INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

IN 1963 Berne (4) and Gerlach et al. (9) proposed that adenosine mediates local metabolic control of coronary blood flow, whereby coronary flow and myocardial oxygen consumption are kept in balance. The hypothesis is that adenosine released from cardiomyocytes crosses the interstitial space to activate adenosine receptors on coronary vascular smooth muscle cells and cause vasodilation. The postulate is that an increase in myocardial oxygen consumption produces an incipient fall in myocardial oxygen tension that augments adenosine release from cardiac cells and that the ensuing increase in oxygen delivery via increased coronary flow returns the oxygen tension back toward the operating point in a negative-feedback manner.

The increase in myocardial oxygen consumption secondary to pacing tachycardia represents a relatively "pure" local metabolic vasodilator stimulus to the coronary circulation. In contrast to catecholamine infusions, pacing tachycardia avoids the direct vascular effects of both -adrenoceptor-mediated vasoconstriction (7) and -adrenoceptor-mediated vasodilation (30). Furthermore, there is evidence for a link between catecholamines and cardiac adenosine release separate from changes in myocardial oxygen consumption (17, 19, 28, 42, 44).

Accordingly, an experiment was designed in which myocardial oxygen consumption was increased by cardiac pacing in the presence of -adrenergic receptor blockade. The adenosine hypothesis was tested with arterial and coronary venous plasma adenosine measurements before and during adenosine-receptor blockade. Interstitial adenosine concentration (estimated with a distributed mathematical model) did not reach vasoactive levels before or during adenosine-receptor blockade, indicating that adenosine is not important in local metabolic control of coronary blood flow.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

General preparation. Adult male mongrel dogs (n = 10) weighing 22-29 kg were initially sedated with morphine sulfate (3 mg/kg sc). One hour later each dog was fully anesthetized with -chloralose (100 mg/kg iv; Sigma). An additional dose of morphine (40 mg im) was given, and anesthesia was supplemented with 500 mg of -chloralose as needed. The metabolic acidosis associated with this anesthesia was corrected with a continuous intravenous infusion of NaHCO₃ and additional bolus injections as needed. Animals were intubated and ventilated with a volume displacement ventilator (model 607; Harvard Apparatus, South Natick, MA) using oxygen-enriched room air so that arterial oxygen tension was maintained >100 mmHg. End-expiratory CO₂ fraction was monitored continuously (model LB-2; Beckman Instruments, Fullerton, CA) and kept near 5% with appropriate adjustments in the ventilatory rate. A segment of Silastic tubing was placed in the left femoral artery for obtaining arterial samples from freely flowing blood. Arterial blood samples were taken periodically and analyzed (model 1306; Instrumentation Laboratories, Waltham, MA) for pH, PCO₂, PO₂, and base excess. Core temperature was monitored with an esophageal thermistor and controlled at 37°C with a YSI controller (model 73A; Yellow Springs, OH) and heating pads placed under the animal. Blood pressure was measured with a strain-gauge manometer (Statham P23 ID; Gould, Cleveland, OH) (Fig. 1). A catheter-tip manometer (model SPC-350; Millar Instruments, Houston, TX) was placed in the left ventricle via the right femoral artery. A catheter was introduced into the abdominal vena cava via the right femoral vein so that, during its intravenous infusion, 8-phenyltheophylline (8-PT) was immediately diluted in a large volume of flowing blood. Heparin sodium (750 U/kg iv) was administered to prevent coagulation. Ibuprofen (12.5 mg/kg iv) was given to inhibit the formation of cyclooxygenase products, which could have been released as a result of complement and white cell activation caused by the blood flow through artificial tubing. Propranolol (0.5 mg/kg iv) was administered to obtain a low initial heart rate and prevent any reflex changes in catecholamines from altering myocardial oxygen consumption.

