Cardiovascular Research

Cardiovascular Research 1997 35(1):148-157; doi:10.1016/S0008-6363(97)00096-5
© 1997 by European Society of Cardiology

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Copyright © 1997, European Society of Cardiology

Mechanism of coronary microvascular responses to metabolic stimulation

Richard P Embrey^a, Leonard A Brooks^b and Kevin C Dellsperger^b,*

^aDepartment of Surgery, University of Iowa, Iowa City, IA 52242, USA
^bDepartment of Internal Medicine and the Cardiovascular Center, University of Iowa, and VA Medical Center, Iowa City, IA 52246, USA

^* Corresponding author. Tel.: +1 (319) 339-7102; fax: +1 (319) 339 7135.

Received 16 December 1996; accepted 24 February 1997

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Previous studies from our laboratory have shown that coronary microvascular dilation to increased myocardial oxygen consumption (MVO₂) is greater in vessels <100 µm. The mechanism responsible for this response is uncertain. Objectives: We tested the hypothesis that microvascular dilation to increased MVO₂ is mediated by nitric oxide (NO). Since NO release may occur in response to increased shear, we also tested the hypothesis that metabolic byproducts released in response to increases in MVO₂ will stimulate opening of the ATP-sensitive potassium channel. Methods: Changes in epicardial coronary microvascular diameters were measured in 9 dogs given N^G-nitro-L-arginine (LNNA; 100 µM, topically), 7 dogs given glibenclamide (10 µM, topically) and 12 control (C) dogs during increases in metabolic demand using dobutamine (DOB, 10 µg/kg/min, i.v.) with rapid atrial pacing (PAC, 300 bpm). Diameters of arterioles were measured using intravital microscopy coupled to stroboscopic epi-illumination. Results: During the protocol, MVO₂ increased to a similar degree in both experimental groups (LNNA and glibenclamide). Baseline hemodynamics and coronary microvascular diameters were similar between the two experimental groups and their respective control groups. In the presence of LNNA, coronary arteriolar (<100 µm) dilation (% change from baseline) was impaired during the protocol (DOB: vehicle 18±5, LNNA 2±2 [P<0.05]; DOB+RAP: vehicle 40±11, LNNA 6±2% [P<0.05]). In contrast, glibenclamide did not impair coronary microvascular responses to increased MVO₂ despite similar increases in MVO₂. Conclusion: This study indicates that coronary microvascular dilation in response to increased metabolic stimulation using dobutamine in conjunction with rapid pacing is mediated through a nitric-oxide-dependent mechanism and not ATP-sensitive potassium channels. These results may have important implications in pathological disease states where nitric oxide mechanisms are impaired, such as diabetes and hypertension.

KEYWORDS Coronary microcirculation; Dobutamine; EDRF; Arginine analogs; Glibenclamide; Potassium channel, ATP-sensitive; Nitric oxide; Endothelium; Intravital microscopy

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Regulation of coronary blood flow is a complex process, which is governed by changes in microvascular resistance. Coronary microvascular dilation may be caused by accumulation of local metabolites [1], reduction in perfusion pressure [2], and locally released vasoactive substances in response to changes in flow and shear stress [3]. Previous studies by Kanatsuka et al. [4]showed that rapid atrial pacing at 300 bpm causes myocardial oxygen consumption (MVO₂) to double, resulting in a modest dilation of arterial coronary microvessels of all sizes. The magnitude of the dilation was inversely related to the baseline microvascular diameter. Because a majority of total coronary resistance resides within coronary microvessels less than 150 µm [5], the greatest dilation would be expected to occur in vessels of this size in response to agents which may increase MVO₂. Recently, Jones and colleagues [6]demonstrated that arginine-derived nitric oxide (NO) is important in the regulation of coronary microvascular dilation in response to rapid pacing perhaps to a flow-mediated response. Therefore, we postulated that NO inhibition by N^G-nitro-L-arginine (LNNA) would attenuate microvascular dilation of small (<100 µm) coronary arterioles in response to a combination of β-adrenergic stimulation and rapid atrial pacing.

