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Cardiovascular Research 1997 36(1):52-59; doi:10.1016/S0008-6363(97)00160-0
© 1997 by European Society of Cardiology
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Copyright © 1997, European Society of Cardiology

Evidence for a role for both the adenosine A1 and A3 receptors in protection of isolated human atrial muscle against simulated ischaemia

Cornelia S Carra, Roger J Hillb, Hiroko Masamuneb, Scott P Kennedyc, Delvin R Knightb, W.Ross Traceyb and Derek M Yellona,*

aThe Hatter Institute, Department of Academic and Clinical Cardiology, University College London Hospitals, Grafton Way, London WC1E 6DB, UK
bDepartments of Cardiovascular and Metabolic Diseases, University College London Hospitals, London, UK
cMolecular Sciences, Pfizer Inc., Groton, CT, USA

* Corresponding author. Tel.: +44 171 380 9888; Fax: +44 171 388 5095; E-mail: s.bush-cavell@ucl.ac.uk

Received 8 January 1997; accepted 29 May 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Adenosine receptor activation has been implicated in the mechanism of ischaemic preconditioning protection. Evidence suggests adenosine A1 receptor involvement, and possibly A3 receptor involvement in the rabbit. This study investigated the roles of these receptors in human preconditioning. Human A1- and A3-selective compounds were chosen based on Ki values for inhibition of N6-(4-amino-3-[125I]iodobenzyl)adenosine (125I-ABA) binding to stably expressed recombinant human A1 and A3 receptors. Cyclopentyladenosine (CPA), a 194-fold selective A1 agonist, and iodobenzylmethylcarboxamidoadenosine (IBMECA), a 10-fold selective A3 agonist were used alone and in combination with dipropylcyclopentylxanthine (DPCPX) a 62-fold selective A1 antagonist. Methods: Human atrial trabeculae were superfused with oxygenated Tyrode's solution. After stabilisation, muscles underwent one of 8 protocols (n = 6 per group), followed by 90 min of simulated ischaemia and 120 min of reoxygenation. The experimental endpoint was recovery of contractile function, presented as percentage baseline function. Results: 5 nM CPA (52.2±3.1%), 30 nM IBMECA (49.7±3.8%) and preconditioning (55.3±2.5%) produced similar functional recoveries at 120 min of reoxygenation; significantly different to controls (27.7±1.0%; P<0.05, ANOVA). When DPCPX (200 nM) was added prior to 5 nM CPA, protection was lost (31.8±0.9%), but when added prior to 30 nM IBMECA, muscles continued to be significantly protected (41.5±2.3%). Conclusions: In human atrium both A1 and A3 receptor stimulation appears to mimic ischaemic preconditioning. This may represent the first evidence for A3 receptor involvement in ‘pharmacological’ preconditioning of human myocardium.

KEYWORDS Adenosine A1 receptor; Adenosine A3 receptor; Ischemic preconditioning; Human atrium


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
When the heart is subjected to brief periods of ischaemia and reperfusion it becomes resistant to infarction. This endogenous protective mechanism is termed ‘ischaemic preconditioning’ [1], and it has been shown to occur in all animal species studied [2–6].

The mechanisms by which ischaemic preconditioning may protect the heart have been partly characterised in both animals and in the human; these include the involvement of adenosine A1 receptor activation [2], protein kinase C activation (PKC), and the possible opening of ATP-sensitive potassium (KATP) channels [7–9].

Adenosine A1 receptor activation has been implicated in the rabbit [10], the dog [11, 12], and the pig [13], but does not appear to be involved in the rat [14, 15]. The activation of another adenosine receptor subtype, the A3 receptor, has also been implicated in ischaemic preconditioning in the rabbit [16–19]and in the chick [20]. PKC involvement in ischaemic preconditioning has been alluded to in the rabbit [21]and the rat [22], but there is also evidence for a possible interaction between PKC and the KATP channel in the rabbit [23]. Although evidence for the involvement of the KATP channel in ischaemic preconditioning has been shown to exist in the pig [24], and dog [7, 8], studies in the rat [25, 26]and the rabbit [27, 28]have been conflicting.

