Circulation. 1995;92:950-961
(Circulation. 1995;92:950-961.)
© 1995 American Heart Association, Inc.
Beneficial Effects of Inhibition of Angiotensin-Converting Enzyme on Ischemic Myocardium During Coronary Hypoperfusion in Dogs
Masafumi Kitakaze, MD;
Tetsuo Minamino, MD;
Koichi Node, MD;
Kazuo Komamura, MD;
Yoshiro Shinozaki, BS;
Hidezo Mori, MD;
Hiroaki Kosaka, MD;
Michitoshi Inoue, MD;
Masatsugu Hori, MD;
Takenobu Kamada, MD
From The First Department of Medicine (M.K., T.M., K.N., K.K., M.H.,
T.K.), Osaka University School of Medicine, Osaka; Department of Physiology
(Y.S., H.M.), Tokai University School of Medicine, Isehara; Department of
Physiology (H.K.), Osaka University School of Medicine, Osaka; and Department
of Information Science (M.I.), Osaka University School of Medicine, Osaka,
Japan.
Correspondence to Masafumi Kitakaze, MD, PhD, The First Department of
Medicine, Osaka University School of Medicine, 2-2 Yamadaoka, Suita 565,
Japan.
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Abstract
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Background Angiotensin-converting enzyme (ACE)
produces angiotensin
II, causing vasoconstriction of
coronary arteries and reduction
of coronary blood flow.
The present study was undertaken to
test the hypothesis that an ACE
inhibitor increases coronary
blood flow and
improves myocardial metabolic and contractile
functions of
ischemic myocardium.
Methods and Results In 65 open-chest dogs, the left anterior
descending coronary artery was perfused through an
extracorporeal bypass tube from the left carotid artery. When
cilazaprilat (3 µg/kg per minute) was infused into the bypass tube
for 10 minutes after reduction of coronary blood flow due to
partial occlusion of the bypass tube, coronary blood flow
increased from 30±1 to 43±2 mL/100 g per minute despite there
being
no changes in coronary perfusion pressure (43±1 mm Hg). The
ratio of myocardial endocardial flow to epicardial flow increased
during an infusion of cilazaprilat. Both fractional shortening and
lactate extraction ratio of the perfused area were increased
(fractional shortening: 4.1±0.6% to 8.9±0.6%,
P<.001;
lactate extraction ratio: -55.7±3.3% to -36.7±3.9%,
P<.001). During an infusion of cilazaprilat, the bradykinin
concentration of coronary venous blood was markedly increased.
The increased coronary blood flow due to cilazaprilat was
attenuated by HOE-140 (an inhibitor of bradykinin
receptors; coronary blood flow: 35±2 mL/100 g per minute), and
by N
-nitro-L-arginine methyl
ester (an inhibitor of nitric oxide synthase;
coronary blood flow: 34±2 mL/100 g per minute).
Intracoronary administration of bradykinin mimicked the
beneficial effects of cilazaprilat. Cyclic GMP content of the
coronary artery was increased by cilazaprilat compared with the
untreated condition in the ischemic myocardium. In
the denervated hearts, the increased coronary blood flow due to
cilazaprilat was not attenuated. On the other hand, CV11974, an
inhibitor of angiotensin II receptors, slightly
increased coronary blood flow to 34±2 from 30±1 mL/100 g per
minute.
Conclusions We conclude that an inhibitor of ACE can
increase coronary blood flow and ameliorate myocardial
ischemia, primarily due to accumulation of bradykinin and
production of nitric oxide from the ischemic
myocardium. Inhibition of angiotensin II
production due to inhibition of ACE partially contributes to
coronary vasodilation in the ischemic
myocardium.
Key Words: angiotensin-converting inhibitor • nitric oxide • cilazaprilat • myocardial ischemia • bradykinin
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Introduction
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Angiotensin-converting enzyme (ACE) is
known to produce angiotensin
II,
1 which may
cause potent coronary vasoconstriction. In ischemic
hearts,
ACE inhibitors may increase coronary blood
flow (CBF) and attenuate
the extent of myocardial ischemia
through the inhibition of
receptors of angiotensin
II.
2 Furthermore, ACE inhibitors are
reported
to inhibit the degradation of bradykinin by inhibiting
kininase
II.
3 4 Because bradykinin mediates the generation
of
nitric oxide (NO) through B
2 receptor
activation
5 6 7 8 and
bradykinin directly causes
coronary vasorelaxation via B
1 receptor
activation,
9 ACE inhibitors may further
increase CBF.
10 According to one report,
11 in
the renal artery an ACE inhibitor
mediates renal
vasodilation through both inhibition of angiotensin
II
production and activation of bradykinin B
2
receptors. However,
there is no clear consensus as to whether and how
ACE inhibitors
modify CBF and affect the myocardial
contractile and metabolic
functions in ischemic
hearts.
Thus, in the present study, to test the effect of an ACE
inhibitor on myocardial ischemia, we infused
cilazaprilat into the coronary artery during coronary
hypoperfusion and measured CBF and regional myocardial contractile and
metabolic functions. Furthermore, to examine the
possibility that this beneficial effect of cilazaprilat is attributable
to increases in NO release through accumulation of bradykinin, we
observed the changes in CBF and the regional contractile and
metabolic functions during HOE-14012 and
N-nitro-L-arginine methyl ester
(L-NAME) treatments.13 Finally, to investigate the
cellular mechanism of this phenomenon, we measured cyclic GMP of the
coronary smooth muscles with and without the administration of
cilazaprilat during myocardial ischemia.14
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Methods
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Instrumentation
Sixty-five mongrel dogs weighing 14 to 23 kg
were used. In six
dogs,
5 days before the experimental instrumentation, systemic chemical
sympathectomy
was performed by intravenous
injection of 50 mg/kg
6-hydroxydopamine.
15 16 17 Deleterious
side
effects of 6-hydroxydopamine were prevented
by
intravenous injections of propranolol (1 mg/kg)
and phentolamine
(1 mg/kg). Three doses of
6-hydroxydopamine (10, 20, and 20
mg/kg) were
administered separately over a period of 24
hours.
