1566

Differential Effects of Captopril and Enalaprilon Tissue Renin-Angiotensin Systems in

Experimental Heart Failure

Alan T. Hirsch, MD; Chris E. Talsness, Angela D. Smith, Heribert Schunkert, MD;Julie R. Ingelfinger, MD; and Victor J. Dzau, MD

Backgrund. Angiotensin converting enzyme (ACE) inhibitor therapy elicits beneficial responses frompatients with heart failure. We hypothesized that a major site of action of these drugs is tissue ACE andthat ACE inhibitors might differ in their ability to inhibit tissue ACE. To test this hypothesis, we assessedthe effects of captopril and enalapril on blood pressure and renal function and on serum and tissue ACEactivities in sham-operated rats and rats with heart failure induced by coronary artery ligation.Methods and Results. During short-term (1-week) treatment, captopril (200 mg kg` - day-') and

enalapril (25 mg * kg` * day-') elicited equipotent effects on blood pressure and inhibition of serum ACEactivity (85%). The effects of long-term treatment (47 days) were then studied beginning 45±5 days aftercoronary ligation in four treatment groups: sham-operated, vehicle (n=14); heart failure, vehicle (n=10);heart failure, captopril (n=8); and heart failure, enalapril rats (n=7). During long-term treatment,captopril and enalapril caused comparable falls of 12-18 mm Hg in blood pressure (p<0.01 comparedwith vehicle treatment). There was no change in urine volume or sodium or potassium excretion in vehicle-or captopril-treated heart failure rats; in contrast, enalapril-treated heart failure rats demonstrated 83%and 10% increases in urine volume and daily sodium excretion, respectively, compared with vehicle-treated rats (bothp <0.01). No significant changes in blood urea nitrogen or creatinine were observed witheither treatment. Enalapril but not captopril elicited a significant decrease in serum and lung ACEactivities. Captopril but not enalapril inhibited aortic ACE activity. Both agents caused a comparableinhibition of renal ACE activity. The magnitude of inhibition of renal ACE activity but not serum andvascular (aortic) ACE activities correlated with the long-term blood pressure response. Enalapril but notcaptopril normalized renal angiotensinogen expression; the magnitude of this effect correlated with theincrease in daily urinary sodium excretion (r= -0.43; p<O.OOS).

Conclusions. These data suggest that chronic treatment with these two agents elicits differential effectson tissue ACE activities and renal angiotensinogen regulation. The differential renal effects of these agentsmay be important in the treatment of heart failure. (Circulation 1992;86:1566-1574)KEY WORDs * angiotensin converting enzymes

angiotensin II

A ngiotensin II is produced in vivo in the systemiccirculation or at local tissue sites. In responseto decreases in cardiac function, as in acute or

decompensated heart failure, the endocrine renin-an-giotensin system is activated. Increased circulating lev-els of angiotensin II may serve a vasoconstrictor role orpromote renal sodium retention. Additionally, acuteinhibition of circulating angiotensin converting enzyme

From the Cardiovascular Division, University of MinnesotaMedical School, and the Division of Cardiovascular Medicine,Cardiovascular Research Center, Stanford University School ofMedicine.A.T.H. is a recipient of individual National Research Service

Award F32-HL-07702-02. H.S. is a recipient of a grant of theDeutsche Forschungsgemeinschaft. Supported by grants HL-35610, HL-35792, HL-35252, HL-40210, and HL-42663 from theNational Heart, Lung, and Blood Institute and a grant from theBristol-Myer-Squibb Institute for Pharmaceutical Research.Address for reprints: Alan T. Hirsch, MD, Cardiovascular

Division, University of Minnesota Medical School, Box 508, 420Delaware Street, S.E., Minneapolis, MN 55455.

Received June 6, 1991; revision accepted July 24, 1992.

* angiotensinogen * myocardial infarction *

(ACE) mediates immediate hypotensive and natriureticresponses. In contrast, circulating levels of angiotensinII are not usually increased in the stable, compensatedphase of heart failure.1-4 Despite normal circulatingrenin and angiotensin II levels in this state, ACEinhibitors have been demonstrated to be efficacious,improving ventricular function and prolonging surviv-al.5-8 We have recently demonstrated that tissue renin-angiotensin systems are activated selectively in the heartand kidney in chronic stable experimental heart failureand cardiac hypertrophy.49'10 It has been hypothesizedthat ACE inhibitors might exert their beneficial effectsprimarily by inhibiting tissue ACE and thereby alteringtissue angiotensin II levels. No studies to date, however,have investigated the effects of ACE inhibitors on tissuerenin-angiotensin systems during chronic treatment inexperimental heart failure or whether the effects varybetween different ACE inhibitors.

Accordingly, in the present study, we hypothesizedthat long-term treatment with captopril and enalapril,the most widely used ACE inhibitors, would cause

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Hirsch et al Tissue Converting Enzyme in Experimental Heart Failure 1567

differential effects on the circulating and tissue renin-angiotensin systems. We further hypothesized that thehemodynamic and renal effects of ACE inhibitor treat-ment during chronic drug administration in rats withstable, compensated heart failure might correlate betterwith tissue ACE than with circulating ACE. The resultsof this study demonstrate that 1) chronic treatment withthese agents elicits differential tissue-specific inhibitionof ACE; 2) the magnitude of inhibition of serum andaortic ACE activities is not related to the long-termhypotensive response; and 3) differential effects of theseagents on renal sodium and water excretion may bemediated by effects on components of the intrarenalrenin-angiotensin system and thereby the local produc-tion of angiotensin II.

