(Circulation. 1995;92:2636-2644.)
© 1995 American Heart Association, Inc.
Articles |
Chronic Inhibition of Endothelium-Derived Nitric Oxide Synthesis Causes Coronary Microvascular Structural Changes and Hyperreactivity to Serotonin in Pigs
From the Research Institute of Angiocardiology and Cardiovascular Clinic and the First Department of Pathology (K.S.), Kyushu University School of Medicine, Fukuoka, Japan.
Correspondence to Kensuke Egashira, MD, PhD, The Research Institute of Angiocardiology and Cardiovascular Clinic, Kyushu University School of Medicine, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812, Japan.
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Abstract |
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 Top Abstract IntroductionMethods Results Discussion References |
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Background Endothelium-derived nitric oxide (NO) is believed to regulate myocardial perfusion and structural changes in the vascular wall. Our objective was to determine whether chronic inhibition of NO synthesis causes structural and functional changes in coronary arteries.
Methods and Results Coronary vasomotor response was
studied in pigs before and after chronic oral administration of the NO
synthesis antagonist
N
-nitro-L-arginine methyl ester
(L-NAME) 30 mg · kg-1 · d-1
for 2
weeks. Chronic L-NAME treatment increased (P<.01)
arterial pressure but did not alter baseline
coronary blood flow (CBF), epicardial coronary
diameter, or heart rate. Chronic L-NAME treatment augmented
(P<.01) the decrease in CBF in response to
intracoronary serotonin (30 µg/kg) from
5±14% to 40±5% but did not alter the CBF response to
prostaglandin F2
. The
serotonin-induced decrease in CBF after acute L-NAME
administration was still less before (1.3±0.4%) than after chronic
L-NAME treatment (51±6%). Chronic L-NAME treatment attenuated the
increase in CBF with bradykinin (100 ng/kg) but did not alter the CBF
response to nitroglycerin (10 µg/kg). Compared with
intact pigs without L-NAME treatment, L-NAME–treated pigs had
significant thickening of the media in the microvessels (diameter,
<300 µm) but not in the large epicardial vessels. Chronic
intracoronary infusion of L-NAME at 3
mg · kg-1 · d-1 for 2 weeks,
which
did not produce arterial hypertension, caused similar
microvascular medial thickening.
Conclusions These results indicate that chronic administration of L-NAME caused coronary microvascular structural changes and hyperreactivity to serotonin in pigs in vivo, suggesting an important role of defective NO synthesis in coronary microvascular disorders.
Key Words: microcirculation • endothelium-derived factors • nitric oxide • coronary disease • remodeling
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Introduction |
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TopAbstractIntroductionMethods Results Discussion References |
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The vascular endothelium plays an important role in the regulation of vascular tone, platelet aggregation, thrombus formation, and proliferation/remodeling of the blood vessel wall.1 2 3 NO or a related compound is a major substance that accounts for the vascular effects of endothelium-derived relaxing factor.4 5 NO is synthesized through the metabolism of L-arginine by NO synthase, which is inhibited by L-arginine analogues.1
Recent studies in animals and humans showed that the presence of coronary risk factors and atherosclerosis was associated with endothelial dysfunction of coronary arteries,6 7 8 9 which may alter CBF regulation and thus contribute to myocardial ischemia. It has been shown that NO has antiproliferative effects on the vascular wall in vitro.10 11 12 In addition, defective NO synthesis contributes to vascular structural changes by the release of growth-promoting substances.2 3 However, it is not known whether endothelial dysfunction (defective NO synthesis) causes structural changes in coronary arteries in vivo.
It was recently reported that chronic inhibition of NO synthesis with an L-arginine analogue caused systemic arterial hypertension in normal rats13 14 and accelerated atherosclerotic lesions in cholesterol-fed rabbits.15 However, coronary vascular structural and functional changes associated with the chronic inhibition of NO synthesis in vivo are poorly understood. This study was designed to determine whether chronic inhibition of NO synthesis with L-arginine analogue would cause structural and functional changes in the coronary arteries of pigs in vivo.
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Methods |
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TopAbstractIntroductionMethods Results Discussion References |
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Materials
Healthy male domestic pigs weighing 23 to 28 kg were housed individually and fed a regular diet at the Animal Research Institute of Kyushu University School of Medicine. The protocol of the present study conformed to the "Guide for Care and Use of Laboratory Animals of Kyushu University Faculty of Medicine."
