1566

Differential Effects of Captopril and Enalapril

on 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 from
patients with heart failure. We hypothesized that a major site of action of these drugs is tissue ACE and
that ACE inhibitors might differ in their ability to inhibit tissue ACE. To test this hypothesis, we assessed
the effects of captopril and enalapril on blood pressure and renal function and on serum and tissue ACE
activities 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 ACE
activity (85%). The effects of long-term treatment (47 days) were then studied beginning 455 days after
coronary 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 compared
with vehicle treatment). There was no change in urine volume or sodium or potassium excretion in vehicleor 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 vehicletreated rats (bothp <0.01). No significant changes in blood urea nitrogen or creatinine were observed with
either treatment. Enalapril but not captopril elicited a significant decrease in serum and lung ACE
activities. Captopril but not enalapril inhibited aortic ACE activity. Both agents caused a comparable
inhibition of renal ACE activity. The magnitude of inhibition of renal ACE activity but not serum and
vascular (aortic) ACE activities correlated with the long-term blood pressure response. Enalapril but not
captopril normalized renal angiotensinogen expression; the magnitude of this effect correlated with the
increase in daily urinary sodium excretion (r= -0.43; p<O.OOS).
Conclusions. These data suggest that chronic treatment with these two agents elicits differential effects
on tissue ACE activities and renal angiotensinogen regulation. The differential renal effects of these agents
may be important in the treatment of heart failure. (Circulation 1992;86:1566-1574)
KEY WORDs * angiotensin converting enzymes * angiotensinogen * myocardial infarction *
angiotensin II
-

A ngiotensin II is produced in vivo in the systemic

circulation or at local tissue sites. In response
to decreases in cardiac function, as in acute or
decompensated heart failure, the endocrine renin-angiotensin system is activated. Increased circulating levels of angiotensin II may serve a vasoconstrictor role or
promote renal sodium retention. Additionally, acute
inhibition of circulating angiotensin converting enzyme
From the Cardiovascular Division, University of Minnesota
Medical School, and the Division of Cardiovascular Medicine,
Cardiovascular Research Center, Stanford University School of
Medicine.
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 the
Deutsche Forschungsgemeinschaft. Supported by grants HL35610, HL-35792, HL-35252, HL-40210, and HL-42663 from the
National Heart, Lung, and Blood Institute and a grant from the
Bristol-Myer-Squibb Institute for Pharmaceutical Research.
Address for reprints: Alan T. Hirsch, MD, Cardiovascular
Division, University of Minnesota Medical School, Box 508, 420
Delaware Street, S.E., Minneapolis, MN 55455.
Received June 6, 1991; revision accepted July 24, 1992.

(ACE) mediates immediate hypotensive and natriuretic

responses. In contrast, circulating levels of angiotensin
II are not usually increased in the stable, compensated
phase of heart failure.1-4 Despite normal circulating
renin and angiotensin II levels in this state, ACE
inhibitors have been demonstrated to be efficacious,
improving ventricular function and prolonging survival.5-8 We have recently demonstrated that tissue reninangiotensin systems are activated selectively in the heart
and kidney in chronic stable experimental heart failure
and cardiac hypertrophy.49'10 It has been hypothesized
that ACE inhibitors might exert their beneficial effects
primarily by inhibiting tissue ACE and thereby altering
tissue angiotensin II levels. No studies to date, however,
have investigated the effects of ACE inhibitors on tissue
renin-angiotensin systems during chronic treatment in
experimental heart failure or whether the effects vary
between different ACE inhibitors.
Accordingly, in the present study, we hypothesized
that 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

differential effects on the circulating and tissue reninangiotensin systems. We further hypothesized that the
hemodynamic and renal effects of ACE inhibitor treatment during chronic drug administration in rats with
stable, compensated heart failure might correlate better
with tissue ACE than with circulating ACE. The results
of this study demonstrate that 1) chronic treatment with
these agents elicits differential tissue-specific inhibition
of ACE; 2) the magnitude of inhibition of serum and
aortic ACE activities is not related to the long-term
hypotensive response; and 3) differential effects of these
agents on renal sodium and water excretion may be
mediated by effects on components of the intrarenal
renin-angiotensin system and thereby the local production of angiotensin II.
Methods
Male Sprague-Dawley rats (weight, 200-250 g;
Charles River Laboratories, Wilmington, Mass.) underwent thoracotomy and left coronary artery ligation at 2
months of age. All animals were housed individually in
a 12-hour dark/light cycle controlled room. They were
allowed free access to a 0.4% sodium diet (Purina rat
chow 5001, Purina Mills, St. Louis, Mo.), and water was
provided ad libidum. These studies were approved by
the institutional Standing Committee on Animals and
were performed according to US Public Health Service
guidelines and recommendations of the American Association for the Accreditation of Laboratory Animal
Care and the American Physiological Society.