View larger version (34K): [in this window] [in a new window]   Fig. 1.   Schematic diagram of experimental preparation. Coronary blood flow was measured with a Doppler flowmeter wedged in left circumflex coronary artery. A cardiac pacing electrode was placed in apex of right ventricle via jugular vein. Arterial samples were drawn from femoral artery, and coronary venous samples were drawn from a Sones catheter inserted into coronary sinus via right jugular vein. Rt, right; vent, ventricular.

Coronary sinus cannulation and pacing catheter. A shortened Sones catheter was inserted into the right external jugular vein and advanced into the coronary sinus with the aid of a fluoroscope (Fig. 1). A metal ring surrounded the tip of the catheter to prevent venous collapse during sampling. Placement of the catheter was confirmed by injection of contrast medium, measurement of coronary sinus PO₂, and anatomic location post mortem. To avoid right atrial admixture, the catheter tip was always placed at least 34 mm inside the coronary sinus ostium (21). Blood samples drawn from the coronary sinus catheter were analyzed for PO₂, PCO₂, pH, oxygen content, lactate concentration, and plasma adenosine concentration. Coronary venous samples were drawn at ~15 ml/min through the catheter, which had an internal volume of 0.55 ml; thus the transit time through the catheter was ~2.2 s. A pacing catheter (USCI, Billerica, MA) was placed in the right ventricle via the right jugular vein.

Circumflex coronary artery blood flow. The circumflex coronary artery was cannulated, closed chest, with a wedge-tip stainless steel cannulating flowmeter inserted via the right carotid artery (38). Blood entered the cannula from the ascending aorta and flowed past Doppler ultrasound crystals and into the circumflex coronary artery. The seal between the cannula tip and the circumflex artery was verified by injecting 10 µg of nitroglycerin dissolved in 0.5 ml of saline down a side tube that opened to the outside of cannula just proximal to the cannula tip. With a satisfactory seal, no increase in circumflex flow was observed, indicating that the nitroglycerin was unable to reach the circumflex bed across the wedged cannula tip. At the end of each experiment, crystal violet dye suspended in 10% NH₄OH was injected into the cannula flowmeter and the weight of the stained tissue was used to calculate the flow per gram of perfused myocardium. The flowmeter was calibrated at the end of each experiment by timed volume collections of the animal's blood pumped through the flowmeter.

Lactate measurement. Arterial and coronary venous blood samples were drawn into syringes, immediately transferred into NaF-coated vials, and placed on ice to prevent glycolysis. Lactate concentration was determined with a YSI lactate analyzer (model 23A). The machine was calibrated with standards before and after each experiment. Percent myocardial lactate extraction was calculated as [(arterial concentration  coronary venous concentration)/arterial concentration] × 100.

Oxygen content measurement. Arterial and coronary venous blood samples were drawn anaerobically into chilled glass syringes and placed on ice. Blood samples were analyzed using the fuel-cell method (Lex-O₂-Con; Hospex, Chestnut Hill, MA). Myocardial oxygen consumption (µl O₂ · min¹ · g myocardium¹) was calculated by multiplying the coronary blood flow per gram by the coronary arteriovenous oxygen content difference.

Plasma adenosine measurements. Paired arterial and coronary venous adenosine measurements were made at each time point. Plasma adenosine concentration was measured with a modified version of the method of Herrmann and Feigl (18). Blood samples (3.7 ml) were collected with a two-syringe arrangement that simultaneously mixed ice-cold enzymatic stop solution (5.0 ml) with the blood to prevent metabolism of adenosine (32). The stop solution contained dipyridamole (32 µM), iodotubercidin (ITC; 1 µM), and erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA; 10 µM). Dipyridamole acts as an inhibitor of cellular adenosine uptake. ITC is an inhibitor of adenosine kinase, preventing the incorporation of adenosine into AMP. EHNA is an inhibitor of adenosine deaminase, preventing the degradation of adenosine to inosine. Theophylline (20 µM) was included in the stop solution as an internal recovery standard. Blood samples were centrifuged at 15,000 revolutions per minute (rpm) and 0°C for 2 min, and 5 ml of the supernatant plasma were added to 1.8 ml of 4 N perchloric acid to precipitate plasma proteins. The sample was again centrifuged at 15,000 rpm and 0°C for 10 min. The acid supernatant (5 ml) was then added to 4.35 ml of a neutralizing solution containing 0.4 mM KH₃PO₄ and 0.8 mM KOH. The resultant 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 40% methanol. The samples were then concentrated by evaporation and resuspended in 200 µl of HPLC buffer. The adenosine in each sample was separated on a Hewlett-Packard 1090M HPLC with a C-18 column using an ion-pairing buffer solution of tetrabutylammonium hydrogen sulfate and KH₃PO₄ with an acetonitrile gradient. Adenosine content was determined by comparison with known adenosine standards, and plasma concentration was calculated using the hematocrit, accounting for dilution steps in sample handling, and was normalized for recovery with the theophylline standard in each sample.

