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Nitric oxide production by coronary conductance and resistance vessels in hypercholesterolemia patients
American Heart Journal Founded in 1925
J u n e 1996 Volume 131, Number 6
CLINICAL INVESTIGATIONS
Chronic Coronary Disease
Nitric oxide production by coronary conductance and resistance vessels in hypercholesterolemia patients Nobuo Shiode, MD, Kensyo Nakayama, MD, Nobuyuki Morishima, MD, Togo Yamagata, MD, Hideo Matsuura, MD, and Goro Kajiyama, MD Hiroshima, Japan
NG-monomethyI-L-arginine (L-NMMA), a specific inhibitor of nitric oxide (NO) synthesis, was used to investigate the effects of inhibition of NO synthesis on the coronary conductance and resistance vessels in hypercholesterolemic patients. Acetylcholine (3 and 30 pg/min) was administered to 10 hypercholesterolemic and 10 control patients before and after L-NMMA (25 pmol/min) infusion. Epicardial coronary diameter was measured by quantitative angiography, and coronary blood flow (CBF) was derived from Doppler flowvelocity and coronary diameter measurements. In hypercholesterolemic patients, acetylcholine-induced dilation of epicardial arteries was attenuated, and the percentage increase in CBF caused by acetylcholine was smaller than that in control patients. L-NMMA attenuated acetylcholine-induced dilation of epicardial arteries in control patients. L-NMMA had no effect on CBF responses to acetylcholine in both patient groups. L-NMMA significantly decreased the baseline coronary diameter and CBF in both groups. These results indicated that hypercholesterolemia impaired the acetylcholine-induced dilation of the conductance and resistance coronary vessels. This impairment in the conductance vessels was dependent on NO production; that of resistance vessels was not. The basal release of NO in conductance and resistance vessels was preserved in hypercholesterolemic patients. (Am Heart J 1996; 131:1051-7.)
The endothelium modulates vascular smooth muscle tone by synthesizing and metabolizing vasoactive substances, including an endothelium-derived relax-
From the First Department of Internal Medicine, Hiroshima University School of Medicine. Received for publication July 17, 1995; accepted Oct. 30, 1995. Reprint requests: Nobuo Shiode, MD, The First Department of Internal Medicine, Hiroshima University School of Medicine, 1-2-3 Kasumi-cho, Minami-kn, Hiroshima, 734, Japan. Copyright © 1996 by Mosby-Year Book, Inc. 0002-8703/96/$5.00 + 0 4/1/71348
ing factor (EDRF). 1,2 Recent studies in human beings showed that acetylcholine infusion dilates angiographically normal coronary arteries and constricts arterial segments containing atherosclerotic lesions. 3-6Acetylcholine also increases coronaryblood flow. In addition, atherosclerosis and other coronary risk factors are associated with an impaired coronary blood flow in response to acetylcholine. 7-1° Such findings suggest that endothelial dysfunction develops in the epicardial and resistance coronary arteries in conjunction with coronary risk factors. Hypercholesterolemia is an important risk factor for coronary atherosclerosis and impairs the endothelium-dependent vasorelaxation of the large coronary arteries before the formation of atherosclerotic lesions.e, s, 11 Hypercholesterolemia also is associated with a blunted endothelium-dependent vasodilation of the coronary microcirculation in animals and h u m a n beings. 9, 11-13However, previous studies evaluated only the vascular response to agonists such as acetylcholine in patients with atherosclerosis and coronary risk factors. EDRF was demonstrated more than a decade ago by Furchgott and Zawadzki. 2 Nitric oxide is synthesized from L-arginine by endothelial cells. 14, 15 N Q monomethyl-L-arginine (L-NMMA) inhibits its formation in a concentration-dependent and stereospecific manner. 16 Our study investigated the effects of blocking the production of nitric oxide by L-NMMA on the coronary circulation of hypercholesterolemic patients. We attempted to determine whether the impaired vascular responses to acetylcholine in such patients was associated with impaired production of nitric oxide and whether a basal release of nitric ox1051
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Table I. Plasma lipoprotein levels
Total cholesterol HDL cholesterol LDL cholesterol Triglycerides
Hypercholesterolemic patients (n = 10) (rag /dl)
Control patients (n = 10) (rag /dl)
p Value
266 _+8 40 _+3 194 _+8 159 _+24
172 -- 5 44 -- 2 107 -- 6 111 _+17
<0.01 NS <0.01 NS
D a t a are e x p r e s s e d as m e a n +- SEM.
ide exists in the epicardial a n d resistance c o r o n a r y arteries of p a t i e n t s with hypercholesterolemia.
METHODS Patients. Ten patients with hypercholesterolemia (fasting total cholesterol level >220 mg/dl) and 10 patients without hypercholesterolemia who had atypical chest pain, normal exercise test results, normal left ventricular function (contrast ventriculogram ejection fraction, ->50%)i and normal coronary flow reserve were studied (Table I). These patients had no significant atherosclerotic stenosis (>25%) in the epicardial coronary arteries. Serum total and high-density lip0protein (HDL) cholesterol and triglycerides were determined by the enzyme-assay method. Lowdensity lipoprotein (LDL) was calculated as (total cholesterol - HDL cholesterol - [triglyceride/5]). Hypercholesterolemic patients. The hypercholesterolemic group comprised 10 patients, 5 men and 5 women, mean age 59 _+ 2 years (range, 48 to 68 years). None was taking a cholesterol-lowering agent before this study. Excluded from study were hypercholesterolemic patients with a family history of hypercholesterolemia, thickening of the Achilles tendon, or xanthoma, as well as any patients with secondary hypercholesterolemia. Control patients. The control group comprised 10 patients, 8 men and 2 women, mean age 54 +_ 3 years (range, 40 to 66 years). The total cholesterol level of each patient in this group was <200 mg/dl. No patients had hypertension (defined as systolic blood pressure >160 mm Hg or diastolic blood pressure >95 mm Hg or both) or diabetes mellitus. Two of the hypercholesterolemic patients and 2 of the control patients were smokers (>10 cigarettes/day). These four patients were asked to refrain from smoking for the 72 hours preceding the study. Written informed consent was obtained from all patients before the diagnostic angiogram. This study was approved by the Human Investigation Ethical Committee of the University of Hiroshima School of Medicine. Study design. Antianginal medication was discontinued 48 hours before catheterization, except for sublingual nitroglycerin, which was withheld I hour before catheterization, but no patient in this study had sublingual nitroglycerine 24 hours before catheterization. Patients were brought to the catheterization laboratory in the fasting state after premedication with hydroxyzine (25 mg, IM) and promethazine hydroch]oride (25 mg, IM). Diagnostic right and left heart catheterization and cor-
onary angiography were performed through a standard percutaneous femoral approach. After vascular access had been obtained, 10,000 U of heparin was infused intravenously. A 6F guide catheter was introduced into the left main coronary artery. A 0.014-inch Doppler flow guide wire (FloWire; Cardiometrics, Mountain View, Calif.) was advanced through the guide catheter into the proximal segment of the left anterior descending coronary artery. Protocol. After the diagnostic catheterization, the following interventions were performed: (1) infusion of saline (1 ml/min for 2 minutes); (2) serial infusion of intracoronary acetylcholine, 3 ~g/min and 30 ~g/min (1 ml/min for 2 minutes each); (3) infusion of adenosine, 100 pg/min (1 m]/min for 2 minutes). Next L-NMMA was infused at a dose of 25 ~mol/min (1 ml/min for 5 minutes). Interventions 1 through 3 were repeated and were followed by an infusion of nitroglycerine, 200 pg/min. All drugs were infused into the left coronary ostium through the guide catheter. We waited 5 minutes after each of the infusions of adenosine and acetylcholine before beginning the next infusion. Coronary arteriography was performed immediately after the end of each infusion. The heart rate, arterial pressure, coronary blood flow velocity, and electrocardiogram were continuously monitored during each infusion. Measurements were also recorded under steady-state conditions. Quantitative coronary angiography. Quantitative coronary angiography was used to measure the diameter of the coronary conductance vessels. Coronary cineangiograms were recorded on 35-mm cinefilm (30 frames/sec) with a Siemens (Munich, Germany) cineangiographic system after selecting the best view for left anterior descending coronary artery visualization. Nonionic contrast medium was injected into the left coronary artery at a rate of 5 to 7 mY sec for a total of 7 to 10 ml. A power injector (Medrad, Pittsburgh, Pc.) was used to optimize the quality and reproducibility of the opacification. 17 Angiograms were obtained while the patients held their breath to avoid the artifacts associated with respiration.iS The proximal and the distal segments of left anterior descending coronary artery were selected for quantitative analysis. The diameter of the lumen of each arterial segment was measured with a computer-assisted system for analyzing coronary angiograms. The arterial segments evaluated were video-digitized at end diastole, with data stored in a cardiac imageanalysis system (Cardio 500; Kontron Instruments, Munich, Germany). Automated counter detection was
Volume131,Number6 Shiode et al.
