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Am J Physiol Heart Circ Physiol 280: H868-H875, 2001;
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Vol. 280, Issue 2, H868-H875, February 2001

KATP+ channels, nitric oxide, and adenosine are not required for local metabolic coronary vasodilation

Johnathan D. Tune, Keith Neu Richmond, Mark W. Gorman, and Eric O. Feigl

Department of Physiology and Biophysics, University of Washington School of Medicine, Seattle, Washington 98195-7290


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The role of ATP-sensitive K+ (KATP+) channels, nitric oxide, and adenosine in coronary exercise hyperemia was investigated. Dogs (n = 10) were chronically instrumented with catheters in the aorta and coronary sinus and instrumented with a flow transducer on the circumflex coronary artery. Cardiac interstitial adenosine concentration was estimated from arterial and coronary venous plasma concentrations using a previously tested mathematical model. Experiments were conducted at rest and during graded treadmill exercise with and without combined inhibition of KATP+ channels (glibenclamide, 1 mg/kg iv), nitric oxide synthesis (Nomega -nitro-L-arginine, 35 mg/kg iv), and adenosine receptors (8-phenyltheophylline, 3 mg/kg iv). During control exercise, myocardial oxygen consumption increased ~2.9-fold, coronary blood flow increased ~2.6-fold, and coronary venous oxygen tension decreased from 19.9 ± 0.4 to 13.7 ± 0.6 mmHg. Triple blockade did not significantly change the myocardial oxygen consumption or coronary blood flow response during exercise but lowered the resting coronary venous oxygen tension to 10.0 ± 0.4 mmHg and during exercise to 6.2 ± 0.5 mmHg. Cardiac adenosine levels did not increase sufficiently to overcome the adenosine receptor blockade. These results indicate that combined inhibition of KATP+ channels, nitric oxide synthesis, and adenosine receptors lowers the balance between total oxygen supply and consumption at rest but that these factors are not required for local metabolic coronary vasodilation during exercise.

coronary blood flow; exercise; myocardial oxygen consumption


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A CURRENT HYPOTHESIS is that there are multiple mechanisms responsible for local metabolic coronary vasodilation, and that when one mechanism is inhibited, another may increase in compensation (7, 11, 15, 26). It has been suggested that adenosine levels increase to mediate coronary vasodilation when either ATP-sensitive K+ (KATP+) channels or nitric oxide synthesis is inhibited at rest (6, 7, 11, 15, 16, 26), during pacing tachycardia (15, 26), and during exercise (6, 7, 11). The evidence suggesting that adenosine compensates for the lack of KATP+ channel or nitric oxide function was a decrease in coronary blood flow and/or coronary venous oxygen tension when adenosine receptor blockade was added to an KATP+ channel antagonist or a nitric oxide synthesis inhibitor. These results are confusing because it is well established that KATP+ channel blockade also inhibits adenosine coronary vasodilation (1, 2, 4-7, 17, 19, 23). In addition, interpretation of these results is compromised by the absence of adenosine measurements.

Ishibashi et al. (11) recently observed that coronary hyperemia and myocardial oxygen consumption were severely limited during exercise with triple blockade of KATP+ channels, nitric oxide synthesis, and adenosine receptors. They concluded that KATP+ channels are the main controller of metabolic coronary vasodilation, but when these channels are inhibited, adenosine and nitric oxide levels increase to compensate for the loss of KATP+ channel function.

