© 1995 American Heart Association, Inc.
Articles |
Direct In Vivo Observation of Subendocardial Arteriolar Response During Reactive Hyperemia
Presented in part at the 67th Scientific Sessions of the American Heart Association, November 1994, Dallas, Tex.
From the Department of Medical Engineering and Systems Cardiology, Kawasaki Medical School, Kurashiki, Japan.
Correspondence to Toyotaka Yada, MD, Department of Medical Engineering and Systems Cardiology, Kawasaki Medical School, 577 Matsushima, Kurashiki 701-01, Japan.
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Abstract To study the vasodilatory capacity of subendocardial (ENDO) arterioles, we evaluated the reactive hyperemic responses of ENDO as well as subepicardial (EPI) arterioles in 40 dogs by our needle-probe intravital microscope. We also examined the individual and combined effects of an ATP-sensitive K+ channel blocker (glibenclamide, 200 µg/kg), an inhibitor of nitric oxide synthase (NG-monomethyl-L-arginine [L-NMMA], 2 µmol/min, 20 minutes), and an adenosine-receptor antagonist (8-phenyltheophylline [8PT], 0.75 µmol/min, 15 minutes). The percent increase in end-diastolic diameter of ENDO arterioles was larger (P<.01) than that of EPI arterioles during reactive hyperemia, especially for the arterioles larger than 120 µm (P<.01). The diastolic-to-systolic vascular pulsation amplitude at the peak flow was greater in ENDO than EPI arterioles (25% versus 6%, P<.05). Compared with control conditions, the presence of both glibenclamide and L-NMMA suppressed the vasodilation responses of ENDO arterioles (P<.01 for both) and EPI arterioles (P<.05 for both). The effect of L-NMMA was greater in ENDO arterioles (P<.01), but that of glibenclamide was not different between ENDO and EPI arterioles. 8PT influenced the hyperemic response, although statistical significance was found only in the flow response. The effect of combined infusion of L-NMMA and glibenclamide with or without 8PT was greater than that of individual infusions in both ENDO and EPI arterioles. Conclusions are as follows: (1) The vasodilatory response of ENDO arterioles was even larger than that of EPI arterioles. Thus, the smaller flow reserve of ENDO arterioles may be caused by other factors, including the greater effects of myocardial compression and nitric oxide on the ENDO arterioles. (2) The vascular responses of ENDO and EPI arterioles were modulated by both endothelium-independent and -dependent vasodilative factors, and the effect of each factor including adenosine was associated with the effects of others.
Key Words: reactive hyperemia • coronary circulation • subendocardial arterioles • diastolic-to-systolic pulsation amplitude • needle-probe videomicroscope
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Introduction |
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Subendocardial vulnerability to ischemia is well known and is associated with a coronary flow reserve that is lower in the subendocardium than in the subepicardium.1 2 3 4 5 The conventional explanations for this difference in flow reserve have invoked the differences in intramyocardial pressures in different layers.6 7 However, because of the technical difficulty involved in the direct observation of subendocardial microvessels, to date no one has examined that there might be regional differences in vascular reactivity. Recently, we have succeeded in observing subendocardial microvessels by a novel needle-probe CCD videomicroscope.8 9
The aim of the present study was to evaluate the reactive hyperemic responses of subendocardial arterioles directly by our needle-probe intravital microscope. The results for subendocardial arterioles were compared with those for subepicardial arterioles. We also examined the individual effect of an ATP-sensitive K+ channel blocker (glibenclamide), an inhibitor of nitric oxide synthase (L-NMMA), and an adenosine-receptor antagonist (8PT) as well as the combined effect of these drugs on the subendocardial and subepicardial arteriolar responses during reactive hyperemia.
