Download receptor activation increases contractility in isolated perfused hearts

Am J Physiol Heart Circ Physiol
279: H1472–H1481, 2000.
Adenosine A2a-receptor activation increases
contractility in isolated perfused hearts
THOMAS S. MONAHAN, DARRELL R. SAWMILLER, RICHARD A. FENTON,
AND JAMES G. DOBSON, JR.
Department of Physiology, University of Massachusetts Medical School,
Worcester, Massachusetts 01655
Received 4 January 2000; accepted in final form 17 April 2000
multiple effects in the
heart, including heart rate reduction (37), vasodilation
(3), and cardioprotection (1, 24, 27, 36), mediated by
activation of adenosine A1 (24, 37), A2a (3, 36, 40), and
A3 (1, 27, 36) receptors. One well-characterized effect of
adenosine on myocardial contractile performance is its
ability to reduce the contractile and metabolic responses elicited by ␤1-adrenergic stimulation (8, 31).
This antiadrenergic effect of adenosine is mediated by
A1 receptors, which activate the inhibitory heterotrimeric G protein, Gi, reducing ␤1-adrenergic-elicited
adenylyl cyclase activity, cAMP accumulation, protein
kinase A activity, and myocardial protein phosphorylation (9, 13, 15, 30).
Adenosine also has a stimulatory influence on myocardial contractile performance elicited by activation of
A2a receptors (11, 39, 40). This effect of adenosine is
putatively mediated by activation of the stimulatory G
protein, Gs, and involves presumably a cAMP-dependent pathway (11, 25, 40). However, a cAMP-independent pathway may also be involved (11, 26). Although
the natural nucleoside adenosine has been shown to
increase contractility of papillary muscles isolated
from rats (23), guinea pigs (4), and dogs (6), it was not
determined in these studies whether the increase in
contractile performance was mediated by activation of
A2a receptors. Some reports have suggested that A2areceptor agonists do not increase the contractility of
isolated ventricular myocytes from rats (32), rabbits
(32), or guinea pigs (2, 32, 35). However, other reports
indicate that A2a-receptor agonists increase contractile
performance of rat (11, 40), chick embryonic (25, 26,
39), and human (34) ventricular myocytes. Thus, if the
latter findings are correct, an increase in the contractility of the intact heart should also result via A2areceptor-mediated mechanisms. The purpose of the
present study was to investigate the role of A2a-receptor activation by adenosine in the enhancement of
contractile performance of intact hearts isolated from
rats. The results from this study indicate that adenosine increases ventricular contractile performance in
the intact heart independently of adrenergic stimulation, perfusion pressure, and coronary flow and that
this increase in contractility is prevented by A2a-receptor antagonists. These results further suggest a physiological role of the A2a receptor as a mediator of
positive inotropy.
Address for reprint requests and other correspondence: J. G. Dobson, Jr., Dept. of Physiology, 55 Lake Ave. North, University of
Massachusetts Medical School, Worcester, MA 01655-0127 (E-mail:
[email protected]).
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.
adenosine A1 receptor; ␤1-adrenergic receptor; myocardial contractility; constant-pressure-perfused heart; constant-flow-perfused heart; A1 antagonists; A2a antagonists; hydralazine
ADENOSINE IS KNOWN TO ELICIT
H1472
0363-6135/00 $5.00 Copyright © 2000 the American Physiological Society
http://www.ajpheart.org
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Monahan, Thomas S., Darrell R. Sawmiller, Richard A. Fenton, and James G. Dobson, Jr. Adenosine
A2a-receptor activation increases contractility in isolated
perfused hearts. Am J Physiol Heart Circ Physiol 279:
H1472–H1481, 2000.—Adenosine A2a-receptor activation
enhances shortening of isolated cardiomyocytes. In the
present study the effect of A2a-receptor activation on the
contractile performance of isolated rat hearts was investigated by recording left ventricular pressure (LVP) and the
maximal rate of LVP development (⫹dP/dtmax). With constant-pressure perfusion, adenosine caused concentrationdependent increases in LVP and ⫹dP/dtmax, with detectable increases of 4.1 and 4.8% at 10⫺6 M and maximal
increases of 12.0 and 11.1% at 10⫺4 M, respectively. The
contractile responses were prevented by the A2a-receptor
antagonists chlorostyryl-caffeine and aminofuryltriazolotriazinyl-aminoethylphenol (ZM-241385) but were not
affected by the ␤1-adrenergic antagonist atenolol. The
adenosine A1-receptor antagonist dipropylcyclopentylxanthine and pertussis toxin potentiated the positive inotropic
effects of adenosine. The A2a-receptor agonists ethylcarboxamidoadenosine and dimethoxyphenyl-methylphenylethyl-adenosine also enhanced contractility. With constant-flow perfusion, 10⫺5 M adenosine increased LVP and
⫹dP/dtmax by 5.5 and 6.0%, respectively. In the presence of
the coronary vasodilator hydralazine, adenosine increased
LVP and ⫹dP/dt max by 7.5 and 7.4%, respectively.