The present study was designed to reexamine the hypothesis of multiple compensating mechanisms of local metabolic coronary control by combining adenosine measurements with simultaneous inhibition of KATP+ channels, nitric oxide synthesis, and adenosine receptors during exercise. Cardiac interstitial adenosine concentration was estimated from arterial and coronary venous plasma measurements using a previously described mathematical model to determine whether adenosine levels increased sufficiently to overcome the adenosine receptor blockade. The present results indicate that inhibition of KATP+ channels, nitric oxide synthesis, and adenosine receptors lowers the balance between myocardial oxygen supply and consumption at rest, but that these mechanisms are not required for coronary vasodilation during exercise. Furthermore, adenosine levels do not increase sufficiently to mediate a compensatory coronary vasodilation when these mechanisms are inhibited.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Surgical preparation. Experiments were performed on 10 adult male mongrel dogs (body weight, 23-32 kg) taught to run on a motorized treadmill. The surgical procedures performed in the present study were previously described by Tune et al. (28). Briefly, a splenectomy was performed through a midline abdominal incision to minimize changes in hematocrit during exercise. After this procedure, a left lateral thoracotomy was performed in the fifth intercostal space. With the use of a modified Seldinger technique, a polyurethane catheter was implanted into the descending thoracic aorta to measure aortic blood pressure and obtain arterial blood samples. A second polyurethane catheter 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. No instruments were implanted in the myocardium, and no surgical stitches were placed in the ventricles to avoid injuring tissue that might release adenosine. The animals were allowed at least 10 days for recovery before experiments were conducted.

Pressure and flow measurement. A 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 (8, 10). 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 transit time, perivascular flow transducer (Transonics; Ithaca, NY). The flow transducers were calibrated before and after chronic implantation. The average difference between the before and after 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 (in µl O2 · min-1 · g-1) 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. Plasma adenosine concentration was measured as previously described by Tune et al. (28). Briefly, 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 (18). The stop solution contained dipyridamole (32 µM), iodotubercidin (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. Iodotubercidin 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. 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 then purified by applying the neutralized supernatant to C-18 Sep-Pak cartridges. Each sample was divided into two 100-µl aliquots, and adenosine deaminase (0.1 U, Boeringer) was added to one of the aliquots, which was used as a paired blank.

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). 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 pmol of adenosine, which is equivalent to approximately a 5.5 nM concentration in plasma. The recovery of 100 pmol 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 (12, 14, 24). 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 (14, 24). 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.

Experimental protocol. The hypothesis of multiple compensating mechanisms of local metabolic coronary control was examined at rest and during graded treadmill exercise with and without triple inhibition of KATP+ channels (glibenclamide, 1 mg/kg iv) (Sigma; St. Louis, MO), nitric oxide synthesis [Nomega -nitro-L-arginine (L-NNA), 35 mg/kg iv] (Sigma), and adenosine receptors [8-phenyltheophylline (8-PT), 3 mg/kg iv] (Sigma). Each animal served as its own control. The dose of glibenclamide (1 mg/kg iv) used in this investigation was previously found to effectively block the vasodilating action of the KATP+ channel opener cromakalim and to shift the coronary blood flow dose-response curve to intracoronary infusions of adenosine 10-fold to the right (23). The dose of 8-PT (3 mg/kg iv) was previously found to shift the coronary blood flow dose-response curve to intracoronary infusion of adenosine to the right 12-fold and at least 12-fold for endogenous adenosine (24). The dose of L-NNA (35 mg/kg iv) was previously found to reduce vasodilation to acetylcholine by >60% in conscious dogs (3, 22, 29) and lower coronary venous oxygen tension during exercise (3, 27).

Coronary blood flow, aortic pressure, and heart rate were continuously measured while the dogs were resting in a sling and during three levels of treadmill exercise: 1) 3 mph, 5% grade; 2) 4 mph, 10% grade; and 3) 5 mph, 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 until hemodynamic variables returned to baseline. Control vehicle and triple blockade experiments were conducted on separate days, and the animals were allowed at least 2 days of recovery between experiments.

Drugs. Both glibenclamide (1 mg/kg iv) and 8-PT (3 mg/kg iv) (Sigma) were placed in 1.5-ml equal parts of 1 N NaOH, ethanol, and propylene glycol and then gently warmed until dissolved. The final volumes were adjusted to 30 ml with 5.0% glucose (glibenclamide) or saline (8-PT). L-NNA (35 mg/kg iv) was dissolved in 120 ml of 0.9% saline. All drugs were infused intravenously over a 10-min period. The adenosine stop solution was made in isotonic saline and included 1 µM iodotubercidin (RBI; Natick, MA), 10 µM EHNA (RBI), 32 µM dipyridamole (Sigma), and 20 µM theophylline (Sigma).

