INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

STANDEN ET AL. (40) reported in 1989 an ATP-sensitive potassium (K⁺_ATP) channel in smooth muscle cells from arterial mesenteric arteries. Miyoshi et al. (29) reported that K⁺_ATP channels in coronary vessels are inhibited by glibenclamide. There is evidence that K⁺_ATP channels play a role in regulating basal coronary blood flow (11, 18, 37, 41), although this has not been observed in every case (20). K⁺_ATP channel inhibition with glibenclamide blunts hypoxic coronary vasodilation (9, 32) and coronary reactive hyperemia (2, 6, 11). However, K⁺_ATP channels are not required for controlling coronary blood flow during coronary autoregulation (41).

K⁺_ATP channels are involved in adenosine coronary vasodilation as demonstrated by the inhibitory effect of the K⁺_ATP channel antagonist glibenclamide on adenosine-induced coronary vasodilation (1, 5, 6, 9, 11, 33, 35, 41).

The present study was designed to examine the role K⁺_ATP channels and adenosine play in the control of coronary blood flow secondary to pacing tachycardia before and during K⁺_ATP channel blockade with glibenclamide. Pacing tachycardia represents a relatively "pure" local metabolic vasodilator stimulus to the coronary circulation, avoiding the direct vascular effects of catecholamine infusion, including -adrenoceptor-mediated vasoconstriction (13) and -adrenoceptor-mediated vasodilation (28). In addition, the experiments were performed in the presence of a -adrenergic receptor-blocking agent to further isolate the local metabolic stimulus.

It has been suggested that inhibition of adenosine vasodilation (as occurs with K⁺_ATP channel blockade) causes an increase in cardiac adenosine release sufficient to overcome the inhibition (11, 27). This hypothesis was tested with arterial and coronary venous plasma adenosine measurements before and during K⁺_ATP channel blockade. When glibenclamide is given, coronary flow and coronary venous PO₂ decrease, and if adenosine receptors are subsequently blocked with 8-phenyltheophylline, coronary flow and coronary venous PO₂ decrease further (11). This has been interpreted to mean that adenosine increased to compensate for the blockade of K⁺_ATP channels. This interpretation was tested in the present study by measuring plasma adenosine levels before and after glibenclamide.

The present results demonstrate that K⁺_ATP channels are not required for local metabolic control of coronary blood flow and that adenosine levels do not increase sufficiently to compensate for K⁺_ATP channel blockade with glibenclamide.

	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 (Sigma) (100 mg/kg iv). 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 by a continuous intravenous infusion of NaHCO₃ with additional bolus injections as needed. Animals were intubated and ventilated with oxygen-enriched room air by a variable demand valve and volume displacement ventilator (model 607, Harvard Apparatus, South Natick, MA) to maintain an arterial oxygen tension of over 100 mmHg. End-expiratory CO₂ fraction was monitored continuously (model LB-2, Beckman Instruments, Fullerton, CA) and kept near 5% by appropriate adjustments in the ventilatory rate during the cardiac pacing protocol. This kept arterial PCO₂ constant but increased arterial PO₂. A segment of Silastic tubing was placed in the left femoral artery to obtain arterial samples from freely flowing blood. Arterial blood samples were taken periodically and analyzed for pH, PCO₂, PO₂, and base excess (model 1306, Instrumentation Laboratories, Waltham, MA). Core temperature was monitored with a thermistor in the esophagus and controlled at 37°C with a YSI model 73A controller (Yellow Springs Instruments, 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 the infusion of glibenclamide 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 that may have been released as a result of complement and white cell activation stemming from the blood flowing 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 (37K): [in this window] [in a new window]   Fig. 1.   Schematic diagram of closed-chest 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 left femoral artery, and coronary venous samples were drawn from a Sones catheter inserted into coronary sinus via right jugular vein.

Coronary sinus cannulation and pacing catheter. A Sones catheter was inserted into the right external jugular vein and guided fluoroscopically into the coronary sinus (Fig. 1). A metal ring surrounded the catheter tip to prevent venous collapse during sampling. Catheter placement was confirmed by injection of contrast medium, measurement of coronary sinus PO₂, and anatomical location post mortem. To avoid right atrial admixture, the catheter tip was placed at least 26-mm inside the coronary sinus ostium (22). Blood samples drawn from the coronary sinus catheter were analyzed for PO₂, PCO₂, pH, oxygen content, lactate concentration, and plasma adenosine concentration. A pacing catheter (USCI, Billerica, MA) was placed in the right ventricle via the right jugular vein.

