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(Circulation Research. 1996;79:415-423.)
© 1996 American Heart Association, Inc.

Articles

PD 81,723, an Allosteric Enhancer of the A₁ Adenosine Receptor, Lowers the Threshold for Ischemic Preconditioning in Dogs

Tsuneo Mizumura, John A. Auchampach, Joel Linden, Robert F. Bruns, Garrett J. Gross

the Department of Pharmacology and Toxicology (T.M., G.J.G.), Medical College of Wisconsin, Milwaukee; the Department of Internal Medicine and Molecular Physiology and Biological Physics (J.A.A., J.L.), University of Virginia, Charlottesville; and Central Nervous System Pharmacology Research (R.F.B.), Lilly Research Laboratories, Eli Lilly and Co, Indianapolis, Ind.

Correspondence to Garrett J. Gross, PhD, Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Rd, Milwaukee, WI 53226.

	Abstract

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PD 81,723 (PD) acts allosterically to increase agonist binding to A₁ adenosine receptors and to enhance functional A₁ receptor–mediated responses in the heart and other tissues. To determine if PD lowers the threshold for ischemic preconditioning (PC), pentobarbital-anesthetized dogs were subjected to 60 minutes of left anterior descending coronary artery (LAD) occlusion and 3 hours of reperfusion. Ischemic PC was produced by either 2.5 or 5 minutes of LAD occlusion 10 minutes before the 60-minute occlusion. PD (100 µg/kg total dose, 5 to 50 µmol/L in coronary arterial blood) or vehicle was infused intracoronarily for 17.5 minutes before the 60-minute occlusion period in non-PC dogs or in dogs preconditioned with 2.5 minutes of ischemia. Myocardial infarct size was determined by triphenyltetrazolium staining and expressed as a percentage of the area at risk. Compared with the control group (26.3±3.6%, mean±SEM), infarct size was not significantly affected by 2.5 minutes of PC alone (23.4±4.2%) or by PD alone (26.5±1.7%) but was decreased by PD+PC (14.6±1.7%, P<.05) or by a longer period (5 minutes) of PC alone (12.5±3.3%). The intravenous administration of the selective antagonist of A₁ adenosine receptors, 8-cyclopentyl-1,3-dipropylxanthine (1 mg/kg), or the ATP-sensitive K⁺ channel blocker, glibenclamide (0.3 mg/kg), for 15 minutes before PD+PC blocked the protection (23.6±2.3% or 25.9±3.3%, respectively). None of the compounds studied affected systemic hemodynamics, collateral blood flow, or AAR. To determine which subtypes of canine adenosine receptors were affected by 10 µmol/L PD, radioligand binding studies were conducted using membranes derived from COS-7 cells expressing recombinant canine receptors and agonist radioligands. PD enhanced the binding of [¹²⁵I]N⁶-4-amino-3-iodobenzyladenosine (¹²⁵I-ABA) to A₁ receptors by increasing the t_1/2 for dissociation by 2.18-fold, but PD had no effect on the dissociation kinetics of ¹²⁵I-ABA from A₃ receptors or [¹²⁵I]-[2-(4-amino-3-iodo-phenyl)ethylamino]adenosine from A_2A receptors. Glibenclamide at concentrations up to 10 µmol/L had no effect on the binding of radioligands to recombinant canine A₁, A_2A, or A₃ receptors. These data suggest that PD reduces the amount of time required for ischemia to produce preconditioning by enhancing adenosine binding to its A₁ receptor. Glibenclamide prevents the protection afforded by A₁ receptor activation by a mechanism not involving adenosine receptor blockade.

Key Words: ischemic preconditioning • adenosine receptors • ATP-sensitive K⁺ channels • allosteric enhancers • glibenclamide

	Introduction

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Ischemic PC, which was first described by Murry et al¹ in 1986, has been widely studied by a number of laboratories. Although the mechanism by which brief periods of ischemia protect the heart from a more prolonged period of ischemia is still not fully understood, it is known that PC must be maintained for a minimum period of time (threshold) to be effective. In anesthetized dogs, a single 5-minute coronary artery occlusion followed by a 10-minute reperfusion period is sufficient to produce ischemic PC.² On the other hand, Yao and Gross³ have recently shown that 3 minutes of PC was not sufficient to reduce infarct size in anesthetized dogs. Similarly, Miura et al⁴ demonstrated that 2 minutes of PC was not sufficient to reduce infarct size in anesthetized rabbits unless the rabbits were pretreated with the adenosine uptake inhibitor dipyridamole. They concluded that stimulation of adenosine receptors by endogenous adenosine mediates the infarct size–reducing effect afforded by ischemic PC. Consistent with this finding, adenosine administration has been shown to mimic the effect of ischemic PC to reduce infarct size,⁵ and it is likely that activation of the A₁ adenosine receptor is, at least in part, involved in this phenomenon.⁶ Endogenous adenosine has also been shown to mediate ischemic PC in swine.⁷

