Donate Help Contact The AHA Sign In Home
American Heart Association
Circulation
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Circulation. 1995;92:320-326

This Article
Right arrow Abstract Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Quyyumi, A. A.
Right arrow Articles by Cannon, R. O.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Quyyumi, A. A.
Right arrow Articles by Cannon, R. O., III
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*(L)-ARGININE
*NITRIC OXIDE

(Circulation. 1995;92:320-326.)
© 1995 American Heart Association, Inc.

Articles

Contribution of Nitric Oxide to Metabolic Coronary Vasodilation in the Human Heart

Arshed A. Quyyumi, MD, MRCP; Nader Dakak, MD; Neil P. Andrews, MB, BS, MRCP; David M. Gilligan, MD; Julio A. Panza, MD; Richard O. Cannon, III, MD

From the National Institutes of Health, Cardiology Branch, NHLBI, Bethesda, Md, and the Division of Cardiology, Medical College of Virginia, Richmond (D.M.G.).


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMethods
down arrowResults
down arrowDiscussion
down arrowReferences
 
Background The vascular endothelium contributes to smooth muscle relaxation by tonic release of nitric oxide. To investigate the contribution of nitric oxide to human coronary epicardial and microvascular dilation during conditions of increasing myocardial oxygen requirements, we studied the effect of inhibiting nitric oxide synthesis with NG-monomethyl-L-arginine (L-NMMA) on the coronary vasodilation during cardiac pacing in patients with angiographically normal coronary arteries with and without multiple risk factors for coronary atherosclerosis.

Methods and Results In 26 patients with angiographically normal or near-normal epicardial coronary arteries, metabolic vasodilation was assessed as a change in coronary vascular resistance and diameter during cardiac pacing (mean heart rate, 141 beats per minute). Endothelium-dependent vasodilation was estimated with intracoronary acetylcholine and endothelium-independent dilation with intracoronary sodium nitroprusside and adenosine. These measurements were repeated after 64 µmol/min intracoronary L-NMMA. At rest, L-NMMA produced a 16±25% (mean±SD) increase in coronary vascular resistance (P<.05) and an 11% reduction in distal epicardial coronary artery diameter (P<.01), indicating tonic basal release of nitric oxide from human coronary epicardial vessels and microvessels. Significant inhibition of pacing-induced metabolic coronary vascular dilation occurred with L-NMMA, coronary vascular resistance was 38±56% higher (P<.03), and epicardial coronary dilation during control pacing (9±13%) was converted to constriction after L-NMMA and pacing (-6±9%, P<.04). L-NMMA specifically inhibited endothelium-dependent vasodilation with acetylcholine (coronary vascular resistance was 72% higher [P<.01]) but did not alter endothelium-independent dilation with sodium nitroprusside and adenosine. Nine patients had no major risk factors for atherosclerosis, defined as serum cholesterol >240 mg/dL, hypertension, or diabetes. The remaining 17 patients with one or more of these risk factors had depressed microvascular vasodilation during cardiac pacing (coronary vascular resistance decreased by 13% versus 36% in those without risk factors, P<.05). The inhibitory effect of L-NMMA on pacing-induced coronary epicardial and microvascular vasodilation was observed only in patients without risk factors, whereas those with risk factors had an insignificant change, indicating that nitric oxide contributes significantly to pacing-induced coronary vasodilation in patients free of risk factors and without endothelial dysfunction. Patients with risk factors also had reduced vasodilation with acetylcholine (40±28% versus 68±8% decrease in coronary vascular resistance, P<.01), but the responses to sodium nitroprusside were similar in both groups.

Conclusions During metabolic stimulation of the human heart, nitric oxide release contributes significantly to microvascular vasodilation and is almost entirely responsible for the epicardial vasodilation. This contribution of nitric oxide is reduced in patients exposed to risk factors for coronary atherosclerosis and leads to a net reduction in vasodilation during stress. An important implication of these findings is that reduced nitric oxide bioavailability during stress in patients with atherosclerosis or risk factors for atherosclerosis may contribute to myocardial ischemia by limiting epicardial and microvascular coronary vasodilation.


Key Words: endothelium-derived factors • vasodilation


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
down arrowResults
down arrowDiscussion
down arrowReferences
 
The vascular endothelium plays a pivotal role in modulating smooth muscle function by releasing endothelium-derived relaxing and constricting factors.1 2 3 4 5 One important endothelium-derived relaxing factor is nitric oxide, or a compound closely related to nitric oxide.6 7 8 Nitric oxide release can be stimulated by a variety of pharmacological agents, including acetylcholine.3 5 In the presence of normal endothelial function, stimulation of nitric oxide release by acetylcholine produces vasodilation of the coronary epicardial and microvascular circulation. With development of hypercholesterolemia, hypertension, heart failure, diabetes, aging, or atherosclerosis, however, the vasodilator response to acetylcholine is attenuated, denoting the presence of endothelial dysfunction.9 10 11 12 13 14 15 16 17 Human in vitro experiments and animal studies have demonstrated that release of endothelium-derived relaxing factor is also enhanced by physiological stimuli that increase shear in blood vessels,18 19 20 and appears to be at least partly responsible for coronary vasodilation that occurs with increase in myocardial oxygen demand.21 22 23

During exercise or with cardiac pacing, human coronary arteries dilate and coronary blood flow increases, indicating epicardial and microvascular dilation with increase in myocardial oxygen requirements.14 24 25 26 27 28 Microvascular dilation is generally believed to be secondary to accumulation of local metabolic byproducts such as adenosine, lactate, or hydrogen ions.29 30 31 Recent studies have implicated an association between vascular endothelial function and vasomotion during metabolic stress by demonstrating similarities between the epicardial coronary artery behavior with acetylcholine and vasomotion associated with exercise, pacing, or mental stress.26 27 28 32 However, the contribution of nitric oxide to coronary vascular tone during physiological stress and whether this contribution, if present, is altered in the presence of endothelial dysfunction in humans have not been studied to date.