Since the release of NO in response to increased shear-rate is well established in the coronary circulation [3], we postulate that metabolic byproducts released in response to increases in MVO₂ will stimulate opening of the ATP-sensitive potassium channel (K⁺_[ATP]). This would result in small arteriolar dilation and subsequent flow-induced dilation of the coronary microvascular through a NO-mediated mechanism. The role of K⁺_[ATP] in the coronary circulation and microcirculation is well established. Our group showed that the K⁺_[ATP] importantly modulates coronary microvascular resistance in response to reductions in perfusion pressure [7]. Others have shown that these channels are important in modulating responses to autoregulation [8, 9]and coronary reactive hyperemia [10, 11]. More recent studies have evaluated the role of K⁺_[ATP] during a metabolic stress. Narishige and colleagues [12]showed that K⁺_[ATP] importantly contributed to coronary vasodilation induced by β-adrenoceptor stimulation in dogs while Aversano et al. [13]showed that pacing-induced dilation was unaffected by K⁺_[ATP] blockade. Thus, we tested the hypothesis that coronary microvascular dilation in response to a metabolic stress (β-adrenergic stimulation with pacing) would be dependent upon either NO or K⁺_[ATP].

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
This investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985).

Nonconditioned dogs of either sex (n = 42) weighing 4–10 kg were sedated with ketamine (20 mg/kg and acepromazine (0.2 mg/kg) subcutaneously and anesthetized with -chloralose 50 mg/kg and sodium borate 50 mg/kg, intravenously. Additional doses were given as necessary to maintain a surgical depth of anesthesia. A constant body temperature of 37–38°C was maintained by a homeothermic blanket system. Both femoral arteries were cannulated for measurement of aortic pressure and collection of reference blood samples for determination of myocardial perfusion. A catheter was placed into the superior vena cava via the right cephalic vein for drug administration. The animals were then intubated with a cuffed endotracheal tube and a high-frequency jet ventilator coupled to the cardiac cycle was used to minimize respiratory-induced cardiac motion. The details of this system have been previously described [5]. Briefly, this was accomplished by a pressure regulator and solenoid valve connected to compressed air which was triggered by a signal from left ventricular dP/dt to open the valve. Inspiratory pressure in the distal airway was maintained between 5 and 15 psi, and positive end-expiratory pressure (2–7 cm H₂O) was employed to reduce atelectasis. Arterial blood gases and pH were monitored throughout the procedures and maintained in the physiologic range by altering duration of valve opening, inspiratory pressure and/or end-expiratory pressure.

A left thoracotomy was then performed, and a segment of chest wall between the third and fifth intercostal spaces was resected and the pericardium opened to expose the surface of the left ventricle. The heart was suspended in a pericardial cradle. A polyethylene catheter (PE 200) was then placed via the left internal jugular vein and the tip positioned in the coronary sinus 2–2.5 cm beyond the ostium of the sinus for determination of coronary sinus blood oxygen content. The left atrium was cannulated via the appendage for administration of radiolabeled microspheres. Bipolar pacing electrodes were placed on the left and right atrial appendages and connected to a stimulator. Heart rates were controlled and maintained by a custom computer program coupled with our microvascular imaging system. A solid-state transducer (Millar, Houston, TX) was advanced into the left ventricle from the left atrial appendage for measurement of left ventricular pressure and dP/dt. The physiologic environment of the epicardial surface was maintained by continuously dripping Krebs-Henseleit bicarbonate buffer at 37°, bubbled with 20% O₂, 75% N₂ and 5% CO₂, onto the surface of the left ventricle.

2.1 Microscope and video system
For direct observation of the coronary microvessels in the beating left ventricle, we used an intravital microscope (Zeiss, Thornwood, NY) equipped with a computer controlled xenon strobe (Chadwick-Helmuth, El Monte, CA), which epi-illuminated the cardiac surface at a single point in mid-diastole during each cardiac cycle. Details of this system have been described previously [4, 14, 15]. With this system, fluorescence coronary microangiography was performed by injecting fluorescein isothiocyanate dextran (FITC; molecular weight 487 000, Sigma Chemical Co., St. Louis, MO) into the left atrium as a contrast medium. A 6.3x objective (NA 0.2, Zeiss) was used. Microvascular fluorescent images were transmitted to a silicon-intensified tube video camera (General Electric, Owensborough, KY) via a 1x or 6.3x relay lens and were digitized and displayed a high-resolution monitor (Panasonic, Tokyo, Japan). Spatial resolution was 4 µm (1x relay lens) or 2.5 µm (6.3x relay lens). Edges of the arterioles were traced with a digitizing tablet (Summagraphics, Cambridge, MA), and their internal diameters were calculated. When arterioles <100 µm were observed, we used a 6.3x relay lens. Locations of measurements were assured by measuring microvascular diameters from specific branch points in the microvascular field of interest. Each vessel was measured 3 times using different images of the same vessel.

