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Am J Physiol Heart Circ Physiol 290: H348-H356, 2006. First published September 2, 2005; doi:10.1152/ajpheart.00740.2005
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Contractile effects of adenosine A1 and A2A receptors in isolated murine hearts

Eugene I. Tikh, Richard A. Fenton, and James G. Dobson, Jr.

Department of Physiology, University of Massachusetts Medical School, Worcester, Massachusetts

Submitted 13 July 2005 ; accepted in final form 30 August 2005


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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The adenosine A1 receptor (A1R) inhibits {beta}-adrenergic-induced contractile effects (antiadrenergic action), and the adenosine A2A receptor (A2AR) both opposes the A1R action and enhances contractility in the heart. This study investigated the A1R and A2AR function in {beta}-adrenergic-stimulated, isolated wild-type and A2AR knockout murine hearts. Constant flow and pressure perfused preparations were employed, and the maximal rate of left ventricular pressure (LVP) development (+dp/dtmax) was used as an index of cardiac function. A1R activation with 2-chloro-N6-cyclopentyladenosine (CCPA) resulted in a 27% reduction in contractile response to the {beta}-adrenergic agonist isoproterenol (ISO). Stimulation of A2AR with 2-P(2-carboxyethyl)phenethyl-amino-5'-N-ethylcarboxyamidoadenosine (CGS-21680) attenuated this antiadrenergic effect, resulting in a partial (constant flow preparation) or complete (constant pressure preparation) restoration of the ISO contractile response. These effects of A2AR were absent in knockout hearts. Up to 63% of the A2AR influence was estimated to be mediated through its inhibition of the A1R antiadrenergic effect, with the remainder being the direct contractile effect. Further experiments examined the effects of A2AR activation and associated vasodilation with low-flow ischemia in the absence of {beta}-adrenergic stimulation. A2AR activation reduced by 5% the depression of contractile function caused by the flow reduction and also increased contractile performance over a wide range of perfusion flows. This effect was prevented by the A2AR antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM-241385). It is concluded that in the murine heart, A1R and A2AR modulate the response to {beta}-adrenergic stimulation with A2AR, attenuating the effects of A1R and also increasing contractility directly. In addition, A2AR supports myocardial contractility in a setting of low-flow ischemia.

perfused heart; antiadrenergic; cardiac contractility; adenosine A2A knockout; chlorocyclopentyladenosine; CGS-21680; ischemia

ADENOSINE PLAYS a role in modulating cardiac functions. In the well-oxygenated myocardium, intracellular levels of adenosine are low, but they rapidly increase in response to adrenergic stimulation or pathological conditions, such as ischemia and hypoxia (10, 11). Four adenosine receptor subtypes have been identified and cloned (13). The adenosine A1 and A2A receptors (A1R and A2AR, respectively) are thought to be particularly important in the regulation of cardiac contractility (4, 7, 9, 19, 26). A2R are also known to be responsible for coronary vasodilation, with A2AR considered the main contributor to this effect (27, 31). A2B receptors are considered more important to fibroblast regulation (3). The role of adenosine A3 receptors is less certain, although they have been linked to preconditioning (24) and have recently been shown to activate PKB in newborn rat cardiomyocytes (14). The A1R is known to exhibit an antiadrenergic action reducing the contractile responsiveness of the myocardium to adrenergic stimulation (1, 5, 6, 12). A2AR, on the other hand, appears to have a direct inotropic effect on the myocardium (4, 26, 34) by facilitating a greater response to adrenergic stimulation (19, 33) and an indirect effect by inhibiting the action of the A1R (28, 34).

Both A1R and A2AR are G protein-linked receptors coupled to Gi and Gs, respectively (13). The effects of these receptors are thought to be mediated either through the modulation of adenylyl cyclase activity (4, 19) with subsequent activation of PKA (8, 13) or via activation of phosphatidylinositol 3-kinase with the subsequent activation of endothelial nitric oxide synthase (eNOS) (17, 32, 35). A cAMP independent Gs-mediated mechanism has been proposed for the stimulatory effects of A2AR as well (4, 20). Recently PKC{epsilon} has been shown to be important in the antiadrenergic effect of A1R (25). Other recent studies (21, 22) have suggested that a significant component of the antiadrenergic effect of A1R is a result of protein phosphatase activation.

