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Am J Physiol Heart Circ Physiol 277: H1895-H1905, 1999;
0363-6135/99 $5.00
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Vol. 277, Issue 5, H1895-H1905, November 1999

A3 adenosine receptor activation attenuates neutrophil function and neutrophil-mediated reperfusion injury

James E. Jordan1, Vinod H. Thourani1, John A. Auchampach2, Jill A. Robinson1, Ning-Ping Wang1, and Jakob Vinten-Johansen1

1 Cardiothoracic Research Laboratory, Carlyle Fraser Heart Center, Crawford Long Hospital of Emory University, Atlanta, Georgia 30365; and 2 Division of Cardiology, University of Louisville, Louisville, Kentucky 40292


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study tested the hypothesis that A3 adenosine receptors inhibit neutrophil (PMN) function and PMN-mediated reperfusion injury. 2-Chloro-N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (Cl-IB-MECA), an A3 agonist, did not attenuate superoxide production or myeloperoxidase release from stimulated PMNs. However, Cl-IB-MECA reduced platelet-activating factor-stimulated PMN adherence to coronary endothelium at low concentrations: 52 ± 27, 45 ± 10, and 87 ± 23 PMNs/mm2 at 0.1, 1.0, and 10 nM vs. 422 ± 64 PMNs/mm2 with platelet-activating factor alone. This inhibition was not blocked by A1 (5 µM KW-3902) or A2a (5 µM KF-21326) antagonists: 44 ± 3 and 43 ± 2 PMNs/mm2, respectively. Endothelial pretreatment with 1 nM Cl-IB-MECA reduced PMN adherence, which was reversed by the A3 antagonist MRS-1220 (100 nM). PMN-mediated reperfusion injury was initiated in isolated rabbit hearts by infusion of 28 × 106 PMNs/min for 10 min early in reperfusion. PMNs caused a significant decrease in recovery of left ventricular developed pressure and positive and negative time derivatives of pressure (23 ± 3, 25 ± 3, and 23 ± 3% of baseline, respectively) vs. buffer-perfused hearts (43 ± 7, 44 ± 7, and 45 ± 6%, respectively). Cl-IB-MECA (10 nM) given at reperfusion attenuated the PMN-mediated loss of contractile recovery (40 ± 3, 46 ± 5, and 42 ± 4% of baseline). Cl-IB-MECA reduced myeloperoxidase release activity (5.3 ± 0.6 absorbance units/min) and CD18-positive cells (54 ± 9 cells/slide) compared with the untreated PMN group (17.9 ± 1.7 absorbance units/min and 183 ± 68 cells/slide). We conclude that Cl-IB-MECA attenuates reperfusion injury by decreasing PMN-endothelial cell interactions. These results suggest that the A3 adenosine receptor may be a novel therapeutic target for treatment of myocardial ischemia and reperfusion.

endothelial adherence; superoxide; myeloperoxidase; CD18


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ADENOSINE IS RECOGNIZED as a potent cardioprotective autacoid that exerts its effects during ischemia and reperfusion through receptor-mediated actions (18, 37). Until recently, most of the cardioprotective actions of adenosine were thought to be mediated through A1 and A2a receptors. A1 receptors exert their protective effects before or during ischemia by reducing metabolic demand and by activating ATP-sensitive K+ (KATP) channels (2, 26). A2a receptor activation exerts protection primarily during reperfusion by inhibiting neutrophil (PMN) function (superoxide radical production) and reducing the adherence of PMNs to the vascular endothelium (40, 42).

In the early 1990s, a new adenosine receptor, the A3 receptor, was cloned first from rats by Meyerhof et al. (27) and by Zhou et al. (43), then from sheep (22), humans (31), and rabbits (12), and more recently from dogs (3). Although much work has focused on the ability of A3 receptors to exert preconditioning (1, 23, 30, 34) and to cause mast cell degranulation (11, 34), only recently have its effects on myocardial ischemia and reperfusion injury been tested. Tracey et al. (35) demonstrated that pretreatment with the selective A3 receptor agonist N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA) protected isolated rabbit hearts from injury induced by 30 min of regional ischemia and crystalloid reperfusion. Recently, pretreatment with A3 adenosine receptor agonists have been shown to elicit cardioprotection through the stimulation of myocardial KATP channels (36). Furthermore, Auchampach et al. (4) demonstrated that pretreatment with IB-MECA attenuated myocardial stunning and reduced infarct size after regional ischemia and reperfusion in conscious rabbits. This later study also demonstrated that IB-MECA was beneficial in the absence of hemodynamic changes in vivo, suggesting that A3 adenosine receptor therapies may be more useful in the clinical setting than other adenosine analogs with vasodilator properties.