Estimation of interstitial adenosine concentration. Estimates of interstitial adenosine concentration were made using a four-region (capillary, endothelial cell, interstitial space, and parenchymal cell) axially distributed mathematical model (22, 25, 39). The model describes the effects of blood flow, adenosine transport and exchange between tissue regions, and cellular production and consumption on the relations 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 (25, 39). Briefly, the model accounts for myocardial flow heterogeneity and for the change in heterogeneity that occurs with changes in blood flow. Also, the model is constrained with previously obtained estimates of capillary adenosine transport and metabolism adjusted for the level of flow. The estimate of interstitial adenosine concentration is obtained by using the measured values of coronary blood flow, hematocrit (to obtain coronary plasma flow), and arterial plasma adenosine concentration. Cellular adenosine production in the model is adjusted iteratively until the model coronary venous plasma adenosine concentration equals the experimentally measured venous concentration. Estimates of interstitial adenosine were made during baseline conditions and during paired pacing before and after administration of 8-PT.

Experimental protocol. The role of adenosine in the control of coronary blood flow during paired pacing was studied by comparing hemodynamic, metabolic, and adenosine data before and after treatment with the adenosine-receptor antagonist 8-PT (3 mg/kg iv). This dose of 8-PT was previously shown to shift the adenosine coronary vasodilation dose-response curve 12-fold to the right (39). Initially, two sets of arterial and coronary venous blood samples were drawn at the spontaneous heart rate without electrical pacing. These samples are reported as basal. Next, paired ventricular pacing was begun at a heart rate of 120 beats/min. The interval between the two paired stimuli was adjusted while the electrocardiogram and left ventricular pressure were monitored to obtain two depolarizations with a single left ventricular contraction (Fig. 2). During pacing, cardiac carbon dioxide production increases, and the ventilation rate was increased to keep end-expiratory carbon dioxide, and thus arterial PCO₂, as constant as possible. Arterial and coronary venous blood samples were drawn 60, 180, and 300 s after stable paired pacing was achieved. After the 5-min samples were taken, the animal was allowed to return to basal conditions. 8-PT (3 mg/kg iv) was then infused via a catheter in the abdominal vena cava over 10 min with a syringe pump. After an additional 10 min, the same sampling protocol was repeated. At each time point, heart rate, arterial blood pressure, and coronary blood flow were recorded.

View larger version (39K): [in this window] [in a new window]   Fig. 2.   Left: example of original data recordings of coronary blood flow, mean coronary blood flow, electrocardiogram (ECG), and left ventricular pressure from 1 dog. Right: during paired pacing, delay between electrical pulses was adjusted so that 2 depolarizations (ECG trace) resulted in a single fused cardiac contraction.