The individual effects of A1R and A2AR on cardiac contractility at the cellular level have been fairly well characterized. However, their individual actions and the interaction between them in the intact heart remain topics of active interest. The main purpose of this study was to examine the interaction of A1R and A2AR in the perfused murine heart subjected to {beta}-adrenergic stimulation. The study also examined the actions of the A1R in the absence of A2AR with the use of an A2AR knockout (A2ARKO) mouse heart and the effect of A2AR activation in the absence of {beta}-adrenergic stimulation. In addition, the influence of A2AR on ischemic heart function was investigated.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
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Experimental Animals

Six- to eight-week-old wild-type C57BL/6 male mice (WT) were purchased from Sprague-Dawley. A2ARKO mice were obtained from a colony maintained by our laboratory. The animals in this study were maintained and used in accordance with recommendations in the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1996) and evaluated and approved according to the guidelines of the Institutional Animal Care and Use Committee of the University of Massachusetts Medical School, Worcester, MA.

A2ARKO Generation and Verification

The progenitors for the A2ARKO–/– mice were obtained as a generous gift from Dr. J. F. Chen of Boston University Medical Center (Boston, MA) and were generated as described previously by others (2). The homozygous knockout animals used in the present study were offspring of heterozygous (+/–) breeders. Animals were validated by using DNA isolated from tail tissue with the use of Qiagen DNEasy tissue kit. ISOlated DNA was amplified by PCR using Qiagen Taq DNA polymerase and primers: 1) AGC CAG GGG TTA CAT CTG TG, 2) TAC AGA CAG CCT CGA CAT GTG, 3) TCG GCC ATT GAA CAA GAT GG, and 4) GAG CAA GGT GAG ATG AGA GG. Primers 1 and 2 correspond to the WT A2AR sequence, whereas primers 3 and 4 correspond to the A2ARKO sequence. Products of PCR were resolved by using 1% agarose gel electrophoresis in Tris-boric acid-EDTA buffer and visualized with ethidium bromide staining under UV light (WT, 180 bp; A2ARKO, 330 bp). The agarose gels were used to verify the genotype of the mice (Fig. 1).



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  		Fig. 1. Agarose gel electrophoresis of PCR products used to determine genotype of mice. WT, wild-type (+/+), 180 bp; knockout (KO), A2A-receptor KO (A2ARKO) (–/–), 330 bp; heterozygous breeders (+/–), both 180 and 330 bp; MW, molecular weight marker (100 bp fragment) with 500–100 bands visible top to bottom.

 
Isolated Heart Preparation

Mice were euthanized by decapitation, and hearts were excised. After the excision, the hearts were rapidly rinsed in saline at room temperature, mounted on the perfusion apparatus, and perfused via the aorta with a physiological saline solution (37°C; PSS) containing (in mM) 118.4 NaCl, 4.7 KCl, 2.5 CaCl2, 25 NaHCO3, 1.2 KH2PO4, 1.2 MgSO4, and 10 dextrose. The pH of the PSS was maintained at 7.4 by bubbling continuously with a 95% O2-5% CO2 gas mixture. The developed left ventricular pressure (LVP) was monitored by a pressure transducer and a canula tipped with a water-filled polyethylene balloon that was inserted through the mitral valve after a left atriotomy. Perfusion pressure was monitored by using a transducer connected to a sidearm of the perfusion cannula. The heart was paced with 3 V at 480 times/min via leads on the perfusion cannula and the pulmonary artery. All agents were delivered into the perfusion canula using infusion pumps (model 22, Harvard Apparatus, Holliston, MA) at the rate required (1.0% of perfusate flow rate) to achieve the final desired concentration in the perfusion fluid. The maximal rates of LVP development (+dP/dtmax) and relaxation (–dP/dtmax) were determined by differentiation of the LVP signal. All data were recorded by using a model RS-3400 Gould polygraph (Chandler, AZ).

Protocols

General. For constant flow experiments, the flow rate was adjusted to achieve a LVP of at least 40 mmHg. Flow rates ranged from 2.5 to 2.9 ml/min. For constant pressure experiments, the perfusion pressure was held constant at 60 mmHg, and perfusate flow rate was determined volumetrically. Hearts were allowed to stabilize for at least 15 min before the initiation of experimental protocols. In both constant pressure and constant flow experiments, hearts failing to demonstrate a LVP of at least 40 mmHg on stabilization were excluded from further study. In experiments with {beta}-adrenergic stimulation, isoproterenol (ISO) was infused for 30 s to achieve a final perfusate concentration of 10–8 M. Adenosine receptor agonists and antagonists were infused to achieve a final perfusate concentration of 10–7 M. Preliminary experiments conducted with the same concentration of ISO at infusion durations of 30 s, 1 min, and 2 min with 15 min washout did not indicate desensitization of {beta}-adrenergic responsiveness with multiple ISO infusions (data not shown).

