INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

ADENOSINE EXERTS its well-described cardiovascular effects through interaction with at least four known adenosine receptor subtypes: A₁, A_2A, A_2B, and A₃ adenosine receptors (2, 8, 18, 26, 32, 35). Whereas A₁ adenosine receptor activation results in negative chronotropic, dromotropic, and inotropic effects as well as protective antiadrenergic responses (2, 8, 32), activation of A_2A receptors is largely responsible for coronary vasodilation (1, 3, 13, 16, 26, 27, 33, 35, 36). It has been speculated that A_2B receptor activation contributes to adenosine-induced coronary vasodilation (13, 22), and the recent demonstration of A_2B receptors in the coronary endothelium (31) supports this possibility. However, the lack of potent and selective A_2B adenosine receptor agonists and antagonists leaves only indirect evidence of A_2B-mediated coronary vasodilation (17, 22, 38), and the relative roles of the A₂ receptor subtypes in regulating coronary flow and myocardial function remain incompletely understood (35).

Recent data indicate that coregulatory interactions between the different adenosine receptor subtypes are more complex than previously appreciated. For instance, the demonstration that A_2A adenosine receptors are present on ventricular myocytes (7, 20, 23, 37) has led to strong evidence that A_2A receptor activation attenuates A₁ receptor-mediated antiadrenergic activity (28) and increases myocardial contractility in isolated hearts independent of effects on coronary flow (24). It is also becoming clear that A_2A adenosine receptor activation protects against ischemia-reperfusion (particularly during reperfusion) in the heart (5, 21) and other tissues (12, 29, 30), and the mechanisms involved are being actively investigated. It is not known whether A_2B receptor activation modulates these protective effects, but tissue distribution of this receptor subtype suggests a coregulatory role.

The demonstration that A_2B adenosine receptor activation causes vasodilation in other tissues (14, 34) and augments contractility in isolated ventricular myocytes (20) suggests that A_2B receptors may indeed modify these cardiac effects. Yet, without pharmacological ligands that are both selective and potent at A_2B receptors, isolating the unique effects of A_2B activation has remained difficult. The advent of targeted gene deletion allows more precise examination of these receptor-mediated responses than previously possible through agonist/antagonist studies alone. With the combination of receptor knockout technology and the traditional receptor-ligand approach, the physiological roles of A₂ adenosine receptor subtypes can be elucidated despite the unavailability of selective and potent A_2B receptor agonists and antagonists.

The present study examines coronary flow and functional responses (left ventricular developed pressure and heart rate) to currently available adenosine analogs in isolated Langendorff-perfused mouse hearts. Comparison is made between hearts from wild-type (WT) and A_2A adenosine receptor knockout (A_2AKO) mice to single out A_2A-mediated responses and "uncover" any contribution to these effects by A_2B adenosine receptor activation. We hypothesized that adenosine-mediated coronary vasodilation and functional responses would be attenuated in murine A_2AKO hearts.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

A_2AKO mice. All animals were cared for in accordance with protocol approved by the Animal Care and Use Committee of the Brody School of Medicine at East Carolina University. A_2AKO and WT mice were raised in the Institute of Experimental Medicine (19). To homogenize the genetic background of the mice, the first generation heterozygotes were bred for 14 generations on a CD1 (Charles River, France) outbred background, with selection for the mutant A_2A gene at each generation by PCR. Fourteenth-generation heterozygotes were bred together to generate the A_2AKO and WT. All animals used in a given experiment originated from the same breeding series and were matched for age and weight. Standard laboratory food and water were available ad libitum. Temperature was held constant at 23 ± 2°C; humidity was 60 ± 10%. An inverted light-dark cycle of 12:12 h was used (lights off at 1700 h). Experiments were conducted in accordance with national legislation and with the Declaration of Helsinki regarding the use of experimental animals.