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Table II. H e m o d y n a m i c c h a n g e s Group
Saline
Hypercholesterolemia HR 64 _+ 3 mBP 98 ± 3 RPP 9021 ± 414 Control HR 63 ± 2 mBP 99 ± 4 RPP 8172 +_ 514
A Ch 30 Ftgrn/min
Adenosine 100 ~tgm/min
L-NMMA 25 #mol/min
A Ch (30 ~tgrn ~rain)
Adenosine 100 Itgm/min
NTG 200 ~tgm ~rain
64 ± 3 98 -+ 4 9283 ± 620
64 ± 3 96 ± 4 9160 ± 589
62 ± 3 105 ± 4* 9593 -+ 542
62 ± 3 103 ± 4 9471 ± 525
62 ± 3 102 ± 4 t 9568 ± 606
71 _+ 3* 91 +_ 5* 9079 ± 581
64 ± 3 97 ± 4 8262 ± 647
64 ± 3 96 ± 4 8214 ± 646
61 +_ 3 102 ± 4* 8389 ± 714
62 ± 3 102 ± 4 8444 ± 660
63 ± 3 100 ± 4~ 8385 ± 616
72 ± 5* 92 ± 4* 8090 -+ 550
Data are expressed as mean ± SEM. HR, Heart rate (beats/min); mBP, mean arterial blood pressure (mm Hg); RPP, rate-pressure product (ram Hg beats/min); ACh, acetylcholine; L-NMMA, NQmonomethyl:L-arginine; NTG, nitroglycerine. *p < 0.05 compared with baseline. tp < 0.05 compared with data before L-NMMA treatment.
Table III. C h a n g e i n c o r o n a r y blood flow ( C B F ) Hypercholesterolemic patients (n = 10) Before L-NMMA CBF (ml / min) Baseline 61.0 _+ 7.9 Acetylcholine 3 ~g/min 88.4 ± 14.5 30 ~g/min 142.9-+ 20.4 Adenosine 251.0 ± 37.6
ACBF ~ baseline)
Control patients (n = 10)
After L-NMMA CBF (ml / min)
Before L-NMMA
ACBF (X baseline)
53.3 _+ 8.5* 1.44 ± 0.10 2.40 ± 0.18 4.14 ± 0.25
70.8 ± 12.0t 120.7 _+ 21.6" 248.8 ± 39.8
CBF (ml / min)
ACBF (X baseline)
52.0 ± 6.8 1.36 ± 0.08 2.28 _+ 0.23 4.88 ± 0.46*
101.2 ± 24.0 213.3 _+ 34.2 226.0 ± 29.3
After L-NMMA CBF (ml / min)
ACBF ~ baseline)
42.9 ± 5.1t 1.80 ± 0.21 4.05 ± 0.35 4.37 ± 0.26
75.3 ± 14.0 144.0 ± 16.0" 234.9 ± 31.5
1.72 _+ 0.16 3.44 ± 0.19 5.46 ± 0.43t
Data are expressed as mean -+ SEM. *'1o< 0.05. tp < 0.01 compared with data before L-NMMA.
performed by a geometric edge differentiation technique s i m i l a r to t h a t d e s c r i b e d b y R e i b e r e t al. 19 T h e d i a m e t e r of t h e s e g m e n t of i n t e r e s t w a s m e a s u r e d , a n d t h e a v e r a g e v a l u e of t r i p l i c a t e m e a s u r e m e n t s w a s u s e d for a n a l y s i s . A 6 F J u d k i n s c a t h e t e r w a s u s e d to c a l i b r a t e t h e a r t e r i a l diameter in millimeters. Arterial diameter was measured in b l i n d f a s h i o n b y t w o i n v e s t i g a t o r s (K. N. a n d N. M.), w h o h a d n o k n o w l e d g e of t h e p a t i e n t s ' c l i n i c a l c h a r a c t e r i s t i c s . Measurements of coronary blood flow velocity and estimation of coronary blood flow. A Doppler guide wire w i t h a 12 M H z p i e z o e l e c t r i c t r a n s d u c e r a t t h e t i p (FloWire; Cardiometrics), was used in measuring the coronary a r t e r y blood-flow velocity. T h i s t e c h n i q u e w a s p r e v i o u s l y v a l i d a t e d . 2° C o n t i n u o u s flow-velocity p r o f i l e s f r o m t h e 12 MHz pulsed Doppler velocimeter (F]oMap; Cardiometrics), together with simultaneous electrocardiograms and aortic pressures, were displayed on a videomonitor and continuo u s l y r e c o r d e d o n 0 . 5 - i n c h V H S v i d e o t a p e . C h a n g e s i n coro n a r y b l o o d flow i n r e s p o n s e to a d m i n i s t r a t i o n of t h e vas o a c t i v e a g e n t s w e r e e s t i m a t e d f r o m t h e p r o d u c t of t h e m e a n c o r o n a r y blood-flow v e l o c i t y a n d t h e c r o s s - s e c t i o n a l a r e a 2 to 3 m m d i s t a l to t h e t i p of t h e D o p p l e r flow g u i d e wire. T h i s d i s t a n c e w a s s e l e c t e d b e c a u s e t h e D o p p l e r
t r a n s d u c e r h a d a r a n g e g a t e d e p t h of 4:2 m m . C h f l i a n e t al. 21 s h o w e d t h a t c o r o n a r y blood flow is d e t e r m i n e d pred o m i n a n t l y b y s m a l l r e s i s t a n c e v e s s e l s (<200 ~ m i n d i a m eter), so w e m e a s u r e d c o r o n a r y b l o o d flow to e v a l u a t e res p o n s e s of c o r o n a r y r e s i s t a n c e v e s s e l s to v a s o a c t i v e a g e n t s . Drug preparations. A c e t y l c h o l i n e ( D a i i c h i P h a r m a c e u tical, Tokyo), w a s dissolved, i m m e d i a t e l y b e f o r e use, i n p h y s i o l o g i c s a l i n e a t a c o n c e n t r a t i o n of 3 ~ g / m l or 30 p g / m l . A d e n o s i n e ( S i g m a C h e m i c a l , St. Louis, Mo.) a n d L - N M M A ( S i g m a ) w e r e d i s s o l v e d i n physiologic s a l i n e a t c o n c e n t r a t i o n s of 100 ~ g / m l a n d 25 ~ m o l / m l , r e s p e c t i v e l y . Nitroglyce r i n e ( N i h o n k a y a k u , Tokyo, J a p a n ) w a s d i s s o l v e d i n p h y s iologic s a l i n e a t a c o n c e n t r a t i o n of 2 0 0 ~g/ml. E a c h d r u g w a s i n f u s e d w i t h a n i n f u s i o n p u m p ( C F V 3000, Nih o n k o d e n , Tokyo, J a p a n ) a t a r a t e of 1 m l / m i n . Statistics. D a t a a r e e x p r e s s e d a s m e a n _+ S E M . Twow a y a n a l y s i s of v a r i a n c e ( A N O V A ) for r e p e a t e d m e a s u r e s w a s u s e d to c o m p a r e t h e d o s e - r e s p o n s e to a c e t y l c h o l i n e b e f o r e a n d a f t e r L-NMMA, w i t h i n or b e t w e e n g r o u p s . T h e d i f f e r e n c e s b e t w e e n t h e effects of a c e t y l c h o l i n e b e f o r e a n d after L-NMMA in both groups were compared by using the p e r c e n t a g e c h a n g e s f r o m b a s e l i n e b e c a u s e of t h e b a s e l i n e d i f f e r e n c e s i n c o r o n a r y d i a m e t e r s a n d c o r o n a r y b l o o d flow
1054
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Shiode et al.
AmericanHeartJournal
-5
.c~
10-
*
t.