{beta}-Adrenergic-stimulated hearts.
EFFECT OF A2AR ACTIVATION ON THE ANTIADRENERGIC EFFECT OF A1R. After stabilization, hearts were subjected to one 30-s ISO administration, and the peak contractile responses were recorded. After the hearts returned to steady state, infusion of the A1R agonist 2-chloro-N6-cyclopentyladenosine (CCPA) commenced. After 5 min of CCPA infusion, two additional ISO responses were elicited with a return to steady state between each administration. CCPA infusion was terminated after the second ISO response. CCPA and the A2AR agonist 2-P(2-carboxyethyl)phenethyl-amino-5'-N-ethylcarboxyamidoadenosine (CGS-21680) were then administered together for 5 min, whereupon two more ISO responses were elicited while continuing the CCPA and CGS-21680 infusion. This protocol was conducted with both WT and A2ARKO hearts. To confirm the response to CCPA as A1R specific, in some preparations a combination of CCPA and the A1R antagonist 1,3-dipropyl-8-cyclopentyl-xanthine (DPCPX) was administered.


EFFECT OF A2AR ACTIVATION IN PRESENCE AND ABSENCE OF A1R INHIBITION. In the first group, after stabilization, hearts were subjected to one 30-s ISO administration, and contractile responses were recorded. On return to baseline, the A2AR agonist CGS-21680 was administered for 5 min, and three 30-s ISO responses were elicited, allowing a return to baseline between stimulations.

In the second group, after stabilization, hearts were subjected to one 30-s ISO administration, and contractile responses were recorded. On return to baseline, the A1R antagonist DPCPX was administered for 5 min, and two 30-s ISO responses were elicited, allowing a return to baseline after each stimulation. Subsequently, a combination of DPCPX and A2AR agonist CGS-21680 was administered for 5 min, and two 30-s ISO responses were elicited as before.

Reduced flow experiments. For flow reduction experiments, the hearts were initially perfused at a rate of 3.0 ml/min. The response to a 1 ml/min flow rate decrease was examined under several experimental conditions. These protocols are depicted in Fig. 7.



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  		Fig. 7. Schematic representation of protocol used to determine effect of A2AR activation with CGS on contractility change occurring with flow decrease (A) and anticipated perfusion pressure response (B and C). Schematic representation of protocol where perfusion flow was decreased to simulate pressure drop observed with CGS (D) and anticipated perfusion pressure response (E). Baseline flow at stabilization is 3.0 ml/min. RF1, flow decrease of 1 ml/min, 1 min duration; RF2, flow decrease sufficient to lower perfusion pressure by 40–45 mmHg. Concentrations used are the following: CGS, 10–7 M; 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM or ZM-241385), and A2AR antagonist, 10–7 M.

 

EFFECT OF A2AR ACTIVATION ON CONTRACTILE RESPONSE TO FLOW REDUCTION. After stabilization, hearts were subjected to three periods of flow reduction, each of 1 min duration separated by periods of normal perfusion (3 ml/min) for ~3 min, allowing a return to baseline contractility (Fig. 7A). At this time, either CGS-21680 or CGS-21680 with the A2AR antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM-241385) was administered for 5 min, whereupon the flow-reduction cycle was repeated. Expected changes in perfusion pressures are depicted in Fig. 7, B and C.


EFFECT OF CGS-LIKE DECREASE IN PERFUSION PRESSURE ON THE CONTRACTILE RESPONSE TO FLOW REDUCTION. The contractile effects of CGS-21680 were further differentiated from the vasodilatory effects of CGS-21680. The response to a 1-ml flow decrease was examined while using a manual reduction in flow to achieve a decrease in perfusion pressure comparable to that occurring in response to CGS-21680 administration. After initial stabilization, the heart was subjected to three 1-min periods of flow reduction separated by periods of normal perfusion (3.0 ml/min) for ~3 min, allowing a return of contractile function to preflow reduction levels. The flow was then reduced to the extent required to produce the same decrease in perfusion pressure as observed with CGS-21680 administration (a decrease of 40–45 mmHg). After stabilization at this new baseline, three more periods of an additional flow decrease of 1 ml/min were administered. Changes in flow with expected changes in perfusion pressure are depicted in Fig. 7, D and E, respectively.

Data and Statistical Analysis

When multiple ISO administrations or flow reductions were used, the responses were averaged. Data are presented as means ± SE. Data were analyzed with the use of Prism (GraphPad Software, San Diego, CA) software. Additional statistical analysis was done by using StatMost (Dataxiom, Los Angeles, CA) software. Data were analyzed using one-way ANOVA, Student-Newman-Keuls multiple comparison test, and two-tailed t-test where appropriate. Values were taken to indicate a statistically significant difference at P < 0.05.

