown that atrial natri-uretic peptide (ANP) has potent vasorelaxant,diuretic, and natriuretic actions and shows

neurohumoral effects that inhibit renin and aldoste-rone secretion. In congestive heart failure (CHF), theincrease in ANP in the plasma may contribute to thecompensatory regulation of hemodynamics, body fluidbalance, and interaction of neurohumoral factors.Although the plasma ANP level remains elevatedabove basal values, the cardiac pressure increases,volume overload develops, and the renin-angiotensin-aldosterone system and sympathetic nervous activityare significantly elevated during the progression ofchronic CHF.1,2 In CHF, the response to ANP infusionis less sensitive with regard to the renal, hormonal, andhemodynamic changes compared with healthy controlsubjects.3-5 The compensatory effects of ANP appearto be attenuated in advanced CHF. We have shownpreviously that plasma levels of ANP and cGMP, anintracellular second messenger of ANP, become ele-

Received December 4, 1993; revision accepted February 4,1994.From the First Department of Internal Medicine, Shiga Univer-

sity of Medical Science, and Kyowa Hakko Kogyo Co (Y.M.).Correspondence to Atsuyuki Wada, MD, The First Department

of Internal Medicine, Shiga University of Medical Science, Tsuki-nowa, Seta, Ohtsu 520-21, Japan.

vated with the progression of mild CHF. However, theplasma cGMP level reached a plateau despite theelevated level of ANP in chronic severe CHF. There-fore, we suggested that this is due to downregulationof guanylate cyclase (GC)-coupled ANP receptors invascular beds.6 Several previous studies using antibod-ies against ANP have been performed to clarify therole of endogenous ANP activity in CHF.7,8 However,the exact role of endogenous ANP in CHF has not yetbeen determined, and whether its compensatorymechanism is actually attenuated in CHF remainsunknown because no specific antagonist for ANP isavailable. We used a specific antagonist of GC-cou-pled ANP receptors, HS-142-1 (HS),9,10 to determinewhether elevated endogenous ANP plays a compensa-tory role in advanced CHF. Hirata et all' used thisANP antagonist, HS, in DOC acetate (DOCA)-salthypertensive rats and suggested that endogenous ANPis involved in the regulation of blood pressure andbody fluid volume. In the present study, we examinedthe extent of the pathophysiological role of endoge-nous ANP in the regulation of the systemic hemody-namics, body fluid volume balance, and neurohumoralfactors in the pacing-induced canine severe heartfailure model system using HS in a dose-responsestudy.

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Wada et al Endogenous ANP in Dogs With Heart Failure 2233

MethodsAnimal Preparation

Conditioned mongrel dogs of both sexes, weighing 11 to 15kg, were used for all experiments. Approval was obtained fromthe Animal Research Committee of Shiga University of Med-ical Science, and all dogs were preconditioned to the studyenvironment for 4 weeks before experiments. The dogs werefasted overnight but allowed water ad libitum until the start ofthe experiment. Anesthesia was induced with pentobarbitalsodium (25 mg/kg), with supplemental doses administered asnecessary to maintain anesthesia. Dogs were intubated andventilated with an Aika R-60 respirator. Through a leftthoracotomy incision at the fourth intercostal space, the heartwas exposed, the pericardium was opened, and two cardiacunipolar pacemaker leads (Matsuda M-23) were sutured ontothe right ventricular apex. After the incision was closed, theleads were tunneled to the back and connected to the externalpacemaker (model 540, Seamed). The left femoral vein wasthen exposed and a 7F Swan-Ganz thermodilution catheter(model T-047-03, Goodtec) was advanced into the pulmonaryartery for measurement of right atrial pressure (RAP), meanpulmonary arterial pressure (MPAP), pulmonary capillarywedge pressure (PCWP), and cardiac output (CO). CO wasassessed by the thermodilution technique in triplicate with 5mL of ice-cold 0.9% saline injected into the right atrium. Thesubsequent thermodilution curves were integrated by a GouldCO computer (SP1435, Statham). A Tygon catheter (flexibleplastic tubing, Norton) was placed in the descending thoracicaorta via the right carotid artery for measurement of meanarterial pressure (MAP) with a pressure transducer (BaxterMP5100). The right jugular vein was also catheterized and theTygon catheter advanced into the vena cava for drug admin-istration. The central filling pressure was recorded on apolygraph (RM-6000, Nihon Kohden). The pacemaker andthe ends of the catheters and pacing wires were securelyfastened in a small bag worn on the back of the animal. Thedogs were allowed to recover from surgery for at least 14 daysbefore control study measurements, during which time theywere trained to lie quietly, unrestrained, on the experimentaltable on a heat mat. Control hemodynamic and venous plasmasamples for neurohumoral determinations were obtained be-fore programming of the pacemaker to an asynchronous modeat a rate of 270 beats per minute. After 14 to 36 days of rapidventricular stimulation (average of 22 days), the dogs werejudged to be in severe CHF on the basis of elevation of centralfilling pressures, evidence of fluid retention, apathy, anorexia,and symptomatic signs of dyspnea. All measurements were

