Journal of the American College of Cardiology
© 2001 by the American College of Cardiology
Published by Elsevier Science Inc.
Vol. 38, No. 5, 2001
ISSN 0735-1097/01/$20.00
PII S0735-1097(01)01582-0
An Inhibitor of Inducible Nitric Oxide
Synthase Decreases Forearm Blood Flow
in Patients With Congestive Heart Failure
Yutaka Ishibashi, MD,* Toshio Shimada, MD,* Yo Murakami, MD,* Nobuyuki Takahashi, MD,*
Takeshi Sakane, MD,* Takashi Sugamori, MD,* Shuzo Ohata, MD,* Shin-ichi Inoue, MD,*
Yoko Ohta, MD,* Ko Nakamura, MD,* Hiromi Shimizu, MD,* Harumi Katoh, MD,*
Michio Hashimoto, MD†
Izumo, Japan
The functional activation of inducible nitric oxide synthase (iNOS) was evaluated as a source
of nitric oxide (NO) in the forearm of patients with heart failure.
BACKGROUND Although endogenous NO is normally produced by constitutive NO synthase (cNOS) in
patients with congestive heart failure (CHF), expression of iNOS provides an additional
source of NO. However, there are no in vivo studies showing functional activation of iNOS
in humans.
METHODS
A nonselective NOS inhibitor, NG-monomethyl-L-arginine (L-NMMA), and a selective
inhibitor of iNOS, aminoguanidine, were administered intra-arterially in graded doses into
the brachial arteries of 13 patients with CHF and 10 normal control subjects. Forearm blood
flow (FBF) was measured simultaneously in the infused and noninfused arms by plethysmography. Arterial and venous plasma concentrations of nitrite/nitrate (NOx) were measured
at baseline and at the highest dose of each drug.
RESULTS
L-NMMA significantly reduced the FBF ratio between the infused and noninfused arms in
both the control and patient groups (35 ⫾ 12% and 34 ⫾ 10%, respectively; both p ⬍ 0.001).
Aminoguanidine at the same concentration significantly reduced the ratio in the patient
group (15 ⫾ 9%, p ⬍ 0.01), with no change in the control group. The arterial NOx
concentration was not affected by either drug; however, venous NOx concentrations were
significantly decreased in both the control and patient groups by L-NMMA (18 ⫾ 5% and
18 ⫾ 17%, respectively; both p ⬍ 0.05) and in the patient group only by aminoguanidine (7 ⫾
6%, p ⬍ 0.05).
CONCLUSIONS These findings suggest that NO production in the forearms of patients with CHF is induced
partly by iNOS activation, whereas in normal subjects, it can be ascribed to cNOS activation.
(J Am Coll Cardiol 2001;38:1470 – 6) © 2001 by the American College of Cardiology
OBJECTIVES
Endothelium-derived nitric oxide (NO) plays a pivotal role
in the regulation of the vasomotor tone of the peripheral
vessels. In patients with congestive heart failure (CHF),
agonist-induced, NO-mediated vasodilation in response to
muscarinic stimulation is impaired in the peripheral circulation (1–3), indicating endothelial dysfunction. In contrast,
conflicting results variously show that basal production and
release of NO decreases (4), remains normal (5) or increases
(6 – 8) in patients with CHF, as compared with normal
subjects. In a recent study, we demonstrated that the
different results of these previous studies might be related to
differences in the severity of heart failure, and that basal NO
production is enhanced in patients with the most severe
heart failure (9). Nitric oxide is synthesized from L-arginine
by NO synthases (NOS), a family of isoenzymes with
distinct functional, biochemical and regulatory properties
(10,11). Two of these enzymes are calcium-dependent
constitutive NO synthases (cNOS): endothelial NOS and
neuronal NOS. The third enzyme, calcium-independent
From the *Fourth Department of Internal Medicine and the †Department of
Physiology, Shimane Medical University, Izumo, Japan.
Manuscript received February 12, 2001; revised manuscript received June 18, 2001,
accepted July 12, 2001.
inducible NO synthase (iNOS), is capable of producing
large amounts of NO once induced by mediators such as
endotoxin and cytokines, resulting in depressed myocardial
contractility (12) and exercise intolerance (13). Clinical in
vitro studies have suggested that enhanced NO production
in patients with CHF is caused by iNOS (14 –20). However, no in vivo studies have been conducted in humans.
