Acute Closed-Chest Coronary Perfusion Preparation

Male dogs (23–27 kg) were premedicated with morphine (80 mg sc) and anesthetized with α-chloralose (100 mg/kg iv). Additional morphine (40 mg im) was given after 1 h, and chloralose (500 mg iv) was given approximately each hour. The animals were ventilated and given intravenous NaHCO₃ to maintain blood gases within normal limits. Aortic pressure was measured via a fluid-filled catheter introduced via the right femoral artery. Without opening the chest, a stainless steel cannula was advanced via the right carotid artery and wedged into the circumflex coronary artery (41). The circumflex coronary artery was perfused with heparinized (750 U/kg iv) blood from the left femoral artery. A servo-controlled pump (32) maintained circumflex coronary artery pressure constant at 100 mmHg while circumflex blood flow was measured with an inline ultrasonic transit-time flow transducer (Transonics, Ithaca, NY). Dogs were treated with ibuprofen (12.5 mg/kg iv) to retard pump-induced white cell activation and propranolol (1 mg/kg iv) to prevent tachycardia. Intracoronary bolus drug injections were made just proximal to the coronary cannula lumen. At the conclusion of experiments additional anesthetic was given, and the circumflex artery perfusion territory was stained with intracoronary crystal violet dye just before euthanasia with intravenous KCl. The stained myocardium was cut out and weighed. Coronary blood flow was normalized per gram of perfused myocardium.

Intracoronary ATP Dose-Response Experiments

The purpose of the acute closed-chest experiments was to determine the purinergic receptor types involved in adenine nucleotide coronary vasodilation using selective purinergic agents in the canine coronary circulation. A baseline ATP dose-response curve was obtained via bolus intracoronary ATP injections (50 μl, Hamilton syringe) at increasing concentrations. The response to each injection was defined as the peak increase in mean coronary blood flow above baseline. Coronary flow was allowed to return to baseline after each injection. To avoid irreversible changes in the preparation, ATP doses resulting in coronary flows greater than approximately five times baseline flow were not administered. Two dose-response curves were obtained in each animal. In experiments where the additive effect of multiple blocking agents was tested, one or two purinergic receptor antagonists were administered before the first ATP dose-response curve.

Conscious Exercising Dog Preparation

Experiments were performed on seven purpose-bred adult mongrel dogs (25–30 kg) taught to run on a motorized treadmill. The surgical procedures have been previously described by Tune et al. (43). Briefly, under isoflurane anesthesia, a left thoracotomy was performed in the fourth intercostal space. A modified Seldinger technique was used to implant a polyurethane catheter into the descending thoracic aorta to obtain arterial blood samples and pressure. A second polyurethane catheter for coronary venous blood sampling was placed in the coronary sinus via an incision in the right atrial appendage. A flow transducer was placed around the circumflex coronary artery. Antibiotic (cefazolin, 20 mg/kg iv) was administered 1 h before surgery and again during surgery. Pain was controlled via an intercostal nerve block in the second to sixth intercostal spaces (bupivacaine, up to 2 mg/kg) before incision. Postoperative pain was controlled with oxymorphone (1 mg/kg im), buprenorphine (0.02 mg/kg sc), and fentanyl (75 μg/h transdermal patch for 3 days). Dogs also received carprofen (2.2 mg/kg po) twice daily for 5 days after surgery. Beginning 24 h after surgery, dogs received daily doses of aspirin (81 mg po) and clopidogrel (75 mg po) to help maintain catheter patency. The arterial catheter was flushed daily and filled with heparin (1,000 U/ml). The coronary venous catheter was flushed continuously with heparinized saline (5 U/ml, 2 ml/h) via a balloon pump (I-Flow, Lake Forest, CA) stored in a jacket worn by the dogs (AKCMA, Hawthorne, CA). Initial experiments were conducted after a recovery period of at least 10 days following surgery.

Flow and Pressure Measurement

Coronary blood flow was continuously measured throughout the experimental protocol with an ultrasonic transit-time perivascular flow transducer (Transonics) calibrated before implantation. After experiments were completed, the animals were euthanized with pentobarbital sodium, and the circumflex artery 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. Arterial pressure was measured using a 3F catheter tip manometer (Millar Instruments, Houston, TX) inserted into the arterial catheter. Data were digitized and recorded continuously using Windaq software (Akron, OH).

