Enhanced iNOS (inducible nitric oxide synthase) activity may contribute to vascular dysfunction in patients with heart failure. In the present study, we aimed to determine whether iNOS activity contributes to the maintenance of vascular tone in patients with symptomatic heart failure with the use of the highly selective iNOS inhibitor 1400W {N-[3-(aminomethyl)benzyl] acetamidine}. Bilateral forearm blood flow was measured using venous occlusion plethysmography in 12 patients with New York Heart Association class II–IV heart failure and eight matched healthy control subjects during intra-brachial infusion of 1400W (0.1–1 μmol/min), L-NMMA (NG-monomethyl-L-arginine; a non-selective NOS inhibitor; 2–8 μmol/min) and noradrenaline (control vasoconstrictor; 60–480 pmol/min). In both patients and controls, intra-brachial infusion of L-NMMA and noradrenaline caused a dose–dependent reduction in infused forearm blood flow (P<0.05 for both): peak reduction of 32±6% and 37±4% during L-NMMA and 52±6% and 49±5% during noradrenaline respectively (P values were not significant when patients were compared with controls). In contrast, 1400W had no effect on blood flow at 1 μmol/min [−3±4% in patients (95% confidence intervals, −11 to 5%) and 3±8% in controls; P value was not significant]. In conclusion, we have demonstrated that intrabrachial selective iNOS inhibition does not influence forearm blood flow in patients with heart failure. We conclude that iNOS activity does not contribute to peripheral vascular tone in patients with symptomatic heart failure.

INTRODUCTION

NO (nitric oxide), a key mediator of vascular tone, is synthesized from L-arginine by a triad of isozymes, the NOSs (NO synthases). nNOS and eNOS (neuronal and endothelial NOS respectively) are both constitutively expressed under normal physiological conditions. In contrast, iNOS (inducible NOS) is activated in response to inflammatory mediators, is uncoupled from G-protein-receptor activation and is capable of sustained production of large quantities of NO within endothelial and vascular smooth muscle cells [1]. Excessive generation of NO by iNOS may account for the vascular dysfunction evident in a number of inflammatory conditions, including sepsis and endotoxaemia [2,3].

Heart failure is a chronic inflammatory state [4] associated with endothelial dysfunction [5,6] and, more specifically, impairment of NO-mediated endothelium-dependent vasodilatation [711]. However, controversy remains as to whether there is altered basal NO release. Although some studies suggest that there is enhancement of basal NO release [8,1214], others show evidence that there may be no change [15] or even a reduction [1618]. Induction of iNOS in heart failure may account for alterations in vascular generation of NO [14].

Until recently, it has not been possible to distinguish clearly between constitutive and inducible NOS activity due to the lack of specific inhibitors. 1400W {N-[3-(aminomethyl)benzyl] acetamidine} is a novel selective inhibitor of human iNOS that has become available for clinical use [19,20]. It competes with L-arginine to bind irreversibly with iNOS and is at least 5000-fold more selective for iNOS than eNOS, making it the most selective iNOS inhibitor to date [19]. The present study aimed to assess the contribution of functional iNOS activity to peripheral vascular tone in patients with symptomatic heart failure by determining the effect of direct local intra-arterial 1400W on peripheral blood flow.

METHODS

Subjects and patients

The protocol was undertaken with the approval of the local Research Ethics Committee and in accordance with the Declaration of Helsinki. Written informed consent of each subject was obtained before entry into the study. Twelve patients with stable NYHA (New York Heart Association) class II–IV congestive heart failure and objective evidence of LV (left ventricular) impairment (LV ejection fraction <35%, shortening fraction <20% or LV end-diastolic diameter >5.5 cm) and eight age- and sex-matched control subjects were enrolled in the study. Patients were excluded if they had significant valvular heart disease, or hepatic or renal impairment. Subjects were non-smokers and abstained from alcohol for 24 h before, and from food and caffeine-containing drinks on, the day of the study. Diuretics were withheld on the morning of the study for patient comfort. All studies were performed in a quiet temperature-controlled room.

Intra-arterial drug administration

The brachial artery of the non-dominant arm was cannulated with a 27-standard wire gauge steel needle (Cooper's Needle Works) under local anaesthesia. The cannula was attached to a 16-gauge epidural catheter (Portex) and patency was maintained by infusion of 0.9% saline (Baxter Health Care) via an IVAC P1000 syringe pump. The rate of intra-arterial infusions was maintained constant throughout all studies at 1 ml/min. Pharmaceutical grade 1400W (Merck Biosciences), L-NMMA (NG-monomethyl-L-arginine; Merck Biosciences) and noradrenaline (Levophed; Sanofi-Winthrop) were dissolved in saline on the day of the study.