Experimental Heart Failure Model

The chronic compensated heart failure model was
prepared by standard methods.1112 Under ether anesthesia, a thoracotomy was performed, the heart exteriorized, and the left anterior coronary ligated with 6-0
prolene suture. Surgical attrition from this procedure is
approximately 40%. One week after surgery, the surviving animals were subgrouped by a modified 10-lead
ECG into sham-operated (SO) or heart failure (HF)
study cohorts. Group stratification was in all cases
confirmed postmortem by both gross pathology and
quantitative left ventricular histopathology.
Experimental Protocol
The first protocol was designed to evaluate equipotent doses of captopril and enalapril with regard to the
magnitude of blood pressure reduction and inhibition of
serum ACE activity in this normotensive strain. The
short-term hemodynamic and serum ACE activity doseresponse relations for captopril and enalapril were thus
evaluated in 52 heart failure animals studied approximately 1.5 months after coronary ligation.
The baseline indirect tail cuff blood pressure was
studied daily for 7 days. The individual daily and/or
diurnal drinking behavior was assessed to determine the
time of peak drug ingestion to serve as a guide for when
to kill the animals (Figure 1). Tail-artery blood was
collected at the end of the baseline period for assessment of the pretreatment serum ACE activity. In these
studies, rats then received either captopril for 1 week at
10, 25, 100, or 200 mg * kg`1 day` or enalapril at 15 or
25 mg* kg` * day-1 doses based on individual drinking
behavior (n =8-10 in each cohort). At the end of each
weekly dosing period, indirect blood pressure was de-

1567

IS.

i30

{
I

20

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

FIGURE 1. Graph showing the diurnal drinking behavior of

a subset of adult, male Sprague-Dawley rats used in these
investigations (n=8). Based on this behavior, all serum and
tissue collections were performed within S hours of peak
drinking.

termined and blood was collected by tail bleed for

measurement of serum ACE activity. Equipotent effects
on blood pressure and serum ACE activity were
achieved at the captopril 200 mg * kg`1 day-1 and enalapril 25 mg * kg`1 day-1 doses.
The effect of chronic ACE inhibitor treatment was
then evaluated in a second protocol. The goal of this
protocol was to evaluate the hemodynamic and renal
effects of captopril and enalapril in animals with chronic
heart failure at doses that had been demonstrated to
have equipotent short-term effects on blood pressure
and circulating ACE. The effects of these treatments on
tissue ACE and components of the intrarenal reninangiotensin system were also assessed. Animals were
stratified into four study groups: 1) sham-operated rats
treated with tap water vehicle (SO-V, n=14); 2) heart
failure rats treated with vehicle (HF-V, n = 10); 3) heart
failure rats treated with captopril, 200 mg * kg- 1. day`
(HF-C, n=8); and 4) heart failure rats treated with
enalapril, 25 mg * kg- 1 day` (HF-E, n=7). All animals
were allowed to recover without treatment for 45+5
days after surgery to establish chronic heart failure.
Assessment of baseline blood pressure and heart rate
was performed, and animals were maintained in individual metabolic cages for assessment of drinking behavior and for urine collection. The concentrations of
captopril (kindly provided by E.R. Squibb and Sons,
Princeton, N.J.) and enalapril maleate (kindly provided
by Merck, Sharp and Dohme, West Point, Pa.) were
then individually adjusted every other day to ensure
adequate administration of each agent despite differing
drinking behaviors. Approximate drug concentrations
for enalapril and captopril were 0.2 mg/ml and 2 mg/ml
of drinking water, respectively.
At the end of the baseline period, blood (400 gul) was
obtained by tail bleed, snap frozen, and stored at -70C
for later ACE activity assay. Chronic treatment was
maintained for 472 days, and hemodynamic and metabolic assessments were repeated during the final 10
days of study. Animals in each study cohort were treated
for an identical duration and were killed at a comparable age.

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1568

Circulation Vol 86, No 5 November 1992

At the end of treatment, all rats were killed in the

early morning by decapitation. Trunk blood was collected for determination of serum ACE activity during
treatment, and all tissues were collected within 3 minutes of death. The heart was removed en bloc, and the
atria were dissected free from remaining heart at the
atrioventricular ring. The right ventricle was dissected
from the left ventricle, and ventricular masses were
determined. A 2.0-mm (40-mg) specimen of interventricular septal tissue was obtained, and right ventricle
and interventricular septal specimens were used for
tissue ACE determinations. The entire left ventricle was
fixed in 10% buffered formalin. The thoracic aorta was
stripped of its adventitia. The lung was dissected to
exclude major pulmonary trunks. The kidneys were
dissected free of their capsule and bisected; half was
used for assay of tissue ACE, and half was flash frozen
for later messenger RNA (mRNA) determinations. All
tissues used for assessment of tissue ACE activity were
immediately placed in ice-cold potassium phosphate
buffer and processed without delay.