Langendorff isolated heart model. Isolated heart experiments were performed in accordance with the methods of Matherne and Headrick as previously described (11, 15, 25, 38). Animals were anesthetized with intraperitoneal pentobarbital sodium (50 mg/kg), a thoracotomy was performed, and the hearts were excised into heparinized (5 U/ml) ice-cold perfusate. The aorta was rapidly cannulated with a 20-gauge, blunt-ended needle, and retrograde coronary perfusion was initiated at constant pressure of 80 mmHg with modified Krebs bicarbonate buffer containing (in mM) 120 NaCl, 25 NaHCO₃, 4.7 KCl, 1.2 KH₂PO₄, 2.5 CaCl₂, 1.2 Mg₂SO₄, 15 glucose, 2 pyruvate, and 0.05 EDTA. The perfusate was equilibrated with 95% O₂-5% CO₂ at 37°C, giving a pH of 7.4 and PO₂ of ~550 mmHg. The left atrium was removed, and the left ventricle was vented with a small polyethylene apical drain. A fluid-filled balloon constructed of plastic film was inserted into the left ventricle across the mitral valve and connected to a pressure transducer permitting continuous measurement of left ventricular pressure. Hearts were immersed in a water-jacketed bath maintained at 37°C, and the ventricular balloon was filled to yield a left ventricular diastolic pressure of 2-5 mmHg. Coronary flow was continuously monitored via a Doppler flow probe (Transonic Systems; Ithaca, NY) located in the aortic perfusion line. Aortic and left ventricular developed pressures were recorded on a MacLab multichannel data acquisition system (ADInstruments; Castle Hill, Australia) connected to a MacIntosh G4 computer. The ventricular pressure signal was digitally processed (using MacLab Chart software version 3.5.6, ADInstruments) to yield diastolic and systolic pressures as well as heart rate.

Experimental protocol. All hearts were equilibrated for 30 min before experimentation. Coronary flow, heart rate, and developed pressure (systolic pressure minus diastolic pressure) were examined in WT and A_2AKO hearts, and baseline data for these parameters were sampled at the end of equilibration. Dose-response relationships were constructed for adenosine, 5'-N-ethyl-carboxamidoadenosine (NECA, a nonselective adenosine receptor agonist), and 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethyl-carboxamidoadenosine (CGS-21680, a selective A_2A adenosine receptor agonist) by infusing these agonists into the coronary perfusate through an injection port directly proximal to the aortic cannula. Infusion rate was controlled to a maximum of 1% of coronary flow with a microinjection infusion pump (Harvard Apparatus; Holliston, MA). After baseline data were acquired, each heart was exposed to progressively increasing concentrations of a single agonist to develop a dose-response relationship. Each concentration of agonist was infused for 5 min (with plateau effect being achieved uniformly between 3 and 5 min), and data were sampled at the end of the 5-min infusion period. After the infusion of each concentration of agonist, a minimum of 5 min of perfusion was allowed for complete drug washout (with recovery to baseline parameters occurring uniformly between 3 and 5 min). Data were again sampled at the end of each washout period and used as a reference for normalizing response to subsequent agonist concentration. Notably, the washout period was sufficient to fully replace the volume of superfusate present in the water-jacketed bath, assuring complete clearance of each preceding dose before infusing a subsequent concentration of agonist.

Antagonist experiments. A subset of experiments was performed in A_2AKO hearts to block the observed NECA-induced changes in coronary flow and developed pressure. Alloxazine, a selective A_2B adenosine receptor antagonist, was used for these experiments. A_2AKO hearts were dissected, cannulated, and equilibrated as described above. After baseline data for each heart were sampled at the end of the 30-min equilibration period, 100 nM NECA was infused at 1% of coronary flow for 5 min, and the plateau effect on coronary flow, heart rate, and left ventricular developed pressure was recorded. This initial infusion of NECA was allowed to wash out for 10 min and then 10 µM alloxazine was infused at 1% of coronary flow for 15 min. Ten minutes into the alloxazine infusion, data were sampled and normalized as a new "baseline," and 100 nM NECA was added to the coronary perfusate at 1% of coronary flow for the remaining 5 min of the alloxazine infusion. Data were sampled at the end of this two-drug infusion for comparison with data resulting from infusion of NECA alone. After alloxazine and NECA were allowed to wash out for 10 min, a repeat infusion of 100 nM NECA was performed for comparison with the first NECA infusion.