Circumflex coronary artery blood flow. Without opening the chest, the circumflex coronary artery was cannulated with a wedge-tip stainless steel cannulating flowmeter inserted via the right carotid artery (38). Blood flowed from the ascending aorta into the flowmeter 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 the 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% ammonium hydroxide 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 masurement. Arterial and coronary venous blood samples were drawn into syringes and immediately transferred into NaF-coated vials and placed on ice to prevent glycolysis. Lactate concentration was determined with a YSI model 23A lactate analyzer. 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 Herrmann and Feigl method (17). 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 (34). The stop solution contained dipyridamole (32 µM), iodotubercidin (ITC, 1 µM), and erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA, 10 µM) dissolved in cold 0.9% saline. Dipyridamole inhibits cellular adenosine uptake. ITC inhibits adenosine kinase, preventing the incorporation of adenosine into AMP. EHNA inhibits 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 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. Five millilters of the acid supernatant were then added to 4.35 ml of a neutralizing solution containing 0.4 mM potassium phosphate (KH₃PO₄) and 0.8 mM potassium hydroxide (KOH). The resultant pH was 7.0. An additional 10-min centrifugation 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 distilled water and divided into two 100-µl aliquots. Adenosine deaminase (10 U, Boehringer Mannheim) was added to one of the aliquots to be used as a paired blank. Samples were incubated at room temperature for 1 h. After incubation the adenosine deaminase was deactivated with 100% methanol and heated at 75°C for 1 h. Samples were concentrated by evaporation and resuspended in 100 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 potassium phosphate with an acetonitrile gradient. The paired chromatograms were superimposed using HP Chemstation software, and the blank was subtracted from the unknown. The resulting chromatogram was integrated, adenosine content was determined by comparison with known adenosine standards, and plasma concentration was calculated by accounting for dilution steps in sample handling, hematocrit, and normalized for recovery with the theophylline standard in each sample.

Estimation of interstitial adenosine concentration. Estimates of interstitial adenosine were made using a four-region (capillary, endothelial cell, interstitial space, parenchymal cell), axially distributed mathematical model (23, 25, 42). 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 (25, 42). Briefly, the model accounts for myocardial flow heterogeneity and the change in heterogeneity that occurs with changes in blood flow. Furthermore, the model is constrained with previous estimates of capillary adenosine transport and metabolism adjusted for the level of flow. Interstitial adenosine concentration is estimated using the measured values of coronary blood flow, hematocrit (to obtain coronary plasma flow), and arterial plasma adenosine concentration by adjusting cellular adenosine production in the model to fit the measured venous plasma adenosine concentration. Estimates of interstitial adenosine were made during both baseline conditions and paired pacing before and after administration of glibenclamide.

Experimental protocol. The role K⁺_ATP channels play in modulating coronary blood flow during paired pacing was studied by comparing hemodynamic, metabolic, and adenosine data before and after treatment with the K⁺_ATP channel antagonist glibenclamide. 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 baseline. Next, paired ventricular pacing was begun at a heart rate of 120 beats/min. The interval between paired stimuli was adjusted while monitoring the ECG and left ventricular pressure to obtain two depolarizations with a single left ventricular contraction (Fig. 2). During pacing, cardiac carbon dioxide production increased, 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. Glibenclamide (1 mg/kg iv) was then infused via a catheter in the abdominal vena cava over 10 min with a syringe pump. This dose of glibenclamide effectively blocks the vasodilating action of the K⁺_ATP channel opener cromakalim and shifts the dose-response curve for adenosine coronary vasodilation 10-fold to the right (41). This dose of glibenclamide was chosen because a larger dose of 3 mg/kg iv results in oscillations in coronary blood flow (31, 41). The 1 mg/kg iv dose of glibenclamide is less than the 2 mg/kg iv dose that Belloni and Hintze (5) demonstrated may be safely given to unanesthetized dogs. Injections of the glibenclamide vehicle intravenously (0.5 ml 1 N NaOH, 0.5 ml ethanol, 0.5 ml propylene glycol in 28.5 ml of 0.9% saline) were without detectable effects in other experiments. After an additional 10 min the sampling protocol was repeated.

View larger version (36K): [in this window] [in a new window]   Fig. 2.   An example of original recordings of coronary blood flow, mean coronary blood flow, electrocardiogram (ECG), and left ventricular pressure from one dog. During paired pacing, delay between electrical pulses was adjusted so that two depolarizations (ECG trace) resulted in a single fused cardiac contraction. Two baseline periods were averaged and compared with 1-, 3-, and 5-min time points in all subsequent figures.