In humans, there is evidence for the adenosine A1 receptor being involved in preconditioning [29–31]; however, there have been no studies to date investigating the possible involvement of the adenosine A3 receptor. Evidence for the involvement of PKC [9]and the KATP channel [9, 32]also exists. Using the isolated human atrial model, adenosine A1 receptor stimulation [30], PKC activation and the opening of the KATP channels [9]have all been implicated in human preconditioning.

The aim of this study was to determine whether selective adenosine A1 and A3 receptor activation protects isolated human atrial muscle from ischaemia. Human A1 and A3 selective compounds, and their respective concentrations, were based on their Ki values for inhibition of 125I-ABA binding to stably expressed recombinant human A1 and A3 receptors. Ki values determined from recombinant receptors have previously been used in the design of a similar study in rabbits [33]. To examine the role of A1 receptors, the 194-fold selective A1 agonist, CPA, was used alone and in combination with the 62-fold selective A1 antagonist, DPCPX. The role of A3 receptors was similarly examined using the 10-fold selective A3 agonist, IBMECA, such that any protection remaining in the presence of DPCPX could be attributed to activation of the A3 receptor.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Recombinant human A1 and A3 cell lines and Ki data experiments
2.1.1 Materials
Full-length human adenosine A1 and A3 receptor cDNAs subcloned into the eukaryotic expression vector pRcCMV (Invitrogen) were obtained from Dr. Peter R. Schofield (The Garvan Institute, Sydney, Australia). Chinese hamster ovary (CHO-K1) cells were obtained from the American Type Tissue Culture Collection (Rockville, MD, USA). DMEM and DMEM/F12 culture media and foetal calf serum were obtained from Gibco-BRL (Grand Island, NY, USA). 125I-ABA was prepared by New England Nuclear (Boston, MA, USA). IBMECA was synthesized at Pfizer Inc. (Groton, CT, USA). Adenosine deaminase (ADA) was obtained from Boehringer Mannheim (Indianapolis, IN, USA). CPA and DPCPX were obtained from Research Biochemicals International (Natick, MA, USA).

2.1.2 Expression studies
For stable expression studies, adenosine receptor A1 and A3 expression plasmids (20 µg) were transfected into CHO-K1 cells, or HEK 293s cells, respectively, grown in DMEM/F12 (CHO) or DMEM (HEK 293s), with 10% foetal calf serum media, using a calcium phosphate mammalian cell transfection kit (5'–3'). Stable transfectants were obtained by selection in complete media containing 500 µg/ml (CHO) or 700 µg/ml (HEK 293s) active neomycin (G418) and screened for expression by 125I-ABA binding. Stable cell lines expressing either human A1 and A3 receptors were each initially generated from a single neomycin resistant cell, therefore the stable cell lines uniformly expressed a single adenosine receptor subtype. An identical procedure for expressing rabbit A1 or A3 receptors has previously been published [34].

2.1.3 Receptor particulate fraction preparation
Cells stably expressing either human A1 or human A3 receptors were collected by centrifugation at 300xg for 5 min, the supernatant was discarded and the cell pellet was resuspended in cell buffer consisting of (mmol/l): HEPES (10), MgCl2 (5), PMSF (0.1), bacitracin (100 µg/ml), leupeptin (10 µg/ml), DNase I (100 µg/ml), ADA (2 U/ml), pH 7.4. Crude cell membranes (particulate fraction) were prepared by repeated aspiration through a 21-gauge needle, collected by centrifugation at 60 000xg for 10 min and stored in cell buffer at –80°C.