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 and coronary venous oxygen tension), statistical testing was directed to overall treatment effects (control vehicle vs. triple blockade). These tests were chosen for their specificity to the hypotheses and to avoid inappropriate multiple comparisons. All analyses accounted for the effects of drug and dog. Multiple linear regression was used to compare slopes of the two treatments for the response variables versus myocardial oxygen consumption relation (SAS, proc glm). Analysis of covariance (ANCOVA) was employed to adjust the response variables for linear dependence on myocardial oxygen consumption after testing for parallel regression lines (SAS, proc glm). 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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  		Table 1.   Hemodynamic and metabolic variables at rest and during graded treadmill exercise


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Hemodynamic and metabolic data for the 10 dogs are given in Table 1. Resting values before vehicle or drug administration are presented in Table 1 but were not included in the statistical analysis of drug and exercise effects. Figure 1 shows the resting and exercise results for heart rate and mean aortic pressure with and without triple blockade of KATP+ channels, nitric oxide synthesis, and adenosine receptors. Systemic administration of L-NNA resulted in a significant increase in mean aortic pressure (Fig. 1B) and a significant decrease in heart rate (Fig. 1A), consistent with previous studies (3, 22, 27, 29).


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  		Fig. 1.   Relationship between heart rate (A) and mean aortic pressure (B) vs. myocardial oxygen consumption with and without triple inhibition of ATP-sensitive K+ (KATP+) channels, nitric oxide synthesis, and adenosine receptors. Triple blockade significantly increased mean aortic pressure (B) (ANCOVA, P < 0.001) and significantly decreased heart rate (A) (ANCOVA, P < 0.001) at rest and during exercise. This increase in blood pressure and reflex reduction of heart rate were most likely the result of systemic administration of Nomega -nitro-L-arginine (L-NNA) (3, 22, 27, 29).

During control vehicle, myocardial oxygen consumption increased ~2.9-fold and coronary blood flow increased ~2.6-fold from rest to the highest level of exercise (Fig. 2). The relationship between coronary blood flow and myocardial oxygen consumption was shifted downward in a parallel manner by triple blockade (P < 0.001). Triple blockade decreased coronary venous oxygen tension (P < 0.001) at rest and during exercise, but the slope of the relationship between coronary venous oxygen tension and myocardial oxygen consumption did not become more negative (Fig. 3). Furthermore, triple blockade did not limit myocardial oxygen consumption (Table 1). These findings indicate that inhibition of KATP+ channels, nitric oxide synthesis, and adenosine receptors lowers the balance between myocardial oxygen supply and consumption at rest, but that these factors are not required for coronary vasodilation during exercise.


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  		Fig. 2.   Relationship between coronary blood flow and myocardial oxygen consumption with and without triple inhibition of KATP+ channels, nitric oxide synthesis, and adenosine receptors. Triple blockade shifted the relationship downward in a parallel manner (ANCOVA, P < 0.001) but did not attenuate the increase in myocardial oxygen consumption during exercise. These findings indicate that KATP+ channels, nitric oxide, and adenosine are not required for local metabolic coronary vasodilation during exercise.



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  		Fig. 3.   Relationship between coronary venous oxygen tension and myocardial oxygen consumption with and without triple inhibition of KATP+ channels, nitric oxide synthesis, and adenosine receptors. Triple blockade significantly decreased coronary venous oxygen tension at rest and during exercise in a parallel manner (ANCOVA, P < 0.001) but did not make the slope more negtive. These findings indicate that inhibition of KATP+ channels, nitric oxide synthesis, and adenosine receptors lowers the balance between coronary blood flow and myocardial oxygen consumption at rest, but that these factors are not required for exercise coronary vasodilation.