In this study, we investigated the role of nitric oxide in determining coronary epicardial and microvascular vasodilation that accompanies cardiac pacing, and we further determined whether the presence of multiple risk factors for coronary atherosclerosis altered the contribution of endothelium-derived nitric oxide to pacing-induced vasodilation. NG-monomethyl-L-arginine (L-NMMA) an analogue of L-arginine that specifically inhibits nitric oxide synthase,33 was used to inhibit production of nitric oxide, and its effect on the vasodilator response of the coronary vasculature to cardiac pacing was studied.


*    Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
down arrowDiscussion
down arrowReferences
 
Patients
We studied 26 patients with angiographically normal or near-normal (<5% narrowing) coronary arteries who were undergoing diagnostic cardiac catheterization for investigation of chest pain or abnormal noninvasive tests. None of the patients had vasospasm of epicardial coronary arteries in response to either ergonovine or acetylcholine. These patients formed part of a larger group of patients reported previously.17 Patients with previous myocardial infarction or valvular heart disease were excluded. The mean age was 48±12 years; there were 10 men (38%) and 16 women. Eleven patients were hypertensive (blood pressure >140/90 mm Hg) but had no echocardiographic evidence of left ventricular hypertrophy. Hypercholesterolemia (total cholesterol >240 mg/dL) was present in 8 patients, and 6 had diabetes (fasting blood glucose >140 mg/dL and on pharmacological antidiabetic therapy). Nine patients had none of these risk factors, 10 were exposed to one factor, and 7 were exposed to two or more. Cardiac medications were withdrawn for at least 48 hours before and aspirin a week before the study. The study was approved by the National Heart, Lung, and Blood Institute Investigational Review Board, and informed written consent was obtained from all patients.

Protocol
After completion of diagnostic coronary arteriography, a 6F guide catheter was introduced into the left main coronary artery and blood flow velocity was measured with an 0.018-in wire equipped with a Doppler crystal at its tip (Cardiometrics Flowire, Cardiometrics, Inc).34 35 The Doppler flow wire was advanced into either the left main (n=4) or the proximal segment of a major epicardial coronary artery (left anterior descending coronary artery in 18 patients and circumflex coronary artery in 2). The wire tip was carefully positioned in a straight segment of the vessel that was free of any major branches within 1 cm from the tip, produced an adequate flow velocity signal, and could be imaged without overlap from other vessels, thus allowing for quantitative measurements of the coronary artery diameter. All drugs were infused directly into the left main coronary artery via the guide catheter at infusion rates ranging from 1 to 2 mL/min. A 7F multipurpose A2 catheter was inserted via the right internal jugular vein into the mid coronary sinus for blood sampling. Oxygen saturation of arterial and coronary sinus venous blood was measured with an oximeter in 19 patients at baseline and after cardiac pacing with and without L-NMMA.

After a 5-minute infusion of dextrose 5% at 1 mL/min, baseline coronary blood flow velocity was measured and coronary angiography performed (Fig 1Down). Rapid atrial pacing was performed in 22 patients at heart rates ranging from 115 to 150 beats per minute (bpm). Pacing from the right ventricle was performed at 150 bpm in the remaining 4 patients, who developed atrioventricular Wenckebach at rates <115 bpm. Thus, the mean cardiac pacing rate was 141±11 bpm. Blood flow velocity and coronary sinus oxygen measurements and angiography were repeated after 2 minutes of pacing.



View larger version (11K):
[in this window]
[in a new window]
  		Figure 1. Protocol design. ACh 1, 2, and 3 denote the dose-response curves with acetylcholine. ACh 4 and 5 denote the two peak vasodilating doses of ACh that were reinfused after NG-monomethyl-L-arginine (L-NMMA).

Endothelium-dependent vasodilation was estimated by performance of a dose-response curve with incremental infusions of intracoronary acetylcholine (Sigma Ltd) starting at 3 µg/min for 2 minutes. This was followed by 2-minute infusions of 30, 100, and 300 µg/min of intracoronary acetylcholine, with measurement of Doppler flow velocity and angiography after each increment. The dose of acetylcholine was not increased further once an infusion either produced reduced blood flow velocity or severely (>50%) narrowed the epicardial coronary vessels. Thus, all patients received the 30-µg/min dose, 14 received doses up to 100 µg/min, and 12 up to 300 µg/min. The peak flow response with acetylcholine was achieved at the 30-µg/min dose in 17 patients, at the 100-µg/min dose in 8, and at the 300-µg/min dose in 1.

Five minutes after the dose-response curve with acetylcholine was performed, endothelium-independent function was estimated with sodium nitroprusside and adenosine (Fig 1Up). Intracoronary sodium nitroprusside was given at 40 µg/min for 3 minutes, followed by measurement of blood flow velocity and coronary angiography. This was followed by administration of intracoronary adenosine at 2.2 mg/min for 2 minutes.

After a 10-minute interval, while dextrose 5% infusion was continued, repeat baseline measurements of flow velocity, oxygen saturations, and angiography were made (Fig 1Up). This was followed by infusion of L-NMMA (Calbiochem), a specific inhibitor of nitric oxide.32 L-NMMA was infused at 32 µmol/min (0.5 mL/min) for 5 minutes and then increased to 64 µmol/min (1 mL/min) for another 5 minutes.

While the infusion of L-NMMA at 64 µmol/min was continued, cardiac pacing was repeated at the same rate as during the control study. Acetylcholine was readministered at the two highest vasodilating doses in 25 patients for 2 minutes. Eighteen patients had repeat infusion of 40 µg/min sodium nitroprusside for 3 minutes, and 2.2 mg/min adenosine was reinfused in 15 patients for 2 minutes (Fig 1Up). Blood flow velocity was measured and coronary angiography performed after each intervention.

Estimation of Coronary Blood Flow and Diameter
Coronary blood flow was estimated from measurement of coronary blood flow velocity and diameter measurements by the formula {pi}xaverage peak velocityx0.125xdiameter2. Coronary vascular resistance was calculated as mean arterial pressure divided by coronary blood flow.

