Proc. Natl. Acad. Sci. USA Vol. 95, pp. 6995–6999, June 1998 Medical Sciences A physiological role of the adenosine A3 receptor: Sustained cardioprotection BRUCE T. LIANG*† AND KENNETH A. JACOBSON‡ *Department of Medicine, Cardiovascular Division, University of Pennsylvania Medical Center, Philadelphia, PA 19104; and ‡Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes, Digestive and Kidney Disease, National Institutes of Health, Bethesda, MD 20892 Communicated by John W. Daly, National Institute of Diabetes, Bethesda, MD, April 2, 1998 (received for review January 14, 1998) ABSTRACT Adenosine released during cardiac ischemia osine released during a 5-min exposure to simulated ischemia exerts a potent, protective effect in the heart. A newly recog- protected the cardiac cells against injury sustained during a nized adenosine receptor, the A3 subtype, is expressed on the second 90-min period of ischemia (5, 9). Both A1 and A3 cardiac ventricular cell, and its activation protects the ven- receptors coexist on these cardiac ventricular cells. Prior tricular heart cell against injury during a subsequent expo- activation of either receptor with a selective agonist can sure to ischemia. A cultured chicken ventricular myocyte protect the cardiac cells during a subsequent period of pro- model was used to investigate the cardioprotective role of a longed ischemia (5). novel adenosine A3 receptor. The protection mediated by prior The presence of functional A1 and A3 receptors capable of activation of A3 receptors exhibits a significantly longer mediating cardioprotection in both the cardiac cell and the duration than that produced by activation of the adenosine A1 intact heart models suggests that the cell model represents a receptor. Prior exposure of the myocytes to brief ischemia also useful system to delineate the specific physiological role of protected them against injury sustained during a subsequent each receptor in mediating the protection. Further, a number exposure to prolonged ischemia. The adenosine A3 receptor- of advantages of the cardiac cell model enabled determination selective antagonist 3-ethyl 5-benzyl-2-methyl-6-phenyl-4- and characterization of the physiological function(s) of each phenylethynyl-1,4-(6)-dihydropyridine-3,5-dicarboxylate adenosine receptor. First, the cultures contain predominantly (MRS1191) caused a biphasic inhibition of the protective cardiac cells and are devoid of vascular, circulating blood cells effect of the brief ischemia. The concomitant presence of the or neurons. Potential confounding effects arising from activa- A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine tion of adenosine receptors on these other cells were avoided. (DPCPX) converted the MRS1191-induced dose inhibition Second, the spontaneously beating nature of these cardiac cells curve to a monophasic one. The combined presence of both suggests that they likely had metabolic requirements more akin antagonists abolished the protective effect induced by the brief to those of beating cardiac cells in the intact heart. Third, the ischemia. Thus, activation of both A1 and A3 receptors is extent of cardioprotection can be readily quantitated. The required to mediate the cardioprotective effect of the brief objective of the present study was to determine the role of ischemia. Cardiac atrial cells lack native A3 receptors and adenosine A1 and A3 receptors in the protection achieved by exhibit a shorter duration of cardioprotection than do ven- the brief initial ischemia and to investigate whether the tricular cells. Transfection of atrial cells with cDNA encoding cardioprotective function mediated by the A1 receptor differs the human adenosine A3 receptor causes a sustained A3 from that mediated by the A3 subtype. agonist-mediated cardioprotection. The study indicates that cardiac adenosine A3 receptor mediates a sustained cardio- protective function and represents a new cardiac therapeutic MATERIALS AND METHODS target. Materials. The adenosine analogs 2-chloro-N6-cyclopenty- ladenosine (CCPA) and 8-cyclopentyl-1,3-dipropylxanthine Adenosine is released in large amounts during myocardial (DPCPX) were from Research Biochemicals (Natick, MA). ischemia and can exert potent, protective effects in the heart 2-Chloro-N6-(3-iodobenzyl)adenosine-59-N-methyluronamide (1). Thus, adenosine released during a brief ischemic episode (Cl-IB-MECA) and 3-ethyl 5-benzyl-2-methyl-6-phenyl-4- can protect the heart against injury sustained during a subse- phenylethynyl-1,4-(6)-dihydropyridine-3,5-dicarboxylate quent period of prolonged ischemia, causing a reduction in the (MRS1191) were synthesized as described (13, 14). Full-length infarct size (2–5). A brief prior exposure of the heart to cDNA encoding