Author + information
- Received February 12, 2001
- Revision received June 18, 2001
- Accepted July 12, 2001
- Published online November 1, 2001.
- 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* and
- Michio Hashimoto, MD†
- ↵*Reprint requests and correspondence:
Dr. Yutaka Ishibashi, Fourth Department of Internal Medicine, Shimane Medical University, 89-1 Enyacho, Izumo City, 693-8501, 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.
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.
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.
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).
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.
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 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-L-arginine (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 acetylcholine to identify the selectivity of iNOS inhibition, because the reported selectivity of this drug has been controversial (23,24).
Thirteen patients admitted to our hospital with CHF due to idiopathic dilated cardiomyopathy and 10 age- and 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.
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 (RA-4122, 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 mercury-filled 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).
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.
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 μg/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 μmol/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, 5, 10 and 20 μmol/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 high-performance 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 μg/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 μmol/min) and L-NMMA (20 μmol/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.
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 ttest 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 two-way (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.
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 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-μmol/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-μmol/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 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).
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 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 (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 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.
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 the use of selective iNOS inhibitors for the treatment of CHF.
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.
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.
- atrial natriuretic peptide
- B-type natriuretic peptide
- congestive heart failure
- constitutive nitric oxide synthase
- forearm blood flow
- inducible nitric oxide synthase
- NG-nitro-L-arginine methyl ester
- nitric oxide
- nitric oxide synthase
- tumor necrosis factor
- Received February 12, 2001.
- Revision received June 18, 2001.
- Accepted July 12, 2001.
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