For calculating flow, coronary artery diameter was measured in a 0.5-cm segment of vessel beginning 0.25 cm beyond the tip of the flow wire. Coronary angiograms were recorded with a cineangiographic system (Toshiba, Inc). Quantitative angiography was performed with ARTEK software (Quantim 200I, STATVIEW, ImageComm Systems, Inc). In addition to measurement of the diameter at the level of the Doppler flow wire, 0.5- to 1-cm segments of the proximal and distal segments of the epicardial coronary arteries were also measured by quantitative coronary angiography by readers blinded to the interventions.

Statistical Analysis
Data are expressed as mean±SD in the text and mean±SEM in figures. Differences between means were compared by paired or unpaired Student's t test, as appropriate. The effects of L-NMMA on the two doses of acetylcholine were compared by ANOVA for repeated measures using a multiple regression model that included dummy variables to correct for between-subject variability.36 The differences between the effects of L-NMMA in patients with and in those without risk factors were compared by use of the percent change from baseline for all parameters because of the baseline differences in regional flow in the two subgroups. All probability values are two-tailed, and a value of P<.05 was considered of statistical significance.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
*Results
down arrowDiscussion
down arrowReferences
 
Effect of L-NMMA on Resting Coronary Vascular Tone
Coronary vascular resistance increased by 16% (4.1± 3.8 to 4.6±4.1 mm Hg · mL-1 · min, P<.05) with 64 µmol/min intracoronary L-NMMA, indicating tonic release of nitric oxide from the coronary microvasculature under resting conditions. This was accompanied by a 6% (P<.01) increase in mean arterial pressure. Epicardial coronary arteries constricted with L-NMMA, resulting in a mean 8% reduction (2.8±0.8 to 2.5±0.8 mm, P<.01) in the proximal and 11% reduction (1.8±0.6 to 1.6±0.6 mm, P<.01) in the distal segments, indicating, as previously reported,17 tonic basal release of nitric oxide from the coronary epicardial vessels.

Effect of L-NMMA on Response to Cardiac Pacing
L-NMMA produced significant inhibition of pacing-induced coronary epicardial and microvascular dilation (Figs 2Down and 3Down). Thus, during the control study, cardiac pacing at 141±11 bpm produced a mean 50% increase in blood flow, 21% reduction in coronary vascular resistance, and a 9% increase in proximal and distal coronary artery diameters (Figs 2Down and 3Down). After L-NMMA, cardiac pacing at 140±12 bpm produced significantly less microvascular vasodilation; the 23% increase in coronary blood flow and the 5% reduction in coronary vascular resistance were not significant. Epicardial vasodilation with cardiac pacing during the control study (18% increase in proximal epicardial coronary artery diameter) was converted to vasoconstriction after L-NMMA (13% reduction in diameter, Figs 2Down and 3Down). Thus, the baseline constriction that occurred with L-NMMA at rest was not overcome by pacing (2.8 mm at baseline, 2.5 mm after L-NMMA [P<.01], and 2.5 mm after L-NMMA and pacing). The reduced vasodilation during cardiac pacing with L-NMMA was also confirmed by the changes in arteriovenous oxygen differences; a 6±14% narrowing of the arteriovenous oxygen difference during the control pacing study was converted to a 5±12% widening (P=.03) of the difference, denoting reduced microvascular vasodilation during pacing after L-NMMA.



View larger version (107K):
[in this window]
[in a new window]
  		Figure 2. A, Data from a patient without any risk factors for atherosclerosis. The left side of each panel depicts the coronary flow velocity signal and the right side, a segment of the left anterior descending coronary artery. Recordings at baseline, after pacing, and after sodium nitroprusside are demonstrated during the control study with dextrose 5%. There is an increase in epicardial artery diameter and flow velocity with both pacing and sodium nitroprusside. APV indicates average peak velocity. B, Data from the same patient after administration of NG-monomethyl-L-arginine (L-NMMA), after L-NMMA and pacing, and after L-NMMA and sodium nitroprusside. Compared with A, there is epicardial constriction with L-NMMA at rest. There is also inhibition of the pacing-induced increases in flow velocity and diameter with L-NMMA. However, there is no inhibition of the sodium nitroprusside–induced dilation with L-NMMA.



View larger version (17K):
[in this window]
[in a new window]
  		Figure 3. Graphs showing effect of NG-monomethyl-L-arginine (L-NMMA) on the pacing-induced changes in coronary vascular resistance, flow, and proximal and distal epicardial coronary artery diameters. Control study is shown by {bullet}, post–L-NMMA study by {circ} (n=26). Data represent mean±SEM. *P<.05, **P<.01, difference between baseline and pacing. Comparisons of changes in these parameters with pacing before versus after L-NMMA are stated as control vs L-NMMA.

Effect of L-NMMA on Response to Acetylcholine
During the control study, there was progressive increase in blood flow and reduction in coronary vascular resistance, indicating microvascular dilation with increasing doses of acetylcholine. Epicardial coronary artery dimension did not change significantly compared with baseline (Fig 4Down). After L-NMMA, repeat infusions of the same doses of acetylcholine produced significant inhibition of microvascular vasodilation and vasoconstriction of epicardial coronary arteries; coronary vascular resistance was 72% greater at the higher dose of acetylcholine after L-NMMA compared with the control study (Fig 4Down). Thus, as previously reported from our laboratory,17 L-NMMA inhibited both epicardial and microvascular vasodilation in response to acetylcholine.



View larger version (18K):
[in this window]
[in a new window]
  		Figure 4. Graphs showing effects of increasing doses of intracoronary acetylcholine on coronary vascular resistance, flow, and percent change in proximal and distal epicardial coronary artery diameters before (control, {bullet}) and after ({circ}) NG-monomethyl-L-arginine (L-NMMA). Two vasodilating doses of acetylcholine were reinfused after L-NMMA (n=25), and the P values denote results of ANOVA comparing the response to acetylcholine before vs after L-NMMA. Data represent mean±SEM.