Analogues of L-arginine, such as NG-monomethyl-Larginine (L-NMMA) and NG-nitro-L-arginine methyl ester (L-NAME), inhibit NOS and have proved to be
valuable in investigating the role of endogenous NO (11).
These inhibitors are nonselective and cause similar inhibition of cNOS and iNOS. In contrast, aminoguanidine, a
nucleophilic hydrazine compound, has been shown to be a
stronger inhibitor of iNOS than of cNOS, with 10- to
100-fold selectivity (21,22).
The present study is a comparative study between a
nonselective NOS inhibitor, L-NMMA, and a selective
iNOS inhibitor, aminoguanidine, in which we examined the
question of whether iNOS contributes to NO production
and peripheral circulation in patients with CHF. Furthermore, we examined the effects of aminoguanidine on
endothelium-derived, NO-mediated vasodilation by using
JACC Vol. 38, No. 5, 2001
November 1, 2001:1470–6
Abbreviations and Acronyms
ANP
⫽ atrial natriuretic peptide
BNP
⫽ B-type natriuretic peptide
CHF
⫽ congestive heart failure
cNOS
⫽ constitutive nitric oxide synthase
FBF
⫽ forearm blood flow
iNOS
⫽ inducible nitric oxide synthase
L-NAME ⫽ NG-nitro-L-arginine methyl ester
L-NMMA ⫽ NG-monomethyl-L-arginine
NO
⫽ nitric oxide
NOS
⫽ nitric oxide synthase
Nox
⫽ nitrite/nitrate
TNF
⫽ tumor necrosis factor
acetylcholine to identify the selectivity of iNOS inhibition,
because the reported selectivity of this drug has been
controversial (23,24).
METHODS
Subjects. Thirteen patients admitted to our hospital with
CHF due to idiopathic dilated cardiomyopathy and 10 ageand gender-matched healthy control volunteers were enrolled in this study. The patient group included eight men
and five women (mean age 56 ⫾ 18 years [range 28 to 78
years]). The control group included six men and four
women (mean age 52 ⫾ 22 years [range 28 to 78 years])
who showed no abnormalities on physical examination,
electrocardiography, chest radiography or routine blood
testing, including fasting blood sugar and serum cholesterol.
Current smokers and subjects who had smoked within two
years were excluded from the study.
All of the patients underwent cardiac catheterization to
measure cardiac hemodynamic variables and subsequently
underwent left ventriculography and coronary angiography.
All coronary angiograms were normal. The mean left
ventricular ejection fraction obtained by left ventriculography was 26 ⫾ 12%. The cardiac index was 2.22 ⫾ 0.79
l/min/m2, and the mean pulmonary capillary wedge pressure
was 21 ⫾ 5 mm Hg. As for the clinical severity of heart
failure, the patients were in New York Heart Association
functional classes I to III. The durations of medication use
were ⬎3 years (to 7 years) in 4 patients, ⬎1 year in 7
patients and ⬍1 year (to 6 months) in 2 patients. All of the
patients had been clinically stable for at least two weeks
before the study, with no signs of pulmonary congestion or
peripheral edema. Background therapies were digoxin (n ⫽
3), diuretics (n ⫽ 11), angiotensin-converting enzyme
inhibitors (n ⫽ 9), nitrates (n ⫽ 5), beta-blockers (n ⫽ 5),
calcium antagonists (n ⫽ 4) and anticoagulants (n ⫽ 5). All
medications were stopped on the day of the study.
All patients and control subjects gave written, informed
consent to participate in the study, which was approved by
the Human Subjects Research Committee of Shimane
Medical University Hospital.
Ishibashi et al.
Effects of iNOS Inhibitor on Forearm Blood Flow
1471
Procedures. All participants were instructed to abstain
from eating food and drinking caffeinated beverages for at
least 12 h before the study. The study was performed with
the patient in the supine position in an air-conditioned
room at a temperature of 25 to 26°C. Forearm blood flow
(FBF; ml/min/100 ml forearm tissue volume) was determined in the dominant and nondominant arms by venous
occlusion plethysmography, as described elsewhere (9).