2.2 Measurement of oxygen saturation, hemoglobin, and myocardial perfusion
At the time of coronary microvascular diameter measurement, arterial and coronary sinus blood samples were collected into heparinized syringes. Oxygen saturation and hemoglobin concentrations were immediately determined using a co-oximeter (model 182, Instrumentation Laboratory, Lexington, MA).

Radioactive microspheres were used to measure myocardial perfusion. Microspheres (15 µm diameter, 7–14 mCi/g), labeled with ⁴⁶Sc, ⁹⁵Sr, ¹¹³Sn, ¹⁴¹Ce, ⁵¹Cr, ⁵⁷Co, or ⁹⁵Nb, were agitated and injected into the left atrium and the cannula immediately cleared with 2 ml of warmed saline. Prior to and for 90 s after injection, reference blood samples were withdrawn from the femoral artery at a constant rate of 1.91 ml/min. After completion of the study, the heart was removed and the left ventricular wall from the microvascular area of interest was excised. This was then divided into two samples: endocardium and epicardium. Tissue and reference blood samples were placed into a germanium crystal gamma counter (Canberra) for measurement of nuclide activity [15]. Myocardial blood flow (MBF) was calculated by the following equation:

where C_m is the sample counts per gram of myocardial tissue, W_r is the withdrawal rate of the reference sample (ml/min) and C_r is the nuclide activity of the reference blood sample (counts). MVO₂ was calculated from arterial and coronary sinus oxygen saturation, hemoglobin and myocardial perfusion.

2.3 Experimental protocols
2.3.1 Protocol 1
To study the effects of increased myocardial oxygen consumption on the diameter of coronary arterioles, baseline hemodynamics, myocardial perfusion, MVO₂ and microvascular diameters were measured in 9 dogs. These measurements were repeated after a 10 min infusion of dobutamine (10 µg/kg/min, i.v.). While the dobutamine infusion continued, rapid atrial pacing (300 beats/min) was initiated and all measurements were repeated 10 min after the addition of pacing. A total of 17 vessels ranging in size from 98 to 184 µm were measured in the 9 animals (1–2 per dog).

To determine whether the increases in microvascular dilator responses seen with dobutamine were due to β₁- or β₂-adrenergic effects [16, 17], 7 of these animals were allowed to recover for 1 h after termination of pacing and dobutamine infusion, and a second set of control measurements of hemodynamics, myocardial perfusion, MVO₂ and microvascular diameters were made. The dogs received the selective β₁-adrenergic antagonist, atenolol (1 mg/kg, i.v.) [18]. Hemodynamics, myocardial perfusion, MVO₂ and microvascular diameters were again measured, before and 10 min after initiating an infusion of dobutamine.

2.3.2 Protocol 2
In order to determine the role of arginine-derived nitric oxide in metabolically stimulated coronary microvascular dilation, control measurements of hemodynamics, myocardial perfusion, MVO₂ and microvascular diameters were made in 14 dogs. N-Nitro-L-arginine (LNNA; 100 µM, n = 9) or its vehicle (n = 5; saline) was continuously suffused onto the cardiac surface and hemodynamics, and myocardial perfusion, MVO₂ and microvascular diameters were measured. Then a 10 min infusion of dobutamine (10 µg/kg/min, i.v.) was started and measurements were repeated. Finally, while the dobutamine infusion continued, atrial pacing at 300 beats per minute was initiated and measurements repeated as described in Protocol 1. A total of 9 vessels ranging in size from 80 to 175 µm were measured in the 5 animals receiving vehicle (1–2 vessels per dog), and 19 vessels between 75 and 159 µm in diameter were measured in the 9 animals in the LNNA group (2–3 vessels per dog).

2.3.3 Protocol 3
To study the effectiveness of blockade of K⁺_[ATP] by glibenclamide, control measurements and responses to aprikalim, a selective K⁺_[ATP] opener [19–21], was studied before and after administration of glibenclamide (10 µM, topically; n = 5 dogs). After baseline hemodynamic measurements and control microvascular diameters, either glibenclamide or vehicle (0.1 vol% DMSO) was given for 30 min. Then, aprikalim (0.1–1.0 µM, topically) was administered and measurements repeated 10 min after each dose. A 30 min washout period allowed baseline diameters to return to control dimensions and vehicle or glibenclamide was given for 30 min and the aprikalim doses were repeated. The order of glibenclamide and vehicle was randomized. Following the last dose of aprikalim, nitroprusside (100 µM, topically) was given to ensure that the preparation was viable.