Experimental Protocols
Protocol 1. The effects of chronic oral administration of L-NAME on coronary vasomotion were studied. Pigs were sedated with intramuscular ketamine hydrochloride (12.5 mg/kg) and anesthetized with intravenous sodium pentobarbital (20 mg/kg). They were then intubated and ventilated with a respirator. Under aseptic conditions, a left thoracotomy was performed, and an ultrasonic transit-time flow probe (Transonic Systems Inc) was placed at the midportion of the LAD. A telemetry transmitter system with a catheter-type pressure manometer (D70, Primetech Inc) was implanted at the subcutaneous space of the neck, and the tip of the manometer was placed in the descending aorta through the left carotid artery. The chest was then closed, and the animals were allowed to recover from the surgery.
Before and 2 weeks after the pigs received a
regular diet supplemented
by L-NAME at doses of 30
mg · kg-1 · d-1, the following
experiments were repeated in eight anesthetized
closed-chest pigs. An 8F Kifa catheter was introduced into the
carotid artery and advanced to the orifice of the left coronary
artery. Physiological saline, serotonin
(3, 10, and 30 µg/kg), adenosine (30 µg/kg), bradykinin
(100 ng/kg), PGF2 (50 µg/kg), and
nitroglycerin (10 µg/kg) were administered into the
left coronary artery via the catheter while CBF and other
hemodynamic variables were measured. In some pigs,
serotonin at doses of 30 µg/kg was administered before
and 10 minutes after intravenous infusion of 1 mg/kg
L-NAME. All these drugs were dissolved with
physiological saline. We waited at least 10 minutes
before injecting the next drug to allow all hemodynamic
variables to return to the basal level. Coronary
arteriography using a nonionic contrast medium was performed before and
3 minutes after infusion of each drug.
Before and during oral L-NAME treatment, arterial pressure and heart rate were measured with the telemetry system without anesthesia.
Protocol 2. The effects of chronic intracoronary infusion of L-NAME at doses of 3 mg · kg-1 · d-1 on histopathological changes in coronary arteries were examined in five pigs. Using the anesthesia and sterile surgery, we inserted a 3F catheter into the obtuse marginal branch. A telemetry transmitter for the measurement of arterial pressure was also implanted. After the pigs had recovered from the surgery, L-NAME at doses of 3 mg · kg-1 · d-1 was administered into the LCx via catheter for 2 weeks with a disposable osmotic infusion pump (Infuser, Baxter Inc). Before and during intracoronary L-NAME treatment, arterial pressure and heart rate were measured by the telemetry system without anesthesia.
Protocol 3. Changes in regional myocardial blood flow in response to serotonin were studied in four L-NAME–treated pigs. After the anesthesia, a left thoracotomy was performed, and a Kifa catheter was advanced to the orifice of the left coronary artery. Through the Kifa catheter, a 2F radiopaque catheter was introduced superselectively into the midportion of the LAD for drug infusion. A 6F catheter was inserted into the left atrium for injection of nonradioactive colored microspheres. An 8F catheter was inserted into the right femoral artery to withdraw the reference blood.
Before and 5 minutes after superselective administration of serotonin at 30 µg/kg, microspheres were injected into the left atrium for measurement of myocardial blood flow.
Measurements
Before and during oral administration of L-NAME
(protocol 1) or
intracoronary infusion of L-NAME (protocol 2),
arterial pressure and heart rate were measured by a
telemetry receiver system (model RA1310, Primetech Inc) after pigs had
rested quietly in their cages at least 5 minutes without
anesthesia.
In protocol 1, CBF was measured by connecting the flowprobe to an ultrasonic transit-time flowmeter (Transonic T201D, Transonic System Inc). In protocols 1 and 3, arterial pressure was measured with a pressure transducer (Nihon-Kohden Inc). Heart rate was measured by means of a heart rate counter (AT 600G, Nihon Koden). These hemodynamic variables and ECGs (I, II, III, V1, and V6 leads) were monitored and recorded by a polygraph system.
In protocol 1, coronary arteriography was performed with a cineangiography system (KXO-1250/CAS/CA, Toshiba Medical Inc) as previously described.16 17 The angle of the projection, the posture of the animal, and the positions of the x-ray focus and the image intensifier were carefully kept constant during each experiment and before and after L-NAME administration. Coronary angiograms were recorded on 35-mm cinefilm. End-diastolic images were selected and printed. The diameters of the coronary arteries were measured with a digital caliper and calibrated by determination of the size of the angiographic catheter. The readily identifiable branch points were regarded as reference markers to accommodate serial changes in the diameter within the same arterial site. With this technique, excellent correlations between repeated measurements (r=.99) and between different observers (r=.98) were confirmed in the 0.9- to 5.0-mm–diameter range. The percent changes in luminal diameter were reported.
