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Cardiovascular Research 1998 40(1):138-145; doi:10.1016/S0008-6363(98)00112-6
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

Selective activation of adenosine A3 receptors with N6-(3-chlorobenzyl)-5'-N-methylcarboxamidoadenosine (CB-MECA) provides cardioprotection via KATP channel activation

W.Ross Traceya,*, William Mageea, Hiroko Masamuneb, Joseph J. Oleyneka and Roger J. Hilla

aDepartment of Cardiovascular and Metabolic Diseases, Central Research Division, Pfizer Inc., Groton, CT 06340, USA
bDepartment of Medicinal Chemistry, Central Research Division, Pfizer Inc., Groton, CT 06340, USA

* Corresponding author. Tel.: +1 (860) 441 1761; Fax: +1 (860) 441 5719; E-mail: w_ross_tracey@groton.pfizer.com

Received 5 November 1997; accepted 10 March 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: The aim of this study was to characterize the adenosine A3 receptor agonist, N6-(3-chlorobenzyl)-5'-N-methylcarboxamidoadenosine (CB-MECA), evaluate its ability to reduce myocardial ischemia/reperfusion injury and determine the role of KATP-channel activation in A3 receptor-mediated cardioprotection. Methods: Binding affinities and adenylate cyclase inhibition were examined in CHO cells expressing rabbit recombinant adenosine A1 or A3 receptors. Infarct size (normalized for area-at-risk;% IA/AAR) was measured in buffer-perfused rabbit hearts exposed to 30-min regional ischemia and 120 min of reperfusion. Results: CB-MECA was 100-fold selective for A3 vs. A1 receptors (A3 Ki: 1 nM; A1 Ki: 105 nM). Five-min perfusion with CB-MECA before ischemia/reperfusion elicited a concentration-dependent reduction in infarct size (EC50: 0.3 nM). The CB-MECA-dependent cardioprotection (control: 58±2; CB-MECA: 21±3% IA/AAR) was unchanged by an A1-selective concentration of the antagonist, BWA1433, but was completely prevented (P<0.05) by a nonselective (A1/A3) concentration (55±6% IA/AAR). The KATP channel inhibitors, glibenclamide and 5-HD, had no effect on control infarct size, yet significantly (P<0.05) blunted the CB-MECA-dependent cardioprotection (glibenclamide: 49±6; 5-HD: 58±4% IA/AAR). Conclusions: CB-MECA is a novel 100-fold A3 receptor-selective agonist which should prove useful for elucidating A3-dependent mechanisms in the rabbit heart. Selective stimulation of adenosine A3 receptors with CB-MECA reduces myocardial ischemia/reperfusion injury via a mechanism which involves activation of KATP channels.

KEYWORDS Rabbit; Heart; Adenosine; Infarction; KATP channel; Preconditioning; Receptors; Reperfusion


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Ischemic preconditioning [1]refers to a phenomenon in which a brief ischemic event protects the myocardium from a subsequent prolonged ischemic insult. The ability of adenosine to provide cardioprotection from ischemic injury was first proposed by Ely et al. [2], while subsequent studies by Downey and colleagues [3–6]and others [7–9]indicated adenosine's cardioprotective effect was mediated by activation of adenosine A1 and/or A3 receptors. Selective stimulation of A3 receptors was recently demonstrated by our laboratory to provide cardioprotection from ischemic injury in the isolated rabbit heart [10], which was subsequently confirmed in vivo by Auchampach et al. [11]. Furthermore, the data of Auchampach et al. [11]demonstrated that unlike for A1 receptors, A3 receptor-mediated cardioprotection is achieved in vivo in the absence of hemodynamic side-effects, and is therefore therapeutically more promising. Recent reports have described the presence of A3 receptor mRNA in isolated rabbit cardiomyocytes [12], as well as protection of isolated cardiomyocytes from simulated ischemia by selective A3 receptor stimulation [13, 14].

KATP channel activation protects the myocardium from ischemia/reperfusion injury; compounds which open these channels have been demonstrated to significantly reduce infarct size [15–17]or enhance postischemic recovery of contractile function [15, 18–20], while KATP channel inhibitors prevent ischemic preconditioning in several species [21–25], including man [26, 27]. Furthermore, these channels are involved in mediating the cardioprotection observed following adenosine A1 receptor stimulation [28–30]. Although A3 and A1 receptors appear to share similar signal transduction pathways [31], the pathways responsible for mediating A3 receptor-dependent cardioprotection have not been elucidated, possibly due to the lack of a potent, selective rabbit A3 receptor agonist.