Hemodynamic Studies
Indirect systolic blood pressure (in millimeters of
mercury) was determined by the tail-cuff method and a
piezoelectric palpatator. Reported values represent the
mean of individual recordings performed at the same
time of day by the same investigator on five consecutive
days. The correlation of indirect blood pressures with
direct femoral arterial pressure measurements in our
laboratory achieves a correlation coefficient of r=0.98
and is comparable to data reported previously.'3 Heart
rate (beats per minute) was calculated from the mean
RR interval derived from 10 consecutive cardiac cycles
of the indirect blood pressure tracing.
Metabolic Studies
Animals were maintained in individual cages with
minimal manipulation. Reported values represent the
mean of data obtained over 5 days and represent
steady-state values. As noted, drug doses were calculated individually to provide the same daily dose of the
drug based on the drinking behavior of each rat. Urine
was collected for determination of daily urine volume
(UV, ml * kg` * day-1), sodium excretion (UNa4V,
meq * kg`1 day-'), and potassium excretion (UKWV,
meq- kg`1 day-'), which were indexed to body weight.
Urinary sodium and potassium concentrations were
determined by flame photometry (Model 443, Instrumentation Laboratories, Inc., Lexington, Mass.). Blood
urea nitrogen (BUN) and serum creatinine concentrations (in milligrams per deciliter) were determined by
spectrophotometric methods via autoanalyzer (Ektachem 700 Clinical Analyzer, Eastman Kodak, Rochester, N.Y.).14
Biochemical Studies
Plasma renin concentration (PRC) was determined as
previously described.1'516 Serum and tissue ACE measurements were performed by a modified fluorimetric
method.'7,18 Baseline and treatment blood samples were
collected into chilled Eppendorf tubes and cold centrifuged for 5 minutes, and serum was then aliquoted for
either storage at -70C (baseline sample) or immediate
assay (sample at death). Baseline and treatment serum

ACE determinations from individual animals were performed within a single assay to minimize interassay
variability. Tissues were washed with cold potassium
phosphate buffer to remove any contaminating blood.
All tissues were assayed immediately the morning the
animal was killed to minimize possible dissociation of
each ACE inhibitor from ACE. Because we and others
have observed that captopril can dissociate ex vivo from
ACE during storage or during assay procedures,19 all
serum samples and tissue homogenates were kept ice
cold until incubation. Additional control rats were also
treated with either captopril (50 mg/kg) or vehicle by
gavage and were killed 1 hour later (synchronously with
chronically treated rats), and tissues were processed
concurrently to ensure sustained ex vivo inhibition of
ACE during assay. Each tissue was immediately homogenized and incubated for 10 minutes at 37C, pH 7.50, in
the presence of Hip-His-Leu; the reaction was stopped
by the addition of NaOH, pH 8.5. His-Leu product was
then tagged with 0.1% phthaldialdehyde and quantified
fluorometrically (SLM 8000C, SLM Instruments, Inc.,
Urbana, Ill.) at an excitation wavelength of 386 nm and
emission wavelength of 436 nm. This assay is linear
between 0.02 and 15 mM of His-Leu product.
RNA Studies
Renal tissue was snap-frozen in liquid nitrogen within
3 minutes and stored at -70C until it was studied.
Tissue homogenization and RNA extraction were performed as described.20 Comparisons of relative mRNA
levels were made in reference to the same amount of
total RNA applied per sample and quantified by absorbance at 260 nm in duplicate. For Northern blot analysis, aliquots of total RNA were run by the formaldehyde-agarose method.21
Gels were transblotted onto nylon filters by capillary
action with 10 SSC (1 SSC equals 0.15 M sodium
chloride, 0.015 M sodium citrate, pH 7.0) for 16 hours.
Efficiency of transfer was confirmed by ethidium bromide stain and examined before and after transfer.
RNA on nylon filters was crosslinked by UV light. For
additional quantification, slot blot hybridization analyses were performed. Samples were formaldehyde denatured and then serially diluted in 15 SSC. Three concentrations from each sample (8, 4, and 2 ,g) were
blotted in duplicate to nylon filters with a slot blot
apparatus (Schleicher and Schuell, Peterborough,
N.H.). After prehybridization for 4 hours, the blots were
hybridized overnight in a buffer to which a-2p complementary DNA (cDNA) probes were added.2'
After hybridization, blots were washed in 0.2 SSC
with 0.1% sodium dodecyl sulfate at room temperature
for 10 minutes, then three times at 65C for 30 minutes.
Blots were exposed for 5 days to x-ray film (Kodak
XAR, Eastman Kodak). All studies of angiotensinogen
mRNA were performed with a full-length rat liver
angiotensinogen cDNA (pRang 1650). For renin
mRNA determinations, we used renin cDNA pDD
1D-2, a full-length mouse submaxillary gland renin
cDNA that readily crosshybridizes with rat renal renin
mRNA.16 To control for possible sample variability,
identical slot blots were performed and hybridized with
a control (,8-actin) cDNA probe.

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Hirsch et al Tissue Converting Enzyme in Experimental Heart Failure

Quantification of mRNA
Autoradiograms generated by slot blots were scanned
with an LKB microdensitometer (Paramus, N.J.) with
background set to zero for each autoradiograph. Regression lines were calculated from the integral values
obtained by scanning the serial concentrations of each
sample. The relative signals of the specific mRNA were
estimated from the slope of the regression line, and only
values of r20.90 were accepted. Slopes of specific
mRNA for each condition were compared as relative
ratios. Sufficient material was available to study specimens with multiple sample applications. Our intrablot
and interblot coefficients of variation were 8% and 9%,
respectively.