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Materials and Methods |
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Animal Preparation
The experimental design was approved by the animal research committee of Kawasaki Medical School. Forty adult mongrel dogs (16 to 25 kg) of either sex were anesthetized with ketamine (10 mg/kg IM) and sodium pentobarbital (25 mg/kg IV). After intubation, each animal was artificially ventilated with room air supplemented by 100% oxygen at a rate sufficient to maintain arterial oxygen and carbon dioxide tensions in the physiological range (pH, 7.35 to 7.45; PCO2, 25 to 40 mm Hg; and PO2, >70 mm Hg). A high-frequency jet ventilator was used to minimize the influence of the lung motion on the visualization of cardiac microcirculation. Each animal was placed on a heating blanket to maintain body temperature at 37°C. The right carotid artery and the right jugular vein were catheterized for hemodynamic and arterial blood gas measurements and for fluid administration. Heparin sodium (750 U/kg IV) was administered to prevent coagulation. Ascending aortic pressure and left ventricular pressure were measured with an 8F pigtail double-lumen manometer catheter (microtipped catheter transducer, model SPC-784A, Millar). After a median sternotomy and a left thoracotomy through the fifth intercostal space, the heart was exposed and suspended in a pericardial cradle. The proximal portion of the LAD was isolated for the placement of an electromagnetic flow probe and an occluder around the vessel. A 24-gauge catheter was introduced into a diagonal branch of the LAD for intracoronary injection of indocyanine green or drug.
An ECG was recorded by standard leads. Heart rate was kept constant at 100 bpm during the experiment by right ventricular pacing after suppressing atrioventricular node activity with an injection of 40% formaldehyde into the region of the atrioventricular node.
Needle-Probe Videomicroscope With a CCD Camera
Details of the needle-probe videomicroscope with a CCD camera
have been previously described.8 9 Briefly, the microscope
system (VMS 1210, Nihon-Kohden) consists of a needle probe, a camera
body containing a CCD camera, a lens system and light guide, a control
unit, a light source, a monitor, and a videocassette recorder
(VTS1000, Hitachi). The camera body was equipped with a 1/2-in CCD
image sensor, 
250 thousand pixels, and 330-line horizontal
resolution. The needle probe (diameter, 4.5 mm; length, 180 mm)
contained a gradient-index (two-pitch) lens surrounded by 18 annularly
arranged light-guide fibers. One end of the needle probe was attached
to the camera body with a focus ring. The image passed through the
gradient-index lens and was focused on the CCD image sensor. Images on
the CCD were monitored and recorded on a videotape every 33
milliseconds. The tissue was illuminated through the light-guide fibers
by light from a halogen lamp (150 W). A green filter was used to
accentuate the contrast between the images of the blood in the vessel
and surrounding tissue. The spatial resolution of this system was 5
µm for x200 magnification.8 The maximum depth of field
was 250 µm.
The needle probe was enclosed in a Silastic 14F double-lumen sheath. A doughnut-shaped balloon on the tip of the sheath avoided direct compression of the vessels by the needle tip. To obtain a clear image of vessels, blood between the tip of the needle probe and endocardial surface inside the doughnut was flushed away with a warm (37°C) Krebs-Henseleit buffer solution injected through a microtube of the sheath.
Measurements of Arteriolar Diameters
The sheathed needle probe was introduced into the left ventricle
through an incision in the left atrial appendage via the mitral valve.
After the surgical procedure and instrumentation, at least 30 minutes
was allowed for stabilization of the monitored variables. The
doughnut-shaped balloon was inflated and then gently placed on the
endocardial surface of the septum between anterior and posterior
papillary muscles. The position of the needle probe was moved slowly
and carefully to search for arterioles with appropriate sizes of 50 to
250 µm by continuing to flush the intervening blood away with the
buffer solution. When a clear image of an arteriole was obtained, the
operator kept the probe position on the vessel manually by monitoring
the image. Then the vascular image was stored on the videocassette
recorder. The arterioles were differentiated from venules by an
injection of indocyanine green (5 to 10 mg/mL) from a diagonal branch
of the LAD. The vascular image with indocyanine green was
analyzed without a green filter. After the injection of dye,
the image of arterioles appeared before venules. The vascular imaging
by indocyanine green was also used to identify the arteriole as a
tributary of the LAD. If the identification of arterioles was difficult
by this method, arterioles and venules were identified during a long
diastole induced by a vagal stimulation, in which the
diameter of subendocardial arterioles decreased and that of venules
increased.9
At the end of the experiment, the time sequential images were
transferred to a Macintosh II fx computer (Apple Computer Inc) for
diameter measurements using appropriate software (IMAGE
1.49, National Institute of Health Research Services Branch). The
end-diastolic and end-systolic diameters of arterioles were
defined as the diameters in the image appearing just before the onset
of the R wave of the ECG and just before the aortic notch,
respectively. The vascular images at end systole and end
diastole were analyzed with a freeze-frame modality
with a time resolution of 30 pictures per second.8 The
diameters of five scan lines neighboring each other were averaged, and
then the vascular diameters at end diastole and end systole
for five consecutive heart beats were ensemble-averaged. We
recorded ascending aortic pressure, left ventricular
pressure, and ECG simultaneously with the vascular
image.