We have previously reported on the species differences in the affinities of adenosine analogues for adenosine A1 and A3 receptors and the impact of these differences on studies of adenosine receptor function in the heart [32]. Thus far, the most selective compound identified for the rabbit A3 receptor is IB-MECA, with 15-fold selectivity vs. the A1 receptor [32]. This limited degree of selectivity potentially hampers the design/interpretation of both in vitro and in vivo studies; thus, a rabbit A3 receptor-selective agonist of greater selectivity vs. the A1 receptor would be of benefit. During the course of evaluation of several compounds reported to possess selectivity for the rat A3 receptor [33], we identified N6-(3-chlorobenzyl)-5'-N-methylcarboxamidoadenosine (CB-MECA) as a potent and selective agonist for the rabbit A3 receptor. We now report the kinetic and functional characteristics of CB-MECA, using cell lines which express rabbit recombinant adenosine A1 and A3 receptors [32], and the isolated rabbit heart [10]. Given the therapeutic potential of A3 receptor-mediated cardioprotection, the increased selectivity of CB-MECA for the rabbit A3 receptor (100-fold vs. A1) makes this compound a potentially useful experimental tool to examine the associated signal transduction pathways. Thus, we also used CB-MECA to elucidate the role of KATP channel activation in A3 receptor-mediated cardioprotection.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Receptor membrane preparation
Chinese hamster ovary (CHO-K1) cells stably transfected with either rabbit A1 or A3 receptors [32]were washed with Ca/Mg-free phosphate-buffered saline (PBS) and were collected by centrifugation at 300xg for 5 min. The supernatant was discarded and the cell pellet was resuspended in incubation buffer consisting of (mM) Tris (50), NaCl (120), MgCl2 (10), KCl (5), CaCl2 (2), PMSF (0.1) bacitracin (100 µg/ml), leupeptin (10 µg/ml), DNase I (100 µg/ml), adenosine deaminase (ADA, 2 U/ml), pH 7.4. Crude cell membranes were prepared by repeated aspiration through a 21-gauge needle, collected by centrifugation at 60 000xg for 10 min and stored in incubation buffer at –80°C.

2.2 125I-ABA binding
Binding reactions (10–20 µg membrane protein) were carried out for one hour at room temperature in 250 µl of incubation buffer containing 0.1 nM 125I-ABA (2200 Ci/mmol) and the appropriate concentration of compound. The reaction was stopped by rapid filtration with ice-cold PBS, through glass-fiber filters (presoaked in 0.6% polyethylenimine) using a Tomtec 96-well harvester (Orange, CT). Filters were counted in a Wallac Microbeta liquid scintillation counter (Gaithersberg, MD). Nonspecific binding was determined in the presence of 5 µM I-ABA. Affinity constants (Ki) were calculated for each individual experiment by fitting binding data via nonlinear least-squares regression analysis to the equation:% Inhibition=100/[1+(10C/10X)D], where X=log [drug concentration], C (IC50)=log [drug concentration at 50% inhibition], and D=the Hill coefficient. At the 0.1 nM concentration of 125I-ABA used in competition experiments (70-fold<125I-ABA KD for rabbit A1, 90-fold<125I-ABA KD for rabbit A3 [32], IC50~=Ki [34].

2.3 cAMP accumulation
CHO-K1 cells stably transfected with either rabbit A1 or A3 receptors were washed with PBS and then detached with 1.0 mM EDTA/PBS. Cells were collected by centrifugation at 300xg for 5 min and the supernatant discarded. The cell pellet was dispersed and resuspended in cell buffer (DMEM/F12 containing 10 mM HEPES, 20 µM RO-20-1724 and 1 U/ml ADA). Following preincubation of cells (100 000/well) for 10 min at 37°C, 5 µM forskolin, with or without the appropriate concentration of test compound, was added and the incubation was continued for 10 min. Reactions were terminated by the addition of 0.1 N HCl followed by centrifugation at 2000xg for 10 min. Sample supernatants were removed and cAMP levels determined by radioimmunoassay (New England Nuclear,). The basal and control forskolin-stimulated cAMP accumulation (pmol/ml/100 000 cells) was routinely 1 and 30, and 1 and 15 for cells containing rabbit A1 or rabbit A3 receptors, respectively.