Dipropylcyclopentylxanthine potentiated the adenosine contractile responses with constant-flow perfusion in the absence and presence of hydralazine. These increases in contractile performance were also antagonized by chlorostyrylcaffeine and ZM-241385. The results indicate that adenosine
increases contractile performance via activation of A2a receptors in the intact heart independent of ␤1-adrenergic receptor
activation or changes in coronary flow.
MYOCARDIAL CONTRACTILITY AND ADENOSINE A2a RECEPTORS
METHODS AND MATERIALS
Preparation of Isolated Perfused Hearts
Experimental Protocols
In the first six protocols, hearts were constant-pressure
perfused at 65 mmHg. This procedure resulted in coronary
flows of 16 ⫾ 1 ml/min (12 ⫾ 1 ml 䡠 min⫺1 䡠 g wet wt⫺1). In
protocols 7–9, hearts were constant-flow perfused, unless
otherwise indicated, at 16 ⫾ 1 ml/min, resulting in a perfusion pressure of 65 mmHg.
Protocol 1. The effect of adenosine on left ventricular
contractility was determined. Adenosine was infused into the
perfusion PS at 1% of the coronary flow rate for 3 min
yielding final PS adenosine concentrations of 10⫺8 –10⫺4 M.
Concentrations were administered randomly. The maximum
contractile response (LVP, ⫹dP/dtmax, and ⫺dP/dtmax) to
each concentration of adenosine was achieved within 1 min
after the beginning of the infusion. Coronary flow was determined, and coronary resistance was calculated by dividing
the perfusion pressure (65 mmHg) by the coronary flow.
Protocol 2. The effects of the A2a-receptor antagonists
chlorostyryl-caffeine (CSC) and aminofuryltriazolotriazinylaminoethylphenol (ZM-241385) on the contractile response to
adenosine were determined. The antagonists were infused to
produce a final concentration of 10⫺7 M in the PS, and the
contractile response to a 3-min administration of 10⫺5 M
adenosine was determined periodically throughout this infusion. The contractile response to a 3-min administration of
10⫺5 M adenosine was also determined periodically throughout a 60-min period in the presence of 0.01% DMSO, the
vehicle for CSC and ZM-241385. Because of the lability of
CSC, it was prepared fresh immediately before use.
Protocol 3. The effect of the A1-receptor antagonist dipropylcyclopentylxanthine (DPCPX) at 10⫺7 M on the adenosine-elicited contractile response was assessed. The antagonist was administered 5 min before and during a 3-min
exposure to 10⫺5 M adenosine.
Protocol 4. The effects of the A2a-receptor agonists ethylcarboxamido-adenosine (NECA) and dimethoxyphenyl-methylphenylethyl-adenosine (DPMA) at 10⫺6 M and carboxyethylphenethyl-aminoethylcarboxamido-adenosine (CGS-21689) at
10⫺7–10⫺5 M on left ventricular contractility were determined.
The agonists were administered for 5 min.
Protocol 5. The effect of pertussis toxin on the adenosineelicited contractile responses was assessed. Rats received
activated pertussis toxin (25 ␮g/kg ip; Research Biochemicals, Natick, MA) 48 h before initiation of experiments. The
toxin was activated by incubation in 50 mM KH2PO4 (pH
7.5), 1 mg/ml ovum albumin, and 250 mM dithiothreitol at
30°C for 18 h.
Protocol 6. The effect of the ␤-adrenergic antagonist atenolol (10⫺6 M) on the contractile responses elicited by 10⫺5 M
adenosine and 10⫺9 M isoproterenol (a ␤1-adrenergic agonist)
was determined. The antagonist was infused for 5 min, and
adenosine or isoproterenol was administered during the final
3 min.