recorded at the elevated heart rate, and all subsequent studieswere performed with animals in the conscious state. Systemicvascular resistance (SVR) and pulmonary vascular resistance(PVR) were calculated as follows: SVR=(MAP-RAP)/COx 80; PVR=(MPAP-PCWP)/CO x 80.

Experimental ProtocolsStudy 1: Effects ofHS on Plasma and Urinary cGMPProduction in Dogs Without PacingThe effects of HS on suppression of endogenous ANP

activity were evaluated by measuring plasma and urinarycGMP concentrations. Eight healthy conscious dogs wereprepared as described without pacing. The urinary bladderwas catheterized with a 7F Cliny balloon drainage tube(Create Medic Co) under short thiopental sodium anesthesia(4 mg/kg). After a 60-minute equilibration period, HS (KyowaHakko Kogyo Co) dissolved in 20 mL saline was injected as abolus into the right atrium at a dose of 0.3 (n=4) or 1.0 mg/kg(n=4). Blood and urine samples were drawn every 30 minutesfor 60 minutes. An equal volume of saline was injected toreplace the volume lost by blood withdrawal, and changes inplasma and urinary cGMP concentrations were measured. Atthe same time, changes in cardiac pressures were measured

every 10 minutes, and those in other neurohumoral factors,including ANP, plasma renin activity (PRA), aldosterone, andnorepinephrine, were determined every 30 minutes.

Study 2: Cardiovascular, Neurohumoral, and RenalEffects ofHS in Severe Heart FailureTo evaluate the role of endogenous ANP in severe CHF, we

divided the dogs with CHF induced by rapid pacing into twogroups; one group (n=6) received HS and the other (n=5)received only saline as time controls. The dogs' bladders werecatheterized as in study 1, and 45 minutes later a priming doseof 50 mg/kg creatinine and 8 mg/kg para-aminohippurate(PAH) dissolved in 10 mL saline solution was infused into thepulmonary artery over a 15-minute period according to themethod of Riegger et a112 with some modifications. This wasfollowed by a constant infusion (0.75 mL/min) of 1.0mg. kg-' * min-t creatinine and 0.3 mg* kg`-l min` PAHthroughout the experimental period. After a 60-minute equil-ibration period, when plasma concentrations were at a steadystate, the bladder of each dog was completely emptied andflushed five times with sterile distilled water before the first oftwo clearance periods, each 30 minutes in duration, was begun.After stable basal hemodynamic records were obtained, HSwas administered stepwise as a bolus at three doses of 0.3, 1.0,and 3.0 mg/kg dissolved in 20 mL saline or vehicle only (saline20 mL) to exclude any temporal effects, injected at intervals of30 minutes into the right atrium. Pressure measurements wererepeated every 10 minutes for 90 minutes after injection. Thecollected pressure data included MAP, RAP, MPAP, andPCWP. CO was determined before and every 30 minutes afterHS administration. Blood was drawn for analysis of plasmaANP level, cGMP concentration, PRA, aldosterone, and nor-epinephrine levels. Blood specimens were centrifuged at 40C,and the plasma was frozen at -300C until assay. After the HSinjection, three additional 30-minute urine collections weremade. Urine for analysis of urinary cGMP excretion wascollected at the end of each clearance period, and urinarycGMP specimens were frozen at -30°C until assay.

