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1 Department of Physiology and Biophysics, University of Washington School of Medicine, Seattle, Washington 98195; and 2 Department of Internal Medicine, University of South Florida College of Medicine, Tampa, Florida 33612
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The purpose of this investigation was to quantitatively evaluate the role of adenosine in coronary exercise hyperemia. Dogs (n = 10) were chronically instrumented with catheters in the aorta and coronary sinus, and a flow probe on the circumflex coronary artery. Cardiac interstitial adenosine concentration was estimated from arterial and coronary venous plasma concentrations using a previously tested mathematical model. Coronary blood flow, myocardial oxygen consumption, heart rate, and aortic pressure were measured at rest and during graded treadmill exercise with and without adenosine receptor blockade with either 8-phenyltheophylline (8-PT) or 8-p-sulfophenyltheophylline (8-PST). In control vehicle dogs, exercise increased myocardial oxygen consumption 4.2-fold, coronary blood flow 3.8-fold, and heart rate 2.5-fold, whereas mean aortic pressure was unchanged. Coronary venous plasma adenosine concentration was little changed with exercise, and the estimated interstitial adenosine concentration remained well below the threshold for coronary vasodilation. Adenosine receptor blockade did not significantly alter myocardial oxygen consumption or coronary blood flow at rest or during exercise. Coronary venous and estimated interstitial adenosine concentration did not increase to overcome the receptor blockade with either 8-PT or 8-PST as would be predicted if adenosine were part of a high-gain, negative-feedback, local metabolic control mechanism. These results demonstrate that adenosine is not responsible for local metabolic control of coronary blood flow in dogs during exercise.
8-phenyltheophylline; 8-sulfophenyltheophylline; myocardial blood flow
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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THE ADENOSINE HYPOTHESIS proposes that adenosine is the vasodilatory metabolite that links coronary blood flow to myocardial metabolism (2, 12). The hypothesis predicts that increases in oxygen consumption decrease myocardial oxygen tension to stimulate adenosine release. The increased concentration of adenosine in the cardiac interstitium results in arteriolar vasodilation and augmented oxygen delivery by activating adenosine receptors on coronary vascular smooth muscle cells. The augmented oxygen delivery restores myocardial oxygen tension to a normal operating level, thereby decreasing cardiac adenosine production in a negative feedback manner. In this way, the interstitial concentration of adenosine would control coronary blood flow to match myocardial oxygen supply with myocardial oxygen consumption and maintain myocardial oxygen tension in a narrow range (3, 5).
Despite various investigations, the role of adenosine in local metabolic control of coronary blood flow during exercise has not been clearly defined. Previous studies found that the release of adenosine into the coronary venous plasma increased in dogs during exercise, suggesting interstitial adenosine concentration is increased when myocardial oxygen consumption is elevated (10, 23). However, blockade of adenosine receptors with 8-phenyltheophylline (8-PT) has failed to alter the relationship between coronary blood flow and myocardial oxygen consumption during exercise (1, 6). One potential explanation for these conflicting results is that interstitial adenosine concentration increases sufficiently during exercise to overcome the competitive receptor blockade with 8-PT as would be predicted by a high gain feedback system (14, 16, 24). This laboratory recently reported (34) that interstitial adenosine concentration did not increase to overcome the receptor blockade by 8-PT when myocardial oxygen consumption was doubled by cardiac paired-pacing in anesthetized closed-chest dogs. However, this does not exclude the possibility that adenosine contributes to local metabolic flow regulation when myocardial oxygen consumption is further increased during exercise. Accordingly, the present study was designed to examine the role of adenosine in exercise coronary vasodilation by determining the relationship between myocardial oxygen metabolism and interstitial adenosine concentration with and without adenosine receptor blockade. Additional experiments were done with an endothelin receptor antagonist (Ro-61-0612) to determine whether adenosine receptor blockade reduced coronary venous oxygen tension by unmasking endothelin-mediated vasoconstriction (33). Experiments were also conducted with the hydrophilic adenosine receptor antagonist 8-p-sulfophenyltheophylline (8-PST).