Materials

ISO was dissolved in 0.1% sodium metabisulfite and diluted to the infusion concentration of 10–6 M with MilliQ-treated water. CCPA, CGS-21680, ZM-241385, and DPCPX were prepared as 10 mM stock solutions in 100% DMSO and diluted with water to 10–5 M that was used for infusion into the perfusion fluid. The resultant perfusion fluid DMSO concentration was not >0.05%. Buffer salts were purchased from Fisher Scientific (Fairlawn, NJ). ISO and adenosine receptor agents CCPA, DPCPX, and CGS-21680 were obtained from Sigma-RBI (St. Louis, MO), and the A2AR antagonist ZM-241385 was purchased from Tocris (Ellisville, MO). Custom primers and DNA reference ladder were acquired from Invitrogen (Carlsbad, CA). Precast 1% agarose minigels with ethidium bromide were obtained from Bio-Rad (Hercules, CA). Qiagen DNEasy tissue kit and Qiagen Taq DNA polymerase Core kit were purchased from Qiagen (Valencia, CA).


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
{beta}-Adrenergic-Stimulated Hearts

Effects of A1R and A2AR activation in {beta}-adrenergic-stimulated hearts. In constant flow-perfused WT hearts, the administration of the A1R agonist CCPA reduced the contractile response to ISO stimulation (Fig. 2). Although CCPA reduced the ISO-induced increase in LVP by 15%, this reduction was not statistically significant (Fig. 2A). However, the A1R agonist significantly attenuated the ISO-induced increase in +dP/dtmax by 22% (Fig. 2B). The ISO responses in the presence of CGS-21680 (reductions of 23% and 28% for LVP and +dP/dtmax, respectively) remained significantly below those observed in the presence of ISO alone. These values were not significantly different from the CCPA plus ISO responses.



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  		Fig. 2. Effect of A1R and A2AR activation on left ventricular pressure (LVP; A) and maximum rate of LVP development (+dP/dtmax; B) responses to {beta}-adrenergic stimulation of the isolated, constant flow-perfused murine heart. ISO, isoproterenol (10–8 M); CCPA, 2-chloro-N6-cyclopentyladenosine, A1R agonist (10–7 M); CGS, 2-P(2-carboxyethyl)phenethyl-amino-5'-N-ethylcarboxyamidoadenosine (10–7 M; CGS-21680). Data are means ± SE for 10 experiments. *Statistically significant difference from ISO value.

 
Experiments were repeated with constant perfusion pressure rather than constant flow (Fig. 3). CGS-21680 administration resulted in an attenuation of the antiadrenergic effect of A1R. With CCPA treatment the values for LVP (Fig. 3A) and +dP/dtmax (Fig. 3B) with ISO stimulation were each reduced by 27%. The antiadrenergic effect of CCPA was fully reversed by an addition of the A1R antagonist DPCPX (data not shown). CGS-21680 attenuated the antiadrenergic effect of CCPA. In the presence of CGS-21680, CCPA only reduced the ISO-induced LVP and +dP/dtmax responses by 12% and 18%, respectively. These values were significantly different from ISO responses observed with CCPA in the absence of CGS-21680. It is concluded that the absence of a CGS-21680 effect in the constant flow preparation (Fig. 2) resulted from the possible influence of a decrease in perfusion pressure resulting from A2AR-induced vasodilation. Although CCPA had no effect on perfusion pressure, CGS-21680 administration resulted in a 35% decrease in perfusion pressure (from 100 mmHg) compared with values for the control and CCPA. The data were further examined as a percentage increase in +dP/dtmax in response to ISO stimulation (Fig. 4). With data expressed in this manner, attenuation of the antiadrenergic effect of CCPA by CGS-21680 was observed for both constant flow and constant pressure preparations. CCPA reduced the ISO response by 34% and 53% for constant pressure and flow, respectively. However, the ISO responses were decreased only by 10% and 35%, respectively, in the presence of both CGS-21680 and CCPA.



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  		Fig. 3. Effect of A1R and A2AR activation on LVP (A) and +dP/dtmax (B) responses to {beta}-adrenergic stimulation of the isolated, constant pressure-perfused murine heart. Concentrations used are the following: ISO, 10–8 M; CCPA, 10–7 M; and CGS, 10–7 M. Data are means ± SE for 5 experiments. *Statistically significant difference from ISO value; §statistically significant difference from CCPA+ISO value.