Morphometric Analysis of Infarct Size

The left ventricle was sectioned transversely in at
least five planes, stained with Masson's trichrome and
hematoxylin/eosin, and projected for quantitative
planimetry of infarct size.22 The mean of the endocardial and epicardial scar circumferences was compared
with total left ventricular circumference to calculate
total infarct size (in percent). All infarctions were
transmural and involved only the free wall of the left
ventricle. Only rats with infarcts greater than 10% of the
left ventricular circumference were included in the
heart failure groups.

Statistical Analysis
Data are presented as mean+SEM. Mean slopes of
slot blot autoradiographs were compared by an unpaired Student's t test. Hemodynamic and plasma ACE
responses were compared by a Student's t test for paired
data. Intergroup variables (SO-V, HF-V, HF-C, and
HF-E) were compared by a randomized ANOVA with a
Newman-Keuls post hoc test for significant differences
between individual groups. Linear regression analysis
was performed to evaluate the relations between selected variables. Significance was accepted at the 95%
confidence limit (p<0.05).
Results
In our initial investigation, we determined equipotent
doses of captopril and enalapril during short-term (1week) treatment of HF rats based on the magnitude of
the hypotensive effect and degree of inhibition of serum

ACE. During the second protocol, the effects of chronic

(7-week) ACE inhibitor treatment with these drugs
were assessed in SO and HF rats to determine the
relevance of the tissue renin-angiotensin system to the
chronic hemodynamic and renal responses.

Protocol 1
In HF-V rats, there were no changes in blood pressure (129+2 versus 125+5 mm Hg, baseline versus
treatment, respectively; p=NS) or serum ACE activity
(89+10 versus 9115 nM * ml-l' min-m; p=NS) before
or during treatment (Table 1). At lower doses (10 and
25 mg . kg` day-'), captopril treatment caused a fall of
approximately 20 mm Hg in blood pressure in all rats
(p=NS between these two doses); the serum ACE
response was not assessed in these treatment subsets.
Captopril treatment at 100 and 200 mg - kg` . day-1
caused blood pressure to fall by an amount comparable

1569

TABLE 1. Dose-Titration Studies With Oral Captopril and

Enalapril in Heart Failure Rats

7-Day treatment
Baseline
BP
BP
Serum ACE
Inhibition
Group (mm Hg) (mm Hg) (nM * ml` * min-1)
(%)
129+2
V
125+5
9115
127+2 101+6*
C1o
NA
...
1233 103+4*
C25
NA
...
C100
122+5 1074*
508
45+9*
C200
1224 1053*
185
815t
E15
1195
926*
264
71+4t
E25
117+8 105+2*
15+4
84+4t
BP, tail cuff blood pressure; ACE, angiotensin converting enzyme 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`1 day` for 1 week. Percent
ACE inhibition during treatment was calculated as (baseline
serum ACE activity-treatment serum ACE activity)/(baseline
serum ACE activity). NA, not available. Data are presented as
mean+SEM.
*p<0.05 compared with baseline value; tp<0.005 compared
with baseline value.
to that observed at the lower two doses. Serum ACE
was inhibited by 45% and 81% at the 100- and 200mg - kg` * day-1 doses, respectively (each,p<0.005 compared with baseline).
Enalapril treatment for 7 days also caused a hypotensive response of 12-27 mm Hg, and a stepwise increase
in serum ACE inhibition was demonstrated. The magnitudes of the hypotensive and serum ACE inhibition
responses were comparable for captopril at 200
mg- kg`-l day`1 and for enalapril at 25 mg - kg' - day-1
(p=NS for both responses between drugs).

Protocol 2
At baseline, animals in each of the four study groups
were comparable with regard to age at operation (61
days), age at the initiation of treatment (108 days), and
age at the time of death (154 days). Baseline weights
and urinary volume and sodium and potassium excretion were also not different between groups (Table 2).
The mean infarct size in the total study group was
244%. SO rats had a mean infarct size of 3.00.3%.
The mean infarct sizes in the HF-V, HF-C, and HF-E
groups 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 Chronic
Treatment Group
HF-V HF-C
SO-V
HF-E
40411 394+13 38812 388+10
Weight (g)
UV (ml/day)
42+4
44+2
433
45+4
UNa+V (meq - kg BW-1 5.9+0.3 6.4+0.3 6.2+0.4 6.60.5

*day1)
UK+V (meq. kg BW`

10.20.5 10.6+0.5 9.90.7 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, and
enalapril, respectively; UV, daily urinary volume; UNaWV, daily
urinary sodium excretion; UK+V, daily urinary potassium excretion; BW, body weight. Data are presented as mean+SEM. No
intergroup dillerences achieved statistical significance.