Briefly, under local anesthesia with 1% lidocaine, the brachial artery of the nondominant arm (the left arm) was
cannulated with a 22-gauge polyethylene catheter (RA4122, Arrow Inc., Reading, Pennsylvania) for blood pressure monitoring, blood sampling and drug infusion. A
24-gauge polyethylene catheter was placed in the basilica
vein for venous blood sampling. The arm was slightly
elevated above the level of the right atrium, and a mercuryfilled silicone strain gauge was placed in the widest part of
the forearm. The strain gauge was connected to a Hokanson
EC-5R Plethysmograph (Hokanson Inc., Bellevue, Washington) that was calibrated to measure the percent change in
volume, and this was connected to a chart recorder to record
the flow measurements. For each measurement, a cuff
placed around the upper arm was inflated to 40 mm Hg
with a rapid cuff inflator (E-10, Hokanson Inc.) to occlude
venous outflow from the extremity. A wrist cuff was inflated
to suprasystolic pressure 1 min before each measurement to
exclude the hand circulation. Flow measurements were
recorded for 5 s every 15 s, and four readings were obtained
for each mean value. Systemic blood pressure was measured
by a cuff sphygmomanometer placed on the contralateral
arm, and arterial pressure on the side of the FBF measurements was continuously monitored with a Life Scope 12
polygraph (Nihonkoden Inc., Tokyo, Japan).
Protocol. After a 20-min rest period, blood samples were
obtained for measurements of baseline plasma atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP)
levels.
ENDOTHELIUM-DEPENDENT VASODILATION. To compare
endothelium-dependent vasodilation between the control
and CHF groups, a dose-response curve was obtained
during infusion of four doses of acetylcholine (2.5, 5, 10 and
20 ␮g/min) into the nondominant brachial artery. Each
dose was infused for 4 min, and FBF was measured during
the last 2 min.
RESPONSES OF FBF TO AMINOGUANIDINE AND L-NMMA.
After completion of the measurements taken during incremental infusion of acetylcholine, the study participants
rested until FBF returned to baseline (⬃40 min). Then, to
assess the effects of NOS inhibitors on FBF, aminoguanidine (Clinalfa Co., Läufelfingen, Switzerland) was first
infused into the nondominant brachial artery (2.5, 5, 10 and
20 ␮mol/min). After completion of FBF measurements at
each dose for the first drug, a rest period of ⬃60 min was
allowed for FBF to return again to baseline, after which the
FBF response to L-NMMA (Clinalfa Co.) was tested (2.5,
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Ishibashi et al.
Effects of iNOS Inhibitor on Forearm Blood Flow
5, 10 and 20 ␮mol/min). Each dose was infused for 7 min,
and FBF was measured during the last 2 min. Arterial and
venous blood sampling for measurement of the plasma
nitrite/nitrate (NOx) concentration was performed at baseline and just before FBF measurement at the highest dose of
each drug. The NOx level was measured with an highperformance liquid chromatography system (ENO-10,
Eicom, Japan), using anion-exchange chromatography,
which is described in detail elsewhere (25). The detectable
lower limit was 0.3 pmol for both nitrite and nitrate.
EFFECTS OF AMINOGUANIDINE AND L-NMMA ON
ENDOTHELIUM-DEPENDENT VASODILATION. To test the
magnitude and selectivity of the selective iNOS and nonselective NOS inhibitors used in the present study, a
dose-response curve with acetylcholine (2.5, 5, 10 and 20
␮g/min) was obtained during co-infusion of saline, aminoguanidine or L-NMMA in an additional five healthy
control volunteers. The infusions of saline, aminoguanidine
(20 ␮mol/min) and L-NMMA (20 ␮mol/min) were started
5 min before the administration of the first dose of acetylcholine and were continued during each dose-response
curve study. Each dose of acetylcholine was infused for
4 min, and FBF was measured during the last 2 min.
Statistical analysis. The ratio of flow between the infused
and noninfused arms was calculated for each time point and
expressed as the percent change from baseline. Data are
expressed as the mean value ⫾ SD, unless otherwise
indicated. Intergroup differences were analyzed by use of the
chi-square test or unpaired t test for baseline characteristics,
except for ANP, BNP and NOx levels. Kruskall-Wallis
analysis of variance (ANOVA), followed by the Scheffé’s
post hoc test, was used to compare the nonparametric
variables of ANP, BNP and NOx. Responses of FBF to
aminoguanidine and L-NMMA were compared with twoway (group and drug treatment) ANOVA for repeated
measures. When significant differences were observed, a
comparison within groups or drug treatments was performed with one-way ANOVA followed by the Scheffé’s
test. Changes in NOx levels during infusion of aminoguanidine and L-NMMA were analyzed by use of the Wilcoxon signed rank test. A p value ⬍0.05 was considered
statistically significant.
