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(Circulation. 1995;91:1314-1319.)
© 1995 American Heart Association, Inc.

Articles

Nitric Oxide Is Responsible for Flow-Dependent Dilatation of Human Peripheral Conduit Arteries In Vivo

Robinson Joannides, MD; Walter E. Haefeli, MD; Lilly Linder, MD; Vincent Richard, PhD; El Hassan Bakkali, BSc; Christian Thuillez, MD, PhD; Thomas F. Lüscher, MD

From the Department of Pharmacology, VACOMED, IFRMP, Rouen University Medical School and Rouen Hospital (France) (R.J., V.R., E.H.B., C.T.) and the Department of Clinical Pharmacology, Basel University Hospital (Switzerland) (W.E.H., L.L., T.F.L.).

Correspondence to T.F. Lüscher, MD, Cardiology, University Hospital, CH 4031, Bern, Switzerland.

	Abstract

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Background Experimental evidence suggests that flow-dependent dilatation of conduit arteries is mediated by nitric oxide (NO) and/or prostacyclin. The present study was designed to assess whether NO or prostacyclin also contributes to flow-dependent dilatation of conduit arteries in humans.

Methods and Results Radial artery internal diameter (ID) was measured continuously in 16 healthy volunteers (age, 24±1 years) with a transcutaneous A-mode echo-tracking system coupled to a Doppler device for the measurement of radial blood flow. In 8 subjects, a catheter was inserted into the brachial artery for measurement of arterial pressure and infusion of the NO synthase inhibitor N^G-monomethyl-L-arginine (L-NMMA; 8 µmol/min for 7 minutes; infusion rate, 0.8 mL/min). Flow-dependent dilatation was evaluated before and after L-NMMA or aspirin as the response of the radial artery to an acute increase in flow (reactive hyperemia after a 3-minute cuff wrist occlusion). Under control conditions, release of the occlusion induced a marked increase in radial blood flow (from 24±3 to 73±11 mL/min; P<.01) followed by a delayed increase in radial diameter (flow-mediated dilatation; from 2.67±0.10 to 2.77±0.12 mm; P<.01) without any change in heart rate or arterial pressure. L-NMMA decreased basal forearm blood flow (from 24±3 to 13±3 mL/min; P<.05) without affecting basal radial artery diameter, heart rate, or arterial pressure, whereas aspirin (1 g PO) was without any hemodynamic effect. In the presence of L-NMMA, the peak flow response during hyperemia was not affected (76±12 mL/min), but the duration of the hyperemic response was markedly reduced, and the flow-dependent dilatation of the radial artery was abolished and converted to a vasoconstriction (from 2.62±0.11 to 2.55±0.11 mm; P<.01). In contrast, aspirin did not affect the hyperemic response nor the flow-dependent dilatation of the radial artery.

Conclusions The present investigation demonstrates that NO, but not prostacyclin, is essential for flow-mediated dilatation of large human arteries. Hence, this response can be used as a test for the L-arginine/NO pathway in clinical studies.

Key Words: blood flow • endothelium-derived factors • L-NMMA • arteries • dilation

	Introduction

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Large arteries accommodate changes in blood flow by increasing their internal diameter (ID). Such a flow-dependent dilatation represents a fundamental mechanism that opposes neurogenic and myogenic vasoconstriction, increases conductance during exercise, and maintains shear stress within physiological values. In animals, flow-dependent dilatation is endothelium dependent and mediated by nitric oxide (NO) and/or prostacyclin.^{1 2 3 4 5} However, whether a similar mechanism also occurs in human arteries remains uncertain. This is especially critical because flow-dependent dilatation of large arteries in vivo has been used as an index of endothelial function in humans and is impaired in atherosclerosis.^{6 7 8} Thus, the present study was designed to assess whether NO or prostacyclin contributes to flow-dependent dilatation of peripheral arteries in humans.

	Methods

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Subjects
The study was performed in 16 healthy volunteers (10 men and 6 women; age, 24±1 years). All subjects were normotensive and nonsmokers and were not taking any medication at the time of the experiment. Blood cholesterol and blood glucose were 6.05±0.36 mmol/L and 4.32±1.07 mmol/L, respectively. The protocol was approved by the Basel Hospital ethical committee, and written informed consent was obtained from all participants.