We also measured the phasic diameter change in subepicardial arterioles with the same instrument.8 9 The camera body with needle probe was suspended by an elastic rubber cord and steadied by hand. After inflation of the doughnut balloon, we placed the needle probe gently on the subepicardial arterioles. Krebs-Henseleit buffer solution was dripped continuously on the epicardial surface. Measurements of subendocardial and subepicardial arterioles were performed separately, because it was very difficult to measure them simultaneously.
Experimental Protocol
The following experiments were performed (1) to compare the
reactive hyperemic response between subendocardial and
subepicardial arterioles under control conditions and (2) to elucidate
the individual effects of glibenclamide, L-NMMA, and 8PT as well as the
combined effect of these drugs on the reactive hyperemic
response of the arterioles. Different dogs were used for each
experimental protocol to avoid a possible interference between
protocols, although there was an overlap between the control group and
drug administration groups. Of the 40 dogs, we used 13 to evaluate the
changes in the LAD flow responses during infusion of acetylcholine (1
µg/kg) and adenosine (1 µg/kg) after the administration of
L-NMMA and 8PT according to the protocol described below. The effect of
glibenclamide on nitroglycerin-induced vasodilation was
also evaluated to exclude the nonspecific inhibitory effect
of glibenclamide on smooth muscle relaxation in subepicardial and
subendocardial arterioles. Aspirin (17 mg/kg in the jugular vein) was
injected 20 to 30 minutes before the beginning of each protocol to
prevent the effects of prostaglandin on the vascular
response.
Comparison of the Reactive Hyperemic Response Between
Subendocardial and Subepicardial Arterioles Under Control Conditions
(15 Dogs)
The reactive hyperemic responses of subendocardial
arterioles (n=25 vascular segments) and subepicardial arterioles (n=19)
after a 20-second occlusion of the LAD were analyzed for 3
minutes by measuring their vascular diameters and the LAD flows. These
observations were used as the control data to evaluate the effect of
drug administration on reactive hyperemic responses.
Effect of Glibenclamide on Reactive Hyperemic Response
(8 Dogs)
To evaluate the role of the ATP-sensitive K+ channel
in reactive hyperemia, glibenclamide (200 µg/kg) was slowly
infused in the LAD for 1 minute.10 Three minutes after the
cessation of glibenclamide infusion, the LAD was occluded for 20
seconds and then released, and the reactive hyperemic responses
of the subendocardial arterioles (n=15) and subepicardial arterioles
(n=22) were observed for 3 minutes with the LAD flow responses (Fig 1
).
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Effect of L-NMMA on Reactive Hyperemic Response (9
Dogs)
The role of nitric oxide in reactive hyperemia was
examined. Nitric oxide synthase was inhibited by using L-NMMA solution,
which was continuously infused into the diagonal branch of the LAD at a
rate of 0.5 mL/min (2 µmol/min) for 20 minutes11 with a
syringe pump (Terumo STC 525). Reactive hyperemic responses of
subendocardial (n=12) and subepicardial (n=19) arterioles were
evaluated immediately after L-NMMA infusion was stopped (see Fig 1
).
Effect of 8PT on Reactive Hyperemic Response (5
Dogs)
To evaluate the role of adenosine on reactive
hyperemia, 8PT solution was continuously infused into the LAD
at a rate of 0.5 mL/min (0.75 µmol/min) for 15 minutes using a
syringe pump.11 The observation of reactive
hyperemia of subendocardial (n=11) and subepicardial (n=11)
arterioles was started immediately after the cessation of 8PT and
continued for 3 minutes (see Fig 1).