RESULTS
Clinical characteristics of the study group. The baseline
clinical characteristics of the study participants are shown in
Table 1. Blood pressure and heart rate did not differ
between the two groups. The patients with CHF showed
evidence of hormonal activation in the form of distinctly
higher plasma ANP and BNP levels, as compared with the
control subjects (both p ⬍ 0.001). The venous plasma
concentration of NOx was also significantly higher in the
CHF group than in the control group (p ⬍ 0.001).
Responses of FBF to acetylcholine. Baseline FBF was
2.66 ⫾ 0.99 ml/min/100 ml in the control group and
JACC Vol. 38, No. 5, 2001
November 1, 2001:1470–6
Table 1. Clinical Characteristics
Gender (M/F)
Age (yrs)
Total cholesterol (mg/dl)
HDL cholesterol (mg/dl)
LDL cholesterol (mg/dl)
Triglycerides (mg/dl)
Glucose (mg/dl)
Heart rate (beats/min)
Systolic BP (mm Hg)
Diastolic BP (mm Hg)
ANP (pg/ml)
BNP (pg/ml)
Control Group
(n ⴝ 10)
CHF Group
(n ⴝ 13)
6/4
52 ⫾ 22
184 ⫾ 23
57 ⫾ 19
122 ⫾ 15
67 ⫾ 49
102 ⫾ 14
62 ⫾ 10
126 ⫾ 15
74 ⫾ 14
14.3 ⫾ 5.2
10.5 ⫾ 12.6
8/5
56 ⫾ 7
157 ⫾ 31
34 ⫾ 19
110 ⫾ 20
117 ⫾ 32
105 ⫾ 10
59 ⫾ 9
126 ⫾ 22
62 ⫾ 15
76.4 ⫾ 82.4*
192.3 ⫾ 217.3*
*p ⬍ 0.001 vs. corresponding control value. Data are presented as the mean value ⫾
SD.
ANP ⫽ atrial natriuretic peptide; BNP ⫽ brain natriuretic peptide; BP ⫽ systemic
blood pressure; CHF ⫽ congestive heart failure; HDL ⫽ high density lipoprotein;
LDL ⫽ low density lipoprotein.
2.59 ⫾ 0.76 ml/min/100 ml in the CHF group (p ⫽ 0.76).
Administration of acetylcholine did not alter the heart rate
or blood pressure in either group, but it significantly
increased the FBF ratio between the infused and noninfused
arms, from 0.99 ⫾ 0.14 at baseline to 6.57 ⫾ 2.92 (p ⬍
0.001) at the highest dose in the control group, and from
1.02 ⫾ 0.40 to 4.32 ⫾ 2.92 (p ⬍ 0.001) in the CHF group.
The peak percent change in FBF during infusion of acetylcholine was significantly lower in the CHF group than in
the control group (310 ⫾ 184% vs. 568 ⫾ 280%, p ⬍
0.001).
Responses of FBF to aminoguanidine and L-NMMA.
The FBF ratios between the infused and noninfused arms
during infusion of aminoguanidine and L-NMMA are
shown in Table 2, and individual data on the percent change
from baseline in the CHF group are shown in Figure 1.
Neither aminoguanidine nor L-NMMA altered the heart
rate or blood pressure in either group. Aminoguanidine did
not cause any change in FBF in the control group; however,
it significantly reduced FBF in the CHF group, in which
the averaged percent change in the FBF ratio between the
infused and noninfused arms was ⫺15 ⫾ 9% (p ⫽ 0.005) at
the 20-␮mol/min dose. In contrast, administration of
L-NMMA caused FBF to decrease significantly in both
groups in a dose-dependent manner. The averaged percent
changes in the FBF ratio were ⫺34 ⫾ 10% (p ⬍ 0.001) in
the CHF group and ⫺35 ⫾ 12% (p ⫽ 0.001) in the control
group at the 20-␮mol/min dose (CHF vs. control, p ⫽
0.98).