Analysis of Blood and Urine SamplesBlood for the determination of plasma ANP, cGMP, PRA,

aldosterone, and norepinephrine levels was collected from thepulmonary artery. Blood for ANP assay was collected in tubescontaining aprotinin (500 kallikrein inhibitory units/mL) andEDTA (1 mg/mL) and immediately placed on ice. Aftercentrifugation at 3000 rpm at 4°C, aliquots of plasma weremeasured by radioimmunoassay as previously described."3Cross-reactivity was assumed to be 100% with dog ANPbecause the amino acid sequences are identical. The intra-assay and interassay coefficients of variation were 6.3% and9.6%, respectively. Plasma and urinary concentrations ofcGMP were determined by radioimmunoassay with a commer-cial kit (Yamasa Syoyu Co Ltd),14 and intra-assay and interas-say coefficients of variation were 2.4% and 8%, respectively.PRA was measured by radioimmunoassay (Gamma CoatRenin Kit, Clinical Assay). Plasma aldosterone was measuredby radioimmunoassay with a SPAC-S Aldosterone Kit (Dai-ichi Isotope Laboratories). Estimation of plasma norepineph-rine concentration was made by high-performance liquid chro-matography using an autoanalyzer (model HLC-8030, TosohCo). Serum and urine creatinine and sodium concentrationswere measured by an autoanalyzer (model 736, Hitachi).Serum and urine PAH concentrations were determined by thedimethylaminocinnamaldehyde method. Creatinine clearanceand PAH clearance were calculated from standard formulasand were equated with glomerular filtration rate (GFR) andrenal plasma flow (RPF), respectively. Filtration fraction (FF)was calculated as GFRIRPF.

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2234 Circulation Vol 89, No S May 1994

TALE 1. Hormonal and Hemodynamic Response to the Injection of HS-142-1 In Dogs WithoutHeart Failure

Time, minDose of

Parameter HS-142-1, mg/kg 0 30 60

ANP, pg/mL 0.3 61±7 65±5 61±3

1.0 53±16 63±11 75±10

cGMP, pmol/mL 0.3 10.8±2.8 8.1±2.0* 8.5±1.8

1.0 11.6±2.5 6.7±1.1* 7.3±1.3

PRA, ng. mL-1 C 0.3 1.3±0.4 1.1±0.4 1.8±0.2

1.0 1.8±0.2 1.6±0.3 1.5±0.5

Aid, pg/mL 0.3 120.5±51.3 86.5±33.9 68.8±26.4

1.0 58.8±19.4 48.8±19.1 28.5±12.1

NE, pglmL 0.3 235±47 229±48 199±40

1.0 247±27 263±28 214±28

U-cGMP, pmoi/mL 0.3 1688±226 1300±108* 1450±96

1.0 1919±480 1373±377* 1230±336*

PCWP, mm Hg 0.3 4.9±1.4 3.8±1.2 4.2±1.7

1.0 5.1±0.7 5.6±0.9 5.3±1.1

MAP, mm Hg 0.3 103.8±1.8 102.0±1.5 109.0±0.6

1.0 104.5±1.7 103.0±2.4 104.5±2.1

ANP indicates atrial natriuretic peptide; PRA, plasma renin activity; Aid, aldosterone; NE, norepinephrine;U-cGMP, urinary cGMP excretion; PCWP, pulmonary capillary wedge pressure; and MAP, mean arterial pressure.Values are mean±SEM (n=4).*P<.05 vs value at time 0.

Statistical AnalysisAll values are given as mean±SEM. ANOVA for repeated

measurements was used to determine the significance ofchanges during time-dependent multiple observations. Com-parisons with control values were analyzed by Dunnett's testafterANOVA for repeated measurements. Student's t test wasused for analysis of the significance of single comparisons.Differences at a value of P<.05 were considered to be statis-tically significant.

ResultsStudy 1: Effects of HS Alone on Plasma andUrinary cGMP Levels in Dogs Without PacingAs shown in Table 1, at doses of 0.3 and 1.0 mg/kg,

HS significantly reduced plasma and urinary cGMPconcentrations in dogs without pacing. However, theplasma ANP level, levels of other neurohumoral factors,and hemodynamic parameters were not significantlychanged. That is, HS at 0.3 and 1.0 mg/kg IV alone didnot influence basal hemodynamics or endocrine secre-tion, with the exception of cGMP level.