Interstitial adenosine concentration was estimated in chronically instrumented dogs from arterial and coronary venous measurements using a previously tested, axially distributed, mathematical model (21, 30). Coronary venous and estimated interstitial adenosine concentrations were little changed when myocardial oxygen consumption was increased fourfold during exercise. Adenosine receptor blockade did not alter the coronary blood flow response during exercise, and estimated interstitial adenosine did not increase to overcome the receptor blockade. These findings demonstrate that adenosine is not responsible for local metabolic control of coronary blood flow in dogs during exercise.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Surgical Preparation
Experiments were performed on adult male mongrel dogs weighing 23-34 kg taught to run on a motorized treadmill. Preanesthesia (acepromazine 0.05 mg/kg + atropine 0.06 mg/kg sc) was administered 30 min before induction of anesthesia with ketamine 5.75 mg/kg + diazepam 0.3 mg/kg iv. A surgical plane of anesthesia was maintained by mechanical ventilation with 0.5 to 3.0% isoflurane gas. With the use of sterile technique, a splenectomy was performed through a midline abdominal incision to minimize changes in hematocrit during exercise. After this procedure, a left lateral thoractomy was performed in the fifth intercostal space. With the use of a modified Seldinger technique, a polyurethane catheter was implanted (see Catheters) into the descending thoracic aorta to measure aortic blood pressure and to obtain arterial blood samples. A second polyurethane catheter (see Catheters) was placed in the coronary sinus via a purse-string suture in the right atrial appendage for coronary venous blood sampling. The circumflex coronary artery was dissected free, and a flow transducer (see Pressure and Flow Measurement) was placed around the artery (Fig. 1). No instruments were implanted in the myocardium, and no surgical stitches were placed in the ventricles to avoid injured tissue, which would release adenosine. A chest tube was placed to evacuate the pneumothorax, and the chest was closed in layers. The catheters and the flow transducer wire were tunneled subcutaneously and exteriorized between the scapulae. Both the abdominal and thoracic incisions were infiltrated with 2.5% bupivacaine, and buprenorphine (Buprenex, 0.01 mg/kg im) was administered to minimize postoperative pain. Antibiotic (cefazolin, 20 mg/kg im) was administered twice daily for 5 days. The animals received a multivitamin, baby aspirin (81 mg), and a dietary iron supplement (324 mg) daily. A nylon jacket (Alice King Chatham, Hawthorne, CA) was placed on the animals to protect the catheters and the flow transducer wire. The animals were allowed to recover for at least 10 days before experiments were conducted.
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Catheters
Polyurethane catheters (0.105 in. OD, 0.065 in. ID) used for both aortic and coronary sinus catheters were commercially coextruded (Putnam Plastics, Dayville, CT) to produce a catheter with a soft biocompatable exterior surface (EG 80A) and a hard interior surface (EG 65D) with a low coefficient of friction. The aortic catheter consisted of a 65-cm length of coextruded polyurethane tubing in which the tip was tapered by heating and stretching the tubing over a piece of 21-gauge, thin-walled metal tubing (0.032 in. OD). The catheter was inserted with an 18-gauge, thin-walled needle (6.5 cm in length, 0.05 in. OD) connected to a stainless steel cable (79 cm in length, 0.03 in. OD) placed inside the catheter. After the catheter was in place, the needle was removed from the aorta by pulling the needle out the opposite end of the catheter. A small piece of surgical felt was glued (Silastic Medical Adhesive) to the catheter 1.5 cm from the tapered end so the catheter could be secured in place.The coronary sinus catheter consisted of a 65-cm length of coextruded polyurethane tubing with a Silastic sleeve (20 cm in length, 0.095 in. OD, 0.062 in. ID) that was placed over the coextruded tubing and positioned so that 3 mm of Silastic tubing extended beyond the tip of the catheter. This Silastic ending was used to decrease trauma to the wall of the coronary sinus.
The catheters were flushed daily with a high viscosity solution of 50% glucose containing penicillin G (65,000 U/ml) and heparin (1,300 U/ml).
Pressure and Flow Measurement
The coextruded polyurethane catheter was used in the aorta so that a high-fidelity Mikro-tip catheter pressure transducer (3-Fr, SPR-524, Millar Instruments, Houston, TX) could be inserted at the time of the experiment to measure aortic blood pressure (11, 15). The pressure transducer was introduced into the aortic catheter through a hemostatic control valve (Tuohy-Borst adapter, Mallinckrodt Medical, St. Louis, MO), which allowed arterial blood samples to be withdrawn while maintaining a fluid-tight seal.Coronary blood flow was continuously measured throughout the experimental protocol (see Experimental Protocols) with an ultrasonic, perivascular flow transducer (Transonics, Ithaca, NY). The flow transducers were calibrated before and after chronic implantation. The average difference between the slopes for the flow calibrations was 5 ± 1% (±SE, n = 9). After all experiments were completed, the animals were euthanized with pentobarbital sodium, and the circumflex perfusion territory was dyed with india ink. The weight of the dyed tissue was used to calculate coronary blood flow per gram of perfused myocardium.