--o- ] 1" p < 0.01
" bas''
"
-15:
-20:
11.P
]] NS
-25T baseline
3
3()
ACh (l~g/min)
Fig, 1, Percentage change in diameter of proximal and distal segments of left anterior descending coronary artery in response to serial infusions of acetylcholine (ACh). ACh, 3 and 30 ~g/min, was administered to hypercholesterolemic and control patients, before and after L-NMMA (25 ~mol/min). Open circles, Control patients before L-NMMA; solid circles, control patients after L-NMMA;open squares, hypercholesterolemic patients before L-NMMA; solid squares, hypercholesterolemic patients after L-NMMA; NS, not significant. Data are expressed as mean -+ SEM. *p < 0.05; **p < 0.01 compared with baseline; tp < 0.01 between patients with and without hypercholesterolemia as determined by analysis of variance. in the two groups. Serial changes in blood flow, coronary diameter, and hemodynamic variables in response to graded doses of acetylcholine were compared by one-way ANOVA. If the ANOVA showed a significant difference between the mean values, the level of statistical significance was determined by contrast. Paired data were compared by paired t tests. A level ofp < 0.05 was accepted as statistically significant. RESULTS Clinical characteristics and hemodynamic variables,
Plasma lipoprotein levels of the patients studied are shown in Table I. Intracoronary infusions of L-NMMA had no effect on the h e a r t rate or the ratepressure product. L-NMMA produced a significant increase in m e a n arterial pressure in both the patients with hypercholesterolemia and the control patients (Table II). Nitroglycerine reduced the m ean arterial pressure and increased the h e a r t rate in both groups. Serial infusions of acetylcholine produced no significant change in m ean blood pressure, h e a r t rate, or rate-pressure product before or after LNMMA t r e a t m e n t in either group (Table II).
--o- Control L-NMMA (-) ~ ContrL-NMMAL'NMMA(-) OIHc (+) j ] "-=-- HC L-NMMA (+)
1" P < 0.01
Fig. 2. Percentage change in coronary blood flow. The change in coronary blood flow (ACBF), before and after LNMMA administration, in response to serial infusions of acetylcholine (ACh) at 3 and 30 ~g/min in hypercholesterolemic and control patients. Open circles, Control patients before L-NMMA; solid circles, control patients after LNMMA; open squares, hypercholesterolemicpatients (HC) before L-NMMA; solid squares, hypercholesterolemic patients after L-NMMA. Data are expressed as mean -+ SEM. tp < 0.01 between patients with and without hypercholesterolemia as determined by analysis of variance. eter of the distal segment of the epicardial coronary arteries (Fig. 1). However, a dose of acetylcholine, 30 pg/min, abolished the vasodilating effect on these arteries. Acetylcholine also produced a dose-dependent increase in coronary blood flow (Table III). In patients with hypercholesterolemia, an acetylcholine infusion, 30 ~g/min, reduced the diameter of both the proximal and distal segments of the epicardial coronary arteries. Conversely, acetylcholine, 3 pg/min, had no effect. Acetylcholine caused a dosedependent increase in coronary blood flow, but the percentage increase in coronary blood flow in the hypercholesterolemic patients was significantly smaller t h a n t h a t in control patients (Fig, 2). Effects of L-NMMA on epicardial artery and coronary blood flow under basal conditions. L-NM~MA, 25 ~mo]]
min, significantly decreased the baseline coronary blood flow from 61.0 _+ 7.9 ml/min to 53.3 _+ 8.5 mY min (p < 0.05) in hypercholesterolemic patients and from 52.0 _+ 6.8 ml/min to 42.9 _+ 5.5 ml/min (p < 0.01) in the control patients. After L-NMMA t reat m ent , the diameters of the proximal and distal segments of the epicardial coronary arteries decreased significantly in both groups (Fig. 3).
Effects of acetylcholine on epicardial coronary arteries and coronary blood flow. In control patients, acetyl-
Effect of L-NMMA on vascular response to acetylcholine. In control patients, by the intracoronary infu-
choline, 3 ~g/min, significantly increased the diam-
sion of L-NMMA, the acetylcholine (3 ~g/min)-in-
Volume 131, Number 6 American Heart Journal
duced dilation of the distal epicardial arteries was attenuated. In both groups, acetylcholine, 3 pg/min, significantly decreased the diameter of the distal segment. Acetylcholine, 30 pg/min, significantly decreased the diameters of the proximal and distal segments of the epicardial coronary arteries (Fig. 1). Acetylcholine produced a dose-dependent increase in coronary blood flow after L-NMMA administration in the patients with and without hypercholesterolemia. The percentage increase in coronary blood flow after L-NMMA treatment was similar to that before LNMMA treatment in both groups (Fig. 2). Effect of adenosine on coronary blood flow. T h e en-
dothelium-independent vasodilator adenosine significantly increased the coronary blood flow in both groups, regardless of L-NMMA addition (Table III). The percentage increase in coronary blood flow induced by adenosine did not differ between the two groups. Effect of nitroglycerine on the diameter of epicardial coronary arteries. Nitroglycerine significantly in-
Shiode et al.
40
1055
Proximal Segment
30 20 E ;.=, ~.~
10 0 .......,
o o~ -10
oc~ 40
~, (b
Distal Segment
3o 2o
10
ol
.loi baseline
L-NMMA 25 limol/min
N:rG
Fig. 3. Effects of intracoronary infusion of L-NMMA (25 ~mol/min) and nitroglycerine (NTG; 200 pg/min) on the baseline coronary diameter of the proximal and the distal left anterior descending coronary artery. Open circles, Control patients; open squares, hypercholesterolemic patients. Data are expressed as mean _+SEM. *p < 0.05; **p < 0.01 compared with baseline.
creased the diameter of epicardial coronary arteries to the same degree in both groups (Fig. 3). DISCUSSION
Intravascular Doppler ultrasound techniques have recently become available for the study of function in the coronary circulation in vivo. Intravascular Doppler transducers provide beat-to-beat information on coronary blood-flow velocity, 22 and used in combination with quantitative coronary angiography data on cross-sectional area, yield measurements of coronary blood flow. 9, io Simultaneous information on the effects of pharmacologic agents on luminal diameter and coronary blood flow allow analysis of their differential effects on the epicardial arteries and the microcirculation. In this study, we measured changes in epicardial coronary diameter and coronary bloodflow velocity to define the pharmacologic action of each agent on the conductance and resistance coronary vessels. Lefroy eta]. 23 showed that L-NMMA, at a dose of 25 pmol/min, significantly reduced the distal, but not the proximal, left anterior descending coronary artery diameter along with a decrease in coronary venous oxygen saturation. These authors concluded that there was a basal release of nitric oxide in the epicardial coronary arteries and the resistance vessels. In our study, L-NMMA administration reduced the basal diameter of the epicardial arteries and reduced the basal coronary blood flow in control patients, indicating a basal release of nitric oxide in both the epicardial coronary arteries and the resistance vessels.
Sudhir et al. 24 reported that the vasodilator response to acetylcholine in dogs was inhibited by Nnitro-L-arginine methyl ester (L-NAME), a inhibitor of nitric oxide synthetase, in the epicardial coronary arteries but not in the resistance vessels. This suggests that acetylcholine-induced, endothelium-dependent relaxation in coronary resistance vessels may not be mediated by nitric oxide alone. In a clinical study, Lefroy et al. 2~ reported that L-NMMA had no effect on the acetylcholine-induced increase in coronary blood flow. Our findings in control patients resemble these of Lefroy et al. and indicate that acetylcholine-induced dilation of the epicardial arteries is mediated by nitric oxide, whereas acetylcholineinduced dilation of resistance vessels is not. This is the first report of the effects of an inhibitor of nitric oxide synthesis on the coronary circulation of patients with hypercholesterolemia. The acetylcholine-induced dilation of the epicardial arteries and of the resistance vessels was reduced in such patients, consistent with previous reports that hypercholesterolemia impairs the endothelium-dependent vasorelaxation of the large coronary arteries and the resistance vessels before the development of the atherosclerotic lesions. 6, s;9, 11-13,25 The vasodilator action of acetylcholine is mediated through EDRFs. Simultaneously, this compound exerts a vasoconstrictor action on the smooth muscle of the epicardial coronary arteries. 25 Evidence from studies in animals and h u m a n beings indicates that atherosclerosis is associated with enhanced vasocon-
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Shiode et al.
