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Am J Physiol Heart Circ Physiol 282: H49–H57, 2002. Comparison of the vascular effects of adenosine in isolated mouse heart and aorta M. A. HASSAN TALUKDER,1 R. RAY MORRISON,2 AND S. JAMAL MUSTAFA1 Departments of 1Pharmacology and 2Pediatrics, The Brody School of Medicine, East Carolina University, Greenville, North Carolina 27858-4354 Received 31 May 2001; accepted in final form 17 September 2001 vasodilation in a majority of mammalian vascular beds (12, 24, 26, 38). The cardiovascular effects of adenosine are mediated by activation of four known cell surface receptors (adenosine A1, A2A, A2B, and A3), and are dictated by receptor subtype distribution, agonist affinity, and efficiency of cell signaling mechanisms (6, 8, 25, 33). It is now established that vasodilatory effects of adenosine and its analogs are mediated by A2 receptors in different species, including bovine, canine, porcine, rat, guinea pig, and humans (1, 9, 16, 21, 27, 34). However, the relative contribution of A2 adenosine receptor subtypes (A2A and A2B) in modulating coronary and peripheral vascular responses is not fully understood. Because adenosine receptors are widely distributed throughout the body, understanding differences in regional vascular responses mediated by each receptor subtype will be necessary for the development of future adenosinergic therapies. Characterization of adenosine receptor-mediated vascular responses and their specific subcellular mechanisms will be best achieved by combining a traditional pharmacological approach with newly available gene-modified animal models. Indeed, a murine model with targeted deletion of A2A adenosine receptor has been developed and is known to be hypertensive, but the effects of this deletion on the coronary circulation are not yet known (15). Before investigating effects of adenosine in this and other yet-to-be-developed gene-modified models, it is necessary to examine adenosine-induced effects on the coronary and aortic vasculature in mice, because there is limited information in this species (11). The isolated perfused mouse heart is a well-established model to examine pharmacological and physiological responses in vitro (37). Moreover, although resistance vessels are more directly related to the regulation of systemic blood pressure, aortic preparations are routinely used to investigate the functional effects of vasodilator substances and their receptor-mediated mechanisms of action in vitro (5, 7, 32). Therefore, parallel experiments were performed in isolated hearts and aortic rings to assess adenosine-induced responses in these physiologically distinct vascular beds. A selective A2A adenosine receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5⬘-N-ethylcarboxamidoadenosine (CGS-21680) and a selective antagonist 5-amino-7(-phenylethyl)-2-(8-furyl)pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (SCH-58261) were used to directly examine A2A receptor-mediated vascular effects in this study. CGS-21680 is the most potent and selective A2A adenosine receptor agonist known, causing coronary vasodilation at low nanomolar concentrations (39) and virtually no effect at A2B receptors with inhibitor constant (Ki) values at A2A and A2B receptors of 15 nM and ⬎100 Address for reprint requests and other correspondence: S. Jamal Mustafa, Dept. of Pharmacology, School of Medicine, East Carolina University, Greenville, NC 27858-4354 (E-mail: mustafas@mail. ecu.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. coronary flow; adenosine A2A receptor; aortic relaxation; adenosine A2B receptor ADENOSINE PRODUCES POTENT http://www.ajpheart.org 0363-6135/02 $5.00 Copyright © 2002 the American Physiological Society H49 Downloaded from http://ajpheart.physiology.org/ by 10.220.33.4 on May 12, 2017 Talukder, M. A. Hassan, R. Ray Morrison, and S. Jamal Mustafa. Comparison of the vascular effects of adenosine in isolated mouse heart and aorta. Am J Physiol Heart Circ Physiol 282: H49–H57, 2002.—The present study was designed to characterize and compare the vascular effects of adenosine and its analogs in the murine heart and aorta. Mouse hearts perfused under constant pressure in standard Langendorff fashion demonstrated concentration-dependent increases in coronary flow to adenosine, 2-chloradenosine (CAD), 5⬘-(N-ethyl-carboxamido)-adenosine (NECA), and 2-p-(2-carboxyethyl)phenethylamino-5⬘-N-ethylcarboxamidoadenosine (CGS-21680). All agonists produced comparable increases in coronary flow with the following order of potency: CGS-21680 ⫽ NECA ⬎⬎ CAD ⱖ adenosine. In lphenylephrine hydrochloride (phenylephrine) precontracted aortic rings, all nonselective agonists (NECA, CAD, and adenosine) produced marked concentration-dependent relaxation, whereas the adenosine A2A selective agonist CGS21680 did not. Adenosine receptor agonists were ⬎100 times more potent for coronary vasodilation than