In protocol 3, myocardial blood flow was measured with nonradioactive colored microspheres (EZ Track) with a mean size of 15 µm. The microsphere solutions were dispersed by vortex for 1 minute. Each 0.5-mL aliquot (1x107 microspheres/mL) was taken up in a 10-mL syringe, mixed with 9.5 mL saline, and infused over 10 seconds through the catheter inserted into the left atrium. The reference blood sample was withdrawn from the catheter inserted into the right femoral artery at a rate of 10 mL/min beginning 10 seconds before and ending 70 seconds after the microsphere injection. To prevent coagulation, each sample was transferred into a 50-mL test tube containing EDTA-Na (12.5 mg). After the experiments with microspheres were completed, the animals were killed with a lethal dose of sodium pentobarbital, and their hearts were excised. After the LAD was ligated at the proximal portion, Evans blue dye was injected into the LAD to determine the area of perfusion of the myocardium. After right ventricular tissue had been removed, hearts were cut into five or six transverse slices parallel to the atrioventricular groove. Tissue sections were cut from the center of the LAD bed and subdivided into the subepicardial, midmyocardial, and subendocardial segments. Myocardial blood flow in these segments was then determined by the technique previously described by Hale et al.18
To determine whether the basal release of NO was inhibited by oral administration of L-NAME, serum nitrite (NO3-) concentration was measured19 before and after oral L-NAME administration in five pigs in protocol 1. Blood was sampled from the jugular vein. Sulfosalicylic acid (35%, 100 µL) and serum sample (500 µL) were mixed and centrifuged at 10 000g for 15 minutes. Supernatant (200 µL) was mixed with 5% NH4Cl (300 µL) and 5% NaOH (60 µL) for analysis. Griess reagent (0.1% naphthylethylenediamine dihydrochloride and 5% sulfanilamide) was added to the sample. After 10 minutes, the supernatant was removed, and the nitrite concentration was measured with a spectrophotometer.
Histopathological Examination
Pigs treated with chronic oral
administration of L-NAME (n=6)
and intracoronary infusion of L-NAME (n=5) and control pigs
without L-NAME administration (n=6) were killed with a lethal dose of
sodium pentobarbital. For fixation of the coronary arteries
under vasodilation, their hearts were excised, and the left
coronary artery was flushed with 10 mL saline containing
nitroglycerin 30 µg/kg and adenosine 10
µg/kg. The hearts then were perfused with 6% formaldehyde solution
at a pressure of 90 mm Hg. Heart weight, left ventricular
weight, and left ventricular weight per body weight were
measured. Hearts were then fixed in 6% formaldehyde for a few days and
cut transversely from base to apex serially at 1-cm intervals. Each
slice was divided into 2 to 4 blocks perfused via the LAD or the LCx,
and the blocks were sectioned at a thickness of 5 µm. These sections
were stained with Masson's trichrome, elastic van Gieson's, and
hematoxylin-eosin stains. Pictures of intramyocardial small
arteries and epicardial coronary arteries (50 to 1800 µm in
diameter) were taken at (20 to (400 magnification with a
photomicroscopic analysis system, and pathological changes were
analyzed. The inner border of the intimal layer and the outer
border of the middle layer of the vessel were traced, and the areas
encircled by the tracings were calculated automatically. For
quantification, nonround vessels due to oblique transection or
branching were excluded, and only round vessels were studied. For the
evaluation of narrowing of the arteries, the wall-to-lumen
ratios of microvessels and large epicardial arteries were calculated by
the formula (O–I)/I, where O and I are the diameters of the areas
encircled by the outer and inner borders of the vessel. Microvessels
were included in the measurement of the area of perivascular fibrosis,
and the perivascular fibrosis index (area of perivascular fibrosis/area
of vessel wall) also was calculated. In each heart, 20 to 30
microvessels and 3 to 10 large epicardial vessels were studied to
exclude possible sampling errors, and averaged values in each size of
vessel were used for analysis. To characterize microvascular
structural changes, the wall-to-lumen ratio was plotted against
its diameter, and the slope of the two variables was determined by
a simple linear regression analysis. The perivascular fibrosis
index of large epicardial arteries was not assessed because of the
difficulty of differentiating the area of fibrosis in the adventitia
from that affected by perivascular fibrosis in those arteries.