 


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  		Fig. 4. Effects of A1R and A2AR activation on {beta}-adrenergic-induced contractile responses in the murine heart. Concentrations used are the following: ISO, 10–8 M; CCPA, 10–7 M; and CGS, 10–7 M. Data are means ± SE for 10 experiments using constant (Const.) flow (Fig. 2) and 5 experiments using constant pressure (Fig. 3) perfusion. *Statistically significant difference from respective control (ISO) value; §statistically significant difference from respective CCPA value.

 
Effects of A1R activation in WT versus A2ARKO hearts. The response to A1R activation, along with {beta}-adrenergic stimulation in the absence and presence of A2AR stimulation, was compared between WT and A2ARKO hearts (Fig. 5). In the WT heart, ISO stimulation resulted in a 244% increase in +dP/dtmax. In the presence of CCPA, ISO only increased +dP/dtmax by 123%. CGS-21680 attenuated this antiadrenegic action, as evidenced by an ISO+CCPA response of 151%.



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  		Fig. 5. Effect of A1R and A2AR activation on the contractile response to {beta}-adrenergic stimulation of constant flow-perfused WT and A2ARKO murine hearts. Concentrations used are the following: ISO, 10–8 M; CCPA, 10–7 M; and CGS, 10–7 M. Data are means ± SE for 6 experiments. *Statistically significant difference from ISO value; §statistically significant difference from CCPA+ISO value.

 
In the A2ARKO heart, ISO stimulation resulted in a 253% increase in +dP/dtmax. In the presence of CCPA, ISO only increased +dP/dtmax by 169%. CGS-21680 in the presence of CCPA resulted in an ISO response of only 125%. The ISO-induced increase in +dP/dtmax after administration of both CCPA and CGS-21680 was not significantly different between A2ARKO and WT hearts.

Direct versus indirect effect of A2AR in WT hearts. To estimate the extent of the A2AR effect achieved by a direct increase in contractility, as opposed to its indirect effect through the inhibition of A1R, A2AR activation in the presence and absence of A1R inhibition was examined (Fig. 6). ISO alone resulted in a 308% increase in +dP/dtmax (Fig. 6A). In the presence of CGS-21680, this response was increased to 490%, reflecting a 182 percentage-point ({Delta}1) increase from ISO alone. This response is attributable to both the direct and indirect effects of the A2AR. In the presence of an A1R blockade by DPCPX, CGS-21680 resulted in an increase of only 67 percentage points ({Delta}2) above that of the ISO response (Fig. 6B). It is assumed that the increase in the ISO response with CGS-21680 in the presence of A1R blockade by DPCPX is due to the direct effect of A2AR on contractility and that the direct and indirect effects of CGS-21680 are independent and additive. The indirect effect of A2AR acting on the A1R can be estimated by subtracting the direct effect from the value representing both direct and indirect influences ({Delta}3, Fig. 6C). Determined in this manner, the indirect effect of the A2AR through the inhibition of A1R is ~63% of the total A2AR effect.



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  		Fig. 6. A: effect of A2AR activation on the contractile response to {beta}-adrenergic stimulation of constant flow-perfused murine heart. {Delta}1, total effect of A2AR. B: effect of A2AR activation on contractile response to {beta}-adrenergic stimulation in presence of A1R blockade. {Delta}2, direct contractility effect of A2AR. C: approximation of A2AR effect attributable to its indirect action via A1R. {Delta}3, {Delta}1 – {Delta}2, indirect effect of A2AR via A1R. Concentrations used are the following: ISO, 10–8 M; 1,3-dipropyl-8-cyclopentyl-xanthine (DPCPX; 10–7 M), an A1R antagonist; and CGS, 10–7 M. Data are means ± SE for 6 experiments. *Statistically significant difference from ISO value; §statistically significant difference from DPCPX value.

 
Reduced Flow Experiments

In the absence of {beta}-adrenergic stimulation, the contractile response to A2AR activation in an intact heart is relatively small (5–10%) compared with that at baseline (26). Furthermore, contractile activity is affected by the fall in perfusion pressure inherent with A2AR-induced vasodilation in the constant flow preparation. As described in MATERIALS AND METHODS, flow reduction experiments were designed to investigate the effects of A2AR activation in the absence of {beta}-adrenergic stimulation (Fig. 7, A–C). Additional experiments were designed to examine the effect of the perfusion pressure decrease that occurs with CGS-21680-induced vasodilation on contractile function (Fig. 7, D and E). A 3.0 ml/min rate of flow was chosen as baseline to permit maximal oxygenation and contractile function without the risk of edema that is caused by higher flow rates.

