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Circulation. 1997;95:104-110

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(Circulation. 1997;95:104-110.)
© 1997 American Heart Association, Inc.

Articles

Coronary Vascular Nitric Oxide Activity in Hypertension and Hypercholesterolemia

Comparison of Acetylcholine and Substance P

Arshed A. Quyyumi, MD, MRCP; David Mulcahy, MD, MRCPI; Neil P. Andrews, MD; Syed Husain, MD; Julio A. Panza, MD; Richard O. Cannon, III, MD

the National Institutes of Health, Cardiology Branch, NHLBI, Bethesda, Md.


*    Abstract
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 *Abstract
down arrowIntroduction
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 down arrowResults
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Background Whether the abnormal responses of the human coronary circulation to acetylcholine in patients with hypertension and hypercholesterolemia extend to other, nonmuscarinic stimulators of the endothelium and whether this signifies a specific abnormality of NO is not known.

Methods and Results We studied 26 patients with angiographically normal coronary arteries, 10 without risk factors, and 16 with either hypertension (n=9) and/or hypercholesterolemia (n=10). Dose-response curves were performed with acetylcholine, substance P, and sodium nitroprusside before and after NG-monomethyl-L-arginine (L-NMMA). Substance P produced predominantly epicardial coronary dilation, whereas the dilating effect of acetylcholine was mainly microvascular. There was no correlation between the responses to the two drugs. L-NMMA did not affect the response to sodium nitroprusside, but it suppressed dilation in response to both substance P and acetylcholine, suggesting that the latter promote bioavailability of NO from the coronary vascular endothelium. Compared with patients without risks, those with hypercholesterolemia and hypertension had significantly reduced vasodilation with substance P: 21% versus 12.6% (P=.004) increase in epicardial coronary diameter and 35% versus 19% (P<.05) decrease in vascular resistance. Similar differences were noted with acetylcholine but not with sodium nitroprusside or adenosine. Epicardial and microvascular dilations with substance P or acetylcholine after L-NMMA were similar in patients with and without risk factors, indicating that the reduced effect of endothelium-dependent vasodilators in those with hypertension and hypercholesterolemia is due to diminished NO activity.

Conclusions (1) Substance P– and acetylcholine-induced coronary vasodilation, like that to acetylcholine, is at least partly due to stimulation of NO activity, indicating that the dysfunction of the coronary vascular endothelial cell layer is not restricted to muscarinic receptors. (2) Hypertension and hypercholesterolemia are associated with depression of both basal and pharmacologically stimulated bioavailability of NO.


Key Words: endothelium • hypercholesterolemia • acetylcholine • endothelium-derived factors • hypertension


*    Introduction
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 *Introduction
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The vascular endothelium modulates smooth muscle tone by releasing a variety of dilating and constricting factors.1 2 3 4 Studies of human endothelial function have relied largely on the use of acetylcholine as a probe for stimulating endothelial production of nitric oxide. Vasodilation in response to acetylcholine is considered to represent normal endothelial function, and constriction or reduced dilation to be a sign of dysfunctional endothelium.5 6 However, the vasodilation in response to acetylcholine may not be entirely due to release of endothelium-derived nitric oxide. For example, acetylcholine is known to release endothelium-derived hyperpolarizing factor3 and prostacyclin in some species.7 In addition, NG-monomethyl-L-arginine (L-NMMA), an inhibitor of nitric oxide synthesis, does not completely abolish acetylcholine-induced microvascular dilation.8 9 10 Moreover, acetylcholine may also stimulate production of endothelium-derived contracting factor,11 and in addition, it causes direct smooth muscle constriction.1 2 3 4 5 6 Thus, the net effect of acetylcholine on the vasculature is due to a combination of its effects described above. Furthermore, some investigations have illustrated discrepancies between the coronary vascular effects of acetylcholine and those of substance P,12 13 14 15 16 17 a nonmuscarinic endothelium-dependent vasodilator that acts via stimulating tachykinin receptors on the endothelial cell surface.

We performed this investigation in humans to examine (1) whether the abnormal coronary vascular responses previously described in hypertension and hypercholesterolemia with acetylcholine also extend to other, nonmuscarinic endothelial surface receptors and (2) whether substance P releases nitric oxide from the human coronary circulation.


*    Methods
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 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
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Patients
We studied 26 patients, 8 male, with angiographically normal epicardial coronary arteries who were undergoing cardiac catheterization for investigation of chest pain or abnormal noninvasive tests (TableDown). Patients with moderately severe left ventricular dysfunction (ejection fraction <35%) and those with valvular heart disease were excluded. Sixteen patients had either hypercholesterolemia (total cholesterol >250 mg/dL or HDL <30 mg/dL, n=10) or hypertension (blood pressure >140/90 mm Hg, n=9). The remaining 10 patients did not have hypercholesterolemia or hypertension, were <60 years old, and had normal left ventricular function. The clinical characteristics of the two groups are shown in the TableDown. All cardiac medications were withdrawn at least 48 hours before the study, and aspirin and other cyclooxygenase inhibitors were withdrawn for at least 7 days before.