MethodsMale Sprague-Dawley rats (weight, 200-250 g;

Charles River Laboratories, Wilmington, Mass.) under-went thoracotomy and left coronary artery ligation at 2months of age. All animals were housed individually ina 12-hour dark/light cycle controlled room. They wereallowed free access to a 0.4% sodium diet (Purina ratchow 5001, Purina Mills, St. Louis, Mo.), and water wasprovided ad libidum. These studies were approved bythe institutional Standing Committee on Animals andwere performed according to US Public Health Serviceguidelines and recommendations of the American As-sociation for the Accreditation of Laboratory AnimalCare and the American Physiological Society.

Experimental Heart Failure ModelThe chronic compensated heart failure model was

prepared by standard methods.1112 Under ether anes-thesia, a thoracotomy was performed, the heart exteri-orized, and the left anterior coronary ligated with 6-0prolene suture. Surgical attrition from this procedure isapproximately 40%. One week after surgery, the surviv-ing animals were subgrouped by a modified 10-leadECG into sham-operated (SO) or heart failure (HF)study cohorts. Group stratification was in all casesconfirmed postmortem by both gross pathology andquantitative left ventricular histopathology.

Experimental ProtocolThe first protocol was designed to evaluate equipo-

tent doses of captopril and enalapril with regard to themagnitude of blood pressure reduction and inhibition ofserum ACE activity in this normotensive strain. Theshort-term hemodynamic and serum ACE activity dose-response relations for captopril and enalapril were thusevaluated in 52 heart failure animals studied approxi-mately 1.5 months after coronary ligation.The baseline indirect tail cuff blood pressure was

studied daily for 7 days. The individual daily and/ordiurnal drinking behavior was assessed to determine thetime of peak drug ingestion to serve as a guide for whento kill the animals (Figure 1). Tail-artery blood wascollected at the end of the baseline period for assess-ment of the pretreatment serum ACE activity. In thesestudies, rats then received either captopril for 1 week at10, 25, 100, or 200 mg * kg`1 day` or enalapril at 15 or25 mg* kg` * day-1 doses based on individual drinkingbehavior (n =8-10 in each cohort). At the end of eachweekly dosing period, indirect blood pressure was de-

IS.

i30 {

20 I8:00-14:00 20:00-2:00

14:00-20:00 2:00-8:00Tlme Period of Observation

FIGURE 1. Graph showing the diurnal drinking behavior ofa subset of adult, male Sprague-Dawley rats used in theseinvestigations (n=8). Based on this behavior, all serum andtissue collections were performed within S hours of peakdrinking.

termined and blood was collected by tail bleed formeasurement of serum ACE activity. Equipotent effectson blood pressure and serum ACE activity wereachieved at the captopril 200 mg * kg 1̀ day-1 and enal-april 25 mg * kg 1̀ day-1 doses.The effect of chronic ACE inhibitor treatment was

then evaluated in a second protocol. The goal of thisprotocol was to evaluate the hemodynamic and renaleffects of captopril and enalapril in animals with chronicheart failure at doses that had been demonstrated tohave equipotent short-term effects on blood pressureand circulating ACE. The effects of these treatments ontissue ACE and components of the intrarenal renin-angiotensin system were also assessed. Animals werestratified into four study groups: 1) sham-operated ratstreated with tap water vehicle (SO-V, n=14); 2) heartfailure rats treated with vehicle (HF-V, n= 10); 3) heartfailure rats treated with captopril, 200 mg * kg-1. day`(HF-C, n=8); and 4) heart failure rats treated withenalapril, 25 mg * kg- 1 day` (HF-E, n=7). All animalswere allowed to recover without treatment for 45+5days after surgery to establish chronic heart failure.Assessment of baseline blood pressure and heart ratewas performed, and animals were maintained in indi-vidual metabolic cages for assessment of drinking be-havior and for urine collection. The concentrations ofcaptopril (kindly provided by E.R. Squibb and Sons,Princeton, N.J.) and enalapril maleate (kindly providedby Merck, Sharp and Dohme, West Point, Pa.) werethen individually adjusted every other day to ensureadequate administration of each agent despite differingdrinking behaviors. Approximate drug concentrationsfor enalapril and captopril were 0.2 mg/ml and 2 mg/mlof drinking water, respectively.At the end of the baseline period, blood (400 gul) was

obtained by tail bleed, snap frozen, and stored at -70°Cfor later ACE activity assay. Chronic treatment wasmaintained for 47±2 days, and hemodynamic and met-abolic assessments were repeated during the final 10days of study. Animals in each study cohort were treatedfor an identical duration and were killed at a compara-ble age.

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1568 Circulation Vol 86, No 5 November 1992

At the end of treatment, all rats were killed in theearly morning by decapitation. Trunk blood was col-lected for determination of serum ACE activity duringtreatment, and all tissues were collected within 3 min-utes of death. The heart was removed en bloc, and theatria were dissected free from remaining heart at theatrioventricular ring. The right ventricle was dissectedfrom the left ventricle, and ventricular masses weredetermined. A 2.0-mm (40-mg) specimen of interven-tricular septal tissue was obtained, and right ventricleand interventricular septal specimens were used fortissue ACE determinations. The entire left ventricle wasfixed in 10% buffered formalin. The thoracic aorta wasstripped of its adventitia. The lung was dissected toexclude major pulmonary trunks. The kidneys weredissected free of their capsule and bisected; half wasused for assay of tissue ACE, and half was flash frozenfor later messenger RNA (mRNA) determinations. Alltissues used for assessment of tissue ACE activity wereimmediately placed in ice-cold potassium phosphatebuffer and processed without delay.