2.4 Langendorff model
This investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985). Male New Zealand White rabbits (3–4 kg; Hazelton Research Products, Denver, PA) were anesthetized by i.v. administration of sodium pentobarbital (30 mg/kg; marginal ear vein), followed by intubation and ventilation with 100% O2 using a positive pressure ventilator. A left thoracotomy was performed, the heart exposed, and a snare (2-0 silk) placed loosely around a branch of the left coronary artery. The heart was rapidly removed from the chest, mounted on a Langendorff apparatus, and maintained by retrograde perfusion (nonrecirculating) with a modified Krebs solution (NaCl 118.5 mM, KCl 4.7 mM, MgSO4 1.2 mM, KH2PO4 1.2 mM, NaHCO3 24.8 mM, CaCl2 2.5 mM and glucose 10 mM) at a constant pressure of 80 mmHg and a temperature of 37°C. Perfusate pH was maintained at 7.4–7.5 by bubbling with 95% O2/5% CO2. The temperature of the hearts was maintained by suspending them in heated, water-jacketed organ baths. A fluid-filled latex balloon was inserted in the left ventricle and connected by stainless steel tubing to a pressure transducer; the balloon was inflated to provide a systolic pressure of 80–120 mmHg, and a diastolic pressure ≤10 mmHg. Heart rate (HR) and left ventricular diastolic and systolic pressures were recorded using a PO-NE-MAH Data Aquisition and Archive System (Gould Instrument Systems, Valley View, OH); left ventricular developed pressures (LVDP) were calculated by subtracting the left ventricular diastolic pressure from the left ventricular systolic pressure. Coronary flow rates (CF) were determined using an in-line flow probe (Transonic Systems, Inc., Ithaca, NY); all coronary flows were normalized for heart weight. These parameters were continuously monitored for the duration of the experiment. Hearts were allowed to equilibrate for 30 min; if stable left ventricular pressures within the parameters outlined above were not observed, the heart was discarded. Hearts were not paced, unless the heart rate fell below 180 bpm prior to the 30-min period of regional ischemia; in this case, the heart was paced at 200 bpm, which was the average spontaneous rate observed.

2.5 Langendorff experimental protocols
CB-MECA was perfused through the heart for 5 min, followed by a 10-min wash-out (Fig. 1A). Thirty min of regional ischemia was then produced by tightening the snare around the branch of the coronary artery. At the end of this period, the snare was released and the heart reperfused for an additional 120 min. In experiments in which either the A1 receptor-selective antagonist, BWA1433 (Table 1), or the KATP channel antagonists, glibenclamide or 5-HD, were used, the antagonists were perfused through the heart for a total of 15 min, beginning 5 min before the CB-MECA perfusion and continuing until 5 min after CB-MECA perfusion was stopped (Fig. 1B). A 5-min wash-out period followed before the ischemia/reperfusion. Control experiments were performed by perfusing only the antagonists through the heart for 15 min, without CB-MECA present. All drugs were added to dedicated drug perfusate reservoirs.


Figure 1
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  		Fig. 1 Langendorff experimental protocol. A. Hearts were perfused for 5 min with CB-MECA. This was followed by 30 min of regional ischemia and 120 min of reperfusion. B. When an A1 receptor antagonist (BWA1433) or KATP channel inhibitor (glibenclamide, 5-HD) was used, it was perfused through the heart for 15 min, either alone or in combination with a 5-min perfusion period of CB-MECA. This was followed by 30 min of regional ischemia and 120 min of reperfusion.

 

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  		Table 1 Ki and IC50 values for reference compounds

 
2.6 Determination of infarct size
At the end of the 120-min reperfusion period, the coronary artery snare was tightened and a 0.5% suspension of fluorescent zinc cadmium sulfate particles (1–10 µM) was perfused through the heart to delineate the area-at-risk (nonstained) for infarct development. The heart was removed from the Langendorff apparatus, blotted dry, weighed, wrapped in aluminum foil and stored overnight at –20°C. Frozen hearts were sliced into 2-mm transverse sections and incubated with 1% triphenyl tetrazolium chloride in phosphate-buffered saline for 20 min at 37°C to delineate noninfarcted (stained) from infarcted (nonstained) tissue. The infarct area and the area-at-risk were calculated for each slice of left ventricle using a precalibrated image analyzer (Optomax V Image Analyzer, AMS, Burlington, MA), followed by adding the values for each tissue slice to obtain the total infarct area and total area-at-risk for each heart. To normalize the infarct area for differences in the area-at-risk between hearts, the infarct size was expressed as the ratio of infarct area vs. area-at-risk (% IA/AAR).