Haemodynamic measurements

Blood flow was measured in both forearms by venous occlusion plethysmography as described previously [2123]. Blood pressure and heart rate were monitored in the non-infused arm at intervals throughout each study with a semi-automated non-invasive oscillometric sphygmomanometer.

Study design

Subjects attended on a single occasion at 09.00 hours and were recumbent throughout the study. Strain gauges and cuffs were applied, and the brachial artery of the nondominant arm was cannulated. FBF (forearm blood flow) was measured every 6–10 min. Following a 30 min equilibration period, subjects received intra-arterial infusions of 1400W (a selective iNOS inhibitor; at 0.1 and 0.3 μmol/min for 6 min at each dose, and 1 μmol/min for 18 min), L-NMMA (a non-selective NOS inhibitor; at 2 and 4 μmol/min for 6 min, and 8 μmol/min for 18 min) and noradrenaline (a control vasoconstrictor; at 60, 120, 240 and 480 pmol/min for 6 min at each dose) with 30 min saline washout periods between drugs.

Data analysis and statistics

Plethysmographic data were extracted from the Chart data files, and the last five linear recordings in each measurement period were averaged. FBFs were calculated from the plethysmographic data as described previously [24,25]. Percentage changes in the infused FBF were calculated [23,24] as follows:

 
formula

where Ib and NIb are the infused and non-infused FBFs at baseline (time 0) respectively, and It and NIt are the infused and non-infused FBFs at a given time point (t) respectively.

Data were examined, where appropriate, by ANOVA with repeated measures and two-tailed Student's t test using Microsoft Excel 2002. Data are expressed as means±S.E.M. Statistical significance was taken at the 5% level.

RESULTS

Patients and controls were generally well matched, although patients had lower systolic and diastolic blood pressures (Table 1). Heart rate, blood pressure and non-infused FBF were unchanged throughout the study in both groups.

Table 1
Subject baseline characteristics

Data are means±S.E.M. *P<0.01 compared with control subjects. DCM, dilated cardiomyopathy; IHD, ischaemic heart disease.

 Patient group (n=12) Control group (n=8) 
Age (years) 64±3 57±3 
Sex (male/female) 8/4 7/1 
Aetiology (DCM/IHD) 4/8 − 
NYHA class (II/III/IV) 7/4/1 − 
Medication (n  
 Aspirin 11 − 
 ACE inhibitor 11 − 
 Diuretic 11 − 
 β-Blocker 10 − 
 Spironolactone − 
 Angiotensin receptor antagonist − 
 Nitrate − 
LV ejection fraction (%) 31±4 − 
LV shortening fraction (%) 15±2 − 
LV end-diastolic diameter (mm) 60±3 − 
Heart rate (beats/min) 58±2 59±3 
Systolic blood pressure (mmHg) 118±6* 143±5 
Diastolic blood pressure (mmHg) 67±3* 81±3 
Baseline infused FBF (ml·100 ml−1·min−12.5±0.3 2.1±0.1 
Baseline non-infused FBF (ml·100 ml−1·min−12.2±0.2 2.2±0.3 
 Patient group (n=12) Control group (n=8) 
Age (years) 64±3 57±3 
Sex (male/female) 8/4 7/1 
Aetiology (DCM/IHD) 4/8 − 
NYHA class (II/III/IV) 7/4/1 − 
Medication (n  
 Aspirin 11 − 
 ACE inhibitor 11 − 
 Diuretic 11 − 
 β-Blocker 10 − 
 Spironolactone − 
 Angiotensin receptor antagonist − 
 Nitrate − 
LV ejection fraction (%) 31±4 − 
LV shortening fraction (%) 15±2 − 
LV end-diastolic diameter (mm) 60±3 − 
Heart rate (beats/min) 58±2 59±3 
Systolic blood pressure (mmHg) 118±6* 143±5 
Diastolic blood pressure (mmHg) 67±3* 81±3 
Baseline infused FBF (ml·100 ml−1·min−12.5±0.3 2.1±0.1 
Baseline non-infused FBF (ml·100 ml−1·min−12.2±0.2 2.2±0.3 

FBF responses

Infusion of both L-NMMA and noradrenaline produced a dose-dependent reduction in FBF in both patients (basal blood flow, 2.3±0.3 and 1.8±0.2 ml·100 ml−1·min−1 respectively; peak reduction, 32±6% and 52±6% respectively) and healthy control subjects [basal blood flow, 1.7±0.1 and 1.6±0.3 ml·100 ml−1·min−1 respectively; peak reduction, 37±4% and 49±5% respectively; P=ns (not significant) when patients were compared with controls; Figure 1]. In contrast, infusion of 1400W had no effect on FBF in either group (P=ns): −3±4% (95% confidence intervals, −11 to 5%) in patients and 3±8% (95% confidence intervals, −13 to 19%) in controls at 1 μmol/min.