Study 2: Response to Rapid Ventricular PacingRapid right ventricular pacing induced CHF in both

experimental groups (HS versus vehicle only). In the HSgroup, MAP was significantly reduced below the pre-pacing value of 105.9±4.1 to 87.6±4.4 mm Hg in CHF(P<.05) and CO from 2.78±0.23 to 1.52±0.13 Llmin(P<.001). However, PCWP and RAP rose progressivelyto peak levels of 19.0±3.1 mm Hg (P<.01) and 7.3+0.9mm Hg (P<.001), respectively. Plasma ANP concentra-tions increased to about sixfold higher than that at basallevel (from 77±9 to 395±67 pg/mL, P<.001). The

plasma level of cGMP was also significantly elevatedabove the prepacing value of 11.1+2.3 to 33.7+6.5pmol/mL in CHF (P<.01). The neurohormone levelsincreased during the development of CHF: PRA in-creased from the prepacing value of 1.2±0.3 to 5.2+2.0ng. mLU . h` in CHF (P<.05), aldosterone level from51.7±21.1 to 364.5+121.1 pg/mL (P<.01), and norepi-nephrine level from 162±37 to 539±100 pg/mL(P<.01). When the basal hemodynamic and hormonaldata of all experimental animals were pooled, thedifferences between the HS and vehicle groups were notsignificant.

cGMP Studies in CHFWe investigated the changes in plasma and urinary

cGMP concentrations as a marker of endogenous ANPactivity. HS reduced plasma cGMP concentration dosedependently from 33.7+6.5 pmol/mL before injection to10.2±+1.4 pmol/mL 90 minutes after injection (P<.001).After injection of HS at 0.3 and 1.0 mg/kg, the plasmacGMP value was reduced to 67.6% and 44.5% of thebasal value, respectively, and at the maximal dose, HSlowered the cGMP concentration to 32.0% (Fig 1).Urinary cGMP excretion was also inhibited signifi-cantly, from 5129±+1542 to 1950±665 pmol/mL, dosedependently (P<.01). Percent changes in urinary cGMPexcretion were reduced to 81.1% of the average basalvalue by 0.3 mg/kg HS, to 48.4% by 1.0 mg/kg HS, andto 37.3% by 3.0 mg/kg HS (Fig 2).

Hemodynamic Changes in CHFFig 3 shows the hemodynamic changes associated

with stepwise administration of HS at three different

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120-

1004

a.

0

0.

0 0.3 1.0 3.0HS-142-1(mg/kg) - HS

....*... Vehice

FIG 1. Une graph showing percent changes in plasma cGMPlevels with administration of vehicle (o, n=5) or HS-142-1 (HS; o,n=6) in dogs with heart failure. HS 0.3, 1.0, and 3.0 mg/kg or thesaline vehicle was administered stepwise at 30-minute intervals.Values are mean±SEM. oooP<.001 compared with basal val-ues; ttP<.001 compared with values with vehicle.

dose levels in dogs with severe CHF. We observed no

significant changes in the systemic hemodynamic responseto HS. MAP, PCWP, and RAP throughout the experi-mental period ranged from 87.2±5.1 to 93.0±4.7mm Hg,from 19.0±3.1 to 21.7±3.7 mm Hg, and from 7.3±0.9 to7.8±1.3 mm Hg, respectively. CO ranged from 1.52±0.13to 1.49±0.15 L/min. PVR and SVR ranged from275.2±43.0 to 365.3±72.7 dynes 5s1 cm-5 and from4342.9±394.0 to 4655.3±403.5 dynes * s`* cm-5, respec-tively. These values did not change significantly duringany experimental procedure. The control saline injectionalso did not affect these hemodynamic parameters.

Plasma Hormonal Changes in CHFFig 4 shows the hormonal changes with administration

of HS in dogs with CHE. Administration of HS caused a

dose-dependent reduction in plasma cGMP concentra-tion, with a significant increase in plasma ANP level from

a.