strictor response to cathecholamines, serotonin, histamine, and ergonovine. 20, 27-29 It is possible that in patients with hypercholesterolemia, impairment of epicardial artery dilation in response to acetylcholine is caused by increased sensitivity of the smooth muscle to acetylcholine. However, after L-NMMA treatment, we observed that acetylcholine, 3 and 30 ~g/min, decreased the diameter of the epicardial coronary arteries in patients with or without hypercholesterolemia. This suggests that in hypercholesterolemic patients the impairment of acetylcholineinduced dilation of the epicardial coronary arteries is caused by lack of endothelial nitric oxide production in response to acetylcholine, not by increased sensitivity of smooth muscle to acetylcholine. Acetylcholine-induced increases in coronary blood flow were reduced in the hypercholesterolemic patients. L-NMMA had no effect on the acetylcholineinduced change in coronary blood flow regardless of lipid status. These results indicate that hypercholesterolemia impairs other endothelial functions in resistance vessels separate from nitric oxide formation. Gilligan et al. 3° reported that L-NMMA increased basal forearm vascular resistance to similar degrees in patients with and without hypercholesterolemia. These authors concluded that hypercholesterolemic patients had normal nitric oxide bioavailability. In our study ofhypercholesterolemic patients, L-NMMA decreased in the basal diameter of the large epicardial coronary arteries and reduced coronary blood flow. The extent of the decrease did not differ between patient groups, indicating that the basal release of nitric oxide in patients with hypercholesterolemia was preserved in the epicardial arteries and the resistance vessels. Limitations. We used a 25 pmol/min infusion of L-NMMA for 5 minutes. Lefroy et al. 23 reported that infusions of L-NMMA (4, 10, and 25 ~mol/min, each for 5 minutes) abolished the acetylcholine-induced dilation of h u m a n epicardial coronary arteries. We also observed that the endothelium-dependent vasodilation induced by a 3 ~g/min infusion of acetylcholine was reduced after L-NMMA treatment. Thus at the dose chosen, L-NMMA attenuated the epicardial coronary dilation caused by agonist-induced nitric oxide production, although the acetylcholine-induced increases in coronary blood flow were not reduced after the L-NMMA infusions. In our study, higher doses of L-NMMA may have attenuated the acetylcholine-induced increase in coronary blood flow. We did not administer such higher doses because of the risk of an increase in systemic blood pressure and concomitant vasoconstriction in other vascular beds. 31, 32
June 1996 American Heart Journal
We observed an unaltered basal release of nitric oxide in the large epicardial coronary arteries and resistance vessels of our hypercholesterolemic pa: tients. All of these patients had no angiographically significant stenotic lesions, suggesting that endothelial function was not sufficiently impaired to reduce the basal nitric oxide release. Panza et a l Y showed that the effect of L-NMMA on basal vascular tone was reduced in hypertensive patients compared with normal control subjects. This finding is consistent with a diminished basal release of nitric oxide in hy: pertensive patients. In this study, if the hypercholesterolemic patients had detectable stenotic lesions in their epicardial coronary arteries along with greater endothelial dysfunction, the basal release of nitric oxide would be diminished. Finally, we studied the only small sample size. In this study, the mean age of the patients in each group did not differ significantly, but the hypercholesterolemic patients tended to be older than the controls. Several studies 12, 34 have shown that advanced age is a significant independent predictor of impaired endothelium-dependent dilation of the coronary resistance vessels, regardless of the presence or absence of epicardial artery atherosclerosis. Therefore, the impairment of coronary bloodflow response to acetylcholine seen in our hypercholesterolemic patients may have been influenced by their age. Moreover, the hypercholesterolemic patient group included many more women than the control group. Reis et al. 35 showed that ethinyl estradiol acutely attenuated abnormal coronary vasomotor responses to acetylcholine. However, because only one woman in the hypercholesterolemic group was menstruating and all the other women in both groups were postmenopausal, the sex mismatch might have little influence on our results. At least, it was unlikely that the different sex distribution had a bad influence in the hypercholesterolemic group. Conclusions. Results of this study indicate that the basal release of nitric oxide from endothelial cells of coronary conductance and resistance vessels is preserved in patients with hypercholesterolemia. However, the production of nitric oxide induced by acetylcholine in coronary conductance and resistance vessels is impaired in these patients. This impairment of conductance arteries was dependent on a decrease in nitric oxide production, but that of the resistance vessels was not. We thank Drs. Yuji Yasunobu, Masaya Kato, Koichi Tanaka, Hitoshi Fujiwara, Akito Hiraoka, and Shinji Karakawa and the nurses and technicians of the catheterization laboratory at Hiroshima University Hospital.
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Shiode et al.
REFERENCES
1. Vane RE, Anggard EE, Betting RM. Regulatory functions of the vascular endothelium. N Engl J Med 1990;323:27-36. 2. Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 1980; 288:373-6. 3. 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;315:1046-51. 4. Ross R. The pathogenesis 0f atherosclerosis--an update. N Engl J Med 1986;314:488-500. 5. Yasue H, Matsuyama K, Matsuyama K, Okumura K, Morikami Y, Ogawa H. Responses of angiographically normal h u m a n coronary arteries to intracoronary injection of acetylcholiue by age and segment. J Am Cell Cardiol 1990;81:482-90. 6. Vita J, 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. J Am Cell Cardiol 1990;81:491-7. 7. Creager MA, Cooke JP, Mendelsohn ME, Gallagher SJ, Coleman SM, Loscalzo J, Dzau VJ. Hypercholesterolemia attenuates endotheliummediated vasodilation in man. J Clin Invest 1990;86:228-34. 8. Zeiher AM, Drexler H, Wollschlyger H, Just H. Modulation of coronary vascular tone in humans: progressive endothelial dysfunction with different early stages of coronary atherosclerosis. Circulation 1991;83:391401. 9. Egashira K, Inou T, Hirooka Y, Yamada A, Maruoka Y, Kai H, Sugimacgi M, Suzuki S, TakeshitaA. Impaired coronaryblood flow response to acetylcholine in patients with coronary risk factors and proximal atherosclerotic lesions. J Clin Invest 1993;91:29-37. 10. Zeiher AM, Drexler H, Saurbier B, Just H. Endothelium-mediated coronary blood flow modulation in humans. J Clin Invest 1993;92:652-62. 11. Lucher TF, Richard V, Tschudi M, Yang Z, Boulanger C. Endothelial control of vascular tone in large and small coronary arteries. J Am Col] Cardiol 1990;15:512-27. 12. Zeiher AM, Drexier H, Saurbier B, Just H. Effects of age, atherosclerosis, hypercholesterolemia, and hypertension: endothelium-mediated coronary blood flow modulation in humans. J Clin Invest 1993;92:65262. 13. Sellke FW, Armstrong ML, Harrison DG. Endothelium-dependent vascular relaxation is abnormal in the coronary microcirculation of atherosclerotic primates. Circulation 1990;81:1586-93. 14. Palmer RM, Ferrige AG, Moncada S. Nitric oxide accounts for the biological activity of endothelium-derived relaxing factor. Nature 1987;327'.524-6. 15. Palmer RM, Ashton DS, Moncada S. Vascular endothelial cells synthesize nitric oxide from L-arginine. Nature 1988;333:664-6. 16. Rees DD, Palmer RMJ, Hodson HF, Moncada S. A specific inhibitor of nitric oxide formation from L-arginine attenuates endothelium-dependent relaxation. Br J Pharmacol 1989;96:418-24. 17. Gardiner GA, Meyerovitz MF, Boxt LM, Harrington DP, Taus RH, Kandarpa KR, Ganz P, Selwyn AP. Selective coronary angiography using a power injector. Am J Radiol 1986;146:831-3. 18. Mancini GBJ, Simon SB, McGfllen J, LeFree M, Friedman H, Vogel RA. Automated quantitative coronary arteriography: morphologic and physiologic validation in rive of a rapid digital angiographic method. Circulation 1987;75:452-60. 19. Reiber JHC, Serruys PW, Kooijman CJ, Wijus W, Slager CJ, Gerbrands J J, Schuurbiers JCH, Boer AD, Hugenholtz PG. Assessment of short-,