Hemodynamic StudiesIndirect systolic blood pressure (in millimeters of

mercury) was determined by the tail-cuff method and apiezoelectric palpatator. Reported values represent themean of individual recordings performed at the sametime of day by the same investigator on five consecutivedays. The correlation of indirect blood pressures withdirect femoral arterial pressure measurements in ourlaboratory achieves a correlation coefficient of r=0.98and is comparable to data reported previously.'3 Heartrate (beats per minute) was calculated from the meanRR interval derived from 10 consecutive cardiac cyclesof the indirect blood pressure tracing.

Metabolic StudiesAnimals were maintained in individual cages with

minimal manipulation. Reported values represent themean of data obtained over 5 days and representsteady-state values. As noted, drug doses were calcu-lated individually to provide the same daily dose of thedrug based on the drinking behavior of each rat. Urinewas collected for determination of daily urine volume(UV, ml * kg` * day-1), sodium excretion (UNa4V,meq * kg 1̀ day-'), and potassium excretion (UKWV,meq- kg 1̀ day-'), which were indexed to body weight.Urinary sodium and potassium concentrations weredetermined by flame photometry (Model 443, Instru-mentation Laboratories, Inc., Lexington, Mass.). Bloodurea nitrogen (BUN) and serum creatinine concentra-tions (in milligrams per deciliter) were determined byspectrophotometric methods via autoanalyzer (Ek-tachem 700 Clinical Analyzer, Eastman Kodak, Roch-ester, N.Y.).14

Biochemical StudiesPlasma renin concentration (PRC) was determined as

previously described.1'516 Serum and tissue ACE mea-surements were performed by a modified fluorimetricmethod.'7,18 Baseline and treatment blood samples werecollected into chilled Eppendorf tubes and cold centri-fuged for 5 minutes, and serum was then aliquoted foreither storage at -70°C (baseline sample) or immediateassay (sample at death). Baseline and treatment serum

ACE determinations from individual animals were per-formed within a single assay to minimize interassayvariability. Tissues were washed with cold potassiumphosphate buffer to remove any contaminating blood.All tissues were assayed immediately the morning theanimal was killed to minimize possible dissociation ofeach ACE inhibitor from ACE. Because we and othershave observed that captopril can dissociate ex vivo fromACE during storage or during assay procedures,19 allserum samples and tissue homogenates were kept icecold until incubation. Additional control rats were alsotreated with either captopril (50 mg/kg) or vehicle bygavage and were killed 1 hour later (synchronously withchronically treated rats), and tissues were processedconcurrently to ensure sustained ex vivo inhibition ofACE during assay. Each tissue was immediately homog-enized and incubated for 10 minutes at 37°C, pH 7.50, inthe presence of Hip-His-Leu; the reaction was stoppedby the addition of NaOH, pH 8.5. His-Leu product wasthen tagged with 0.1% phthaldialdehyde and quantifiedfluorometrically (SLM 8000C, SLM Instruments, Inc.,Urbana, Ill.) at an excitation wavelength of 386 nm andemission wavelength of 436 nm. This assay is linearbetween 0.02 and 15 mM of His-Leu product.

RNA StudiesRenal tissue was snap-frozen in liquid nitrogen within

3 minutes and stored at -70°C until it was studied.Tissue homogenization and RNA extraction were per-formed as described.20 Comparisons of relative mRNAlevels were made in reference to the same amount oftotal RNA applied per sample and quantified by absor-bance at 260 nm in duplicate. For Northern blot anal-ysis, aliquots of total RNA were run by the formalde-hyde-agarose method.21

Gels were transblotted onto nylon filters by capillaryaction with 10 SSC (1 SSC equals 0.15 M sodiumchloride, 0.015 M sodium citrate, pH 7.0) for 16 hours.Efficiency of transfer was confirmed by ethidium bro-mide stain and examined before and after transfer.RNA on nylon filters was crosslinked by UV light. Foradditional quantification, slot blot hybridization analy-ses were performed. Samples were formaldehyde dena-tured and then serially diluted in 15 SSC. Three con-centrations from each sample (8, 4, and 2 ,g) wereblotted in duplicate to nylon filters with a slot blotapparatus (Schleicher and Schuell, Peterborough,N.H.). After prehybridization for 4 hours, the blots werehybridized overnight in a buffer to which a-2p comple-mentary DNA (cDNA) probes were added.2'

After hybridization, blots were washed in 0.2 SSCwith 0.1% sodium dodecyl sulfate at room temperaturefor 10 minutes, then three times at 65°C for 30 minutes.Blots were exposed for 5 days to x-ray film (KodakXAR, Eastman Kodak). All studies of angiotensinogenmRNA were performed with a full-length rat liverangiotensinogen cDNA (pRang 1650). For reninmRNA determinations, we used renin cDNA pDD1D-2, a full-length mouse submaxillary gland renincDNA that readily crosshybridizes with rat renal reninmRNA.16 To control for possible sample variability,identical slot blots were performed and hybridized witha control (,8-actin) cDNA probe.