2.7 Data expression and analysis
Data are expressed as the mean±SE. Comparisons of Ki values and hemodynamic variables were performed by t-test, while group% IA/AAR values were compared using a Mann-Whitney test with a Bonferroni correction for multiple comparisons. A P value of less than 0.05 was considered statistically significant.

2.8 Drugs and drug preparation
125I-ABA was prepared by New England Nuclear (Boston, MA). N6-(4-amino-3-iodobenzyl)adenosine (I-ABA), N6-(3-chlorobenzyl)-5'-N-methylcarboxamidoadenosine (CB-MECA) and 8-(4-carboxyethenylphenyl)-1,3-dipropylxanthine (BWA1433) were synthesized at Pfizer Central Research (Groton, CT). Adenosine deaminase (ADA) was obtained from Boehringer Mannheim (Indianapolis, IN). 4-[(3-Butoxy-4-methoxyphenyl)methyl]-2-imidazolidinone (RO-20-1724), glibenclamide and 5-hydroxydecanoate (5-HD) were obtained from Research Biochemicals International (Natick, MA). All drugs administered to the isolated hearts were dissolved in DMSO and diluted in buffer; the final DMSO concentration was less than 0.1%, which had no effect on infarct size [10]. DMEM/F12 culture media and fetal calf serum were obtained from Gibco-BRL (Grand Island, NY).


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
Fig. 2A illustrates the concentration-dependent inhibition of 125I-ABA binding to rabbit A1 and A3 receptors by CB-MECA. CB-MECA demonstrated selective inhibition of binding to rabbit A3 receptors (100-fold, P<0.05) with Ki values (n=5) of 105±44 nM and 1.0±0.7 nM for A1 and A3 receptors, respectively (Table 1). The Hill coefficients for adenosine inhibition of 125I-ABA binding to A1 and A3 receptors were 0.97±0.1 and 1.1±0.1, respectively.


Figure 2
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  		Fig. 2 A. CB-MECA inhibition of 125I-ABA binding to rabbit A1 ({blacktriangleup}) and A3 (bullet) receptors. 125I-ABA binding to CHO-K1 cells stably transfected with rabbit A1 or A3 receptors was measured as described in Section 2. Data are presented as mean±S.E. of 6 individual experiments. Smooth curves were fitted to the data via nonlinear least-squares regression analysis as described in Section 2. B. CB-MECA inhibition of forskolin-stimulated cAMP accumulation in CHO-K1 cells expressing rabbit A1 ({blacktriangleup}) or A3 (bullet) receptors. cAMP measurement was as described in Section 2. Data are presented as mean of duplicate determinations of a representative experiment. Smooth curves were fitted to the data via nonlinear least-squares regression analysis to the equation:% Forksolin-stimulated cAMP=100/[1+(10X/10C)D], where X=log [drug concentration], C (IC50)=log [drug concentration at 50% inhibition], and D=the Hill slope.

 
The functional activity of CB-MECA was characterized via measurement of forskolin-stimulated cAMP accumulation in CHO-K1 cells stably transfected with either rabbit A1 or A3 receptors. CB-MECA produced a concentration-dependent inhibition of forskolin-stimulated cAMP accumulation with IC50 values of 186 nM and 2 nM for rabbit A1 and A3 receptors, respectively (Fig. 2B, Table 1), indicating CB-MECA is a potent and selective A3 receptor agonist.

Baseline heart rate-, coronary flow- and left ventricular-developed pressure values for each of the treatment groups were similar prior to the regional ischemia and are shown in Table 2. Left ventricular-developed pressures were significantly (P<0.05) reduced in all groups by occlusion of the coronary artery, confirming that ischemia was achieved in all groups. Similarly, coronary flows were significantly reduced in all groups, except those treated with glibenclamide (Table 2); this may reflect a direct vasoconstrictor effect of glibenclamide on the coronary vessels. Area-at-risk expressed as a percent of left ventricular area was 47±3% (n=8) for the control group; other groups did not differ significantly (P<0.05) from the control group (Table 3).