Effect of L-NMMA on Responses to Sodium Nitroprusside and Adenosine
During the control study, there was significant epicardial and microvascular vasodilation in response to both sodium nitroprusside and adenosine; coronary vascular resistance decreased by 53±19% with sodium nitroprusside and by 73±22% with adenosine in the control study. After L-NMMA, reinfusion of sodium nitroprusside and adenosine at the same intracoronary concentrations produced similar vasodilation of both coronary microvascular and epicardial vessels; coronary vascular resistance was 56±14% lower with sodium nitroprusside and 69±25% lower with adenosine after L-NMMA (both P=NS compared with control). Proximal epicardial coronary artery diameters with sodium nitroprusside before and after L-NMMA (3±1 to 3±1 mm) and with adenosine before and after L-NMMA (2.9±0.9 to 2.7±0.9 mm) were similar (P=NS). Thus, L-NMMA did not inhibit coronary vasodilation in response to the endothelium-independent vasodilators sodium nitroprusside and adenosine.

Impact of Risk Factors for Coronary Atherosclerosis on Response to Cardiac Pacing
Microvascular vasodilation with pacing was greater in patients without risk factors (36% reduction in coronary vascular resistance) compared with those with risk factors (13% reduction, P<.05) (Fig 5Down). The mean pacing heart rate was 137±13 bpm in those without and 146±7 bpm in those with risk factors (P=NS). Epicardial dilation with pacing tended to be greater in patients without risks (Fig 5Down), but the difference did not reach statistical significance (18% increase in proximal diameter in patients without risks versus 5% in those without, P=.09).



View larger version (19K):
[in this window]
[in a new window]
  		Figure 5. Graphs showing percent change in coronary vascular resistance, flow, and proximal and distal epicardial coronary diameters during control pacing and on pacing after NG-monomethyl-L-arginine (L-NMMA). Patients with risk factors (n=17) for coronary atherosclerosis (hypercholesterolemia, hypertension, diabetes) are shown by {circ}, and those without risk factors (n=9) by {bullet}. *P<.05, **P<.01 comparing control pacing vs pacing after L-NMMA in each group. P<.05 compares responses (from baseline to pacing) in patients with vs those without risks. Results are expressed as mean±SEM.

Impact of Risk Factors for Coronary Atherosclerosis on Response to Acetylcholine
The peak vasodilator response with acetylcholine was greater in the 9 patients without risk factors than in those with risk factors for atherosclerosis; coronary vascular resistance decreased by 68±8% in patients with no risk factors, compared with a decrease of 40±28% in patients with risk factors (P<.01). However, the vasodilator response to sodium nitroprusside was not statistically significant (63±13% versus 55±13%, P=NS, no risk factors versus risk factors, respectively).

Effect of L-NMMA on Pacing Response in Patients With and Without Multiple Risk Factors
The coronary epicardial and microvascular vasoconstriction during pacing after L-NMMA, compared with the initial pacing sequence, was observed only in patients without multiple risk factors (Fig 5Up). Thus, proximal epicardial coronary arteries that dilated by 18% during control pacing constricted by 13% on pacing after L-NMMA in patients without risk factors. Similarly, coronary vascular resistance that fell by 36% during control pacing did not change significantly on pacing after L-NMMA (Fig 5Up), indicating that nitric oxide contributes to pacing-induced coronary vascular dilation in this group. In contrast, the changes in epicardial and microvascular dilation with L-NMMA in patients with risk factors were not statistically significant, suggesting that the contribution of nitric oxide to pacing-induced vasodilation is minimal in these patients.

Epicardial and coronary microvascular dilation with pacing after L-NMMA were similar in patients with and without risk factors (Fig 5Up), suggesting that the increased dilation with pacing observed during the control pacing sequence is due to increased nitric oxide activity in patients without risk factors.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
up arrowResults
*Discussion
down arrowReferences
 
Contribution of Nitric Oxide to Metabolic Coronary Vasodilation
Metabolic coronary vasodilation in response to increased myocardial oxygen requirements has long been recognized to be mediated by a variety of local, humoral, and neural factors.30 31 32 Microvascular dilation is believed to be primarily due to accumulation of local metabolites, including adenosine, prostaglandins, carbon dioxide, and hypoxia, and partly due to circulating catecholamines and withdrawal of sympathetic tone. Epicardial vessel dilation, on the other hand, is considered to be secondary not only to the humoral and neural factors stated above but more importantly to flow-mediated dilation. Recent experimental studies have shown that flow-mediated conductance vessel dilation is due to release of endothelium-derived relaxing factor.18 19 20 37 38 Studies in human coronary arteries have also demonstrated flow-mediated dilation of epicardial coronary arteries that is abolished in atherosclerosis.39 40 That the endothelium may also contribute to microvascular vasodilation during a metabolic stimulus such as cardiac pacing was suggested from our previous study in a similar population of patients with normal coronary angiograms.14 In that investigation, patients with a depressed vasodilator response to acetylcholine also had a depressed dilator response with atrial pacing, a finding that was confirmed in this study. To investigate whether the apparent relation between metabolic and endothelium-dependent vasodilation observed previously was due to stimulation of release of endothelium-derived nitric oxide during stress, we conducted this study using L-NMMA as a specific inhibitor of nitric oxide synthesis. Our findings demonstrate that nitric oxide contributes significantly to both epicardial and coronary microvascular vasodilation, which accompanies conditions that increase myocardial oxygen requirements. In patients without risk factors for atherosclerosis, coronary vascular resistance during pacing after L-NMMA was significantly higher compared with pacing alone, and pacing-induced epicardial vessel dilation was abolished by L-NMMA. These findings are compatible with our previous report on the contribution of nitric oxide to exercise-induced vasodilation in the forearm.41 Forearm vascular resistance was 26% higher during exercise after L-NMMA compared with exercise alone. Taken together, these studies suggest that endothelium-derived release of nitric oxide from both the coronary and peripheral vasculatures contributes to metabolic vasodilation.