Drugs
L-NAME, serotonin, adenosine, bradykinin
(Sigma Chemical Co), nitroglycerin (Nihon-Kayaku
Pharmaceutical Co), and PGF2 (Ono Pharmaceutical Co)
were used. All drugs were dissolved in
physiological saline immediately before use.
Statistical Analysis
Data are expressed as mean±SEM.
Student's t test
was used in comparison of paired or unpaired data. Serial changes in
hemodynamic variables were compared by one-way
ANOVA followed by Bonferroni's multiple comparison tests. Changes in
CBF in response to serotonin at the graded doses before and
after L-NAME were compared by two-way ANOVA followed by the
multiple comparison tests. A level of P<.05 was considered
statistically significant.
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Results |
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TopAbstractIntroductionMethods Results Discussion References |
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Effects of Chronic Administration of L-NAME on Arterial Pressure and Heart Rate in Conscious Pigs (Protocols 1 and 2)
Chronic oral administration of L-NAME (30 · kg-1 ·d-1) (protocol 1) increased arterial pressure but did not alter heart rate. Mean arterial pressures before L-NAME administration and 2, 4, 8, 12, and 14 days after L-NAME were 93±3, 132±8, 114±7, 128±7, 130±7, and 132±5 mm Hg, respectively (P<.01 versus before L-NAME for each). Heart rates before L-NAME and 2, 4, 8, 12, and 14 days after L-NAME were 123±8, 96±10, 103±10, 116±9, 113±8, and 115±6 beats per minute, respectively (P=NS). Since there were no apparent diurnal changes in arterial pressure and heart rate after L-NAME administration in our preliminary experiments, these variables were measured between 2 and 4 PM and used for analysis.
Chronic intracoronary infusion of L-NAME at doses of 3 mg · kg-1 · d-1 (protocol 3) did not alter arterial pressure (94±7 mm Hg before L-NAME and 93±8 mm Hg 14 days after L-NAME) or heart rate (85±5 beats per minute before and 80±1 beats per minute after L-NAME).
Effects of Chronic Administration of L-NAME on Coronary
Vasomotor Responses in Anesthetized Closed-Chest Pigs
(Protocol 1)
Chronic oral administration of L-NAME tended
(P=.08) to
increase mean arterial pressure. No significant differences
were observed before or after chronic oral administration of L-NAME for
2 weeks in terms of mean arterial pressure, heart rate,
CBF, or coronary artery diameter in the baseline conditions
(Table 1
).
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Before L-NAME, intracoronary serotonin at 3
and
10 µg/kg mildly but significantly (P<.05) increased CBF,
but serotonin at 30 µg/kg did not alter it.
Serotonin at the same three doses did not alter
arterial pressure or heart rate. After chronic L-NAME
administration for 2 weeks, serotonin decreased CBF in a
dose-dependent manner without altering arterial
pressure or heart rate. The percent decreases in CBF in response to
serotonin after 2 weeks of administration of L-NAME were
significantly greater than those before L-NAME (Fig 1).
In three of the eight pigs, serotonin at 30 µg/kg caused
ischemic ST segment elevation in leads V1 and
V6 after 2 weeks of administration of L-NAME, whereas it
had no such effect before L-NAME.
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P<.01 by
multiple-comparison t tests.
Effects of acute L-NAME
administration on the CBF responses to
serotonin at 30 µg/kg were studied in pigs before and
after chronic L-NAME treatment for 2 weeks (n=4, Table
2). Before and after chronic L-NAME treatment,
acute intravenous administration of L-NAME (1 mg/kg
within 5 minutes) increased basal mean aortic pressure but did not
alter heart rate, CBF, or their responses to serotonin. The
percent decrease in CBF in response to serotonin after
acute L-NAME administration was still less before than after chronic
L-NAME treatment.
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Intracoronary PGF2 at 50 µg/kg
decreased
CBF without altering arterial pressure and heart rate; the
percent change in CBF in response to PGF2 did not differ
before (-64±12%) or after (-47±16%) chronic L-NAME
treatment (Fig 1
). The percent increase in CBF in response to
intracoronary bradykinin at 100 ng/kg was less after
(125±39%) than before (286±56%, P<.05) chronic
L-NAME
treatment, whereas the responses of CBF to adenosine at 30
µg/kg (from 220±30% to 244±46%) and nitroglycerin
at 10 µg/kg (from 113±35% to 76±44%) did not differ before
and
after chronic L-NAME treatment (Fig 2).