Lk

120

1004

0 0.3 1.0 3.0

HS-142-1 (mg/kg) Z0 HS-. 1 ... Vehicle

FIG 2. Une graph showing percent changes in urinary cGMPlevels with administration of vehicle (e, n=5) or HS-142-1 (HS; o,

n=6) in dogs with heart failure. HS 0.3, 1.0, and 3.0 mg/kg or thesaline vehicle was administered stepwise at 30-minute intervals.Urine samples were obtained at the end of each 30-minuteperiod. Basal values are the average of the two basal phasevalues. Values are mean±SEM. oP<.05, oooP<.001 comparedwith basal values; ftP<.01, "tP<.001 compared with valueswith vehicle.

the preinjection value of 395±67 to 550±58 pg/mL, 90minutes after injection (P<.01). PRA and plasma aldo-sterone and plasma norepinephrine levels also increasedfrom 5.2±2.0 to 12.2±3.3 ng* mL' * h` (P<.01), from364.5±121.1 to 651.7+184.1 pg/mL (P<.01), and from538.8±99.7 to 1358.8±322.8 pg/mL (P<.01), respec-tively, with injection of HS. There were no significantchanges in these values throughout the experimentalperiod in the vehicle-only group.

Changes in Renal Function in CHFTable 2 and Fig 5 show the changes in renal variables

after HS injection. Urinary flow rate was significantlyreduced, to 0.32±0.11 mL/min, from the average basalvalue of 0.55±0.18 mL/min (P<.01), and absolute uri-nary sodium excretion was reduced to 7.6±2.8 ,uEq/minfrom the average basal value of 41.5±17.2 liEq/min(P<.05), although there were no significant changes inGFR, RPF, or FF. None of these variables changedsignificantly in the vehicle group.

DiscussionTo elucidate the pathophysiological roles of endoge-

nous ANP in severe CHF, we administered the antag-onist of GC-coupled ANP receptors, HS, to dogs withheart failure induced by rapid ventricular pacing. Weinvestigated the extent to which endogenous ANP actu-ally contributes to the regulation of the hemodynamic,neurohumoral function, and body fluid balance in se-vere CHF by HS administration. HS dose dependentlylowered both plasma and urinary cGMP concentrationswhile increasing PRA and aldosterone and norepineph-rine levels. Renal excretion of sodium and water wasreduced, with no associated changes in GFR or RPF,although cardiac hemodynamics remained unchangedthroughout the experimental period. Plasma and uri-nary cGMP levels have been demonstrated to serve asbiological markers of endogenous ANP activity.15-18Although cGMP is produced by the activation of twodifferent isoenzyme forms of GC, soluble and particu-late GC, ANP is well known to interact specifically onlywith particulate GC. Conversely, nitric oxide (NO)activates soluble GC and in turn increases the cGMPlevels in endothelial and vascular smooth muscle cells.The NO system may alter the renin-angiotensin-aldo-sterone system and sodium excretion.1920 HS specificallyblocks the actions of ANP without inhibiting othercGMP-linked substances such as sodium nitroprussideand acetylcholine, which are known to activate solubleGC and stimulate NO production. HS does not inhibitthe sodium nitroprusside-induced accumulation ofcGMP observed in porcine kidney epithelial LLC-PK1cells and rabbit aortic rings10,21 or prevent the vasodila-tive response to acetylcholine observed in isolated ratrenal artery."1 Thus, HS specifically blocks ANP-in-duced cGMP production through specific blockade ofthe particulate GC-linked receptor. The present studyshowed that HS blocks the biological activities of en-dogenous ANP and is suitable for estimating the patho-physiological role of endogenous ANP in CHF.

Hemodynamic ChangesANP is considered to play a significant role in vascu-

lar tone regulation via its vasodilative activity.22,23 How-ever, the extent of the role of endogenous ANP in the

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cE

0U

10 20 30 40 50 60 70 80 90 100

7000

5000

E 3000-Co

0 10 20 30 40 50 60 70 80 90 100

Time (min) Z HS4 Vehicle

-10 0

{...... .........- f.........

0 10 20 30 40 50 60 70 80 90 100

.i................{..... .. ....

FIG 3. Graphs showing the timecourse of changes in hemodynam-ics with administration of vehicle (a,n=5) or HS-142-1 (HS; o, n=6) indogs with heart failure. HS 0.3, 1.0,and 3.0 mg/kg or the saline vehiclewas administered stepwise at 30-minute intervals. Pressure mea-surements were repeated every 10minutes from 0 to 90 minutes afteradministration, and cardiac output(CO) was determined before andevery 30 minutes after administra-tion. Systemic vascular resistancewas calculated at these times.PCWP indicates pulmonary capil-lary wedge pressure; MAP, meanarterial pressure; and SVR, sys-temic vascular resistance. Valuesare mean±SEM.

. . . . . . .