Blood Sampling
Arterial and coronary venous blood samples were collected simultaneously in heparinized glass syringes that were immediately sealed and placed on ice. The samples were analyzed for hydrogen ion concentration, carbon dioxide tension, and oxygen tension with an Instrumentation Laboratories 1306 pH/blood gas analyzer (Waltham, MA). Oxygen content was determined using the fuel-cell method (Total O2X, Hospex, Chestnut Hill, MA). In addition, a portion of the arterial and coronary venous blood samples was transferred into NaF-coated vials to prevent glycolysis, and lactate concentration was determined with a YSI model 1500 lactate analyzer (Yellow Springs Instruments, Yellow Springs, OH). Myocardial oxygen consumption (µl O2 · min
1 · g1)
was calculated by multiplying coronary blood flow per gram of perfused
tissue by the arterial-coronary venous difference in oxygen content.
Percent myocardial lactate extraction was calculated as the difference
in arterial and coronary venous lactate concentration divided by the
arterial lactate concentration.
Plasma Adenosine Measurement
Arterial and coronary venous adenosine measurements were made at rest and during steady-state conditions at each exercise level (see Experimental Protocols). Plasma adenosine concentration was measured with a modified version of the Herrmann and Feigl method (18). With a two-syringe arrangement, blood samples (3.7 ml) were collected and simultaneously mixed with an ice-cold enzymatic stop solution (5.0 ml) to prevent any further metabolism of adenosine (27). The stop solution contained dipyridamole (32 µM), iodotubercin (1 µM), and erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA, 10 µM) dissolved in cold 0.9% saline. Dipyridamole was used to inhibit cellular adenosine uptake. Iodotubercin was used to inhibit adenosine kinase, preventing incorporation of adenosine into AMP. EHNA inhibits adenosine deaminase, preventing degradation of adenosine to inosine. Theophylline (20 µM) was included in the stop solution as an internal recovery standard. Anticoagulation of the samples was not necessary because of the dilution with stop solution. Blood samples were immediately centrifuged at 15,000 rpm and 0°C for 2 min. Then 5 ml of the plasma-stop solution supernatant were rapidly added to 1.8 ml of 4 N perchloric acid to precipitate plasma proteins. The samples were again centrifuged at 15,000 rpm and 0°C for 10 min; 5 ml of acid supernatant were then added to 4.35 ml of a neutralizing solution containing 0.4 mM KH3PO4 and 0.8 mM KOH and kept on ice for 30 min. The resulting pH was ~7.0. An additional centrifugation for 10 min at 15,000 rpm and 0°C precipitated most of the salt. The samples were then purified by applying the neutralized supernatant to C-18 Sep-Pak cartridges. Adenosine and theophylline were eluted into test tubes with 2 ml of 40% methanol. The samples were evaporated to dryness in a Buchler vortex evaporator (Buchler Instruments, Fort Lee, NJ) and resuspended in 200 µl of distilled water. Each sample was divided into two 100-µl aliquots, and adenosine deaminase (0.1 U, Boehringer Mannheim) was added to one of the aliquots, which was used as a paired blank. The blank samples were incubated at room temperature for 15 min. After incubation, 250 µl of 100% methanol were added to each sample to inactivate the adenosine deaminase, and the samples were again incubated at room temperature for 15 min and then heated at 75°C for 15 min. The samples were evaporated to dryness and resuspended in 100 µl of HPLC buffer.The adenosine in each sample was separated on a Hewlett-Packard 1100 HPLC with a C-18 column (5 µm, 220 × 2.1 mm, Perkin Elmer, Norwalk, CT) using a buffer solution of 4 mM KH2PO4 in 2% acetonitrile with a linear acetonitrile gradient to 23% in 20 min with a flow rate of 0.3 ml/min. The diode array optical detector recorded absorbance at 260 nm (bandwidth = 16 nm) with a reference spectrum of 320-380 nm throughout the separation. The adenosine peak was identified by comparison with plasma samples spiked with adenosine, adenosine standards, and spectral analysis. The paired chromatograms were superimposed using HP Chemstation software, and the blank was subtracted from the unknown. The chromatogram peaks were integrated, and adenosine content was determined by comparison with known adenosine standards. Plasma adenosine concentration was calculated by accounting for dilution steps in sample handling and hematocrit and was normalized for recovery with the theophylline standard in each sample. The detection limit of the assay is 1.5 pmoles of adenosine, which is equivalent to approximately a 5.5 nM concentration in plasma. The recovery of 100 pmoles of adenosine added to an initial blood sample and carried through the entire assay was 86 ± 10% (±SD, n = 20).