aortic vasorelaxation. The selective A2A receptor antagonist 5-amino-7-(phenylethyl)-2-(8-furyl)pyrazolo-[4,3-e]-1,2,4-triazolo-[1,5c]pyrimidine (SCH-58261) blocked both CGS-21680- and NECA-induced increases in coronary flow, whereas the A2B receptor antagonist benzo[g]pteridine-2,4(1H,3H)-dione (alloxazine) inhibited NECA-induced aortic relaxation. These data indicate a differential response to adenosine agonists in murine coronary vasculature and aorta where coronary vasodilation is mediated predominantly by activation of A2A adenosine receptors. H50 VASCULAR EFFECTS OF ADENOSINE IN MOUSE MATERIALS AND METHODS All of the experimental protocols were performed according to the guidelines of the Animal Care and Use Committee at East Carolina University. Langendorff-perfused heart preparation. Hearts were isolated from male Balb/c mice (Charles River) of 10 to 12 wk of age as previously described (10, 37). Briefly, mice were deeply anesthetized with pentobarbital sodium (100 mg/kg ip), a thoracotomy was performed, and hearts were quickly excised en bloc with the mediastinum and placed in heparinized ice-cold buffer to arrest cardiac contraction. After lungs, trachea, and extraneous connective tissues were removed, the aorta was carefully tied to an aortic cannula made from a 20-gauge blunted needle. Hearts were retrogradely perfused at a constant pressure of 80 mmHg with warmed KrebsHenseleit buffer in standard Langendorff fashion and allowed to beat spontaneously. The composition of the modified Krebs-Henseleit buffer was composed of (in mM) 118 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, 2.5 CaCl2, 0.5 Na2EDTA, 11 glucose, and 2.0 pyruvate. The buffer was prefiltered to particle size of ⬍0.22 m and bubbled continuously with 95% O2-5% CO2 at 37°C (pH 7.4). The left ventricle was vented with a small polyethylene apical drain, and a water-filled balloon made of plastic wrap was inserted into the left ventricle across the mitral valve through a left atriotomy. The balloon was connected to a fluid-filled pressure transducer by polyethylene tubing for continuous measurement of left ventricular developed pressure (LVDP). Hearts were then immersed in perfusate maintained at 37°C and the ventricular balloon was inflated to yield a left ventricular end-diastolic pressure of 2 to 5 mmHg. Coronary flow was continuously measured using an ultrasonic flow probe (T106, Transonic Systems; Ithaca, NY) placed in the aortic perfusion line, and aortic pressure was recorded via a pressure transducer attached to the sidearm of an aortic cannula. All of the transducers and an ultrasonic flowmeter were coupled to a PowerLab/4sp data acquisition system (ADInstruments; Castle Hill, Australia) and functional data were recorded on an Apple G4 Power Mac computer using PowerLab Chart 3.5.6 software (ADInstruments). Baseline coronary flow, LVDP, and heart rate (derived from the ventricuAJP-Heart Circ Physiol • VOL lar pressure tracing) were monitored for an initial 30-min equilibration period. Hearts with persistent arrhythmias or poor LVDP (⬍50 mmHg) during equilibration were excluded from the study. Protocol for isolated heart experiments. When hearts reached a steady-state coronary flow, increasing concentrations of adenosine and its analogs were infused by a Harvard infusion pump (Harvard Apparatus) into the aortic cannula immediately above the heart at a rate of 1% of the basal flow to achieve the desired concentration in the perfusate. All agonist concentration-response curves (CRCs) were constructed noncumulatively and one CRC was performed on each heart. The concentration of agonist was tested in steps of 0.5-log units. Infusion of each agonist concentration was maintained until coronary flow demonstrated a new steady state (typically within 5 min), and a washout period of at least 5 min was allowed before the administration of the next (higher) concentration. It is reported (14) that successive addition of agonist in the same heart did not have any blunted response on repetition in the rat, and we also did not observe any blunted response on repetition of agonist concentration in the same heart (data not shown). Therefore, the influence of an antagonist was investigated on the same heart in a paired manner for only one agonist concentration. When responses to antagonists are examined, the effect of agonist was first determined in the absence of the antagonist (control). After complete washout of control response (when coronary flow returned to baseline value), the antagonist was infused into the perfusion line and allowed to equilibrate for at least 10 min before adding the same dose of the agonist in the perfusion. The antagonist remained present during agonist administration until steady-state response was achieved. Changes in coronary flow, heart rate, and LVDP were expressed as percent change from predrug baseline value. Preparation of isolated aortic rings. Male Balb/c