Recovery of FBF after infusion of aminoguanidine. The
effect of aminoguanidine on FBF remained for at least
10 min after administration of the last dose, and FBF
returned to baseline within 15 min (Fig. 2).
Changes in NOx during infusion of aminoguanidine and
L-NMMA. The arterial plasma concentration of NOx was
not affected by administration of either aminoguanidine or
L-NMMA (Table 3). With administration of aminoguanidine, the venous plasma NOx concentration decreased
Ishibashi et al.
Effects of iNOS Inhibitor on Forearm Blood Flow
JACC Vol. 38, No. 5, 2001
November 1, 2001:1470–6
1473
Table 2. Forearm Blood Flow Ratio Between Infused and Noninfused Arms During Infusion of
Aminoguanidine and L-NMMA
Aminoguanidine
Dose
(␮mol/min)
Baseline
2.5
5
10
20
L-NMMA
Control
Group
CHF
Group
Control
Group
CHF
Group
1.03 ⫾ 0.23
1.02 ⫾ 0.24
1.01 ⫾ 0.25
1.02 ⫾ 0.22
1.05 ⫾ 0.24
1.05 ⫾ 0.29
1.03 ⫾ 0.27
1.01 ⫾ 0.25
0.96 ⫾ 0.25*‡
0.89 ⫾ 0.30†§
1.02 ⫾ 0.15
0.99 ⫾ 0.09
0.85 ⫾ 0.20*
0.72 ⫾ 0.14†
0.64 ⫾ 0.12†
1.04 ⫾ 0.23
0.90 ⫾ 0.21*
0.80 ⫾ 0.20†
0.77 ⫾ 0.22†
0.68 ⫾ 0.22†
*p ⬍ 0.01 and †p ⬍ 0.001 vs. corresponding baseline value. ‡p ⬍ 0.01 and §p ⬍ 0.001 vs. corresponding control value. Data
are presented as the mean value ⫾ SD.
CHF ⫽ congestive heart failure; L-NMMA ⫽ NG-monomethyl-L-arginine.
significantly by 7 ⫾ 6% in the CHF group (p ⫽ 0.017), but
did not change in the control group (2 ⫾ 4%, p ⫽ 0.18).
L-NMMA significantly reduced the venous plasma NOx
concentration by 18 ⫾ 17% in the CHF group (p ⫽ 0.017)
and by 18 ⫾ 5% in the control group (p ⫽ 0.027).
Administration of drugs did not have any adverse effects
on hemodynamic variables or blood tests performed immediately after, the day after and three days after the study.
Effects of aminoguanidine and L-NMMA on the FBF
response to acetylcholine. L-NMMA caused a 30 ⫾ 5%
(p ⫽ 0.002) reduction in the rest FBF ratio and markedly
attenuated the increases in FBF produced by co-infusion of
acetylcholine. In contrast, aminoguanidine did not alter
FBF either at rest or during co-infusion of acetylcholine
(Fig. 3).
DISCUSSION
This study shows that enhanced production and release of
NO in patients with CHF is partly caused by functional
activation of iNOS in the forearm.
Functional activation of iNOS in peripheral vessels.
Although endothelial function is impaired in patients with
CHF (1–3), previous investigations have showed that basal
NO production and release are more greatly enhanced in
patients with CHF than in normal subjects, based on the
results of measurements of NO metabolites (8) and vasoconstrictive responses to nonselective NOS inhibitors such
as L-NMMA and L-NAME (6,7,9). It has been suggested
in experimental and human studies that enhanced NO
production in heart failure is induced by iNOS (14 –20,26).
Recent human studies have clearly demonstrated that expression of iNOS messenger ribonucleic acid is increased in
skeletal myocytes (17,18), monocytes (20), cardiac myocytes
(14,15) and myocardial vasculature (endothelial cells and
smooth muscle cells) (27) in patients with severe heart
failure. In the present study, the vasoconstrictive response to
the iNOS inhibitor possibly indicates expression of iNOS in
the vascular wall of peripheral resistance vessels. This
possibility is supported by recent experimental investigations of ischemic heart failure in the rat model, which
demonstrated increased L-arginine uptake and iNOS activity in the aortas of rats with heart failure (26) and iNOS
Figure 1. Percent change from baseline in the forearm blood flow (FBF) ratio between the infused and noninfused arms during intra-arterial infusion of
aminoguanidine (left) and NG-monomethyl-L-arginine (L-NMMA) (right) in individual patients with congestive heart failure.