Estimation of Cardiac Interstitial Adenosine Concentration
Cardiac interstitial adenosine concentration was estimated using a four-region (plasma, endothelial cell, interstitial space, parenchymal cell), axially distributed mathematical model (19, 21, 30). The model describes the effects of blood flow, adenosine transport, and exchange between tissue regions, as well as cellular production and consumption on the relationship among arterial, venous, and interstitial adenosine concentrations. This model has been used previously to estimate interstitial adenosine concentrations in vivo, and the constraints and assumptions have been described extensively (21, 30). The model accounts for myocardial blood flow heterogeneity and the change in heterogeneity that occurs with changes in blood flow. In addition, the model is constrained with previous estimates of capillary adenosine transport and metabolism adjusted for the level of coronary blood flow. Interstitial adenosine concentration was estimated using the measured values of coronary plasma flow and arterial plasma adenosine concentration by adjusting cellular adenosine production in the model to fit the measured coronary venous plasma adenosine concentration. Interstitial adenosine concentration was calculated at rest and during steady-state exercise conditions with and without administration of adenosine receptor antagonists.Experimental Protocols
8-PST dose-response experiments. Experiments to determine the effects of 8-PST on adenosine-mediated coronary vasodilation were conducted in four
-choralose-anesthetized closed-chest dogs. The
left circumflex coronary artery was cannulated with a stainless steel
wedge-tipped cannula via the right common carotid artery and perfused
at a constant pressure of 100 mmHg with a servo-controlled roller pump
(32). Exogenous adenosine was infused into the circumflex coronary
artery over a range of 2.67-267 ng/min before and 40 min after
intravenous administration of 8-PST (3 mg/kg + 1 mg/kg supplemental
dose administered 20 min after initial dose). The timing of the dose
response with 8-PST was based on the typical 40-min length of the
exercise protocol. This dose of 8-PST was found to shift the
dose-dependent coronary blood flow response to intracoronary infusions
of adenosine to the right fivefold.
Exercise experiments. The role of adenosine in control of coronary blood flow was examined at rest and during graded treadmill exercise in three groups: 1) control vehicle (n = 10); 2) 8-PT (n = 8); and 3) 8-PST (n = 6). Each animal received either 8-PT or 8-PST, and four received both. Each animal served as its own control. The dose of 8-PT (3 mg/kg iv) used in this investigation was previously found to shift the coronary blood flow dose-response curve to intracoronary infusions of adenosine to the right 12-fold and at least 12-fold for endogenous adenosine (30).
Coronary blood flow, aortic pressure, and heart rate were continuously measured in all groups while the dogs were resting in a sling and during three levels of treadmill exercise: 1) 3 miles/h, 5% grade; 2) 4 miles/h, 10% grade; and 3) 5 miles/h, 15% grade. Arterial and coronary venous blood samples were collected when hemodynamic variables stabilized at each level. Each exercise period was ~2 min in duration, and the animals were allowed to rest between each level for hemodynamic variables to return to baseline. Control and adenosine receptor blockade experiments were conducted on separate days, and the animals were allowed at least 2 days of recovery between experiments.
Endothelin experiments. Additional experiments were done with the endothelin receptor antagonist Ro-61-0612 (1 mg/kg iv) followed by 8-PT (3 mg/kg iv) in five dogs to determine whether adenosine receptor blockade reduced coronary venous oxygen tension by unmasking endothelin-mediated vasoconstriction. The exercise protocol described above was followed in these experiments. The 1 mg/kg dose of Ro-61-0612 used in this investigation was previously shown to be an effective endothelin ETA and ETB receptor antagonist (28).