A2AR effect on contractile function with reduced flow. In the absence of CGS-21680, a flow reduction of 1 ml/min (RF1) resulted in a 32% decrease in perfusion pressure (Fig. 8A), which in turn led to significant 33%, 30%, and 43% decreases in LVP, +dP/dtmax, and –dP/dtmax, respectively, compared with control values (Fig. 8, B and C). In the presence of CGS-21680, a vasodilation elicited a drop in perfusion pressure of 45 mmHg. This represents a 50% decrease from the level observed in the absence of the A2AR agonist. This value was significantly lower than that observed after the flow decrease (RF1) in the absence of CGS-21680. In the presence of CGS-21680, the LVP and ±dP/dtmax were significantly decreased from control values. However, these contractile parameters with CGS-21680 were significantly higher than those seen during RF1 without CGS-21680, despite the lower perfusion pressure. In the presence of CGS-21680, a 1 ml/min flow decrease resulted in a 32% decrease in perfusion pressure compared with the A2AR agonist alone (Fig. 8A). This reduction in perfusion pressure was associated with decreases of 27% in LVP (Fig. 8B), 23% in +dP/dtmax, and 39% in –dP/dtmax (Fig. 8C).



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  		Fig. 8. Effect of A2AR activation on effects of a brief flow reduction (RF1) on perfusion pressure (A), LVP (B), and ±dP/dtmax (C) of the constant flow-perfused murine heart. CGS concentration was 10–7 M. Data are means ± SE for 6 experiments. *Statistically significant difference from corresponding control value; §statistically significant difference from RF1 value observed in absence of CGS.

 
To verify the CGS-21680 response as A2AR specific, experiments were repeated by using the A2AR antagonist ZM-241385. The previously observed effects of CGS-21680 were blocked by ZM-241385 (data not shown). These results indicate that the enhanced contractility observed, despite a decrease in perfusion pressure, is A2AR specific.

Effect of A2AR-comparable perfusion pressure reduction on contractile function. Experiments were conducted to ascertain whether the changes in contractile response to flow reduction were due to the effect of CGS-21680 on contractility or as a result of the A2AR agonist-induced decrease in perfusion pressure. A protocol was designed with a similar sequence of flow decreases (RF1). However, instead of CGS-21680 administration, the flow was manually reduced (RF2) to the extent required to simulate the decrease in perfusion pressure of 40–45 mmHg that occurs with CGS-21680 (Fig. 8). The changes observed in LVP and ±dP/dtmax correlated closely with the decreases in perfusion pressure (Fig. 9). The flow reduction RF2 decreased perfusion pressure, LVP, +dP/dtmax, and –dP/dtmax by 36%, 32%, 32%, and 46%, respectively. The decreases in these four parameters showed no significant differences from those observed during RF1.



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  		Fig. 9. Effect of a CGS-like perfusion pressure drop (RF2) in conjunction with a brief flow reduction (RF1) on perfusion pressure (A), LVP (B), and ±dP/dtmax (C) in the constant flow-perfused murine heart. Data are means ± SE for 6 experiments. *Statistically significant difference from control value; **statistically significant difference from RF2 value alone.

 
Further analysis of the data presented in Figs. 8 and 9 reveals that the contractile depression occurring with RF1 is attenuated by CGS-21680. In the absence of CGS-21680, RF1 resulted in decreases of 870 (36%) and 763 mmHg/s (43%) in +dP/dtmax and –dP/dtmax, respectively (Fig. 8C). However, after CGS-21680 administration RF1 resulted in decreases of 559 (23%) and 535 mmHg/s (38%) in +dP/dtmax and –dP/dtmax, respectively. A significant decrease in response to RF1 in the presence of CGS-21680 may also be observed when examining the percentage decrease in ±dP/dtmax, resulting from RF1 (Fig. 10A). The decreases (in %) presented in Fig. 10 are the means of the appropriate differences obtained from Figs. 8 and 9. Attenuation of the contractile response to RF1 observed in the presence of CGS-21680 (Fig. 10A) did not occur in preparations using a manual flow reduction to simulate CGS-21680-induced decrease in perfusion pressure (Fig. 10B). The attenuation of RF1 contractile depression with CGS-21680 was prevented by the A2AR antagonist ZM-241385 (Fig. 10C).