Although evidence suggesting a protective role for the A3 receptor during myocardial ischemia and reperfusion is accumulating, the mechanisms involved in this protection remain unknown. One proposed mechanism suggests that A3 receptors are expressed in cardiomyocytes and that activation of these receptors is cardioprotective by a mechanism similar to that of A1 adenosine receptors. This hypothesis is based on studies in which activation of A3 receptors protects isolated cardiomyocytes against injury induced by simulated ischemia and reperfusion (23, 34). However, a potential mechanism that has not been explored is that activation of A3 adenosine receptors may inhibit PMN functions, including superoxide anion generation and adherence to endothelium, thereby reducing PMN-mediated reperfusion injury. Accordingly, the goal of the present study was to determine the effect of stimulating A3 adenosine receptors with the selective A3 receptor agonist 2-chloro-IB-MECA (Cl-IB-MECA) on isolated canine PMN superoxide production, degranulation, and adherence to coronary artery endothelium. Furthermore, we tested the hypothesis that Cl-IB-MECA, given at the onset of reperfusion, reduces reperfusion injury by inhibiting the accumulation of PMNs in myocardium subjected to ischemia-reperfusion.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The protocols were approved by the Institutional Animal Care and Use Committee of Emory University. All experiments complied with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals [DHHS Publication No. (NIH) 85-23, Revised 1996, Office of Science and Health Reports, Bethesda, MD 20892] and the guidelines set forth in the "Guiding Principles for Research Involving Animals and Human Beings," approved by the Council of the American Physiological Society.

Radioligand binding. Radioligand binding analysis with recombinant canine A3 and A1 adenosine receptors was used to determine the selectivity of the drugs used in the following studies and was performed as previously described (3). Briefly, membranes were prepared from COS-7 cells expressing canine A3 or A1 adenosine receptors. The full coding region of the receptor cDNAs was subcloned into the expression vector CLDN10B and transiently expressed (60 h) in COS-7 cells by the diethylaminoethyl-dextran method (8). Transfected cells were washed in PBS, homogenized in 10 mM EDTA, 10 mM Na-HEPES (pH 7.4), and 0.1 mM benzamidine, and centrifuged at 40,000 g for 20 min. Pellets were resuspended and washed in the same buffer with 10% (wt/vol) sucrose at a membrane concentration of 1 mg/ml. Protein concentrations were determined using fluorescamine with bovine albumin as standard, as previously described (33). Membranes were frozen in aliquots and stored at -80°C.

Competition binding studies were performed by incubating membranes (10-25 µg) with 0.5-1 nM N6-(4-amino-3-[125I]iodobenzyl)adenosine ([125I]ABA) and a range of concentrations of competitors. The incubations included 5 mM MgCl2 and 5 U/ml adenosine deaminase in a final volume of 0.1 ml. After incubation for 3 h at 21°C, bound [125I]ABA was separated from the reaction by rapid filtration over glass fiber filters (Whatman GF/C) with a Brandell cell harvester. Radioactivity trapped by the filters was counted in a gamma counter. IC50 and dissociation constant (Ki) values were calculated for canine A3 and A1 receptors, as previously described in detail (3), by fitting the data to one (A1 receptor) or two (A3 receptor) binding sites (3).

Canine PMN and endothelial segment isolation. Seventeen microfilaria-free dogs, weighing 18-35 kg, were premedicated with morphine sulfate (4 mg/kg im). Induction of deep anesthesia was attained with pentobarbital sodium (20 mg/kg iv), and an endotracheal tube was inserted for ventilation. PMNs were isolated from whole arterial blood by use of the Ficoll-Paque technique, as described previously (42). This method yielded a PMN suspension that was 90.4 ± 0.7% pure and 97 ± 0.4% viable.

Coronary arteries (left anterior descending and left circumflex) were carefully isolated from the ex vivo heart in a bath of cold Krebs-Henseleit (KH) buffer. Once free of the myocardium, the coronary arteries were carefully cleaned of fat and connective tissue, cut into 2- to 3-mm segments, split open to expose the endothelium, and stored in cold, oxygenated KH buffer until the assay began.