Measurements of Radial Artery Hemodynamics
Measurements were performed while subjects were supine in a quiet, air-conditioned room maintained at a constant temperature (22°C to 24°C). Radial artery ID was measured continuously with a high-precision A-mode echo-tracking device (NIUS 02, Asulab) with a resolution on the diameter evolution of <1 µm.^{9 10 11} Briefly, a 10-MHz focused transducer was positioned over the radial artery. The probe was set perpendicular to the artery by a stereotaxic arm with micrometric screws, while proper positioning was adjusted with a stereo Doppler mode. After switching to A-mode, the echoes from both anterior and posterior walls of the artery were visualized on a screen and tagged by electronic trackers, allowing continuous recording of the artery ID as shown previously.⁹ The processed radiofrequency line was visualized on a computer screen, and the operator selected the peaks corresponding to the interfaces, after which the exact position of each selected peak was determined by an interpolation technique. Finally, radial artery blood velocity was continuously recorded by an 8-MHz Doppler probe (Doptek 2002, Deltex). Radial artery flow was calculated from the measurements of velocity and ID. Positioning of the probes and of the electronic trackers was always performed by the same investigator, after which calculation of radial parameters was performed automatically without any further intervention by the investigator.

Fig 1 shows a representative tracing of the measured parameters, ie, radial diameter, radial blood flow, and arterial blood pressure throughout five consecutive cycles, showing that the echo tracking technique allows continuous tracking of the diameter signal throughout the cardiac cycle, even though the systolic-diastolic differences in diameter are, in the present case, <20 µm.

View larger version (30K): [in this window] [in a new window]   Figure 1. Typical recording of radial artery diameter (RAD, mm), radial blood flow (RBF, mL/min), and arterial pressure (AP, mm Hg) obtained in one subject at baseline in the absence of NG-monomethyl-L-arginine.

In experiments involving L-NMMA, an 18-gauge catheter was inserted under local anesthesia (lidocaine 1%) into the left brachial artery for continuous measurement of arterial pressure (Statham P23Pb)¹² and infusion of L-NMMA.¹³

Study Protocol
Subjects were allowed to rest 30 minutes after instrumentation, then heart rate, arterial pressure, radial artery flow, and diameter were recorded for 5 minutes. After baseline measurements, an arterial occlusion cuff placed at the wrist (ie, distal to the site of radial artery measurements) was inflated for 3 minutes at 180 mm Hg and was deflated to allow reactive hyperemia. All parameters were recorded continuously throughout cuff inflation and during hyperemia until radial artery diameter had returned to baseline (ie, within 7 minutes after release of the cuff).

Subsequently, subjects received L-NMMA (Clinalfa; 8 µmol/min for 7 minutes; infusion rate, 0.8 mL/min) or aspirin 1 g PO. Five minutes after L-NMMA or 2 hours after aspirin, all parameters were again measured at baseline, and the same occlusion-hyperemia protocol was repeated. All measurements represent means of 15 cardiac cycles. Parameters were measured at baseline, peak increase in radial blood flow during hyperemia, and peak increase in radial artery ID. In addition, since experiments performed in dogs suggested that L-NMMA could reduce the duration of the hyperemic phase,^{14 15} we also calculated parameters characteristic of the duration of hyperemia, ie, the area under the curve of the flow response and the time elapsing between peak hyperemia and return to 50% of this peak (t_1/2). Results are expressed as mean±SEM for 8 subjects in each group. Results were analyzed by repeated-measures ANOVA, followed, when ANOVA was significant, by a Tukey test, and a value of P<.05 was considered statistically significant.

	Results

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Stability of Hemodynamic Measurements and Reproducibility of Hyperemic Test Under Control Conditions
The stability of the measured hemodynamic parameters was assessed by two consecutive measurements over 30 minutes (t₀ and t₃₀) in 8 subjects who did not receive any treatment and in whom the probes were left in place throughout the observation period. Results are shown in Fig 2. There were no changes in arterial pressure, radial artery flow (t₀, 31±7 and t₃₀, 30±8 mL/min), or radial artery diameter (t₀, 3.06±0.15 and t₃₀, 3.05±0.14 mm) throughout the observation period. From these experiments, intraobserver variability was calculated to be 5.7±2.6% for radial flow and 1.3±0.4% for radial diameter.