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  		Table 1. Patient Characteristics

Protocol
After diagnostic coronary angiography was performed, a 6F guide catheter was introduced into the proximal segment of the left coronary artery, and blood flow velocity was measured with a 0.018-in wire equipped with a Doppler crystal at its tip (Cardiometrics Flowire, Cardiometrics, Inc).10 18 The Doppler wire was advanced into either the left main coronary artery (n=3), the proximal segment of the left anterior descending coronary artery (n=12), or the circumflex coronary artery (n=4). The wire tip was carefully positioned in a segment of the vessel that was straight and free of any major branches within 1 cm from the tip, that produced an adequate flow velocity signal, and that 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 between 1 and 2 mL/min.

After a 5-minute infusion of dextrose 5% at 1 mL/min, baseline coronary blood flow velocity and coronary angiography were performed (Fig 1Down). Endothelium-dependent vasodilation was estimated by dose-response curves performed with incremental 2-minute infusions of intracoronary acetylcholine and substance P. The order of the two agents was randomized so that 12 patients received acetylcholine first and the remaining subjects received substance P first. Acetylcholine was started at 30 µg/min, increasing to 100 and 300 µg/min. The dose of acetylcholine was not increased further once the infusion either reduced blood flow velocity or severely (>50%) narrowed the epicardial coronary tree. Thus, in all patients, the 30-µg/min and the 100-µg/min doses were given, and 6 patients received up to 300 µg/min of acetylcholine. Substance P was started at 5 pmol/min in 19 patients and at 10 pmol/min in 7 patients and was increased to 20 pmol/min in all patients and to 40 pmol/min in 6 patients. Thus, all patients received the 10- and 20-pmol/min infusions. Because a systemic effect was observed at the 40-pmol/min dose, this dose was not given to all patients. All infusions were given for 2 to 2.5 minutes. Measurement of Doppler flow velocity and angiography were performed after each infusion.



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  		Figure 1. Protocol design. L-NMMA indicates NG-monomethyl-L-arginine.

Endothelium-independent function was estimated with sodium nitroprusside, and flow reserve was measured with adenosine. Intracoronary sodium nitroprusside was given at 40 µg/min for 3 to 4 minutes, followed by measurement of flow velocity and coronary angiography (n=20). This was followed by administration of intracoronary adenosine at 2.2 mg/min for 2 minutes (n=25). Peak flow velocity measurement and angiography were repeated.

After a 10-minute interval, while dextrose 5% infusion was continued, repeat baseline measurements of flow velocity and angiography were made. This was followed by infusion of L-NMMA (Clinalfa AG), a specific inhibitor of nitric oxide synthesis from L-arginine (n=23). L-NMMA was infused at 32 µmol/min (0.5 mL/min) for 5 minutes and increased to 64 µmol/min (1 mL/min) for another 5 minutes. Blood flow velocity and angiography were performed at each stage.

While the infusion of L-NMMA at 64 µmol/min was continued, the peak dose of acetylcholine was readministered (30 µg/min in 12 patients, 100 µg/min in 9, and 300 µg/min in 1). Substance P was repeated at 20 pmol/min in 21 patients. Twenty-one patients had repeat infusions of 40 µg/min sodium nitroprusside for 3 to 4 minutes. Blood flow velocity was measured and coronary angiography performed after each intervention.

The study was approved by the Institutional Review Board of the National Heart, Lung, and Blood Institute.

Measurement of Coronary Blood Flow and Diameter
Coronary blood flow was derived from the coronary blood flow velocity and diameter measurements by the formula {pi}xaverage peak velocityx0.125xdiameter2.18 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 Doppler wire. Coronary angiograms were obtained with low-osmolar, low-ionic-contrast agent (Hexabrix, Mallinckrodt Medical Inc) and were obtained immediately after aspiration of the infused drug from the guide catheter. Quantitative angiography was performed with ARTEK software (Quantim 2001, Statview, ImageComm Systems, Inc). In addition to measurement of the diameter at the level of the Doppler wire, 0.5- to 1-cm segments of mid and distal regions of the epicardial coronary arteries were also measured. A total of 78 segments (3 in each patient) were measured.

There were no significant differences between the mean baseline diameter or regional coronary vascular resistance in patients with and without risk factors. Peak vasodilator responses with substance P were observed at similar concentrations in the two groups: 17.5±5 pmol/min in patients without risks and 16.7±6 pmol/min in patients with risks. Similarly, the peak microvascular vasodilating dose of acetylcholine was similar in the two groups: 74±82 µg/min in patients without risks and 68±39 µg/min in patients with risks.

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 differences between the effects of L-NMMA in patients with and without risk factors were compared by use of the percent change from baseline for all parameters because of the baseline differences in regional diameters and flows in the two groups. All probability values were two-tailed, and a value of P<.05 was considered of statistical significance. When the effects of two doses of L-NMMA on coronary hemodynamics were compared with baseline, a paired t test was performed with a Bonferroni adjustment to {alpha}. Correlations between the effects of acetylcholine and substance P were made by use of the Pearson correlation coefficient.