PD 81,723 is an allosteric enhancer of the A₁ adenosine receptor.⁸ This compound has been found to increase radioligand binding and functional activation of A₁ receptors in rat brain and heart, guinea pig heart, and human myocardium and in CHO cells stably transfected with recombinant human A₁ receptors.^{8 9 10 11 12} The enhancer has been shown to stabilize agonist receptor–G protein complexes.¹² Thus, in the present study, we wished to determine whether PD 81,723 lowers the threshold for ischemic PC in intact dogs. Second, by use of radioligand analysis with recombinant adenosine receptors, we determined whether PD 81,723 acts selectively on A₁ receptors or whether it also influences other adenosine receptor subtypes. Furthermore, since the A₁ adenosine receptor has been shown to be coupled to the K_ATP channel via a G_i protein¹³ and opening of myocardial K_ATP channels results in ischemic PC,¹⁴ we determined whether the effect of PD 81,723 is also blocked by the sulfonylurea K_ATP antagonist glibenclamide. Finally, since glibenclamide blocks responses to adenosine in several tissues in a manner resembling a competitive receptor antagonist,¹⁵ we determined whether glibenclamide competes for radioligand binding to recombinant canine A₁, A_2A, and A₃ adenosine receptors.

	Materials and Methods

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Animal Experiments
General Preparation of Dogs
All experiments conducted in the present study were in accordance with the Position of the American Heart Association on Research and Animal Use, adopted in 1984 by the American Heart Association, and the guidelines of the animal care committee of the Medical College of Wisconsin. The Medical College of Wisconsin is accredited by the American Association of Laboratory Animal Care.

Adult mongrel dogs of either sex, weighing 19.0 to 28.0 kg, were fasted overnight, anesthetized with a combination of sodium barbital (200 mg/kg) and sodium pentobarbital (15 mg/kg), and ventilated with room air supplemented with 100% oxygen. Atelectasis was prevented by maintaining an end-expiratory pressure of 5 to 7 cm H₂O with a trap. Arterial blood pH, PCO₂, and PO₂ were monitored at selected intervals by an AVL automatic blood gas system and maintained within normal physiological limits (pH 7.35 to 7.45; PCO₂, 30 to 35 mm Hg; and PO₂, 85 to 100 mm Hg) by adjusting the respiration rate and oxygen flow or by intravenous administration of 1.5% sodium bicarbonate if necessary. Body temperature was maintained at 38±1°C with a heating pad. Aortic blood pressure and left ventricular pressure were monitored by inserting a double-pressure transducer–tipped catheter (model PC 771, Millar) into the aorta and left ventricle via the left carotid artery. Left ventricular dP/dt was recorded by electronic differentiation of the left ventricular pressure pulse, and heart rate was determined by a tachometer. The right femoral vein and artery were cannulated for drug administration and for analyses of blood gases and myocardial tissue blood flow, respectively. A left thoracotomy was performed at the fifth intercostal space, the lung was carefully retracted, and the pericardium was incised. The heart was then suspended in a pericardial cradle. A proximal portion of the LAD distal to the first diagonal branch was isolated from surrounding tissue, and a calibrated electromagnetic flow probe (Statham SP 7515) was placed around the vessel. A flowmeter (Statham 2202) was used to measure blood flow. A mechanical occluder was placed distal to the flow probe such that there were no branches between the flow probe and the occluder. The occluder was used to zero the flow probe (20 minutes before the 60-minute experimental occlusion, the LAD was occluded for 10 seconds), to occlude the vessel, and to reperfuse the myocardium. If the basal heart rate was <150 bpm, the heart was paced at that rate with rectangular pulses of 4-millisecond duration and a voltage twice threshold via bipolar electrodes clipped to the left atrial appendage. Pacing was not used in the few animals with initial rates of >150 bpm. Hemodynamic variables, heart rate, and coronary blood flow were monitored and recorded by a polygraph throughout the experiment. The left atrium was cannulated via the appendage for radioactive microsphere injection.