Drugs. -Chloralose (Sigma) was dissolved in warm 0.9% saline. Heparin (SoloPak Laboratories, Franklin Park, IL) was given as an intravenous bolus dose of 750 U/kg. Ibuprofen (Sigma) was dissolved in 0.2 M NaHCO₃ at a concentration of 25 mg/ml (pH was adjusted to 7.5-8.0 with 1 N HCl) and given as an intravenous bolus dose of 12.5 mg/kg. Propranolol (Sigma) was dissolved in 0.9% saline and given as an intravenous bolus dose of 0.5 mg/kg. 8-PT (3 mg/kg; Sigma) was placed in 1.5 ml of equal parts 1 N NaOH, ethanol, and propylene glycol and gently warmed until dissolved. Final volume was adjusted to 30 ml with warm 0.9% saline. The adenosine stop solution was made in cold isotonic saline and included 1 µM ITC (Research Biochemicals), 10 µM EHNA (Sigma), 32 µM dipyridamole (Sigma), and 20 µM theophylline (Sigma).

Data analysis. Hemodynamic variables were recorded with Windaq data analysis software (Dataq Instruments). 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, heart rate, and mean aortic pressure were averaged over a 15-s period for baseline and paired-pulse stimulation time points.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Data tracings from one dog appear in Fig. 2. Hemodynamic and metabolic data for all 10 dogs are summarized in Table 1. During control conditions (before 8-PT), there was a 92% increase in coronary blood flow during the first minute of cardiac paired pacing (Fig. 3A). During adenosine-receptor blockade with 8-PT (3 mg/kg), coronary blood flow was not altered during baseline conditions and increased by 81% in response to pacing. Control myocardial oxygen consumption increased 104% during the first minute of pacing (Fig. 3B). After 8-PT administration, baseline myocardial oxygen consumption was not significantly different from that during control conditions and increased 94% during the first minute of pacing. Mean baseline heart rate during control conditions was 55 ± 4 beats/min, rising to a baseline value of 62 ± 4 beats/min after 8-PT (Fig. 3C). Mean aortic blood pressure was little changed throughout the experiment (Fig. 3D).

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Hemodynamic and metabolic responses to pacing before and after adenosine-receptor blockade with 8-phenyltheophylline (8-PT). Cardiac pacing increased coronary blood flow (A), myocardial oxygen consumption (B), and heart rate (C) similarly before and after 8-PT. Cardiac pacing had little effect on mean arterial blood pressure (D) during control and after 8-PT. P values are from a Student-Newman-Keuls test after Bonferroni adjustment for 10 variables. B, baseline measurement.

During control conditions the baseline coronary venous PO₂ was 20 ± 2 mmHg, and after 8-PT the baseline value fell to 17 ± 1 mmHg (P < 0.05) (Fig. 4A). Myocardial lactate extraction remained positive and >45% throughout the experiments, indicating that the myocardium was not ischemic (Fig. 4B). Ischemic myocardium is known to release large amounts of adenosine and would have confounded the adenosine results (24).

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Coronary venous PO2 and lactate extraction before and after adenosine-receptor blockade (8-PT). During control, coronary venous PO2 (A) and lactate extraction (B) were not significantly different before and after pacing. After 8-PT, venous PO2 was significantly lower than during control. P values are from a Student-Newman-Keuls test after Bonferroni adjustment for 10 variables. B, baseline measurement.

Arterial and venous plasma adenosine measurements and estimates of interstitial adenosine concentration are shown in Figs. 5 and 6. Arterial plasma adenosine concentration was little changed throughout the experiment (Fig. 5A). During control conditions, coronary venous plasma adenosine concentration increased from 16 nM during baseline conditions to 25 nM during the first minute of pacing, decreasing to 20 nM for the remainder of pacing. Correspondingly, the baseline interstitial adenosine concentration of 44 nM increased to 72 nM during the first minute (Fig. 6) but remained below the threshold concentration necessary for coronary vasodilation (Fig. 7) (39). After adenosine-receptor blockade with 8-PT, baseline adenosine levels were not significantly different from that of control (Fig. 6). The absence of an increase in adenosine concentration after adenosine-receptor blockade does not support the hypothesis that adenosine levels rise to overcome receptor blockade.