the human adenosine A3 receptor was kindly adenosine, instead of brief ischemia, can induce a similar provided by Peter Schofield (Garvan Medical institute, Syd- protective effect. The mechanism by which adenosine causes ney, Australia). Adenosine was obtained from Sigma. The the cardioprotective effect is incompletely understood. Al- vector pcDNA3 was obtained from Invitrogen. Embryonated though both A1 and A3 receptors can mediate cardioprotection chicken eggs were from SPAFAS (Norwich, CT). (5–8), the specific protective function mediated by each re- Preparation of Cardiac Myocyte Model of Simulation of ceptor has not been delineated. Each receptor may serve a Ischemia. Atrial and ventricular cells were cultured from distinct role in cardiac tissue. chicken embryos 14 days in ovo and maintained in culture as A cultured ventricular cell model has been developed (5, 9, described previously (9, 10, 15). All experiments were per- 10) that exhibits adenosine-mediated cardioprotection, char- formed on day 3 in culture, at which time the medium was acteristic of that found in the intact heart (4, 6–8, 11, 12). changed to a Hepes-buffered medium containing 139 mM Exposure of the cardiac cells to hypoxia in glucose-free medium has been used to simulate ischemia (5, 9, 10). Aden- Abbreviations: CCPA, 2-chloro-N6-cyclopentyladenosine; DPCPX, 8-cyclopentyl-1,3-dipropylxanthine; MRS1191, 3-ethyl 5-benzyl-2- 6 The publication costs of this article were defrayed in part by page charge methyl-6-phenyl-4-phenylethynyl-1,4-( )-dihydropyridine-3,5- dicarboxylate; Cl-IB-MECA, 2-chloro-N6-(3-iodobenzyl)adenosine- payment. This article must therefore be hereby marked ‘‘advertisement’’ in 59-N-methyluronamide; CK, creatine kinase. accordance with 18 U.S.C. §1734 solely to indicate this fact. †To whom reprint requests should be addressed at: 504 Johnson © 1998 by The National Academy of Sciences 0027-8424y98y956995-5$2.00y0 Pavilion, University of Pennsylvania Medical Center, 3610 Hamilton PNAS is available online at http:yywww.pnas.org. Walk, Philadelphia, PA 19104. e-mail: [email protected]. 6995 Downloaded by guest on October 3, 2021 6996 Medical Sciences: Liang and Jacobson Proc. Natl. Acad. Sci. USA 95 (1998) NaCl, 4.7 mM KCl, 0.5 mM MgCl2, 0.9 mM CaCl2,5mM Hepes (N-2-hydroxyethylpiperazine-N9-2-ethanesulfonic acid) and 2% fetal bovine serum, pH 7.4, 37°C, before exposing the myocytes to simulated ischemia. Simulated ischemia was in- duced by exposing the myocytes to 90 min of hypoxia and glucose deprivation in a hypoxic incubator (NuAire) where O2 was replaced by N2 as described previously (9, 10). The extent of myocyte injury was determined at the end of the 90-min ischemia, at which time myocytes were taken out of the hypoxic incubator and reexposed to room air (normal percentage of O2). Aliquots of the medium then were obtained for creatine kinase activity measurement, which is followed by quantitation of the number of viable cells, as determined by the ability to exclude trypan blue (5). Measurement of the basal level of cell injury was made after parallel incubation of control cells under normal percentage of O2. The extent of ischemia-induced injury was determined quantitatively by the percentages of FIG. 1. Effect of 8-cyclopentyltheophylline on the CCPA- and cells killed and by the amount of creatine kinase (CK) released Cl-IB-MECA-induced cardioprotective effect. Cultured ventricular into the medium according to a previously described method cells were prepared and exposed to either CCPA (10 nM) or Cl-IB- MECA (10 nM) in the presence and absence of varying concentrations (9). The amount of CK was measured as enzyme activity y of the adenosine A1 receptor antagonist 8-cyclopentyltheophylline. (unit mg), and increases in CK activity above the control level Media were changed to that lacking agonist or antagonist. Cells were were determined. The percentage of cells killed was calculated then exposed to room air at 37°C for 10 min before being exposed to as the number of cells obtained from the control group simulated ischemia for 90 min. The percentage of cells killed and the (representing cells not subjected to any hypoxia or drug amount of creatine kinase released were determined after the 90-min treatment) minus the number of cells from the treatment ischemic period. Data represented the means and standard errors of group divided by number of cells in control group multiplied four experiments. by 100%. Gene Transfer into Cardiac Myocytes. Cardiac atrial myo- by the A3 receptor, antagonists selective at the A3 receptor (10, cytes were transfected with pcDNA3 or with pcDNA3 con- 13, 14) were used. The antagonist MRS1191, previously shown taining the full-length cDNA encoding the human adenosine