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1570

Circulation Vol 86, No

November 1992

TABLE 3. Cardiac Weights in Each Chronic Treatment Group

HF-C
HF-E
SO-V
HF-V
LV, mg/kg BW 2.040.04 2.300.12 2.110.08 2.250.19
RV, mg/kg BW 0.470.02 0.550.06 0.580.06 0.640.07

SO-V, sham-operated rats treated with vehicle; HF-V, HF-C,

HF-E, heart failure rats treated with vehicle, captopril, and enalapril, respectively; LV, left ventricle; RV, right ventricle; BW, body
weight. No intergroup differences reached statistical significance.

comparable and did not differ statistically

(p=NS).
These observations are confirmed by the measurement of cardiac weights as displayed in Table 3. The left
ventricular and right ventricular weights were slightly
greater in the HF-V rats compared with the SO group,
but this did not reach statistical significance. This is
probably because of the smaller infarct size in the HF-V
animals. The left and right ventricular weights of rats
chronically treated with captopril or enalapril were not
significantly different from those of vehicle-treated
animals.
Figure 2 displays the blood pressure responses in
these groups at baseline and at the end of treatment. As
depicted, there was no change in the blood pressure of rats
in the SO-V and HF-V groups (from 1242 to 125+3
mm Hg in HF-V; from 1263 to 1292 mm Hg at baseline and during treatment in each group, respectively;
p=NS). There was a comparable hypotensive response of
12-18 mm Hg in the HF-C (from 1203 to 1023
mm Hg; p<0.01) and HF-E (from 1164 to 1042
mm Hg; p<0.01) groups from baseline to treatment. The
magnitude of the hypotensive response did not correlate
with 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 rate
did not change significantly in any of the treatment
groups: SO-V (from 417 7 to 4068 beats per minute),
HF-V (from 39410 to 40515 beats per minute),
HF-C (from 38812 to 37712 beats per minute), and
HF-E (from 38810 to 39216 beats per minute).
During vehicle treatment, there was no significant
change in the daily urine volume (Figure 3), urinary
sodium excretion, or urinary potassium excretion in SO
or HF rats (Table 4). Similarly, there was no change in
these responses in the HF-C rats. In contrast, HF-E
groups were

140.

o0o SO-V
*-* HF-V
A-A

-v0

Dl.

70-

HF-C

v-v HF-E

50-

30
Base

i
Rx

FIGURE 3. Graph showing the daily urine volume (UV.

ml* kg` * day-') excreted by rats at baseline and at the end of
treatment (Rx) in each group. SO-V, sham-operated rats
treated with vehicle; HF-V, HF-C, HF-E, heart failure rats
treated with vehicle, captopril, or enalapril, respectively.
**p<0.01 compared with baseline.

animals demonstrated a modest diuretic and natriuretic

response to treatment (Table 4 and Figure 3). Although
there was a tendency for the BUN to increase in the
ACE inhibitor-treated animals, this did not reach statistical significance. The serum creatinine was unchanged during treatment.
The PRC was not different in HF-V versus SO-V rats
(Table 5), as we have previously demonstrated4; treatment with captopril and enalapril caused PRC to rise by
comparable amounts compared with both vehicletreated control groups (p<0.01 for each). Treatment
with these two ACE inhibitors resulted in differential
effects on serum and tissue ACE activities. Serum ACE
activity (Figure 4) did not change during vehicle treatment in the SO and HF rats (serving as a time control).
During chronic enalapril treatment, serum ACE was
inhibited by approximately 55% compared with baseline
values. In contrast, there was no significant inhibition of
serum ACE in the HF-C animals after 47 days of
treatment. Table 6 displays the effects of captopril and
enalapril treatment on tissue ACE activities. Captopril
elicited a decrease in aortic ACE activity (31% inhibition), but this was not achieved during enalapril treatment. Both drugs elicited comparable decreases in renal
ACE activity (56% during captopril and 44% during
enalapril treatment, respectively). Captopril treatment
was associated with an induction in pulmonary ACE
activity, whereas enalapril treatment elicited a 33% fall
in ACE activity in the lung. Neither drug caused a

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

TABLE 4. Urine Electrolyte Excretion, Blood Urea Nitrogen,

and Serum Creatinine at End of Treatment

130

120-oCL110--

0-0

8
in * =

1(00-

90

so-v

HF-Y
HF-C
HF-E

Rx

FIGURE 2. Graph showing the baseline and treatment blood

pressures in sham-operated rats treated with vehicle
open circles) and heart failure rats treated with vehicle

(SO-V,
(HF-V,

closed circles), captopril (HF-C, upright triangles), or enalapril (HF-E, inverted triangles). Rx, end of treatment.
**p<0.01 compared with baseline.

SO-V
HF-V
UNa+V(meq.kgBW-1 4.9+0.1 5.3+0.1
* day 1)
9.3 +0.3 9.8+0.4
UK+V (meq * kg BW
* day 1)
BUN (mg/dl)
19+0.5 190.8
Creatinine (mg/dl)
0.50.01 0.50.01

HF-C
HF-E
5.10.2 5.8+0.4*
8.5 0.5 9.6+0.5

22+2
298
0.50.01 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, and
enalapril, 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<0.01
compared with SO-V.