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medium-, and long-term variations in arterial dimensions from computer-assisted quantification of coronary cineangiograms. Circulation 1985;71:280-8. Schroeder JS, Bolen JL, Quint RA, Clark DA, Hayden WG, Higgins CB, Wex]er L. Provocation of coronary spasm with ergonovine maleate. Am J Cardiol 1977;40:487-91. ChiIian WM, Eastham CL, Marcus ML. Microvascular distribution of coronary vascular resistance in beating lef~ ventricle. Am J Physio] 1986;251:H779-86. Wilson RF, Laughlin DE, Ackell PH, Chilian WM, Holida MD, Hartley CJ, Armstrong ML, Marcus ML, White CW. Transluminal, subselective measurement of coronary artery blood flow velocity and vasodflator reserve in man. Circulation 1995;72:82-92. Lefroy DC, Crake T, Uren NG, Davies GJ, Maseli A. Effect of inhibition of nitric oxide synthesis on epicardial coronary artery caliber and coronary blood flow in humans. Circulation 1993;88:43-54, Sudhir K, MacGregor JS, Amidon TM, Guptta M, Yock PG, Chatterjee K. Differential contribution of nitric oxide to regulation of vascular tone in coronary conductance and resistance arteries: intravascular ultrasound studies. Am Heart J 1994;127:858-65. Zeiher AM, Drexler H, Wollschlager H, Just H. Endothelial dysfunction of the coronary microvasculature is associated with impaired coronary blood flow regulation in patients with early atherosclerosis. Circulation 1991;84:1984-92. Hodgson JM, Marshall J. Direct vasoconstriction and endothelium-dependent vasodilation: mechanisms of acetylcholine effects on coronary flow and arterial diameter in patients with nonstenotic coronary arteries. Circulation 1989;79:1043-51. Freiman PC, Mitchell GG, Heistad DD, Armstrong ML, Harrison DD. Atherosclerosis impairs endothelium-dependent vascular relaxation to acetylcholine and thrombin in primates. Circ Res 1986;58:783-9. Heistad DD, Armstrong ML, Marcus ML, Piegors DJ, Mark AL. Augmented responses to vasoconstrictor stimuli in hypercholesterolemic and atherosclerotic monkeys. Circ Res 1984;54:711-8. Shimakawa H, Tomoike H, Nabeyama S, Yamamoto H, Araki H, Nakamura M. Coronary artery spasm induced in atherosclerotic miniature swine. Science 1983;221:560-2. Gilligan DM, Guetta V, Panza JA, Garcia CE, Quyyumi AA, Cannon RO. Selective loss of microvascular endothelial function in h u m a n hypercholesterolemia. Circulation 1994;90:35-4L Rosenblum WI, Nishimura H, Nelson GH. Endothelium-dependent LArg- and L-NMMA-sensitive mechanisms regulate tone of brain microvessels. Am J Physiol 1990;259:H1396-401. Kovach AGB, Szabo C, Benyo Z, Csaki C, Greenberg JH, Reivich M. Effects of N-nitro-L-arginine and L-arginine on regional cerebral blood flow in the cat. J Physiol 1992;449:183-96. Panza JA, Casino PR, Kilcoyne CM, Quyyumi AA. Impaired endotheIium-dependent vasodilation in patients with essential hypertension: evidence that the abnormality is not at the muscarinic receptor level. J Am Cell Cardiol 1994;23:1610-6. Egashira K, Inou T, Hireoka Y, Kai H, Sugimachi M, Suzuki S, Kuga T, Urabe Y, Takeshita A. Effects of age on endothelium-dependent vasodilation of resistance coronary artery by acetylcholine in humans. Circulation 1993;88:77-81. Reis SE, Gloth ST, Blumenthal RS, Resar JR, Zarur HA, Gerstenblith G, Brinker JA. Ethinyl estradiol acutely attenuates abnormal coronary vasomotor responses to acetylcholh~e in postmenopausa] women. Circulation 1994;89:52-60.
J u n e 1996 Volume 131, Number 6
CLINICAL INVESTIGATIONS
Chronic Coronary Disease
Nitric oxide production by coronary conductance and resistance vessels in hypercholesterolemia patients Nobuo Shiode, MD, Kensyo Nakayama, MD, Nobuyuki Morishima, MD, Togo Yamagata, MD, Hideo Matsuura, MD, and Goro Kajiyama, MD Hiroshima, Japan
NG-monomethyI-L-arginine (L-NMMA), a specific inhibitor of nitric oxide (NO) synthesis, was used to investigate the effects of inhibition of NO synthesis on the coronary conductance and resistance vessels in hypercholesterolemic patients. Acetylcholine (3 and 30 pg/min) was administered to 10 hypercholesterolemic and 10 control patients before and after L-NMMA (25 pmol/min) infusion. Epicardial coronary diameter was measured by quantitative angiography, and coronary blood flow (CBF) was derived from Doppler flowvelocity and coronary diameter measurements. In hypercholesterolemic patients, acetylcholine-induced dilation of epicardial arteries was attenuated, and the percentage increase in CBF caused by acetylcholine was smaller than that in control patients. L-NMMA attenuated acetylcholine-induced dilation of epicardial arteries in control patients. L-NMMA had no effect on CBF responses to acetylcholine in both patient groups. L-NMMA significantly decreased the baseline coronary diameter and CBF in both groups. These results indicated that hypercholesterolemia impaired the acetylcholine-induced dilation of the conductance and resistance coronary vessels. This impairment in the conductance vessels was dependent on NO production; that of resistance vessels was not. The basal release of NO in conductance and resistance vessels was preserved in hypercholesterolemic patients. (Am Heart J 1996; 131:1051-7.)
The endothelium modulates vascular smooth muscle tone by synthesizing and metabolizing vasoactive substances, including an endothelium-derived relax-
From the First Department of Internal Medicine, Hiroshima University School of Medicine. Received for publication July 17, 1995; accepted Oct. 30, 1995. Reprint requests: Nobuo Shiode, MD, The First Department of Internal Medicine, Hiroshima University School of Medicine, 1-2-3 Kasumi-cho, Minami-kn, Hiroshima, 734, Japan. Copyright © 1996 by Mosby-Year Book, Inc. 0002-8703/96/$5.00 + 0 4/1/71348
ing factor (EDRF). 1,2 Recent studies in human beings showed that acetylcholine infusion dilates angiographically normal coronary arteries and constricts arterial segments containing atherosclerotic lesions. 3-6Acetylcholine also increases coronaryblood flow. In addition, atherosclerosis and other coronary risk factors are associated with an impaired coronary blood flow in response to acetylcholine. 7-1° Such findings suggest that endothelial dysfunction develops in the epicardial and resistance coronary arteries in conjunction with coronary risk factors. Hypercholesterolemia is an important risk factor for coronary atherosclerosis and impairs the endothelium-dependent vasorelaxation of the large coronary arteries before the formation of atherosclerotic lesions.e, s, 11 Hypercholesterolemia also is associated with a blunted endothelium-dependent vasodilation of the coronary microcirculation in animals and h u m a n beings. 9, 11-13However, previous studies evaluated only the vascular response to agonists such as acetylcholine in patients with atherosclerosis and coronary risk factors. EDRF was demonstrated more than a decade ago by Furchgott and Zawadzki. 2 Nitric oxide is synthesized from L-arginine by endothelial cells. 14, 15 N Q monomethyl-L-arginine (L-NMMA) inhibits its formation in a concentration-dependent and stereospecific manner. 16 Our study investigated the effects of blocking the production of nitric oxide by L-NMMA on the coronary circulation of hypercholesterolemic patients. We attempted to determine whether the impaired vascular responses to acetylcholine in such patients was associated with impaired production of nitric oxide and whether a basal release of nitric ox1051
June 1996 American Heart Journal
1052 Shiode et al.
Table I. Plasma lipoprotein levels
Total cholesterol HDL cholesterol LDL cholesterol Triglycerides
Hypercholesterolemic patients (n = 10) (rag /dl)
Control patients (n = 10) (rag /dl)
p Value
266 _+8 40 _+3 194 _+8 159 _+24
172 -- 5 44 -- 2 107 -- 6 111 _+17
<0.01 NS <0.01 NS
D a t a are e x p r e s s e d as m e a n +- SEM.
ide exists in the epicardial a n d resistance c o r o n a r y arteries of p a t i e n t s with hypercholesterolemia.