Drugs
8-PST was prepared (by R. A. Olsson) as previously described by Daly et al. (4). The retention time on reverse-phase HPLC (mobile phase, formic acid:methanol:water 1:49:50) and ultraviolet light spectrum of the product were identical to those of an authentic sample, and the 1H NMR spectrum was consistent with the putative structure. 8-PST (4 mg/kg) was dissolved in 0.9% saline (5 mg/ml) and titrated to pH 8.0 with 0.1 N NaOH. 8-PT (3 mg/kg, Sigma, St. Louis, MO) was placed in 1.5-ml equal parts of 1 N NaOH, ethanol, and propylene glycol and gently warmed until dissolved. Final volume was adjusted to 30 ml with warm 0.9% saline. Both adenosine receptor antagonists were infused intravenously over a 10-min period. The endothelin receptor antagonist Ro-61-0612 (1 mg/kg; Actelion, Allschwil, Switzerland) was dissolved in 0.9% saline (2 mg/ml) and infused intravenously over a 10-min period. The adenosine stop solution was made in isotonic saline and included 1 µM iodotubercin (RBI, Natick, MA), 10 µM EHNA (Sigma), and 32 µM dipyridamole (Sigma).Data Analysis
Calculation of arterial plasma adenosine concentration. During intracoronary arterial adenosine infusions, arterial plasma adenosine concentration was calculated using the following equation
![[Ado]<SUB>art, plasma</SUB> = <FR><NU>Ado infusion rate</NU><DE>Blood flow × (1 − Hct)</DE></FR> × [Ado]<SUB>infusate</SUB>](/content/vol278/issue1/fulltext/H74/img001.gif)
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(1) |
Hill equation fitting to dose responses. The adenosine
dose-response data were fit with the Hill equation
![F = F<SUB>min</SUB> + <FR><NU>(F<SUB>max</SUB> − F<SUB>min</SUB>) × [Ado]<SUP><IT>H</IT></SUP></NU><DE>[Ado]<SUP><IT>H</IT></SUP> + ED<SUP><IT>H</IT></SUP><SUB>50</SUB></DE></FR>](/content/vol278/issue1/fulltext/H74/img002.gif)
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Statistical analyses. Hemodynamic variables were recorded with Windaq data analysis software (Dataq Instruments, Akron, OH). Analog signals from the recording instruments were digitized and stored on disk at a rate of 200 samples/s. The values for mean coronary blood flow, mean aortic pressure, and heart rate at rest and during exercise were averaged over a 30-s period.
For the key postulated response variables (coronary blood flow,
coronary venous oxygen tension, and coronary venous and interstitial adenosine concentrations), statistical testing was directed to overall
treatment effects (vehicle, 8-PT, 8-PST). These tests were chosen for
their specificity to the hypothesis and to avoid inappropriate multiple
comparisons. All analyses accounted for the effects of drug and dog.
Analysis of covariance (ANCOVA) was employed to adjust coronary venous
oxygen tension for linear dependence on myocardial oxygen consumption
after testing for parallel regression lines (SAS, proc
glm). Multiple linear regression was used to compare
slopes for the three treatments in the interstitial adenosine concentration versus myocardial oxygen consumption relation (SAS, proc
glm). The same procedure was used for the coronary blood flow and
coronary venous plasma adenosine concentration versus myocardial oxygen
consumption relations. Table 1 presents
means by exercise level and does not include P values because
the P values pertain to the overall treatment effects as shown
in the figures. Data are presented as means ± SE unless otherwise
noted.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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8-PST Dose-Response Studies
The effects of 8-PST (3 mg/kg + 1 mg/kg iv supplemental dose) on the coronary blood flow response to intracoronary adenosine infusions are shown in Fig. 2. 8-PST produced a fivefold increase in the ED50. These data demonstrate that the intravenous dose of 8-PST used in this investigation significantly attenuates adenosine-mediated coronary vasodilation. As previously noted, 8-PT (3 mg/kg iv) was found to shift the coronary blood flow response to adenosine to the right 12-fold (30).
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Exercise Studies
Hemodynamic and metabolic data for the 10 dogs are given in Table 1. The hemodynamic response to graded treadmill exercise from one dog is shown in Fig. 3. In the vehicle group, myocardial oxygen consumption increased 4.2-fold and coronary blood flow increased 3.8-fold, from rest to the highest level of exercise (Fig. 4A). The slope of the coronary blood flow versus myocardial oxygen consumption relationship was not significantly altered by adenosine receptor blockade with either 8-PT or 8-PST. Heart rate increased ~2.5-fold from rest to the highest level of exercise in all groups, whereas mean aortic pressure was unchanged (Fig. 4, B and C). Arterial carbon dioxide tension gradually decreased with the graded exercise in all three groups, indicating hyperventilation (Table 1). Carbon dioxide tension and hydrogen ion concentration were decreased by 8-PT treatment in both arterial and coronary venous blood, as previously described by Duncker et al. (6) (Table 1).
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The relationship between myocardial oxygen consumption and coronary
venous oxygen tension is shown in Fig. 5.