Specificity of the Response to L-NMMA
As reported previously in other vascular beds and in the coronary circulation,17 42 43 44 45 L-NMMA inhibited both the epicardial and microvascular vasodilator responses to acetylcholine. This inhibition was specific for nitric oxide, because dilation in response to sodium nitroprusside and adenosine were not affected by L-NMMA. In light of the specificity of the effect of L-NMMA on endothelium-dependent dilation, the attenuation of pacing-induced vasodilation by L-NMMA indicates that cardiac pacing produced vasodilation of epicardial and coronary microvessels at least in part by an endothelium-dependent mechanism. Thus, the increase in coronary vascular resistance and epicardial coronary constriction during pacing after L-NMMA indicates that nitric oxide contributes to coronary vascular dilation that accompanies conditions associated with increases in myocardial oxygen demands.

Contribution of Non–Nitric Oxide Mediators to Metabolic Coronary Vasodilation
Our study also demonstrates that there is persistent coronary microvascular dilation with cardiac pacing after administration of L-NMMA; coronary vascular resistance decreased from 4.6±4 mm Hg · mL-1 · min after L-NMMA at rest to 4.0±4.1 mm Hg · mL-1 · min after L-NMMA and pacing, confirming the presence of non–nitric oxide–related mechanisms that contribute to metabolic vasodilation of the coronary microvasculature in humans. An alternative explanation for this finding is that L-NMMA, a competitive antagonist of nitric oxide synthase, does not completely block production of nitric oxide from the microvascular endothelium in the dose given. In contrast to the microcirculation, epicardial coronary arteries did not dilate with pacing after L-NMMA compared with pacing alone, suggesting that coronary epicardial vasodilation during metabolic stimulation of the human heart is likely to be mediated entirely by the endothelium-derived release of nitric oxide.

Influence of Risk Factors for Coronary Atherosclerosis
Patients exposed to multiple risk factors for atherosclerosis had a depressed vasodilator response to acetylcholine compared with those without risk factors, a finding that is compatible with previous studies.11 14 15 16 Whether the abnormality of pharmacological release of endothelium-derived relaxing factor is indicative of diminished nitric oxide activity during physiological stress was the subject of the present study. In this study, we not only demonstrate that endothelium-derived nitric oxide contributes to metabolic vasodilation but also show that this contribution is significant only in patients without risk factors. These patients had suppression of pacing-induced vasodilation with L-NMMA. Indeed, the similarity in vascular responses to pacing after L-NMMA in both groups suggests that the greater vasodilation in patients without risks during control pacing was due to higher nitric oxide activity in these patients. These observations suggest that endothelial dysfunction manifested in patients with risk factors leads to a net reduction in coronary vasodilation during stress due to diminished nitric oxide activity. Diminished vasodilation during stress may, in turn, contribute to the development of myocardial ischemia in patients with endothelial dysfunction.

Our results are compatible with those of previous studies demonstrating that segments of epicardial coronary arteries that have a constrictor response to intracoronary acetylcholine also react abnormally in response to stress such as atrial pacing, cold pressor test, and mental stress26 27 28 29 30 and extend these findings to the coronary microcirculation. Moreover, this study, for the first time, specifically links the previously observed association between stress-induced epicardial coronary dilation and the acetylcholine response to nitric oxide activity in the human coronary epicardial and microvascular circulation.

Limitations
The exact mechanism of the depression in nitric oxide activity observed in our study cannot be determined from our findings. It may be due to factors that affect the signal transduction pathway,46 to a defect in the nitric oxide synthase enzyme itself, or to a lower rate of synthesis of nitric oxide, which may in turn be a result of substrate deficiency.47 48 Alternatively, it may be secondary to increased breakdown of normally produced nitric oxide by superoxide anions.49 50 We are also unable to determine, in this relatively small group of patients, whether any one or more of the risk factors are more or less important in precipitating the abnormality in bioavailability of nitric oxide during stress.

Implications
This study defines the physiological role of endothelium-derived nitric oxide released from coronary epicardial vessels and microvessels during metabolic stimulation of the human heart. Although abnormalities in the response to acetylcholine in epicardial coronary arteries10 11 12 13 26 27 28 29 and in microvessels51 has been noted in patients with atherosclerosis, we demonstrate reduced coronary vascular nitric oxide bioavailability, both at rest and after stress, in patients with angiographically normal-appearing coronary arteries who have been exposed to multiple risk factors for atherosclerosis. These findings may have important implications: reduced coronary vasodilation in patients with multiple risk factors and normal epicardial coronary arteries may precipitate ischemia and might account for chest pain in some patients with microvascular angina.14 52 53 In patients with established atherosclerosis, also known to be associated with endothelial dysfunction, coronary blood flow increase during stress not only is limited by epicardial stenoses but also may be additionally compromised by diminished microvascular dilation. Moreover, reduced nitric oxide activity from the coronary vasculature may contribute to accelerated progression of atherosclerosis due to an absence in the antiproliferative effects of nitric oxide.54 55 56


*    Acknowledgments
 
The authors are grateful for the technical assistance of William Schenke, Gregory Johnson, and Rita Mincemoyer, RN.


*    Footnotes
 
Reprint requests to Arshed A. Quyyumi, MD, National Institutes of Health, Cardiology Branch, NHLBI, Bldg 10, Room 7B-15, 10 Center Dr, MSC 1650, Bethesda, MD 20892-1650.

Received November 14, 1994; revision received January 19, 1995; accepted January 28, 1995.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
up arrowResults
up arrowDiscussion
*References
 