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Changes in
epicardial coronary artery diameters in response to
serotonin, PGF2, bradykinin, and
nitroglycerin are summarized in Fig 3.
The diameters of two or three pairs of proximal and distal
coronary artery segments of the LAD or LCx were measured in
each animal. The identical sites were carefully determined before and
after L-NAME administration. Data were excluded when the measurement
site could not be identified. The decreases in the proximal epicardial
arterial diameter in response to 30 µg/kg
serotonin were comparable before L-NAME (from 2.5±0.1 to
2.0±0.1 mm; percent decrease, 20±3%) and after 2 weeks of L-NAME
administration (from 2.3±0.1 to 1.9±0.1 mm; percent decrease,
20±3%), whereas the decreases in the distal epicardial
arterial diameter in response to serotonin were
greater (P<.01) after L-NAME (from 1.0±0.1 to
0.7±0.1 mm;
percent decrease, 39±6%) than before L-NAME (from 1.1±0.1 to
0.9±0.1 mm; percent decrease, 16±6%). The changes in the
proximal
and distal epicardial arterial diameter in response to
PGF2 at doses of 50 µg/kg, bradykinin 100 ng/kg, and
nitroglycerin 10 µg/kg were similar before and after
L-NAME.
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Chronic oral administration of L-NAME for 2 weeks significantly decreased serum nitrite concentrations from 68±10 to 28±2 µmol/L (n=5, P<.01).
Effects of Chronic Administration of L-NAME on Histopathological
Changes in Coronary Arteries
There were no significant differences
between control pigs without
L-NAME treatment and pigs with chronic oral L-NAME treatment in terms
of heart weight (131±10 and 146±12 g, respectively), left
ventricular weight (74±5 and 86±5 g), and left
ventricular weight per body weight (2.8±0.2 and 3.2±0.2
g/kg).
Histological examinations revealed that pathological
structural changes were noted in coronary microvessels but not
in large epicardial coronary vessels in pigs after oral chronic
L-NAME treatment (Fig 4). A smooth layer of
endothelial cell lining was noted in control pigs,
whereas an irregular luminal layer of endothelial cell
lining was noted in pigs with chronic L-NAME treatment. In all of the
vessels in either group, the internal elastic lamina was round, without
wrinkles. Fig 5 shows a relation between the
wall-to-lumen ratio and vessel diameter in a control pig and an
L-NAME–treated pig. There was a significant (P<.01)
negative correlation between the wall-to-lumen ratios and the
vessel diameters in all of the L-NAME–treated pigs (mean slope value,
-3.6±1.0x10-3), but no such significant
(P=.62) correlation was noted in control pigs
(-0.2±0.2x10-3). Fig 6
illustrates the
averaged wall-to-lumen ratios in microvessels with a diameter
of <300 µm and large epicardial vessels with a diameter of >500
µm. The wall-to-lumen ratio in microvessels was significantly
greater in pigs with chronic L-NAME treatment than in control pigs,
whereas the ratio in large epicardial vessels was comparable between
the two groups. The wall-to-lumen ratio was comparable between
microvessels located in the subendocardial and subepicardial layers
(data not shown). Intimal thickening of the vessel wall was rarely
noted. The perivascular fibrosis index in microvessels was greater in
pigs with L-NAME treatment than in control pigs.
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In pigs receiving
chronic intracoronary infusion of L-NAME,
the wall-to-lumen ratio of microvessels in
myocardium perfused via the LCx, into which L-NAME was
infused, was significantly greater than the ratio in
myocardium perfused via the LAD, which received no L-NAME
infusion (Fig 7). The wall-to-lumen ratio of
large epicardial vessels was comparable in the myocardium
perfused via the LCx and that perfused via the LAD. The perivascular
fibrosis index in microvessels was greater in myocardium
perfused via the LCx than that perfused via the LAD. In our preliminary
study, chronic intracoronary administration of saline did
not cause such pathological structural changes of the vascular wall
(n=3, data not shown).