*    Results
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 *Results
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Coronary Vascular Effects of Substance P and Acetylcholine
Intracoronary infusion of substance P produced progressive epicardial and microvascular coronary vasodilation in all patients (Fig 2Down). After a 20-pmol/min concentration of substance P, epicardial coronary diameter increased by 12.9% and coronary vascular resistance was 21% lower, but there was no change in heart rate or arterial blood pressure. With 40 pmol/min substance P (n=6), although there was further dilation of epicardial arteries (15%) and microvessels (24% reduction in coronary vascular resistance), this was accompanied by a significant reduction in arterial blood pressure. Therefore, for comparison between groups, the peak response obtained during infusions up to 20 pmol/min was used.



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  		Figure 2. Changes in coronary blood flow, coronary vascular resistance, and epicardial coronary artery diameter with incremental intracoronary infusions of substance P (solid line, solid circles) and acetylcholine (dashed line, open circles) in all 26 patients. Dose-response curves were analyzed by ANOVA. *P<.05, {dagger}P<.001 compared with baseline.

Acetylcholine infusions up to 100 µg/min produced progressive microvascular dilation in all patients (Fig 2Up), as reported previously, without changes in heart rate or arterial blood pressure. At the 300-µg/min infusion (n=7), there was no further dilation. Epicardial diameter did not change at the 30-µg/min dose but constricted at the 100-µg/min dose (Fig 2Up).

The peak epicardial and microvascular dilating effects of substance P were noted at 17.5±5 pmol/min, and with acetylcholine, peak blood flow increase occurred at a mean concentration of 69±59 µg/min. The effects of substance P were predominantly on the epicardial coronary arteries, with a modest microvascular dilating action, whereas the effects of acetylcholine were predominantly on the coronary microvessels. Thus, there was a 51±26% reduction in coronary vascular resistance with acetylcholine, compared with a peak 23±20% reduction with substance P. In contrast, epicardial coronary artery diameter increased by 16±13% with substance P and by only 0.5±10% with acetylcholine at the 30-µg/min dose. There was no correlation between the magnitude of epicardial coronary artery diameter change with substance P and acetylcholine in the 78 segments in which the responses were tested: r=.22, P=NS. Similarly, there was no correlation between the coronary microvascular effects in response to the two agents; the correlation coefficient between the decrease in coronary vascular resistance with acetylcholine and substance P was r=.11, P=NS.

Effects of Inhibition of Nitric Oxide Synthesis With L-NMMA on the Responses to Substance P, Acetylcholine, and Sodium Nitroprusside
At rest, L-NMMA produced progressive epicardial and microvascular coronary constriction. Thus, at the 64-µmol/min dose of L-NMMA, coronary blood flow was 12±15% lower (P<.01), coronary vascular resistance was 26±24% higher (P<.01), and coronary epicardial diameter was reduced by 10±10% (P<.001).

As shown in Fig 3Down, L-NMMA also suppressed epicardial and coronary microvascular dilation in response to both substance P and acetylcholine, but the vasodilator response to sodium nitroprusside was unaffected by L-NMMA.



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  		Figure 3. Effect of inhibition of nitric oxide synthesis with NG-monomethyl-L-arginine (L-NMMA) on the coronary vascular responses to substance P (20 pmol/min), acetylcholine (71±12 µg/min), and sodium nitroprusside (40 µg/min). *P<.01, **P<.001.

Effect of Risk Factors on the Responses to Acetylcholine, Substance P, Sodium Nitroprusside, and Adenosine
The presence of hypercholesterolemia and/or hypertension was associated with lower epicardial and microvascular dilator responses with both endothelium-dependent dilators, acetylcholine and substance P, but not with the endothelium-independent dilator, sodium nitroprusside (Fig 4Down). Diminished epicardial and microvascular responses were seen with both the 10- and 20-pmol/min infusions of substance P and at the 30 and 100 µg/min concentrations of acetylcholine, but the maximal responses are shown in Fig 4Down. Additionally, there was no significant difference between the 77±7% reduction in coronary vascular resistance with adenosine in patients without risks, compared with the 70±12% decrease in resistance in those with risk factors.



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  		Figure 4. Effects of substance P, acetylcholine, and sodium nitroprusside on coronary vascular responses in patients without (open bars) and those with (solid bars) hypertension and/or hypercholesterolemia. Baseline regional coronary vascular resistance and diameters were not significantly different between groups (2.8±1 vs 3.52 mm Hg·min-1·mL-1 and 1.9±0.5 vs 1.8±0.6 mm Hg·min-1·mL-1 in patients without vs those with risks). *P<.05, **P<.01, risk factors vs no risk factors.