Experimental Design
Animals were assigned to one of seven groups, and all dogs were subjected to 60 minutes of LAD occlusion followed by 3 hours of reperfusion (Fig 1). Approximately 17.5 minutes before the 60-minute occlusion, either PD 81,723 (100 µg/kg) or an equivalent volume of vehicle (0.5 mL polyethylene glycol and Emulphor EL 620) was administered intracoronarily distal to the occlusion site and continued to the time of occlusion in the group administered PD 81,723 (PD group) and the control group, respectively. This dose of PD 81,723 was chosen so that 5 to 50 µmol/L of the drug was present in the coronary circulation during coronary perfusion and occlusion, since previous studies^{9 10 11 12} have shown that 5 to 50 µmol/L of PD 81,723 produces an optimal enhancement of A₁ receptor binding for adenosine or selective A₁ agonists, whereas at concentrations above 50 µmol/L, PD 81,723 produces a competitive blockade. In the next two groups, 2.5' PC was elicited before the longer occlusion, and the same dose of PD 81,723 (PD+PC group) or vehicle (vehicle PC group) was given 5 minutes before 2.5' PC and continued to the time of the longer occlusion. In two other groups, either 1 mg/kg of the selective adenosine A₁ receptor antagonist DPCPX (D+PD+PC group) or 0.3 mg/kg of the K_ATP channel antagonist glibenclamide (G+PD+PC group) was administered intravenously 15 minutes before intracoronary PD 81,723 infusion. These doses of DPCPX and glibenclamide have been shown by our laboratory to block ischemic PC in dogs without affecting infarct size in the absence of PC.^{14 16} For purposes of comparison with the 2.5' PC group, a 5' PC group was also included. In all groups, hemodynamics, blood gas analyses, and myocardial blood flow measurements were performed at 30 minutes into the 60-minute occlusion period. After reperfusion, hemodynamics were measured every hour, and myocardial blood flow was determined at the end of the 3-hour reperfusion period. Finally, at the end of the experiment, the hearts were electrically fibrillated, removed, and prepared for infarct size determination and regional myocardial blood flow measurements.

View larger version (39K): [in this window] [in a new window]   Figure 1. Schematic diagrams of the experimental protocols. All animals were subjected to 60 minutes of LAD occlusion (Occ) followed by 3 hours of reperfusion (Rep). PD 81,723 (PD, 100 µg/kg, total dose) or saline was infused intracoronarily for 17.5 minutes before the 60-minute Occ (60'-Occ) in the PD or control group, respectively. In the next two groups, 2.5' PC was elicited 5 minutes after PD or vehicle infusion was started in the PD+PC or vehicle group, respectively. In two other groups, DPCPX (D, 1 mg/kg) or glibenclamide (G, 0.3 mg/kg) was administered intravenously 15 minutes before the combination of 2.5' PC plus PD 81,723 in the D+PD+PC or G+PD+PC group, respectively. In the 5' PC group, 5' PC was elicited before the longer occlusion.

Infarct Size Determination
At the end of the 3-hour reperfusion period, the LAD was cannulated. To determine the anatomic AAR and the nonischemic area, 5 mL of Patent blue dye and 5 mL of saline were injected at equal pressure into the left atrium and LAD, respectively. The heart was then immediately fibrillated and removed. The left ventricle was dissected and sliced into serial transverse sections 6 to 7 mm in width. The nonstained ischemic area and the blue-stained normal area were separated, and both regions were incubated at 37°C for 15 minutes in 1% TTC (Sigma Chemical Co) in 0.1 mol/L phosphate buffer adjusted to pH 7.4. The TTC stains the noninfarcted myocardium a brick red color, indicating the presence of a formazan precipitate that results from the reduction of TTC by dehydrogenase enzymes in viable tissue. After storage overnight in 10% formaldehyde, infarcted and noninfarcted tissues within the AAR were separated and determined gravimetrically. Infarct size was expressed as a percentage of the AAR.

Regional Myocardial Blood Flow Measurement
Regional myocardial blood flow was measured by the radioactive microsphere technique as previously described in this laboratory.^{14 16} Microspheres were administered at 30 minutes into the prolonged 60-minute occlusion period as well as at the end of reperfusion. Carbonized plastic microspheres (15-µm diameter, New England Nuclear) labeled with ¹⁴¹Ce or ⁹⁵Nb were suspended in isotonic saline with 0.01% Tween-80 added to prevent aggregation. The microspheres were sonicated for 5 minutes and vortexed for another 5 minutes before injection. One milliliter of the microsphere suspension (2 to 4x10⁶ spheres) was given via the left atrial catheter and flushed by 5 mL of saline. A reference blood flow sample was withdrawn from the right femoral artery at a constant rate of 9.4 mL/min starting 30 seconds before the microsphere injection and continuing for 3 minutes. On the following day, the tissue slices were sectioned into subepicardium, midmyocardium, and subendocardium of nonischemic (three pieces) and ischemic (five pieces) regions. Transmural pieces were obtained from the center of several transverse sections used to determine the AAR and were at least 1 cm from the perfusion boundaries as indicated by Patent blue dye. All samples were counted in a gamma counter (Tracor Analytic 1195) to determine the activity of each isotope in each sample. The activity of each isotope was also determined in the reference blood flow samples. Myocardial blood flow was calculated by using a preprogrammed computer to obtain the true activity of each isotope after correction for overlap of the two isotopes in individual samples, and tissue blood flow was calculated from the following equation: Qm=QrxCm/Cr, where Qm is myocardial blood flow (milliliters per minute per gram of tissue), Qr is the rate of withdrawal of the reference blood flow (9.4 mL/min), Cr is the activity of the reference blood flow sample (counts per minute), and Cm is the activity of the tissue sample (counts per minute per gram). Transmural blood flow was calculated as the weighted average of the three layers in each region.