View larger version (33K): [in this window] [in a new window]   Figure 2. Bar graphs showing radial artery flow (mL/min) and radial artery diameter (mm) measured at baseline (Base) and during reactive hyperemia (RH) at two different times in eight subjects in whom two consecutive hyperemic tests were performed 30 minutes apart (t0 and t30). The area under the curve (AUC, arbitrary units) of the flow response during hyperemia and the time elapsing between peak hyperemia and return to 50% of this peak (t1/2, seconds) are also shown. Results are mean±SEM. **P<.01 vs baseline.

The reproducibility of the hyperemic test was also assessed in the same 8 subjects as two consecutive ischemia-hyperemia cycles without any treatment. Results are shown in Fig 2. There were no differences between the two hyperemic tests, whether in terms of peak radial flow (t₀, 100±17 and t₃₀, 102±13 mL/min), peak radial diameter (t₀, 3.18±0.15 and t₃₀, 3.22±0.15 mm), or duration of the hyperemic phase (area under the curve: t₀, 6.8±1.2 and t₃₀, 6.4±1.0 arbitrary units [AU]; t_1/2: t₀, 32±6 and t₃₀, 31±5 seconds).

Effect of L-NMMA on Baseline Parameters and on Flow-Dependent Dilatation
In the absence or in the presence of L-NMMA, hyperemia was not associated with any changes in heart rate or blood pressure, and administration of L-NMMA did not induce any changes in heart rate or arterial blood pressure (Table).

View this table: [in this window] [in a new window]   Table 1. Heart Rate and Arterial Pressure1

In control conditions, deflation of the occluding cuff increased radial artery flow (from 24±3 to 73±11 mL/min; percent change from baseline, 214±34%; P<.01; Figs 3 and 4) and radial artery diameter (from 2.67±0.10 to 2.77±0.12 mm; percent change from baseline, 3.6±0.8%; P<.01; Figs 3 and 4). However, the increase in diameter was delayed compared with the increase in blood flow; indeed, the time to peak increase in flow was 7.1±2.4 seconds, whereas the time to peak increase in diameter was 77.1±15.8 seconds (P<.01).

View larger version (32K): [in this window] [in a new window]   Figure 3. Bar graphs showing radial artery flow (mL/min) and radial artery diameter (mm) measured at baseline (Base) and during reactive hyperemia before and after infusion of NG-monomethyl-L-arginine (L-NMMA). All results are the mean±SEM of eight subjects. **P<.01 vs Base; P<.05 and P<.01 vs corresponding control value.

View larger version (29K): [in this window] [in a new window]   Figure 4. Plots showing individual and mean±SEM percent changes from baseline of radial blood flow and radial diameter during reactive hyperemia (RH). L-NMMA indicates NG-monomethyl-L-arginine. **P<.01 vs corresponding control value.

At baseline (ie, before cuff wrist occlusion), L-NMMA decreased radial flow (from 24±3 to 13±3 mL/min; P<.05; Fig 3) and increased radial resistance (from 2.98±0.41 to 6.14±0.97 mm Hg · min/mL; P<.01) but did not significantly affect radial artery diameter (from 2.67±0.10 to 2.62±0.11 mm; P=NS). After L-NMMA, deflation of the occluding cuff was associated with an increase in radial artery flow to 76±12 mL/min (P<.01 versus baseline, Fig 3). These values obtained at peak hyperemia were not different from those obtained in the absence of L-NMMA. However, the percent increase in flow during hyperemia was higher than that observed in the absence of L-NMMA (Fig 4; control, 214±34%; L-NMMA, 493±53%; P<.01). In addition, L-NMMA reduced the area under the curve (control, 6.8±1.2 and L-NMMA, 4.0±0.7 AU; P<.01; Fig 5) as well as t_1/2 (control, 31.6±6.2 and L-NMMA, 13.3±1.3 seconds; P<.05; Fig 5). Thus, despite a similar peak increase in flow, L-NMMA markedly reduced the duration of hyperemia. After L-NMMA, hyperemia was associated with a significant decrease in radial artery diameter, to 2.55±0.11 mm (percent change from baseline, -2.8±0.4%; P<.01; Figs 3 and 4). This value was lower than that obtained in the absence of L-NMMA (2.77±0.12 mm; P<.01). Thus, L-NMMA abolished flow-dependent dilatation of the radial artery and converted it to a vasoconstriction.