Because patients with risk factors were significantly older than those free of risk factors (TableUp), we examined subgroups of patients between 40 and 60 years old. There were significant differences in the microvascular dilation in response to both endothelium-dependent vasodilators; in 8 patients without risks, with a mean age of 48 years, the 69% reduction in resistance with acetylcholine and the 39% reduction with substance P were greater than the changes in the 9 patients with risk factors (mean age, 51 years), in whom the reductions in coronary vascular resistance were 29% and 21%, respectively, with acetylcholine and substance P (both P<.01).

Effect of Risk Factors on the Response to L-NMMA
As reported previously, under resting conditions, the inhibitory effects of L-NMMA on coronary vascular resistance and diameter were greater in patients without risk factors than in those with risk factors10 : coronary vascular resistance increased by 39±23% in patients without risk factors and by 18±22% (P<.01) in those with hypercholesterolemia and hypertension at the 64-µmol/min dose. Similarly, there was a strong trend toward a significantly greater epicardial vessel constriction with L-NMMA in patients free of risk factors compared with those with risk factors: epicardial diameter constricted by 12±10% in those without risks and by 8.1±9.3% in those with risk factors (P=.058).

Coronary microvascular dilation in response to substance P was inhibited by L-NMMA in both patient groups, such that there was no significant difference between the changes in coronary vascular resistance after L-NMMA between the groups, suggesting that the greater vasodilation in patients without risks during the control study is due to greater nitric oxide bioavailability with substance P (Fig 5Down). Similarly, the inhibitory effect of L-NMMA on the epicardial coronary artery response to substance P was significant only in patients free of risk factors (Fig 5Down). As described previously, L-NMMA inhibited acetylcholine-induced coronary microvascular and epicardial dilation in patients both with and without risk factors10 (Fig 5Down). In contrast, epicardial and microvascular vasodilation in response to sodium nitroprusside was unaffected after L-NMMA in both patient groups.



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  		Figure 5. The effect of NG-monomethyl-L-arginine (L-NMMA) on coronary vascular responses in patients with (dashed line, open circles) and without (solid line, solid circles) hypertension and/or hypercholesterolemia. *P<.05, **P<.01 before vs after L-NMMA.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
The major findings of this investigation are that (1) substance P–induced coronary vascular dilation, like that with acetylcholine, is at least partly due to release of nitric oxide, because its effects were inhibited by L-NMMA; and (2) hypertension and hypercholesterolemia, even in the presence of angiographically normal coronary arteries, are associated with depression of coronary epicardial and microvascular dilation in response to substance P, a finding previously noted with acetylcholine. This suggests that dysfunction of the stimulatory capacity of the endothelial cell layer is not restricted to the muscarinic receptors and extends to other, nonmuscarinic receptors on the coronary vascular endothelium.

Specificity of the Endothelial Effects of Acetylcholine
Ever since it was demonstrated that acetylcholine is capable of releasing endothelium-derived relaxing factor, it has been commonly used as an agent to test endothelial function in human studies. However, the net effect of acetylcholine in the intact circulation appears to be due to a combination of its many vasoactive effects. Thus, an abnormal vascular response to acetylcholine may be a sign of defective nitric oxide, prostacyclin, or endothelium-derived hyperpolarizing factor release, or it could be indicative of increased smooth muscle cell sensitivity to muscarinic stimulation or to excessive release of endothelium-derived contracting factor, a finding recently reported in heart failure.1 2 3 4 5 6 7 8 9 10 11 19 These concerns regarding the specificity of the effects of acetylcholine on the vascular endothelium were reinforced by in vitro and in vivo studies in the human coronary circulation.14 15 16 20 21 22 Given the complexities of the vascular effects of acetylcholine and thereby the ambiguity in assigning a specific mechanistic interpretation based on its effects alone, we used a nonmuscarinic endothelium-dependent vasodilator and a specific antagonist of nitric oxide synthesis in this study to investigate the basal and stimulatory capacity of the endothelium.

Substance P and the Endothelium
Substance P is an 11-amino-acid neuropeptide that is present in the autonomic nervous system, the peripheral sensory neurons, and the perivascular regions of myocardial arterioles in humans.23 24 25 In vitro, it appears to specifically release nitric oxide, because its effect is inhibited by L-NMMA,26 27 28 but it may also evoke release of endothelium-derived hyperpolarizing factor and histamine in some species.29 30 However, unlike that with acetylcholine, vasodilation with substance P is not affected by cyclooxygenase inhibitors, and no constrictor action or direct smooth muscle dilator effects of substance P have been reported.31 In vitro, human coronary arteries relax in response to substance P, and this is partially attenuated in atherosclerotic strips.27 28 32 In vivo, substance P causes forearm microvascular vasodilation in humans and both epicardial and microvascular coronary vasodilation.33 34 35 36 Few patients with atherosclerosis have been studied with substance P, and the results have been inconclusive.37 Patients with transplant vasculopathy appear to have a depressed response to substance P,38 39 and microvascular but not epicardial vessel abnormalities have been reported in hypertensive patients.40 Most of the coronary vascular studies in humans have not compared the effects of substance P with those of acetylcholine in the same patients.12 Moreover, none of the studies have investigated the release of nitric oxide from the human coronary circulation with substance P, and finally, the impact of hypercholesterolemia with or without concomitant hypertension on nonmuscarinic endothelium-dependent vasodilation of the coronary vasculature has not been studied before.