mice of 8 to 10 wk old were used in this study. The preparation of isolated mouse aorta was similar to that described by Pomerleau et al. (32). Under deep anesthesia with pentobarbital sodium (100 mg/kg ip), a thoracotomy was performed and the thoracic aorta was gently removed. After removal of fat and connective tissues, the aorta was cut transversely into 2 to 3 rings of 3 to 4 mm in length. The rings were mounted vertically between two stainless steel wire hooks with extreme care not to damage the endothelium, and suspended in 10-ml organ baths containing Krebs-Henseleit solution continuously gassed with 95% O2-5% CO2 (37°C, pH 7.4). Aortic rings were allowed to equilibrate for 90 min with an initial resting tension of 1 g (the length-tension relationship determined separately), and the bathing solution was changed at 15-min intervals. Composition of the Krebs-Henseleit solution was (in mM) 118 NaCl, 4.8 KCl, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, 2.5 CaCl2, and 11 glucose. Changes in isometric tension were measured with a fixed-range precision force transducer (model TSD 125C, BIOPAC Systems) connected to a differential amplifier (model DA 100B, BIOPAC Systems). Data were recorded on a Dell computer using MP 100 WSW BIOPAC Systems digital acquisition system and analyzed using Acqknowledge 3.2 Software (BIOPAC Systems). Protocol for aortic relaxation experiments. After equilibration, the responsiveness and stability of each individual ring was checked by the successive administration of a submaximally effective concentration of l-phenylephrine hydrochloride (phenylephrine) (1 M). The integrity of the vascular endothelium was assessed pharmacologically by acetylcholine-induced relaxation of phenylephrine-precontracted rings. Tissues that did not elicit reproducible and stable contraction with phenylephrine, and did not relax ⬎50% to 282 • JANUARY 2002 • www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.33.4 on May 12, 2017 M, respectively (6, 8). SCH-58261 is a potent and highly selective competitive antagonist at adenosine A2A receptors both in vivo and in vitro at nanomolar concentrations, and it has little or no affinity (up to the micromolar range) for adenosine A2B and A3 receptors (30, 31, 41). A2B receptor-mediated responses have been demonstrated by using a nonselective agonist 5⬘-(N-ethyl-carboxamide)-adenosine (NECA) in concert with a putative A2B receptor antagonist benzo[g]pteridine-2,4(1H,3H)-dione (alloxazine) (17, 36). NECA has affinity for both A2A and A2B receptors with Ki values of 3 to 20 nM and 0.5 to 5 M, respectively (6, 8). Likewise, alloxazine is the only nonxanthine antagonist reported to have ninefold greater selectivity for A2B receptors than A2A receptors (4, 6). The purpose of this study was, therefore, twofold: 1) to characterize the responses to adenosine and its analogs in the coronary and aortic vasculature, and 2) to determine which adenosine receptor subtype(s) are involved in these effects using different antagonists. We hypothesized that the vasodilatory response to adenosine would differ in the coronary and aortic vasculature in mice. H51 VASCULAR EFFECTS OF ADENOSINE IN MOUSE (Natick, MA). All other chemicals were of the highest grade available and were purchased from Sigma. NECA, CGS21680, and adenosine antagonists were dissolved in 100% DMSO as 10 mM stock solution, followed by serial dilutions in 50% DMSO and distilled water. All other chemicals were dissolved in distilled water. RESULTS Baseline functional parameters in isolated mouse hearts. Baseline functional parameters for male Balb/c mice were recorded at the end of the 30-min equilibration period for each heart before beginning the experimental protocol. The coronary flow (normalized to the wet weight of each heart), heart rate, and LVDP at equilibrium were 9.42 ⫾ 0.23 ml 䡠 min⫺1 䡠 g⫺1, 353 ⫾ 3.5 beats/min, and 87.5 ⫾ 3.1 mmHg, respectively (n ⫽ 61). Vascular effects of adenosine and its analogs on isolated hearts and aortic rings. Adenosine and its analogs CAD, NECA, and CGS-21680, applied noncumulatively, produced concentration-dependent increases in the coronary flow (vasodilation) in isolated mouse hearts perfused at constant pressure (Fig. 1A). CGS21680 and NECA displayed greater potency for coronary vasodilation than CAD and adenosine. The percent increase in coronary flow caused by 100 nM concentration of adenosine, CAD, NECA, and CGS21680 was 128.98 ⫾ 4.87, 185.55 ⫾ 4.46, 398.92 ⫾ 21.19, and 441.74 ⫾ 21.98%, respectively. Comparing the EC50 values for coronary vasodilation (Table 1), the relative order of agonist potency was CGS-21680 ⫽ NECA ⬎⬎ CAD ⱖ adenosine. In isolated aorta experiments, adenosine, CAD, and NECA relaxed the phenylephrine-precontracted mouse aortic rings in a concentration-dependent manner (Fig. 1B). The maximal responses achieved by these agonists varied (Table 1). Only CAD produced the most complete