View larger version (26K): [in this window] [in a new window]   Figure 5. Bar graphs showing area under the curve (AUC, arbitrary units) of the radial flow response during hyperemia and the time elapsing between peak hyperemia and return to 50% of this peak (t1/2, seconds) before and after infusion of NG-monomethyl-L-arginine (L-NMMA). Results are mean±SEM. *P<.05 and **P<.01 vs control.

Effect of Aspirin on Baseline Parameters and Flow-Dependent Dilatation
Administration of aspirin did not induce any changes in heart rate (control, 65±3 and aspirin, 62±2 beats per minute) or mean arterial pressure (control, 75±3 and aspirin, 76±3 mm Hg). The effect of aspirin on reactive hyperemia and flow-dependent dilatation is shown in Fig 6. Aspirin did not affect radial blood flow or radial diameter at baseline (radial flow: control, 31±7 and aspirin, 28±7 mL/min; radial diameter: control, 3.06±0.15 and aspirin, 3.06±0.15 mm) or during hyperemia (radial flow: control, 100±17 and aspirin, 89±13 mL/min; radial diameter: control, 3.18±0.15 and aspirin, 3.18±0.15 mm). Furthermore, aspirin did not affect the duration of the hyperemia (area under the curve: control, 6.8±1.2 and aspirin, 5.9±1.2 AU; t_1/2: control, 34±6 and aspirin, 31±8 seconds).

View larger version (32K): [in this window] [in a new window]   Figure 6. Bar graphs showing radial artery flow (mL/min) and radial artery diameter (mm) measured at baseline (Base) and during reactive hyperemia (RH) before and 2 hours after administration of aspirin (1 g PO). The area under the curve (AUC, arbitrary units) of the flow response during hyperemia and the time elapsing between peak hyperemia and return to 50% of this peak (t1/2, seconds) are also shown. All results are the mean±SEM of eight subjects. **P<.01 vs Base.

	Discussion

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The main results of the present study performed in the human radial artery are that (1) local administration of the NO synthase inhibitor L-NMMA did not affect the peak increase in flow during hyperemia but reduced the duration of the hyperemia; (2) in the presence of L-NMMA, flow-dependent dilatation of the radial artery was abolished and converted to a vasoconstriction; and (3) in contrast to L-NMMA, cyclooxygenase inhibition with aspirin did not affect the reactive hyperemic response or the flow-dependent dilatation of the radial artery. These results demonstrate that NO is essential for the flow-mediated dilatation of the human radial artery in vivo.

Administration of L-NMMA decreased radial blood flow but did not affect radial diameter. The decrease in radial flow after L-NMMA is consistent with the existence of a basal release of NO in this vascular bed, in agreement with previous results obtained with plethysmographic techniques.^{13 16} In contrast, the modest effect of L-NMMA on radial diameter would suggest that basal release of NO is minimal at the level of these large peripheral arteries. In recent experiments in which L-NMMA was injected into the human left anterior descending (LAD) coronary artery, the L-arginine analogue also did not induce any change in proximal LAD diameter, although it induced a small decrease in distal LAD diameter, again suggesting that basal release of NO does not contribute significantly to the tone of large conduit arteries in humans.¹⁷ Hence, as shown previously in isolated arteries, the basal release of NO is not uniform in the circulation and may differ in arteries of different sizes^{18 19} as well as of different anatomic origin.²⁰ Most likely, the basal release of NO is larger in the forearm microcirculation than in conduit vessels like the the radial artery.