Comparison of the Effects of Acetylcholine With Those of Substance P
There appears to be a preponderance of substance P receptors in the epicardial coronary arteries, and conversely, muscarinic receptors are preponderant in the coronary microvessels. This is evident from the fact that compared with acetylcholine, at any given dose, greater epicardial versus microvascular dilation was observed with substance P (Fig 2Up). By comparing the two vasodilators in the same patients, our study demonstrates that there is a poor correlation between the magnitude of epicardial coronary artery and microvascular dilation with acetylcholine and substance P. Thus, segments that either constricted or dilated with acetylcholine dilated with substance P to approximately the same extent. Similarly, there was no correlation between the microvascular responses to the two agents. Thus, although patients with hypertension and/or hypercholesterolemia had reduced vasodilation in response to both endothelium-dependent agents, the magnitude of reduction in the response to the two agents was variable. This suggests that in individual patients and in individual segments of coronary arteries, the impact of risk factors on different endothelial cell receptor–mediated pathways is variable. These observations are similar to findings in patients with variant angina, in whom segments of coronary arteries that were spastic in response to acetylcholine dilated normally with substance P13 41 and are also compatible with the results in epicardial coronary arteries of patients with hypertension.40

The lack of concordance in the effects of acetylcholine and substance P in the coronary epicardial vessels and microvessels may be because when dysfunction of the endothelium develops, the degree to which different receptors or their signal transduction pathways are affected is variable. Alternatively, it may be because of the potential difficulties in the measurement of endothelial function in vivo in humans; we may not have administered the maximum dose of the two agents because of the limitations posed by systemic effects that occurred at the higher doses.

Impact of Risk Factors on the Responses to Substance P and Acetylcholine
Previous studies from our institution have demonstrated depressed forearm vasodilator responses with substance P and acetylcholine in patients with hypertension and hypercholesterolemia.35 36 In this study, we show that human epicardial coronary arteries and microvessels also have depressed vasodilator responses to substance P and acetylcholine in hypertension and hypercholesterolemia, despite the lack of angiographic atherosclerosis. These findings are compatible with a recent report demonstrating re-duced coronary microvascular response to substance P in patients with hypertension.40 Depressed acetylcholine-induced coronary vascular dilation in the presence of risk factors for coronary atherosclerosis has been reported previously10 42 43 44 and was confirmed in our study.

Our patient group with risk factors was not large enough to determine whether hypertension, hypercholesterolemia, or their combination is more likely to produce greater dysfunction of one receptor subtype, although studies in the forearm microvessels suggest that substance P and acetylcholine responses are depressed in both conditions.34 35

Nitric Oxide Release at Rest and With Substance P
Human epicardial coronary arteries and microvessels release nitric oxide under resting conditions, and its contribution to resting coronary vasodilator tone is reduced in patients with risk factors for atherosclerosis.10 As we previously reported, acetylcholine-induced coronary epicardial and microvascular dilation is at least partly due to stimulation of release of nitric oxide, and the difference in dilation in the groups with and without risk factors is due to a reduction in nitric oxide release in patients with risk factors.10 The new finding of this study is that in vivo, substance P–induced coronary epicardial and microvascular dilation is also at least partly due to stimulation of release of nitric oxide. Second, because dilation with substance P after L-NMMA was similar in both groups, there is evidence to suggest that the greater baseline dilation in patients free of risk factors is due to higher production of nitric oxide in these patients compared with those with risk factors.

The results of our study demonstrate reduced activity of nitric oxide at rest and after pharmacological stimulation in patients with hypertension and hypercholesterolemia but do not allow us to distinguish between the possible mechanisms of dysfunction in the nitric oxide pathway that might be responsible. These include (1) substrate deficiency, described in patients with hypercholesterolemia45 46 ; (2) abnormalities in the signal transduction pathways47 48 ; (3) reduced activity of nitric oxide synthase; and (4) increased breakdown of normally or excessively produced nitric oxide by reactive oxygen intermediates.49 50

Limitations of the Study
Although greater coronary vasodilation was observed with the 40-pmol/min infusion of substance P than with the 20-pmol/min dose, there was systemic vasodilation with the higher dose, and therefore, this dose was not given to all patients in this study. Thus, the maximal effect of substance P may not have been measured. Nevertheless, all patients received infusions up to 20 pmol/min, the dose that was not associated with a significant systemic response, and comparison between groups was made using the maximal dilation that was achieved during substance P infusions between 5 and 20 pmol/min.