Infused FBF during incremental doses of 1400W, L-NMMA and noradrenaline in patients with congestive heart failure and control subjects

Figure 1
Infused FBF during incremental doses of 1400W, L-NMMA and noradrenaline in patients with congestive heart failure and control subjects

(○) Patients with congestive heart failure (n=12); (■) control subjects (n=8). *P<0.05 (ANOVA) for dose–response in both patients and control subjects. Norepinephrine, noradrenaline.

Figure 1
Infused FBF during incremental doses of 1400W, L-NMMA and noradrenaline in patients with congestive heart failure and control subjects

(○) Patients with congestive heart failure (n=12); (■) control subjects (n=8). *P<0.05 (ANOVA) for dose–response in both patients and control subjects. Norepinephrine, noradrenaline.

DISCUSSION

There has been controversy surrounding the importance of iNOS activity in the vasculature of patients with heart failure. Using the highly selective iNOS inhibitor 1400W, we have demonstrated for the first time that selective intrabrachial iNOS inhibition does not alter FBF in patients with heart failure. We therefore conclude that there is no evidence of iNOS activity in the peripheral vasculature of patients with symptomatic heart failure.

Evidence for iNOS in the peripheral vasculature

iNOS expression [26] and activity [27] has been demonstrated in the vasculature of rodent models of heart failure. Vascular iNOS activity is associated with functional changes and, more specifically, altered vascular responsiveness to adrenergic stimulation, which can be reversed by selective iNOS inhibition [26,28].

In patients, iNOS is present in a number of tissues, including the myocardium, intramyocardial vasculature and skeletal muscle [2935]; however, evidence for functional iNOS in the peripheral vasculature remains unconvincing. Despite the presence of iNOS in the aorta [27] and mesenteric vessels [26] of rodent models of heart failure, there are no animal or human studies of heart failure that have isolated either iNOS mRNA or protein from the peripheral vasculature.

Indirect evidence of functional iNOS activity in the peripheral vasculature of patients with heart failure comes from an in vivo study [14]. Ishibashi and co-workers [14] reported a modest reduction in FBF during infusion of aminoguanidine, a NOS inhibitor with some selectivity for iNOS. Aminoguanidine is, however, an extremely weak inhibitor of iNOS and complete inhibition of iNOS is only achieved with high (approaching millimolar) concentrations [20]. Furthermore, aminoguanidine is only 9-fold more selective for iNOS compared with eNOS [20,36]. A reduction in FBF during aminoguanidine infusion only becomes evident at higher doses (10–20 μmol/min) when effective end-organ concentrations would be in the order of between 400–800 μmol/l [14]. During infusion of such high doses, aminoguanidine would be expected to have considerable action on eNOS [20,36].

In contrast with patients with heart failure, aminoguanidine has no effect on FBF in healthy control subjects [14]. This suggests that patients with heart failure have altered NOS activity compared with controls. Aminoguanidine has an augmented inhibitory action on eNOS in the presence of excess tetrahydrobiopterin, the requisite cofactor for NOS [37]. Inflammatory stimuli enhance tetrahydrobiopterin synthesis by induction of GTP cyclohydrolase I [38,39]. As heart failure is an inflammatory disorder, there may be increased availability of tetrahydrobiopterin as a result of increased activity of GTP cyclohydrolase I. We therefore speculate that, consistent with the work of Vallance's group [40], the purported in vivo inhibition of iNOS seen with aminoguanidine simply reflects an enhanced inhibition of eNOS in the presence of up-regulated GTP cyclohydrolase I. The possibility that these patients may have augmented (or ‘induced’) eNOS activity as a consequence of enhanced tetrahydrobiopterin availability merits further investigation.

Considering the lack of specificity of aminoguanidine as an inhibitor of iNOS and the possibility that eNOS activity may be up-regulated during inflammation, it is likely that the effects of aminoguanidine are a consequence of inhibition of eNOS rather than iNOS. In the present study, we have been able to use a highly selective inhibitor of iNOS to demonstrate that functional iNOS activity does not appear to play a major role in the regulation of peripheral vascular tone in heart failure patients.

1400W is a highly selective inhibitor of iNOS

The present study is the first to use the iNOS inhibitor 1400W in patients with heart failure in vivo. 1400W is a monoamidine monoamine analogue that competes with L-arginine to bind tightly and irreversibly to iNOS with a rapid onset of action (<5 min) [19]. It is >5000-fold more potent against purified human iNOS than eNOS and thus is the most selective inhibitor of iNOS reported to date [19]. The selectivity of 1400W, both in vitro and in vivo, makes it an attractive tool for assessing the contribution of iNOS [20,36]. As the primary aim of the present study was to determine the contribution of iNOS activity to the maintenance of vascular tone and considering that 1400W irreversibly inhibits iNOS whereas L-NMMA inhibits both iNOS and eNOS, intra-arterial drug order was not randomized. Instead, the effects of selective iNOS inhibition on FBF were observed first, followed by non-selective NOS inhibition with L-NMMA and finally, intra-arterial administration of the control vasoconstrictor noradrenaline. As a consequence, noradrenaline was observed to have a rather blunted (albeit significant) effect on FBF, which is probably explained by a persistent vasoconstrictive effect of L-NMMA.

