Direct In Vivo Observation of Subendocardial Arteriolar Response During Reactive Hyperemia
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.
- reactive hyperemia
- coronary circulation
- subendocardial arterioles
- diastolic-to-systolic pulsation amplitude
- needle-probe videomicroscope
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.
Materials and Methods
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 ×200 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.
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⇓).
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.
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.
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.
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.
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.
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.
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).
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%).
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.
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⇑).
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.
Selected Abbreviations and Acronyms
|EDRF||=||endothelium-derived relaxing factor|
|LAD||=||left anterior descending coronary artery|
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.
Reprint requests to Prof Fumihiko Kajiya, MD, PhD, Department of Medical Engineering and Systems Cardiology, Kawasaki Medical School, 577 Matsushima, Kurashiki 701-01, Japan.
Presented in part at the 67th Scientific Sessions of the American Heart Association, November 1994, Dallas, Tex.
- Received January 3, 1995.
- Accepted May 31, 1995.
- © 1995 American Heart Association, Inc.
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