As myocardial oxygen consumption was increased, coronary venous oxygen
tension fell in all three groups, indicating that the coronary
vasodilator response did not completely match the increase in oxygen
consumption. The slope of the coronary venous oxygen tension versus
myocardial oxygen consumption relationship was not significantly
altered by either 8-PT or 8-PST, demonstrating that adenosine is not
required for exercise-induced coronary vasodilation. Coronary venous
oxygen tension fell significantly with both adenosine receptor
antagonists at rest and during exercise.
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Measurements of arterial and coronary venous plasma adenosine
concentration are shown in Fig. 6. Arterial
plasma adenosine concentration was not altered by the increase in
myocardial oxygen consumption or by adenosine receptor blockade (Fig.
6A). In the vehicle group, coronary venous plasma and estimated
interstitial adenosine concentrations were little changed when
myocardial oxygen consumption was increased approximately fourfold at
the highest level of exercise (Figs. 6B and
7). The estimated interstitial adenosine
concentration remained well below the threshold concentration necessary
for coronary vasodilation (117 nM) (30). The slopes of the relationship
between estimated interstitial adenosine concentration and myocardial
oxygen consumption during control vehicle and adenosine receptor
blockade did not differ significantly, and the estimated slope did not
differ significantly from zero (Fig. 7). The same is true for the
slopes of the coronary venous adenosine concentration versus myocardial
oxygen consumption relationships (Fig. 6B).
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Endothelin Receptor Blockade Studies
The relationship between coronary venous oxygen tension and myocardial oxygen consumption during endothelin receptor blockade with Ro-61-0612 is shown in Fig. 8. Administration of Ro-61-0612 failed to inhibit the fall in coronary venous oxygen tension with 8-PT at rest or during exercise, indicating adenosine receptor blockade does not reduce coronary venous oxygen tension by unmasking endothelin-mediated vasoconstriction. Compared with the vehicle or 8-PT alone, the slope of the coronary venous oxygen tension versus myocardial oxygen consumption relationship was not significantly altered by Ro-61-0612 plus 8-PT, demonstrating that neither endothelin nor adenosine are required for exercise-induced coronary vasodilation.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The present study is the first to combine adenosine measurements with adenosine receptor blockade during coronary exercise hyperemia. This is important because if, as postulated, adenosine is part of a high-gain negative feedback system, then adenosine levels will increase to overcome the competitive receptor blockade, and little change in the coronary blood flow response will be observed.
The major findings of the present study are the following. 1) coronary venous (Fig. 6B) and calculated myocardial interstitial (Fig. 7) adenosine concentrations were little changed when myocardial oxygen consumption was augmented more than fourfold during exercise. 2) Adenosine receptor blockade did not alter the coronary blood flow response during exercise (Fig. 4). 3) Importantly, coronary venous (Fig. 6B) and estimated interstitial (Fig. 7) adenosine concentrations did not increase to overcome the blockade. These findings demonstrate that adenosine is not responsible for local metabolic control of coronary blood flow in dogs during exercise.
Adenosine Levels During Exercise
Simple examination indicates that coronary venous plasma adenosine concentration changed little during exercise (Fig. 6B). The methods used in the present experiments are capable of detecting elevations in coronary venous adenosine concentration as occur during hypoxia (17), coronary autoregulation (29), intracoronary norepinephrine infusion (31), and the release of endogenous adenosine by inhibiting adenosine kinase and adenosine deaminase (30). Thus it is very likely that the present methods are adequate for detecting significant changes in coronary venous plasma adenosine concentration during exercise. The references cited above also demonstrate that increases in myocardial interstitial adenosine concentration are reflected in coronary venous adenosine concentration despite avid uptake of adenosine by coronary vascular endothelium (21, 26).Two previous studies measured coronary venous adenosine concentration during exercise, and both studies supported a role for adenosine in exercise hyperemia (10, 23). However, there is reason to question the adenosine assay methods in both cases. McKenzie et al. (23) reported arterial adenosine as ~100 nM and coronary venous as ~116 nM in resting unanesthetized dogs. Those values are much higher than the 6-15 nM values found in the present study. McKenzie et al. (23) precipitated whole blood samples for adenosine assay with perchloric acid, and this is likely to release a variable amount of adenosine from cells in the blood. Ely et al. (10) reported arterial plasma adenosine as 480 nM and coronary venous as 380 nM in resting unanesthetized dogs. These investigators treated 3 ml of blood with 0.25 ml of stop solution containing dipyridamole, EHNA, and 5% methanol in saline. The blood and stop solution mixture was kept on ice for 20 min before centrifugation for 10 min to separate plasma from cells. The method used by Ely et al. (10) was compared with the present method in parallel experiments. The blood-handling method used by Ely et al. was duplicated, and the separated plasma was then analyzed using the HPLC method of the present study. The method used by Ely et al. resulted in an arterial plasma adenosine concentration of 236 ± 36 nM (±SE, n = 6 from 2 dogs) compared with 12 ± 5 nM (±SE, n = 6 from 2 dogs) using the present methods for samples drawn at the same time. These results indicate that the bulk of the adenosine reported by Ely et al. (10) came from formed blood elements and does not reflect cardiac adenosine levels.