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  		Fig. 10. Percentage decrease of ±dP/dtmax in response to a brief flow reduction (RF1) in the presence of CGS (A; data from Fig. 8), manual flow decrease sufficient to achieve a perfusion pressure drop similar to CGS (RF2) (B; data from Fig. 9), or a combination of CGS and ZM (C). Concentrations used are the following: CGS, 10–7 M; and ZM, 10–7 M. RF1+2, combined flow decrease of RF2 and RF1. Data are means ± SE for 6 experiments. *Statistically significant difference from corresponding RF1 value.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
A2AR and A1R Activation in Presence of {beta}-Adrenergic Stimulation

The main finding of this study is that A2AR increases the contractile performance of the myocardium through both direct and indirect mechanisms in the {beta}-adrenergic-stimulated mouse heart. The actions of A2AR also support myocardial contractility during reduced flow (low-flow ischemia). These results extend the previously reported observations that the A2AR can increase contractility in intact hearts and cardiomyocytes that are obtained from rats (4, 19, 26, 34). The A1R and A2AR interaction has also been reported previously in the rat heart (28) and isolated rat cardiomyocytes (34), although the exact mechanism by which this occurs remains unknown. The antiadrenergic action of A1R is thought to be mediated through multiple signaling mechanisms involving a decrease in adenylyl cyclase activity (18), reduction in calcium transients (12, 28), and increased PKC{epsilon} translocation (25). The inhibition of the A1R effect by the A2AR may occur by a modulation of any of these processes. Postulated mechanisms for the effects of the A2AR have included an activation of adenylyl cyclase (19), calcium-dependent and -independent mechanisms (4, 7, 33), as well as cAMP-independent mechanisms (4, 20). Based on the data presented, the effects of the A2AR can be considered as both direct and indirect. Direct effects involve the improvement of contractile performance through positive effects, such as an enhanced activation of adenylyl cyclase, whereas indirect effects involve the inhibition of the antiadrenergic effects of A1R. The direct effects of A2AR activation in the intact heart can be observed independently of {beta}-adrenergic stimulation as described by Monahan et al. (26). However, the effect of A2AR activation becomes more pronounced when examined in the presence of {beta}-adrenergic stimulation.

In both constant flow and constant pressure preparations, A2AR stimulation was observed to attenuate the antiadrenergic effects of A1R activation. The direct increase in contractile performance observed with the activation of the A2AR was consistent with previous reports (26) in which rat hearts were used. Interestingly, in the constant perfusion pressure preparation, A2AR activation appeared to have a greater effect than with constant flow. Although CGS-21680 significantly attenuated the effects of CCPA in both constant flow and constant pressure preparations, the +dP/dtmax returned to control levels in the constant pressure preparation but remained significantly below control level in the constant flow preparation after treatment with CGS-21680 (Fig. 4). This observation is likely due to the increased perfusion flow and vascular filling seen with constant pressure perfusion. Increased regional tissue distension as a result of increased vascular filling may result in ventricular myocytes experiencing enhanced preload conditions (Water hose/Gregg effect; 16, 30).

With respect to the attenuation of the A1R effect by A2AR, the findings of the present study confirm those reported by Norton et al. (28) in the rat heart. It is possible, however, to further delineate the functional aspect of the A2AR effect into direct and indirect components. To estimate the extent to which each of these mechanisms of A2AR action occurs in the adrenergic-stimulated mouse heart, the contractile response to CGS-21680 stimulation of the A2AR was compared in the presence and absence of A1R inhibition. The ISO response in the presence of CGS-21680 is presumed to be affected by both mechanisms in an additive fashion, i.e., A2AR is able to both inhibit the manifestation of A1R effects and increase contractility directly. The direct action of A2AR can be revealed by pretreating the heart with the A1R antagonist DPCPX before A2AR stimulation. The resulting increase in the contractile response to ISO in this case is due to the direct effect of the A2AR on contractility. The observed increase in the ISO response with CGS-21680 was reduced after pretreatment with DPCPX (Fig. 6). The additional increase in the ISO response was approximately a third of the response seen when CGS-21680 was administered without prior A1R inhibition ({Delta}1 vs. {Delta}2; Fig. 6). These calculations suggest that a major part of the A2AR effect is mediated through the inhibition of the A1R (indirect effect), as opposed to its direct effect on contractility. This conclusion is consistent with the observation that A2AR activation only has a minor effect on contractility (5–10%) in the absence of adrenergic stimulation. In the presence of adrenergic stimulation where the A1R plays a significant role in attenuating the adrenergic response, the A2AR exerts a more profound inhibitory effect on the antiadrenergic action of A1R. Thus the interaction of the A2AR and the A1R is of greater importance in the adrenergic-stimulated heart.