16 17
These dogs were anesthetized with
pentobarbital sodium (30
mg/kg IV). The trachea was intubated, and the animal was ventilated
with room air mixed with oxygen (1 L/min). The chest was opened through
the left fifth intercostal space, and the heart was suspended in a
pericardial cradle. The left anterior descending coronary
artery (LAD) was cannulated and perfused with blood via the left
carotid artery through an extracorporeal bypass tube. Coronary
perfusion pressure (CPP) was monitored at the tip of the
coronary arterial cannula, and CBF of the perfused
area was measured with an electromagnetic flow probe attached at the
bypass tube. A small, short collecting tube (1-mm diameter and 7-cm
length) was inserted into a small coronary vein near the center
of the perfused area to sample coronary venous blood. The
drained venous blood was collected in the reservoir placed at the level
of the left atrium and was returned to the jugular vein. High-fidelity
left ventricular (LV) pressure was measured with a
micromanometer (Konigsberg P-5) placed in the LV
cavity through the apex. A pair of ultrasonic crystals were placed in
the inner third of the myocardium 
1 cm apart to measure
myocardial segment length with an ultrasonic dimension gauge (5 MHz,
2-mm diameter; Schuessler). In five dogs, an electromagnetic flow probe
was attached at the root of the ascending aorta to measure cardiac
output.
Experimental Protocols
Protocol 1: Effects of
Cilazaprilat on Myocardial Ischemia
Produced by Coronary Hypoperfusion
Forty-one dogs were used in this
protocol. First, in 13 dogs,
after hemodynamic stabilization, coronary
arterial and venous blood samples were taken for blood gas
analysis and determination of lactate,
norepinephrine, and bradykinin concentrations and plasma
ACE activity. Hemodynamic parameters, ie,
LV pressure (LVP), dP/dt, and segment length of the perfused area, were
measured. End-diastolic length (EDL) was determined at the
R wave of the ECG, and end-systolic length (ESL) was determined at the
minimal dP/dt.18 Fractional shortening (FS) was calculated
by (EDL-ESL)/EDL as an index of myocardial
contractility of the perfused area. In 5 of 13 dogs,
cardiac output was measured. With an occluder attached at the
extracorporeal bypass tube, CPP was reduced so that CBF decreased to
one third of the control CBF. After a low CPP was determined, the
occluder was adjusted exactly to keep CPP constant at the low level.
All hemodynamic parameters were measured 3,
5, 7, and 10 minutes after the onset of hypoperfusion, and both
coronary arterial and venous blood for the
metabolic parameters were sampled at 10
minutes. After these measurements, cilazaprilat (3 µg/kg per minute;
Nippon Roche KK) was infused into the LAD, and all
hemodynamic and metabolic
parameters were measured again. Ten minutes later,
cilazaprilat infusion was discontinued, and the
hemodynamic and metabolic
parameters were obtained when hemodynamic
parameters were stabilized (n=13). For assessment of the
ratio of endocardial flow to epicardial flow (endo/epi flow ratio),
microspheres were injected before and 10 (ischemia
without cilazaprilat), 20 (ischemia with cilazaprilat), and 30
minutes (ischemia after withdrawal of cilazaprilat) after the
onset of coronary hypoperfusion. In the preliminary study, we
tested four dosages of cilazaprilat (0.33, 1, 3, and 9 µg/kg per
minute IC) during coronary hypoperfusion (n=3 in each dosage in
3 dogs). In the groups receiving cilazaprilat 0.33 to 3 µg/kg per
minute, CBF increased to 31±1, 37±2, and 42±2 from
29±2 mL/100 g
per minute during coronary hypoperfusion, and 9 µg/kg per
minute cilazaprilat increased CBF to 41±2 mL/100 g per minute.
Therefore, we decided to perform the experiments using 3 µg/kg per
minute cilazaprilat.
Second, to test that the effect of cilazaprilat is
related to
activation of the B2 receptors of bradykinin (n=6), we
infused cilazaprilat into the LAD during hypoperfusion during an
intracoronary infusion of HOE-140 (0.5 ng/kg per minute), a
selective antagonist of bradykinin B2
receptors. The administration of HOE-140 was initiated 10 minutes
before the coronary hypoperfusion.
Third, to test the direct effect of
bradykinin on ischemic
myocardium, we infused bradykinin instead of cilazaprilat
into the LAD during hypoperfusion. During coronary
hypoperfusion, all hemodynamic parameters
were measured 3, 5, 7, and 10 minutes after the onset of hypoperfusion,
and both coronary arterial and venous blood for the
metabolic parameters were sampled at 10
minutes. After these measurements, bradykinin (20 ng/kg per minute) was
infused into LAD, and all hemodynamic and
metabolic parameters were measured 10 minutes
later. After the measurements, bradykinin infusion was discontinued,
and the hemodynamic and metabolic
parameters were obtained when hemodynamic
parameters were stabilized (n=5).
Fourth, since bradykinin is
reported to increase the release of NO, we
tested the idea that cilazaprilat increases NO and thus CBF via the
activation of bradykinin receptors (n=8). Cilazaprilat was infused into
the LAD during hypoperfusion during intracoronary infusion
of L-NAME (3 µg/kg per minute), an inhibitor of NO
synthase, to inhibit release of NO. Administration of L-NAME was
initiated 10 minutes before coronary hypoperfusion.
Fifth, in six other
denervated dogs, the identical procedures and
measurements of all variables were performed in experiments with
cilazaprilat. We confirmed that the norepinephrine
concentrations in the myocardium in systemically denervated
(n=5) and innervated control (n=5) dogs were 21±6 and
398±21 pg/mg (P<.001), respectively.