Protocol 7. The effect of 10⫺5 M adenosine on left ventricular contractile performance was determined under constant-flow conditions. This was considered because, during
constant-pressure perfusion, the vasodilation elicited by
adenosine may have resulted in an increased flow that, on its
own, could have augmented contractility. After achieving
steady state under constant-pressure perfusion, the hearts
were switched to constant-flow perfusion at their natural
flow rate of 16 ⫾ 1 ml/min. Subsequently, adenosine was
administered at 10⫺8–10⫺4 M for 3 min, and LVP, ⫹dP/dtmax,
and coronary perfusion pressure were recorded.
Protocol 8. The effect of the A1-receptor antagonist DPCPX
(10⫺7 M) on the adenosine-elicited contractile response was
assessed under constant-flow conditions. The antagonist was
administered 5 min before and during a 3-min exposure to
10⫺5 M adenosine.
Protocol 9. Hydralazine, a known vasodilator (33), was
employed in constant-flow experiments to vasodilate the
hearts so that the addition of adenosine would contribute
little to the total vasodilation. Pretreatment of hearts with
10⫺4 M hydralazine attenuated the reduction of coronary
perfusion pressure (⬃1–5 mmHg) mediated by adenosineinduced vasodilation. Some hearts were also treated with
10⫺7 M DPCPX 5 min before and during the 3-min administration of 10⫺5 M adenosine to prevent the possible involvement of A1 receptors. The contractile response to adenosine
was determined before and after treatment of the hearts with
10⫺7 M CSC for 60 min or 10⫺7 M ZM-241385 for 5 min and
then after CSC or ZM-241385 was allowed to wash out from
the heart for an additional 30 min.
During the course of this study, the adenosine-induced
contractile response was reduced or entirely absent in hearts
from hyperexcited rats compared with hearts from tranquil
rats. If it was deemed necessary, the rats were moved to a
quiet room for 2–3 days before experimentation. All animals
were exposed to a 12:12-h light-dark cycle and given food and
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Male Sprague-Dawley rats (310 ⫾ 7 g; Harlan Sprague
Dawley, Indianapolis, IN) were initially anesthetized with
pentobarbital sodium (40 mg/kg ip). The hearts were rapidly
excised, immersed in ice-chilled physiological saline (PS),
and immediately perfused via the aortic cannula with nonrecirculated PS at 37°C. A perfusion pump maintained a
column of PS at a height of 88 cm in the perfusion apparatus,
providing a constant coronary perfusion pressure of 65
mmHg. In constant-flow preparations, the perfusion pump
delivered PS directly to the perfusion apparatus at a desired
constant flow. The PS was prepared daily and contained (in
mM) 120 NaCl, 4.7 KCl, 2.5 CaCl2, 25 NaHCO3, 1.2 MgSO4,
1.2 KH2PO4, and 10 glucose. The pH was maintained at 7.4
by gassing the PS with 95% O2-5% CO2. Coronary perfusion
pressure was recorded by a pressure transducer connected
via a side tube to the aortic perfusion cannula. Left ventricular pressure (LVP) was determined using a water-filled,
latex balloon-tipped polyethylene cannula (1.5 mm ID) attached to a strain-gauge manometer (Micro-Med, Louisville,
KY). The balloon (Hugo Sachs, Hugstetten, Germany) was
inserted into the lumen of the left ventricle. The diastolic
pressure was set at 5–10 mmHg and held constant. The
hearts were paced at 5 Hz with a stimulator (model SD9,
Grass Instruments, Quincy, MA) via platinum wire electrodes inserted into the right and left atria. The voltage was
10% above threshold, and the pulse duration was 5 ms. LVP,
maximum rates of LVP development (⫹dP/dtmax) and relaxation (⫺dP/dtmax), heart rate, and end-diastolic pressure
were assessed continuously by an analog-to-digital converter
(Heart Performance Analyzer, Micro-Med, Louisville, KY) at
a frequency of 500 Hz. These data were averaged over 0.5-s
intervals and stored on a personal computer (Gateway 2000,
North Sioux City, SD). A system of dual outputs allowed the
signal from the Heart Performance Analyzer to be simultaneously recorded on a multichannel polygraph (model 7758A,
Hewlett-Packard, Waltham, MA). Coronary flow was determined volumetrically. The hearts were allowed to stabilize
for ⱖ45 min before an experimental protocol was begun.