It has been suggested from in vitro studies that tachyphylaxis may develop in arteries after 3 to 10 minutes of exposure to substance P.31 Whether this occurred in our patients is difficult to measure, because all patients had 2-minute infusions of substance P given in incremental concentrations with 2- to 3-minute intervals between infusions to obtain angiograms. In a pilot study to investigate vascular effects of substance P, we found no difference in vasodilation during the first and second administrations of substance P in the femoral circulation (unpublished results). The observation that a single 20-pmol/min dose of substance P after L-NMMA produced less vasodilation than the same dose given during the control study when it was given as the second or third dose suggests that desensitization was not a significant problem with repeated substance P infusions in this study.

Our population consists predominantly of female patients who presented with chest pain and normal coronary arteries and who were undergoing cardiac catheterization, but the difference between patients with and those without risk factors for coronary atherosclerosis could not be accounted for by differences in sex distribution in the two groups, because approximately one third of both groups were men. Similarly, to exclude the impact of age differences between patients with and without risk factors, we analyzed age-matched subgroups to demonstrate that the reduced dilation in response to both substance P and acetylcholine is due to presence of hypertension and hypercholesterolemia in patients with risks. Additionally, the response to endothelium-dependent dilators was not significantly different in the two smokers compared with the other nonsmokers in the normal patient group.

We did not perform intravascular ultrasound in our patients and therefore cannot rule out intimal thickening in the angiographically smooth-appearing coronary arteries in patients with risk factors. Indeed, it is possible that various degrees of intimal thickening were in fact present but were angiographically invisible in these patients as described in previous studies,51 but there appears to be controversy related to the possible relationship of intimal thickening to the responses observed with acetylcholine.52

Implications
Substance P is present in the perivascular neurons and has been demonstrated by autoradiography to bind to human coronary endothelium.25 37 Furthermore, it is also released from the vascular endothelium in response to conditions of increased blood flow in the rat hind limb.53 Although the precise role of substance P in modulating endothelium-dependent coronary vasodilator tone in humans is not known, given the evidence above, it is possible to speculate that resting vascular tone may be modulated by local production (from endothelial cells) of substance P. Exposure to hypertension and hypercholesterolemia may depress local endothelial cell production of substance P, leading to lower basal production of nitric oxide. In addition, risk factors may reduce or damage endothelial substance P and muscarinic receptors, accounting for the depressed response to these vasodilators. Further investigations, perhaps using specific inhibitors of substance P, are warranted to clarify these issues.


*    Acknowledgments
 
The authors acknowledge the technical assistance of Gregory Johnson, William H. Schenke, and Rita Mincemoyer, RN.


*    Footnotes
 
Reprint requests to Arshed A. Quyyumi, MD, National Institutes of Health, Cardiology Branch, NHLBI, Bldg 10, Room 7B15, 10 Center Dr, MSC 1650, Bethesda, MD 20892-1650.

Received March 13, 1996; revision received August 19, 1996; accepted August 22, 1996.


