Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Cardiac contractility modulation wikipedia , lookup
Quantium Medical Cardiac Output wikipedia , lookup
History of invasive and interventional cardiology wikipedia , lookup
Jatene procedure wikipedia , lookup
Arrhythmogenic right ventricular dysplasia wikipedia , lookup
Management of acute coronary syndrome wikipedia , lookup
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 Downloaded from http://ajpheart.physiology.org/ by 10.220.33.6 on May 12, 2017 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 Downloaded from http://ajpheart.physiology.org/ by 10.220.33.6 on May 12, 2017 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. H1473 H1474 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 Downloaded from http://ajpheart.physiology.org/ by 10.220.33.6 on May 12, 2017 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 H1475 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. Downloaded from http://ajpheart.physiology.org/ by 10.220.33.6 on May 12, 2017 Adenosine-Elicited Contractile Response Is Unaffected by the 1-Adrenergic Receptor Antagonist Atenolol H1476 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. Downloaded from http://ajpheart.physiology.org/ by 10.220.33.6 on May 12, 2017 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 H1477 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. Downloaded from http://ajpheart.physiology.org/ by 10.220.33.6 on May 12, 2017 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. H1478 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. Downloaded from http://ajpheart.physiology.org/ by 10.220.33.6 on May 12, 2017 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- Downloaded from http://ajpheart.physiology.org/ by 10.220.33.6 on May 12, 2017 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. H1479 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). REFERENCES 1. Armstrong S and Ganote CE. Adenosine receptor specificity in preconditioning of isolated rabbit cardiomyocytes: evidence of A3 receptor involvement. Cardiovasc Res 28: 1049–1056, 1994. 2. Behnke N, Muller W, Neumann J, Schmitz W, Scholz H, and Stein B. Differential antagonism by 1,3-dipropylxanthine8-cyclopentylxanthine and 9-chloro-2-(2-furanyl)-5,6-dihydro1,2,4-triazolo(1,5-c)quinazolin-5-imine of the effects of adenosine derivatives in the presence of isoprenaline on contractile response and cyclic AMP content in cardiomyocytes. Evidence for the coexistance of A1- and A2-adenosine receptors on cardiomyocytes. J Pharmacol Exp Ther 254: 1017–1023, 1990. 3. Berne RM. The role of adenosine in the regulation of coronary blood flow. Circ Res 47: 807–813, 1980. 4. Bruckner R, Fenner A, Meyer W, Nobis T-M, Schmitz W, and Scholz H. Cardiac effects of adenosine and adenosine analogs in guinea-pig atrial and ventricular preparations: evidence against a role of cyclic AMP and cyclic GMP. J Pharmacol Exp Ther 234: 766–774, 1985. 5. Burnstock G and Meghji P. The effect of adenyl compounds on the rat heart. Br J Pharmacol 79: 211–218, 1983. 6. Chiba S and Himori N. Different inotropic responses to adenosine on the atrial and ventricular muscle of the dog heart. Jpn J Pharmacol 25: 489–491, 1975. 7. Dijkman MA, Heslinga JW, Sipkema P, and Westerhof N. Perfusion-induced changes in cardiac contractility depend on capillary perfusion. Am J Physiol Heart Circ Physiol 274: H405– H410, 1998. 8. Dobson JG Jr. Reduction by adenosine of the isoproterenolinduced increase in cyclic adenosine 3⬘,5⬘-monophosphate formation and glycogen phosphorylase activity in rat heart muscle. Circ Res 43: 785–792, 1978. 9. Dobson JG Jr. Mechanism of adenosine inhibition of catecholamine-induced elicited responses in heart. Circ Res 52: 151– 160, 1983. 