10 20 30 40 50 60 70 80 90 100Time (min) Z HS

----- Voehc

control of cardiac hemodynamics in advanced CHF hasnot been determined. The stepwise administration ofHS in the present study demonstrated that the regula-tion of blood pressure by endogenous ANP was atten-uated in severe CHF. Since administration of HS instepwise doses reduced the plasma cGMP concentra-tion to 32% of the basal level, we expected that thecentral filling pressure would be elevated during theexperiment. However, no statistically significantchanges were observed. HS has been shown to preventANP-induced relaxation of isolated rabbit thoracic aor-ta21 and inhibit the depressor response to human ANP

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400'~~~~~~~~~~~~~~200 1000

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infusion in rats.24 When ANP antiserum was used in ratswith CHF, different results were reported concerningthe hemodynamic regulation by endogenous ANP.However, plasma cGMP levels were not estimated inthese previous studies.78 Recently, Hirata et al1' alsoadministered HS to DOCA-salt hypertensive rats andshowed that a higher dose ofHS (8.0 mg/kg) induced anincrease in MAP associated with a 77% reduction inplasma cGMP levels. The differences in the hemody-namic responses in these various studies could be due todifferences in the level and duration of elevation ofendogenous ANP concentration and/or differences in

otT i

Tf I FIG 4. Bar graphs showing changes in

plasma hormone levels with administra-tion of vehicle (open bars, n=5) or HS-142-1 (HS; hatched bars, n=6) in dogs

0.3 1.0 3.0 with heart failure. HS 0.3, 1.0, and 3.0mg/kg or the saline vehicle was admin-istered stepwise at 30-minute intervals.

00 ANP indicates atrial natriuretic peptide;° T Ald, aldosterone; PRA, plasma renin ac-

tivity; and NE, norepinephrine. Valuesare mean+SEM. oP<.05, ooP<.01compared with basal values; tP<.05compared with values with vehicle.

U *0 0.3 1.0 3.0

H5-142-1(m9/k9) E HS02 vehc

120

110

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100

90

80

70

-10 0

40

35

30

25

20'

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0.3 1.0 3.0HS-142-1(mg/k9) U HS

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TABLE 2. Changes in Renal Function After Administration of HS-142-1 In Dogs With Heart Failure

Dose of HS-142-1, mg/kg

Parameter 0 0.3 1.0 3.0U-cGMP, pmol/mL HS 5129±1542 4402±1396 2720±975* 1950±66514

Vehicle 4657±843 4764±803 4292±642 4594±859U-V, m/min HS 0.55±0.18 0.38±11* 0.36±0.10* 0.32±0.11**

Vehicle 0.56±0.12 0.66±0.16 0.66±0.14 0.67±0.15

U-Na, gEq/min HS 41.5±17.2 23.9±9.7 13.7±5.3* 7.6±2.8**Vehicle 26.6±12.8 29.0±13.3 33.1±15.6 41.7±19.1

GFR, m/min HS 33.6±6.1 37.5±5.2 37.4±5.6 31.4±6.2Vehicle 30.2±5.7 31.1±7.1 39.1±9.2 38.8±9.6

RPF, mUmin HS 91.0±17.3 94.4±15.5 87.4±15.2 76.0±12.1Vehicle 99.1±22.7 89.0±11.9 108.3±15.8 106.8±22.8

FF HS 0.47±0.12 0.45±0.08 0.48±0.07 0.44±0.06Vehicle 0.33±0.06 0.37±0.06 0.36±0.05 0.37±0.06

U-cGMP indicates urinary cGMP excretion; U-V, urine flow rate; U-Na, urinary sodium excretion; GFR, glomerularfiltration rate; RPF, renal plasma flow; and FF, filtration fraction. After two 30-minute basal clearances were obtained,HS 0.3, 1.0, and 3.0 mg/kg or the saline vehicle was administered at 30-minute intervals. Three 30-minuteclearances were performed after administration of each dose. Basal values are the average obtained during thepreceding basal period. Values are mean±SEM for the HS (n=6) and vehicle (n=5) groups.