After the stabilization period, hearts that did not have an
LVP ⱖ75 mmHg were not utilized.
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MYOCARDIAL CONTRACTILITY AND ADENOSINE A2a RECEPTORS
water ad libitum. In addition, because it was difficult to
assess the contractile response in hearts displaying a high
degree of ventricular arrhythmic activity, data from arrhythmic hearts are not included.
Animals
The animals were maintained and used in accordance with
recommendations in the Guide for the Care and Use of Laboratory Animals [Institute of Laboratory Animals Resources,
National Research Council (NIH Publication), vol. 25, no. 28,
revised 1996] and the guidelines of the Institutional Animal
Care and Use Committee of the University of Massachusetts
Medical School.
Materials
Statistical Treatments
Contractile data (LVP and ⫾dP/dtmax) were recorded via
the Heart Performance Analyzer immediately before each
treatment with adenosine, NECA, DPMA, CGS-21680, or
vehicle and during the peak responses to the agents. In some
studies the concentration-dependent effects of adenosine on
all three indexes of left ventricular contractility (LVP, ⫹dP/
dtmax, and ⫺dP/dtmax) were determined. These studies indicated that the effect of adenosine on contractile performance
is adequately assessed through measurements of LVP and
⫹dP/dtmax. Therefore, in most studies, LVP and ⫹dP/dtmax
were utilized as the indexes of contractility. Values are
means ⫾ SE. Statistical analysis was performed by randomized block ANOVA, which allowed the contractile responses
to each treatment to be compared with a control value generated from the same heart. P ⬍ 0.05 was accepted as
indicating a statistically significant difference.
RESULTS
Adenosine Enhances Contractile Performance
With Constant-Pressure Perfusion
Adenosine at 10⫺5 M increased LVP, ⫹dP/dtmax, and
⫺dP/dtmax within 3 min (Fig. 1). On termination of the
adenosine administration, the contractile variables returned to control levels within 5 min. Concentrationresponse curves revealed increases for LVP, ⫹dP/dtmax,
and ⫺dP/dtmax of 4.1, 4.8, and 4.6% at 10⫺6 M adenosine,
increases of 8.0, 8.7, and 9.4% at 10⫺5 M adenosine, and
maximum increases of 12.0, 11.1, and 14.3% at 10⫺4 M
adenosine, respectively (Fig. 2). The hearts demonstrated
contractile responses to 3-min infusions of adenosine every 10 min for 90 min. Repeated infusion of the vehicle for
adenosine (PS or water) did not significantly change
contractile performance.
Although 10⫺6 –10⫺4 M adenosine significantly increased contractility of the constant-pressure-perfused
hearts, only 10⫺4 M adenosine significantly increased
coronary flow and decreased coronary resistance (Fig.
3). With 10⫺4 M adenosine, the increase in coronary
flow was 8.5% and the decrease in coronary resistance
was 7.5%. Thus, 10⫺6 –10⫺5 M adenosine produced an
increase in the contractile variables without significantly affecting coronary flow or resistance.
Adenosine-Elicited Increase in Contractile
Performance Is Prevented by A2a-Receptor Antagonists
The A2a-receptor antagonists CSC and ZM-241385,
when administered alone at 10⫺7 M, did not significantly affect LVP and ⫹dP/dtmax (Fig. 4). However,
both antagonists prevented the 8.8 and 8.1% increase
in LVP and ⫹dP/dtmax, respectively, caused by 10⫺5 M
adenosine. A 60-min, continuous infusion of CSC was
required to prevent the adenosine-elicited contractile
response, whereas only a 5-min infusion of ZM-241385
was required for the same inhibitory effect. A 60-min
infusion of 0.001–0.005% DMSO, the vehicle for CSC
and ZM-241385, did not alter the contractile response
to adenosine.
A1-Receptor Antagonist, DPCPX, Potentiates
the Adenosine-Elicited Increase
in Contractile Performance
DPCPX was used in some studies to ascertain
whether A1-receptor stimulation could affect the
adenosine-elicited response. In the absence of DPCPX,
10⫺5 M adenosine increased LVP by 9.5% and
⫹dP/dtmax by 9.8% (Fig. 5). However, in the presence of
10⫺7 M DPCPX, the adenosine-elicited increases in
LVP and ⫹dP/dtmax were significantly potentiated to
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Adenosine (Boehringer Mannheim, Indianapolis, IN) and
NECA (Research Biochemicals, Natick, MA) were prepared
as stock solutions of 10⫺2 M and 3 ⫻ 10⫺3 M, respectively, in
PS or deionized water (MilliQ water system, Millipore, Bedford, MA). CSC (Research Biochemicals) was prepared as a
stock solution of 10⫺3 M in 50% DMSO, and isoproterenol
(Sigma Chemical, St. Louis, MO) was prepared as a stock
solution of 10⫺2 M in 0.1% (wt/vol) sodium metabisulfite.