A1R and A2AR Stimulation Response: Comparing Knockout and WT Hearts

There were two findings of interest when comparing the responses of A1R and A2AR activation in WT and A2ARKO hearts. First, the observed response to A1R activation was greater in the WT than in the A2ARKO hearts (Fig. 5). The ISO responses observed were not markedly different between WT and A2ARKO hearts, whereas there was approximately a 50 percentage point difference between the average ISO response levels in the presence of CCPA. These findings conflict with previous reports indicating that in a WT rat heart, the inhibition of A2AR with ZM-241385 increased the antiadrenergic effects of A1R activation (28). The absence of the A2AR in the A2ARKO heart should result in a situation similar to that where the A2ARs are inhibited pharmacologically. It would be expected that the A2ARKO would be more responsive to the antiadrenergic effect of A1R. However, the opposite was observed in the present experiments. It is possible that this is due to a modification of A1R signaling in the A2ARKO. However, the study of vascular smooth muscle responses to adenosine analogs in the A2ARKO mouse suggested no adaptations (29). The reason for the presently observed enhancement of the A1R response in A2ARKO hearts remains to be explored.

The second notable difference between the WT and A2ARKO hearts was the response to the CGS-21680 and CCPA combination. In the WT hearts, CGS-21680 produced an increase in contractile response to ISO from that observed in the presence of CCPA, as expected. However, in the A2ARKO hearts, there was a further decrease in ISO contractile response after treatment with CCPA and CGS-21680 together. A possible explanation is an interaction between CGS-21680 and the A1R. A binding of CGS-21680 to tissues in an A1R-dependent manner has been reported in the mouse brain (15, 23). The exact nature of this interaction between CGS-21680 and A1R remains unknown. The observed decrease in the ISO contractile response seen with CGS-21680 beyond that with CCPA alone may occur in the A2ARKO hearts, because CCPA together with CGS-21680 activates the A1R to a greater extent.

A2AR Supports Myocardial Contractility With Low-Flow Ischemia

To study the importance of A2AR activation in providing contractile support in a nonadrenergic-stimulated heart and the role of vasodilation in the contractile effects of A2AR activation, an experimental protocol was used where the response to brief periods of low-flow ischemia was examined. The effect of A2AR on contractility in the absence of adrenergic stimulation is small. In the current protocol, where a decrease in flow rather than adrenergic stimulation was applied, it was possible to examine the effect of A2AR activation on contractility even in the absence of adrenergic stimulation. There were two main findings in this series of experiments. First, A2AR activation resulted in a higher level of observed contractile performance at a given perfusion pressure. After the administration of CGS-21680, vasodilation resulted in a perfusion pressure significantly lower than that observed with the l-ml flow decrease (RF1, Fig. 8A). Despite this result, the observed LVP and ±dP/dtmax were both higher than that observed after RF1 in the absence of CGS-21680. This indicates that in the presence of A2AR stimulation, myocardial contractility is enhanced as evidenced by the increased LVP and ±dP/dtmax at a given perfusion pressure. This effect was fully reversible with the A2AR antagonist ZM-241385 and thus was attributable to A2AR.

The second observation of interest was that the actual decrease in contractility observed during the administered periods of low-flow ischemia (RF1) was significantly less in the presence of A2AR stimulation with CGS-21680. This difference was most clearly visible with respect to ±dP/dtmax (Fig. 10). To determine whether this observation was due to A2AR contractile effects, as opposed to the result of the decreased perfusion pressure resulting from CGS-21680-induced vasodilation, another series of experiments used a manual decrease in flow (RF2, Fig. 9) to simulate the decrease in perfusion pressure caused by CGS-21680 administration. This manual perfusion pressure decrease did not attenuate the extent of contractile function reduction seen with flow drop RF1. In fact, the attenuation of response to flow reduction was only seen with CGS-21680 and was prevented by the A2AR antagonist ZM-24138.

In summary, this study has found that the A2AR enhances the contractile response to {beta}-adrenergic stimulation in the murine heart directly through an effect on contractility and indirectly by an attenuation of the antiadrenergic actions of the A1R. In addition, A2AR activation supports contractile function during low-flow ischemia, resulting in an increased contractile function at the given reduced perfusion pressure.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was made possible by the National Institutes of Health (NIH) Grants AG-11491 and HL-66045. The contents of this study are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.


   ACKNOWLEDGMENTS
 
A2ARKO breeders were generously donated by Dr. J. F. Chen of Boston University Medical School. Portions of this study have been presented in abstract form (Tikh EI, Fenton RA, and Dobson JG Jr, FASEB 18: A1245, 2004.) We thank Izi Obokhare for initial work performed on cardiac adenosine A2A receptors in the A2ARKO mice.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. G. Dobson, Jr., Dept. of Physiology, Univ. of Massachusetts Medical School, 55 Lake Ave. N., Worcester, MA 01655 (e-mail: james.dobson{at}umassmed.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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