2.1.4 125I-ABA binding
Receptor particulate fractions were resuspended in incubation buffer consisting of (mmol/l): HEPES (10), EDTA (1), MgCl2 (5), pH 7.4. Binding reactions (10–20 µg particulate fraction protein) were carried out for 1 h at room temperature in 250 µl 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 fibre filters (presoaked in 0.6% polyethylenimine) using a Tomtec 96-well harvester (Orange, CT, USA). Filters were counted in a Wallac Microbeta liquid scintillation counter (Gaithersberg, MD, USA). Non-specific binding was determined in the presence of 5 µM I-ABA. Compound inhibitory constants (Ki) were calculated by fitting binding data via non-linear 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 slope. At the concentration of radioligand used in the present study (10-fold<KD), IC50=Ki [35].

2.2 Atrial experimental design
Atrial trabeculae were derived from right atrial appendages harvested from patients undergoing cardiopulmonary bypass during coronary artery bypass graft (CABG) surgery for chronic stable angina. Patient exclusion criteria for this study included pre-existing arrhythmias, right ventricular failure, oral anti-arrhythmic or oral hypoglycaemic medication. Prior ethical approval for this study was granted by the Ethics and Clinical Investigations Panel of the Middlesex Hospital.

The transport and subsequent handling of the atrial specimens was conducted in an identical manner to that previously described [9]. The time interval between harvesting of the appendage and the start of superfusion was 20 min, during which time they were transported to the laboratory in preoxygenated Tyrode's solution. In brief, the trabeculae were dissected out, mounted and superfused in an organ bath and connected to a force transducer [9].

Once the trabecula had been suspended in the organ bath, it was paced by field stimulation at 1 Hz. Platinum pacing electrodes were positioned in the organ bath on either side of the trabecula, and pacing was driven by an isolated stimulator (Digitimer DS2, Hertfordshire, UK). The pulse width was fixed at 2 ms, and the pulse amplitude was set at twice threshold (30 V). The output of the force transducer was recorded on paper (Gould RS3400 chart recorder, Ohio, USA).

At the end of the experiment, the width and length of the trabeculae were measured with an eyepiece graticule in an overhead microscope (Prior, Cambridge, UK); subsequently all specimens were weighed.

2.2.1 Simulated ischaemia
Simulated ischaemia was accomplished by superfusing the trabecula with substrate – free modified Tyrode's solution bubbled with 95% N2/5% CO2 (pH 7.24–7.34) to render it hypoxic, and rapid pacing at 3 Hz. In order to maintain constant osmolarity, whilst yielding a substrate-free superfusate, 7.0 mM choline chloride was substituted for glucose and pyruvic acid. Reoxygenation consisted of re-exposure to oxygenated normal Tyrode's solution and pacing at 1 Hz, as for the stabilisation period.

2.2.2 Measured end points
The only end point that was taken was contractile function (force of contraction as a percentage of the baseline) which was measured at intervals throughout the experiment.

2.2.3 Materials
The adenosine A1 receptor agonist CPA was dissolved in dimethylsulphoxide (DMSO) to give a stock solution which was stored at –70°C. Aliquots of this stock solution were subsequently added to 100 ml of Tyrode's solution to give a final concentration of 5 nM.

The adenosine A3 receptor agonist IBMECA was dissolved in DMSO to give a stock solution which was stored at –20°C. Aliquots of this stock solution were subsequently added to 100 ml of Tyrode's solution to give final concentrations of 10 and 30 nM.

The adenosine A1 receptor antagonist DPCPX was dissolved in DMSO to give a stock solution which was stored at –70°C. Aliquots of this stock solution were subsequently added to 100 ml of Tyrode's solution to give a final concentration of 200 nM.

We did not perform control experiments with DMSO alone, as previous work by Speechly-Dick (unpublished) at this laboratory has shown that the amount of DMSO used as vehicle in these experiments had no independent protective or detrimental effect on human atrial tissue.

2.2.4 Experimental protocol
Following suspension in the organ bath, trabeculae were paced at 1-Hz unstretched for 15 min to allow time for recovery following transport and handling. They were then gradually stretched in a stepwise manner over approximately 15 min until the maximum force of contraction was achieved. Following a further 45-min period of stabilisation, the specimens were then randomised to one of eight groups (Fig. 1).