2.3.4 Protocol 4
To study the role of K⁺_[ATP] in coronary microvascular dilation in response to metabolic stimulation, control measurements of hemodynamics, myocardial perfusion, MVO₂ and microvascular diameters were made in 14 dogs. Glibenclamide (10 µM, n = 7) or its vehicle (0.1 vol%, DMSO; n = 7) was continuously suffused onto the surface of the heart and hemodynamics, myocardial perfusion, MVO₂ and microvascular diameter were measured before and following 10 min of a dobutamine infusion (10 µg/kg/min, i.v.) and subsequently with the addition of atrial pacing at 300 bpm, as described in Protocol 2. A total of 18 vessels ranging in size from 49 to 141 µm were measured in the 7 animals receiving vehicle (2–3 vessels per dog), and 17 vessels between 42 and 112 µm in diameter were measured in the 7 animals in the glibenclamide group (2–3 vessels per dog).

2.4 Criteria for data acceptance
Experiments were included for data analysis if mean arterial pressure was >70 mmHg, arterial blood gases were within the physiologic range (pH 7.35–7.45; pCO₂ 30–45 mmHg; pO₂ >70 mmHg), and if mean arterial pressure during dobutamine infusion and/or rapid atrial pacing varied by less than 10 mmHg from control level. Also, microvascular diameters had to return to within 10% of control after termination of dobutamine and atrial pacing. Microvascular fields were selected only if high-quality images of arterial vessels could be obtained in a reproducible manner.

2.5 Statistical analysis
All data are expressed as the mean±s.e.m. In general, 1–3 vessels per dog were used for analysis. When more than 1 vessel within 25 µm of each other was used, the average of the 2 diameters were used as a single vessel measurement. For statistical purposes, the degrees of freedom were the number of dogs studied. One-way analysis of variance with repeated measures was used to evaluate changes in hemodynamic variables, regional coronary blood flow, blood gases, myocardial oxygen consumption and microvascular diameters. When the analysis of variance showed differences among the groups, individual group comparisons were made using a Scheffé F-test. Student's t-test for paired or unpaired samples with Bonferroni correction was used for individual comparisons where appropriate (before and after atenolol). The probability level for statistical significance was taken as P<0.05.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
3.1 Arterial blood gases, hemodynamics, myocardial perfusion and MVO₂
3.1.1 Protocol 1
Baseline measures of arterial blood gases, hemoglobin, hemodynamics, myocardial perfusion and oxygen saturation were within the physiological range (Table 1). Before atenolol, during dobutamine infusion, there was an increase in heart rate (153±4 to 184±4 bpm, P<0.05), left ventricular dP/dt (2793±237 to 5337±309 mmHg/s, P<0.05), myocardial perfusion (123±11 to 221±16 ml/min/100 g, P<0.05) and myocardial oxygen consumption (13.2±1.4 to 23±1.1 ml/min/100 g, P<0.05). Throughout the protocol, there was no significant change in arterial blood gas, hemoglobin, mean arterial pressure, aortic and coronary sinus oxygen saturations. With the addition of rapid atrial pacing, there was a further increase in heart rate (304±5 bpm, P<0.05), myocardial perfusion (343±24 ml/min/100 g, P<0.05) and myocardial oxygen consumption (35±2.9 ml/min/100 g, P<0.05). The left ventricular dP/dt was elevated from control but similar to that during the dobutamine infusion (5007±357 mmHg/s, P<0.05). During the second control period, there were no significant changes in the monitored variables from the first control period in the 7 dogs studied. With the administration of atenolol (1 mg/kg), there were no changes in any of the measured variables. In the presence of atenolol, during the dobutamine infusion there were no changes in the monitored variables from baseline. Thus, the β₁-adrenergic antagonist, atenolol, blocked the metabolic responses to dobutamine. Rapid atrial pacing at 300 bpm could not be performed after the administration of atenolol since AV block occurred at heart rates of approximately 175–200 bpm.

View this table: [in this window] [in a new window]   Table 1 Arterial blood gases, hemoglobin, hemodynamics, myocardial perfusion, and myocardial O2 consumption in dogs undergoing dobutamine infusion and rapid pacing before and after atenolol

 
3.1.2 Protocol 2
Baseline measurements of arterial blood gases, heart rate, mean arterial pressure, left ventricular dP/dt, myocardial perfusion and myocardial oxygen consumption are shown in Table 2. There were no differences between the LNNA or vehicle groups during control conditions. Arterial blood gases and mean arterial pressure did not change throughout the protocol in either group. During dobutamine, there were significant and similar increases in left ventricular dP/dt, myocardial perfusion and myocardial oxygen consumption in both groups. With the addition of atrial pacing, there was a further rise in myocardial perfusion and myocardial oxygen consumption, but importantly these parameters were similar between the groups.