Blood Sampling

Arterial and coronary venous blood samples were collected simultaneously during rest and exercise in heparinized glass syringes that were immediately sealed and placed in ice. The samples were analyzed for hydrogen ion concentration, carbon dioxide tension, and oxygen tension with an Instrumentation Laboratories Gem Premier 3000 blood gas analyzer (Waltham, MA). Arterial and coronary venous oxygen content was determined using the fuel-cell method (Total O₂X; Hospex, Chestnut Hill, MA). Myocardial oxygen consumption (in μl O₂·min⁻¹·g⁻¹) was calculated by multiplying mean coronary blood flow per gram of perfused tissue by the arterial-coronary venous difference in oxygen content. Hemoglobin saturation was calculated from the measured pH, Pco₂, and Po₂ using the formula for dog blood developed by Reeves et al. (37).

Exercise Protocol

Coronary blood flow, arterial pressure, and heart rate were continuously measured while the dogs were standing in a sling and during two levels of treadmill exercise, 1) 3 mph, 5% grade and 2) 4 mph, 10% grade in ascending order. The exercise periods were continued beyond the time when heart rate and coronary blood flow became stable. During the stable hemodynamic period, simultaneous arterial and coronary venous samples were drawn as exercise continued. Coronary blood flow, heart rate, and mean arterial pressure were averaged during 20–30 s of this stable period. The average exercise period was 2.7 min. The animals were allowed to rest between each exercise level.

Each dog performed an exercise study under control conditions (vehicle administration only) and during purinergic blockade on separate days. Purinergic blockade consisted of treatment with P1 and P2Y₁ receptor antagonists and a nitric oxide synthesis inhibitor. Blood samples were drawn under resting conditions before and after drug or vehicle administration, as well as during exercise.

Drugs

ATP (A7699; Sigma, St. Louis, MO) was dissolved in isotonic saline for intracoronary injections. Purinergic P1 receptor responses were inhibited with 8-phenyltheophylline (P2276, Sigma) (8-PT, 3 mg/kg iv) dissolved in 1.5 ml of equal parts 1 N NaOH, ethanol, and propylene glycol and diluted to 25 ml with saline. P2Y₁ receptors were inhibited with 2-iodo-N⁶-methyl-(N)-methanocarba-2′-deoxyadenosine-3′,5′-biphosphate (2159; Tocris, Ellisville, MO) (MRS 2500, 0.1 mg/kg iv) (4). MRS 2500 was dissolved in isotonic saline. Nitric oxide synthesis was inhibited by l-nitroarginine (N5501; Sigma) (LNA, 35 mg/kg iv) dissolved in warmed saline and infused through a 0.22-μm syringe tip filter. The weak but highly selective P2Y₂ agonist uridine 5′-tetraphosphate δ-phenyl ester (triethyl ammonium salt) (MRS 2768) (25) was dissolved in saline for intracoronary injections. MRS 2768 was provided by the Molecular Recognition Section, NIDDK National Institutes of Health. The same drugs with the same doses were used in the exercise experiments and the acute coronary ATP dose-response studies.

Outline of the Adenine Nucleotide Hypothesis

A sensor is required for a negative feedback control mechanism, and hemoglobin is the oxygen sensor in the adenine nucleotide hypothesis. The hemoglobin oxygen saturation in coronary venous capillaries reflects the balance between oxygen delivery and oxygen consumption at the local microvascular unit. Thus the controlled variable in the nucleotide hypothesis is the coronary venous hemoglobin oxygen saturation. On the basis of the work of Ellsworth and colleagues (13) the assumption is that red blood cells are the primary source of ATP. However, the ATP could be released by any cell type in the heart. Even white blood cells release ATP when activated (14). The hypothesis is diagrammed in Fig. 4. The initial element in the hypothesis is that, when oxygen is unloaded from hemoglobin, deoxyhemoblogin facilitates the release of ATP from red blood cells. The ATP then activates purinergic P2Y₁ receptors on capillary endothelial cells, which results in a retrograde conducted signal that produces vasodilation of the upstream arteriole. Nitric oxide is involved, presumably in the endothelial cell to vascular smooth muscle transmission. ATP in the plasma is broken down by nucleotidases in the plasma and on the surface of endothelial cells to ADP, AMP, and adenosine. ADP acts on endothelial P2Y₁ receptors to produce a retrograde conducted vasodilator response. ADP also acts on P2Y₁₃ receptors on red blood cells to inhibit the further release of ATP from red blood cells. AMP acts on endothelial P1 receptors to initiate a conducted vasodilator response. Adenosine is avidly taken up by endothelial and red blood cells to enter the purine salvage pathway and replenish ATP.

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Fig. 4.