METHODS Patients. Ten patients with hypercholesterolemia (fasting total cholesterol level >220 mg/dl) and 10 patients without hypercholesterolemia who had atypical chest pain, normal exercise test results, normal left ventricular function (contrast ventriculogram ejection fraction, ->50%)i and normal coronary flow reserve were studied (Table I). These patients had no significant atherosclerotic stenosis (>25%) in the epicardial coronary arteries. Serum total and high-density lip0protein (HDL) cholesterol and triglycerides were determined by the enzyme-assay method. Lowdensity lipoprotein (LDL) was calculated as (total cholesterol - HDL cholesterol - [triglyceride/5]). Hypercholesterolemic patients. The hypercholesterolemic group comprised 10 patients, 5 men and 5 women, mean age 59 _+ 2 years (range, 48 to 68 years). None was taking a cholesterol-lowering agent before this study. Excluded from study were hypercholesterolemic patients with a family history of hypercholesterolemia, thickening of the Achilles tendon, or xanthoma, as well as any patients with secondary hypercholesterolemia. Control patients. The control group comprised 10 patients, 8 men and 2 women, mean age 54 +_ 3 years (range, 40 to 66 years). The total cholesterol level of each patient in this group was <200 mg/dl. No patients had hypertension (defined as systolic blood pressure >160 mm Hg or diastolic blood pressure >95 mm Hg or both) or diabetes mellitus. Two of the hypercholesterolemic patients and 2 of the control patients were smokers (>10 cigarettes/day). These four patients were asked to refrain from smoking for the 72 hours preceding the study. Written informed consent was obtained from all patients before the diagnostic angiogram. This study was approved by the Human Investigation Ethical Committee of the University of Hiroshima School of Medicine. Study design. Antianginal medication was discontinued 48 hours before catheterization, except for sublingual nitroglycerin, which was withheld I hour before catheterization, but no patient in this study had sublingual nitroglycerine 24 hours before catheterization. Patients were brought to the catheterization laboratory in the fasting state after premedication with hydroxyzine (25 mg, IM) and promethazine hydroch]oride (25 mg, IM). Diagnostic right and left heart catheterization and cor-
onary angiography were performed through a standard percutaneous femoral approach. After vascular access had been obtained, 10,000 U of heparin was infused intravenously. A 6F guide catheter was introduced into the left main coronary artery. A 0.014-inch Doppler flow guide wire (FloWire; Cardiometrics, Mountain View, Calif.) was advanced through the guide catheter into the proximal segment of the left anterior descending coronary artery. Protocol. After the diagnostic catheterization, the following interventions were performed: (1) infusion of saline (1 ml/min for 2 minutes); (2) serial infusion of intracoronary acetylcholine, 3 ~g/min and 30 ~g/min (1 ml/min for 2 minutes each); (3) infusion of adenosine, 100 pg/min (1 m]/min for 2 minutes). Next L-NMMA was infused at a dose of 25 ~mol/min (1 ml/min for 5 minutes). Interventions 1 through 3 were repeated and were followed by an infusion of nitroglycerine, 200 pg/min. All drugs were infused into the left coronary ostium through the guide catheter. We waited 5 minutes after each of the infusions of adenosine and acetylcholine before beginning the next infusion. Coronary arteriography was performed immediately after the end of each infusion. The heart rate, arterial pressure, coronary blood flow velocity, and electrocardiogram were continuously monitored during each infusion. Measurements were also recorded under steady-state conditions. Quantitative coronary angiography. Quantitative coronary angiography was used to measure the diameter of the coronary conductance vessels. Coronary cineangiograms were recorded on 35-mm cinefilm (30 frames/sec) with a Siemens (Munich, Germany) cineangiographic system after selecting the best view for left anterior descending coronary artery visualization. Nonionic contrast medium was injected into the left coronary artery at a rate of 5 to 7 mY sec for a total of 7 to 10 ml. A power injector (Medrad, Pittsburgh, Pc.) was used to optimize the quality and reproducibility of the opacification. 17 Angiograms were obtained while the patients held their breath to avoid the artifacts associated with respiration.iS The proximal and the distal segments of left anterior descending coronary artery were selected for quantitative analysis. The diameter of the lumen of each arterial segment was measured with a computer-assisted system for analyzing coronary angiograms. The arterial segments evaluated were video-digitized at end diastole, with data stored in a cardiac imageanalysis system (Cardio 500; Kontron Instruments, Munich, Germany). Automated counter detection was
Volume131,Number6 Shiode et al.
American Heart Journal
1053
Table II. H e m o d y n a m i c c h a n g e s Group
Saline
Hypercholesterolemia HR 64 _+ 3 mBP 98 ± 3 RPP 9021 ± 414 Control HR 63 ± 2 mBP 99 ± 4 RPP 8172 +_ 514
A Ch 30 Ftgrn/min
Adenosine 100 ~tgm/min
L-NMMA 25 #mol/min
A Ch (30 ~tgrn ~rain)
Adenosine 100 Itgm/min
NTG 200 ~tgm ~rain
64 ± 3 98 -+ 4 9283 ± 620
64 ± 3 96 ± 4 9160 ± 589
62 ± 3 105 ± 4* 9593 -+ 542
62 ± 3 103 ± 4 9471 ± 525
62 ± 3 102 ± 4 t 9568 ± 606
71 _+ 3* 91 +_ 5* 9079 ± 581
64 ± 3 97 ± 4 8262 ± 647
64 ± 3 96 ± 4 8214 ± 646
61 +_ 3 102 ± 4* 8389 ± 714
62 ± 3 102 ± 4 8444 ± 660
63 ± 3 100 ± 4~ 8385 ± 616
72 ± 5* 92 ± 4* 8090 -+ 550
Data are expressed as mean ± SEM. HR, Heart rate (beats/min); mBP, mean arterial blood pressure (mm Hg); RPP, rate-pressure product (ram Hg beats/min); ACh, acetylcholine; L-NMMA, NQmonomethyl:L-arginine; NTG, nitroglycerine. *p < 0.05 compared with baseline. tp < 0.05 compared with data before L-NMMA treatment.
Table III. C h a n g e i n c o r o n a r y blood flow ( C B F ) Hypercholesterolemic patients (n = 10) Before L-NMMA CBF (ml / min) Baseline 61.0 _+ 7.9 Acetylcholine 3 ~g/min 88.4 ± 14.5 30 ~g/min 142.9-+ 20.4 Adenosine 251.0 ± 37.6
ACBF ~ baseline)
Control patients (n = 10)
After L-NMMA CBF (ml / min)
Before L-NMMA
ACBF (X baseline)
53.3 _+ 8.5* 1.44 ± 0.10 2.40 ± 0.18 4.14 ± 0.25
70.8 ± 12.0t 120.7 _+ 21.6" 248.8 ± 39.8
CBF (ml / min)
ACBF (X baseline)
52.0 ± 6.8 1.36 ± 0.08 2.28 _+ 0.23 4.88 ± 0.46*
101.2 ± 24.0 213.3 _+ 34.2 226.0 ± 29.3
After L-NMMA CBF (ml / min)
ACBF ~ baseline)
42.9 ± 5.1t 1.80 ± 0.21 4.05 ± 0.35 4.37 ± 0.26
75.3 ± 14.0 144.0 ± 16.0" 234.9 ± 31.5
1.72 _+ 0.16 3.44 ± 0.19 5.46 ± 0.43t
Data are expressed as mean -+ SEM. *'1o< 0.05. tp < 0.01 compared with data before L-NMMA.