Drugs. -Chloralose (Sigma) was dissolved in warm 0.9% saline. Heparin (SoloPak, Franklin Park, IL) was given as a bolus dose of 750 U/kg iv. Ibuprofen (Sigma) dissolved in 0.2 M sodium carbonate at a concentration of 25 mg/ml (pH was adjusted to 7.5-8 with 1 N HCl) was given as a bolus dose of 12.5 mg/kg iv. Propranolol (Sigma) was dissolved in 0.9% saline and given as a bolus dose of 0.5 mg/kg iv. Glibenclamide (1 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 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 a disk at a rate of 200 samples/s. The values for coronary blood flow, heart rate, and arterial pressure were averaged over a 15-s period for baseline and paired-pulse stimulation time points.

The data are expressed as means ± SE for 10 dogs. The data in Figs. 3-6 are plotted as the individual mean ± SE for each time point. For statistical comparisons, the two basal points before pacing were averaged and called baseline for each variable. The effect of pacing was tested by comparing the average baseline values to pacing values within a condition (before or after glibenclamide) with the Student-Newman-Keuls test for multiple comparisons (SigmaStat, SPSS). A Bonferroni correction for multiple comparisons was made for the 10 response variables shown in the figures and table (arterial PO₂ and PCO₂ were not considered response variables). The effect of glibenclamide was evaluated by comparing baseline points before and after glibenclamide and by comparing the changes from baseline to pacing before and after glibenclamide, also using the Student-Newman-Keuls test and Bonferroni correction. Significant differences were accepted with P < 0.05 (15).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Hemodynamic and metabolic data for the 10 dogs are shown in Table 1. Examples of recordings of coronary blood flow (phasic and mean), electrocardiogram, and left ventricular pressure are shown in Fig. 2. During paired pacing two depolarizations elicit a single fused ventricular contraction.

Glibenclamide decreased baseline coronary blood flow in 9 of 10 experiments (significant before correction for multiple comparisons) by an average of 11%. During control conditions myocardial oxygen consumption increased 88% during the first minute of pacing (Fig. 3A), and after glibenclamide treatment, it increased 92% during the first minute of pacing. Mean baseline heart rate (Fig. 3B) during control was 57 beats/min and was similar after glibenclamide (56 beats/min). After 1 min of pacing, coronary blood flow (Fig. 3C) increased 76% during control and 77% with glibenclamide, with little change in blood pressure (Fig. 3D).

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Hemodynamic and metabolic responses to pacing before and after ATP-sensitive K+ channel (K+ATP) receptor blockade with glibenclamide. Cardiac pacing increased myocardial oxygen consumption (A), heart rate (B), and coronary blood flow (C) similarly before and after glibenclamide. Cardiac pacing had little effect on mean arterial blood pressure (D) during control or after glibenclamide. Two plots are superimposed in B because of identical values. B, baseline.

Coronary venous PO₂ during control baseline conditions was 19 ± 1 mmHg and fell during the first minute of pacing to 17 ± 1 mmHg (Fig. 4A). After glibenclamide, coronary venous PO₂ fell to 13 ± 1 mmHg during baseline and after 1 min of pacing decreased further to 11 ± 1 mmHg. Coronary venous PO₂ after glibenclamide was significantly lower than control at all time points, demonstrating that inhibition of K⁺_ATP channels causes a consistent diminution of flow in relation to myocardial oxygen consumption.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Coronary venous PO2 before and after K+ATP receptor blockade with glibenclamide (A). During cardiac pacing there was a significant decrease in coronary venous PO2 after 1 min for both control and during K+ATP receptor blockade. Coronary venous PO2 was significantly lower than control after glibenclamide during baseline and cardiac pacing. Lactate extraction (B) was relatively constant throughout experiment.

Arterial and venous plasma adenosine concentrations are shown in Fig. 5. Arterial plasma adenosine concentration was unchanged throughout the experiment (Fig. 5A). Control venous plasma adenosine concentration was not altered by pacing. After glibenclamide, venous plasma adenosine concentration was unchanged during baseline measurements but increased significantly from baseline after 1 min of pacing. Estimated interstitial adenosine concentration (Fig. 6) correspondingly increased significantly after 1 min of pacing with glibenclamide treatment, but the concentration was well below the threshold concentration for coronary vasodilation (Fig. 7).

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Arterial and venous plasma adenosine concentration during control and after K+ATP receptor blockade. Arterial plasma adenosine concentration (A) was unchanged throughout experiment. There was no change in coronary venous plasma adenosine concentration (B) with pacing during control protocol. After glibenclamide, baseline coronary venous adenosine concentration was unchanged, but with pacing there was a significant (P < 0.05) increase in venous plasma adenosine after 1 and 3 min.