The effects of myocardial oxygen consumption and coronary venous oxygen tension on coronary venous adenosine concentration are plotted in Fig. 4. Triple blockade increased coronary venous adenosine concentration as myocardial oxygen consumption was increased (Fig. 4A) and as coronary venous oxygen tension fell (Fig. 4B). However, the estimated interstitial adenosine concentration remained well below the threshold value necessary for coronary vasodilation with adenosine receptor blockade (Fig. 5). The estimated interstitial adenosine concentration would have had to increase at least 12-fold to reach vasoactive levels, because adenosine receptor blockade with 8-PT alone was previously found to shift the coronary blood flow dose-response curve to adenosine 12-fold to the right (24). It is likely that the adenosine dose-response curve would have been shifted even further to the right, because glibenclamide also inhibits adenosine-mediated coronary vasodilation (23). Therefore, although coronary venous and estimated interstitial adenosine concentrations were increased during exercise with triple blockade, they did not increase sufficiently to overcome the blockade.


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  		Fig. 4.   Effects of myocardial oxygen consumption (A) and coronary venous oxygen tension (B) on coronary venous plasma adenosine concentration with and without triple inhibition of KATP+ channels, nitric oxide synthesis, and adenosine receptors. Triple blockade increased coronary venous adenosine concentration as myocardial oxygen consumption was increased and as coronary venous oxygen tension was decreased, indicating that the myocardium became ischemic.



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  		Fig. 5.   Effects of myocardial oxygen consumption (A) and coronary venous oxygen tension (B) on estimated interstitial adenosine concentration with and without triple inhibition of KATP+ channels, nitric oxide synthesis, and adenosine receptors. Triple blockade increased the estimated interstitial adenosine concentration as myocardial oxygen consumption was increased and as coronary venous oxygen tension was decreased. However, the estimated interstitial adenosine concentration remained well below the threshold value necessary for coronary vasodilation in the presence of adenosine receptor blockade. 8-PT, 8-phenyltheophylline.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study is the first to combine plasma adenosine measurements with triple inhibition of KATP+ channels, nitric oxide synthesis, and adenosine receptors. Although inhibition of these factors decreased the balance between myocardial oxygen supply and consumption at rest (Figs. 2 and 3), it did not make the relationship between coronary venous oxygen tension and myocardial oxygen consumption more negative (Fig. 3). Therefore KATP+ channels, nitric oxide, and adenosine do not act as local metabolic vasodilators in the usual sense of coupling coronary blood flow to myocardial oxygen consumption. Triple blockade increased coronary venous and estimated interstitial adenosine concentrations as myocardial oxygen consumption increased and coronary venous oxygen tension decreased, but the adenosine levels did not increase sufficiently to overcome the adenosine receptor blockade (Figs. 4 and 5). The present results indicate that KATP+ channels, nitric oxide, and adenosine are not required for local metabolic coronary vasodilation during brief (~2 min) exercise.

Relationship between coronary blood flow and myocardial oxygen consumption. Under normal physiological conditions, coronary blood flow is closely matched with the myocardial metabolic rate (9). The ability of the coronary circulation to adjust flow to meet the oxygen requirements of the tissue is extremely important due to the limited oxygen extraction reserve of the heart. A sensitive way to determine the relationship between coronary blood flow and myocardial metabolism is the plot of coronary venous oxygen tension versus myocardial oxygen consumption (Fig. 3). Coronary venous oxygen tension is an index of tissue oxygenation that reflects the balance between coronary oxygen delivery and consumption. Influences on baseline flow will shift the relationship in a parallel manner, whereas an intervention that affects local metabolic coronary vasodilation will change the slope. In the present study, the small but significant parallel downward shift in coronary blood flow versus myocardial oxygen consumption (Fig. 2) with triple blockade resulted in a similar significant parallel downward shift in the coronary venous oxygen tension versus myocardial oxygen consumption relationship shown in Fig. 3. However, the slope did not become more negative, indicating that KATP+ channels, nitric oxide, and adenosine are not required for local metabolic control. Furthermore, triple blockade did not limit myocardial oxygen consumption (Table 1), as might be expected if local metabolic vasodilation were attenuated.