*P<.05, tP<.01 vs basal values; tP<.05 vs values with vehicle.

experimental animals used and basal diseases studied.Previously, we reported that the effect of attenuation ofendogenous ANP activity on hemodynamics observedin patients with severe chronic CHF may be due to thedownregulation of ANP receptors in the vascularbeds.6,14 In addition, the mechanism of the reduction invasodilative action of endogenous ANP in this studymay be related to a defect in intracellular mechanismsother than those involved in the production of cGMP,because the hemodynamics were not altered signifi-cantly when the plasma cGMP level was reduced belowthe basal level.32526 However, there is an alternativepossibility that the reduced level of plasma cGMP doesnot originate mainly from the smooth muscle but ratherfrom the vascular endothelium. Porter et a127 recentlyreported that the potency of ANP in the induction ofcGMP production is even higher in endothelial than insmooth muscle cells. Therefore, inhibition of the ANPreceptor in smooth muscle may not be sufficient to elicitsignificant hemodynamic changes.

Plasma Hormonal ChangesIn study 2, the plasma ANP concentrations were

significantly elevated after HS administration. AlthoughHS very selectively blocks the biologically active recep-tor (B-receptor) that is coupled to GC, HS does notaffect the binding of ANP to the metabolic clearancereceptor, which lacks GC activity. 10,2829 The attenuationof ANP metabolism may be due to impaired internal-ization of ANP-receptor complexes in CHF30-32 How-ever, the increase in plasma ANP levels observed in thepresent study may be a consequence of HS displacingANP from the B-receptor or blocking its binding to theB-receptor, since we found that HS also tended toinduce an increase in plasma ANP levels in dogs withoutCHF in study 1.

We observed rapid and marked elevation of PRA,aldosterone, and norepinephrine levels despite the lackof overall deterioration in systemic hemodynamics. Thisis, to the best of our knowledge, the first demonstrationof a significant role of endogenous ANP in counteract-ing these vasoconstrictive/antidiuretic hormones inCHF. In the present study, PRA increased to 226% ofthe control level when the cGMP level had decreased to32%. Several previous studies have shown that exoge-nous ANP, administered intravenously, suppresses re-nin secretion, despite a sustained decrease in arterialpressure.3'5'33'34 However, whether endogenous ANPhas an inhibitory effect on renin release remains con-troversial. After administration of ANP antiserum in anormal rat model, Naruse et a135 observed an elevationin PRA, whereas Rudd et a136 reported no change inPRA. In the present study, we observed a significantelevation of the already activated PRA compared withthe prepacing values when endogenous ANP activitywas suppressed by HS. Although ANP-mediated renininhibition may involve a macula densa mechanism, theresults of the present study do not allow us to distin-guish between this and a direct receptor-mediatedmechanism.4'37

Secretion of aldosterone is known to be inhibited byexogenous ANP, both in vivo and in vitro.38M39 In thepresent study, plasma aldosterone level increased to179% of the control level; plasma angiotensin II levelalso increased, but not significantly (data not shown).We consider the increase in aldosterone secretion not tobe stimulated by angiotensin II but rather to be a resultof the release from direct ANP-mediated inhibition.Oda et aW40 demonstrated a positive correlation betweenthe restoration of ANP-induced inhibition of aldoste-rone production and the reduction of intracellularcGMP level by administration of HS to adrenal glo-merulosa cells. Our findings in the present study indi-

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2238 Circulation Vol 89, No 5 May 1994

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HS-142-1 (mg/kg) "Z HS

.* VehiceFIG 5. Graphs showing percent changes in urine flow rate (U-V)and urinary sodium excretion (U-Na) with administration ofvehicle (e, n=5) or HS-142-1 (HS; o, n=6) in dogs with heartfailure. After two 30-minute basal urine collections were ob-tained, HS 0.3, 1.0, and 3.0 mg/kg or the saline vehicle wasadministered stepwise at 30-minute intervals, and three experi-mental urine collections were performed for each 30 minutesafter administration. Basal values are the average obtainedduring the preceding two 30-minute basal periods. Values aremean+SEM. oP<.05, ooP<.01 compared with basal values;tP<.05, 1tP<.01 compared with values with vehicle.

cate that endogenous ANP inhibits the secretion ofaldosterone through a GC-linked pathway in our severecanine CHF model.We found a significant increase in plasma norepi-

nephrine level to 252% of that in the control. ANP cansuppress the release of sympathetic neurotransmitters41and is thought to inhibit norepinephrine release byacting on the central or ganglionic sympathetic outflowand on catecholamine-secreting cells in the adrenalmedulla.2'42'43 Endogenous ANP modulates muscle andrenal sympathetic nervous activity and may contributeto the hypotensive and natriuretic activity.