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Hirsch et al Tissue Converting Enzyme in Experimental Heart Failure

TABLE 5. Effect of Treatment With Captopril vs. Enalapril on
Renal Angiotensinogen and Renal Renin Expression and Plasma
Renin Concentration
ANG mRNA
Renin mRNA
PRC (ng AI * ml-l hr-1)

SO-V
181
13710
7.5+1.0

HF-V
24+2*
10927
9.9+2.2

HF-C
HF-E
26+7* 16+2t
229+32* 24825*
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 enalapril, respectively; ANG, angiotensinogen; mRNA, messenger RNA;
PRC, plasma renin concentration; AI, angiotensin I mRNA data
presented as arbitrary densitometric unitsx 103. Data are presented
as mean+SEM.
*p<0.05 compared with SO-V; tp<0.05 compared with HF-V.

significant change in either right ventricular or interventricular septal ACE activities. Serum and tissues from
acutely gavaged rats, assayed concurrently with those
from chronically treated animals, demonstrated greater
than 85% inhibition compared with vehicle gavage.
The long-term hypotensive response (472 days of
oral treatment) to ACE inhibitor therapy did not correlate with the serum or aortic ACE activities during
treatment (r=0.08 and p=NS for serum ACE activity
and r=0.15 and p=NS for aortic ACE activity); a
moderate correlation was present between the treatment renal ACE activity and the hypotensive response
(r=0.48, pcO.05).
The intrarenal renin-angiotensin system was differentially affected by induction of heart failure as well as
by chronic ACE inhibitor treatment (Table 6). HF rats
demonstrated a 33% induction in renal angiotensinogen
expression compared with SO control rats (p<0.05).
Renin mRNA expression was unchanged in HF rats
compared with SO control rats. This is consistent with
the PRC results and confirms our previous data on rats
with chronic compensated heart failure. Chronic captopril treatment did not change the renal angiotensinogen
expression mRNA level but elicited an increase in renin
mRNA expression compared with HF-V rats (67% and
110% compared with SO-V and HF-V, respectively;
both p<0.05); PRC increased significantly (to 96+24 ng
AI ml-l hr-1; p<O.OS compared with HF-V) during
captopril treatment. In contrast, enalapril treatment

1571

TABLE 6. Effect of Treatment on Tissue ACE Activities in Heart

Failure Rats

HF-V
HF-C
HF-E
Aorta
0.350.05
0.240.06*
0.310.06
Kidney
0.320.03
0.14+0.03*
0.180.05*
Lung
61
102*
41*
RV
0.080.01
0.060.01
0.090.02
IVS
0.070.01
0.060.01
0.100.01
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 meanSEM.
*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 also
stimulated PRC (to 5820 ng AI * ml- *. hr- ; p< .05
compared with HF-V).

Discussion
It has been demonstrated that ACE inhibitors are
particularly effective in the treatment of severe heart
failure, in which the plasma renin-angiotensin system is
activated. Recent data also demonstrate that ACE
inhibitors are effective in mild-to-moderate congestive
heart failure and in left ventricular dysfunction without
overt heart failure.5-8.23 Because these conditions are
characterized by normal plasma renin activity,2,24 the
mechanisms underlying the efficacy of ACE inhibitors
remain poorly understood. We and others have hypothesized that these agents might modulate tissue reninangiotensin system activity and thereby beneficially alter end-organ physiological responses.
The coronary ligated rat model of experimental heart
failure is characterized by a stable compensated lowoutput state in association with regional blood flow
redistribution, renal sodium avidity, and increased mortality.6'25 In this model, as in chronic, stable heart
failure, plasma renin activity and serum ACE activity
are normal in the compensated state4,26; nevertheless,
ACE inhibition normalizes regional blood flow, induces
natriuresis, and improves survival.6,2728 Thus, the physiological and neurohormonal status of this model simulates those of human chronic heart failure. This model
also demonstrates tissue-specific activation of cardiac
ACE in proportion to the size of the myocardial
infarction.4
1;ca~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
The present investigation contributes to our under100 .I
standing of the role of tissue ACE in heart failure by
demonstrating that 1) the hypotensive response to
chronic ACE inhibition is not predicted by effects on
IN.
circulating ACE activity; 2) the magnitude of chronic
50tissue ACE inhibition is not predicted by short-term
O so-V
*-* HF-V
serum ACE inhibition; 3) chronic treatment may elicit
25- A-A HF-C
the induction of tissue and serum ACE activities; and 4)
n-v
chronic treatment by these two commonly used ACE
inhibitors can result in differential responses of the
Bane
intrarenal renin-angiotensin system, which may influFIGURE 4. Graph showing the serum ACE activity reence sodium and water excretion.
sponses in sham-operated rats treated with vehicle (SO-V,
Blood Pressure Response
open circles) and heartfailure rats treated with vehicle (HF-V,
closed circles), captopril (HF-C, upright triangles), or enalaDuring chronic (7-week) treatment, captopril and
pril (HF-E, inverted triangles). Rx, end of treatment.
enalapril caused blood pressure to fall comparably in
HF rats. The magnitude of the hypotensive response at
**p<0.01 compared with baseline.
'zo-