Combined Effects of L-NMMA and Glibenclamide With or Without 8PT on
Reactive Hyperemic Response (4 Dogs)
The combined effects of L-NMMA and glibenclamide with or
without 8PT on reactive hyperemic response were examined. A
constant infusion of L-NMMA solution into the LAD at a rate of 0.5
mL/min (2 µmol/min) was started. In one experiment
(L-NMMA+glibenclamide), glibenclamide was infused into the LAD (200
µg/kg) 16 minutes after the initiation of L-NMMA infusion (see Fig 1). In the other experiment (L-NMMA+8PT+glibenclamide), 8PT solution
was infused into the LAD at a rate of 0.5 mL/min (0.75 µmol/min) 5
minutes after the infusion of L-NMMA, and then glibenclamide was
infused into the LAD (200 µg/kg) 16 minutes after the initiation of
L-NMMA. The number of vessels examined was 11 for both subendocardial
and subepicardial arterioles in the experiment without 8PT, 11 for
subendocardial arterioles, and 10 for subepicardial arterioles in the
experiment with 8PT.
Statistical Analysis
Data were reported as mean±SEM. The difference of
time-dependent changes between two groups and the difference of percent
diameter changes against control diameters between two groups were
assessed by ANOVA. The relation between variables was evaluated by
a simple correlation analysis. Student's t test was
used for both paired and unpaired comparisons. The criterion for
statistical significance was P<.05.
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Hemodynamics
Systolic and diastolic blood pressure and left ventricular end-diastolic pressure before and after each drug administration are shown in Table 1
. The
change in each hemodynamic value was not significant
statistically, indicating that each drug administration had no effect
on the hemodynamics. The LAD flow responses to
acetylcholine and adenosine were significantly attenuated after
L-NMMA and 8PT infusion; the percent increase in peak hyperemic
flow decreased from 304±50% (versus preocclusion state) to 115±13%
with L-NMMA and from 323±48% to 168±29% with 8PT infusion. It was
also observed that the flow suppression effect of L-NMMA was quickly
reversed by the administration of L-arginine (298±35%).
The vasodilation of nitroglycerin was not affected by
glibenclamide.
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Comparison of Arteriolar Diameter Responses Between Subendocardium
and Subepicardium During Reactive Hyperemia
Fig 2 shows representative images
of subendocardial arterioles before the LAD occlusion and during peak
reactive hyperemia under control conditions (without any drug
administration). The subendocardial arterioles were dilated remarkably,
and their pulsation amplitudes between end diastole and end
systole were enhanced during reactive hyperemia (25% versus
13% before LAD occlusion, P<.01). Subepicardial arteriolar
diameters were also dilated, but diastolic-to-systolic
pulsation amplitudes were small even during peak reactive
hyperemia (6% versus 2% before LAD occlusion,
P=NS). Fig 3 exhibits the time course of
end-diastolic arteriolar diameter responses in both
subendocardium and subepicardium during reactive hyperemia. The
time courses were similar to each other, but the percent
end-diastolic diameter increment in subendocardial
arterioles during reactive hyperemia was larger than that in
subepicardial arterioles (P<.01, Fig 3 and Table 2), indicating that the magnitude of the vasodilatory
response of subendocardium is even greater than that of
subepicardium.
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Fig 4 shows the percent diameter increase in
subendocardial and subepicardial arterioles at end diastole
during peak reactive hyperemic flow against the
end-diastolic diameter before LAD occlusion. There is a
significant difference in the vascular responses between subendocardial
and subepicardial arterioles (P<.01). The difference was
significant for arterioles of >120 µm (P<.01) but not
for the smaller arterioles. This comparison was made because there were
no arterioles between 100 and 120 µm in subendocardium observed in
the present study.