In summary, coronary venous plasma adenosine concentration changed little during exercise in the present study, and there is reason to question previous measurements of coronary venous adenosine concentration.
Myocardial interstitial adenosine concentration was calculated from coronary blood flow, hematocrit, and the adenosine concentrations in arterial and venous plasma using a mathematical model. The model accounts for adenosine uptake by vascular endothelial and parenchymal cells, flow heterogeneity as a function of flow, and paracellular diffusion between the interstitium and plasma in an axially distributed manner. The model has been extensively tested and described (21). Previous experiments using both exogenous and endogenous adenosine demonstrated that the threshold interstitial adenosine concentration for coronary vasodilation is ~117 nM (30). The estimated interstitial adenosine concentration in the present experiments remained well below the threshold value at rest and during exercise, as shown in Fig. 7.
It may be asked if the model parameter values that were determined in anesthetized, closed-chest dogs are valid in dogs during exercise. The most sensitive parameter relating interstitial adenosine concentration to the measured venous plasma adenosine concentration is the paracellular adenosine diffusion in the cleft between vascular endothelial cells [permeability-surface area product of interendothelial gaps (PSg) in the model]. Other model parameters will change interstitial and venous plasma adenosine levels in an equivalent manner, and because venous adenosine is a measured variable, any such parameter changes will largely be accounted for. There is no a priori reason to postulate a change in PSg in addition to its known dependence on flow that is included in the model. Catecholamines do not change coronary PSg studied in buffer-perfused guinea pig hearts (13). Adenosine infusion also does not change the value of PSg (J. B. Bassingthwaighte, personal communication). If a highly unlikely twofold decrease in PSg is modeled, the estimated interstitial adenosine concentration increases ~20% (nonlinear function) in the present experiments. Such a 20% increase in interstitial adenosine concentration does not change any of the interpretations in the present experiments.
Effects of Adenosine Receptor Blockade on Hemodynamics and Adenosine Levels
Adenosine receptor blockade with either 8-PT or 8-PST had little effect on coronary blood flow or other cardiovascular variables at rest or during exercise (Fig. 4). Adenosine receptor blockade also had little effect on coronary venous or estimated interstitial adenosine concentrations (Figs. 6B and 7). If, as postulated, adenosine is part of a high-gain, negative feedback system that keeps myocardial oxygen tension in a narrow range (3, 5), then the failure of adenosine receptor blockade to blunt coronary vasodilation during exercise could be explained by an increase in adenosine levels to overcome the competitive receptor blockade. In the present experiments this would have required a 12-fold increase in adenosine concentration for 8-PT and a 5-fold increase for 8-PST. This clearly did not occur (Figs. 6B and 7).Bache et al. (1) used 8-PT (5 mg/kg iv) in dogs, and Duncker et al. (6) used 8-PT (5 mg/kg iv) in pigs to evaluate the adenosine hypothesis and found no diminution of coronary blood flow during rest or exercise. The present results confirm the hemodynamic findings of Bache et al. and Duncker et al. Edlund and co-workers (8, 9) used intravenous theophylline to block adenosine receptors in humans during exercise. Using coronary sinus thermodilution, they determined coronary blood flow during exercise was diminished by theophylline and concluded that adenosine contributes to exercise coronary hyperemia. However, theophylline treatment also decreased myocardial oxygen consumption, and a replotting of Edlund et al.'s data (8, 9) of coronary flow versus myocardial oxygen consumption reveals that the points before and after theophylline lie close to the same line, similar to Fig. 4A in the present paper. Thus the flow data of Edlund et al. do not support a role for adenosine coronary vasodilation during exercise.