CGS-21680, DPMA, DPCPX (Research Biochemicals), and
ZM-241385 (Tocris Cookson, Ballwin, MO) were prepared in
stock solutions of 10⫺2 M in DMSO. All solutions were further diluted in PS or water before infusion. Pentobarbital
sodium was obtained from Abbott Laboratories (North Chicago, IL), and all other salts, glucose, and solvents were of
certified grade (Fisher Scientific, Boston, MA, or J. T. Baker,
Phillipsburg, NJ).
Fig. 1. A typical recording illustrating the effect of adenosine
(Ado) on left ventricular pressure (LVP) and maximal rates of LVP
development and relaxation (⫾dP/dtmax). Ado was infused at 10⫺5
M for 3 min. The heart was perfused at a constant-pressure (65
mmHg) and paced at 300 contractions/min, and coronary flow was
16 ml/min.
MYOCARDIAL CONTRACTILITY AND ADENOSINE A2a RECEPTORS
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Pertussis Toxin Potentiated the Adenosine-Elicited
Increase in Contractile Performance
Because the A1-receptor antagonist DPCPX potentiated the contractile response of adenosine, pertussis
toxin was used as an alterative approach to eliminate
the influence of A1-receptor stimulation, which is manifest via inhibitory G protein action. Treatment with
pertussis toxin increased the 10⫺6, 10⫺5, and 10⫺4 M
adenosine-elicited increases in LVP from 6.0, 9.5, and
13.7% to 10.4, 15.9, and 21.0%, respectively (Fig. 6).
The increases in ⫹dP/dtmax caused by these adenosine
concentrations were enhanced from 6.0, 9.8, and 14.0%
to 10.4, 16.4, and 18.0%, respectively. Thus pertussis
toxin, like DPCPX, potentiated the adenosine-elicited
increases in the contractile variables by 31–74%.
Atenolol was used in some studies to assess whether
catecholamines endogenously released could play a
role in the adenosine-elicited increase in contractility.
Atenolol alone had no effect on LVP and ⫹dP/dtmax and
did not affect the 10⫺5 M adenosine-elicited
10.4–10.8% increases in these contractile variables
Fig. 2. Effect of 10⫺8 –10⫺4 M Ado on LVP (A), ⫹dP/dtmax (B), and
⫺dP/dtmax (C) of constant-pressure-perfused hearts. Values are
means ⫾ SE for 7 hearts. * Statistically significant increase from zero
adenosine.
15.2 and 15.4%, respectively. DPCPX alone did not
affect the basal level of LVP or ⫹dP/dtmax.
A2a-Receptor Agonists, NECA and DPMA, Increase
Left Ventricular Contractile Performance
As with adenosine, NECA and DPMA at 10⫺6 M
increased LVP and ⫹dP/dtmax (Fig. 5). NECA increased
LVP by 4.5% and ⫹dP/dtmax by 4.2%. DPMA increased
LVP and ⫹dP/dtmax by 4.1 and 3.5%, respectively. The
A2a-receptor agonist CGS-21680 at 10⫺7 –10⫺5 M did
not increase LVP and ⫹dP/dtmax (data not shown).
Furthermore, at these concentrations, CGS-21680 did
not significantly increase coronary flow. Infusion of the
appropriate vehicle (DMSO or water) did not affect
LVP or ⫹dP/dtmax.
Fig. 3. Effect of 10⫺9 –10⫺4 M Ado on coronary flow (A) and coronary
resistance (E; B) of constant-pressure-perfused hearts. Values are
means ⫾ SE for 7 hearts. * Statistically significant difference from
zero adenosine.