Protocol 2: Effects of an Antagonist of
Angiotensin II Receptors on Myocardial Ischemia
Produced by Coronary Hypoperfusion
Nine dogs were used in this
protocol. After
hemodynamic stabilization, coronary
arterial and venous blood were sampled for blood gas
analysis and determination of lactate and
norepinephrine concentrations. Hemodynamic
functions were measured as in protocol 1. With an occluder attached at
the extracorporeal bypass tube, CPP was reduced so that CBF decreased
to one third of the control CBF. After a low CPP was determined, the
occluder was adjusted exactly to keep CPP constant at the low level.
All hemodynamic parameters were measured 3,
5, 7, and 10 minutes after the onset of hypoperfusion. After these
measurements, CV11974 (10 µg/kg per minute; Takeda Pharmaceutical Co)
was infused into the LAD, and all hemodynamic
parameters were measured again (n=6). Ten minutes later,
CV11974 infusion was discontinued, and hemodynamic
parameters were obtained when they were stabilized. In the
preliminary study, we tested the effects of 3.3, 10, and 33 µg/kg per
minute of CV11974 on CBF during coronary hypoperfusion. Three
dosages of CV11974 increased CBF to 31±1, 35±1, and 34±2
from 29±1
mL/100 g per minute (n=3 for each dosage) with constant CPP
(CPP=42±1
mm Hg). Thus, we decided to use 10 µg/kg per minute of CV11974 in
this protocol.
Protocol 3: Effects of Cilazaprilat on
Coronary Circulation
in the Nonischemic Myocardium
To examine the effects of cilazaprilat
on CBF in the normoxic
condition, we infused three dosages of cilazaprilat (1, 3, and 9
µg/kg per minute) into five dogs. After the
hemodynamic stabilization, CBF was measured before and
during infusion of each dose of cilazaprilat. Coronary
arterial and venous blood were sampled for the measurement
of bradykinin concentration.
Protocol 4: Effects of
Cilazaprilat on Cyclic GMP Content of
Epicardial Coronary Artery in Ischemic
Hearts
We tested whether cilazaprilat increases cyclic GMP content of
the coronary artery in the ischemic
myocardium. With an occluder attached at the extracorporeal
bypass tube, CPP was reduced so that CBF decreased to one third of the
control CBF. After a low CPP was determined, the occluder was adjusted
to keep CPP constant at the low level. After the low CPP was maintained
for 10 minutes, cilazaprilat (3 µg/kg per minute) was infused into
the LAD for 10 minutes, and we rapidly removed the epicardial LAD
(ischemic region) and left circumflex coronary artery
(nonischemic control region) (n=5) with the use of
precooled stainless steel scissors and tongs. We rapidly stored samples
in liquid nitrogen. In five other dogs, CPP was reduced so that CBF
decreased to one third of the control CBF for 20 minutes, and the
epicardial LAD (ischemic region) and left circumflex
coronary artery (nonischemic control region) were
rapidly removed and stored in liquid nitrogen.
Measurements of Regional Coronary Blood Flow
Regional
myocardial blood flow was determined by the
microsphere technique as previously reported19
with the use of nonradioactive microspheres (Sekisui Plastic
Co) made of inert plastic labeled with different types of stable heavy
elements as described in detail previously.16 In the
present study, microspheres labeled with Nb, Br, Zr, and I
were used. The mean diameter was 15 µm, and specific gravity was 1.32
for Nb, 1.34 for Br, 1.36 for Zr, and 1.60 for I. Microspheres
were suspended in isotonic saline with 0.01% Tween 80 to prevent
aggregation. The microspheres were ultrasonicated for 5 minutes
followed by 5 minutes of voltexing immediately before injection.
Approximately 1 mL of the microsphere suspension (2 to
4x105 spheres) was injected into the left atrium followed
by several warm (37°C) saline flushes (5 mL).
The x-ray
fluorescence activity of stable heavy elements was
measured by a wavelength dispersive spectrometer (PW 1480, Phillips
Co). Specification of this x-ray fluorescence spectrometer has
been described previously. In brief, when the microspheres are
irradiated by the primary x-ray beam, the electrons fall back to a
lower orbit and emit measurable energy. This energy level of the x-ray
fluorescence depends on the characteristics for each element.
Therefore, it was possible to qualify the x-ray fluorescence of
several differently labeled microspheres in the mixture. The
endo/epi flow ratio was calculated as the ratio of each tissue content
normalized with the wet weight of the sampled
myocardium.
Chemical Analysis
M
O2 (mL/100 g per
minute) was
calculated as: CBF (mL/100 g per minute)xcoronary
arterial and venous blood oxygen difference (mL/dL).
Lactate was assessed by the enzymatic assay, and lactate extraction
ratio (LER) was obtained by multiplying the coronary
arteriovenous difference in lactate concentration by 100 and dividing
by arterial lactate concentration.
Norepinephrine Measurement
The method of norepinephrine measurement has been
described previously.20 Five milliliters of
coronary arterial and venous blood taken into a
tube containing EDTA was immediately placed in ice water and
centrifuged for 20 minutes. The plasma was kept at -80°C.
Within 2 weeks, plasma norepinephrine was adsorbed on
alumina and separated by high- performance liquid
chromatography (HPLC) (pump, LC-3A; column Zpax-SCX;
Shimazu Seisakusho Co). Plasma norepinephrine was
determined spectrofluorometrically by the trihydoxyindole (THI) method
(spectrofluorophotometer RF-500LCA, Shimazu). In this system,
sensitivity of the assay is 10 pg/mL plasma and the intra-assay
coefficient of variation is 6.8%.20
Bradykinin
Measurement
The method of bradykinin measurement has been described
previously.21 One milliliter of blood withdrawn from the
sampling tube was squirted rapidly into siliconized polyethylene tubes
containing 4 mL of 96% ethanol, which was centrifuged at
2500g at 4°C for 15 minutes. The supernatant was decanted
into a siliconized 250-mL round-bottomed flask. The precipitate was
resuspended in 20 mL 75% ethanol and recentrifuged. This
supernatant was combined with the previous supernatant. After 0.5 mL of
octanol was added to prevent frothing, the ethanol was removed, and the
volume was reduced to 2 mL through evaporation at 60°C under the
reduced pressure. The residual solution was acidified with 5 mL of 0.01
mol/L HCl and partitioned twice with 20 mL of diethyl ether. This
procedure was performed in the same flask as the original evaporation,
with the ether supernatant being removed by suction after each
partitioning. The aqueous phase remaining in the flask after the ether
extractions was subsequently reduced to dryness using a rotary
evaporator. These dried samples were stored at -80°C before assay.