One possibility to explain the apparent lack of effect of L-NMMA on radial diameter would be that the number of subjects on whom experiments were performed was insufficient to detect a significant reduction in radial diameter after L-NMMA. However, it should be noted that inclusion of three additional subjects who received L-NMMA according to the same protocol but were not subjected to hyperemia did not affect the outcome of the results on radial diameter (baseline, 2.93±0.11 and L-NMMA, 2.90±0.14 mm; percent change from baseline, -1.5±1.7%; P=.49; data not shown). Another possibility is that the dose of L-NMMA used was not sufficient to block the endothelial synthesis of NO. Although we have not assessed the effect of an NO-dependent vasodilator (such as acetylcholine) in the present experiments, it must be noted that the dose of L-NMMA used (8 µmol/min for 7 minutes) is higher than that previously shown by different investigators to blunt the forearm response to acetylcholine.¹³

In the present experiments, aspirin administered at a dose known to be effective in blocking vascular cyclooxygenase²¹ did not affect reactive hyperemia or flow-dependent dilatation of the radial artery. The lack of effect of aspirin on postischemic flow is not in agreement with the study of Carlsson and Wennmalm,²² in which reactive hyperemia of the forearm was inhibited by aspirin as well as other inhibitors of cyclooxygenase. There are, however, several possible explanations for these different results. First, it must be noted that, in our experiments, blood flow was measured with a Doppler system, whereas in the study of Carlsson and Wennmalm, flow was measured by plethysmography. Indeed, the hyperemia-induced increase in flow, as assessed by plethysmography, is caused by dilatation of both small arteries and veins.²³ Since the venous ischemic vasodilatation is mediated primarily by cyclooxygenase products and reduced after aspirin blockade,²⁴ the aspirin-induced decrease in postischemic hyperemia observed with the plethysmographic method may be explained at least in part by the decrease in venodilatation in this model. The effect of aspirin in our experiments may be minimal because of the small contribution of veins in our model, and this could explain the discrepancy observed. Second, it is possible that the contribution of the venous vasodilatation and/or of cyclooxygenase products may have increased with the duration of ischemia.²³ In this context, the lack of effect of aspirin in our experiments may be explained by the relatively short period of ischemia used in the present experiments. Third, it must also be noted that, in our study, occlusion was performed at the level of the wrist, whereas in that of Carlsson and Wennmalm, ischemia involved the whole forearm. Thus, it is possible that the differences in the effect of cyclooxygenase inhibitors may simply be due to differences in the regulation of hand and forearm circulation.

Our results show that local infusion of L-NMMA did not affect the peak but markedly reduced the duration of the hyperemic response, as evidenced by the decrease in both the area under the curve and the time to return to 50% of peak hyperemia. To the best of our knowledge, this is the first study in which the effect of local administration of L-NMMA on hyperemic response was investigated in humans. In dogs, intracoronary administration of NO synthase inhibitors also reduces excess flow and repayment of flow debt after brief ischemia, suggestive of a reduction in the duration of the hyperemic phase.^{14 15} Although we cannot exclude the possibility that this shortening of the hyperemic phase is the consequence of a nonspecific action of L-NMMA secondary to its effect on baseline blood flow, these results in human peripheral arteries confirm results obtained in dogs and would suggest that the synthesis of NO from L-arginine plays a role primarily in the maintenance of reactive hyperemia. On the other hand, the absence of effect of L-NMMA on peak hyperemic response suggests that the initial phase of hyperemia is independent of NO and is probably the consequence of ischemia-induced reduction in oxygen tension in vascular smooth muscle cells, which in turn causes relaxation; alternatively, the decreased myogenic forces secondary to the reduction in perfusion pressure during ischemia may be responsible for the initial increase in flow immediately after release of the cuff.²⁵

The mechanism by which NO could modulate the delayed phase of reactive hyperemia is not clear. One possibility is that NO is released in response to adenosine, which is believed to be a major contributor of reactive hyperemia.²⁶ Indeed, the coronary flow response to adenosine is inhibited by L-NMMA in dogs.¹⁵ Another possibility is that the delayed phase of hyperemia is at least in part the consequence of a flow-mediated vasodilatation at the level of small resistance arteries. In isolated resistance arteries, increases in flow velocity indeed induce endothelium-dependent vasodilatation.^{27 28 29} However, the existence of a similar flow-dependent dilatation of human resistance vessels has not been investigated.