View this table: [in this window] [in a new window]   Table 2 Arterial blood gases, hemodynamics, myocardial perfusion, and myocardial O2 consumption in dogs undergoing dobutamine infusion and rapid pacing with LNNA or vehicle

 
3.1.3 Protocol 4
Arterial blood gases, heart rate, mean arterial pressure, left ventricular dP/dt, myocardial perfusion and myocardial oxygen consumption are shown in Table 3 for the glibenclamide protocol. These parameters were similar between the vehicle and glibenclamide-treated dogs, but mean arterial pressure tended to be lower in the glibenclamide-treated dogs at baseline (vehicle 96±4; glibenclamide 85±4 mmHg, P = n.s.). During the administration of vehicle or glibenclamide, this difference became significant (vehicle 102±4; glibenclamide 85±5 mmHg, P<0.05). However, the mean arterial pressure did not change within each group throughout the protocol. During dobutamine infusion in the vehicle group, there was a substantial increase in left ventricular dP/dt, myocardial perfusion and myocardial oxygen consumption, which was similar to that seen in the glibenclamide group. With the addition of rapid atrial pacing, there was a significant increase in heart rate, left ventricular dP/dt, myocardial perfusion and myocardial oxygen consumption, which were similar in magnitude in the vehicle and glibenclamide-treated dogs.

View this table: [in this window] [in a new window]   Table 3 Arterial blood gases, hemodynamics, myocardial perfusion, and myocardial O2 consumption in dogs undergoing dobutamine infusion and rapid pacing with glibenclamide or vehicle

 
3.2 Microvascular diameters
3.2.1 Protocol 1
In 9 dogs, the dobutamine/pacing protocol was studied. Without atenolol, coronary microvascular responses during dobutamine infusion and during dobutamine infusion with rapid atrial pacing resulted in marked coronary microvascular dilation in all coronary microvessels and the magnitude of dilation was inversely related to the control diameter (Fig. 1). There was substantial coronary microvascular dilation in vessels <100 µm during dobutamine infusion, which further increased with the addition of rapid pacing (Fig. 2). Rapid pacing did not affect small coronary artery diameter (>100 µm) beyond that of the dobutamine infusion alone.

View larger version (15K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Scatter plot showing percent change in diameter of coronary arterioles versus control microvascular diameter during dobutamine infusion alone and with atrial pacing (DOB+RAP). Coronary arteriolar dilation was heterogeneous with the magnitude of the dilation inversely related to control microvascular diameter.

 

View larger version (12K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Bar graph showing percent change in diameter for different sized coronary microvessels during dobutamine infusion alone and dobutamine plus pacing (DOB+RAP). Coronary microvascular responses were significantly greater in the coronary arterioles (<100 µm, left panel, n = 7, baseline diameter = 86±3 µm) compared to the responses in the small coronary arteries (>100 µm, right panel, n = 10, baseline diameter = 161±13 µm).

 
To evaluate the selectivity of dobutamine to the β₁-adrenergic receptor, the selective β₁-adrenergic antagonist, atenolol, was studied. If coronary microvascular dilation occurred through β₂-adrenergic-mediated dilation, atenolol might inhibit hemodynamic changes without substantially affecting coronary microvascular diameter responses. Fig. 3 demonstrates the hemodynamic and diameter responses in 7 dogs with and without atenolol. As seen in this figure, there is a total inhibition of the changes in left ventricular dP/dt, myocardial perfusion and myocardial oxygen consumption, in addition to an inhibition of coronary microvascular dilation.

View larger version (13K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Effect of atenolol on left ventricular dP/dt, myocardial perfusion (MBF), myocardial oxygen consumption (MVO2) and microvascular dilation (baseline diameter = 107±12, range = 69–184 µm) during dobutamine infusion (n = 7). β1-Adrenergic blockade attenuated all response to dobutamine. *P<0.01 vs. without atenolol.

 
3.2.2 Protocol 2
To evaluate the role of arginine-derived nitric oxide, LNNA was superfused onto the coronary microvascular field of interest. Fig. 4 demonstrates coronary arteriolar and small coronary artery responses to dobutamine and dobutamine plus rapid atrial pacing. As shown in Fig. 4, LNNA completely inhibited coronary arteriolar responses in vessels <100 µm to the dobutamine/pacing protocol. This response is in contrast to what is seen in small coronary arteries (>100 µm) where LNNA had no effect.