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Hirsch et al Tissue Converting Enzyme in Experimental Heart Failure 1569

Quantification ofmRNAAutoradiograms generated by slot blots were scanned

with an LKB microdensitometer (Paramus, N.J.) withbackground set to zero for each autoradiograph. Re-gression lines were calculated from the integral valuesobtained by scanning the serial concentrations of eachsample. The relative signals of the specific mRNA wereestimated from the slope of the regression line, and onlyvalues of r20.90 were accepted. Slopes of specificmRNA for each condition were compared as relativeratios. Sufficient material was available to study speci-mens with multiple sample applications. Our intrablotand interblot coefficients of variation were 8% and 9%,respectively.

Morphometric Analysis of Infarct SizeThe left ventricle was sectioned transversely in at

least five planes, stained with Masson's trichrome andhematoxylin/eosin, and projected for quantitativeplanimetry of infarct size.22 The mean of the endocar-dial and epicardial scar circumferences was comparedwith total left ventricular circumference to calculatetotal infarct size (in percent). All infarctions weretransmural and involved only the free wall of the leftventricle. Only rats with infarcts greater than 10% of theleft ventricular circumference were included in theheart failure groups.

Statistical AnalysisData are presented as mean+SEM. Mean slopes of

slot blot autoradiographs were compared by an un-paired Student's t test. Hemodynamic and plasma ACEresponses were compared by a Student's t test for paireddata. Intergroup variables (SO-V, HF-V, HF-C, andHF-E) were compared by a randomized ANOVA with aNewman-Keuls post hoc test for significant differencesbetween individual groups. Linear regression analysiswas performed to evaluate the relations between se-lected variables. Significance was accepted at the 95%confidence limit (p<0.05).

ResultsIn our initial investigation, we determined equipotent

doses of captopril and enalapril during short-term (1-week) treatment of HF rats based on the magnitude ofthe hypotensive effect and degree of inhibition of serumACE. During the second protocol, the effects of chronic(7-week) ACE inhibitor treatment with these drugswere assessed in SO and HF rats to determine therelevance of the tissue renin-angiotensin system to thechronic hemodynamic and renal responses.

Protocol 1In HF-V rats, there were no changes in blood pres-

sure (129+±2 versus 125+±5 mm Hg, baseline versustreatment, respectively; p=NS) or serum ACE activity(89±+10 versus 91±15 nM * ml-l' min-m; p=NS) beforeor during treatment (Table 1). At lower doses (10 and25 mg . kg` day-'), captopril treatment caused a fall ofapproximately 20 mm Hg in blood pressure in all rats(p=NS between these two doses); the serum ACEresponse was not assessed in these treatment subsets.Captopril treatment at 100 and 200 mg - kg` . day-1

TABLE 1. Dose-Titration Studies With Oral Captopril andEnalapril in Heart Failure Rats

Baseline 7-Day treatment

BP BP Serum ACE InhibitionGroup (mm Hg) (mm Hg) (nM * ml` * min-1) (%)

V 129+2 125+5 91±15C1o 127+2 101+6* NA ...

C25 123±3 103+4* NA ...

C100 122+5 107±4* 50±8 45+9*C200 122±4 105±3* 18±5 81±5tE15 119±5 92±6* 26±4 71+4tE25 117+8 105+2* 15+4 84+4t

BP, tail cuff blood pressure; ACE, angiotensin converting en-zyme activity. Animals were allowed free access to drinking water(V, vehicle); captopril (C) at 10, 25. 100, or 200 mg - kg`. day-1;or enalapril (E) at 15 or 25 mg * kg 1̀ day` for 1 week. PercentACE inhibition during treatment was calculated as (baselineserum ACE activity-treatment serum ACE activity)/(baselineserum ACE activity). NA, not available. Data are presented asmean+SEM.

*p<0.05 compared with baseline value; tp<0.005 comparedwith baseline value.

to that observed at the lower two doses. Serum ACEwas inhibited by 45% and 81% at the 100- and 200-mg - kg` * day-1 doses, respectively (each,p<0.005 com-pared with baseline).

Enalapril treatment for 7 days also caused a hypoten-sive response of 12-27 mm Hg, and a stepwise increasein serum ACE inhibition was demonstrated. The mag-nitudes of the hypotensive and serum ACE inhibitionresponses were comparable for captopril at 200mg- kg`-l day`1 and for enalapril at 25 mg - kg' - day-1(p=NS for both responses between drugs).

Protocol 2At baseline, animals in each of the four study groups

were comparable with regard to age at operation (61days), age at the initiation of treatment (108 days), andage at the time of death (154 days). Baseline weightsand urinary volume and sodium and potassium excre-tion were also not different between groups (Table 2).The mean infarct size in the total study group was24±4%. SO rats had a mean infarct size of 3.0±0.3%.The mean infarct sizes in the HF-V, HF-C, and HF-Egroups were 17+3%, 33+4%, and 25 +3%, respectively.The mean infarct sizes of the captopril and enalapril HF

TABLE 2. Baseline Characteristics of Rats in Each ChronicTreatment Group

SO-V HF-V HF-C HF-E

Weight (g) 404±11 394+13 388±12 388+10UV (ml/day) 45+4 42+4 43±3 44+2UNa+V (meq - kg BW-1 5.9+0.3 6.4+0.3 6.2+0.4 6.6±0.5*day1)UK+V (meq. kg BW` 10.2±0.5 10.6+0.5 9.9±0.7 10.2+0.6*day 1)SO-V, sham-operated rats treated with vehicle; HF-V, HF-C,