Effect of Adenosine Receptor Blockade on Coronary Venous Oxygen Tension
Adenosine receptor blockade with either 8-PT or 8-PST decreased coronary venous oxygen tension at rest and during exercise as shown in Fig. 5. If adenosine were involved in exercise coronary vasodilation, then a divergence from control oxygen tension values would be expected at high exercise levels; that is, the slopes of the regression lines in Fig. 5 during adenosine receptor blockade would be steeper than the control slope; i.e., a larger drop in coronary venous oxygen tension at high exercise levels. The parallel slopes observed in Fig. 5 indicate adenosine is not involved in exercise coronary vasodilation.The mechanism for the decrease in resting coronary venous oxygen tension was examined with additional experiments. 8-PT is lipophyllic and thus readily enters cells where it may have effects in addition to blockade of adenosine receptors on the outer cell surface. 8-PST is strongly anionic at physiological pH and thus does not enter cells. However, a similar decrease in coronary venous oxygen tension was observed with 8-PST (Fig. 5).
Velasco et al. (33) demonstrated that adenosine inhibits the release of endothelin during reperfusion of ischemic myocardium; thus a possible mechanism for the decrease in resting coronary venous oxygen tension with adenosine receptor blockade is that low tonic nonvasoactive levels of adenosine inhibit the release of the vasoconstrictor peptide endothelin. Therefore, blockade of endothelial cell adenosine receptors would lead to the release of endothelin. This idea was tested by pretreatment with an endothelin receptor-blocking agent (Ro-61-0612, 1 mg/kg iv), but this was ineffective (Fig. 8).
A decrease in resting coronary venous oxygen tension with adenosine receptor blockade was observed by Edlund et al. (8, 9), using theophylline in humans, and in previous experiments from this laboratory, using 8-PT in dogs (31, 34). Duncker et al. (6) also observed a decrease in coronary venous oxygen tension with 8-PT in pigs. Unexpectedly, Bache et al. (1) did not observe a significant decrease in resting canine coronary venous oxygen tension using a larger dose of 8-PT (5 mg/kg iv) than in the present experiments (3 mg/kg iv).
The simple interpretation of a decrease in resting coronary venous oxygen tension with adenosine receptor blockade and a decrease parallel to control during exercise is that there is resting adenosine-mediated coronary vasodilation but no additional adenosine release during exercise. This simple interpretation seems unlikely because of experiments where adenosine deaminase was used to decrease cardiac adenosine levels. Kroll and Feigl (20), Merrill et al. (25), and Mallet et al. (22) each used intracoronary infusions of adenosine deaminase in unstressed dogs and found no decrease in coronary venous oxygen tension.
Thus when theophylline or its derivatives 8-PT and 8-PST are used to block adenosine receptors, a fall in coronary venous oxygen tension is observed, but this effect is not observed when adenosine deaminase is used to lower cardiac adenosine levels. This indicates that theophylline and its 8-PT and 8-PST derivatives have an action in addition to blocking adenosine receptors coupled to coronary vasodilation.
The Bache group (7) observed that intracoronary infusion of the ATP-sensitive potassium (K+ATP) channel inhibitor glibenclamide lowered coronary venous oxygen tension at rest and during exercise and that the addition of adenosine receptor blockade with 8-PT (5 mg/kg iv) further lowered coronary venous oxygen tension during rest and exercise. From these observations and the previous negative finding with 8-PT by Bache et al. (1), they concluded that an increase in adenosine levels compensates for the loss of K+ATP function. The present experiments are focused on the role of adenosine during normal exercise without K+ATP channel inhibition and did not test the role of adenosine after glibenclamide.
In conclusion, coronary venous plasma adenosine concentration and estimated interstitial adenosine concentration changed little during exercise that increased myocardial oxygen consumption more than fourfold above resting values. Adenosine receptor blockade with 8-PT or 8-PST did not blunt coronary vasodilation during exercise. Adenosine receptor blockade did not augment coronary venous or estimated interstitial adenosine concentration to overcome the blockade as would be predicted if adenosine were part of a high-gain, negative feedback local metabolic control mechanism. Therefore, the present results do not support the hypothesis that adenosine is responsible for local metabolic control of coronary blood flow during exercise.
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ACKNOWLEDGEMENTS |
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We thank Pamela Campbell for expert technical and editorial assistance in all phases of this research. The endothelin-blocking agent Ro-61-0612 was generously provided by Dr. Jean-Paul Clozel of Actelion Pharmaceuticals. We thank Dr. William Chilian for the suggestion that tonic low levels of adenosine might have inhibited endothelial cell endothelin release.
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FOOTNOTES |
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This study was supported by National Institutes of Health Grants RR-01243, HL-49822, and HL-07403.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: E. O. Feigl, Dept. of Physiology and Biophysics, University of Washington School of Medicine, Box 357290, Seattle, WA 98195-7290.
Received 8 June 1999; accepted in final form 29 July 1999.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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