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  		Table 2 Hemodynamic data from isolated rabbit hearts

 

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  		Table 3 % Area-at-risk/left ventricle (% AAR/LV) data from isolated rabbit hearts

 
CB-MECA elicited a concentration-dependent reduction in infarct size in the isolated rabbit hearts (Fig. 3), with an EC50 of 0.3 nM. The maximum reduction in infarct size was 67% (control: 58±2; 20 nM CB-MECA: 19±5% IA/AAR). At an A1 receptor-selective concentration (50 nM) [10, 32], BWA1433 had no effect on the cardioprotection elicited by 2 nM CB-MECA (31±5 and 21±3% IA/AAR for ±BWA1433, respectively). However, at a higher, nonselective concentration (5 µM), which blocks both A1 and A3 receptors [10, 32], BWA1433, completely prevented the CB-MECA-dependent reduction in infarct size (55±6 and 21±3% IA/AAR for ±BWA1433, respectively). We have previously demonstrated that BWA1433 alone has no effect on infarct size in this model [10].


Figure 3
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  		Fig. 3 Effect of CB-MECA on% IA/AAR in isolated rabbit hearts. CB-MECA was perfused through the hearts for 5 min, in the presence or absence of BWA1433, followed by 30 min of regional ischemia and 120 min of reperfusion, as described in Section 2. Infarct areas and area-at-risk were determined by image analysis and infarct area was normalized for area-at-risk (% IA/AAR). CB-MECA elicited a concentration-dependent reduction in infarct size, which was not altered by an A1 receptor-selective concentration (50 nM) of BWA1433. A higher, nonselective (A1/A3) concentration (5 µM) of BWA1433 significantly (P<0.05) reduced the CB-MECA-dependent cardioprotection. Data from each heart are presented, along with the mean±SE for each group. n=4–9; * significantly different (P<0.05) from control; {dagger} significantly different (P<0.05) from 2 nM CB-MECA.

 
Control experiments indicated the KATP channel inhibitors, glibenclamide and 5-HD, had no significant effect on infarct size when administered alone to isolated hearts (Figs. 4 and 5Go). When combined with 2 nM CB-MECA however, both glibenclamide and 5-HD significantly (P<0.05) inhibited the cardioprotection provided by the A3 receptor-selective compound (CB-MECA: 21±3;+glibenclamide: 49±6;+5-HD: 58±4% IA/AAR) (Figs. 4 and 5Go).


Figure 4
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  		Fig. 4 Effect of glibenclamide on CB-MECA-dependent cardioprotection in isolated rabbit hearts. CB-MECA (2 nM) was perfused through the hearts for 5 min, in the presence or absence of 1 µM glibenclamide, followed by 30 min of regional ischemia and 120 min of reperfusion, as described in Section 2. Infarct areas and area-at-risk were determined by image analysis and infarct area was normalized for area-at-risk (% IA/AAR). Glibenclamide significantly (P<0.05) inhibited the CB-MECA-dependent cardioprotection. Data from each heart are presented, along with the mean±SE for each group. n=5–9; * significantly different (P<0.05) from control; {dagger} significantly different (P<0.05) from 2 nM CB-MECA.

 

Figure 5
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  		Fig. 5 Effect of 5-HD on CB-MECA-dependent cardioprotection in isolated rabbit hearts. CB-MECA (2 nM) was perfused through the hearts for 5 min, in the presence or absence of 20 µM 5-HD, followed by 30 min of regional ischemia and 120 min of reperfusion, as described in Section 2. Infarct areas and area-at-risk were determined by image analysis and infarct area was normalized for area-at-risk (% IA/AAR). 5-HD significantly (P<0.05) inhibited the CB-MECA-dependent cardioprotection. Data from each heart are presented, along with the mean±SE for each group. n=6–9; * significantly different (P<0.05) from control; {dagger} significantly different (P<0.05) from 2 nM CB-MECA.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The present study identifies CB-MECA as the most potent and selective rabbit A3 receptor agonist reported to date, confirms A3 receptors mediate protection from myocardial ischemia/reperfusion injury, and provides the first evidence supportive of KATP channel mediation of A3 receptor-dependent cardioprotection.

CB-MECA was found to both displace 125I-ABA binding from rabbit recombinant A3 receptors and inhibit A3 receptor-mediated adenylate cyclase activity with high affinity/potency (Ki/IC50: 1–2 nM). Furthermore, 100-fold selectivity vs. A1 receptors was observed in both assays. Thus CB-MECA is a potent and highly selective rabbit A3 agonist and represents a 6–7-fold improvement in A3 receptor selectivity over IB-MECA [32].