HF-E, heart failure rats treated with vehicle, captopril, andenalapril, respectively; UV, daily urinary volume; UNaWV, dailyurinary sodium excretion; UK+V, daily urinary potassium excre-tion; BW, body weight. Data are presented as mean+SEM. No

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1570 Circulation Vol 86, No 5 November 1992

TABLE 3. Cardiac Weights in Each Chronic Treatment Group

SO-V HF-V HF-C HF-E

LV, mg/kg BW 2.04±0.04 2.30±0.12 2.11±0.08 2.25±0.19RV, mg/kg BW 0.47±0.02 0.55±0.06 0.58±0.06 0.64±0.07

SO-V, sham-operated rats treated with vehicle; HF-V, HF-C,HF-E, heart failure rats treated with vehicle, captopril, and enala-pril, respectively; LV, left ventricle; RV, right ventricle; BW, bodyweight. No intergroup differences reached statistical significance.

groups were comparable and did not differ statistically(p=NS).These observations are confirmed by the measure-

ment of cardiac weights as displayed in Table 3. The leftventricular and right ventricular weights were slightlygreater in the HF-V rats compared with the SO group,but this did not reach statistical significance. This isprobably because of the smaller infarct size in the HF-Vanimals. The left and right ventricular weights of ratschronically treated with captopril or enalapril were notsignificantly different from those of vehicle-treatedanimals.

Figure 2 displays the blood pressure responses inthese groups at baseline and at the end of treatment. Asdepicted, there was no change in the blood pressure of ratsin the SO-V and HF-V groups (from 124±2 to 125+±3mm Hg in HF-V; from 126±3 to 129±2 mm Hg at base-line and during treatment in each group, respectively;p=NS). There was a comparable hypotensive response of12-18 mm Hg in the HF-C (from 120±3 to 102±3mm Hg; p<0.01) and HF-E (from 116±4 to 104±2mm Hg; p<0.01) groups from baseline to treatment. Themagnitude of the hypotensive response did not correlatewith the size of the previous infarct (r=0.03 for HF-C rats,and r=0.36 for the HF-E rats, both p=NS). Heart ratedid not change significantly in any of the treatmentgroups: SO-V (from 417± 7 to 406±8 beats per minute),HF-V (from 394±10 to 405±15 beats per minute),HF-C (from 388±12 to 377±12 beats per minute), andHF-E (from 388±10 to 392±16 beats per minute).

During vehicle treatment, there was no significantchange in the daily urine volume (Figure 3), urinarysodium excretion, or urinary potassium excretion in SOor HF rats (Table 4). Similarly, there was no change inthese responses in the HF-C rats. In contrast, HF-E