The dried samples were redissolved in 2.5 mL of 0.1 mol/L Tris-HCl
buffer containing 0.2% gelatin, 0.1% neomycin, and 0.01 EDTA,
adjusted to pH 7.4. The incubation mixture for radioimmunoassay
consisted of 0.1 mL of 0.01 mol/L 1,10-phenanthroline HCl, diluent
buffer of 0.5 mL containing the unknown or standard bradykinin, 0.1 mL
antiserum diluted 1:600 with diluent buffer, and 0.1 mL
(125I-Try8)-bradykinin (approximately 8000 cpm)
dissolved in normal saline. It was incubated in a polyethylene tube at
4°C for 24 hours, and dextran-coated charcoal was used to separate
the free labeled antigen from that bound to antibody. Three replicate
tubes containing only buffer, phenanthroline, and
(125I-Try8)-bradykinin were incubated and
treated with coated charcoal to determine the amount of labeled antigen
that remained in the supernatant in the absence of antibody. The mean
value of this measurement was subtracted from supernatant radioactivity
after centrifugation of the antibody-containing tubes,
and the resultant value was used to calculate the proportion of label
bound to antibody.
Cyclic GMP Measurement
The
method of cyclic GMP measurement in tissue has been
previously described.22 After removal of the adventitial
connective tissues in the coronary arteries (20 to 40 mg), the
frozen tissue was powdered, homogenized at 4°C in 1 mL of
ice-cold 6% trichloroacetic acid, and centrifuged at
2500g for 20 minutes. The supernatant fluid was removed,
extracted three times with 3 mL of diethyl ether saturated with water,
and stored in the freezer (-80°C). Cyclic GMP concentration in the
supernatant fluid was measured by the radioimmunoassay
method22 within 7 days. Briefly, we used 100 µL of
dioxane-triethylamine mixture containing succinic acid anhydride
succinylated cyclic GMP in the supernatant (100 µL). After a
10-minute incubation, the reaction mixture was added to 800 µL of 0.3
mol/L imidazole buffer (pH 6.5). One hundred microliters of succinyl
cyclic GMP tyrosine methyl ester iodinated with
125I (15 000 to 20 000 cpm in <10-14 mol/L)
was added to the assay mixture containing 100 µL of the supernatant
and 100 µL of diluted antisera in the presence of chloramine
T22 ; the mixture was kept at 4°C for 24 hours. A cold
solution of dextran-coated charcoal (500 µL) was added to the mixture
in an ice-cold water bath. The charcoal was spun down, and 0.5 mL of
the supernatant was counted for radioactivity in a gamma spectrometer.
The amount of cyclic GMP was normalized by protein content of
coronary artery assayed by the Lowry
method.23
Measurement of Plasma ACE Activity
The method of measurement of the plasma ACE activity has been
described previously.24 Three milliliters of
coronary arterial and venous blood taken into a
polyethylene tube was immediately placed in ice water and
centrifuged for 20 minutes. The plasma was kept at -80°C.
Within 7 days, plasma ACE activity was assessed by measuring the
production of hippuric acid from the substrate (Hip-His-Leu).
The concentration of hippuric acid was measured by
colorimetric methods24 using HPLC with a
Model Tri-Rotor (Japan Spectroscopic Co).
Statistical Analysis
Statistical analysis was performed with
paired
t tests adjusted by the modified Bonferroni's multiple
comparison.25 26 All values were expressed as
mean±SEM,
with P<.05 considered statistically significant.
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Results
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Table 1
depicts systolic and diastolic
blood pressures and heart
rate for all groups. Before and during
coronary hypoperfusion
with and without pharmacological
interventions did not affect
these systemic hemodynamic
parameters. In the denervated dogs,
although systolic and
diastolic blood pressures were not different,
baseline
heart rate was reduced compared with the cilazaprilat
group.
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Table 1. Sequential Changes in Systolic and
Diastolic Pressures and Heart Rate Before, During, and
After 90 Minutes of Ischemia
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Effects of Inhibition of ACE on Myocardial
Ischemia
Table 2 shows the changes in CPP, CBF, and the
bradykinin concentration of coronary arterial and
venous blood during infusions of cilazaprilat (0.33, 1, 3, and 9
µg/kg per minute) in the nonischemic condition.
Intracoronary infusions of 0.33, 1, and 3 µg/kg per
minute of cilazaprilat did not change CBF but slightly increased the
bradykinin concentration of coronary venous blood at 3 µg/kg
per minute of cilazaprilat. An intracoronary infusion of 9
µg/kg per minute of cilazaprilat slightly increased CBF with a
further increment of the bradykinin concentration.
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Table 2. Changes in Coronary Perfusion Pressure,
Coronary Blood Flow, and Coronary Arteriovenous
Differences in Bradykinin Concentrations With and Without
Administration of Cilazaprilat in the
Nonischemic Condition
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Table
3 shows the coronary
hemodynamic and metabolic
parameters before the reduction of CPP. There were no
significant differences in the baseline coronary
hemodynamic and metabolic
parameters of the nonischemic condition.
Neither L-NAME nor HOE-140 altered the coronary
hemodynamic and metabolic
parameters. In the denervated myocardium,
MO2 in the baseline
condition was
significantly reduced compared with the baseline condition in the
cilazaprilat group. Fig 1 shows the effect of
cilazaprilat on CBF and FS during coronary hypoperfusion. After
an abrupt reduction of CPP, CBF decreased from 91±2 to 30±1
mL/100 g
per minute, and FS decreased within 1 minute keeping a steady state for
10 minutes (5.1±0.6%). Within 1 minute, once CPP was set, CPP was
kept constant at 43±1 mm Hg. After the initiation of an
intracoronary infusion of cilazaprilat, both CBF and FS
gradually increased. Ten minutes after the onset of the cilazaprilat
infusion, both CBF and FS increased to 43±2 mL/100 g per minute and
8.9±0.6%, respectively, indicating that an intracoronary
infusion of cilazaprilat increases both CBF and FS of the
ischemic myocardium. Table 4 depicts
cardiac output before and during coronary hypoperfusion.