75-

0-

HF-E

Rx

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1572

Circulation Vol 86, No 5 November 1992

the target doses (captopril, 200 mg. kg`1day-l and

enalapril, 25 mg * kg`1 day-') was approximately equal
during 1 week and 7 weeks of treatment. It is notable
that this dose of captopril is approximately the same as
that used in the chronic survival studies performed by
Pfeffer et a16 (2 g/l). Nevertheless, the magnitude of
inhibition of serum ACE during short-term (1-week)
treatment was substantially greater than that observed
after chronic treatment. Both captopril and enalapril
elicited an 85% fall in serum ACE activity during 1
week of treatment. In contrast, despite comparable
hypotensive effects, only enalapril elicited a sustained
inhibition of serum ACE activity. Thus, serum ACE
activity was a poor predictor of the systemic hemodynamic response.
The magnitude of the hypotensive effect was not
predicted by the severity of heart failure as assessed by
the size of the infarcted myocardium. Additionally, the
magnitude of inhibition of serum and vascular (aortic)
ACE activity did not correlate with the fall in blood
pressure in HF rats. Previous investigations have also
observed that the magnitude of serum ACE inhibition
poorly predicts the hypotensive response in normotensive and hypertensive animal models.29-34 Chronic treatment with both enalapril and captopril has been shown
to cause induction of total circulating ACE activity;
therefore, this lack of correlation is not unexpected.35-37
In our study, the long-term blood pressure response
correlated best with renal ACE activity. The nature of
this relation is not known. It is interesting to speculate
that the reduction in renal ACE activity results in
reduced intrarenal angiotensin II concentration and
enhanced sodium excretion. Thus, the long-term effect
on plasma volume may determine the blood pressure
response with chronic therapy.

Chronic ACE Inhibitor Treatment: Effects on the

Intrarenal Renin-Angiotensin System
The present study demonstrates differing patterns of
renal angiotensinogen expression and sodium and volume excretion during treatment with captopril and
enalapril. We have recently reported from a separate
investigation that chronic experimental heart failure in
this model causes a tissue-specific induction of renal
angiotensinogen expression.10 The present study demonstrates that the increased renal angiotensinogen
mRNA level is normalized by chronic enalapril treatment but is not affected by long-term captopril therapy.
Renal renin mRNA expression and plasma renin concentration during treatment with these two drugs were
comparable. Because the blood pressure was not different between the two treatment groups, differences in
intrarenal mechanisms of action of these two drugs are
presumed to underlie these different renal angiotensinogen responses. The nature of these differences in
intrarenal action is unclear at this time.
The physiological consequences of intrarenal angiotensin production may be extrapolated from the results
of the present study. HF-C rats displayed a lower daily
excretion of both sodium and water than HF-E rats.
Because renal perfusion pressure (arterial pressure)
was not different between the two drug treatment
groups, an intrarenal effect must differentiate these
markedly disparate renal responses; such an intrarenal
effect may be the level of angiotensin II. Because the

12

0 so-v
* HF-V

9.

'ia

~z
+o

0
00

Cp eO

00
3-

10

10-

A&HF-C
v

20

5( D

40

30

HF-E

8E

B~~~~~~~
A

+5

10
20
30
40
ANG mRNA (Arbitrary D.nmltom*IM Unit X 10

50

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

magnitude of renal ACE inhibition was comparable

between these two treatment groups, and because renin
was present in excess, we speculate that angiotensinogen levels may determine the rate of intrarenal angiotensin synthesis. Intrarenal angiotensin II has been
shown to stimulate sodium and water retention via a
direct proximal tubular effect. The relation between
angiotensinogen mRNA levels and renal sodium excretion is displayed in Figure 5. As shown, there was no
correlation between the angiotensinogen mRNA level
and sodium excretion during vehicle treatment (r=0.09,
p=NS). In contrast, drug-treated HF rats displayed a
fairly striking inverse relation, as increased angiotensinogen expression was associated with lower sodium excretion (r= -0.43,p<0.05). Although such a correlation
does not delineate either a cause-and-effect mechanism
or a common cofactor that directionally alters both
angiotensinogen expression and sodium excretion, this
finding provides an initial clue to suggest that such links
exist. It should be noted that the present 24-hour
urinary sodium measurements determined during the
final week of treatment may not reflect cumulative
changes in chronic sodium balance that likely occur in
this model with chronic treatment with either ACE
inhibitor.
These data should also be interpreted in the context
of the study of Packer et a138 that demonstrated that
enalapril resulted in a higher BUN and serum creatinine than captopril under the conditions of their experiments. In the present study, we did not observe a

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Hirsch et al Tissue Converting Enzyme in Experimental Heart Failure

difference in renal function between the two drug

treatments under the conditions of our experiment.
Unlike Packer et al, we did not observe a difference in
systemic blood pressures between the two drugs, as
expected from the design of our protocol. We did,
however, observe a difference in angiotensinogen
mRNA expression that may reflect a shorter intrarenal
duration of action of captopril. Because we have shown
that intrarenal angiotensin II directly stimulates angiotensinogen mRNA expression, the higher renal angiotensinogen mRNA in the captopril rats is consistent
with a lesser reduction in angiotensin II in the kidney.
Because angiotensin II can also directly stimulate proximal tubule sodium reabsorption, this differential effect
on intrarenal angiotensin may also explain the differences in urinary sodium excretion between the two
drugs.