*    References
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 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. Moncada S, Palmer RMJ, Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev.. 1991;43:109-142.[Medline] [Order article via Infotrieve]
  5. 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]
  6. Hodgson JM, Marshall JJ. Direct vasoconstriction and endothelium-dependent vasodilation: mechanism of acetylcholine effects on coronary flow and arterial diameter in patients with nonstenotic coronary arteries. Circulation.. 1989;79:1043-1051.[Abstract/Free Full Text]
  7. Forstermann U, Hertting G, Neufang B. The role of endothelial and non-endothelial prostaglandins in the relaxation of isolated blood vessels of the rabbit induced by acetylcholine and bradykinin. Br J Pharmacol.. 1986;87:521-532.[Medline] [Order article via Infotrieve]
  8. Panza JA, Casino PR, Kilcoyne CM, Quyyumi AA. Role of endothelium-derived nitric oxide in the abnormal endothelium-dependent vascular relaxation of patients with essential hypertension. Circulation.. 1993;87:1468-1474.[Abstract/Free Full Text]
  9. Casino PR, Kilcoyne CM, Quyyumi A, Hoeg JM, Panza JA. The role of nitric oxide in endothelium-dependent vasodilation of hypercholesterolemic patients. Circulation.. 1993;88:2541-2547.[Abstract/Free Full Text]
  10. 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.
  11. Luscher TF, Vanhoutte PM. Endothelium-dependent contractions to acetylcholine in the aorta of spontaneously hypertensive rats. Hypertension.. 1990;8:344-348.[Abstract/Free Full Text]
  12. Egashira K, Inou T, Yamada A, Hirooka Y, Urabe Y, Nagasawa K, Takeshita A. Heterogenous effects of the endothelium-dependent vasodilators acetylcholine and substance P on the coronary circulation of patients with angiographically normal coronary arteries. Coron Artery Dis.. 1992;3:945-952.
  13. Yamamoto H, Yoshimura H, Noma M, Kai H, Suzuki S, Tajimi T, Sugihara M, Kikuchi Y. Preservation of endothelium-dependent vasodilation in the spastic segment of the human epicardial coronary artery by substance P. Am Heart J.. 1992;128:298-303.
  14. Forstermann U, Mugge A, Frolich J. Endothelium-dependent relaxation of human epicardial coronary arteries: frequent lack of effect of acetylcholine. Eur J Pharmacol.. 1986;128:277-281.[Medline] [Order article via Infotrieve]
  15. Forstermann U, Mugge A, Alheid U, Haverich A, Frolich JC. Selective attenuation of endothelium-mediated vasodilation in atherosclerotic human coronary arteries. Circ Res.. 1988;62:185-190.[Abstract/Free Full Text]
  16. Toda N, Okamura T. Endothelium-dependent and -independent responses to vasoactive substances of isolated human coronary arteries. Am J Physiol.. 1989;257:H988-H995.[Abstract/Free Full Text]
  17. Christie MI, Griffith TM, Lewis MJ. A comparison of basal and agonist-stimulated release of endothelium-derived relaxing factor from different arteries. Br J Pharmacol.. 1989;98:397-406.[Medline] [Order article via Infotrieve]
  18. 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]
  19. Katz SD, Schwartz M, Yuen J, LeJemtel TH. Impaired acetylcholine-mediated vasodilation in patients with congestive heart failure. Circulation. 1993;88:55-61.[Abstract/Free Full Text]
  20. Miwa K, Goto M, Lee JD, Matsuyama F, Shimizu H, Kato T, Hara A, Nakamura T. Supersensitivity of coronary arteries in variant angina to spasm induced by intracoronary acetylcholine. Am J Cardiol.. 1988;61:77-82.[Medline] [Order article via Infotrieve]
  21. Horio Y, Yasue H, Okumura K, Takaoka K, Matsuyama K, Goto K, Minoda K. Effects of intracoronary injection of acetylcholine on coronary arterial hemodynamics and diameter. Am J Cardiol.. 1988;62:887-891.[Medline] [Order article via Infotrieve]
  22. Nellessen U, Lee TC, Fischell TA, Ginsburg R, Masuyama T, Alderman EL, Schroeder JS. Effects of acetylcholine on epicardial coronary arteries after cardiac transplantation without angiographic evidence of fixed graft narrowing. Am J Cardiol.. 1988;62:1093-1097.[Medline] [Order article via Infotrieve]
  23. Hokfelt T, Kellerth J-O, Nilson G, Pernow B. Experimental immunohistochemical studies on the localisation and distribution of substance P in cat primary sensory neurons. Brain Res.. 1975;100:235-252.[Medline] [Order article via Infotrieve]
  24. Lundberg J, Hokfelt T, Kewenter J, Pettersson G, Ahlman H, Edin R, Dahlstrom A, Nilsson G, Terenius L, Uvnas-Wallensten K, Said S. Substance P-, VIP-, and enkephelin-like immunoreactivity in the human vagus nerve. Gastroenterology.. 1979;77:468-471.[Medline] [Order article via Infotrieve]
  25. Weihe E, Reinecke M, Opherk D, Forssmann W. Peptidergic innervation (substance P) in the human heart. J Mol Cell Cardiol.. 1981;13:331-333.[Medline] [Order article via Infotrieve]
  26. Whittle BJR, Lopez-Belmonte J, Rees DD. Modulation of the vasodepressor actions of acetylcholine, bradykinin, substance P and endothelin in the rat by a specific inhibitor of nitric oxide formation. Br J Pharmacol.. 1989;98:646-652.[Medline] [Order article via Infotrieve]
  27. Bossaller C, Habib GB, Yamomoto H, Williams C, Wells S, Henry PD. Impaired muscarinic endothelial-dependent relaxation and cyclic guanosine 5-monophosphate formation in atherosclerotic human coronary artery and rabbit aorta. J Clin Invest.. 1980;79:170-174.