Chemicals. A stock solution of adenosine (Sigma, St. Louis, MO) was prepared using distilled water. Stock solutions of NECA (Sigma), CGS-21680 (Sigma), and alloxazine {benzo[g]pteridine-2,4 (1H,3H)-dione; RBI} were prepared in dimethyl sulfoxide (DMSO, Sigma), and serial dilutions to desired concentrations were made in distilled water. (A subset of experiments in hearts from balb/c mice demonstrated that the effect of DMSO at 1% of coronary flow for 5 min elicited less than a 5% increase in baseline coronary flow; data not shown.) All stated chemical concentrations reflect actual drug concentration when delivered as 1% of coronary flow.

Statistical analysis. Baseline functional data for WT and A_2AKO groups were analyzed by t-test. Differences in dose response between WT and A_2AKO groups at individual agonist concentrations were analyzed by one-way ANOVA with Bonferroni's correction for multiple comparisons applying Student-Neuman-Keuls post hoc test. Differences in dose response within each group were analyzed by one-way repeated-measures ANOVA with Bonferroni's correction for multiple comparisons. EC₅₀ values were derived as previously described (11, 15) by plotting responses for individual hearts in each group and fitting the following single-site, four-parameter logistic equation to the data
where A is the response at zero dose (baseline preinfusion value), B is the response at infinite dose (maximal response), [D] is the drug concentration, EC₅₀ is the effective concentration mediating 50% maximal response, and slope factor describes the steepness of the dose-response curve. The equation was fit to raw data using KaleidaGraph (Abelbeck Software; Reading, PA), and individual EC₅₀ values were derived from each curve fit. Resultant EC₅₀ values were compared by t-test. Coronary flow, developed pressure, and heart rate responses to antagonist infusion for WT and A_2AKO hearts were compared by t-test. For all tests, statistical significance was accepted at P < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Baseline function in isolated WT and A_2AKO hearts. Table 1 summarizes baseline functional parameters (coronary flow, heart rate, and developed pressure) after 30 min of equilibration in isolated mouse hearts perfused at constant pressure. The average body weight and heart weight were significantly higher in age-matched A_2AKO mice compared with WT mice. Whereas this is consistent with previous findings in the A_2AKO knockout model (19), the heart weight-to-body weight ratios of WT and A_2AKO knockout animals were not different. There were no significant differences in baseline coronary flow, heart rate, or developed pressure between WT and A_2AKO hearts (Table 1).

Effects of adenosine on coronary flow in WT and A_2AKO hearts. Adenosine and its analogs NECA and CGS-21680 caused concentration-dependent increases in coronary flow (vasodilation) in isolated WT hearts perfused at constant pressure (Fig. 1, A-C). For both WT and A_2AKO hearts, maximal response to each agonist varied, as did the potency for producing coronary vasodilation. In A_2AKO hearts, concentration-response curves for adenosine- and NECA-induced coronary vasodilation were shifted to the right and demonstrated attenuated maximal responses compared with WT hearts (Fig. 1, A and B).

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Concentration-response curves for coronary flow (expressed as percent change from baseline) for adenosine (A), NECA (B), and CGS-21680 (C) in isolated perfused mouse hearts. Open circles represent wild-type (WT) hearts and closed squares represent A2A receptor knockout (KO) hearts. Values are means ± SE. *Significant differences between WT and A2AKO groups, P < 0.05.

Effects of adenosine on developed pressure in WT and A_2AKO hearts. In WT hearts, adenosine and its analogs NECA and CGS-21680 caused concentration-dependent increases in left ventricular developed pressure with a similar maximal response (Fig. 2, A-C). The maximum percent change in developed pressure in WT hearts for adenosine, NECA, and CGS-21680 was 167 ± 3% (n = 6), 177 ± 9% (n = 7), and 176 ± 7% (n = 7) of baseline, respectively (not significant; Fig. 2, A-C). In A_2AKO hearts, the selective A_2A receptor agonist CGS-21680 had no effect on developed pressure (Fig. 2C). EC₅₀ values for agonist-induced increases in left ventricular developed pressure are summarized in Table 2. Adenosine (10 µM) elicited the same maximal increase in developed pressure for WT and A_2AKO hearts (167 ± 3% and 170 ± 15% of baseline, respectively; not significant; Fig. 2A), although the EC₅₀ for adenosine-induced increase in developed pressure was lower in WT versus A_2AKO hearts (P < 0.05, Table 2). Likewise, 500 nM NECA produced the same maximal response in WT and A_2AKO hearts (177 ± 9% and 181 ± 8% of baseline, respectively; not significant; Fig. 2B); however, the EC₅₀ for developed pressure could not be determined for NECA due to terminal bradyarrhythmias at 1 µM.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Concentration-response curves for developed pressure (expressed as percent change from baseline) for adenosine (A), NECA (B), and CGS-21680 (C) in isolated perfused mouse hearts. Values are means ± SE. *Significant differences between WT and KO groups, P < 0.05.