Exclusion Criteria
Dogs were excluded if (1) heartworms were found after the animals were killed, (2) transmural collateral blood flow was >0.16 mL/min per gram, (3) heart rate was >180 bpm at the beginning of the experiment, or (4) more than three consecutive attempts were needed to convert ventricular fibrillation with low-energy DC pulses (15 to 20 J) applied directly to the heart.

Radioligand Binding Studies
Chemicals
PD 81,723 (also called LY 202472) was synthesized at Eli Lilly and Co, R-PIA was obtained from Research Biochemicals, and all cell culture reagents were obtained from GIBCO BRL. Canine A₁ and A_2A receptor cDNAs were kind gifts of Dr Gilbert Vassart (Brussels, Belgium), and the A₃ receptor was previously cloned as described by our laboratory.^{17 125}I-ABA¹⁸ and ¹²⁵I-APE¹⁹ were synthesized as described previously. All other reagents were purchased from Sigma.

Expression of Adenosine Receptors in COS-7 Cells
The canine A₁, A_2A, and A₃ adenosine receptor cDNAs were subcloned into the mammalian expression vector CLDN-10B (a gift of Dr Mitch Reff, SKF Laboratories). The A₁ adenosine receptor cDNA (RDC7, 1.3 kb) was introduced between the Kpn I–HindIII sites, and the A_2A receptor cDNA (RDC7, 2.4 kb) was subcloned between the Kpn I–EcoRV sites within the polycloning region of CLDN. The A₃ adenosine receptor cDNA was inserted into an EcoRI site of a modified version of CLDN, with additional restriction sites placed into the polycloning cassette. The vectors were individually transformed into Escherichia coli (JM109, Promega), and large quantities of plasmid DNAs were isolated (Maxi-Preps, Promega). The plasmid DNA was subsequently introduced into COS-7 cells using the DEAE-dextran method.²⁰ Transfected cells were grown in DMEM with 10% fetal calf serum, 100 U/mL penicillin G, and 100 µg/mL streptomycin on 150-cm² cell culture plates for 60 hours.

Membrane Preparation and Radioligand Binding
Crude membrane preparations were made from transfected COS-7 cells and used for radioligand binding analysis. Cells were initially washed in phosphate-buffered saline (pH 7.4), homogenized in 10 mmol/L EDTA, 10 mmol/L sodium HEPES (pH 7.4), and 0.1 mmol/L benzamidine, and then centrifuged at 20 000g for 20 minutes. Pellets were resuspended and washed twice in HE buffer (10 mmol/L sodium HEPES and 1 mmol/L EDTA, pH 7.4) and were resuspended in the same buffer with 10% (wt/vol) sucrose at a membrane protein concentration of 5 mg/mL. Membranes were frozen in aliquots at -20°C. For radioligand binding analysis, triplicate aliquots of cell membranes were diluted to 1 mg of protein/mL and incubated in 0.1 mL with 2.5 (dissociation assays) or 5 mmol/L (competition assays) MgCl₂, 2.5 U/mL adenosine deaminase, and 0.1 to 1 nmol/L ¹²⁵I-ABA or ¹²⁵I-APE for 3 hours. ¹²⁵I-ABA was used as a radioligand for A₁ and A₃ receptors,^{17 18} and ¹²⁵I-APE was used as a radioligand for A_2A receptors.¹⁹ The bound and free radioligands were separated by rapid filtration through Whatman GF/C filters using a Brandel cell harvester. Nonspecific binding was measured using 5 µmol/L nonradioactive I-ABA or I-APE. To measure dissociation kinetics, radioligand was bound to equilibrium (3 hours) in a water bath (at 21°C or 37°C), and then 20 µmol/L R-PIA was added alone or in combination with 10 µmol/L PD 81,723 for various times before filtration. Data were fit to the following biexponential decay equation:

where B₁ and B₂ are specific binding at equilibrium to two binding sites, B is total binding at time t after the addition of R-PIA, _-1 and _-2 are the dissociation rate constants, and NS is nonspecific binding. The values for t_1/2 of dissociation were calculated by dividing 0.693 by the rate constant. Three separate experiments were performed in triplicate for each receptor subtype.

Competition by Glibenclamide
The ability of glibenclamide to compete for radioligand binding was determined in equilibrium binding assays. Glibenclamide was added to test tubes with radioligands and incubated for 3 hours before filtration and receptor binding assays, which were performed as described above (n=3, each receptor subtype).