Cardiac output was reduced due to coronary hypoperfusion, and
cilazaprilat administration increased cardiac output during
coronary hypoperfusion. Systemic vascular resistance (SVR) was
decreased due to coronary hypoperfusion; however, SVR did not
change regardless of whether cilazaprilat was administered during
coronary hypoperfusion. Fig 2 shows the endo/epi
flow ratio during coronary hypoperfusion with and without
administration of cilazaprilat. Before the reduction in CPP, endo/epi
flow ratio was 1.17±0.07. The endo/epi flow ratio decreased to
0.74±0.03, and administration of cilazaprilat significantly increased
endo/epi flow ratio to 0.81±0.01. This result indicates that
cilazaprilat increases CBF in the endocardium more than the epicardium.
This beneficial effect of cilazaprilat was demonstrated from the aspect
of myocardial metabolism. Fig 3 shows the
changes in LER, MO2, and
coronary arterial and venous concentrations of
norepinephrine during ischemia with and without
cilazaprilat. Cilazaprilat increased LER, indicating that myocardial
anaerobic metabolism was improved by the
cilazaprilat infusion, and thus
MO2
increased. There were no significant differences in
norepinephrine concentrations in the coronary
venous blood. Norepinephrine release (coronary
arteriovenous differences in norepinephrine multiplied by
CBF [ng/100 g per minute]) in the control condition and in the
ischemic myocardium before, during, and after
administration of cilazaprilat were 2.44±0.83, 1.12±0.75,
0.71±1.45,
and 1.07±1.03 ng/100 g per minute, respectively. There were no
significant differences in norepinephrine release among
these four conditions. pH in coronary venous blood was
7.38±0.01 at baseline and decreased (P<.001) to
7.22±0.02
during coronary hypoperfusion. However, the cilazaprilat
infusion improved (P<.005) pH in coronary venous
blood (7.31±0.01). pH of coronary arterial blood
did not change throughout this study (7.40±0.02). There were no
significant differences in plasma ACE activity in the coronary
arterial blood in the control condition and in the
ischemic myocardium before, during, and after
administration of cilazaprilat (5.6±0.9, 5.8±0.8,
5.6±0.9, and
5.4±0.8 IU/L, respectively). Plasma ACE activities in the
coronary venous blood in the control and ischemic
myocardium were 5.5±0.9 and 5.9±0.5 IU/L, respectively.
Cilazaprilat administration markedly reduced plasma ACE activity in the
coronary venous blood to 0.2±0.1 IU/L during coronary
hypoperfusion (P<.001), and plasma ACE activity returned to
5.0±1.0 IU/L 10 minutes after withdrawal of cilazaprilat
infusion.
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Table 3. Coronary Hemodynamic and
Metabolic Parameters Before and After HOE-140
and L-NAME Administrations in the Nonischemic Hearts
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Figure 1. Plots of changes in coronary perfusion
pressure (top), coronary blood flow (middle), and fractional
shortening (bottom) during the intracoronary infusion of
cilazaprilat and withdrawal of cilazaprilat infusion during
coronary hypoperfusion. From 3 to 5 minutes after cilazaprilat
infusion, coronary blood flow was gradually increased and
fractional shortening recovered, indicating that
intracoronary infusion of cilazaprilat improves mechanical
myocardial function during ischemia. During this protocol,
reduced coronary perfusion pressure was kept constant.
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Table 4. Sequential Changes in Systemic
Hemodynamic Parameters With and Without
Cilazaprilat Administration Before and During
Coronary Hypoperfusion
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Figure 2. Plot of endothelium-to-epithelium
(endo/epi [End/Epi]) flow ratio during coronary hypoperfusion
with and without administration of cilazaprilat. After the onset of
myocardial ischemia, endo/epi flow ratio was reduced from
1.17±0.07 (nonischemic control condition) to 0.74±0.03.
Cilazaprilat significantly increased endo/epi flow ratio during
ischemia.
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Figure 3. Plots of changes in lactate extraction ratio (n=13),
myocardial oxygen consumption
(MO2) (n=13), and
norepinephrine (NE) concentrations in the coronary
arterial (A, n=8) and venous blood (V, n=8) before, during,
and after intracoronary infusions of cilazaprilat.
Intracoronary infusions of cilazaprilat significantly
improved lactate extraction ratio, indicating that the cilazaprilat
infusion improves metabolic function of ischemic
myocardium. With this improvement in metabolic
function, myocardial oxygen consumption also increased. Cilazaprilat
did not affect NE concentrations in the coronary
arterial and venous blood.
|
|
Mechanisms by Which Cilazaprilat Increases CBF and Improves
Myocardial Ischemia
Fig 4 shows the coronary
arteriovenous
differences in the concentration of bradykinin during coronary
hypoperfusion with and without cilazaprilat administration. In the
nonischemic condition, the coronary arteriovenous
difference in the bradykinin concentration was -2.1±2.5 pg/mL. In
the
ischemic condition, the coronary arteriovenous
difference in the bradykinin concentration increased, and
administration of cilazaprilat further increased the coronary
arteriovenous difference in the bradykinin concentration. Fig 5
shows the effect of bradykinin on CBF and FS during
coronary hypoperfusion. After an abrupt reduction of CPP
associated with CBF, FS decreased within 1 minute, keeping a steady
state for 10 minutes. Within 1 minute, once CPP was set, it was kept
constant. Ten minutes after the onset of the bradykinin infusion, both
CBF and FS increased to 46±4 mL/100 g per minute and 9.7±1.0%,
respectively, indicating that an intracoronary infusion of
bradykinin can increase both CBF and FS of the ischemic
myocardium. Fig 6 shows the changes in LER,
MO2, and coronary
arterial and venous concentrations of
norepinephrine during ischemia with and without
bradykinin administration. Bradykinin increased LER, indicating that
myocardial anaerobic metabolism was improved by
the bradykinin infusion, and thus
MO2
increased. There were no significant differences in
norepinephrine concentrations in the coronary
venous blood. There also were no differences between
norepinephrine release in the control condition and in the
ischemic myocardium before, during, and after
administration of bradykinin (2.10±0.98, 1.40±0.93,
1.73±1.39, and
1.38±0.98 ng/100 g per minute, respectively).