performed by a geometric edge differentiation technique s i m i l a r to t h a t d e s c r i b e d b y R e i b e r e t al. 19 T h e d i a m e t e r of t h e s e g m e n t of i n t e r e s t w a s m e a s u r e d , a n d t h e a v e r a g e v a l u e of t r i p l i c a t e m e a s u r e m e n t s w a s u s e d for a n a l y s i s . A 6 F J u d k i n s c a t h e t e r w a s u s e d to c a l i b r a t e t h e a r t e r i a l diameter in millimeters. Arterial diameter was measured in b l i n d f a s h i o n b y t w o i n v e s t i g a t o r s (K. N. a n d N. M.), w h o h a d n o k n o w l e d g e of t h e p a t i e n t s ' c l i n i c a l c h a r a c t e r i s t i c s . Measurements of coronary blood flow velocity and estimation of coronary blood flow. A Doppler guide wire w i t h a 12 M H z p i e z o e l e c t r i c t r a n s d u c e r a t t h e t i p (FloWire; Cardiometrics), was used in measuring the coronary a r t e r y blood-flow velocity. T h i s t e c h n i q u e w a s p r e v i o u s l y v a l i d a t e d . 2° C o n t i n u o u s flow-velocity p r o f i l e s f r o m t h e 12 MHz pulsed Doppler velocimeter (F]oMap; Cardiometrics), together with simultaneous electrocardiograms and aortic pressures, were displayed on a videomonitor and continuo u s l y r e c o r d e d o n 0 . 5 - i n c h V H S v i d e o t a p e . C h a n g e s i n coro n a r y b l o o d flow i n r e s p o n s e to a d m i n i s t r a t i o n of t h e vas o a c t i v e a g e n t s w e r e e s t i m a t e d f r o m t h e p r o d u c t of t h e m e a n c o r o n a r y blood-flow v e l o c i t y a n d t h e c r o s s - s e c t i o n a l a r e a 2 to 3 m m d i s t a l to t h e t i p of t h e D o p p l e r flow g u i d e wire. T h i s d i s t a n c e w a s s e l e c t e d b e c a u s e t h e D o p p l e r
t r a n s d u c e r h a d a r a n g e g a t e d e p t h of 4:2 m m . C h f l i a n e t al. 21 s h o w e d t h a t c o r o n a r y blood flow is d e t e r m i n e d pred o m i n a n t l y b y s m a l l r e s i s t a n c e v e s s e l s (<200 ~ m i n d i a m eter), so w e m e a s u r e d c o r o n a r y b l o o d flow to e v a l u a t e res p o n s e s of c o r o n a r y r e s i s t a n c e v e s s e l s to v a s o a c t i v e a g e n t s . Drug preparations. A c e t y l c h o l i n e ( D a i i c h i P h a r m a c e u tical, Tokyo), w a s dissolved, i m m e d i a t e l y b e f o r e use, i n p h y s i o l o g i c s a l i n e a t a c o n c e n t r a t i o n of 3 ~ g / m l or 30 p g / m l . A d e n o s i n e ( S i g m a C h e m i c a l , St. Louis, Mo.) a n d L - N M M A ( S i g m a ) w e r e d i s s o l v e d i n physiologic s a l i n e a t c o n c e n t r a t i o n s of 100 ~ g / m l a n d 25 ~ m o l / m l , r e s p e c t i v e l y . Nitroglyce r i n e ( N i h o n k a y a k u , Tokyo, J a p a n ) w a s d i s s o l v e d i n p h y s iologic s a l i n e a t a c o n c e n t r a t i o n of 2 0 0 ~g/ml. E a c h d r u g w a s i n f u s e d w i t h a n i n f u s i o n p u m p ( C F V 3000, Nih o n k o d e n , Tokyo, J a p a n ) a t a r a t e of 1 m l / m i n . Statistics. D a t a a r e e x p r e s s e d a s m e a n _+ S E M . Twow a y a n a l y s i s of v a r i a n c e ( A N O V A ) for r e p e a t e d m e a s u r e s w a s u s e d to c o m p a r e t h e d o s e - r e s p o n s e to a c e t y l c h o l i n e b e f o r e a n d a f t e r L-NMMA, w i t h i n or b e t w e e n g r o u p s . T h e d i f f e r e n c e s b e t w e e n t h e effects of a c e t y l c h o l i n e b e f o r e a n d after L-NMMA in both groups were compared by using the p e r c e n t a g e c h a n g e s f r o m b a s e l i n e b e c a u s e of t h e b a s e l i n e d i f f e r e n c e s i n c o r o n a r y d i a m e t e r s a n d c o r o n a r y b l o o d flow
1054
June 1996
Shiode et al.
AmericanHeartJournal
-5
.c~
10-
*
t.
--o- ] 1" p < 0.01
" bas''
"
-15:
-20:
11.P
]] NS
-25T baseline
3
3()
ACh (l~g/min)
Fig, 1, Percentage change in diameter of proximal and distal segments of left anterior descending coronary artery in response to serial infusions of acetylcholine (ACh). ACh, 3 and 30 ~g/min, was administered to hypercholesterolemic and control patients, before and after L-NMMA (25 ~mol/min). Open circles, Control patients before L-NMMA; solid circles, control patients after L-NMMA;open squares, hypercholesterolemic patients before L-NMMA; solid squares, hypercholesterolemic patients after L-NMMA; NS, not significant. Data are expressed as mean -+ SEM. *p < 0.05; **p < 0.01 compared with baseline; tp < 0.01 between patients with and without hypercholesterolemia as determined by analysis of variance. in the two groups. Serial changes in blood flow, coronary diameter, and hemodynamic variables in response to graded doses of acetylcholine were compared by one-way ANOVA. If the ANOVA showed a significant difference between the mean values, the level of statistical significance was determined by contrast. Paired data were compared by paired t tests. A level ofp < 0.05 was accepted as statistically significant. RESULTS Clinical characteristics and hemodynamic variables,
Plasma lipoprotein levels of the patients studied are shown in Table I. Intracoronary infusions of L-NMMA had no effect on the h e a r t rate or the ratepressure product. L-NMMA produced a significant increase in m e a n arterial pressure in both the patients with hypercholesterolemia and the control patients (Table II). Nitroglycerine reduced the m ean arterial pressure and increased the h e a r t rate in both groups. Serial infusions of acetylcholine produced no significant change in m ean blood pressure, h e a r t rate, or rate-pressure product before or after LNMMA t r e a t m e n t in either group (Table II).
--o- Control L-NMMA (-) ~ ContrL-NMMAL'NMMA(-) OIHc (+) j ] "-=-- HC L-NMMA (+)
1" P < 0.01
Fig. 2. Percentage change in coronary blood flow. The change in coronary blood flow (ACBF), before and after LNMMA administration, in response to serial infusions of acetylcholine (ACh) at 3 and 30 ~g/min in hypercholesterolemic and control patients. Open circles, Control patients before L-NMMA; solid circles, control patients after LNMMA; open squares, hypercholesterolemicpatients (HC) before L-NMMA; solid squares, hypercholesterolemic patients after L-NMMA. Data are expressed as mean -+ SEM. tp < 0.01 between patients with and without hypercholesterolemia as determined by analysis of variance. eter of the distal segment of the epicardial coronary arteries (Fig. 1). However, a dose of acetylcholine, 30 pg/min, abolished the vasodilating effect on these arteries. Acetylcholine also produced a dose-dependent increase in coronary blood flow (Table III). In patients with hypercholesterolemia, an acetylcholine infusion, 30 ~g/min, reduced the diameter of both the proximal and distal segments of the epicardial coronary arteries. Conversely, acetylcholine, 3 pg/min, had no effect. Acetylcholine caused a dosedependent increase in coronary blood flow, but the percentage increase in coronary blood flow in the hypercholesterolemic patients was significantly smaller t h a n t h a t in control patients (Fig, 2). Effects of L-NMMA on epicardial artery and coronary blood flow under basal conditions. L-NM~MA, 25 ~mo]]
min, significantly decreased the baseline coronary blood flow from 61.0 _+ 7.9 ml/min to 53.3 _+ 8.5 mY min (p < 0.05) in hypercholesterolemic patients and from 52.0 _+ 6.8 ml/min to 42.9 _+ 5.5 ml/min (p < 0.01) in the control patients. After L-NMMA t reat m ent , the diameters of the proximal and distal segments of the epicardial coronary arteries decreased significantly in both groups (Fig. 3).
Effects of acetylcholine on epicardial coronary arteries and coronary blood flow. In control patients, acetyl-
Effect of L-NMMA on vascular response to acetylcholine. In control patients, by the intracoronary infu-
choline, 3 ~g/min, significantly increased the diam-
sion of L-NMMA, the acetylcholine (3 ~g/min)-in-
Volume 131, Number 6 American Heart Journal
duced dilation of the distal epicardial arteries was attenuated. In both groups, acetylcholine, 3 pg/min, significantly decreased the diameter of the distal segment. Acetylcholine, 30 pg/min, significantly decreased the diameters of the proximal and distal segments of the epicardial coronary arteries (Fig. 1). Acetylcholine produced a dose-dependent increase in coronary blood flow after L-NMMA administration in the patients with and without hypercholesterolemia. The percentage increase in coronary blood flow after L-NMMA treatment was similar to that before LNMMA treatment in both groups (Fig. 2). Effect of adenosine on coronary blood flow. T h e en-
dothelium-independent vasodilator adenosine significantly increased the coronary blood flow in both groups, regardless of L-NMMA addition (Table III). The percentage increase in coronary blood flow induced by adenosine did not differ between the two groups. Effect of nitroglycerine on the diameter of epicardial coronary arteries. Nitroglycerine significantly in-
Shiode et al.