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Adenosine-Elicited Contractile Response Is Unaffected
by the ␤1-Adrenergic Receptor Antagonist Atenolol
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MYOCARDIAL CONTRACTILITY AND ADENOSINE A2a RECEPTORS
A1-Receptor Antagonist DPCPX Potentiates the
Adenosine-Elicited Contractile Performance
Increase in Constant-Flow-Perfused Hearts
In constant-flow-perfused hearts, DPCPX potentiated the 10⫺5 M adenosine-induced increases in LVP
and ⫹dP/dtmax (Fig. 9). DPCPX potentiated the
adenosine-elicited increases in LVP and ⫹dP/dtmax
from 5.6 and 5.9% to 8.8 and 8.6%, respectively. The
potentiation was similar to that observed in the constant-pressure-perfused hearts (Fig. 5).
Adenosine Elicits an Increase in Contractile
Performance in the Maximally Dilated
Constant-Flow-Perfused Hearts
Fig. 4. Effect of 10⫺7 M chlorostyryl-caffeine (CSC) or ZM-241385
(ZM) on the increase of LVP (A) and ⫹dP/dtmax (B) elicited by 10⫺5 M
adenosine in constant-pressure-perfused hearts. Values are means ⫾
SE of 6 hearts. * Statistically significant increase in the contractile
variable from zero adenosine with no additions.
(Fig. 7). However, the adrenergic antagonist did prevent the 17.8 and 18.3% increases in LVP and
⫹dP/dtmax, respectively, caused by the ␤-adrenergic
agonist isoproterenol at 10⫺9 M. Isoproterenol was
employed at this concentration because it produced a
contractile response similar in magnitude to that
caused by 10⫺5 M adenosine. Isoproterenol at 10⫺8 M
produced a twofold increase in contractility (data not
shown), as previously reported for a perfused rat heart
preparation (9).
Adenosine Increased Contractile Performance
With Constant-Flow Perfusion
In constant-flow-perfused hearts having a coronary
flow of 16 ⫾ 1 ml/min, 10⫺5 M adenosine increased
LVP, ⫹dP/dtmax, and ⫺dP/dtmax by 5.5, 6.0, and 6.5%,
respectively (Fig. 8). At 10⫺4 M adenosine the increases were 8.7, 8.9, and 9.4%, respectively, for these
contractile variables. The coronary perfusion pressure
was only significantly decreased by 5.2% at 10⫺4 M
adenosine. This indicated that the increase in the contractile variables at 10⫺5 M adenosine was most likely
independent of a change in perfusion pressure.
Fig. 5. Effect of 10⫺5 M Ado, 10⫺7 M dipropylcyclopentylxanthine
(DPCPX), 10⫺6 M ethylcarboxamido-adenosine (NECA), and 10⫺6 M
dimethoxyphenyl-methylphenylethyl-adenosine (DPMA) on LVP (A)
and ⫹dP/dtmax (B) of constant-pressure-perfused hearts. Values are
means ⫾ SE from 5 hearts. * Statistically significant increase in the
contractile variable from control (Cont). † Significant increase from
the Ado value.
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In constant-flow-perfused hearts, hydralazine, a
vasodilator, was administered at 10⫺4 M to maximally dilate the coronary vasculature. The coronary
flow increased and ranged from 18 to 20 ml/min to
maintain a perfusion pressure of 65 mmHg. In the
presence of hydralazine, 10⫺5 M adenosine increased
LVP and ⫹dP/dtmax by 7.5 and 7.4%, respectively
(Fig. 10). In the presence of DPCPX, the adenosineinduced increases in these contractile variables were
MYOCARDIAL CONTRACTILITY AND ADENOSINE A2a RECEPTORS
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Fig. 6. Effect of pertussis toxin (PT) on the Ado-elicited increase
in LVP (A) and ⫹dP/dtmax (B) of constant-pressure-perfused
hearts. Experiments were performed in the absence (E) and presence (●) of pertussis toxin. Values are means ⫾ SE for 5 hearts.
* Statistically significant increase from zero Ado. † Significantly
different from the corresponding Ado value in the absence of
pertussis toxin.
12 and 11.2%, respectively. CSC at 10⫺7 M prevented
these increases in LVP and ⫹dP/dtmax. After a 30min washout of CSC, 10⫺5 M adenosine in the absence of DPCPX increased LVP and ⫹dP/dtmax by 6.8
and 6.7%, respectively. ZM-241385, like CSC, at
10⫺7 M prevented the adenosine-induced increase in
LVP and ⫹dP/dtmax (data not shown). The adenosine-elicited contractile response also returned on
washout of ZM-241385. Overall the above results
indicate that the adenosine-elicited increase in contractility of the perfused heart is independent of
changes in coronary flow.