140.

~~~~~~~~~~~~~~~T

E 130

120-o-

CL110-- 0-0 so-v

8 HF-Yin * = HF-C

1(00- HF-E

90Rx

FIGURE 2. Graph showing the baseline and treatment bloodpressures in sham-operated rats treated with vehicle (SO-V,open circles) and heartfailure rats treated with vehicle (HF-V,closed circles), captopril (HF-C, upright triangles), or enala-pril (HF-E, inverted triangles). Rx, end of treatment.**p<0.01 compared with baseline.

-v0Dl.

Base Rx

FIGURE 3. Graph showing the daily urine volume (UV.ml* kg` * day-') excreted by rats at baseline and at the end oftreatment (Rx) in each group. SO-V, sham-operated ratstreated with vehicle; HF-V, HF-C, HF-E, heart failure ratstreated with vehicle, captopril, or enalapril, respectively.**p<0.01 compared with baseline.

animals demonstrated a modest diuretic and natriureticresponse to treatment (Table 4 and Figure 3). Althoughthere was a tendency for the BUN to increase in theACE inhibitor-treated animals, this did not reach sta-tistical significance. The serum creatinine was un-

changed during treatment.The PRC was not different in HF-V versus SO-V rats

(Table 5), as we have previously demonstrated4; treat-ment with captopril and enalapril caused PRC to rise bycomparable amounts compared with both vehicle-treated control groups (p<0.01 for each). Treatmentwith these two ACE inhibitors resulted in differentialeffects on serum and tissue ACE activities. Serum ACEactivity (Figure 4) did not change during vehicle treat-ment in the SO and HF rats (serving as a time control).During chronic enalapril treatment, serum ACE wasinhibited by approximately 55% compared with baselinevalues. In contrast, there was no significant inhibition ofserum ACE in the HF-C animals after 47 days oftreatment. Table 6 displays the effects of captopril andenalapril treatment on tissue ACE activities. Captoprilelicited a decrease in aortic ACE activity (31% inhibi-tion), but this was not achieved during enalapril treat-ment. Both drugs elicited comparable decreases in renalACE activity (56% during captopril and 44% duringenalapril treatment, respectively). Captopril treatmentwas associated with an induction in pulmonary ACEactivity, whereas enalapril treatment elicited a 33% fallin ACE activity in the lung. Neither drug caused a

TABLE 4. Urine Electrolyte Excretion, Blood Urea Nitrogen,and Serum Creatinine at End of Treatment

SO-V HF-V HF-C HF-E

UNa+V(meq.kgBW-1 4.9+0.1 5.3+0.1 5.1±0.2 5.8+0.4** day 1)UK+V (meq * kg BW 9.3 +0.3 9.8+0.4 8.5 ±0.5 9.6+0.5* day 1)BUN (mg/dl) 19+0.5 19±0.8 29±8 22+2Creatinine (mg/dl) 0.5±0.01 0.5±0.01 0.5±0.01 0.5+0.01

SO-V, sham-operated rats treated with vehicle; HF-V, HF-C,HF-E, heart failure rats treated with vehicle, captopril, andenalapril, respectively; UNa+V, daily urinary sodium excretion;UK+V, daily urinary potassium excretion; BW, body weight; BUN,blood urea nitrogen. Data presented as mean+SEM. *p<0.01compared with SO-V.

o0o SO-V*-* HF-VA-A HF-C Tv-v HF-E

70-

50-

30 i

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TABLE 5. Effect of Treatment With Captopril vs. Enalapril on

Renal Angiotensinogen and Renal Renin Expression and PlasmaRenin Concentration

SO-V HF-V HF-C HF-E

ANG mRNA 18±1 24+2* 26+7* 16+2tRenin mRNA 137±10 109±27 229+32* 248±25*PRC (ng AI * ml-l hr-1) 7.5±+1.0 9.9+2.2 96+24t 58-20t

SO-V, sham-operated rats treated with vehicle; HF-V, HF-C,HF-E, heart failure rats treated with vehicle, captopril, and enala-pril, respectively; ANG, angiotensinogen; mRNA, messenger RNA;PRC, plasma renin concentration; AI, angiotensin I mRNA datapresented as arbitrary densitometric unitsx 103. Data are presentedas mean+SEM.

*p<0.05 compared with SO-V; tp<0.05 compared with HF-V.

significant change in either right ventricular or interven-tricular septal ACE activities. Serum and tissues fromacutely gavaged rats, assayed concurrently with thosefrom chronically treated animals, demonstrated greaterthan 85% inhibition compared with vehicle gavage.The long-term hypotensive response (47±2 days of

oral treatment) to ACE inhibitor therapy did not cor-

relate with the serum or aortic ACE activities duringtreatment (r=0.08 and p=NS for serum ACE activityand r=0.15 and p=NS for aortic ACE activity); a

moderate correlation was present between the treat-ment renal ACE activity and the hypotensive response(r=0.48, pcO.05).The intrarenal renin-angiotensin system was differ-

entially affected by induction of heart failure as well as

by chronic ACE inhibitor treatment (Table 6). HF ratsdemonstrated a 33% induction in renal angiotensinogenexpression compared with SO control rats (p<0.05).Renin mRNA expression was unchanged in HF ratscompared with SO control rats. This is consistent withthe PRC results and confirms our previous data on ratswith chronic compensated heart failure. Chronic capto-pril treatment did not change the renal angiotensinogenexpression mRNA level but elicited an increase in reninmRNA expression compared with HF-V rats (67% and110% compared with SO-V and HF-V, respectively;both p<0.05); PRC increased significantly (to 96+24 ngAI ml-l hr-1; p<O.OS compared with HF-V) duringcaptopril treatment. In contrast, enalapril treatment

c

E

IN.

'zo-1;ca~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

100 .I

75-

50- 0- O so-V

*-* HF-V

25- A-A HF-C

n-v HF-E

n

Bane Rx

FIGURE 4. Graph showing the serum ACE activity re-

sponses in sham-operated rats treated with vehicle (SO-V,open circles) and heartfailure rats treated with vehicle (HF-V,closed circles), captopril (HF-C, upright triangles), or enala-pril (HF-E, inverted triangles). Rx, end of treatment.**p<0.01 compared with baseline.

TABLE 6. Effect of Treatment on Tissue ACE Activities in HeartFailure Rats

HF-V HF-C HF-E

Aorta 0.35±0.05 0.24±0.06* 0.31±0.06Kidney 0.32±0.03 0.14+0.03* 0.18±0.05*Lung 6±1 10±2* 4±1*RV 0.08±0.01 0.06±0.01 0.09±0.02IVS 0.07±0.01 0.06±0.01 0.10±0.01

HF-V, HF-C, HF-E, heart failure rats treated with vehicle,captopril, and enalapril, respectively; RV, right ventricle; IVS,interventricular septum. Data are presented as mean±SEM.

*p<0.05 compared with vehicle treatment.

normalized the renal angiotensinogen mRNA level(p<0.01 compared with both HF-V and HF-C) and alsostimulated PRC (to 58±20 ng AI * ml- *. hr- ; p< .05compared with HF-V).

DiscussionIt has been demonstrated that ACE inhibitors are