Schematic diagram of the adenine nucleotide hypothesis of negative feedback coronary control. Oxygen (O₂) is released from hemoglobin (Hb) in coronary capillaries in response to myocardial oxygen consumption. Deoxyhemoglobin facilitates the release of ATP from red blood cells (RBC) that acts on endothelial purinergc P2Y₁ receptors to initiate a retrograde conducted response via gap junctions connecting endothelial cells. The conducted signals dilate the upstream arteriole smooth muscle cells via gap junctions and the release of nitric oxide (NO). Nucleotidases (NTase) in the plasma and on the surface of endothelial cells break ATP down to ADP, which activates P2Y₁ receptors. ADP also activates P2Y₁₃ receptors on the surface of RBC to inhibit the further release of ATP from RBC. ADP is broken down to AMP, which activates P1 receptors to generate a retrograde conducted vasodilator response. AMP is degraded to adenosine (ADO) that is rapidly taken up by RBC and endothelial cells.

The Evidence for the Sequential Steps in the Adenine Nucleotide Hypothesis

Criteria for a Feedback Transmitter

In 1983 Feigl (16) suggested the following six criteria for a feedback transmitter controlling coronary blood flow.

Feedforward Vs. Feedback Control

Feedforward control is also called parallel control, yoked control, or open-loop control. A typical example is when two variables are controlled by a common input. In the coronary circulation, parallel feedforward control has been demonstrated, where β-adrenoceptor activation on cardiac cells increases myocardial oxygen consumption and simultaneously dilates coronary vascular smooth muscle cells via β-adrenoceptor activation (8, 10, 22, 30). Another example of feedforward open-loop control is the hydrogen peroxide hypothesis (39). According to this hypothesis cardiac mitochondria generate superoxide in proportion to the rate of oxygen consumption. Superoxide is converted to hydrogen peroxide that diffuses to coronary arterioles to produce vasodilation. In this way coronary blood flow is coupled to myocardial oxygen consumption without an oxygen sensor.

In a negative feedback control system there is a sensor for the regulated variable that is used to generate an error signal. In the adenine nucleotide hypothesis hemoglobin in coronary capillaries acts as an oxygen sensor.

The disadvantage of feedforward control is that small discrepancies will accumulate over time and may lead to very large errors. This is because there is no self-checking component in feedforward control. The disadvantage of negative feedback control is that there is a tradeoff between accuracy, stability, and response time. The higher the feedback gain, the faster the response and the smaller the error signal. However, increasing gain beyond a stability threshold leads to oscillations and ultimately to dynamic instability. However, Miyashiro and Feigl (31) modeled the coronary circulation and found that a combination of adrenergic feedforward and local metabolic feedback control resulted in a rapid and accurate response without oscillations. The adenine nucleotide negative-feedback control revealed in the present experiments plus the previously documented adrenergic feedforward control (8, 10, 22) demonstrates combined control of coronary blood flow during exercise.

In the adenine nucleotide hypothesis the regulated variable is the coronary venous hemoglobin oxygen saturation, which reflects that balance between oxygen delivery and myocardial oxygen consumption at the local microvascular unit level. The prediction of the adenine nucleotide hypothesis is that blocking purinergic vasodilation will decrease the balance between oxygen delivery and consumption as demonstrated in Fig. 2A. More specifically it is that purinergic blockade will increase the feedback error signal (deoxyhemoglobin saturation) to obtain similar flows per minute (Fig. 3B) or flow per heartbeat before and after blockade.

Other Purinergic Receptors

The results in Fig. 1, C, D, and F, indicate that combined blockade of P1 and P2Y₁ purinergic receptors incompletely inhibits coronary vasodilation attributable to adenine nucleotides. This suggests that other purinergic receptors are involved. Because the additional receptors may act through nitric oxide as a downstream mediator, LNA was added to produce a more effective purinergic blockade (Fig. 1D).

Following pretreatment with the P1 blocker 8-PT plus the P2Y₁ blocker MRS 2500, the highly selective but weak P2Y₂ receptor agonist MRS 2768 produces coronary vasodilation in a dose-dependent manner (Fig. 1F). This indicates the presence of P2Y₂ receptors in the canine coronary circulation. The result is in agreement with the work of Wang et al. (45), who found expression of P2Y₁ and P2Y₂ purinergic receptors in endothelial cells from human umbilical veins. The relative potency difference between ATP and MRS 2768 (1.6 log units) at the P2Y₂ receptor is within expected range. The EC₅₀ for ATP acting on P2Y₂ receptors is 0.085 ± 0.012 μM (24), and the EC₅₀ for MRS 2768 is 1.89 ± 1.07 μM (25). These results do not establish that ATP and/or ADP and AMP cause coronary dilation by activating P2Y₂ receptors but do suggest that P2Y₂ receptors may be involved in nucleotide coronary vasodilation. Unfortunately adequate P2Y₂ receptor blocking agents are not presently available to test the role of P2Y₂ receptors in exercise coronary vasodilation.