View larger version (13K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Coronary arterial microvascular responses to dobutamine (DOB) and dobutamine plus pacing (DOB+RAP) in dogs receiving iNG-nitro-L-arginine (LNNA) or its vehicle. LNNA inhibited the coronary arteriolar dilation in response to dobutamine and rapid atrial pacing in vessels less than 100 µm in diameter (left panel: n = 6, vehicle, baseline diameter = 85±5 µm; n = 8, LNNA, baseline diameter =81±5 µm), but no significant effect in small coronary arteries >100 µm (right panel: n = 3, vehicle, baseline diameter = 151±11 µm; n = 11, LNNA, baseline diameter = 147±13 µm). *P<0.05 vs. vehicle.

 
3.2.3 Protocol 3
To establish effectiveness of blockade of K⁺_[ATP] by glibenclamide, responses to aprikalim in the presence or absence of glibenclamide was evaluated. Baseline diameters were 75±6 µm (n = 8) for vessels <100 µm and 135±12 µm (n = 3) for vessels >100 µm. Hemodynamic variables did not change throughout this protocol (data not shown). In vessels <100 µm topical application of glibenclamide produced a reduction in diameter (64±5 µm, P<0.05 vs. baseline). Coronary microvascular responses to aprikalim are shown in Table 4 demonstrating effectiveness of K⁺_[ATP] inhibition by this dose of glibenclamide.

View this table: [in this window] [in a new window]   Table 4 Coronary microvascular diameter responses (% change) to aprikalim in the presence of glibenclamide or vehicle

 
3.2.4 Protocol 4
To evaluate the role of K⁺_[ATP] in mediating metabolically-induced coronary microvascular dilation, glibenclamide was administered topically to the microvascular field of interest. As in Protocol 3, in vessels <100 µm glibenclamide produced a reduction in coronary microvascular diameter (baseline diameter 73±5; with glibenclamide 64±4 µm, P<0.05). In contrast to LNNA, inhibition of K⁺_[ATP] with glibenclamide failed to impair coronary microvascular responses to dobutamine or dobutamine with rapid atrial pacing in either vessels <100 µm or vessels >100 µm (Fig. 5).

View larger version (17K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Coronary arterial microvascular responses to dobutamine (DOB) and dobutamine plus pacing (DOB+RAP) in dogs receiving glibenclamide or its vehicle. Glibenclamide had no significant effect on coronary arteriolar dilation in response to dobutamine and rapid atrial pacing in vessels less than 100 µm in diameter (left panel: n = 11, vehicle, baseline diameter = 74±5 µm; n = 11, glibenclamide, baseline diameter = 73±5 µm) or small coronary arteries >100 µm (right panel: n = 4, vehicle, baseline diameter = 124±12 µm; n = 3, glibenclamide, baseline diameter = 125±6 µm).

 

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
The present study provides evidence that: (1) metabolically-induced coronary microvascular dilation is mediated through an arginine-derived nitro-vasodilator; and (2) the ATP-sensitive potassium channel does not play an important role in coronary microvascular dilation in response to the metabolic stimulus of pacing and β₁-adrenergic stimulation. The data interpretation is dependent upon our methodology for measuring coronary microvascular responses, the appropriateness of our dobutamine/pacing model and the use of our antagonist agents for nitric oxide synthase and K⁺_[ATP]. Each of these areas will be discussed below.

4.1 Critique of methodology
Our system has a spatial resolution of 2.5 µm (6.3x relay lens), and changes in microvascular diameter by the combination of dobutamine and rapid pacing far exceed our minimal resolution. In order to determine the stability of the preparation, control measurements were made before and after our experimental protocol (see Protocol 1 results). If our measured variables did not return to within 10%, the animal would have been eliminated from the study. In our studies, the second control values returned to within 10%. There are apparent discrepancies among the results in vessels >100 µm from the initial protocol (Fig. 2), LNNA protocol (Fig. 4) and glibenclamide (Fig. 5). Fig. 1 shows that microvascular responses are dependent upon baseline diameter. This apparent discrepancy in Fig. 5 is due to a slightly smaller mean diameter in the glibenclamide protocol groups ([µm] glibenclamide 125±6, LNNA 151±11, glibenclamide vehicle 124±11 and LNNA vehicle 161±13) when compared to the other groups of studies.