relaxation, whereas the selective A2A receptor agonist CGS-21680 produced minimal relaxation (Fig. 1B). Higher concentrations of NECA could not be tested because of limited solubility, and a plateau was not reached with adenosine and CAD even at 1 mM Fig. 1. Concentration-dependent vascular effects of adenosine, 2-chloradenosine (CAD), 5⬘-(N-ethyl-carboxamido)adenosine (NECA), and CGS-21680 in isolated perfused mouse heart and mouse aorta. Vehicle control experiments for DMSO were included only in aorta. Each symbol with a vertical bar represents the mean ⫾ SE of 6–12 experiments. Concentration-response curves (CRCs) for coronary flow (A) and aortic relaxation (B) were constructed noncumulatively and cumulatively, respectively. y-axis, Changes in the coronary flow, expressed as percent change from immediate control value, which was considered as 100%, and the aortic relaxation as a percent decrease of l-phenylephrine hydrochloride, (phenylephrine)-induced contraction; x-axis, molar concentration of agonists on a logarithmic scale. AJP-Heart Circ Physiol • VOL 282 • JANUARY 2002 • www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.33.4 on May 12, 2017 10 M acetylcholine were discarded from the study. Preparations were then washed several times with drug-free Krebs-Henseleit solution, and allowed to relax fully for 30 min before the experimental protocol began. To determine the vasodilator responses to adenosine receptor agonists, the aortic rings (with baseline resting tension of 1 g) were precontracted with phenylephrine. The CRCs for aortic relaxation by adenosine and its analogs were obtained by cumulative addition of agonist in the organ bath of rings precontracted with 1 M phenylephrine. The concentration of agonist in the organ bath was increased in steps of either 0.5- or 1-log units. In all cases, agonists were added to yield the next higher concentration only when the response to the lower dose reached a steady state. One CRC was constructed for each ring. To examine the effect of an antagonist, it was added 15 min before contraction of the tissue with phenylephrine, and was present throughout the experiments. Antagonist experiments were performed in parallel using two rings from the same aorta, one serving as control (without antagonist) and one serving as treated (with antagonist). The vasodilator (relaxant) responses were expressed as percent decrease of phenylephrine-induced precontraction where contraction produced by 1 M phenylephrine in each ring from its initial resting tension (1 g) was considered as 100%. In experiments with 2-chloradenosine (CAD), the phenylephrine precontracted rings relaxed ⬎100% by higher concentration of CAD, and this was due to a decrease of phenylephrineinduced contraction in excess to the initial resting tension of 1 g. In the current study, an increase in the coronary flow was considered as vasodilation to reflect qualitatively the vasorelaxation of aortic rings. The terms vasodilation and vasorelaxation have been interchangeably used throughout the manuscript. Data analysis. Experimental values are presented as means ⫾ SE. For each CRC to adenosine and its analogs, the concentration required to produce a 50% response (EC50) in coronary flow was obtained by graphic analysis of individual curve. Significant differences were estimated by Student’s t-test for paired data from the same experiment and unpaired data from different experiments. The difference between two CRCs was estimated by two-way ANOVA for repeated measures. A P value of ⬍0.05 was considered significant. Chemicals. Adenosine, CAD, phenylephrine, and acetylcholine were purchased from Sigma (St. Louis, MO). NECA, CGS-21680, and alloxazine were purchased from RBI H52 VASCULAR EFFECTS OF ADENOSINE IN MOUSE Table 1. Coronary flow and aortic relaxation responses, and associated EC50 values for adenosine and its analogues in mice Langendorff Heart Agonist Maximal CF % n Adenosine CAD NECA CGS-21680 429 ⫾ 15 405 ⫾ 31 419 ⫾ 19 441 ⫾ 22 7 6 6 6 Aorta EC50, nM Maximal relaxation % n EC50 740 335 31 25 80 ⫾ 2 110 ⫾ 1 44 ⫾ 2 14 ⫾ 2 12 12 12 6 ND ND ND ND concentration, therefore, EC50 values could not be calculated. The aortic relaxation produced by 10 M adenosine, CAD, NECA, and CGS-21680 was 4.61 ⫾ 1.01%, 14.82 ⫾ 2.01%, 44.13 ⫾ 2.43%, and 13.84 ⫾ 2.25% of phenylephrine-induced contraction, respectively. To facilitate the comparison of vascular effects of adenosine agonists between coronary and aortic vasculature, data from Fig. 1 were normalized (maximal responses for each agonist were considered as 100%) and plotted in Fig. 2. In isolated hearts, adenosine agonists produced coronary vasodilation at low nanomolar range, whereas in aortic rings, vasorelaxation was seen at micromolar concentration. The CRCs for adenosine agonists for aortic relaxation were shifted ⬃100-fold to the right of coronary flow, implying that adenosine and its agonists were more potent in