1474
Ishibashi et al.
Effects of iNOS Inhibitor on Forearm Blood Flow
Figure 2. Time course of forearm blood flow (FBF) after finishing infusion
of the last dose of aminoguanidine in 13 patients with congestive heart
failure. Data are presented as the mean value ⫾ SD.
expression in the vascular wall (endothelial cells, smooth
muscle cells and the adventitia) of small mesenteric arteries
of rats with heart failure (28). However, there have been no
in vivo studies of patients to support this hypothesis. The
present study is the first in vivo study, to the best of our
knowledge, showing the contribution of iNOS to enhanced
NO production in the forearms of patients with CHF.
Different FBF responses to aminoguanidine and
L-NMMA. The FBF and NOx responses to the two NOS
inhibitors differed between the control and CHF groups.
Thus, although the responses to L-NMMA were similar
between the two groups, the response to aminoguanidine
was observed only in the CHF group, and the decreases in
FBF and NOx with aminoguanidine were relatively modest
as compared with those with L-NMMA. The results in the
control group suggest that under normal conditions, iNOS
is not expressed, whereas the results in the patient group
suggest that NO production in the forearms of patients with
CHF is derived from both iNOS and cNOS. We should
consider, however, some methodologic limitations of the
present study. The doses of two NOS inhibitors used in the
present study might not cause complete NOS inhibition.
However, both drugs appeared to approach a plateau effect
Table 3. Arterial and Venous Plasma Concentrations of Nitrite/
Nitrate During Infusion of Aminoguanidine and L-NMMA
Baseline
Control subjects
Artery (␮mol/l)
Vein (␮mol/l)
Patients with CHF
Artery (␮mol/l)
Vein (␮mol/l)
Aminoguanidine
L-NMMA
21.2 ⫾ 8.0
20.0 ⫾ 7.1
19.8 ⫾ 7.7
19.8 ⫾ 7.1
19.6 ⫾ 7.7
16.2 ⫾ 5.1*
70.0 ⫾ 63.8†
70.0 ⫾ 62.6†
69.0 ⫾ 64.9†
63.0 ⫾ 52.2*†
63.6 ⫾ 59.8†
52.3 ⫾ 38.1*†
*p ⬍ 0.05 vs. corresponding baseline value. †p ⬍ 0.05 vs. corresponding control value.
Data are presented as the mean value ⫾ SD.
Abbreviations as in Table 2.
JACC Vol. 38, No. 5, 2001
November 1, 2001:1470–6
Figure 3. Percent change from baseline in the forearm blood flow (FBF)
ratio between the infused and noninfused arms during intra-arterial
infusion of acetylcholine under co-infusion of saline, aminoguanidine or
NG-monomethyl-L-arginine (L-NMMA) in five normal subjects. *p ⬍
0.001 vs. corresponding saline value.
(Fig. 1), suggesting that this difference does not reflect a
dosing issue. Another possible explanation is that the
biologic inhibitory effect of aminoguanidine on iNOS is not
always equipotent to that of L-NMMA in clinical in vivo
use, although an in vitro study using cultured cells has
demonstrated that aminoguanidine and L-NMMA are
equipotent inhibitors of cytokine-induced nitrite formation
and cyclic guanosine monophosphate accumulation (29).
Finally, we need to consider the possibility of an interaction
between drugs (acetylcholine, aminoguanidine and
L-NMMA), because the order of these drugs was not
randomized in the present study. Although a sufficient
interval was allowed so that flow had returned to baseline
between the interventions, we cannot exclude the possibility
that some aminoguanidine effect persisted at the time that
L-NMMA was administered. However, because
L-NMMA was used to produce nonselective NOS inhibition, the persistence of some degree of iNOS inhibition
would not be expected to affect the results.