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Individual Effects of Glibenclamide, L-NMMA, and 8PT on
Subendocardial and Subepicardial Arteriolar Responses
Fig 5A through 5C shows the individual effects of
glibenclamide, L-NMMA, and 8PT on the vascular responses of
subendocardial arterioles before LAD occlusion, at the end of the
occlusion, and during the reactive hyperemic response. Both
glibenclamide and L-NMMA reduced the degree of vascular diameter
increment significantly during reactive hyperemia
(P<.01 for both). The suppression of the vascular dilation
was significant statistically at the initial part of the reactive
hyperemia (ie, L-NMMA, 5 to 15 seconds after LAD reopening;
glibenclamide, 5 to 10 seconds after the reopening). The effect of 8PT
on the vascular response was not significant statistically, although
there was a tendency to suppression of the vascular dilation. Fig 6 shows the percent diameter changes during peak
reactive hyperemia under control conditions (without any drug)
and after each drug administration for subepicardium (right) compared
with the data for subendocardium (left). Glibenclamide and L-NMMA
suppressed the maximum vascular responses in the subendocardial
arterioles (P<.01 for both), but 8PT did not change them
significantly (Fig 6 and Table 2). The effects of three drugs on the
reactive hyperemic response of subepicardial arterioles showed
tendencies similar to those of subendocardial arterioles.
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The effect of each drug on the LAD flow response is shown in Fig 7. Although the magnitude of peak flows at 5 seconds
after the reopening was not affected by each drug, the flow decay part
of the reactive hyperemia (after 15 to 30 seconds) was reduced
significantly by glibenclamide (P<.01), L-NMMA
(P<.05), and 8PT (P<.01).
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The percent changes in the amplitude of
diastolic-to-systolic diameter pulsation after each drug
during peak hyperemic flow are shown in Fig 8.
Both glibenclamide and L-NMMA decreased the percent changes of
pulsation amplitude (16% and 18%, respectively; P<.05 for
both) from the control condition (25%), but 8PT did not change the
pulsation amplitude significantly (21%).
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Fig 9 shows the percent end-diastolic
diameter changes at the peak flow after the administration of
glibenclamide (A), L-NMMA (B), and 8PT (C) for both subendocardial and
subepicardial arterioles against the end-diastolic
diameters before LAD occlusion. The vasodilation after each drug
administration was inversely linear with the initial diameter of
arterioles; ie, smaller arterioles dilated more than larger arterioles.
The relations between vasodilation and initial diameters were almost
identical between subendocardium and subepicardium for glibenclamide
and 8PT, whereas there was a small but significant difference for
L-NMMA; ie, there was a smaller percent diameter change for
subendocardial arterioles.
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Combined Effect of Glibenclamide and L-NMMA With or Without 8PT
on Reactive Hyperemic Response
The combined effect of L-NMMA+glibenclamide or
L-NMMA+8PT+glibenclamide on the reactive hyperemic responses of
subendocardial arterioles is shown in Fig 5D and 5E. The vascular
responses were suppressed significantly by both L-NMMA+glibenclamide
and L-NMMA+8PT+glibenclamide from the time immediately after the LAD
reopening until 3 minutes after the reopening, except for a few
sampling points. The suppression in percent diameter increases in
subendocardial and subepicardial arterioles during the peak flow is
shown in Fig 6 (L-NMMA+glibenclamide and L-NMMA+8PT+glibenclamide). The
degree of diameter increases in both subendocardial and subepicardial
arterioles by the combined treatments was diminished further from
those by the individual treatment of glibenclamide or L-NMMA, although
a statistical significance was found only between glibenclamide and
L-NMMA+8PT+glibenclamide and between L-NMMA and
L-NMMA+8PT+glibenclamide (P<.05 for both) for the
subendocardial arterioles and between L-NMMA and
L-NMMA+8PT+glibenclamide (P<.05) for the subepicardial
arterioles. Both combined treatments of L-NMMA+glibenclamide and
L-NMMA+8PT+glibenclamide decreased the vasodilatory response of
arterioles, especially that of smaller arterioles in both
subendocardium and subepicardium (Fig 9D and 9E). The decreases in LAD
flow responses by L-NMMA+glibenclamide and L-NMMA+8PT+glibenclamide
were also greater than those by the individual drug treatment (Fig 7D and 7E). The peak reactive hyperemic flows were also suppressed
by both treatments, unlike the response to individual drug (peak flow
was almost unchanged as seen in Fig 7A through 7C), although only the
suppression of the peak flow by L-NMMA+8PT+glibenclamide was
significant statistically. The decrease in the flow responses after
peak flow was significant at all observation points for both
L-NMMA+glibenclamide and L-NMMA+8PT+glibenclamide. The
diastolic-to-systolic percent pulsation change of
subendocardial arterioles by the combined treatments was also slightly
decreased from the individual treatments, but these changes were not
significant statistically (Fig 8).