Effects of adenosine on heart rate in WT and A_2AKO hearts. Neither adenosine nor the selective A_2A receptor agonist CGS-21680 had significant effect on the heart rate of WT and A_2AKO hearts (Fig. 3, A and C). NECA caused a concentration-dependent decrease in heart rate in both WT and A_2AKO hearts beginning at 50 nM (Fig. 3B).

View larger version (8K): [in this window] [in a new window]   Fig. 3.   Concentration-response curves for heart rate (expressed as percent change from baseline) for adenosine (A), NECA (B), and CGS-21680 (C) in isolated perfused mouse hearts. Values are means ± SE. *Significant difference from baseline value within KO group; **significant difference from baseline value within WT group, P < 0.05.

Effects of A_2B adenosine receptor blockade on NECA-induced responses in A_2AKO hearts. The influence of alloxazine on cardiac responses induced by NECA was investigated in a paired manner in A_2AKO hearts. Because 100 nM NECA elicited significantly different coronary vasodilatory responses between WT and A_2AKO hearts (Fig. 1B) while having the same effect on developed pressure between groups (Fig. 2B), this concentration was used for antagonist experiments. Although alloxazine has selectivity favoring antagonism of A_2B adenosine receptors (9), its low potency requires high concentrations to impart receptor blockade, thus a 10 µM concentration was used. At this dose, a 10-min infusion of alloxazine mildly increased coronary flow and developed pressure to 125 ± 5% and 111 ± 3% of baseline, respectively (P < 0.05; n = 6), and this effect was normalized as described in MATERIALS AND METHODS. Alloxazine had no effect on heart rate in A_2AKO hearts (103 ± 1.7% of preinfusion baseline, not significant).

View larger version (6K): [in this window] [in a new window]   Fig. 4.   Influence of A2B adenosine receptor antagonism by alloxazine (Allox) on NECA-induced changes in coronary flow (A), developed pressure (B), and heart rate (C) in A2AKO hearts. Response to 100 nM NECA was first determined in the absence of alloxazine. After washout, hearts were treated with 10 µM alloxazine for 10 min before second exposure to NECA (100 nM). Values are means ± SE. *Significant difference from untreated versus alloxazine-treated groups; n = 5, P < 0.05.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The primary intent of this study was to examine coronary vascular and functional responses to adenosine and its analogs in isolated hearts from WT and A_2AKO mice. The data demonstrate that nonselective agonists adenosine and NECA increase coronary flow in hearts lacking A_2A adenosine receptors, albeit with a rightward shift of the concentration-response curves and attenuated maximal response. The maximal increase in developed pressure induced by adenosine and NECA was the same in WT and A_2AKO hearts despite observed differences in coronary flow between the groups. NECA-induced increases in both coronary flow and developed pressure in A_2AKO hearts were attenuated by alloxazine, an A_2B adenosine receptor antagonist. Taken together, these findings support the conclusion that A_2B adenosine receptor activation increases coronary flow and developed pressure in isolated murine hearts.