  1. Furchgott RF, Zawadzki JV. The obligatory role of the endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature. 1980;288:373-376. [Medline] [Order article via Infotrieve]
  2. Vanhoutte PM. The endothelium: modulator of vascular smooth muscle tone. N Engl J Med. 1988;319:512-513. [Medline] [Order article via Infotrieve]
  3. Feletou M, Vanhoutte PM. Endothelium-dependent hyperpolarization of canine coronary smooth muscle. Br J Pharmacol. 1988;93:515-524. [Medline] [Order article via Infotrieve]
  4. Cocks TM, Angus JA. Endothelium-dependent relaxation of coronary arteries by noradrenaline and serotonin. Nature. 1983;305:627-630. [Medline] [Order article via Infotrieve]
  5. De Mey JG, Claeys M, Vanhoutte PM. Endothelium-dependent inhibitory effects of acetylcholine, adenosine triphosphate, thrombin and arachidonic acid in the canine femoral artery. J Pharmacol Exp Ther. 1982;222:166-173. [Abstract/Free Full Text]
  6. Palmer RM, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature. 1987;327:524-526. [Medline] [Order article via Infotrieve]
  7. Ignarro LJ, Byrns RE, Buga GM, Wood KS. Endothelium-derived relaxing factor from pulmonary artery and vein possesses pharmacologic and chemical properties identical to those of nitric oxide radical. Circ Res. 1987;61:866-879. [Abstract/Free Full Text]
  8. Kelm M, Schrader J. Control of coronary vascular tone by nitric oxide. Circ Res. 1990;66:1561-1575. [Abstract/Free Full Text]
  9. Lefer AM, Ma X-L. Decreased basal nitric oxide release in hypercholesterolemia increases neutrophil adherence to rabbit coronary artery endothelium. Arterioscler Thromb. 1993;13:771-776. [Abstract/Free Full Text]
  10. Ludmer PL, Selwyn AP, Shook TL, Wayne RR, Mudge GH, Alexander RW, Ganz P. Paradoxical vasoconstriction induced by acetylcholine in atherosclerotic coronary arteries. N Engl J Med. 1986;315:1046-1051. [Abstract]
  11. Zeiher AM, Drexler H, Wollschläger H, Just H. Modulation of coronary vasomotor tone in humans: progressive endothelial dysfunction with different early stages of coronary atherosclerosis. Circulation. 1991;83:391-401. [Abstract/Free Full Text]
  12. Hodgson JM, Marshall JJ. Direct vasoconstriction and endothelium-dependent vasodilation: mechanism of acetylcholine effects on coronary flow and arterial diameter in patients with non-stenotic coronary arteries. Circulation. 1989;79:1043-1051. [Abstract/Free Full Text]
  13. Newman CM, Maseri A, Hackett DR, El-Tamimi HM, Davies GJ. Responses of angiographically normal and atherosclerotic left anterior descending coronary arteries to acetylcholine. Am J Cardiol. 1990;66:1070-1076. [Medline] [Order article via Infotrieve]
  14. Quyyumi AA, Cannon RO, Panza JA, Diodati JG, Epstein SE. Endothelial dysfunction in patients with chest pain and normal coronary arteries. Circulation. 1992;86:1864-1871. [Abstract/Free Full Text]
  15. Vita JA, Treasure CB, Nabel EG, McLenachan JM, Fish RD, Yeung AC, Vekshtein VI, Selwyn AP, Ganz P. Coronary vasomotor response to acetylcholine relates to risk factors for coronary artery disease. Circulation. 1990;81:491-497. [Abstract/Free Full Text]
  16. Egashira K, Inou T, Hirooka Y, Yamade A, Maruoka Y, Kai H, Sugimachi M, Suzuki J, Takeshita A. Impaired coronary blood flow response to acetylcholine in patients with coronary risk factors and proximal atherosclerotic lesions. J Clin Invest. 1993;91:29-37.
  17. Quyyumi AA, Dakak N, Andrews NP, Husain S, Arora S, Gilligan DM, Panza JA, Cannon RO III. Nitric oxide activity in the human coronary circulation: impact of risk factors for coronary atherosclerosis. J Clin Invest. 1995;95:1747-1755.
  18. Holtz J, Forstermann U, Pohl U, Giesler M, Bassenge E. Flow-dependent, endothelium-mediated dilatation of epicardial coronary arteries in conscious dogs: effects of cyclooxygenase inhibition. J Cardiovasc Pharmacol. 1984;6:1161-1169. [Medline] [Order article via Infotrieve]
  19. Rubanyi GM, Romero JC, Vanhoutte PM. Flow-induced release of endothelium-derived relaxing factor. Am J Physiol. 1986;250:H1145-H1149.[Abstract/Free Full Text]
  20. Lamontagne D, Pohl V, Busse R. Mechanical deformation of vessel wall and shear stress determine the basal release of endothelium-derived relaxing factor in the intact rabbit coronary vascular bed. Circ Res. 1992;70:123-130. [Abstract/Free Full Text]
  21. Yamabe H, Okumura K, Ishizaka H, Tsuchiya T, Yasue H. Role of endothelium-induced nitric oxide in myocardial reactive hyperemia. Am J Physiol. 1992;263:H8-H14. [Abstract/Free Full Text]
  22. Bassenge E. Endothelium-mediated regulation of coronary tone. In: Drexler H, Zeiher AM, Bassenge E, Just H, eds. Endothelial Mechanisms of Vasomotor Control. Darmstadt, Germany: Steinkopff Verlag; 1991:69-75.
  23. Kuo L, Chilian WM, Davis MJ. Interaction of pressure- and flow-induced responses in porcine coronary resistance vessels. Am J Physiol. 1991;261:H1706-H1715. [Abstract/Free Full Text]
  24. Gage JE, Hess OM, Murakami T, Ritter M, Gramm J, Krayenbuehl HP. Vasoconstriction of stenotic coronary arteries during dynamic exercise in patients with classic angina pectoris: reversibility by nitroglycerin. Circulation. 1986;73:865-876. [Abstract/Free Full Text]
  25. Gaglione A, Hess OM, Corin WJ, Ritter H, Grimm J, Krayenbuehl HP. Is there coronary vasoconstriction after intracoronary beta-adrenergic blockade in patients with coronary artery disease? J Am Coll Cardiol. 1987;10:299-310. [Abstract]
  26. Nabel EG, Selwyn AP, Ganz P. Paradoxical narrowing of atherosclerotic arteries induced by increases in heart rate. Circulation. 1990;81:850-859. [Abstract/Free Full Text]
  27. Gordon JB, Ganz P, Nabel EG, Fish RD, Zebede J, Mudge GH, Alexander RW, Selwyn AP. Atherosclerosis influences the vasomotor response of epicardial coronary arteries to exercise. J Clin Invest. 1989;83:1946-1952.
  28. Nabel EG, Ganz P, Gordon JB, Alexander RW, Selwyn AP. Dilation of normal and constriction of atherosclerotic coronary arteries caused by the cold pressor test. Circulation. 1988;77:43-52. [Abstract/Free Full Text]
  29. Rubio R, Berne RM. Regulation of coronary blood flow. Prog Cardiovasc Dis. 1975;18:105-122. [Medline] [Order article via Infotrieve]