increasing coronary flow than in producing aortic relaxation. Vehicle control experiments for DMSO have been performed and included in Fig. 1B for aorta where it did not effect vascular response. In isolated hearts, Fig. 2. Comparison of the vascular effects of adenosine, CAD, and NECA in isolated perfused mouse heart and mouse aorta. To facilitate the comparison of vascular responses between heart and aorta, data in Fig. 1 were normalized (maximal response for each agonist was considered as 100%) and plotted for NECA (A), CAD (B), and adenosine (C). Each symbol with a vertical bar represents the mean ⫾ SE of 6–12 experiments. A: CRCs for NECA in the heart and aorta; B: CRCs for CAD in the heart and aorta; and C: CRCs for adenosine in the heart and aorta. y-axis, Responses are expressed as percentage of maximum response; x-axis, molar concentration of agonists on a logarithmic scale. AJP-Heart Circ Physiol • VOL 282 • JANUARY 2002 • www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.33.4 on May 12, 2017 All values are means ⫾ SE obtained from Fig. 1, A and B; n, no. of experiments from different hearts or aorta. CF, coronary flow; CAD, 2-chloradenosine; NECA, 5⬘-(N-ethyl-carboxamido)-adenosine; ND, not determined. Maximal CF is expressed as percent change from respective baseline level and maximal aortic relaxation is expressed as percent decrease of phenylephrine-induced contraction. only the last dose of NECA (1 M) was dissolved in 50% DMSO and this 50% DMSO was delivered as 0.5% DMSO in the perfusion line. Perfusion with vehicle (0.5% DMSO) in isolated hearts for 5 min had very little effect and it increased the coronary flow to 104.74 ⫾ 2.71% (n ⫽ 4) from baseline (100%); however, NECA by itself did not further increase the coronary flow at that dose. Therefore, it is conceivable that DMSO, in particular, did not effect the responses of the tissue to agonists in this study. Influence of A2A adenosine receptor blockade on adenosine-induced vascular responses in isolated mouse hearts and aortic rings. The reproducibility of responses induced by CGS-21680 (100 nM) and NECA (100 nM) on the coronary flow was assessed in a subset of three hearts for each agonist (data not illustrated). Each heart was exposed to the same agonist concentration three times (each time for 5 min), separated by at least 10 min of agonist-free perfusion. The increases in coronary flow in three 5-min CGS-21680 (100 nM) infusions were 435.44 ⫾ 8.40, 429.21 ⫾ 17.99, and 421.70 ⫾ 17.99% of baseline, respectively. NECA (100 nM) also produced qualitatively similar and reproducible effects on coronary flow with repeated exposure, and the percent increase in coronary flow for three 5min NECA infusions was 396.86 ⫾ 9.61, 407.97 ⫾ 23.74, and 421.54 ⫾ 12.03% of baseline, respectively. Thus it is evident that adenosine-induced coronary vascular responses in mouse hearts do not exhibit tachyphylaxis with successive dosing as has been reported (14) in isolated rat hearts. The selective A2A adenosine receptor antagonist SCH-58261 was used to determine the extent to which adenosine A2A receptor activation contributes to coronary vasodilation in isolated hearts. We used 100 nM SCH-58261, because this concentration has been shown to selectively and completely block adenosine VASCULAR EFFECTS OF ADENOSINE IN MOUSE H53 A2A receptor-mediated effects in related experiments (3, 30, 41). SCH-58261 alone decreased the coronary flow to 91.15 ⫾ 1.58% of baseline value (n ⫽ 16; P ⬍ 0.05 vs. baseline). CGS-21680 is the most potent, highly selective agonist at A2A receptors and is virtually ineffective at A2B receptors (6, 25). The Ki values for CGS-21680 at A1, A2A, and A2B receptors are ⬎350 nM, 15 nM, and ⬎100 M, respectively (8). After obtaining control response to CGS-21680 in the absence of SCH-58261 and subsequent 10 min agonist-free perfusion, SCH-58261 was infused for at least 10 min before the second administration of CGS-21680 and was present during 5-min agonist infusion. In these experiments (Fig. 3), control infusion of CGS-21680 at 10 nM and 100 nM in the absence of an antagonist increased coronary flow by 218.11 ⫾ 17.01% (n ⫽ 4) and 443.45 ⫾ 14.18% (n ⫽ 6) of baseline, respectively. Figure 3A shows that SCH-58261 inhibited completely the coronary vasodilation (106.62 ⫾ 7.39% of predrug value) induced by 10 nM CGS-21680. In the presence of SCH-58261, infusion of 100 nM CGS-21680 increased coronary flow by only 154.81 ⫾ 12.03% of predrug baseline value (P ⬍ 0.05 vs. respective control), which was markedly lower than that produced by CGS-21680 alone (Fig. 3B). With the use of a similar protocol, contribution of adenosine A2A receptors on NECA-induced increases in the coronary flow was assessed with SCH-58261. In the absence of SCH-58261, NECA increased the coronary flow by 370.45 ⫾ 14.18% of baseline value (n ⫽ 6). Treatment with SCH-58261 also significantly attenuated NECA-induced coronary flow to 