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Effects of Serotonin on Myocardial Blood Flow in
L-NAME–Treated Pigs (Protocol 3)
Intracoronary administration of
serotonin
at doses of 30 µg/kg decreased transmural myocardial blood flow from
1.2±0.2 to 0.8±0.3
mL · min-1 · g-1
(Table 3). The percent decrease in myocardial blood flow
in response to 30 µg/kg serotonin was 40±12%, which was
similar to the response observed in anesthetized
closed-chest pigs in protocol 1. Serotonin-induced
decreases in myocardial blood flow were similar among the three layers
of myocardium. In all four pigs, serotonin
caused transient ischemic ST segment elevation in the
V1 lead.
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Discussion |
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TopAbstractIntroductionMethods Results Discussion References |
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This study revealed the major new finding that chronic L-NAME treatment caused structural and functional abnormalities in coronary microvessels but not in large epicardial coronary arteries in vivo.
Structural Abnormalities of Coronary
Microvessels
Chronic oral or parenteral administration of L-NAME
produced systemic arterial hypertension associated
with the marked decrease in intracellular cGMP levels of vascular
smooth muscle in rats.13 14 Our finding that chronic
oral
administration of L-NAME caused arterial hypertension in
conscious pigs supported the results of the previous
studies.13 14 We measured the serum nitrite
concentrations, which decreased markedly after oral L-NAME treatment
for 2 weeks in our pigs. These results suggest that the basal release
of NO from the endothelium might have been reduced by
the chronic administration of L-NAME in our pigs. However, the degrees
to which L-NAME inhibited the NO synthesis in the
endothelium or in other cell types such as
platelets or macrophage remain unknown.
Endothelial dysfunction (defective NO synthesis) is implicated as playing an important role in vascular structural changes by the release of growth-promoting substances that can influence the status of the growth, migration, and extracellular matrix.2 3 20 A novel finding of the present study is that chronic oral administration of L-NAME for 2 weeks was associated with marked medial thickening and perivascular fibrosis in coronary microvessels but not in large epicardial coronary vessels of pigs. It is likely that the observed histopathological findings were due to structural changes but did not result from the artifact due to vasoconstriction of the vessel wall, because histological analysis was performed after the pressure fixation of the coronary arteries under vasodilation (see the "Methods" section). Because our L-NAME–treated pigs were markedly hypertensive, we must consider the possibility that the observed microvascular structural changes were due to adaptive responses to elevated arterial pressure (vascular remodeling due to rearrangement of existing material).21 22 However, we found in this study that chronic intracoronary infusion of L-NAME that did not affect arterial pressure caused similar microvascular structural changes. We previously reported that microvascular medial thickening induced by chronic oral treatment of L-NAME for 8 weeks was not inhibited by antihypertensive treatment with hydralazine in rats.23 Studies in animals and humans indicate that antihypertensive treatment can reverse vascular remodeling due to arterial hypertension.21 Therefore, arterial hypertension may not be prerequisite to the development of the observed coronary microvascular structural changes. Although Heagerty et al21 emphasized the importance of calculating growth or remodeling index to distinguish between growth and remodeling when the wall-to-lumen ratio is increased, we could not do so because of the difficulty of matching a normal coronary microvessel to an abnormal one in this study. These findings suggest that the development of microvascular structural changes in our L-NAME–treated pigs resulted from the increased production of growth-promoting substances in the vascular wall. Further studies are needed to elucidate the mechanism of microvascular structural abnormality induced by chronic L-NAME treatment.
Functional Abnormalities of Coronary
Microvessels
Another new finding of this study is that chronic oral
administration of L-NAME for 2 weeks was associated with an augmented
serotonin-induced decrease in CBF. Importantly,
ischemic ST segment elevation was noted in response to
serotonin in some pigs after chronic L-NAME administration.
Chronic L-NAME treatment did not alter the decrease in CBF in response
to PGF2, indicating that the altered CBF response
to serotonin was probably not nonspecific.