When perfused through the isolated rabbit heart, CB-MECA elicited a concentration-dependent reduction in infarct size. The maximum cardioprotection was attained at a concentration of 2 nM, with an EC50 of 0.3 nM; this represents a 9-fold increase in potency over IB-MECA (2.7 nM) in this model [10]. The CB-MECA-dependent cardioprotection was confirmed to be A3 receptor-mediated by using the adenosine receptor antagonist, BWA1433. At a concentration of 50 nM, BWA1433 is A1 receptor-selective [10, 32], and did not affect the reduction in infarct size afforded by CB-MECA. However, at a concentration of 5 µM, which blocks both A1 and A3 receptors [10, 32], BWA1433 completely inhibited the CB-MECA-dependent cardioprotection. Thus, CB-MECA selectively stimulates A3 receptors in the isolated rabbit heart and protects the myocardium from ischemic injury, confirming our previous studies with IB-MECA. As we have pointed out previously [10], while it would be desirable to demonstrate blockade of an A3 agonist's cardioprotective effect with a selective A3 receptor antagonist, such a compound has not been identified for the rabbit. Nonetheless, due to the increased A3 receptor selectivity of CB-MECA (vs. IB-MECA), this compound should prove to be a useful tool with which to delineate the A3 receptor-dependent pathways involved in mediating cardioprotection.

One potential effector through which A3 receptors may provide protection from myocardial ischemia/reperfusion injury is the KATP channel. Several studies point to an involvement of KATP channels in both ischemic preconditioning [21–27]and adenosine-mediated cardioprotection [35–37]. A1 receptor stimulation has been reported to either directly [38]or secondarily (following PKC activation [27, 39, 40]) open KATP channels; the opening of these channels mediates A1 receptor-dependent cardioprotection [28–30]. Since A1 and A3 receptors appear to share similar signal transduction pathways [31], we used CB-MECA to investigate whether KATP channel activation was also involved in A3 receptor-dependent cardioprotection.

Two structurally distinct inhibitors of KATP channels, glibenclamide [41]and 5-HD [42, 43], were used in the present study, both of which have been used previously to elucidate the role of KATP channels in A1-mediated cardioprotection [28–30]. While glibenclamide inhibits KATP channels in several tissues or cell types [19, 41, 44], 5-HD appears thus far to be relatively selective for cardiac KATP channels [19, 42, 43]. Our observations would seem to confirm these relative selectivities, as glibenclamide, but not 5-HD, reduced coronary flow in the isolated hearts. When evaluating their effect on infarct size, neither KATP channel inhibitor had a significant influence when administered alone. However, when combined with CB-MECA, both glibenclamide and 5-HD significantly inhibited the cardioprotection elicited by this A3 receptor agonist. Therefore, these data strongly suggest that KATP channel opening mediates A3 receptor-dependent protection from ischemia/reperfusion injury in isolated rabbit hearts.

Although our data provide pharmacological evidence for KATP channel mediation of A3 receptor-dependent cardioprotection in the whole heart, unequivocal demonstration of this mechanism in the cardiomyocyte will require direct channel recordings in the presence of a selective A3 receptor agonist, as has been shown for the A1 receptor in rat ventricular myocytes [38]. It also remains to be determined whether KATP channel opening is a direct effect of A3 receptor stimulation or subsequent to PKC activation (as observed following A1 receptor stimulation); however, a PKC inhibitor has been reported to inhibit A3 receptor-mediated cardioprotection in vivo [11].

In conclusion, the present study confirms the role of the adenosine A3 receptor in protecting the heart from ischemia/reperfusion injury and demonstrates the involvement of KATP channels in A3 receptor-mediated cardioprotection using the novel rabbit A3 receptor-selective agonist, CB-MECA. This compound will be a useful experimental tool to further examine the signal transduction pathways involved in A3 receptor-mediated cardioprotection, including the interaction of A3 receptors and KATP channels with other downstream effectors. Such studies will be of value given the therapeutic potential of A3 receptor-mediated cardioprotection.

Time for primary review 28 days


   Acknowledgements
 
The authors would like to thank Drs. Delvin Knight and Allan Buchholz for critical review of the manuscript and helpful feedback.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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