particularly effective in the treatment of severe heartfailure, in which the plasma renin-angiotensin system isactivated. Recent data also demonstrate that ACEinhibitors are effective in mild-to-moderate congestiveheart failure and in left ventricular dysfunction withoutovert heart failure.5-8.23 Because these conditions arecharacterized by normal plasma renin activity,2,24 themechanisms underlying the efficacy of ACE inhibitorsremain poorly understood. We and others have hypoth-esized that these agents might modulate tissue renin-angiotensin system activity and thereby beneficially al-ter end-organ physiological responses.The coronary ligated rat model of experimental heart

failure is characterized by a stable compensated low-output state in association with regional blood flowredistribution, renal sodium avidity, and increased mor-tality.6'25 In this model, as in chronic, stable heartfailure, plasma renin activity and serum ACE activityare normal in the compensated state4,26; nevertheless,ACE inhibition normalizes regional blood flow, inducesnatriuresis, and improves survival.6,2728 Thus, the phys-iological and neurohormonal status of this model simu-lates those of human chronic heart failure. This modelalso demonstrates tissue-specific activation of cardiacACE in proportion to the size of the myocardialinfarction.4The present investigation contributes to our under-

standing of the role of tissue ACE in heart failure bydemonstrating that 1) the hypotensive response tochronic ACE inhibition is not predicted by effects oncirculating ACE activity; 2) the magnitude of chronictissue ACE inhibition is not predicted by short-termserum ACE inhibition; 3) chronic treatment may elicitthe induction of tissue and serum ACE activities; and 4)chronic treatment by these two commonly used ACEinhibitors can result in differential responses of theintrarenal renin-angiotensin system, which may influ-ence sodium and water excretion.

Blood Pressure ResponseDuring chronic (7-week) treatment, captopril and

enalapril caused blood pressure to fall comparably inHF rats. The magnitude of the hypotensive response at

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1572 Circulation Vol 86, No 5 November 1992

the target doses (captopril, 200 mg. kg`1day-l andenalapril, 25 mg * kg 1̀ day-') was approximately equalduring 1 week and 7 weeks of treatment. It is notablethat this dose of captopril is approximately the same asthat used in the chronic survival studies performed byPfeffer et a16 (2 g/l). Nevertheless, the magnitude ofinhibition of serum ACE during short-term (1-week)treatment was substantially greater than that observedafter chronic treatment. Both captopril and enalaprilelicited an 85% fall in serum ACE activity during 1week of treatment. In contrast, despite comparablehypotensive effects, only enalapril elicited a sustainedinhibition of serum ACE activity. Thus, serum ACEactivity was a poor predictor of the systemic hemody-namic response.The magnitude of the hypotensive effect was not

predicted by the severity of heart failure as assessed bythe size of the infarcted myocardium. Additionally, themagnitude of inhibition of serum and vascular (aortic)ACE activity did not correlate with the fall in bloodpressure in HF rats. Previous investigations have alsoobserved that the magnitude of serum ACE inhibitionpoorly predicts the hypotensive response in normoten-sive and hypertensive animal models.29-34 Chronic treat-ment with both enalapril and captopril has been shownto cause induction of total circulating ACE activity;therefore, this lack of correlation is not unexpected.35-37In our study, the long-term blood pressure responsecorrelated best with renal ACE activity. The nature ofthis relation is not known. It is interesting to speculatethat the reduction in renal ACE activity results inreduced intrarenal angiotensin II concentration andenhanced sodium excretion. Thus, the long-term effecton plasma volume may determine the blood pressureresponse with chronic therapy.

Chronic ACE Inhibitor Treatment: Effects on theIntrarenal Renin-Angiotensin SystemThe present study demonstrates differing patterns of

renal angiotensinogen expression and sodium and vol-ume excretion during treatment with captopril andenalapril. We have recently reported from a separateinvestigation that chronic experimental heart failure inthis model causes a tissue-specific induction of renalangiotensinogen expression.10 The present study dem-onstrates that the increased renal angiotensinogenmRNA level is normalized by chronic enalapril treat-ment but is not affected by long-term captopril therapy.Renal renin mRNA expression and plasma renin con-centration during treatment with these two drugs werecomparable. Because the blood pressure was not differ-ent between the two treatment groups, differences inintrarenal mechanisms of action of these two drugs arepresumed to underlie these different renal angiotensin-ogen responses. The nature of these differences inintrarenal action is unclear at this time.The physiological consequences of intrarenal angio-

tensin production may be extrapolated from the resultsof the present study. HF-C rats displayed a lower dailyexcretion of both sodium and water than HF-E rats.Because renal perfusion pressure (arterial pressure)was not different between the two drug treatmentgroups, an intrarenal effect must differentiate thesemarkedly disparate renal responses; such an intrarenaleffect may be the level of angiotensin II. Because the

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FIGURE 5. Graphs showing the relation between renal an-giotensinogen expression and urinary sodium excretion(UNa'V). Panel A: Data from sham-operated, vehicle-treated rats (SO-V, open circles) and heart failure, vehicle-treated rats (HF-V, closed circles) (r=0.09, p=NS). Panel B:Data from heartfailure, captopril-treated rats (HF-C, uprighttriangles) and heart failure, enalapril-treated rats (HF-E,inverted triangles) (r= -0.43, p<O.05).

magnitude of renal ACE inhibition was comparablebetween these two treatment groups, and because reninwas present in excess, we speculate that angiotensino-gen levels may determine the rate of intrarenal angio-tensin synthesis. Intrarenal angiotensin II has beenshown to stimulate sodium and water retention via adirect proximal tubular effect. The relation betweenangiotensinogen mRNA levels and renal sodium excre-tion is displayed in Figure 5. As shown, there was nocorrelation between the angiotensinogen mRNA leveland sodium excretion during vehicle treatment (r=0.09,p=NS). In contrast, drug-treated HF rats displayed afairly striking inverse relation, as increased angiotensin-ogen expression was associated with lower sodium ex-cretion (r= -0.43,p<0.05). Although such a correlationdoes not delineate either a cause-and-effect mechanismor a common cofactor that directionally alters bothangiotensinogen expression and sodium excretion, thisfinding provides an initial clue to suggest that such linksexist. It should be noted that the present 24-hoururinary sodium measurements determined during thefinal week of treatment may not reflect cumulativechanges in chronic sodium balance that likely occur inthis model with chronic treatment with either ACEinhibitor.These data should also be interpreted in the context