Figure 1
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  		Fig. 1 Experimental protocols. All protocols were preceded by at least 45 min stabilisation. Eight groups were studied. All groups were subjected to 90 min of simulated ischaemia followed by 120 min of reperfusion. Preconditioning was induced by 3 min of simulated ischaemia and 7 min of reperfusion.

 
All groups eventually underwent a period of 90 min of simulated ischaemia followed by 120 min of reoxygenation. The control (C) group did not undergo any other treatment, whereas all other groups underwent treatment prior to the long simulated ischaemia: preconditioned group (PC), 3 min simulated ischaemia and 7 min reoxygenation; CPA group (CPA), 5 min exposure to 5 nM CPA, and 5 min washout with oxygenated Tyrode's solution; DPCPX group (DPCPX), trabeculae were exposed to 200 nM DPCPX for 15 min; CPA+DPCPX group (CPA+DPCPX), 5 min exposure to 200 nM DPCPX, 5 min exposure to 5 nM CPA with 200 nM DPCPX, and 5 min exposure to 200 nM DPCPX; 10 nM IBMECA group (IBMECA 10), 5 min exposure to 10 nM IBMECA, and 5 min washout with oxygenated Tyrode's solution; 30 nM IBMECA group (IBMECA 30), 5 min exposure to 30 nM IBMECA, and 5 min washout with oxygenated Tyrode's solution; and IBMECA+DPCPX group (IBMECA+DPCPX), 5 min exposure to 200 nM DPCPX, 5 min exposure to 30 nM IBMECA with 200 nM DPCPX, and 5 min exposure to 200 nM DPCPX.

The baseline force of contraction was measured just prior to the start of any intervention and was assigned the arbitrary value of 100%. The force of contraction was then measured during preconditioning or drug exposure, just prior to the 90-min simulated ischaemia, at 15-min intervals during the simulated ischaemia, and at 30-min intervals during reoxygenation. For all these time points, the force of contraction is expressed as a percentage of the baseline force of contraction.

2.2.5 Statistics
All results were expressed as mean±s.e.m. Comparisons were made between groups through time using two-way analysis of variance (ANOVA) with repeated measures, and at individual time points using one-way analysis of variance with multiple comparisons. The Fisher's test was used for multiple comparisons between the groups. A value of P≤0.05 was considered significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Human adenosine A1 and A3 Ki data
The Ki values of CPA, IBMECA and DPCPX for inhibition of 125I-ABA binding to human A1 and A3 receptors are summarized in Table 1. The adenosine receptor agonist, CPA, and adenosine receptor antagonist, DPCPX, were 194- and 62-fold selective for human A1 vs. A3 receptors, respectively. The adenosine receptor agonist, IBMECA, was 10-fold selective for A3 vs A1 receptors. Based on these data 5 nM CPA and 200 nM DPCPX were chosen as human A1 selective concentrations, and 30 nM IBMECA was chosen as a moderately selective human A3 concentration.


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  		Table 1 Ki values for human A1 and A3 receptors

 
3.1.1 Atrial trabeculae data
Samples were obtained from 37 patients with chronic stable angina (30 men and 7 women; age range 42–80 years; median age 62 years; similar mean ages in each group). No trabeculae were excluded in this study. If two suitable trabeculae could be dissected from one atrial appendage then each trabecula was allocated to one of eight groups (two sets of apparatus were used simultaneously).

The data show that there was no statistical difference in developed force between the eight groups at the end of the stabilisation period prior to entering the protocol, nor was there any difference in the length, width or weight of the atrial trabeculae used (Table 2).


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  		Table 2 Baseline data

 
3.1.2 Trabeculae controls and preconditioning data
Subjecting the control group to 90 min simulated ischaemia resulted in a dramatic reduction of the developed force which was evident within 15 min of the onset of the simulated ischaemia (Fig. 2). There was also a further reduction in the developed force over the ensuing 30 min of simulated ischaemia. In the control group, the force of contraction then plateaued until the onset of reoxygenation, during which there was a gradual, but small, increase in the force of contraction which reached a maximum of 27.7±1.0% of baseline. All other groups behaved in a similar fashion to the control group during the long period of simulated ischaemia.