Statistical Analysis
In the radioligand binding studies, differences in dissociation rate constants were compared by Student's t test by paired analysis. Differences between groups in hemodynamics at various time points were compared by using a two-way ANOVA for time and treatment with repeated measures and Fisher's least significant difference test if significant F ratios were obtained. Differences in tissue blood flow, AAR, or infarct size were compared by a one-way ANOVA and Fisher's least significant difference test. ANCOVA was used to determine whether the relationship between transmural collateral blood flow and infarct size differed between the control group and drug-treated or preconditioned groups. All values are expressed as mean±SEM. Differences between groups were considered significant at P<.05.

	Results

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Animal Experiments
Mortality and Exclusions
Fifty-three dogs were initially used in the present study. Six dogs were excluded because transmural collateral blood flow was >0.16 mL/min per gram (two dogs in the D+PD+PC group and one each in the control, PD, vehicle PC, and PD+PC groups), and one dog in the control group was excluded because more than three consecutive attempts were needed to convert ventricular fibrillation with DC pulses. Therefore, 46 dogs were used for data analysis: eight dogs for the control and D+PD+PC groups and six each for the remaining groups.

Hemodynamic Responses
The hemodynamic data are shown in Table 1. There were no significant differences in heart rate, mean arterial blood pressure, rate-pressure product, left ventricular dP/dt, and LAD blood flow at baseline between groups, except for the mean arterial blood pressure in the vehicle PC group. In all groups, there were no significant differences in systemic hemodynamics between groups throughout the experiment, except for the baseline mean arterial blood pressure in the 5' PC group.

View this table: [in this window] [in a new window]   Table 1. Hemodynamics in the Different Groups

Myocardial Infarct Size
Myocardial infarct size and AAR data are shown in Fig 2. The anatomic AAR expressed as a percentage of the left ventricle was not significantly different between groups (Fig 2A): control group, 29.5±2.4% (mean±SEM); PD group, 32.9±1.1%; vehicle PC group, 32.7±3.3%; PD+PC group, 35.3±3.6%; D+PD+PC group, 31.6±1.7%; G+PD+PC group, 35.6±1.6%; and 5' PC group, 29.3±0.7%. Myocardial infarct size expressed as a percentage of the AAR (Fig 2B) was significantly reduced in the PD+2.5^' PC group (14.6±1.7%, P<.05) and 5' PC group (12.5±3.3%, P<.01) compared with the control group (26.3±3.6%). There were no significant differences in the infarct size in the PD group (23.4±4.2%), vehicle PC group (26.5±1.7%), D+PD+2.5^' PC group (23.6±2.3%), and G+PD+2.5^' PC group (25.9±3.3%) compared with the control group.

View larger version (77K): [in this window] [in a new window]   Figure 2. Bar graphs illustrating the effects of PD 81,723 (PD) and/or ischemic PC on the AAR expressed as a percentage of the left ventricle (AAR/LV) and infarct size expressed as a percentage of the AAR (IS/AAR). A, There were no significant differences in AAR/LV between groups. B, PD administration combined with a 2.5-minute PC (PD+PC group) resulted in a significant reduction in infarct size (*P<.05 vs control [Cont] group), and the 5' PC group also showed a marked reduction in infarct size (P<.01 vs Cont group). The infarct size–reducing effect of the PD+PC group was completely abolished by pretreatment with glibenclamide (G+PD+PC group) and was attenuated by pretreatment with DPCPX (D+PD+PC group).

Fig 3 shows the relationship between transmural collateral blood flow measured at 30 minutes of occlusion and infarct size expressed as a percentage of the AAR. In all groups, there was an excellent inverse correlation (r=.614 to .914) between these two variables. However, the regression line of the "preconditioned" groups (5' PC and PD+2.5^' PC groups) was shifted downward when compared with that of the control groups by ANCOVA (P<.05). There were no statistically significant differences between any of the other groups and the control group by ANCOVA.

View larger version (24K): [in this window] [in a new window]   Figure 3. A plot of the relationship between transmural collateral blood flow and infarct size (IS) expressed as a percentage of the AAR. There was an excellent inverse correlation (r=.614 to .914) between collateral blood flow and IS/AAR in all groups (A through D). The regression lines for the 2.5' PC and the 5' PC groups were shifted significantly downward compared with that for the control group by ANCOVA (P<.05). There were no other statistically significant differences between the other groups and control line (P.05). PD indicates PD 81,723; GLB, glibenclamide.

Regional Myocardial Blood Flow
Transmural collateral blood flow data in nonischemic (left circumflex coronary artery) and ischemic (LAD) areas are summarized in Table 2. There were no significant differences in transmural collateral blood flow between groups compared with the control group except for the flows in the nonischemic region in the G+PD+2.5^' PC group at 30 minutes of occlusion and in the 5' PC group at 3 hours of reperfusion.