40
1055
Proximal Segment
30 20 E ;.=, ~.~
10 0 .......,
o o~ -10
oc~ 40
~, (b
Distal Segment
3o 2o
10
ol
.loi baseline
L-NMMA 25 limol/min
N:rG
Fig. 3. Effects of intracoronary infusion of L-NMMA (25 ~mol/min) and nitroglycerine (NTG; 200 pg/min) on the baseline coronary diameter of the proximal and the distal left anterior descending coronary artery. Open circles, Control patients; open squares, hypercholesterolemic patients. Data are expressed as mean _+SEM. *p < 0.05; **p < 0.01 compared with baseline.
creased the diameter of epicardial coronary arteries to the same degree in both groups (Fig. 3). DISCUSSION
Intravascular Doppler ultrasound techniques have recently become available for the study of function in the coronary circulation in vivo. Intravascular Doppler transducers provide beat-to-beat information on coronary blood-flow velocity, 22 and used in combination with quantitative coronary angiography data on cross-sectional area, yield measurements of coronary blood flow. 9, io Simultaneous information on the effects of pharmacologic agents on luminal diameter and coronary blood flow allow analysis of their differential effects on the epicardial arteries and the microcirculation. In this study, we measured changes in epicardial coronary diameter and coronary bloodflow velocity to define the pharmacologic action of each agent on the conductance and resistance coronary vessels. Lefroy eta]. 23 showed that L-NMMA, at a dose of 25 pmol/min, significantly reduced the distal, but not the proximal, left anterior descending coronary artery diameter along with a decrease in coronary venous oxygen saturation. These authors concluded that there was a basal release of nitric oxide in the epicardial coronary arteries and the resistance vessels. In our study, L-NMMA administration reduced the basal diameter of the epicardial arteries and reduced the basal coronary blood flow in control patients, indicating a basal release of nitric oxide in both the epicardial coronary arteries and the resistance vessels.
Sudhir et al. 24 reported that the vasodilator response to acetylcholine in dogs was inhibited by Nnitro-L-arginine methyl ester (L-NAME), a inhibitor of nitric oxide synthetase, in the epicardial coronary arteries but not in the resistance vessels. This suggests that acetylcholine-induced, endothelium-dependent relaxation in coronary resistance vessels may not be mediated by nitric oxide alone. In a clinical study, Lefroy et al. 2~ reported that L-NMMA had no effect on the acetylcholine-induced increase in coronary blood flow. Our findings in control patients resemble these of Lefroy et al. and indicate that acetylcholine-induced dilation of the epicardial arteries is mediated by nitric oxide, whereas acetylcholineinduced dilation of resistance vessels is not. This is the first report of the effects of an inhibitor of nitric oxide synthesis on the coronary circulation of patients with hypercholesterolemia. The acetylcholine-induced dilation of the epicardial arteries and of the resistance vessels was reduced in such patients, consistent with previous reports that hypercholesterolemia impairs the endothelium-dependent vasorelaxation of the large coronary arteries and the resistance vessels before the development of the atherosclerotic lesions. 6, s;9, 11-13,25 The vasodilator action of acetylcholine is mediated through EDRFs. Simultaneously, this compound exerts a vasoconstrictor action on the smooth muscle of the epicardial coronary arteries. 25 Evidence from studies in animals and h u m a n beings indicates that atherosclerosis is associated with enhanced vasocon-
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strictor response to cathecholamines, serotonin, histamine, and ergonovine. 20, 27-29 It is possible that in patients with hypercholesterolemia, impairment of epicardial artery dilation in response to acetylcholine is caused by increased sensitivity of the smooth muscle to acetylcholine. However, after L-NMMA treatment, we observed that acetylcholine, 3 and 30 ~g/min, decreased the diameter of the epicardial coronary arteries in patients with or without hypercholesterolemia. This suggests that in hypercholesterolemic patients the impairment of acetylcholineinduced dilation of the epicardial coronary arteries is caused by lack of endothelial nitric oxide production in response to acetylcholine, not by increased sensitivity of smooth muscle to acetylcholine. Acetylcholine-induced increases in coronary blood flow were reduced in the hypercholesterolemic patients. L-NMMA had no effect on the acetylcholineinduced change in coronary blood flow regardless of lipid status. These results indicate that hypercholesterolemia impairs other endothelial functions in resistance vessels separate from nitric oxide formation. Gilligan et al. 3° reported that L-NMMA increased basal forearm vascular resistance to similar degrees in patients with and without hypercholesterolemia. These authors concluded that hypercholesterolemic patients had normal nitric oxide bioavailability. In our study ofhypercholesterolemic patients, L-NMMA decreased in the basal diameter of the large epicardial coronary arteries and reduced coronary blood flow. The extent of the decrease did not differ between patient groups, indicating that the basal release of nitric oxide in patients with hypercholesterolemia was preserved in the epicardial arteries and the resistance vessels. Limitations. We used a 25 pmol/min infusion of L-NMMA for 5 minutes. Lefroy et al. 23 reported that infusions of L-NMMA (4, 10, and 25 ~mol/min, each for 5 minutes) abolished the acetylcholine-induced dilation of h u m a n epicardial coronary arteries. We also observed that the endothelium-dependent vasodilation induced by a 3 ~g/min infusion of acetylcholine was reduced after L-NMMA treatment. Thus at the dose chosen, L-NMMA attenuated the epicardial coronary dilation caused by agonist-induced nitric oxide production, although the acetylcholine-induced increases in coronary blood flow were not reduced after the L-NMMA infusions. In our study, higher doses of L-NMMA may have attenuated the acetylcholine-induced increase in coronary blood flow. We did not administer such higher doses because of the risk of an increase in systemic blood pressure and concomitant vasoconstriction in other vascular beds. 31, 32
June 1996 American Heart Journal
We observed an unaltered basal release of nitric oxide in the large epicardial coronary arteries and resistance vessels of our hypercholesterolemic pa: tients. All of these patients had no angiographically significant stenotic lesions, suggesting that endothelial function was not sufficiently impaired to reduce the basal nitric oxide release. Panza et a l Y showed that the effect of L-NMMA on basal vascular tone was reduced in hypertensive patients compared with normal control subjects. This finding is consistent with a diminished basal release of nitric oxide in hy: pertensive patients. In this study, if the hypercholesterolemic patients had detectable stenotic lesions in their epicardial coronary arteries along with greater endothelial dysfunction, the basal release of nitric oxide would be diminished. Finally, we studied the only small sample size. In this study, the mean age of the patients in each group did not differ significantly, but the hypercholesterolemic patients tended to be older than the controls. Several studies 12, 34 have shown that advanced age is a significant independent predictor of impaired endothelium-dependent dilation of the coronary resistance vessels, regardless of the presence or absence of epicardial artery atherosclerosis. Therefore, the impairment of coronary bloodflow response to acetylcholine seen in our hypercholesterolemic patients may have been influenced by their age. Moreover, the hypercholesterolemic patient group included many more women than the control group. Reis et al. 35 showed that ethinyl estradiol acutely attenuated abnormal coronary vasomotor responses to acetylcholine. However, because only one woman in the hypercholesterolemic group was menstruating and all the other women in both groups were postmenopausal, the sex mismatch might have little influence on our results. At least, it was unlikely that the different sex distribution had a bad influence in the hypercholesterolemic group. Conclusions. Results of this study indicate that the basal release of nitric oxide from endothelial cells of coronary conductance and resistance vessels is preserved in patients with hypercholesterolemia. However, the production of nitric oxide induced by acetylcholine in coronary conductance and resistance vessels is impaired in these patients. This impairment of conductance arteries was dependent on a decrease in nitric oxide production, but that of the resistance vessels was not. We thank Drs. Yuji Yasunobu, Masaya Kato, Koichi Tanaka, Hitoshi Fujiwara, Akito Hiraoka, and Shinji Karakawa and the nurses and technicians of the catheterization laboratory at Hiroshima University Hospital.
Volume 131, Number 6 American Heart Journal
Shiode et al.
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