Figure 2
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  		Fig. 2 Developed force, expressed as a percentage of baseline developed force with time. The comparisons shown are Control vs. PC, 5 nM CPA, 200 nM DPCPX and 5 nM CPA+DPCPX *P<0.05 vs. control (n = 6 per group).

 
Ischaemic preconditioning using simulated ischaemia and reoxygenation resulted in a significant and immediate reduction in the measured force of contraction initially (Fig. 2). In this group, at the end of the preconditioning stimulus, the force of contraction was 32.3±4.5% (P<0.05 vs. all other groups). However, the developed force of contraction underwent a dramatic recovery during the short reoxygenation phase of the preconditioning protocol to 96.8±2.0% (not significantly different to the other groups). Subsequent recovery of function during reoxygenation was significantly different to that of controls, in that at the end of the 120 min of reoxygenation contractile force was 55.3±2.5% (P<0.05 vs. control).

3.1.3 A1-mediated preconditioning
Prior treatment with CPA (52.2±3.1%) resulted in a significant improvement in the force of contraction when compared to the control group (Fig. 2). The DPCPX (28.7±1.2%) on its own showed no protection and behaved in a similar manner to the control group (27.7±1.0%) (Fig. 2). The protection afforded by CPA was completely abolished by the addition of DPCPX, the specific A1 receptor antagonist, (CPA+DPCPX 31.8±0.9% vs. CPA 52.2±3.1%; P<0.05) (Fig. 2).

3.1.4 A3-mediated preconditioning
Prior treatment with 30 nM IBMECA (49.7±3.8%) resulted in a significant improvement in the force of contraction when compared to the control group (Fig. 3). The 10 nM IBMECA group (34.0±3.3%) showed no protection and behaved in a similar manner to the control group (27.7±1.0%). The protection afforded by 30 nM IBMECA was only partially abolished by the addition of DPCPX, with the resulting protection still significantly better than controls (30 nM IBMECA+DPCPX 41.5±2.3%, 30 nM IBMECA 49.7±3.8% vs. control 27.7±1.0%; P<0.05) (Fig. 3).


Figure 3
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  		Fig. 3 Developed force, expressed as a percentage of baseline developed force with time. The comparisons shown are Control vs. PC, 30 nM IBMECA and 30 nM IBMECA+DPCPX *P<0.05 vs. control (n = 6 per group).

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
One of the possible theories of ischaemic preconditioning protection is the stimulation of the adenosine A1 receptor. Although stimulation of the A1 receptor has been shown to play a role in human ischaemic preconditioning [30], involvement of the adenosine A3 receptor remains undefined. In this study we have attempted to establish a link between A3 receptor stimulation and human ischaemic preconditioning protection.

In this study, pretreatment of the trabeculae with CPA resulted in protection which was comparable to that of ischaemic preconditioning. The 5 nM CPA dose was chosen to be the one antagonised by DPCPX, as it provided protection while maintaining A1 selectivity; the addition of DPCPX abolished the protective effects of CPA. These results provide convincing evidence that CPA brought about its protection through stimulation of the adenosine A1 receptor.

IBMECA, a moderately selective adenosine A3 agonist, produced no measurable protection at 10 nM; but at 30 nM, it resulted in protection which was comparable to that of both treatment with CPA, and preconditioning. We attempted to dissociate the effects of adenosine A3 receptor activation with IBMECA, from that of A1 receptor activation, by selectively blocking the A1 receptor with DPCPX. DPCPX (200 nM) could only reduce the protection provided by 30 nM IBMECA, leaving a statistically significant protective effect. These findings suggest that this residual protective action of IBMECA was due to its actions on the adenosine A3 receptor.