  30. Klocke FJ. Coronary blood flow in man. Prog Cardiovasc Dis. 1976;19:117-166. [Medline] [Order article via Infotrieve]
  31. Case RB, Greenberg H. The response of canine coronary vascular resistance to local alterations in coronary arterial pCO2. Circ Res. 1976;39:558-566. [Abstract/Free Full Text]
  32. Yeung AC, Vekshtein VI, Krantz DS, Vita JA, Ryan TJ, Ganz P, Selwyn AP. The effect of atherosclerosis on the vasomotor response of coronary arteries to mental stress. N Engl J Med. 1991;325:1551-1556. [Abstract]
  33. Rees DD, Palmer RM, Hodson HF, Moncada S. A specific inhibitor of nitric oxide formation from L-arginine attenuates endothelium-dependent relaxation. Br J Pharmacol. 1989;96:418-424. [Medline] [Order article via Infotrieve]
  34. Ofili E, Kern MJ, Tatineni S, Deligonul U, Aguirre F, Serota H, Labovitz AJ. Detection of coronary collateral flow by a Doppler-tipped guide wire during coronary angioplasty. Am Heart J. 1991;122:221-225. [Medline] [Order article via Infotrieve]
  35. Doucette JW, Corl D, Payne HM, Flynn AE, Goto M, Nassi M, Segal J. Validation of a Doppler guide wire for intravascular measurement of coronary artery flow velocity. Circulation. 1992;85:1899-1911. [Abstract/Free Full Text]
  36. Glantz SA, Slinker BK. Repeated measures. In: Glantz SA, Slinker BK, eds. Primer of Applied Regression and Analysis of Variance. New York, NY: McGraw-Hill; 1990:381-463.
  37. Kuo L, Davis MJ, Chilian WM. Endothelium-dependent, flow-induced dilation of isolated coronary arterioles. Am J Physiol. 1990;259:H1063-H1070. [Abstract/Free Full Text]
  38. Griffith TM, Edwards DH. Myogenic autoregulation of flow may be inversely related to endothelium-derived relaxing factor activity. Am J Physiol. 1990;258:H1171-H1180. [Abstract/Free Full Text]
  39. Cox DA, Vita JA, Treasure CB, Fish RD, Alexander RW, Ganz OP, Selwyn AP. Atherosclerosis impairs flow-mediated dilatation of coronary arteries in humans. Circulation. 1989;80:458-465. [Abstract/Free Full Text]
  40. Drexler H, Zeiher AM, Wollschlager H, Meinertz T, Just H, Bonzel T. Flow-dependent coronary artery dilation in humans. Circulation. 1989;80:466-474. [Abstract/Free Full Text]
  41. Gilligan DM, Panza JA, Kilcoyne CM, Waclawiw MA, Casino PR, Quyyumi AA. Contribution of endothelium-derived nitric oxide to exercise-induced vasodilation. Circulation. 1994;90:2853-2858. [Abstract/Free Full Text]
  42. Tschudi M, Richard V, Buhler FR, Luscher TF. Importance of endothelium-derived nitric oxide in porcine coronary resistance arteries. Am J Physiol. 1991;260:H13-H20. [Abstract/Free Full Text]
  43. Parent R, Pare R, Lavallee M. Contribution of nitric oxide to dilation of resistance coronary vessels in conscious dogs. Am J Physiol. 1992;262:H10-H16. [Abstract/Free Full Text]
  44. Komarv T, Lamping KG, Eastham CL, Harrison DG, Marcos ML, Dellsperger KC. Effect of an arginine analogue on acetylcholine-induced coronary microvascular dilatation in dogs. Am J Physiol. 1991;261:H2001-H2007. [Abstract/Free Full Text]
  45. Ishizaka H, Okumura K, Yamabe H, Tsuchiya T, Yasue H. Endothelium-derived nitric oxide as a mediator of acetylcholine-induced coronary vasodilation in dogs. J Cardiovasc Pharmacol. 1991;18:665-669. [Medline] [Order article via Infotrieve]
  46. Flavahan NA. Atherosclerosis or lipoprotein-induced endothelial dysfunction: potential mechanisms underlying reduction in EDRF/nitric oxide activity. Circulation. 1992;85:1927-1938. [Free Full Text]
  47. Drexler H, Zeiher AM, Meinertz T, Just H. Correction of endothelial dysfunction in coronary microcirculation of hypercholesterolemic patients by L-arginine. Lancet. 1991;338:1546-1550. [Medline] [Order article via Infotrieve]
  48. Creager MA, Girerd XJ, Gallagher SH, Coleman S, Dzau VJ, Cooke JP. L-Arginine improves endothelium-dependent vasodilation in hypercholesterolemic humans. J Clin Invest. 1992;90:1248-1253.
  49. Ohara Y, Peterson TE, Harrison D. Hypercholesterolemia increases superoxide anion production by the endothelium. Circulation. 1992;86(suppl I):I-222. Abstract.
  50. Minor RL, Myers PR, Guerra R Jr, Bates JN, Harrison DG. Diet-induced atherosclerosis increases the release of nitrogen oxides from rabbit aorta. J Clin Invest. 1990;86:2109-2116.
  51. Zeiher AM, Drexler H, Wollschläger H, Just H. Endothelial dysfunction of the coronary microvasculature is associated with impaired coronary blood flow regulation in patients with early atherosclerosis. Circulation. 1991;84:1984-1992. [Abstract/Free Full Text]
  52. Egashira K, Inou T, Hirooka Y, Kai H, Sugimachi M, Suzuki S, Kuga T, Urabe Y, Takeshita A. Effects of age on endothelium-dependent vasodilation of resistance coronary arteries by acetylcholine in humans. Circulation. 1993;88:77-81. [Abstract/Free Full Text]
  53. Egashira K, Inou T, Hirooka Y, Yamada A, Urabe Y, Takeshita A. Evidence of impaired endothelium-dependent coronary vasodilation in patients with angina pectoris and normal coronary angiograms. N Engl J Med. 1993;328:1659-1664. [Abstract/Free Full Text]
  54. Garg UC, Hassid A. Nitric oxide-generating vasodilators and 8-bromo-cyclic guanosine monophosphate inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells. J Clin Invest. 1989;83:1774-1777.
  55. Cayatte AJ, Palacino JJ, Horten K, Cohen RA. Chronic inhibition of nitric oxide production accelerates neointima formation and impairs endothelial function in hypercholesterolemic rabbits. Arterioscler Thromb. 1994;14:753-759. [Abstract/Free Full Text]
  56. Naruse K, Shimizu M, Muramatsu M, Toki Y, Miyazaki Y, Okumura K, Hashimoto H, Ito T. Long-term inhibition of NO synthesis promotes atherosclerosis in the hypercholesterolemic rabbit thoracic aorta. Arterioscler Thromb. 1994;14:746-752.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 HypertensionHome page
S. Lavi, E. H. Yang, A. Prasad, V. Mathew, G. W. Barsness, C. S. Rihal, L. O. Lerman, and A. Lerman
The Interaction Between Coronary Endothelial Dysfunction, Local Oxidative Stress, and Endogenous Nitric Oxide in Humans
Hypertension, January 1, 2008; 51(1): 127 - 133.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
D. J. Duncker and D. Merkus
Exercise hyperaemia in the heart: the search for the dilator mechanism
J. Physiol., September 15, 2007; 583(3): 847 - 854.
[Abstract] [Full Text] [PDF]