Postulated multiple mechanisms of metabolic coronary vasodilation. Several studies have postulated that KATP+ channels, nitric oxide, and adenosine represent multiple redundant mechanisms of local metabolic coronary vasodilation at rest (6, 7, 11, 15, 16, 26) or during increases in myocardial oxygen consumption with either cardiac pacing (15, 26) or exercise (6, 7, 11). The reason for postulating multiple compensating mechanisms was the observation that the addition of an adenosine receptor antagonist to a prior nitric oxide synthesis inhibition (L-NNA) (15, 26) or a prior KATP+ channel inhibition (glibenclamide) (6, 7, 11) decreased coronary blood flow and/or decreased the relation between coronary venous oxygen tension and myocardial oxygen consumption. The interpretation was that adenosine increased to compensate for the loss of nitric oxide or KATP+ channel vasodilation. This laboratory recently found that inhibition of nitric oxide synthesis did not increase coronary venous plasma or estimated interstitial adenosine concentrations at rest or during exercise in dogs (27). In addition, the estimated interstitial adenosine concentration remained well below the threshold value for coronary vasodilation with or without nitric oxide synthesis inhibition, indicating that adenosine does not mediate a compensatory local metabolic coronary vasodilation when nitric oxide synthesis is inhibited.

The addition of adenosine receptor blockade to a KATP+ channel antagonist is puzzling, because it is well established that KATP+ channel blockade also inhibits adenosine coronary vasodilation (1, 2, 4-7, 17, 19, 23). Therefore, the previous KATP+ channel blockade would have already inhibited adenosine-mediated coronary vasodilation, and it is unclear why the addition of adenosine receptor blockade would make a difference unless massive amounts of adenosine were released from the myocardium to overcome the adenosine receptor blockade. As illustrated in Fig. 5, adenosine levels did not increase sufficiently to overcome the receptor blockade. Thus the present results do not support the hypothesis that KATP+ channels, nitric oxide, and adenosine act as multiple compensating local metabolic vasodilator mechanisms.

Results from the present investigation do not support the findings of Ishibashi et al. (11) in that triple blockade did not attenuate exercise coronary hyperemia (Fig. 2) or make the slope of the coronary venous oxygen tension versus myocardial oxygen consumption relationship more negative (Fig. 3). The major difference between these studies was that glibenclamide and L-NNA were infused intravenously in the present investigation and intracoronary infusion was used in the Ishibashi et al. study (11). Intravenous administration was chosen in preference to intracoronary infusion for several reasons. Intravenous infusion avoids direct intracoronary injection of the harsh alkaline vehicle that is required to get glibenclamide into solution. A 10-min intravenous infusion provides time for equilibration of the blocking agent. A continuous intracoronary infusion is not suitable for steady-state measurements because recirculation of the blocking agent would result in ever-increasing coronary concentrations. This loss of steady state might confound the measurement of myocardial oxygen consumption, a critical factor in these investigations. Aside from the methodological differences noted above, it is not clear what may account for difference between the present results and those of Ishibashi et al. (11). The important new element is that the present adenosine measurements do not support interpretation by Ishibashi et al. that adenosine compensates for the loss of KATP+ channel function.

The intravenous dose of glibenclamide (1 mg/kg) used in the present study is effective as demonstrated by a shift in the dose-response curve to the KATP+ channel opener cromakalim (23) and a decrease in coronary venous oxygen tension at rest and during exercise (20, 21). Duncker et al. (7) observed a decrease of ~2.5 mmHg in resting coronary venous oxygen tension using an intracoronary glibenclamide dose of 50 µg · kg-1 · min-1, compared with a resting 6-mmHg decrease observed with a 1 mg/kg intravenous dose (21), as also used in the present study. This indicates that the KATP+ channel blockade used in the two laboratories is comparable. Similarly, the intravenous dose of L-NNA (35 mg/kg) is effective as demonstrated by blocking cholinergic vasodilation (3, 22, 29) and by lowering coronary venous oxygen tension during exercise (3, 27).