View larger version (24K): [in this window] [in a new window]   Fig. 6.   Effect of cardiac pacing and K+ATP receptor blockade by glibenclamide on estimated interstitial adenosine concentration. Pacing had little effect on interstitial adenosine during control period. Glibenclamide had no effect on baseline levels of interstitial adenosine. During pacing with glibenclamide interstitial adenosine concentration was significantly higher but remained an order of magnitude lower than vasoactive threshold (see Fig. 7).

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Coronary blood flow plotted against estimated interstitial adenosine concentration during control conditions (A) and after glibenclamide (B). Line plot in A is dose-response curve from Stepp et al. (42) aligned vertically so that curve intersects baseline values. During control pacing interstitial adenosine was well below the threshold vasoactive concentration. After glibenclamide, dose-response curve was shifted to the right 10-fold (41). Estimated interstitial adenosine concentration is more than an order of magnitude lower than that necessary for observed flow increase.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The present study was designed to test the role of K⁺_ATP channels in local metabolic coronary vasodilation and determine whether adenosine levels increase to compensate for the loss of K⁺_ATP channel function after their blockade with glibenclamide. Inhibition of K⁺_ATP channels with glibenclamide reduced baseline coronary blood flow in 9 of 10 experiments and significantly decreased coronary venous oxygen tension, demonstrating a decrease in the ratio of oxygen supply to myocardial oxygen consumption. Paired-pace cardiac stimulation in the presence of -adrenoceptor blockade was used to augment myocardial oxygen consumption and obtain local metabolic coronary vasodilation without adrenergic activation. During these conditions, K⁺_ATP channels are not required for the local metabolic control of coronary blood flow. In addition, neither coronary venous nor estimated interstitial adenosine increased sufficiently to overcome the K⁺_ATP channel blockade.

Effect of paired-pulse ventricular pacing. The purpose of using the paired-pulse pacing stimulus was to augment myocardial contractility and oxygen consumption without the use of catecholamines (4, 36). The advantage of using paired-pulse stimulation rather than single-pulse pacing is that a larger increase in myocardial oxygen consumption is possible for the same heart rate. Two closely spaced electrical pulses generate two action potentials for each myocardial contraction that enhance contractility caused by increased cytosolic calcium concentration. Evidence exists showing a link between catecholamines and cardiac adenosine release separate from changes in myocardial oxygen consumption (16, 21, 26, 43, 44). When combined with -adrenoceptor blockade, paired pacing produces a local metabolic stimulus to increase coronary blood flow without catecholamine effects.

Role of K⁺_ATP channels in control of baseline coronary blood flow. The majority of studies that examined the connection between coronary blood flow and K⁺_ATP channel blockade with glibenclamide found that basal flow is reduced by 15-25% (8, 11, 18, 30, 41). However, Katsuda et al. (20) saw no change in baseline coronary blood flow with glibenclamide in anesthetized and conscious dogs. In the present study, there was a small increase in baseline blood pressure (Fig. 3D) and myocardial oxygen consumption (Fig. 3A) and an 11% decrease in baseline coronary blood flow (Fig. 3C) following glibenclamide. The small increase in myocardial oxygen consumption combined with the decrease in coronary blood flow produced a significant decrease in coronary venous PO₂ (Fig. 4A), demonstrating a fall in the balance between oxygen delivery and consumption. Despite the fall in coronary venous PO₂ with K⁺_ATP channel blockade, coronary venous plasma adenosine concentration (Fig. 5B), estimated interstitial adenosine concentration (Fig. 6), and lactate extraction (Fig. 4B) were unchanged during baseline conditions.

Role of K⁺_ATP channels in local metabolic flow regulation. Although K⁺_ATP channels play a role in the regulation of basal coronary blood flow, they are not required for the increase in flow that accompanies an increase in myocardial metabolism based on the results presented here. The increase in coronary blood flow during control pacing and pacing with glibenclamide was the same (76% vs. 77%) (Fig. 3C). Analogous findings have been reported in conscious dogs by Duncker et al. (11). They found that glibenclamide decreased resting coronary blood flow but did not attenuate the increase in flow associated with exercise, which is a more complicated phenomenon involving feedforward -adrenergic coronary vasodilation (10). Duncker et al. (11) observed that glibenclamide lowered coronary venous PO₂ and subsequent adenosine receptor blockade with 8-phenyltheophylline further lowered coronary venous PO₂. They interpreted their results to indicate that an increase in adenosine concentration compensated for the loss of K⁺_ATP channel function. The present results do not confirm the interpretation of Duncker et al. because adenosine concentration did not increase to vasoactive levels following glibenclamide.