In Protocol 1, we investigated the general microvascular responses to dobutamine with rapid atrial pacing as the metabolic stimulus. Since dobutamine at high doses may have β₂-adrenergic coronary microvascular dilator effects [16, 17], we tested the hypothesis that at low dose of dobutamine the responses observed during our protocol were primarily due to increases in myocardial oxygen consumption. To test this hypothesis, we studied the effects of dobutamine on hemodynamic and coronary microvascular effects before and following selective β₁-adrenergic blockade with atenolol. As is demonstrated from Table 1 and Fig. 3, atenolol blocked all of the hemodynamic changes observed during dobutamine infusion. In addition, the diameter effects of dobutamine were completely antagonized by the β₁-adrenergic selective blocker, atenolol. These data suggest that in our study the coronary microvascular responses to dobutamine (10 µg/kg/min) were due to increase in myocardial oxygen consumption.

The effect of arginine analogs on the coronary microcirculation has been well described in preparations similar to those described in this study [6, 22, 23]. From our laboratory using the identical open-chest preparation, Komaru and colleagues [22]demonstrated the selectivity of the arginine analogs on the acetylcholine-induced coronary microvascular dilation (LNNA and N^G-monomethyl-L-arginine). In these studies, Komaru et al. showed that acetylcholine-induced coronary microvascular dilation can be inhibited by the arginine analogs and reversed by subsequent administration of L-arginine. They further demonstrated that nitroprusside-induced coronary microvascular dilation was unaffected by the arginine analogs.

In Protocol 4, the role of K⁺_[ATP] was evaluated in response to dobutamine plus rapid atrial pacing. K⁺_[ATP] was inhibited using the topical application of the sulfonylurea, glibenclamide. Glibenclamide has been demonstrated in our laboratory to inhibit coronary microvascular dilation in response to reductions in coronary perfusion pressure [7]. In that study, glibenclamide had no effect on nitroprusside-induced coronary microvascular dilation. We demonstrated that glibenclamide inhibits K⁺_[ATP] opening induced by aprikalim (Table 4). In addition, Lamping et al. [24]have demonstrated that glibenclamide markedly shifts the dose–response curve to the K⁺_[ATP] channel opener, aprikalim, in both normal microvessels and native collaterals. Thus, in our preparation, glibenclamide appears to be sensitive to the K⁺_[ATP]-channel-mediated responses.

In these studies, both antagonists were administered topically to the surface of the heart. This methodology has been used in our laboratory for several years [7, 15, 22, 25, 26]. This methodology is widely used in other microcirculatory beds such as the mesentery, cheek pouch, cremaster muscle and pial circulations. Our group has previously shown that topical application of agonist may be inhibited by intravenous antagonist and intravenous application of agonist may be inhibited by topical antagonist [25]. The advantage of topical application of antagonists is that activation of neurohumoral responses will not provide confounding factors in the interpretation of our results.

4.2 Role of nitric oxide in coronary regulation
Our study, using slightly different methodologies, showed similar results to that by Jones and colleagues [6]. In their studies, the arginine analog tested was N^G-nitro-L-arginine methyl ester (L-NAME). In addition, in their preparation, animals were pretreated with both propranolol and indomethacin to antagonize β-adrenergic stimulation, as well as cyclo-oxygenase products. They used pacing at 180 bpm to induce the metabolic stimulus. In their studies, they found that pacing-induced coronary microvascular dilation was significantly less than that observed in our studies and dilation was inhibited by L-NAME primarily in vessels <100 µm. These investigators did not measure myocardial oxygen consumption. Others have evaluated the role of nitric oxide in coronary vascular responses to other physiological and pathophysiological stimuli. Smith et al. [27]and Ueeda et al. [28]both demonstrated the importance of nitric oxide in modulating coronary autoregulatory responses in the dog and guinea pig. Other investigators have shown that nitric oxide may play a minor role in mediating coronary vascular responses to reactive hyperemia [29].

4.3 Role of K⁺_[ATP] in coronary regulation
Since the initial description by Daut and colleagues [30]that hypoxic dilation of coronary arteries is mediated by K⁺_[ATP], a number of investigators have studied the role of this potassium channel in coronary flow regulation. Our group was the first to demonstrate that K⁺_[ATP] played an important role in mediating coronary microvascular responses to hypoperfusion [7]. Those results have been subsequently confirmed by Narishige et al. [8]. Dankelman and colleagues [9]demonstrated that glibenclamide reduces the rate of change of coronary resistance in an anesthetized goat preparation. Aversano et al. [10]and Kanatsuka and colleagues [11]have demonstrated that K⁺_[ATP] plays an important role in mediating coronary vascular responses to reactive hyperemia.