168.81 ⫾ 12.03% of predrug baseline value (P ⬍ 0.05 vs. respective control), which was markedly lower than that produced by NECA alone (Fig. 3C). These data demonstrate that selective A2A adenosine receptor blockade with SCH-58261 inhibits both CGS-21680and NECA-induced coronary vasodilation. To examine the role of adenosine A2A receptors in adenosine-mediated aortic relaxation, antagonism of NECA- and CAD-induced vasorelaxation was investiAJP-Heart Circ Physiol • VOL gated with the same concentration of SCH-58261 used in the coronary flow protocol. NECA is a nonselective agonist and has affinity for both A2A and A2B receptors. The Ki values for NECA at A1 and A2A receptors is 3 to 20 nM and at A2B receptors is 0.5 to 5 M, respectively (6, 8). CAD is a stable analog of adenosine and is also capable of activating both A2A and A2B receptors. The Ki values for CAD at A1, A2A, and A2B receptors are 3 to 30 nM, 20 to 200 nM, and 5 to 20 M, respectively (8). Figure 4 shows that selective blockade of A2A receptors with 100 nM SCH-58261 had little or no effect on NECA- and CAD-induced aortic relaxation. Taken together with the inability of the selective A2A receptor agonist CGS-21680 to cause aortic relaxation, these findings suggest that A2A receptor activation has a negligible role in the murine aortic vasculature. Influence of A2B adenosine receptor blockade on adenosine-induced vascular responses in isolated mouse hearts and aortic rings. To examine whether adenosine A2B receptors are involved in regional vascular responses, we investigated the influence of a relatively selective A2B receptor antagonist, alloxazine (4), on adenosine-mediated coronary flow and aortic relaxation using a similar protocol. Alloxazine is the only nonxanthine antagonist reported to have ninefold greater selectivity for A2B receptors than A2A receptors (4, 6) and has been successfully used to identify a role for A2B receptors in chick heart cells and rat pial artery (17, 36). We tested the concentration-dependent effects of alloxazine on baseline coronary flow and on adenosine-induced increases in coronary flow. Alloxazine had direct effect on coronary flow in isolated mouse hearts. At 1 M, alloxazine decreased the coronary flow to 89.44 ⫾ 2.61% of baseline (n ⫽ 5, P ⬍ 0.05 vs. baseline), whereas at 10 M, it increased the coronary flow to 117.92 ⫾ 5.78% of baseline (n ⫽ 5, P ⬍ 0.05 vs. baseline). Presence of 1 M alloxazine did not influence NECA-induced increases in the coronary flow (Fig. 5A). However, in the presence of 10 M alloxazine coronary flow was significantly attenuated to 334.23 ⫾ 23.98% 282 • JANUARY 2002 • www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.33.4 on May 12, 2017 Fig. 3. Influence of selective blockade of adenosine A2A receptor with SCH-58261 on CGS-21680 (A and B) and NECA (C) induced increases in the coronary flow. Control responses to CGS-21680 (10 nM and 100 nM) and NECA (100 nM) were first determined in the absence of SCH-58261. After a 10- to 12-min washout, hearts were pretreated with SCH-58261 (100 nM) for 10 min before a second exposure to CGS-21680 and NECA. Bars represent the means ⫾ SE of 4–6 experiments from different hearts for each concentration of agonist. y-axis, Increases in the coronary flow are expressed as percent change from baseline or washout value that was assigned as 100%; x-axis, molar concentration of agonists. *P ⬍ 0.05 vs. respective control value. H54 VASCULAR EFFECTS OF ADENOSINE IN MOUSE Fig. 4. Influence of selective blockade of adenosine A2A receptor with SCH58261 on NECA- and CAD-induced relaxation of mouse thoracic aorta precontracted with 1 M phenylephrine. Each symbol with a vertical bar represents the mean ⫾ SE of 7–12 experiments. A: CRCs for NECA, control, and SCH-58261 (100 nM). B: CRCs for CAD, control, and SCH-58261 (100 nM). For other details, see Fig. 1. DISCUSSION In the present study of adenosine and its analogs (CAD, NECA, and CGS-21680), in isolated mouse hearts and aortic rings, we observed that: 1) in isolated hearts, adenosine agonists produced concentration-dependent increases in coronary flow with similar maxi- mal response; 2) in aortic preparations, all agonists (except CGS-21680) produced marked concentrationdependent relaxation of phenylephrine-precontracted rings; 3) adenosine and its analogs were ⬃100-fold more potent in increasing coronary flow than in producing aortic relaxation; and 4) adenosine-induced coronary vasodilation was more susceptible to selective A2A receptor blockade than was aortic relaxation. To our knowledge, this is the first study to investigate in parallel the vascular responses of adenosine and its analogs in the mouse heart and aorta. In isolated mouse hearts, the CRCs for adenosine agonists on coronary flow were bell shaped and similar maximal responses were reached with each agonist. In comparison, the CRCs for aortic relaxation by adenosine agonists were steeper and