In our experiments, the abrupt increase in flow during hyperemia was followed by a delayed increase in radial artery diameter, in agreement with results previously obtained in the same arterial bed in humans.^{30 31} In the present experiments, the duration of the ischemic period was limited to 3 minutes. As a result, the hyperemia-induced changes in radial flow and diameter were less marked than those observed in hyperemia after longer periods of ischemia.^{30 31} We chose to limit the duration of ischemia to 3 minutes to obtain modest changes in flow and thus in diameter to avoid losing the tracked echo signal during hyperemia, as might have been the case in the event of larger changes in diameter. The relatively modest increase in hyperemic flow in the present study may also be a consequence of the lower metabolic demand of the hand (in the case of a wrist occlusion like the one used here) compared with the traditionally studied forearm. However, despite this short duration of ischemia and moderate increase in flow during hyperemia, the increase in radial diameter measured in the present experiments was 3.6±0.8%, which corresponds to an average increase of 98±25 µm, a value well above the dynamic resolution of the technique. Indeed, Fig 1 shows that the echo-tracking technique allows continuous tracking of the diameter signal throughout the cardiac cycle, even though the systolic-diastolic differences in diameter are <20 µm, suggesting that the dynamic resolution of the system, in our conditions, is well under this 20-µm value. Furthermore, the stability of the observed diameter response was assessed in experiments in which subjects underwent two consecutive hyperemic tests without treatment. In these experiments, we found no significant changes in baseline radial diameter or radial flow or in the response of the radial artery during hyperemia (Fig 2). Thus, we believe that the echo-tracking technique used allows precise evaluation of the hyperemia-induced changes in radial artery diameter as well as an accurate determination of the effects of acute treatments (such as L-NMMA or aspirin) on radial hemodynamics and on flow-dependent dilatation of the radial artery.

Under these conditions, we observed that the flow-dependent vasodilatation of the radial artery was not affected by aspirin but was abolished by L-NMMA, demonstrating that it was independent of the release of vasoactive prostanoids and entirely mediated by NO. This finding confirms, in humans, results previously obtained in vitro^{5 32} or in conscious, chronically instrumented dogs.³³ One intriguing aspect of our results is that administration of L-NMMA unmasked a flow-mediated vasoconstriction of the radial artery. Such a paradoxical vasoconstriction also occurs during exercise in patients with coronary artery disease.³⁴ Hence, inhibition of NO mimics the response of atherosclerotic arteries to increased shear.^{6 7 8} One possible explanation is that flow stimulates the endothelium to release a vasoconstrictor substance (for example endothelin³⁵ ) whose effects are normally masked by the NO-dependent dilatation. The fast time course of the response, however, argues against a role for endothelin. Indeed, in isolated vascular tissue, endothelin is synthetized on a de novo basis, a process that requires 2 to 4 hours.³⁵ Another possibility is that the vasoconstriction could reflect an unmasking of a hyperemia-induced sympathetic vasoconstriction in the presence of L-NMMA. Indeed, NO inhibits sympathetic nerve activity³⁶ as well as norepinephrine-induced vasoconstriction.²⁰ Hence, flow-mediated vasodilatation continuously opposes adrenergic vasoconstriction, as shown in vitro.^{32 37}

In conclusion, the present investigation demonstrates that NO is essential for flow-mediated dilatation of human radial arteries in vivo. Thus, this test can be used as a reliable noninvasive estimate of the capacity of human endothelial cells to release NO in response to a physiological stimulus as well as an estimate of endothelial dysfunction in diseased states.

	Acknowledgments

 
These studies were supported by the Swiss National Science Foundation (grant 32-36575.92), the Schweizerische Rentenanstalt, the Freiwillige Akademische Gesellschaft Basel, Switzerland, and the Association Charles Nicolle, Rouen, France.

Received November 17, 1994; accepted January 3, 1995.

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