of the study of Packer et a138 that demonstrated thatenalapril resulted in a higher BUN and serum creati-nine than captopril under the conditions of their exper-iments. In the present study, we did not observe a

D

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Hirsch et al Tissue Converting Enzyme in Experimental Heart Failure 1573

difference in renal function between the two drugtreatments under the conditions of our experiment.Unlike Packer et al, we did not observe a difference insystemic blood pressures between the two drugs, asexpected from the design of our protocol. We did,however, observe a difference in angiotensinogenmRNA expression that may reflect a shorter intrarenalduration of action of captopril. Because we have shownthat intrarenal angiotensin II directly stimulates angio-tensinogen mRNA expression, the higher renal angio-tensinogen mRNA in the captopril rats is consistentwith a lesser reduction in angiotensin II in the kidney.Because angiotensin II can also directly stimulate prox-imal tubule sodium reabsorption, this differential effecton intrarenal angiotensin may also explain the differ-ences in urinary sodium excretion between the twodrugs.

Differential Effects of Captopril and Enalaprilon Tissue ACE

It has previously been recognized that ACE inhibitorshave differential effects on the inhibition of tissueACE.39 Previous studies have reported a decrease incardiac hypertrophy and the prevention of ventricularenlargement with ACE inhibition.44' Chronic ACEinhibitor treatment in this study did not decrease eithertotal left or right ventricular mass, however, despiteprolonged drug therapy. This may not be surprising, inthat ACE inhibitors were not initiated until 47 daysafter myocardial infarction. Anversa et a142 have shownthat cardiac myocyte hypertrophy is an early response toinjury, occurring within 3 days of infarction. Addition-ally, it is interesting that cardiac ACE activity in thisstudy did not appear to be suppressed by either capto-pril or enalapril. This is probably because of the induc-tion of cardiac ACE during concomitant ACE inhibi-tion.4 Although both agents may inhibit cardiac ACEwhen administered as a single dose in spontaneouslyhypertensive rats, the chronic responses in normoten-sive experimental models have not been previouslystudied.

Aortic ACE activity was inhibited by captopril but notenalapril in HF rats. Thus, one might speculate thatcaptopril would have greater effects on vascular compli-ance or hypertrophy than enalapril at these adminis-tered doses. Although these would be potentially im-portant end-organ effects, these vascular functionalresponses were not specifically studied in theseprotocols.

Equipotency: Serum versus Tissue ResponsesIn clinical trials, the effects of various ACE inhibitors

on clinical endpoints have been assessed by titration ofdrug doses based on the systemic hemodynamic re-sponse. This study demonstrates that hemodynamicequipotency cannot always be correlated with equipo-tent effects at the tissue level. Whereas captopril andenalapril caused differing patterns of tissue ACE inhi-bition, it is likely that one might achieve comparabletissue ACE inhibition with alternative doses of theseagents. Additionally, the duration of treatment by eachagent may cause variable induction of serum and tissueACE activities. Until better methods of assessing themagnitude of this induction are available, true drug

magnitude of penetration and/or binding of theseagents to tissue ACE is difficult to quantify.

Study LimitationsThis investigation examined primarily tissue ACE

activities; it examined renin and angiotensinogen only inthe kidney. It was not realistic to study every componentof this system in all tissues. Present techniques do notpermit localization of the cell type(s) at which ACE isinhibited. Tissue angiotensin II concentrations were notdetermined. Concentrations of angiotensin II in tissuehomogenates are notoriously difficult to quantify be-cause of the presence of other non-ACE tissue pepti-dases and crossreactivity of most antibodies to angioten-sins I, II, and III. Thus, the magnitude of effect of thesedrugs on angiotensin II biosynthesis must be inferredfrom surrogate markers (e.g., rate-limiting componentsof the system). In addition, ACE inhibitors may alterthe synthesis and metabolism of kinins and prostaglan-dins, which may contribute to their beneficial end-organeffects.

Finally, whereas these data describe the effect ofthese ACE inhibitors at selected doses in a relevantsmall-animal model of heart failure, these findingsshould be extrapolated to clinical investigation cau-tiously. Small-animal pharmacological investigations areoften characterized by greater drug doses on a milligramper kilogram basis. The effect of clinically appropriatedoses of ACE inhibitors on human tissue ACE is notknown.

ConclusionsThese findings demonstrate that circulating and tis-

sue ACE levels are probably altered (induced) bychronic drug therapy. Despite comparable acute hemo-dynamic and serum ACE inhibitory effects, chroniccaptopril and enalapril treatment elicit quite differentpatterns of inhibition of tissue ACE in experimentalheart failure; this finding has relevance in the design offuture clinical or experimental investigations that at-tempt to compare these or other agents of this class.The beneficial effects of ACE inhibitors are probably aresult of the interplay of multiple mechanisms of actionof these agents on target end-organs in heart failure andother pathophysiological states. Some of these effectsmay be attributed to the ability of ACE-inhibitors toalter the expression or activity of tissue renin-angioten-sin systems.

AcknowledgmentsThe authors wish to acknowledge the contributions of Dr.

David Cushman for assistance in ACE activity determinations;Dr. Charles Sweet for assistance with cardiac histopathology;Dr. Art Lage and the staff of the Harvard Animal ResourceCenter for animal care; and Donna MacDonald for prepara-tion of the manuscript.

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A T Hirsch, C E Talsness, A D Smith, H Schunkert, J R Ingelfinger and V J Dzauexperimental heart failure.

Differential effects of captopril and enalapril on tissue renin-angiotensin systems in

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