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Figure 4. Plot of coronary arteriovenous differences
in bradykinin during coronary hypoperfusion with and without
cilazaprilat administration. Cilazaprilat increased the
coronary arteriovenous differences for bradykinin.
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Figure 5. Plots of changes in coronary perfusion
pressure (top), coronary blood flow (middle), and fractional
shortening (bottom) during the intracoronary infusion of
bradykinin and withdrawal of bradykinin infusion during
coronary hypoperfusion. During this protocol, reduced
coronary perfusion pressure was kept constant.
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Figure 6. Plots of changes in lactate extraction ratio,
myocardial oxygen consumption
(MO2), and
norepinephrine (NE) concentrations in the coronary
arterial (A) and venous (V) blood before, during, and after
intracoronary infusions of bradykinin.
Intracoronary infusions of bradykinin significantly
improved lactate extraction ratio, indicating that the bradykinin
infusion improves metabolic function of ischemic
myocardium. With this improvement in metabolic
function, myocardial oxygen consumption was also increased. Bradykinin
did not affect the norepinephrine concentrations in the
coronary arterial and venous blood.
|
|
Fig
7 shows that these beneficial effects of
cilazaprilat are antagonized by concomitant treatment with HOE-140.
Intracoronary infusion of cilazaprilat during
administration of HOE-140 slightly increased both CBF and FS during
coronary hypoperfusion with the constant low CPP. The increases
in CBF (12.8±1.1 versus 2.8±0.9 mL/100 g per minute) and FS
(4.8±0.2% versus 0.7±0.2%) were attenuated by 78% and 85%
relative to the group of the cilazaprilat administration (Fig
1
). Fig 8 shows the changes in LER,
MO2, and coronary
arterial and venous concentrations of
norepinephrine during ischemia with and without
cilazaprilat during administration of HOE-140. Cilazaprilat slightly
increased LER. There were no significant differences in
MO2 and norepinephrine
concentrations in the coronary arterial and venous
blood. There also were no differences between
norepinephrine release in the control condition with and
without HOE-140 administration (1.63±1.13 and 1.95±0.69 ng/100 g
per
minute, respectively) and in the ischemic
myocardium before, during, and after administration of
cilazaprilat (0.79±0.41, 0.25±0.47, and 1.33±0.25 ng/100
g per
minute, respectively). pH in the coronary venous blood was
7.37±0.01 at baseline and decreased to 7.22±0.01 during
coronary hypoperfusion. However, the cilazaprilat infusion did
not improve pH in coronary venous blood (7.24±0.01). These
results indicate that HOE-140 attenuates the beneficial effects of
cilazaprilat on myocardial ischemia by 80%, indicating that
the beneficial effect of cilazaprilat is mainly attributable to the
accumulation of bradykinin in the ischemic
myocardium.
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Figure 7. Plots of changes in coronary perfusion
pressure (top), coronary blood flow (middle), and fractional
shortening (bottom) during the intracoronary infusion of
cilazaprilat and withdrawal of cilazaprilat infusion in
coronary hypoperfusion during HOE-140 treatment. Although
coronary blood flow and fractional shortening increased
slightly due to administration of cilazaprilat, the extent of increase
in coronary blood flow and fractional shortening was attenuated
in the cilazaprilat-only group (Fig 1). During this protocol,
reduced
coronary perfusion pressure was kept constant.
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Because bradykinin is reported to increase NO production in
endothelial cells, we tested whether L-NAME attenuates
the beneficial effects of cilazaprilat in the ischemic
myocardium. Fig 9 shows that L-NAME
treatment attenuates the cilazaprilat-induced increases in CBF
(12.8±1.4 versus 4.0±0.7 mL/100 g per minute) and FS
(4.8±0.2%
versus 1.2±0.4%) during the constant low CPP by 69% and 75%,
respectively. Fig 10 shows the changes in LER,
MO2, and coronary
arterial and venous concentrations of
norepinephrine during coronary hypoperfusion with
and without cilazaprilat during treatment with L-NAME. Cilazaprilat
slightly increased LER and
MO2. There
were no significant differences in norepinephrine
concentrations in the coronary arterial and venous
blood. There also were no differences between
norepinephrine release in the control condition with and
without L-NAME administration (3.01±1.99 and 2.97±1.03 ng/100 g
per
minute, respectively) and in the ischemic
myocardium before, during, and after administration of
cilazaprilat (1.75±0.62, 2.63±0.69, and 1.55±0.72 ng/100
g per
minute, respectively). pH in coronary venous blood was
7.38±0.01 at baseline and decreased to 7.21±0.01 during
coronary hypoperfusion. However, the cilazaprilat infusion did
not improve the pH in coronary venous blood (7.25±0.02). These
results indicate that the beneficial effect of cilazaprilat is mostly
attributable to augmentation of NO release due to the bradykinin
accumulation. To examine whether NO increases CBF of the
ischemic myocardium by its direct coronary
vasodilatory action or by attenuation of norepinephrine
release, we tested the effects of cilazaprilat in the denervated
ischemic myocardium. Fig 11 shows
the effect of cilazaprilat on CBF and FS during coronary
hypoperfusion in the denervated myocardium. After an abrupt
reduction of CPP associated with CBF, FS decreased within 1 minute
keeping a steady state for 10 minutes. Ten minutes after the onset of
the cilazaprilat infusion, both CBF and FS increased to the levels in
the cilazaprilat group in the innervated control
myocardium (Fig 1). Fig 12 shows the
changes in LER, MO2, and
coronary arterial and venous concentrations of
norepinephrine during ischemia with and without
cilazaprilat in the denervated myocardium. Cilazaprilat
increased LER, indicating that myocardial anaerobic
metabolism was also improved in the denervated
myocardium, and thus MO2
increased. There were no significant differences in
norepinephrine concentration in the coronary venous
blood. There also were no differences between
norepinephrine release in the control condition and in the
ischemic myocardium before, during, and after
administration of cilazaprilat (1.50±2.14, 0.49±0.33,
1.61±2.63, and
1.41±0.52 ng/100 g per minute, respectively).