Differential Effects of Captopril and Enalapril

on Tissue ACE
It has previously been recognized that ACE inhibitors
have differential effects on the inhibition of tissue
ACE.39 Previous studies have reported a decrease in
cardiac hypertrophy and the prevention of ventricular
enlargement with ACE inhibition.44' Chronic ACE
inhibitor treatment in this study did not decrease either
total left or right ventricular mass, however, despite
prolonged drug therapy. This may not be surprising, in
that ACE inhibitors were not initiated until 47 days
after myocardial infarction. Anversa et a142 have shown
that cardiac myocyte hypertrophy is an early response to
injury, occurring within 3 days of infarction. Additionally, it is interesting that cardiac ACE activity in this
study did not appear to be suppressed by either captopril or enalapril. This is probably because of the induction of cardiac ACE during concomitant ACE inhibition.4 Although both agents may inhibit cardiac ACE
when administered as a single dose in spontaneously
hypertensive rats, the chronic responses in normotensive experimental models have not been previously

studied.
Aortic ACE activity was inhibited by captopril but not
enalapril in HF rats. Thus, one might speculate that
captopril would have greater effects on vascular compliance or hypertrophy than enalapril at these administered doses. Although these would be potentially important end-organ effects, these vascular functional
responses were not specifically studied in these
protocols.
Equipotency: Serum versus Tissue Responses
In clinical trials, the effects of various ACE inhibitors
on clinical endpoints have been assessed by titration of
drug doses based on the systemic hemodynamic response. This study demonstrates that hemodynamic
equipotency cannot always be correlated with equipotent effects at the tissue level. Whereas captopril and
enalapril caused differing patterns of tissue ACE inhibition, it is likely that one might achieve comparable
tissue ACE inhibition with alternative doses of these
agents. Additionally, the duration of treatment by each
agent may cause variable induction of serum and tissue
ACE activities. Until better methods of assessing the
magnitude of this induction are available, true drug
equipotency may be difficult to define. Finally, the

1573

magnitude of penetration and/or binding of these

agents to tissue ACE is difficult to quantify.

Study Limitations
This investigation examined primarily tissue ACE
activities; it examined renin and angiotensinogen only in
the kidney. It was not realistic to study every component
of this system in all tissues. Present techniques do not
permit localization of the cell type(s) at which ACE is
inhibited. Tissue angiotensin II concentrations were not
determined. Concentrations of angiotensin II in tissue
homogenates are notoriously difficult to quantify because of the presence of other non-ACE tissue peptidases and crossreactivity of most antibodies to angiotensins I, II, and III. Thus, the magnitude of effect of these
drugs on angiotensin II biosynthesis must be inferred
from surrogate markers (e.g., rate-limiting components
of the system). In addition, ACE inhibitors may alter
the synthesis and metabolism of kinins and prostaglandins, which may contribute to their beneficial end-organ
effects.
Finally, whereas these data describe the effect of
these ACE inhibitors at selected doses in a relevant
small-animal model of heart failure, these findings
should be extrapolated to clinical investigation cautiously. Small-animal pharmacological investigations are
often characterized by greater drug doses on a milligram
per kilogram basis. The effect of clinically appropriate
doses of ACE inhibitors on human tissue ACE is not
known.
Conclusions
These findings demonstrate that circulating and tissue ACE levels are probably altered (induced) by
chronic drug therapy. Despite comparable acute hemodynamic and serum ACE inhibitory effects, chronic
captopril and enalapril treatment elicit quite different
patterns of inhibition of tissue ACE in experimental
heart failure; this finding has relevance in the design of
future clinical or experimental investigations that attempt to compare these or other agents of this class.
The beneficial effects of ACE inhibitors are probably a
result of the interplay of multiple mechanisms of action
of these agents on target end-organs in heart failure and
other pathophysiological states. Some of these effects
may be attributed to the ability of ACE-inhibitors to
alter the expression or activity of tissue renin-angiotensin systems.

Acknowledgments
The 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 Resource
Center for animal care; and Donna MacDonald for preparation of the manuscript.

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

experimental heart failure.
A T Hirsch, C E Talsness, A D Smith, H Schunkert, J R Ingelfinger and V J Dzau
Circulation. 1992;86:1566-1574
doi: 10.1161/01.CIR.86.5.1566
Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
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