Home page
 J Am Coll CardiolHome page
B. P. Choudhary, C. Antoniades, A. F. Brading, A. Galione, K. Channon, and D. P. Taggart
Diabetes Mellitus as a Predictor for Radial Artery Vasoreactivity in Patients Undergoing Coronary Artery Bypass Grafting
J. Am. Coll. Cardiol., September 11, 2007; 50(11): 1047 - 1053.
[Abstract] [Full Text] [PDF]

Home page
 CirculationHome page
M. J. Raher, H. Thibault, K. K. Poh, R. Liu, E. F. Halpern, G. Derumeaux, F. Ichinose, W. M. Zapol, K. D. Bloch, M. H. Picard, et al.
In Vivo Characterization of Murine Myocardial Perfusion With Myocardial Contrast Echocardiography: Validation and Application in Nitric Oxide Synthase 3 Deficient Mice
Circulation, September 11, 2007; 116(11): 1250 - 1257.
[Abstract] [Full Text] [PDF]

Home page
 HeartHome page
D. Neglia, R. De Maria, S. Masi, M. Gallopin, P. Pisani, S. Pardini, A. Gavazzi, A. L'Abbate, and O. Parodi
Effects of long-term treatment with carvedilol on myocardial blood flow in idiopathic dilated cardiomyopathy
Heart, July 1, 2007; 93(7): 808 - 813.
[Abstract] [Full Text] [PDF]

Home page
 Vasc MedHome page
J. T. Kuvin, A. Mammen, P. Mooney, A. A. Alsheikh-Ali, and R. H. Karas
Assessment of peripheral vascular endothelial function in the ambulatory setting
Vascular Medicine, February 1, 2007; 12(1): 13 - 16.
[Abstract] [PDF]

Home page
 HypertensionHome page
A. A. Quyyumi and M. Ozkor
Vasodilation by Hyperpolarization: Beyond NO
Hypertension, December 1, 2006; 48(6): 1023 - 1025.
[Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
C. Kusmic, G. Lazzerini, F. Coceani, R. Barsacchi, A. L'Abbate, and G. Sambuceti
Paradoxical coronary microcirculatory constriction during ischemia: a synergic function for nitric oxide and endothelin
Am J Physiol Heart Circ Physiol, October 1, 2006; 291(4): H1814 - H1821.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
Y. Neishi, S. Mochizuki, T. Miyasaka, T. Kawamoto, T. Kume, R. Sukmawan, M. Tsukiji, Y. Ogasawara, F. Kajiya, T. Akasaka, et al.
Evaluation of bioavailability of nitric oxide in coronary circulation by direct measurement of plasma nitric oxide concentration
PNAS, August 9, 2005; 102(32): 11456 - 11461.
[Abstract] [Full Text] [PDF]

Home page
 CirculationHome page
E. Barbato, F. Piscione, J. Bartunek, G. Galasso, P. Cirillo, G. De Luca, G. Iaccarino, B. De Bruyne, M. Chiariello, and W. Wijns
Role of {beta}2 Adrenergic Receptors in Human Atherosclerotic Coronary Arteries
Circulation, January 25, 2005; 111(3): 288 - 294.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
D. J Green, A. Maiorana, G. O'Driscoll, and R. Taylor
Effect of exercise training on endothelium-derived nitric oxide function in humans
J. Physiol., November 15, 2004; 561(1): 1 - 25.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
J. D. Tune, M. W. Gorman, and E. O. Feigl
Matching coronary blood flow to myocardial oxygen consumption
J Appl Physiol, July 1, 2004; 97(1): 404 - 415.
[Abstract] [Full Text] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
H. M. O. Farouque, S. G. Worthley, and I. T. Meredith
Effect of ATP-Sensitive Potassium Channel Inhibition on Coronary Metabolic Vasodilation in Humans
Arterioscler. Thromb. Vasc. Biol., May 1, 2004; 24(5): 905 - 910.
[Abstract] [Full Text]

Home page
 ANGIOLOGYHome page
J. Gellman, J. M. Hare, C. J. Lowenstein, G. Gerstenblith, V. Coombs, P. Langenberg, J. A. Brinker, and J. R. Resar
L-Arginine Ameliorates the Abnormal Sympathetic Response of the Dysfunctional Human Coronary Microvasculature
Angiology, January 1, 2004; 55(1): 1 - 8.
[Abstract] [PDF]