View this table: [in this window] [in a new window]   Table 2. Regional Myocardial Blood Flow

Radioligand Binding Studies
To determine whether PD 81,723 enhances binding to canine A₁ adenosine receptors and to other canine adenosine receptor subtypes (A_2A and A₃), the effect of PD 81,723 on the dissociation kinetics of radioligand binding to recombinant adenosine receptors was examined, and the results are shown in Fig 4. Rates were determined at 37°C for the A₁ and A_2A receptors but at 21°C for A₃ receptors, since binding to this receptor was quickly lost at higher temperatures, possibly because of the activation of a protease or kinase. For all three receptors, dissociation occurs in two kinetically distinct components. The first component, probably reflecting low-affinity binding to uncoupled receptors, was too rapid to measure accurately by the filtration method used in these studies. PD 81,723 (10 µmol/L) significantly increased the t_1/2 of the slow high-affinity component of ¹²⁵I-ABA binding by 2.18-fold from 5.80±0.70 to 12.6±0.69 minutes (n=3, P<.05 by paired analysis). PD 81,723 had no effect (n=3, each group) on the dissociation kinetics of ¹²⁵I-ABA from A₃ receptors (without PD 81,723, 18.5±0.71 minutes; with PD 81,723, 16.5±0.74 minutes) or ¹²⁵I-APE from A_2A receptors (without PD 81,723, 9.0±0.70 minutes; with PD 81,723, 7.1±0.70 minutes).

View larger version (14K): [in this window] [in a new window]   Figure 4. Effect of PD 81,723 on dissociation kinetics of agonist radioligands from recombinant canine adenosine receptor subtypes. Cell membranes (25 µg membrane protein per assay tube) derived from COS-7 cells transfected with A1 (A), A2A (B), or A3 (C) adenosine receptors were incubated with 0.75 to 1.30 nmol/L 125I-ABA (A1 and A3 receptors) or 125I-APE (A2A receptors) for 3 hours at 37°C (A1 and A2A receptors) or 21°C (A3 receptors). R-PIA (20 µmol/L) with or without PD 81,723 (10 µmol/L) was added at various times before filtration. Specific binding is normalized to binding at equilibrium, and the data were fit to a biexponential decay model (see "Materials and Methods"). PD 81,723 consistently increased by twofold the t1/2 of the slow component of 125I-ABA dissociation but had no effect on dissociation kinetics of 125I-ABA or 125I-APE for A3 or A2A receptors, respectively. All values are mean±SEM (n=3 for each subtype), performed in triplicate. Total (T) and nonspecific (NS) binding were as follows (cpm): A1, T=10 211±703, NS=814±10; A2A, T=10 121±469, NS=2713±112; and A3, T=17 565±739, NS=1682±39.

Since glibenclamide completely blocked the protection produced by the combination of PC plus PD 81,723, we reasoned that this sulfonylurea might act by preventing adenosine from binding to A₁ adenosine receptors. Therefore, competition studies were also performed to determine whether glibenclamide competes for radioligand binding to recombinant canine adenosine receptors. As shown in Fig 5, glibenclamide had no influence on agonist binding to the three adenosine receptor subtypes at concentrations as high as 10 µmol/L (n=3, each receptor subtype).

View larger version (20K): [in this window] [in a new window]   Figure 5. Lack of competition by glibenclamide for agonist radioligand binding to recombinant canine adenosine receptors. Transfected COS-7 cell membranes (25 µg membrane protein per assay tube) were incubated with 0.14 to 0.87 nmol/L 125I-ABA (A1 and A3 receptors) or 125I-APE (A2A receptors) for 3 hours in the presence of increasing concentrations of glibenclamide. Binding is plotted as a fraction of control specific binding. All values are mean±SEM (n=3 for each subtype), performed in triplicate. Total (T) and nonspecific (NS) binding were as follows (cpm): A1, T=4998±1226, NS=492±229; A2A, T=1473±79, NS=660±46; and A3, T=3068±1010, NS=480±132.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The major findings of the present study are as follows: (1) PD 81,723 lowers the threshold for ischemic PC, although it had no effect on infarct size by itself; (2) the infarct-limiting effect of PD 81,723 is blocked by the selective A₁ adenosine receptor antagonist, DPCPX, and by the K_ATP channel antagonist, glibenclamide; (3) 10 µmol/L PD 81,723 significantly decreased the slow component of ¹²⁵I-ABA dissociation from canine A₁ adenosine receptors by 2.18-fold in binding studies, whereas PD 81,723 had no effect on the dissociation kinetics of ¹²⁵I-ABA or ¹²⁵I-APE from A₃ or A_2A adenosine receptors, respectively, which demonstrates that PD 81,723 selectively acts on A₁ receptors in dogs; and (4) glibenclamide is not a competitive adenosine receptor antagonist at concentrations as high as 10 µmol/L.