DISCUSSION
Adenosine Enhancement of Contractile Performance
The present study indicates that A2a-receptor activation increases left ventricular contractile performance in intact hearts isolated from rats. Adenosine
increased contractility in a concentration-dependent
fashion, starting at 10⫺6 M (Fig. 2). Only the highest
concentration of adenosine (10⫺4 M) significantly in-
Fig. 7. Effect of 10⫺6 M atenolol (Atn) on the 10⫺5 M Ado- or 10⫺9 M
isoproterenol (Iso)-induced increases in LVP (A) and ⫹dP/dtmax (B) in
constant-pressure-perfused hearts. Values are means ⫾ SE for 6
hearts. * Statistically significant increase from value with no additions.
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creased coronary flow (Fig. 3). The increases in contractility elicited by adenosine were prevented by the A2areceptor antagonists CSC (14, 19) and ZM-241385 (22,
29) at 10⫺7 M (Fig. 4). Because these compounds are
primarily A2a-receptor antagonists, the present findings would suggest that the contractile responses are
probably the result of A2a-receptor stimulation. In addition, the A2a-receptor agonists NECA and DPMA
increased left ventricular contractile performance, further indicating that the increase is mediated by A2areceptor activation (Fig. 5). The increase in left ventricular contractile performance mediated by
adenosine was not antagonized by the ␤-adrenergic
antagonist atenolol, indicating that the contractile response was not mediated by ␤-adrenergic stimulation
(Fig. 7).
The antagonism of the adenosine-elicited contractile response by CSC required ⱖ60 min of continuous
CSC treatment to completely develop. However, the
antagonism of the adenosine-elicited contractile response was observed within 5 min after the administration of ZM-241385. The reason for this difference in the time required to achieve A2a-receptor
blockade is not known.
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MYOCARDIAL CONTRACTILITY AND ADENOSINE A2a RECEPTORS
Potentiation of Adenosine by Pertussis Toxin
and A1-Receptor Antagonism
Pertussis toxin, which is known to prevent A1-receptor-mediated events in the heart (18, 20) by ADPribosylation of Gi protein, potentiated the adenosineelicited increase in contractile performance. This
Fig. 8. Effect of 10⫺8 –10⫺4 M Ado on LVP (A), ⫹dP/dtmax (B),
⫺dP/dtmax (C), and coronary perfusion pressure (D) of constant-flowperfused hearts. Values are means ⫾ SE for 7 hearts. * Statistically
significant increase from zero Ado.
Adenosine Contractile Effects With Constant-Pressure
and Constant-Flow Perfusion
The adenosine-elicited increase in contractile performance was presumably not caused by increased coronary flow elicited by adenosine-induced vasodilation,
because it was also observed under constant-flow perfusion conditions (Fig. 8). An adenosine-elicited contractile response was observed after pretreatment of
hearts with the coronary vasodilator hydralazine (Fig.
10). Moreover, hydralazine dilated the vascular bed,
thereby reducing the vasodilatory effect of 10⫺4 M
adenosine (Fig. 8). This prevented any reduction in
perfusion pressure caused by adenosine. The increase
Fig. 9. Effect of 10⫺7 M DPCPX on the 10⫺5 M Ado-induced increase
in LVP (A) and ⫹dP/dtmax (B) of constant-flow-perfused hearts.
Values are means ⫾ SE for 6 hearts. * Statistically significant increase in the contractile variable from value with no addition. † Significant increase from the Ado value.
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in ventricular contractility elicited by adenosine under
constant-flow perfusion conditions was antagonized by
CSC and ZM-241385, further indicating that this increase in contractility is mediated by activation of the
A2a receptor.
The adenosine-elicited contractile responses were
somewhat greater with constant-pressure than with
constant-flow perfusion. This small difference in the
contractile responses could be a flow-dependent component related to the Gregg phenomenon (7, 16) in
constant-pressure-perfused hearts. The flow-dependent component is assumed to be small, because only
the largest concentration of adenosine administered
caused a significant increase in coronary flow. Thus the
positive inotropic responses elicited by adenosine are
independent of coronary flow changes.