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Discussion |
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The following new findings were elucidated by the present study: (1) Percent end-diastolic diameter increase during reactive hyperemia in subendocardial arterioles was larger than that in subepicardial arterioles. The subendocardial arteriolar dilation was accompanied with the augmentation of diastolic-to-systolic pulsation amplitude. The vasodilation was greater in the smaller arterioles both for subendocardial and subepicardial arterioles, but the larger arteriolar dilation in subendocardium was significantly greater than that in subepicardium. (2) Both glibenclamide and L-NMMA decreased maximal vasodilation of subendocardial and subepicardial arterioles and also decreased pulsation amplitude of the subendocardial vessels, but 8PT did not. In the presence of L-NMMA, there was a small but significant difference in the arterioles, ie, greater suppression of subendocardial arterioles. (3) Reduction in vasodilation and pulsation amplitude after combined infusion of L-NMMA and glibenclamide or L-NMMA+8PT+glibenclamide was greater than that after individual infusion of glibenclamide, L-NMMA, or 8PT.
Critique of Experimental Model and Methodology
The validity of our experimental measurements has previously been
demonstrated extensively by measuring intraballoon pressure before and
after the visualization of a clear vascular image and also by measuring
the pressure waveform near the subendocardium encircled by the
doughnut-shaped balloon.8 However, holding the needle
probe on the same visual field of the subendocardial surface for over 3
minutes is difficult. Because of this temporal limitation, we measured
arteriolar diameters 3 minutes after reopening the LAD. For the same
reason, we chose to observe the reactive hyperemic response
after a 20-second LAD occlusion. Although Kanatsuka et
al10 demonstrated similar vascular responses of reactive
hyperemia between 20- and 30-second LAD occlusion, the vascular
response and the effect of each drug might be different for longer LAD
occlusions.
We used a dose of 200 µg/kg (glibenclamide), 2 µmol/min (L-NMMA,
for 20 minutes), or 0.75 µmol/min (8PT, for 15 minutes) by
intracoronary infusion according to the method of Kanatsuka
et al10 and Yamabe et al.11 Infusion of these
drugs had no significant effects on the hemodynamics
(Table 1). We confirmed that the LAD flow responses to acetylcholine
and adenosine were significantly attenuated after L-NMMA and
8PT infusions. The flow-suppression effect of L-NMMA was quickly
reversed by the administration of L-arginine, as
previously reported by Yamabe et al.11 The specificity of
the ATP-sensitive K+ channel blocker was also validated,
since no influence of glibenclamide on the vasodilation by
nitroglycerin was found.
Subendocardial flow was usually 1.2 to 1.7 times greater than the subepicardium in conscious dogs.7 However, subendocardial flow per unit mass was higher in conscious animals than in anesthetized animals (almost unity across myocardium).7 12 13 In normal hearts, opening the pericardium did not alter the subendocardial-to-subepicardial flow ratio at normal left ventricular diastolic pressures or when they were raised to 20 to 25 mm Hg.14 15 On the other hand, if cardiac function was impaired, the flow ratio rose to 2.3 after opening the pericardium.14 These findings indicate that anesthesia and opening the pericardium might have some influence on the experimental results, although they are necessary procedures in the present study.
The range of vessel diameters sampled in the subendocardium and
subepicardium was not exactly matched in the various protocols. For
example, the range of control diameters in Fig 4 was somewhat wider for
subendocardial arterioles than for subepicardial arterioles. However,
the opposite was true in other protocols, as shown in Fig 9A, 9D, and 9E; thus, it is unlikely that there was a systematic bias in the data.
Furthermore, the relation between percent change in diameter and
initial vessel diameter was found to be linear; thus, small differences
in the vessel diameter sampling ranges are unlikely to change the
interpretation. The possibility of interindividual variation
influencing the results was tested during control conditions (15 dogs)
by ANOVA. No significant difference in the response of subepicardial or
subendocardial arterioles was found among the dogs.