The effect of K⁺_[ATP] in mediating coronary vascular responses to increased metabolic stimulation are more controversial. Narishige and colleagues [12]demonstrated that the administration of glibenclamide prevented coronary vasodilation induced by β-adrenergic stimulation with isoproterenol or denopamine. In the isoproterenol-treated animals, a substantial β₂ coronary flow effect was observed. This confounds the interpretation of their study. The response to denopamine was not challenged with selective β₁-adrenergic blockade, as we did with Protocol 1 in our studies (Table 1, Fig. 1). Thus, it is unclear whether β₂-adrenergic stimulation may have played a role in their responses to denopamine. Lastly, in their study myocardial oxygen consumption only increased modestly, from 1.4 to 1.8 ml/min before glibenclamide with denopamine to 1.3 to 1.6 ml/min after glibenclamide with denopamine. Further, the stimulated (denopamine) myocardial oxygen consumption was lower than the baseline MVO₂ in the isoproterenol protocol. Aversano and colleagues [13]studied the effect of K⁺_[ATP] in a model of elevated blood pressure with phenylephrine and simultaneous atrial pacing at elevated heart rates. They did not measure myocardial oxygen consumption directly, but rather as the product of heart rate and left ventricular systolic pressure. They demonstrated that glibenclamide had no effect on flow responses to phenylephrine plus atrial pacing in dogs. Our results agree with those of Aversano and colleagues since we found no substantial effect on coronary microvascular responses in our dobutamine/pacing protocol (Table 3, Fig. 3). In another study, Dunker and colleagues [31]evaluated the role of K⁺_[ATP] during graded treadmill exercise in chronically instrumented dogs. In their studies, glibenclamide was infused intracoronary and measurements of blood flow, heart rate, mean arterial pressure and left ventricular dP/dt were made. They showed that basal flow was altered but the response to metabolic stress was not effected by inhibition of K⁺_[ATP]. Thus, in the only positive study, no effect on basal coronary blood flow was observed during K⁺_[ATP] inhibition. In the two published negative studies for the role of K⁺_[ATP] in mediating coronary flow responses to metabolic demands [13, 31]and this study, inhibition of K⁺_[ATP] caused a decrease in baseline blood flow [13, 31]or diameter [7]. Therefore, we agree that the ATP-sensitive potassium channel does not play an important role in coronary vascular dilation to increased metabolic dilation.

4.4 Physiological considerations
Our initial hypothesis was that both K⁺_[ATP] and nitric oxide inhibition may play a role in this complex physiological effect from increasing myocardial oxygen consumption with our dobutamine/pacing protocol. It is well established that the coronary vasculature releases nitric oxide as part of a flow-dependent mechanism [3]. Since this observation has also been well established in the coronary microcirculation, we thought that metabolic byproducts could be released to initiate opening of the ATP-dependent potassium channels, subsequently causing coronary microvascular vasodilation with larger arteriolar vasodilation as a result of flow-induced effects. This was not the case in our studies. In contrast to this speculation, our microvascular responses to LNNA inhibition were completely abolished in response to the dobutamine/pacing protocol (Fig. 4) and not affected by glibenclamide (Fig. 5). Our data support a direct coupling of increased metabolic demand with nitric oxide. This coupling has been suggested in studies by Shen et al. [32]. In that study, the authors postulate that NO inhibits myocardial oxygen consumption. The administration of LNNA abolishes the inhibitory effect of NO on tissue metabolism, resulting in enhanced tissue metabolism. Thus, in the presence of LNNA, total oxygen consumption is enhanced. Taken together with our results, the release of NO would not only increase myocardial perfusion through arteriolar dilation, but would decrease the MVO₂ of the myocardium, resulting in a very favorable O₂ balance within the myocardium.

In conclusion, our study indicates that coronary microvascular dilation in response to increased metabolic stimulation using dobutamine in conjunction with rapid pacing is mediated through a nitric-oxide-dependent mechanism and not ATP-sensitive potassium channels. These results may have important implications in pathological disease states where nitric oxide mechanisms are impaired, such as diabetes and hypertension.

Time for primary review 23 days.

	Acknowledgements

 
This work was supported by RO1 HL51308, American Heart Association Grant-in-Aid (91-15050). Dr. Dellsperger is an Established Investigator of the American Heart Association. We acknowledge the secretarial assistance of Ms. Cindy Evans in the preparation of this manuscript.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 

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