An important concern regarding the present study is the possibility that the lack of effect seen with 1400W on FBF reflects a failure of 1400W to inhibit iNOS effectively in vivo at the chosen dose range (0.1–1 μmol/min). 1400W inhibits iNOS with an 8-fold higher potency than L-NMMA (EC50 values of 0.8 μmol/l and 6 μmol/l respectively) [19]. During infusion of 1400W at 1 μmol/min, we would predict effective end-organ concentrations to be between 40–50 μmol/l. This is >50-fold higher than the EC50, and 10-fold higher than concentrations that we have demonstrated to have vascular effects in vitro in rodents [26]. It would therefore be anticipated that this dose is sufficient to achieve inhibition of functional iNOS activity. Indeed, we have recently demonstrated [41] that intra-arterial infusion of 1400W, at the same doses used in the present study, results in a reduction in FBF in patients with liver cirrhosis, an inflammatory condition associated with induction of iNOS [42].

Study limitations

The discordant findings in previous reports on NO generation in heart failure may, in part, be explained by differences in the severity of heart failure, with basal NO production increasing and stimulated release decreasing with progression of disease [43]. In the present study, inclusion criteria were deliberately chosen to encompass a broad range of patients, so that the response to selective iNOS inhibition could be correlated with disease severity. Almost half of the patients (five out of 12) were in NYHA class III or IV and thus had evidence of severe disease. Subgroup analysis confirmed that infusion of 1400W had no effect on FBF in patients in NYHA classes III or IV: −3±5% at 1 μmol/min (95% confidence intervals, −6 to 12%; n=5; P=ns). It is therefore unlikely that the failure to detect iNOS activity was due to inadequate severity of disease in this group. It is noteworthy that almost all of the patients who took part in the present study were prescribed diuretic, ACE (angiotensin-converting enzyme) inhibitor and β-blocker therapy that may have modified vascular NO effects.

Clinical implications

In clinical conditions where iNOS activity is enhanced, one would intuitively expect inhibition of iNOS to result in vasoconstriction, as a consequence of reduced bioavailability of NO, a potent vasodilator. Vasoconstriction, in the context of heart failure, would result in reduced tissue perfusion and increased afterload and, hence, a deterioration in cardiac function. However, the failure to demonstrate functional iNOS activity in the peripheral vasculature may provide an exciting opportunity to explore other potential benefits of iNOS inhibition in heart failure with less concern about the adverse effects of vasoconstriction.

Data from animal models suggest that modulation of iNOS activity may have therapeutic benefits in heart failure. Selective iNOS inhibition improves myocardial oxygen consumption and LV function in dogs [44] and reverses the β-adrenergic hyporesponsiveness of papillary muscle from rats [45] with congestive heart failure. iNOS is present in the myocardium [3032] and skeletal muscle [34,35] of patients with heart failure and excessive generation of NO in these tissues may, in part, explain the impaired myocardial contractility [46] and exercise intolerance [47]. Further clinical studies are necessary to investigate whether systemic inhibition of iNOS is a therapeutic target for improving cardiac function and exercise tolerance in heart failure patients.

Conclusions

We conclude that, in patients with symptomatic congestive heart failure, functional iNOS activity does not contribute to the maintenance of peripheral vascular tone. Further studies may reveal exciting therapeutic potential for the inhibition of both GTP cyclohydrolase I and iNOS in patients with heart failure.

Abbreviations

     
  • 1400W

    N-[3-(aminomethyl)benzyl] acetamidine

  •  
  • ACE

    angiotensin-converting enzyme

  •  
  • FBF

    forearm blood flow

  •  
  • L-NMMA

    NG-monomethyl-L-arginine

  •  
  • LV

    left ventricular

  •  
  • NO

    nitric oxide

  •  
  • NOS

    NO synthase

  •  
  • eNOS

    endothelial NOS

  •  
  • iNOS

    inducible NOS

  •  
  • ns

    not significant

  •  
  • NYHA

    New York Heart Association

This work was supported by a grant from Chest Heart and Stroke Scotland (Res02/A63). A. R. D. and S. C. are supported by the British Heart Foundation (grants PG/02/113/14452 and FS/2001/049). We acknowledge the support of the Wellcome Trust Clinical Research Facility.

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