a plateau was not reached with any agonist even at concentrations as high as 1 mM. In isolated hearts, all of the adenosine agonists increased coronary flow at low nanomolar concentrations, whereas micromolar concentrations were necessary to induce aortic relaxation (Fig. 1). This differential sensitivity of adenosine agonists between Fig. 5. Influence of alloxazine, a putative A2B receptor blocker, on NECA-induced increases in the coronary flow. Control responses to NECA (100 nM) were first determined in the absence of alloxazine. After 10- to 12 min washout, hearts were pretreated either with 1 M alloxazine (A) or 10 M alloxazine (B) for 10 min before second exposure to NECA. Bars represent the means ⫾ SE of 5 experiments from different hearts. *P ⬍ 0.05 vs. respective control value. For other details, see Fig. 3. AJP-Heart Circ Physiol • VOL 282 • JANUARY 2002 • www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.33.4 on May 12, 2017 from 415.91 ⫾ 28.38% of predrug baseline value (P ⬍ 0.05 vs. respective control, Fig. 5B). Pretreatment of aortic preparations with alloxazine had no effect on resting tension or phenylephrineinduced contraction. Alloxazine at 1 M had no influence on NECA-induced relaxation, but at 10 M, it caused a significant rightward parallel shift of the CRC and attenuation of the maximal response to 13.01 ⫾ 2.31% of phenylephrine-induced contraction (P ⬍ 0.05 vs. control, Fig. 6). This observation, in conjunction with the inability of a selective A2A receptor antagonist (SCH-58261) to block NECA- and CAD-induced aortic relaxation (Fig. 4), indicates that adenosine-induced aortic relaxation in mice involves predominantly A2B receptor activation. VASCULAR EFFECTS OF ADENOSINE IN MOUSE coronary flow and aortic relaxation suggests the possible involvement of different receptor subtype(s) in coronary and aortic relaxation. A2 adenosine receptors mediate vasorelaxation in most vascular beds (2, 3, 6, 8, 19, 27, 28, 33, 34, 39). In isolated hearts, CGS-21680 and NECA were equally potent at producing coronary vasodilation (Fig. 1A) and had lower EC50 values for this response than that of both CAD and adenosine (Table 1). This suggests that murine coronary vasodilation is predominantly mediated by adenosine A2A adenosine receptors. Our observation that selective A2A receptor blockade with SCH58261 equally antagonized the coronary vasodilation caused by CGS-21680 and NECA (Fig. 3, B and C) further supports this conclusion. SCH-58261 is a potent and highly selective competitive antagonist at adenosine A2A receptors both in vivo and in vitro, and it has little or no affinity up to the micromolar range for adenosine A2B and A3 receptors (30, 31, 41). Although a detailed analysis of the relationship between the concentration of CGS-21680 and SCH-58261 was not carried out, it appears that the antagonism was of a competitive nature, because no complete inhibition of CGS-21680-induced coronary flow was noted. Our findings are in agreement with the reported results in guinea pig aorta and porcine coronary vessels where adenosine A2A receptors are abundant, and SCH-58261 (100 nM) has been shown to competitively antagonize CGS-21680-induced increases in coronary conductance and vasodilation (3, 30). Both adenosine A2A and A2B receptors have been reported to be present in human and porcine coronary artery endothelial cells (29). The suggestion that A2B adenosine receptors may coregulate coronary flow was initially made by Makujina et AJP-Heart Circ Physiol • VOL al. (18), and this has recently been confirmed in human small resistance-like coronary arteries (13). In the present study, coronary flow changes caused by NECA could be partially reduced using the nonselective antagonist alloxazine (Fig. 5B). Among nonselective adenosine agonists (NECA, adenosine, and CAD), NECA has been used to characterize A2B receptor-mediated effects in vascular and cardiac preparations (6, 13, 17, 19, 20, 35, 36). Although NECA is a relatively potent A2B receptor agonist among these nonselective agents (Ki values at A2B receptors: NECA ⫽ 0.5–5 M; adenosine and CAD ⫽ 5–20 M), it also possesses a 100-fold greater affinity for A2A receptors than A2B receptors (8). Similarly, alloxazine is only ninefold more potent at A2B than A2A receptors (4, 6). Because these available pharmacological agents are nonselective, it is difficult to quantitate the effect of A2B receptor activation in the murine coronary circulation. Here it is noteworthy that in isolated hearts from adenosine A2A receptor knockout mice, CGS-21680 produced no effect on coronary flow and dose-response curves for adenosine-induced coronary vasodilation were significantly shifted to the right with attenuated maximal response compared with wild-type hearts, indicating the involvement of another adenosine receptor subtype in the regulation of coronary flow (23). The findings in aortic ring preparations were markedly