Our present findings suggest that endothelium-dependent increases in CBF in response to bradykinin and serotonin24 25 at the low dose were impaired, but endothelium-independent increases in CBF in response to adenosine and nitroglycerin were preserved after chronic administration of L-NAME. To determine the relative contributions of defective NO release and hyperreactivity of microvascular smooth muscle to the altered CBF response to serotonin after chronic administration of L-NAME, the CBF response to serotonin at 30 µg/kg was studied before and after short-term administration of L-NAME. Short-term administration of L-NAME did not alter the serotonin-induced decrease in CBF before and after long-term L-NAME treatment; the decrease in CBF by serotonin 30 µg/kg after short-term L-NAME administration was still less before than after chronic L-NAME treatment. These findings suggest that the altered CBF response to serotonin was due to hyperreactivity of microvascular smooth muscle to serotonin but did not result from inhibition of NO release per se. This mechanism of hyperreactivity of microvascular smooth muscle may be analogous to that of spasm of large epicardial coronary arteries.17 26
Changes in myocardial blood flow in response to serotonin were examined in pigs after chronic oral administration of L-NAME by the microsphere technique. The serotonin-induced decreases in myocardial blood flow were similar in the subendocardial, midmyocardial, and subepicardial layers of the myocardium in those pigs. These findings suggest that the augmented serotonin-induced decrease in CBF was not associated with altered transmural distribution of myocardial blood flow.
Vasomotor responses of the large epicardial
coronary artery
were examined by coronary arteriography. Vasodilating responses
of the proximal and distal epicardial coronary artery segments
to bradykinin, nitroglycerin, and adenosine did
not differ before or after chronic L-NAME treatment. These findings
suggested that, unlike that of microvessels,
endothelium-dependent vasodilation of large
epicardial coronary arteries with bradykinin were
preserved despite chronic L-NAME treatment. It is possible that a
lack of inhibition of NO synthesis in the large coronary
arteries accounted for the lack of remodeling in those vessels. It is
unclear why the effects of chronic L-NAME treatment on the vasodilating
response to bradykinin differ in the large epicardial coronary
arteries and microvessels. Vasoconstricting responses of the proximal
epicardial coronary artery segments to serotonin at
30 µg/kg were comparable before and after chronic L-NAME treatment,
whereas the responses of the distal coronary artery segments to
serotonin were augmented after chronic L-NAME treatment.
Vasoconstricting responses of the proximal and distal coronary
segments to PGF2 were similar before and after chronic
L-NAME treatment. These results suggest that chronic L-NAME treatment
was associated with augmented vasoconstriction of the distal
coronary artery segments. However, the degree of
serotonin-induced vasoconstriction of the distal
coronary artery segments after chronic L-NAME treatment was
39±6%, so it is unlikely that augmented vasoconstricting responses of
distal coronary arteries to serotonin could
contribute largely to altered CBF response to serotonin. In
physiological conditions, coronary vascular
resistance at rest is regulated primarily by coronary
microvessels at a diameter of 
200 µm.27 Thus, further
studies are needed to determine which coronary vascular
segments are responsible for altered control of coronary
vascular resistance in response to serotonin in
L-NAME–treated pigs.
Clinical Implications
This animal model may have several
clinical implications. First,
this study is the first to demonstrate that chronic oral treatment with
L-NAME causes functional and structural changes in the coronary
microvasculature but not in large epicardial coronary arteries
in pigs in vivo. Our results suggest that defective NO synthesis plays
an important role in mediating coronary microvascular
disorders. Second, we have previously suggested the presence of
endothelial dysfunction of the coronary
microcirculation in angina pectoris and normal coronary
angiograms,6 in which abnormal vasomotion in the
coronary microcirculation contributes to myocardial
ischemia. Furthermore, coronary microvascular
structural changes in our animal model are similar to those seen in
patients with angina pectoris and normal coronary
angiograms.28 Therefore, our animal model may be useful
for study of the relation between defective endothelial
NO synthesis and coronary microvascular disorders. Third, our
results are similar to those of Golino et al,29 who
demonstrated that intracoronary infusion of
serotonin markedly decreased CBF associated with
vasoconstriction of distal epicardial arteries in patients. In this
study, we have demonstrated that augmented microvascular constriction
was noted in response to serotonin in our animal model.
This finding suggests the possible alterations in the serotonergic
receptor and/or signal transduction system for vascular smooth muscle
constriction. Further studies are needed to elucidate the mechanism of
hyperreactivity of coronary microvessels to
serotonin.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by grants-in-aid for scientific research (05454274, 05670617, 05857085, 06670725, and 06404034) from the Ministry of Education, Science, and Culture, Tokyo; the Uehara Memorial Foundation research grant, Tokyo; and a Japan cardiovascular research grant, Osaka, Japan. The authors are grateful to Mika Mizokami for her technical assistance.
Received March 20, 1995; revision received June 7, 1995; accepted June 12, 1995.
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References |
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TopAbstractIntroductionMethods Results Discussion References |
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