10. Dobson JG Jr. Adenosine and adrenergic mediated effects in the heart. In: Purines and Myocardial Protection, edited by Abd-Elfattah AS and Wechsler AS. Norwell, MA: Kluwer, 1996, p. 359–372. 11. Dobson JG Jr and Fenton RA. Adenosine A2a receptor function in rat ventricular myocytes. Cardiovasc Res 34: 337–347, 1997. 12. Felsch A, Stocker K, and Borchard U. Adenosine A1 and A2 receptor agonists alter cardiac functions and prostacyclin release in the isolated guinea-pig heart. Eur J Pharmacol 263: 261–268, 1994. 13. Fenton RA and Dobson JG Jr. Adenosine and calcium alter adrenergic-induced intact heart protein phosphorylation. Am J Physiol Heart Circ Physiol 246: H559–H565, 1984. 14. Fredholm BB, Abbracchio MP, Burnstock G, Daly JW, Harden TK, Jacobson KA, Leff P, and Williams M. Nomenclature and classification of purinoceptors. Pharmacol Rev 46: 143–156, 1994. 15. George EE, Romano FD, and Dobson JG Jr. Adenosine and acetylcholine reduce isoproterenol-induced protein phosphorylation of rat myocytes. J Mol Cell Cardiol 23: 749–764, 1991. 16. Gregg DE. Effect of coronary perfusion pressure or coronary flow on oxygen usage of the myocardium. Circ Res 13: 497–500, 1963. 17. Haleen SJ, Steffen RP, and Hamilton HW. PD 116,948, a highly selective A1 adenosine receptor antagonist. Life Sci 40: 555–561, 1987. 18. Hazeki O and Ui M. Modification by islet-activating protein of receptor-mediated regulation of cyclic AMP accumulation in isolated rat heart cells. J Biol Chem 256: 2856–2862, 1981. 19. Jacobson KA, Nikodijevic O, Padgett WL, Gallo-Rodriguez C, Maillard M, and Daly JW. 8-(3-Chlorostyryl) caffeine (CSC) is a selective A2-adenosine antagonist in vitro and in vivo. FEBS Lett 323: 141–144, 1993. 20. Jakobs KH, Aktories K, and Schultz G. Mechanism of pertussis toxin action on the adenylate cyclase system. Eur J Biochem 140: 177–181, 1984. 21. Jarvis MF, Schulz R, Hutchison AJ, Do UH, Sills MA, and Williams M. [3H]CGS 21680, a selective A2 adenosine receptor agonist, directly labels A2 receptors in rat brain. J Pharmacol Exp Ther 251: 888–893, 1989. 22. Keddie JR, Poucher SM, Shaw GR, Brooks R, and Collis MG. In vivo characterization of ZM241385, a selective adenosine A2a receptor antagonist. Eur J Pharmacol 301: 107–113, 1996. 23. Legssyer A, Poggioli J, Renard D, and Vassort G. ATP and other adenine compounds increase mechanical activity and inositol trisphosphate production in rat heart. J Physiol (Lond) 401: 185–199, 1988. 24. Liang BT. Direct preconditioning of cardiac ventricular myocytes via adenosine A1 receptor and KATP channel. Am J Physiol Heart Circ Physiol 271: H1769–H1777, 1996. 25. Liang BT and Haltiwanger B. Adenosine A2a and A2b receptors in cultured fetal chick heart cells. High- and low-affinity coupling to stimulation of myocyte contractility and cAMP accumulation. Circ Res 76: 242–251, 1995. 26. Liang BT and Morley JF. A new cyclic AMP-independent, Gs-mediated stimulatory mechanism via the adenosine A2a receptor in the intact cardiac cell. J Biol Chem 271: 18678–18685, 1996. 27. Liu GS, Richards SC, Olsson RA, Mullane K, Walsh RS, and Downey JM. Evidence that the adenosine A3 receptor may mediate the protection afforded by preconditioning in the isolated rabbit heart. Cardiovasc Res 28: 1057–1061, 1994. 28. Norton GR, Woodiwiss AJ, McGinn RJ, Lorbar M, Chung ES, Honeyman TW, Fenton RA, Dobson JG Jr, and Meyer TE. Adenosine A1 receptor-mediated antiadrenergic effects are modulated by A2a receptor activation in rat heart. Am J Physiol Heart Circ Physiol 276: H341–H349, 1999. 