Since a number of previous studies have shown that at least a single 5- or 10-minute period of coronary occlusion followed by 10 to 60 minutes of reperfusion is required to produce the beneficial effect of ischemic PC in the canine heart, we initially tested a single 2.5' PC or 5' PC stimulus and found that 2.5 minutes of LAD occlusion was not long enough to reduce infarct size, whereas 5' PC resulted in a significant reduction in infarct size in this canine model. In agreement, Miura et al⁴ reported that a single 2-minute period of coronary artery occlusion with a 5-minute period of reperfusion followed by a subsequent longer occlusion was insufficient to reduce infarct size in rabbit hearts. Recently, results from our laboratory³ also showed that a single 3-minute period of coronary artery occlusion before a subsequent 60-minute period of occlusion was not sufficient to produce the beneficial effect of PC in dogs. On the other hand, Ovize et al²¹ demonstrated that 2.5 minutes of coronary occlusion resulted in a significant reduction in infarct size in anesthetized dogs subjected to 60 minutes of occlusion and 4.5 hours of reperfusion. Thus, although there are small differences in the duration of brief coronary artery occlusion necessary to produce ischemic PC, the results of most studies suggest that 3 to 5 minutes of coronary artery occlusion is the threshold of time necessary to produce ischemic PC in dogs and rabbits. Our data indicate that the presence of PD 81,273 during a brief normally subthreshold period of ischemia reduces the threshold necessary to elicit a PC-like effect in dogs.

A large body of evidence suggests that adenosine can protect the heart against ischemia/reperfusion injury^{5 22 23 24 25} and that activation of A₁ adenosine receptors mimics and blockade of these receptors abolishes the effect of ischemic PC to reduce infarct size.^{6 16} Liu et al⁶ showed that in isolated rabbit hearts intracoronary administration of adenosine for 5 minutes followed by 10 minutes of a drug-free period was as effective as ischemic PC to limit infarct size. PD 81,723 has been previously shown to increase agonist binding to A₁ adenosine receptors and enhance the functional effects of these agonists in tissues derived from rats,^{9 10} guinea pigs,¹¹ and humans.¹² According to those in vitro studies, the maximum effect of PD 81,723 should be obtained at concentrations of 20 µmol/L,^{9 12} and the action of this agent is highly selective for the adenosine receptor.^{9 10} PD 81,723 has been shown to have no effect on agonist binding to -opiate, M₂-muscarinic, or ₂-adrenergic receptors.⁹

The selectivity of PD 81,723 has been shown to extend to the A₁ subtype of adenosine receptor. In previous studies, it has been shown that PD 81,723 enhances the binding of agonist radioligands to rat, guinea pig, and human A₁ adenosine receptors.^{9 10 11 12} In the present study, we show that PD 81,723 also enhances binding of an agonist, ¹²⁵I-ABA, to canine A₁ adenosine receptors. Although not unexpected, such a finding was not ensured, inasmuch as the ligand binding characteristics of canine A₁ adenosine receptors are quite different from the A₁ receptors of other species.²⁶ It also has been reported that PD 81,723 is selective in its enhancement for A₁ over A_2A receptors on the basis of radioligand binding to rat brain.⁹ Such adenosine receptor subtype selectivity also has been demonstrated functionally in the guinea pig heart, since PD 81,723 enhances the negative dromotropic effect of adenosine mediated by A₁ receptors but does not enhance coronary vasodilation, which is mediated by A_2A receptors. In the present study, we have shown that the absence of an effect of PD 81,723 on A_2A receptors can be extended to the canine species; this extrapolation is based on the lack of effect of PD on ¹²⁵I-APE binding to recombinant A_2A adenosine receptors and on the failure of PD 81,723 to influence hemodynamic responses in the dog. Effects of PD on the A₃ subtype of adenosine receptors have not been previously investigated. It seemed possible that PD 81,723 might influence A₃ adenosine receptors, since of the four subtypes of adenosine receptors, only the A₁ and A₃ subtypes couple primarily to G_i/G_o, whereas A_2A and A_2B receptors couple to G_s. Also, A₁ receptors are structurally more similar to A₃ receptors than to A_2A or A_2B receptors.²⁷ For these reasons, our new finding that PD 81,723 does not enhance agonist binding to recombinant canine A₃ receptors implies even greater specificity of the enhancer actions of PD for a distinct binding domain unique to A₁ receptors than does the absence of enhancer effect on A_2A receptors, reported previously.

In the context of the present study, the failure of PD 81,723 to enhance agonist binding to A₃ adenosine receptors is informative for another reason. It has been postulated that the effects of adenosine to produce PC might be mediated by A₃ adenosine receptors.^{28 29} Although our data do not rule out the involvement of A₃ adenosine receptors in the ischemic PC response, the observation that PD 81,723 enhances PC, coupled with the observation that PD 81,723 does not enhance binding of an agonist to A₃ adenosine receptors, implies that ischemic PC in the dog is mediated, at least in part, by A₁ adenosine receptors. Also consistent with this conclusion is the finding that the A₁-selective antagonist DPCPX blocked protection produced by PC plus PD 81,723.