different from those observed in isolated hearts. All nonselective adenosine agonists produced marked relaxation of aortic rings, whereas the selective A2A receptor agonist CGS-21680 was ineffective (Fig. 1B). Agonist-induced aortic relaxation occurred at a higher concentration (micromolar) than coronary vasodilation (nanomolar). In contrast to coronary vasodilation, selective blockade of adenosine A2A receptors with SCH58261 in aortic rings had no influence on agonistinduced relaxation (Fig. 4A), whereas blockade of A2B receptors with alloxazine significantly inhibited NECA-induced relaxation (Fig. 6). The current observation that CGS-21680 is a substantially weaker aortic vasodilator than NECA in mice is consistent with findings from other models, including guinea pig aorta (19), rat renal artery (20), rat mesenteric arterial bed (35), and human small coronary arteries (13). Moreover, in guinea pig aorta, SCH-58261 failed to antagonize NECA-induced relaxation leading to the conclusion that aortic vasodilation results from adenosine A2B receptor activation (41). We observe similar findings in mouse aorta where SCH-58261 failed to antagonize NECA- and CAD-induced relaxation. In mouse aorta, alloxazine caused a much greater inhibition of NECAinduced vasorelaxation than SCH-58261, which is consistent with findings in rat pial arteries in which NECA-induced vasodilation was blocked by alloxazine but not by an A2A receptor antagonist (36). The availability of a selective A2A adenosine receptor agonist (CGS-21680) and antagonist (SCH-58261) has allowed direct examination of A2A receptor-mediated effects (3, 30, 41), but characterizing A2B receptormediated responses is limited by the lack of selective 282 • JANUARY 2002 • www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.33.4 on May 12, 2017 Fig. 6. Influence of alloxazine, a putative A2B receptor blocker, on NECA-induced relaxation of mouse thoracic aorta precontracted with 1 M phenylephrine. Each symbol with a vertical bar represents the mean ⫾ SE of 5–12 experiments. CRCs for NECA, control, and alloxazine (1 M and 10 M). For other details see Fig. 1. * P ⬍ 0.05 vs. respective control value. H55 H56 VASCULAR EFFECTS OF ADENOSINE IN MOUSE AJP-Heart Circ Physiol • VOL itself possesses a higher affinity for A2A (1–20 nM) than for A2B (5–20 M) receptors (8). Shryock et al. (39) recently reported that the high potency of adenosine and its analogs to cause coronary vasodilation in guinea pig heart could be explained by the presence of a large adenosine A2A receptor reserve in the coronary circulation. Although we did not determine whether an A2A receptor reserve exists in our preparations, the different sensitivity of adenosine agonists in producing coronary and aortic vasodilation supports the possibility that different A2 receptor subtypes mediate these effects. In summary, the present study provides the first functional evidence that adenosine-induced vascular responses in mouse heart and aorta are quite different with respect to concentration dependence and response characteristics. In addition, we have demonstrated compelling evidence for the differential involvement of adenosine receptor subtypes in mouse coronary vasodilation and aortic relaxation. We conclude that murine coronary vasodilation is mediated predominantly by activation of A2A adenosine receptors, and aortic relaxation is likely predominated by activation of A2B receptors. A more definitive characterization of adenosine receptors in the regulation of coronary vasodilation awaits studies in A2A and A2B receptor knockout mice and/or the use of selective A2B receptor agents. Understanding the relative contributions to these effects will have important consequences for the development of future adenosinergic therapy. The authors gratefully acknowledge the generous gift of SCH58261 from Dr. A. Monopoli of Shearing Plough, Milan, Italy. This work was supported by National Heart, Lung, and Blood Institute Grant HL-27339 and a postdoctoral fellowship from American Heart Association, Mid-Atlantic Affiliate to M. A. Hassan Talukder and a faculty research grant of East Carolina University, Brody Medical School, to R. Ray Morrison. REFERENCES 1. Abebe W, Makujina SR, and Mustafa SJ. Adenosine receptormediated relaxation of porcine coronary artery in presence and absence of endothelium. Am J Physiol Heart Circ Physiol 266: H2018–H2025, 1994. 2. Balwierczak JL, Sharif R, Krulan CM, Field FP, Weiss GB, and Miller MJS. Comparative effects of a selective adenosine A2 receptor agonist, CGS 21680, and nitroprusside in vascular smooth muscle. Eur J Pharmacol 196: 117–123, 1991. 3. Belardinelli L, Shryock JC, Snowdy S, Zhang Y, Monopoli A, Lozza G, Ongini E, Olsson RA, and Dennis DM. The A2A adenosine receptor mediates coronary vasodilation. J Pharmacol Exp Ther 284: 1066–1073, 1998. 4. Brackett LE and Daly JW. 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