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Figure 11. Plots of changes in coronary perfusion
pressure (top), coronary blood flow (middle), and fractional
shortening (bottom) during the intracoronary infusion of
cilazaprilat and withdrawal of cilazaprilat infusion during
coronary hypoperfusion in the denervated
myocardium. After cilazaprilat infusion, coronary
blood flow was gradually increased and fractional shortening recovered,
indicating that intracoronary infusion of cilazaprilat
improves mechanical myocardial function during ischemia in the
denervated heart. During this protocol, reduced coronary
perfusion pressure was kept constant.
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Figure 12. Plots of changes in lactate extraction ratio,
myocardial oxygen consumption
(MO2), and
norepinephrine (NE) concentrations in the coronary
arterial (A) and venous (V) blood before, during, and after
intracoronary infusions of cilazaprilat in the denervated
myocardium. Intracoronary infusions of
cilazaprilat significantly improved lactate extraction ratio,
indicating that the cilazaprilat infusion improves
metabolic function of ischemic denervated
myocardium. With this improvement of metabolic
function, myocardial oxygen consumption was also increased.
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The remaining 20%
to 30% of the beneficial effects of cilazaprilat
may be attributable to the inhibition of angiotensin II
receptors. Therefore, we tested whether inhibition of
angiotensin II receptors can account for the remaining 30%
of the beneficial effect of cilazaprilat. Fig 13 shows
that CV11974 increases CBF and FS by 4.0±0.5 mL/100 g per minute and
1.7±0.3%, respectively, despite the constant low CPP. CV11974
increased LER from -52.5±4% to -43.2±2.2%
(P<.05) but
did not change MO2
(1.6±0.1 to
1.7±0.1 mL/100 g per minute). CV11974 did not alter the concentrations
of norepinephrine in the coronary
arterial (from 393±21 to 404±20 pg/mL) and venous blood
(from 417±11 to 411±16 pg/mL). There also were no differences
between
norepinephrine release in the control condition and in the
ischemic myocardium before, during, and after
administration of CV11974 (2.79±2.05, 0.80±1.05,
2.84±1.00, and
0.71±0.83 ng/100 g per minute, respectively).
Table
5 represents the cellular basis of the
interaction between cilazaprilat and NO. Without cilazaprilat
treatment, myocardial ischemia (CPP: 105±4 to 42±2 mm Hg,
CBF: 82±3 to 27±2 mL/100 g per minute) increased cyclic GMP
content
of the coronary artery from 69±5 to 126±7 fmol/mg protein
(P<.01). However, treatment with cilazaprilat during
myocardial ischemia further increased (P<.01)
cyclic GMP content of the involved coronary artery to 272±16
fmol/mg protein.
|
Discussion
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|---|
In the present study, we showed that an inhibition of ACE
causes
coronary vasodilation and improves contractile and
metabolic
functions in the ischemic heart, mainly
through accumulation
of bradykinin and NO release and partially through
inhibition
of activation of angiotensin II receptors.
However, before reaching
a conclusion, we should consider several
possibilities of the
effect of an inhibitor of
ACE.
27
Validity of the Experimental Model in the Present
Study
The major assumption in all of the experimental protocols in the
present study was that intracoronary infusion of
chemicals, such as cilazaprilat, L-NAME, HOE-140, and CV11974, does not
have any effects on peripheral vessels, and the observed
changes in the LAD area were due only to local effects on the
coronary vasculature. If pharmacological interventions to the
LAD area also affect systemic hemodynamics, the
beneficial effects of cilazaprilat may be secondary to the systemic
vascular effects such as afterload reduction. However, in the
present study, systolic and diastolic blood pressures
and heart rate did not change during any pharmacological interventions
(Table 1), suggesting that pharmacological interventions to the
LAD
area minimally affect the systemic hemodynamic
parameters in the present study. Furthermore, when
intracoronary administration of 3 µg/kg per minute
cilazaprilat markedly reduced plasma ACE activity in the
coronary venous blood, this administration of cilazaprilat did
not affect the plasma ACE activity in the coronary
arterial blood. Indeed, intracoronary
cilazaprilat administration did not affect SVR (Table 4). These
observations strengthen the idea that the beneficial effects of
cilazaprilat are attributable to the local coronary vascular
and myocardial changes rather than the systemic
hemodynamic changes.
Coronary Vasodilation due to Inhibition of ACE in the
Ischemic Hearts
ACE inhibitors inhibit the accumulation of
angiotensin II and accumulate bradykinin in the
myocardium. First, because angiotensin II is
reported to promote the release of norepinephrine from the
presynaptic vesicles, ACE inhibitors may decrease the
release of norepinephrine from the presynaptic
vesicles,28 and the subsequent withdrawal of
-adrenoceptor activation in the ischemic
myocardium would cause coronary vasodilation.
However, in our experiment there is evidence that cilazaprilat during
coronary hypoperfusion does not alter the
norepinephrine concentration in the coronary venous
blood (Fig 2), and the beneficial effects of cilazaprilat were
not
blunted in the denervated ischemic myocardium (Figs 11 and
12). These observations suggest that withdrawal of sympathetic
nerve activity is not likely for the mechanisms of the coronary
vasodilation in the ischemic myocardium.