Coronary vascular responses in WT and A_2AKO hearts. The diverse cardiovascular effects of adenosine are determined in part by tissue distribution, agonist affinity, and receptor signaling pathways associated with each of the adenosine receptor subtypes (2, 26, 32, 35). High-affinity A_2A and low-affinity A_2B adenosine receptors coexist in coronary endothelial cells (31) and ventricular myocytes (20). Early studies in most mammals indicate that coronary vasodilation is primarily mediated by A_2A adenosine receptor activation (1, 3, 13, 23, 27, 38). Evidence for A_2B receptor-mediated coronary vasodilation is rather indirect because of the lack of potent and selective A_2B adenosine receptor agonists and antagonists (17, 22, 38). Because activation of high-affinity A_2A adenosine receptors results in marked responses, it has been difficult to determine whether and to what extent activation of low-affinity A_2B receptors alters vasoreactive and/or functional responses in the heart. That is, the physiological effects of A_2A receptor activation may overshadow any contributing responses resulting from activation of A_2B adenosine receptors.

Functional responses in WT and A_2AKO hearts. The selective A_2A adenosine receptor agonist CGS-21680 increased developed pressure in WT hearts but had no effect on developed pressure in A_2AKO hearts (Fig. 2C). Adenosine and NECA resulted in the same maximal increase in developed pressure in both WT and A_2AKO hearts (Fig. 2, A and B). This equal increase in contractility occurs despite attenuated vasodilation by these agonists in A_2AKO hearts (Fig. 1, A and B). Therefore, nonselective adenosine agonists increase developed pressure in A_2AKO hearts independent of changes in coronary flow. Monahan et al. (24) recently reported that A_2A receptor activation increases contractility independent of changes in coronary flow in isolated rat hearts perfused at constant pressure. The current data indicate the same is true in isolated mouse hearts lacking A_2A receptors. That is, adenosine and NECA increase contractility in A_2AKO hearts (to an extent not accounted for by changes in coronary flow) through activation of a receptor subtype other than A_2A adenosine receptors.

A_2B receptor blockade in A_2AKO hearts. To establish whether A_2B adenosine receptor activation contributes to coronary vasodilation and/or developed pressure, the effect of alloxazine on NECA-induced responses in A_2AKO hearts was examined. Alloxazine is a nonxanthine A₂ adenosine receptor antagonist with nearly 10-fold greater selectivity for A_2B receptors than A_2A receptors, with dissociation constants of 2.3 and 20 µM, respectively (4), and it has been used to examine A_2B receptor-mediated effects in chick heart cells and the rat pial artery (20, 34). Alloxazine does have a K_i value of 9 µM at A₁ adenosine receptors (6), but there are no reports on whether it has affinity for A₃ adenosine receptors.

Study limitations. Studies examining the physiological consequences of A_2B adenosine receptor activation continue to be limited by the lack of antagonists that are both selective and potent for this receptor. Whereas the selectivity of alloxazine has proven sufficient to examine A_2B receptor-mediated effects in this and other models (20, 34), its lack of potency requires very high doses to elicit receptor blockade. The current work circumvents this issue by examining A_2B receptor antagonism in A_2AKO hearts, but it is recognized that neither the coronary vasodilation nor the increased developed pressure induced by NECA could be completely eliminated by alloxazine (Fig. 4, A and B). Thus, whereas the potency of the antagonist used may remain limiting, its selectivity is sufficient to conclusively establish A_2B-mediated responses in this model. Additionally, contractile function was assessed in the present study using constant pressure perfusion; however, further characterization of adenosine-mediated contractile function in A_2AKO hearts may require studies using constant flow perfusion and/or isolated myocyte preparations (24, 40). Finally, like all recent studies taking advantage of an ever-increasing array of gene-modified animal models, consideration must be given to the possibility that other receptor systems may have been affected by targeted expression or deletion of a single receptor subtype (11). That is, the functional genomics of A_2AKO animals has not yet been examined and will be an important component of future work with this model.

Conclusions and future directions. The present study combines receptor knockout technology with the traditional pharmacological approach to examine A₂ adenosine receptor subtypes in regulation of coronary flow and functional responses in isolated mouse hearts. The results provide the first direct evidence that activation of A_2B adenosine receptors contributes to coronary vasodilation and cardiac contractility. This strategy can now be utilized to further examine the heretofore-elusive nature of A_2B receptor effects and cell-signaling processes in the heart under pathophysiological conditions such as hypoxia or ischemia-reperfusion. Clarifying the relative roles of adenosine receptor subtypes in the cardiovascular system remains an essential step in the development of adenosinergic therapies for the clinical management of heart disease.