We found that pretreatment with glibenclamide blocks the myocardial protection afforded by PC plus PD 81,723. Since 2.5 minutes of PC alone did not precondition, but PC plus PD 81,723 was protective, and since PD is selective for A₁ adenosine receptors in binding assays, we conclude that the protection observed with PC plus PD 81,723 is mediated by A₁ adenosine receptors. Furthermore, since the protection afforded by PC plus PD 81,723 is absent in hearts pretreated with glibenclamide, we conclude that the component of ischemic PC mediated by A₁ adenosine receptors is mediated via a mechanism that is prevented by the sulfonylurea. This effect of glibenclamide clearly is not due to blockade of adenosine receptors, since in our binding studies it was found that glibenclamide is not an antagonist of adenosine receptors (Fig 5). Glibenclamide most likely antagonizes the effects of PD 81,723 by blocking K_ATP channels, which supports our original hypothesis that activation of K_ATP channels is an important step in the mechanism of ischemic PC.¹⁴

Our laboratory first suggested that K_ATP channels may be involved in ischemic PC on the basis of the finding that glibenclamide, at a low dose that is not proischemic, blocks the protective effect of ischemic PC in dogs.¹⁴ This finding has been reproduced in several other laboratories and in several different species.^{30 31 32 33} Subsequently, we found that glibenclamide also blocks the protective effect of adenosine receptor agonists.¹⁶ These findings, in combination with the studies of Kirsch et al¹³ and Ito et al,³⁴ which demonstrated by patch-clamp methodology that K_ATP channels are activated after stimulation of adenosine A₁ receptors though a G_i protein pathway, suggest that adenosine mediates ischemic PC by activating A₁ adenosine receptors, thereby opening K_ATP channels. According to this hypothesis, however, it is difficult to explain the "memory" associated with PC, since activation of a K⁺ channel is an acute short-lived event, whereas PC persists for up to 1 to 2 hours.¹ There are two potential explanations for this apparent discrepancy. First, recent studies suggest that PKC may also be a cellular mediator in ischemic PC.³⁵ Since PKC may remain active for a prolonged period of time or substrate proteins may not be dephosphorylated immediately, the memory of PC may be explained by prolonged protein phosphorylation. In support of this hypothesis, Liu et al³⁶ have recently shown in rabbit ventricular myocytes that there is an important synergistic interaction between adenosine, PKC, and the K_ATP channel, which suggests that these three components may be necessary for PC to occur. Furthermore, in pancreatic beta cells, phorbol esters activate the K_ATP channel, which causes membrane hyperpolarization.³⁷ Thus, in the context of PC, PKC may phosphorylate the K_ATP channel, or perhaps an accessory protein that influences the K_ATP channel, such that the channel opens more readily in response to a subsequent ischemic insult.³⁶ On the other hand, recent work of Przyklenk et al³⁸ has suggested that PKC is not involved in ischemic PC in the canine heart. A second possible explanation regarding the K_ATP channel and the memory of PC comes from the recent studies of Niroomand et al.³⁹ These investigators found that the responsiveness of receptor-mediated G_i protein activation is markedly reduced (38%) within the first 5 minutes of myocardial ischemia in dog hearts. However, in preconditioned hearts, G_i protein responsiveness was actually increased for at least 60 minutes after a single PC stimulus. Thus, communication between adenosine receptors and K_ATP channels, as well as other effector proteins, such as PKC, may be maintained or increased in preconditioned hearts. Finally, since PD 81,723 and PC have been shown to possess similar effects to increase adenosine receptor coupling to G proteins,^{11 39} it will be of interest in future studies to determine whether PD 81,723 added during a prolonged occlusion period can produce myocardial protection in the absence of a PC stimulus.

	Selected Abbreviations and Acronyms

 

125I-ABA = N6-(3-[125I]iodo-4-aminobenzyl)adenosine 125I-APE = 2-[2-(amino-3-[125I]iodophenyl)-ethylamino]adenosine 2.5' PC = 2.5 minutes of LAD occlusion followed by 10 minutes of reperfusion 5' PC = 5 minutes of LAD occlusion followed by 10 minutes of reperfusion AAR = area at risk DPCPX = 8-cyclopentyl-1,3-dipropylxanthine KATP channel = ATP-sensitive K+ channel LAD = left anterior descending coronary artery PC = preconditioning PD 81,723 = (2-amino-4,5-dimethyl-3-thienyl)-[3-(trifluoromethyl)phenyl]methanone PKC = protein kinase C R-PIA = R-N6-phenylisopropyladenosine TTC = 2,3,5-triphenyltetrazolium chloride

	Acknowledgments

 
This study was supported by National Institutes of Health grants HL-08311 and HL-37942. We would like to thank Anna Hsu and Jeannine Moore for their excellent technical assistance and Carol Knapp for outstanding secretarial assistance.

Received January 19, 1996; accepted April 10, 1996.

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