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Arterial and coronary venous plasma adenosine concentration during control and after adenosine-receptor blockade (8-PT). There was no change in arterial plasma adenosine concentration (A) with pacing during control protocol. There was a small but significant increase in venous adenosine concentration (B) 1 min after start of pacing but not at 3 or 5 min. Arterial and venous adenosine concentrations were unchanged after 8-PT for both baseline (B) and pacing period. P values are from a Student-Newman-Keuls test after Bonferroni adjustment for 10 variables.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Effects of pacing and adenosine-receptor blockade (8-PT) on estimated interstitial adenosine concentration. Interstitial adenosine during baseline (B) was not affected by 8-PT. Pacing-induced increase in myocardial oxygen consumption had little effect on interstitial adenosine concentration, except for an increase during control conditions at 1-min time point. This concentration is below the threshold required for coronary vasodilation (see Fig. 7). P values are from a Student-Newman-Keuls test after Bonferroni adjustment for 10 variables.

View larger version (21K): [in this window] [in a new window]   Fig. 7.   Coronary blood flow plotted against estimated interstitial adenosine concentrations during control conditions (A) and after adenosine-receptor blockade (8-PT; B). Line plot in A is dose-response curve from Stepp et al. (39) aligned vertically so that curve intersects baseline points. During control pacing, interstitial adenosine concentration is well below the level necessary to evoke vasodilation, with data falling to left of dose-response curve. Line plot in B indicates how dose-response curve is shifted 12-fold by adenosine-receptor blockade with 8-PT (39) (note 10-fold increase in scale of x-axis). After 8-PT, interstitial adenosine concentration is more than an order of magnitude below that required to elicit observed increase in flow.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The major finding of this study is that adenosine is not essential for local metabolic control of coronary blood flow. Paired-pulse cardiac stimulation in the presence of -adrenoceptor blockade was used to augment myocardial oxygen consumption and obtain local metabolic coronary vasodilation without adrenergic activation. This experimental design was used for local metabolic control, because previous studies suggest a link between catecholamines and adenosine levels inde-pendent of myocardial oxygen consumption (17, 19, 28, 42, 44). The adenosine results in the present study were compared with a previously determined exogenous and endogenous adenosine dose-response curve for the in vivo dog heart (39), and estimated interstitial adenosine did not reach vasoactive concentrations either during control pacing or during pacing after adenosine-receptor blockade by 8-PT. An important new finding is that, after adenosine-receptor blockade, neither coronary venous nor estimated interstitial adenosine levels increased to overcome the blockade, as has been previously suggested (29).

Effect of paired-pulse ventricular pacing. The primary purpose in using the pacing stimulus was to augment myocardial contractility and oxygen consumption (2, 34). Paired-pulse stimulation has an advantage over single-pulse pacing in that larger increases in myocardial oxygen consumption are possible. The two closely spaced electrical pulses cause two action potentials per myocardial contraction and thus augment contractility via increased intracellular calcium concentration. This stimulus, when combined with the -adrenergic blocking agent propranolol, provided the means for achieving increases in coronary blood flow in response to a local metabolic stimulus without catecholamine effects.

Estimates of interstitial adenosine concentration. Simple inspection of the coronary venous plasma adenosine concentration during paired pacing shown in Fig. 5 indicates little or no role for adenosine in local metabolic vasodilation. However, there was a transient increase in coronary venous plasma concentration after 1 min of paired pacing during control conditions before adenosine-receptor blockade. Thus it will be useful to determine whether this reflects a vasoactive interstitial adenosine concentration.

Adenosine hypothesis with adenosine-receptor blockade. Adenosine-receptor blockade has been used in previous studies with cardiac pacing but has not been combined with adenosine concentration measurements. Two studies found a diminished coronary flow response after adenosine-receptor blockade with aminophylline (26, 33), and a diminished response was found with 8-p-sulfophenyltheophylline (27). A study using aminophylline in humans failed to demonstrate a decrement in the coronary flow response to cardiac pacing (35).