29. Poucher SM, Keddie JR, Singh P, Stoggall SM, Caulkett PWR, Jones G, and Collis MG. The in vitro pharmacology of ZM241385, a potent, non-xanthine, A2a selective adenosine receptor antagonist. Br J Pharmacol 115: 1096–1102, 1995. 30. Romano FD, Macdonald SG, and Dobson JG Jr. Adenosine receptor coupling to adenylate cyclase of rat ventricular myocyte Downloaded from http://ajpheart.physiology.org/ by 10.220.33.6 on May 12, 2017 tions to mediate responses via cAMP-dependent and -independent mechanisms (11, 26), possibly involving Ca2⫹ (26) but having only a minimal effect on intracellular Ca2⫹ transients (38). The mechanism(s) involved requires further study. In conclusion, the present study shows that A2areceptor activation increases contractility of the intact heart isolated from rats. This increase in contractility is independent of activation of -adrenergic receptors or the increase in coronary flow mediated by adenosine-induced vasodilation. Although the adenosine A2a-receptor-elicited positive inotropic response is rather small in magnitude, it may be important in providing contractile support of the mechanically depressed hypoperfused heart in which interstitial adenosine levels are increased (10). Furthermore, A2a-receptor activation has been demonstrated to attenuate the antiadrenergic action of adenosine mediated by A1 receptors (28). Thus inhibition of the antiadrenergic effect of adenosine coupled with its intrinsic direct positive inotropic effect suggests that stimulation of A2a receptors is likely to be capable of enhancing and maintaining myocardial contractility. The role of A2a receptors in modulation of heart contractile function should be addressed further. MYOCARDIAL CONTRACTILITY AND ADENOSINE A2a RECEPTORS 31. 32. 33. 34. 35. 36. Strickler J, Jacobson KA, and Liang BT. Direct preconditioning of cultured chick ventricular myocytes. Novel functions of cardiac adenosine A2a and A3 receptors. J Clin Invest 98: 1773–1779, 1996. 37. West GA and Belardinelli L. Sinus slowing and pacemaker shift caused by adenosine in rabbit SA node. Pflügers Arch 403: 66–74, 1985. 38. Woodiwiss AJ, Honeyman TW, Fenton RA, and Dobson JG Jr. Adenosine A2a-receptor activation enhances cardiomyocyte shortening via Ca2⫹-independent and -dependent mechanisms. Am J Physiol Heart Circ Physiol 276: H1434–H1441, 1999. 39. Xu D, Kong H, and Liang BT. Expression and pharmacological characterization of a stimulatory subtype of adenosine receptor in fetal chick ventricular myocytes. Circ Res 70: 56–65, 1992. 40. Xu H, Stein B, and Liang BT. Characterization of a stimulatory adenosine A2a receptor in adult rat ventricular myocyte. Am J Physiol Heart Circ Physiol 270: H1655–H1661, 1996. Downloaded from http://ajpheart.physiology.org/ by 10.220.33.6 on May 12, 2017 membranes. Am J Physiol Heart Circ Physiol 257: H1088– H1095, 1989. Schrader J, Baumann G, and Gerlach E. Adenosine as inhibitor of myocardial effects of catecholamines. Pflügers Arch 372: 29–35, 1977. Shryock J, Song Y, Wang D, Baker SP, Olsson RA, and Belardinelli L. Selective A2-adenosine receptor agonists do not alter action potential duration, twitch shortening, or cyclic AMP accumulation in guinea pig, rat, or rabbit isolated ventricular myocytes. Circ Res 72: 194–205, 1993. Shultz PJ and Raij L. Effect of antihypertensive agents on endothelium-dependent and endothelium-independent relaxations. Br J Clin Pharmacol 23 Suppl 2: 151S–157S, 1989. Stein B, Kiehn J, Borea PA, and Gessi S. Role of adenosine A2a receptors in failing human hearts: a new regulatory mechanism (Abstract)? Circulation 100 Suppl 1: I-559, 1999. Stein B, Schmitz W, Scholz H, and Seeland C. Pharmacological characterization of A2-adenosine receptors in guinea pig ventricular cardiomyocytes. J Mol Cell Cardiol 26: 403–414, 1994. H1481