Increased FBF response to aminoguanidine in patients
with severe heart failure. Individual FBF responses to
aminoguanidine in the patient group varied widely. The
decreases in FBF were small (⬍10%) in 5 of 13 patients
with CHF (Fig. 1). In these five patients, the plasma
concentrations of BNP and the effects of L-NMMA on
FBF were also smaller than those in the other eight patients
(80.0 ⫾ 68.2 pg/ml vs. 292.3 ⫾ 233.7 pg/ml, respectively,
p ⬍ 0.001). Our recent study (9) demonstrated that the
vasoconstrictor response to L-NMMA was enhanced in
proportion to the basal plasma BNP level, indicating that
basal production and release of NO is enhanced in severe
heart failure. Taken together, we could conclude that NO
production in the forearm of patients with severe heart
failure is induced partly by iNOS activation, whereas in
JACC Vol. 38, No. 5, 2001
November 1, 2001:1470–6
patients with mild heart failure, iNOS activation might be
minimal or absent. Future longitudinal studies examining
the relationship between plasma BNP levels and FBF
responses to aminoguanidine and L-NMMA may be useful
in determining the influence of the relative roles of iNOS
and cNOS on the severity and progression of heart failure.
Selectivity of aminoguanidine as an iNOS inhibitor. An
important limitation of this study concerns the selectivity of
iNOS inhibition by aminoguanidine. This compound has
been used as a selective inhibitor of iNOS in animal
(22,24,30 –32) and human (33,34) studies, in doses that did
not inhibit the endothelial NOS-mediated basal relaxant
responses to acetylcholine (31,35) or bradykinin (34,36).
Aminoguanidine can produce relatively selective inhibition
of iNOS at appropriate drug concentrations, but excess
plasma levels can also result in some degree of cNOS
inhibition. Although there are novel agents that have better
selectivity (24) than aminoguanidine, those agents are not
currently applicable to human studies. The dose of aminoguanidine used in the present study did not alter the FBF
responses to acetylcholine, indicating that the drug dose
used did not result in nonselective inhibition of cNOS. This
finding supports our contention that NO produced by
iNOS contributes to regulation of FBF in patients with
CHF.
Clinical implications. Although further efforts should be
made to confirm our findings, aminoguanidine may prove to
be valuable in elucidating the role of iNOS in heart failure,
thus providing new therapeutic insights into heart failure.
We can not assess the precise mechanism of iNOS activation in our study patients; however, recent studies have
suggested that pro-inflammatory cytokines, such as interleukins and tumor necrosis factor (TNF)-alpha, are potent
inducers of iNOS in heart failure (14,20,26,37–39). Recently, therapeutic approaches targeting cytokines or iNOS
have been reported in experimental heart failure models
using animals (40 – 42), as well as in patients with heart
failure (43). Treatment with amlodipine (40), a calcium
channel antagonist, and pimobendan (41), a phosphodiesterase III inhibitor, was shown to increase the survival of
mice with heart failure due to viral myocarditis, which is
associated with inhibition of both pro-inflammatory cytokines (interleukin-1-beta, interleukin-6 and TNF-alpha)
and NO production by iNOS. Furthermore, treatment of
Dahl salt-sensitive rats with hypertensive heart failure with
imidapril (42), an angiotensin-converting enzyme inhibitor,
reduced the expression of iNOS messenger ribonucleic acid,
resulting in improved myocardial and vascular remodeling.
Deswal et al. (43) reported favorable effects of a single
intravenous infusion of etanercept, a specific TNF antagonist, on quality-of-life scores, 6-min walk distance and left
ventricular ejection fraction in 12 patients with heart failure.
These approaches will eventually be tried in larger patient
populations. The present data, suggesting increased iNOS
activity in the peripheral circulation of patients with heart
failure, support the need for future studies that will examine
Ishibashi et al.
Effects of iNOS Inhibitor on Forearm Blood Flow
1475
the use of selective iNOS inhibitors for the treatment of
CHF.
Conclusions. We have found that the enhanced production of NO in patients with CHF is induced partly by iNOS
activation, whereas in normal subjects, it is due to cNOS
activation. Furthermore, the present findings also suggest
that the relative importance of iNOS and cNOS in the
regulation of rest FBF and as sources of NO in patients with
CHF could change according to the severity and stage of
heart failure.
Acknowledgments
We thank Drs. Yoshitsugu Kunizawa and Nobuhiro Kodani
(Shimane Medical University, Cardiovascular Division,
Izumo, Japan) for managing the patients, as well as Pat
Yonemura (Minneapolis, Minnesota) for revising the report.
Reprint requests and correspondence: Dr. Yutaka Ishibashi,
Fourth Department of Internal Medicine, Shimane Medical University, 89-1 Enyacho, Izumo City, 693-8501, Japan. E-mail:
[email protected].
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