Second, angiotensin II also
directly constricts the
coronary smooth muscles,2 which may constitute the
mechanisms for coronary vasodilation in the ischemic
heart. However, CV11974, an inhibitor of
angiotensin II receptors, increased CBF only by 20% to
30% of cilazaprilat-induced increases in CBF, suggesting that
inhibition of angiotensin II accumulation is not the major
factor.
Third, because our results revealed that bradykinin
accumulation due to
cilazaprilat administration is a major factor for coronary
vasodilation and improvement of myocardial contractile and
metabolic functions in the ischemic
myocardium, the direct coronary vasodilation due to
bradykinin may be a primary factor. Bradykinin is reported to relax
vascular smooth muscles,9 and our data revealed that
bradykinin increases CBF during coronary hypoperfusion and
improves ischemic myocardium.
Fourth, bradykinin-induced coronary
vasodilation may be
involved in increases in NO
production5 6 7 8 or
accumulation of prostacyclin.29 The present study
revealed that L-NAME, an inhibitor of NO synthase,
attenuates the cilazaprilat-induced coronary vasodilation to
the same extent as HOE-140, indicating that the coronary
vasodilation due to bradykinin during administration of cilazaprilat is
attributable to NO accumulation. Because NO is known to be a potent
coronary
vasodilator,30 31 32 33 it is
likely that
NO-induced coronary vasodilation is a major cause for the
cilazaprilat-induced coronary vasodilation in the
ischemic myocardium. Furthermore, coronary
arterial content of cyclic GMP, which is increased by NO
via guanylate cyclase activation, was increased by
administration of cilazaprilat during coronary
hypoperfusion.
Aside from the NO-induced coronary vasodilation, NO can
inhibit
platelet aggregation and neutrophil adherence to coronary
vasculature.34 35 36 If this is the case,
increased CBF may
be attributable to the inhibition of progressive platelet
aggregation and neutrophil adherence. However, this may be unlikely,
because even during administration of L-NAME, both CBF and FS remained
constant at low levels during coronary hypoperfusion without
progressive decreases, suggesting that progressive platelet
aggregation and adherence of neutrophils may not occur in the
ischemic hearts even without cilazaprilat in the present
experimental protocols. NO accumulation due to an
intracoronary infusion of cilazaprilat also may open
functional collateral vessels to the ischemic area, which may
reduce the severity of ischemia. We cannot negate this
possibility; however, increases in CBF measured at the bypass tube
suggest that forward flow into the coronary artery is
essentially increased due to cilazaprilat. Taken together, cilazaprilat
increases CBF in the ischemic heart through NO
production via augmentation of accumulation of bradykinin.
Role of Bradykinin for Coronary Vasodilation in the
Ischemic Heart
Results of the present study also indicate that
endo/epi flow
ratio during myocardial ischemia is increased due to
administration of cilazaprilat, suggesting that bradykinin accumulation
or NO production due to bradykinin is more prominent in the
endocardium than the epicardium. In the kidney, the formation of kinins
is reported to be H+ dependent,37 suggesting
that H+ may increase bradykinin concentration in the
tissues. If this is the case in the ischemic heart, endocardium
may produce H+ more than epicardial myocardium
because epicardial flow is slightly less than the endocardium, although
myocardial oxygen consumption in the endocardium is more than in the
epicardium. Bradykinin accumulation measured in the coronary
venous effluent in the ischemic myocardium was more
than twice that in the nonischemic myocardium
when cilazaprilat was administered in the coronary artery. This
difference in the bradykinin accumulation may be attributable to the
differences in H+ accumulation in the
myocardium. Furthermore, it has been reported that
Ca2+ concentration in the endocardium is higher than
that in the epicardium, and ischemia elevates myocardial
Ca2+ concentrations.38 The rise in
Ca2+ may affect release of bradykinin and activity
of NO synthase, which may account for the higher endocardial flow and
higher accumulation of bradykinin in the ischemic
myocardium. In addition, activation of
1-adrenoceptor activation produces a >40-fold increase
in the rate of kallikrein secretion, and the kinin output in the venous
effluent from the submandibular gland becomes 700-fold,39
suggesting that 1-adrenoceptor stimulation, which occurs
during myocardial ischemia, augments the increases in
bradykinin accumulation.
Although our results indicate that coronary
arteriovenous
differences in bradykinin concentrations are increased (Table 1
and Fig 4), this extent of increases in bradykinin may
minimally affect
hemodynamic parameters, because it is
reported that several nanograms per milliliter of bradykinin are
necessary to affect the hemodynamic
parameters.40 However, bradykinin is very
fragile and degrades rapidly in the blood, suggesting that bradykinin
concentration near the vessel wall may be very high to cause
substantial vasodilation. During administration of cilazaprilat in the
nonischemic myocardium (Table 1), although
release of bradykinin was increased, CBF did not increase at 3 µg/kg
per minute cilazaprilat. When CPP varies in the range of
coronary flow autoregulation, coronary vasodilatory or
constrictive response may be overwhelmed by other vasoactive
substances. Another possible explanation for the discrepancy between
the concentrations of bradykinin and CBF in the nonischemic
myocardium may be the shape of their dose-response
relation.
Clinical Implications
It has been reported that ACE
inhibitors are effective
for attenuation of infarct size after myocardial ischemia and
reperfusion.41 42 Martorana et
al41 42
reported that ACE inhibitors attenuate infarct size from
55% to 25%. Furthermore, ACE inhibitors are effective for
promoting remodeling of ventricle after the acute myocardial
infarction, which prevents enlargement of the ventricle.43
The present study helps to clarify the role of bradykinin in the
beneficial effects of ACE inhibitors in the canine
experimental model of ischemic heart disease, although further
basic and clinical research must be performed to investigate the
possible use of ACE inhibitor in the treatment of effort
angina.
|
Acknowledgments
|
|---|
This work was supported by Scientific Research Grant-in-Aid
03670449
from the Ministry of Education, Science, and Culture, Japan.
The
authors gratefully acknowledge Noriko Tamai, Yoshitomo Edahiro,
and
Shinya Suzuki for their technical assistance.
Received January 4, 1995;
accepted February 7, 1995.
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Top
Abstract
Introduction