MYOCARDIAL CONTRACTILITY AND ADENOSINE A2a RECEPTORS
suggests that, in the absence of pertussis toxin, adenosine is also activating myocardial A1 receptors, which
appear to oppose the A2a-receptor-elicited contractile
effects of adenosine. Moreover, in the presence of the
A1-receptor antagonist DPCPX (17), the contractile response to adenosine was greater than in the absence of
the antagonist. This also indicates that with blockade
of A1 receptors the A2a-receptor-mediated action is
enhanced. The interaction of A1 receptors and A2a
receptors has been observed previously where the use
of A2a-receptor antagonists was reported to enhance
the antiadrenergic action of A1-receptor agonists in
perfused hearts (28).
Myocardial Actions of Adenosine
and Adenosine Analogs
The results from this study suggest that A2a-receptor
activation increases contractility in the intact heart, as
previously reported for ventricular myocytes isolated
from rats (11, 40), human myocardium (34), and chick
embryos (25, 26, 39). In addition, these results confirm
previous studies that indicate that stimulation of adenosine receptors increases contractility in isolated papillary muscles from rats (23), guinea pigs (4), and dogs
(6). However, other investigators have shown that
adenosine or A2-receptor agonists do not increase contractility in isolated ventricular myocytes from rats
(32), rabbits (32), or guinea pigs (2, 32, 35). Utilizing
papillary muscle isolated from guinea pigs, Stein et al.
(35) reported that the A2a-receptor agonist CGS-21680
does not increase myocardial contractility. In addition,
Shryock et al. (32) and Felsch et al. (12) reported that
the A2a-receptor agonists CGS-21680, methylphenylethoxy-adenosine (WRC-0090), and NECA do not increase the contractile performance of hearts isolated
from guinea pigs. It has also been shown that adenosine does not increase contractility of rat ventricular
strips (5). The reason for these differences is not
known, but several suggestions can be offered. First,
the effect of A2a-receptor stimulation on myocardial
contractile performance may be lost in studies where
the animals were stressed before experimentation.
During the course of this study, it was found that
adenosine-induced contractile responses were reduced
or absent in hearts isolated from hyperexcited rats
compared with hearts from tranquil rats. Second, the
effect of A2a-receptor stimulation on myocardial contractility might also vary depending on the underlying
level of arrhythmia. In our study a contractile response
to adenosine was not detectable in hearts that displayed a high level of ventricular arrhythmias during
experimentation. In these hearts the contractile response to adenosine was comparable to, or smaller
than, the oscillations in baseline contractility elicited
by these arrhythmias. Third, the variable effects of
A2a-receptor stimulation on myocardial contractility
observed in previous studies might be related to the
experimental system utilized for measuring contractility. It is possible that our experimental system, which
utilized an analog-to-digital converter and computer
software for assessing left ventricular contractility,
facilitated the resolution of the small increases in contractility elicited by adenosine. Other studies that relied solely on polygraphic recordings of myocardial
contractility might have missed this response. Fourth,
the lack of a contractile response to A2a-receptor stimulation in some reports could also be due to differences
in preparations such as those that are inherent in
ventricular myocyte isolation. Finally, the effect of
A2a-receptor activation on myocardial contractility
might vary depending on the specific A2a-receptor agonist utilized. It was found here that the A2a-receptor
agonists NECA and DPMA produced smaller increases
in contractility than adenosine (Fig. 5). However, the
A2a-receptor agonist CGS-21680 (11, 21) did not cause
a significant increase in contractility or coronary flow.
It is likely that different A2a-receptor agonists may
have varying degrees of A2a-receptor availability or
activation/transduction efficiencies in the intact heart.
Mechanism of Adenosine Action
The positive inotropic action of adenosine reported
here appears to occur via A2a receptors. Agonists for
this receptor have been reported in cardiac prepara-
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Fig. 10. Effect of 10⫺7 M DPCPX or CSC on the 10⫺5 M Ado-induced
increase in LVP (A) and ⫹dP/dtmax (B) of constant-flow-perfused
hearts in the presence of 10⫺4 M hydralazine. Values are means ⫾
SE for 6 hearts. * Statistically significant increase in the contractile
variable from value with no additions. † Significant increase from the
Ado value.
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H1480
MYOCARDIAL CONTRACTILITY AND ADENOSINE A2a RECEPTORS
This publication was made possible by National Institutes of
Health Grants AG-11491 and HL-22828. Its contents are solely the
responsibility of the authors and do not necessarily represent the
official view of the National Institutes of Health.
A preliminary report of this work has been presented in abstract
form (FASEB J 13: A110, 1999).
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