Coronary Flow Reserve in Subendocardium and
Subepicardium
It is widely accepted that the coronary flow reserve is
lower in subendocardium than in subepicardium.7 When
coronary perfusion pressure is reduced stepwise while keeping
myocardial oxygen demand constant, autoregulation is maintained, and
transmural flow remains almost constant. As the pressure is lowered
further, subendocardial flow decreases first, and a decrease in
subepicardial flow reserve follows.5
Why is the coronary flow reserve in subendocardium lower than
in subepicardium? The one possible reason is the difference in the
vasodilatory responses between two layers. However, the present
study demonstrated that the percent diameter change at peak reactive
hyperemic flow was greater in subendocardial arterioles than in
subepicardial arterioles (see Fig 3). Thus, the hypothesis that
decreased subendocardial flow reserve may be due to decreased vascular
reactivity was shown to be unfounded. If anything, the vascular
responses increased, perhaps in an attempt to compensate for the
imposed external forces in the subendocardium. Since the larger
arterioles (>120 µm) in subendocardium had already dilated at the
peak reactive hyperemic flow (Fig 4), further vasodilatory
capacity might have been limited compared with larger arterioles in
subepicardium, which dilated only a little. Another possible mechanical
reason may be the much larger diastolic-to-systolic
vascular pulsation amplitude of the subendocardial arterioles. This may
be mainly due to external forces imposed on vessels by increased
vascular volume. The increased pulsatility may cause not only systolic
impediment of coronary inflow into the
endomyocardium but also wasteful retrograde flow from
the endomyocardium to epicardium (coronary
slosh phenomenon).16
The greater inhibition of nitric oxide on subendocardial arterioles
(see Fig 9B) may relate to the difference of coronary flow
reserve between subendocardium and subepicardium. A number of in vitro
studies performed in isolated conduit vessels from other vascular beds
have demonstrated that EDRF is released in response to change in
pulsatility.17 Lamontagne et al18 found that
external compression and decompression of isolated femoral arteries
produced a pronounced increase in effluent cGMP (an indirect assay of
nitric oxide). As for the in vivo study, Hintze and
Vatner19 showed pacing-induced increase in
coronary artery diameter. More recently, Canty and
Schwartz20 observed that the epicardial
arterial diameter change after pacing at a rate of 200 bpm
was attenuated after L-NAME compared with control conditions,
indicating the pulsation dependence of nitric oxide release. These
observations may suggest that the contribution of EDRF to the control
of the coronary flow in a basal state is greater in the
subendocardium, where vascular pulsation is larger, leading to the
greater inhibition of nitric oxide, although the available data are
from conduit arteries but not from small arteries or arterioles.
The difference of myogenic response between subepicardial and subendocardial arterioles may also be involved. Kuo et al21 have demonstrated in an in vitro study using porcine coronary arterioles that subepicardial arterioles exhibit greater myogenic response than do subendocardial arterioles and that subendocardial arterioles respond only passively at a lower perfusion pressure, unlike subepicardial arterioles, which respond actively even at low pressure. Although the present experiment cannot evaluate the involvement of myogenic response specifically, this mechanism may contribute to the lower coronary flow reserve in subendocardium.
Arteriolar Vasodilation During Reactive Hyperemia and the
Role of ATP-Sensitive K+ Channel, Nitric Oxide, and
Adenosine
Glibenclamide is a selective blocker of ATP-sensitive
K+ channel, and 8PT is a nonselective adenosine
receptor antagonist. It was reported that glibenclamide and
8PT reduced the vasodilation response to hypoxic
perfusion.22 23 Nakhostine and Lamontagne24
have demonstrated that 8PT and glibenclamide block the vasodilator
response to adenosine and significantly reduce the
hypoxia-induced vasodilation in the isolated
Langendorff-perfused rabbit heart. They have suggested that the
activation of the ATP-sensitive K+ channel plays a major
role in hypoxia-induced vasodilation and that a major part of
this activation results from the action of adenosine on
receptors closely related to the A1 type. More recently,
Komaru et al25 demonstrated that pertussis
toxin–dependent G protein is crucially involved in microvascular
control during ischemia and autoregulation. Lee et
al26 demonstrated the existence of hypoxia-induced
vasodilation even after 8PT injection in anesthetized dogs.
They suggested that 8PT attenuates but does not abolish the
coronary vasodilatory response to hypoxic
hyperemia.