Adenosine levels during triple blockade. The present results suggest that triple blockade results in incipient myocardial ischemia during exercise. During myocardial ischemia, intracellular (13) and extracellular (23) adenosine levels increase with extracellular adenosine concentration increasing before net cardiac lactate production occurs (23). Coronary venous (Fig. 4) and estimated interstitial (Fig. 5) adenosine concentrations increased when coronary venous oxygen tension fell to ~6 mmHg during exercise following triple blockade. The coronary venous oxygen tension of 6 mmHg is consistent with the critical oxygen tension of ~3 mmHg for adenosine release observed in isolated in vitro cardiac myocytes (25), remembering that there must be an in vivo oxygen tension gradient from blood to tissue for diffusion to occur. The likely interpretation is that triple blockade causes a decrease in resting coronary blood flow and that the added stress of exercise during triple blockade results in beginning subendocardial ischemia. Transmural coronary blood flow was not measured in the present experiments; thus the subendocardial ischemia suggested by the adenosine measurements was not documented by flow measurements.

The present findings are also consistent with earlier studies from this laboratory (21, 27, 28) in that during unblocked control conditions, coronary venous adenosine concentration is little changed with exercise and that the estimated interstitial adenosine concentration remains well below the threshold value for coronary vasodilation. These results indicate that adenosine is not responsible for local metabolic control of coronary blood flow during increases in myocardial oxygen consumption.

The estimate of interstitial adenosine concentration is dependent on a mathematical model that has been extensively tested (14). The model accounts for adenosine uptake by vascular endothelial cells, flow heterogeneity as a function of flow, and paracellular diffusion between the interstitium and the plasma through interendothelial gaps. The most sensitive parameter relating interstitial adenosine concentration to plasma concentration is the permeability-surface area product of interendothelial gaps (PSg). When a highly unlikely twofold decrease in PSg is modeled, the estimated interstitial adenosine concentration increases ~20% (nonlinear function). Such a 20% increase does not change any of the interpretations of the present experiments.

In conclusion and summary, triple inhibition of KATP+ channels, nitric oxide synthesis, and adenosine receptors decreased the balance between myocardial oxygen supply and consumption but did not make the relationship between coronary venous oxygen tension and myocardial oxygen consumption more negative. Therefore, KATP+ channels, nitric oxide, and adenosine do not act as local metabolic vasodilators in the usual sense of coupling coronary blood flow to myocardial oxygen consumption. Furthermore, adenosine levels do not increase sufficiently to mediate a compensatory coronary vasodilation when these mechanisms are inhibited. The present findings indicate that the factor(s) responsible for local metabolic coronary vasodilation during increases in myocardial oxygen consumption remain to be elucidated.


    ACKNOWLEDGEMENTS

We thank Pamela Campbell for expert technical assistance in all phases of this research. We also thank Julie Kleeberger for expert surgical assistance.


    FOOTNOTES

This study was supported by National Institutes of Health Grants HL-49822, HL-49170, HL-07403, RR-01243.

Address for reprint requests and other correspondence: E. O. Feigl, Dept. of Physiology and Biophysics, Univ. of Washington School of Medicine, Box 357290, Seattle, WA 98195-7290.

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. Section 1734 solely to indicate this fact.

Received 5 June 2000; accepted in final form 29 August 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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21.   Richmond, KN, Tune JD, Gorman MW, and Feigl EO. Role of KATP+ channels and adenosine in the control of coronary blood flow during exercise. J Appl Physiol 89: 529-536, 2000[Abstract/Free Full Text].

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Am J Physiol Heart Circ Physiol 280(2):H868-H875
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