Our results confirm the findings of a previously published study by Yellon's group indicating the possible involvement of adenosine A1 receptor stimulation in ischaemic preconditioning protection of isolated human atrial tissue [30]. In this study, the A1 agonist, R-phenyl-isopropyl-adenosine (R-PIA), was used, with a similar degree of protection to that seen with preconditioning. Also the non-selective adenosine antagonist 8-p-sulphophenyltheophylline (SPT) abolished the protective effect of preconditioning without adversely affecting controls [30].

It is interesting to note that although investigators agree on the possible involvement of the adenosine A1 receptor stimulation in the rabbit heart [16], there remains a degree of controversy on whether ischaemic preconditioning is mediated via this receptor in the rat [15, 25, 36]. There is also evidence that ischaemic preconditioning involves the adenosine A3 receptor subtype in the rabbit heart [37]. Adenosine A3 receptor agonists have been shown to be equally protective as adenosine itself [17]and SPT, but not DPCPX, has been shown to block the protection conferred by A1 agonists [17–19].

Human evidence for adenosine A1 receptor involvement in the mechanism of ischaemic preconditioning exists for the atrial model [30]and in isolated cardiomyocytes [29]. Administration of the supernatant from ischaemically preconditioned cells has also been shown to protect non-preconditioned cardiomyocytes, an effect blocked by the administration of SPT [29]. Further human evidence comes from the angioplasty model, where the selective adenosine A1 receptor antagonist, Bamiphylline, abolished the reduction in anginal pain and ST elevation normally seen with the second balloon inflation [31].

Ki values for human adenosine receptor subtypes are optimally determined in a cell background expressing a single human receptor subtype, in contrast to determining Ki values using human tissue, where multiple endogenous adenosine receptor subtypes are likely to be expressed. The recombinant assay system provides Ki values for a single adenosine receptor subtype, free of potential contamination from other adenosine receptor subtypes. Thus, subtype-selective compounds can be identified with confidence and their Ki values can be used to choose appropriate subtype-selective concentrations for subsequent in vitro experiments. It is necessary to extrapolate these recombinant Ki values back to endogenous tissue, and since it has been shown, using cDNAs and Northern blotting, that A3 receptors are expressed in human cardiac tissue [38, 39], we submit that the extrapolation of Ki values in the present study is justified and have therefore interpreted the cardioprotective effects of IBMECA at 30 nM (in the presence of A1 blockade) as supporting evidence for A3-mediated cardioprotection in human atrial tissue.

The A3 receptor shows much lower homology between species at the amino acid level than the A1 receptor. In addition, there are remarkable differences in the affinity of adenosine agonists and antagonists for the A3 receptor of different species [38, 40]. Thus the studies investigating the role of the A3 receptor in preconditioning the rabbit [17, 18], using non-selective concentrations of agonists and antagonists based on different species receptor data (rat and sheep), may require re-examination.

Despite the original definition of ischaemic preconditioning relating to infarct size limitation [1], improved myocardial contractile function appears dependent on infarct size limitation, rather than an anti-stunning effect [41, 42]. In this isolated trabecula model, it is possible that cell death may occur following 90 min of simulated ischaemia. It has been shown to be a reliable model producing consistent results using both animal and human tissues [9, 30, 43].

The data in this study suggest a potentially important role for adenosine A3 receptor stimulation in human pharmacological preconditioning. Further studies in this field are required in order to establish the relevance of adenosine A3 stimulation to ischaemic protection of human myocardium, especially the left ventricle. Defining which specific adenosine receptor subtype brings about ischaemic protection may lead to the development of novel cardioprotective agents.

Time for primary review 27 days.


   Acknowledgements
 
Miss C. Carr FRCS was funded by a British Heart Foundation Junior Research Fellowship. For technical assistance we acknowledge the following people: Joseph Oleynek, Ocean Pellett, Steven Hawrylik, Christopher Hoth. All drugs were provided by Pfizer Inc., Groton, CT, USA. We are grateful to the Hatter Foundation for continued support.


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

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