  28. Chester AH, O'Neill GS, Tadjikarimi S, Palmer RMJ, Moncada S, Yacoub MH. The role of nitric oxide in mediating endothelium-dependent relaxations in human epicardial coronary artery. Int J Cardiol.. 1990;29:305-309.[Medline] [Order article via Infotrieve]
  29. Feletou M, Vanhoutte PM. Endothelium-dependent hyperpolarization of canine coronary artery smooth muscle. Br J Pharmacol. 1988;93:515-524.
  30. Brock JW, Joshua IG. Substance P mechanisms in the regulation of striated muscle microcirculation. Microvasc Res.. 1991;41:24-28.[Medline] [Order article via Infotrieve]
  31. Furchgott RF. The role of endothelium in the responses of vascular smooth muscle. Annu Rev Pharmacol Toxicol.. 1984;24:175-197.[Medline] [Order article via Infotrieve]
  32. Chester AH, O'Neil GS, Moncada S, Tadjkarimi S, Yacoub MH. Low basal and stimulated release of nitric oxide in atherosclerotic epicardial coronary arteries. Lancet.. 1990;336:897-900.[Medline] [Order article via Infotrieve]
  33. McEwan JR, Benjamin N, Larkin S, Fuller RW, Dollery CT, MacIntyre I. Vasodilation by calcitonin gene-related peptide and by substance P: a comparison of their effects on resistance and capacitance vessels of human forearms. Circulation.. 1988;77:1072-1080.[Abstract/Free Full Text]
  34. Casino PR, Kilcoyne CM, Cannon RO III, Quyyumi AA, Panza JA. Impaired endothelium-dependent vascular relaxation in patients with hypercholesterolemia extends beyond the muscarinic receptor. Am J Cardiol.. 1995;75:40-44.[Medline] [Order article via Infotrieve]
  35. Panza JA, Casino PR, Kilcoyne CM, Quyyumi AA. Impaired endothelium-dependent vasodilation in patients with essential hypertension: evidence that the abnormality is not at the muscarinic receptor level. J Am Coll Cardiol.. 1994;23:1610-1616.[Abstract]
  36. Crossman DC, Larkin SW, Fuller RW, Davies GJ, Maseri A. Substance P dilates epicardial coronary arteries and increases coronary blood flow in humans. Circulation.. 1989;80:475-484.[Abstract/Free Full Text]
  37. Crossman DC, Larkin SW, Dashwood MR, Davies GJ, Yacoub M, Maseri A. Responses of atherosclerotic human coronary arteries in vivo to the endothelium-dependent vasodilator substance P. Circulation.. 1991;84:2001-2010.[Abstract/Free Full Text]
  38. Kushwaha SS, Crossman DS, Bustami M, Davies GJ, Mitchell AG, Maseri A, Yacoub MH. Substance P for evaluation of coronary endothelial function after cardiac transplantation. J Am Coll Cardiol.. 1991;17:1537-1544.[Abstract]
  39. Mugge A, Heublein B, Kuhn M, Nolte C, Haverich A, Warnecke J, Forssmann W-G, Lichtlen PR. Impaired coronary dilator responses to substance P and impaired flow-dependent dilator responses in heart transplant patients with graft vasculopathy. J Am Coll Cardiol.. 1993;21:163-170.[Abstract]
  40. Egashira K, Suzuke S, Hirooka Y, Kai H, Sugimachi M, Imaizumi T, Takeshita A. Impaired endothelium-dependent vasodilation of large epicardial and resistance coronary arteries in patients with essential hypertension. Hypertension.. 1995;25:201-206.[Abstract/Free Full Text]
  41. Egashira K, Inou T, Yamada A, Hirooka Y, Takeshita A. Preserved endothelium-dependent vasodilation at the vasospastic site in patients with variant angina. J Clin Invest.. 1992;89:1047-1052.
  42. 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]
  43. Zeiher AM, Drexler H, Wollschlager H, Just H. Modulation of coronary vascular tone in humans: progressive endothelial dysfunction with different early stages of coronary atherosclerosis. Circulation.. 1991;83:291-401.
  44. Egashira K, Inou T, Hirooka Y, Yamada A, Maruoka Y, Kai H, Sugimachi M, Suzuke S, 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.
  45. 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]
  46. 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.
  47. Flavahan NA. Atherosclerotic or lipoprotein-induced endothelial dysfunction: potential mechanisms underlying reduction in EDRF/nitric oxide activity. Circulation.. 1992;85:1927-1938.[Free Full Text]
  48. Panza JA, Garcia CE, Kilcoyne CM, Quyyumi AA, Cannon RO III. Impaired endothelium-dependent vasodilation in patients with essential hypertension: evidence that the nitric oxide abnormality is not localized to a single signal transduction pathway. Circulation.. 1995;91:1732-1738.[Abstract/Free Full Text]
  49. Ohara Y, Peterson TE, Harrison DG. Hypercholesterolemia increases endothelial superoxide anion production. J Clin Invest.. 1993;91:2546-2551.
  50. Minor RL, Myers PR, Guerra R, Bates JN, Harrison DG. Diet-induced atherosclerosis increases the release of nitrogen oxides from rabbit aorta. J Clin Invest.. 1990;86:2109-2116.
  51. Zieher AM, Schachinger V, Hohnloser SH, Saurbier B, Just H. Coronary atherosclerotic wall thickening and vascular reactivity in humans: elevated high-density lipoprotein levels ameliorate abnormal vasoconstriction in early atherosclerosis. Circulation.. 1994;89:2525-2532.[Abstract/Free Full Text]
  52. Nishimura RA, Lerman A, Chesebro JH, Ilstrup DM, Hodge DO, Higano ST, Holmes DR, Tajik J. Epicardial vasomotor responses to acetylcholine are not predicted by coronary atherosclerosis as assessed by intracoronary ultrasound. J Am Coll Cardiol.. 1995;26:41-49.[Abstract]
  53. Ralevic V, Milner P, Hudlica D, Kristek F, Burnstock G. Substance P is released from the endothelium of normal and capsaicin-treated rat hind-limb vasculature, in vivo, by increased blood flow. Circ Res.. 1990;66:1178-1183.[Abstract/Free Full Text]



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