Several studies have demonstrated that the inhibition of the formation
of nitric oxide from L-arginine blunts the reactive
hyperemic response after a transient coronary
occlusion.27 28 29 Yamabe et al11 evaluated the
role of EDRF and adenosine in the coronary blood flow
response during reactive hyperemia by L-NMMA and 8PT infusion.
Both L-NMMA and 8PT reduced repayments of flow debt but did not
attenuate the peak reactive flow. Our data support their results.
Although the effect of L-arginine analogue on the
magnitude of the peak reactive hyperemic flow has been
variable among reports, there was a distinct difference between
arteriolar diameter response and flow response during reactive
hyperemia, especially at the peak flow in the present study
(see Figs 5 and 7). This difference may be explained by the capacitance
flow,30 which implies the inflow into actively and
passively vasodilating vascular segments in the myocardium
during reactive hyperemia. Thus, we should conclude that L-NMMA
attenuates vasodilation in the whole phase of reactive
hyperemia and that 8PT partially attenuates the vascular
dilation.
As for the mechanism involved in the different responses for the
two sizes of arterioles, it was indicated that the dilation of small
vessels plays a more important role in the early phase of reactive
hyperemia and that the dilation of large vessels has a greater
contribution to the late phase.10 In the present
study, both glibenclamide and L-NMMA reduced the arteriolar dilation
especially in the early phase (see Fig 5A and 5B). Thus, both
ATP-sensitive K+ channel and nitric oxide may mainly
contribute to the smaller arteriolar responses in reactive
hyperemia. In isolated coronary arterial
microvessels, potent myogenic response has been observed in vessels of
<100 µm17 but was absent in vessels of >200
µm.31 Therefore, it is also conceivable that the
predominant dilation of smaller arterioles is related to their lowered
vascular intraluminal pressure during the LAD occlusion.
Combined Effect of Drugs on Reactive
Hyperemia
During the combined infusion of L-NMMA and glibenclamide or
L-NMMA+8PT+glibenclamide, the dilation of arterioles and the degree of
the vascular diameter increment were suppressed further in both early
and late phases of the time course. This suggests that the myocardial
reactive hyperemia is modulated additively by both the
endothelium-independent vasodilative metabolite(s) and
the EDRF(s). Yamabe et al11 also reported that
simultaneous infusion of L-NMMA and 8PT further attenuated
the peak reactive flow rate and reduced the repayment of flow debt
compared with the results for a single dose. The combined infusion of
three drugs (L-NMMA+8PT+glibenclamide) further attenuated the
reactive hyperemic flow response compared with the combined
infusion of two drugs (L-NMMA+glibenclamide) (see Fig 7D and 7E),
supporting their observation on the additive effect of
adenosine. However, the similar suppression of vasodilation
between L-NMMA+glibenclamide and L-NMMA+8PT+glibenclamide (see
Fig 9D and 9E) suggested that the vasodilation was mostly mediated by
the ATP-sensitive K+ channel and nitric oxide.
Conclusions are as follows: (1) The smaller flow reserve of subendocardial arterioles was not caused by decreased vasodilatory capacity but by other factors, including the greater myocardial compression in the subendocardium and the difference of the effect of nitric oxide on the vascular responses between endocardial and epicardial arterioles. (2) The vascular response of myocardial reactive hyperemia of endocardial and epicardial arterioles was modulated by both the endothelium-independent vasodilative metabolite(s) and EDRF(s), and each effect including adenosine was associated with the effects of others.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
|---|
This study was supported by grant-in-aid 05454278 for General Scientific Research (B), grant-in-aid 05557043 for Developmental Scientific Research (B) from the Ministry of Education, Science, and Culture, Japan, and by a research project grant (No. 4-107) from Kawasaki Medical School. We are grateful to Prof J.I.E. Hoffman for his valuable comments and English revision. We thank Chikako Tokuda for her expert technical assistance.
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Footnotes |
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Reprint requests to Prof Fumihiko Kajiya, MD, PhD, Department of Medical Engineering and Systems Cardiology, Kawasaki Medical School, 577 Matsushima, Kurashiki 701-01, Japan.
Received January 3, 1995; accepted May 31, 1995.
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