Renal Function StudiesHS has been demonstrated to block the binding of

`PI-ANP to the renal cortex92,8 and to inhibit natriuresisinduced by ANP.24"44A45 Although the renal response toANP has been reported to be reduced in CHE, wefound a significant reduction in urine flow rate andurinary sodium excretion after stepwise administrationof HS. HS induced a reduction in urinary cGMPexcretion to 37% of the basal level. However, there wereno statistically significant differences in GFR or RPF

from basal levels after HS treatment. Urinary cGMPoriginates from renal cells, and its level is correlatedwith the natriuresis induced by ANP. Thus, excretion ofurinary cGMP is assumed to be a biological marker forrenal ANP activity in vivo.17,18 Therefore, HS may haveblocked the renal endogenous ANP activity in thepresent study, and endogenous ANP may have actuallycontributed to the excretion of sodium and water insevere CHF without causing significant changes in GFRor RPF. Pharmacological doses of ANP have beenshown to induce an elevation in the excretion of waterand sodium associated with a significant increase inGFR.-5A6 Marin-Grez et a147 showed that ANP induces apreglomerular vasodilatation and a postglomerular va-soconstriction with enhanced natriuresis. Since no sig-nificant changes in FF were observed in the presentstudy, the vasodilative effect of endogenous ANP on theafferent arteriole seemed to be diminished, as it was onthe systemic vasculature. Low doses of ANP infusion indogs46,4849 have been reported to induce natriuresiswithout changes in GFR or blood pressure. A very highdensity ofANP receptor binding sites in the kidney wasreported over the inner medulla,50'51 and the increase incGMP content in response to ANP is largest in the innermedullary collecting ducts (IMCD) among the renaltubular segments. The threshold ANP concentration foran increase in cGMP accumulation in the IMCD hasbeen reported to be about 100 times higher than that inthe glomeruli.52 The IMCD appears to be a major site ofendogenous ANP action, and ANP-induced natriuresisand diuresis are mainly the result of the effect ofANP inthe inner medulla associated with a reduction in reab-sorption of sodium and water in the IMCD. In addition,ANP acts to reduce proximal tubular reabsorption atphysiological concentrations by inhibition of angiotensin-stimulated sodium and water transport.53 Since HS sig-nificantly elevated the plasma aldosterone level andtended to increase that of angiotensin II, the inhibition ofthese hormones by endogenous ANP may also contributeto natriuresis and diuresis.

Limitations of the StudyRecent studies have shown that the plasma brain

natriuretic peptide (BNP) concentration is elevated inCHF.5455 BNP, in addition to ANP, may play a patho-physiological role in CHF. Although two similar mem-brane-bound GCs (GC-A and GC-B) are known to benatriuretic peptide receptors, GC-A is an ANP andBNP receptor, whereas GC-B is a specific receptor forthe C-type natriuretic peptide (CNP).56,57 HS wasequally effective in inhibiting the increase in cGMPproduction stimulated by BNP, CNP, or ANP.21.58 HSshows almost equal antagonistic activity on GC-A andGC-B. In the present study, we could not measure theplasma concentrations of either BNP or CNP, andtherefore, we cannot exclude the possibility that ourfindings are due to inhibition of these peptides. Furtherinvestigation is required to determine to what extent HSinhibits the effects of BNP or CNP in CHE.

In summary, the increment in endogenous ANPobserved in severe CHF actually suppresses the activa-tion of the renin-aldosterone system and sympatheticnervous activity and inhibits the reabsorption of waterand sodium without inducing changes in GFR or RPF.

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Wada et al Endogenous ANP in Dogs With Heart Failure 2239

However, endogenous ANP exerts no significant hemo-dynamic effect through its vasodilative activity.

AcknowledgmentsWe wish to thank Mikio Nakagawa and Chika Yamamoto

for excellent technical assistance. We also express thanks toDaniel Mrozek for his assistance in preparing the manuscript.

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A Wada, T Tsutamoto, Y Matsuda and M Kinoshitareceptors.

severe congestive heart failure using a specific antagonist for guanylate cyclase-coupled Cardiorenal and neurohumoral effects of endogenous atrial natriuretic peptide in dogs with

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