Drug preparation. Stock solutions of Cl-IB-MECA (10 mM) and MRS-1220 (1 mM) were prepared in DMSO and diluted daily with fresh KH buffer. The final concentration of DMSO in the adherence assay was substantially <0.0001%. KW-3902 and KF-21326 were made fresh and dissolved in small amounts of ethanol before dilution with KH buffer. Previous studies (39) showed no effect with the amount of ethanol or DMSO in the final concentrations of drugs used for this study.

Superoxide production by PMNs. Adherence-independent superoxide radical production by 10 × 106 PMNs/well was determined by measuring the superoxide dismutase (SOD)-inhibitable reduction of ferricytochrome c to ferrocytochrome c, as described previously (42). The PMNs were stimulated with 10 µM platelet-activating factor (PAF), and tests were run simultaneously with and without SOD to correct for nonspecific activity or color generation. Superoxide production was measured spectrophotometrically (optical density at 500 nm), and the results are reported as nanomoles of SOD-inhibitable superoxide produced by a suspension of 10 × 106 PMNs during a 5-min measurement period at 37°C.

PMN degranulation assays. PMN degranulation was assessed by myeloperoxidase (MPO) release from stimulated PMNs by a modification of the method of Ely et al. (9). Test drugs were incubated for 5 min at 37°C before 20 min of stimulation with 10 µM PAF in the presence of cytochalasin B. The supernatant was removed by centrifugation and analyzed for MPO activity as a marker for granule release. The rate of reaction was measured over 5 min, and degranulation was reported as the maximal slope of the optical density vs. time plot. The reaction was started by the addition of a substrate containing o-dianisidine and hydrogen peroxide.

PMN adherence to coronary artery segments. Isolated canine PMNs were labeled with a vital fluorescent dye, as described previously (42). Briefly, a solution of PKH26-GL dye (Sigma Chemical) was added to a suspension of PMNs, and after 3-4 min of labeling, a stop solution of 10% platelet-poor plasma in PBS was added to the mixture. The canine coronary segments were then placed into plastic dishes containing KH buffer in a 37°C water bath. The segments were incubated with or without test drugs for 5 min (antagonists were given 5 min before addition of agonist, where appropriate). After drug exposure, the labeled, unstimulated PMNs (2 × 106 PMNs/well) were added to the baths and immediately stimulated with PAF. After 15 min the segments were removed, washed gently with KH buffer to remove nonadherent PMNs, and mounted on microscope slides. Adherence was measured under epifluorescence (rhodamine filter cube, Olympus) at ×200 magnification by counting the number of adherent PMNs on four separate fields of view and expressed as the number of PMNs adhered per square millimeter of coronary artery endothelium.

In a series of experiments designed to determine whether the inhibitory effect of 1 nM Cl-IB-MECA was exerted directly on the endothelium, only the endothelial segments were exposed to Cl-IB-MECA and activated. To accomplish this, coronary artery segments were coincubated with 1 nM Cl-IB-MECA for 5 min before 20 min of stimulation with the endothelial activator thrombin (2 U/ml). Each segment was then removed from the bath, washed with KH buffer, and placed into a new dish of buffer free of any drugs. Labeled, unactivated PMNs were then added to the baths for 15 min before being counted, as described above.

To confirm that Cl-IB-MECA would inhibit canine PMN adherence to rabbit endothelium, adherence studies were performed using rabbit aorta segments. Aortic segments isolated from rabbits were sliced open to expose the endothelial surface and placed into dishes containing KH buffer. Cl-IB-MECA was introduced into the baths in various concentrations (0.2-200 nM) for 5 min before the addition of 1 × 106 labeled canine PMNs. Subsequently, the PMNs and the endothelium were activated with PAF. After 15 min of coincubation, the aortic segments were removed from the dishes, gently washed with KH buffer, mounted on microscope slides, and quantified as described above.

Isolated, perfused rabbit heart. Rabbits of either gender (3.0-3.8 kg) were anesthetized with ketamine (30 mg/kg im) and xylazine (6 mg/kg im). Supplemental pentobarbital anesthesia was given through an ear vein as necessary. A tracheostomy was performed, and the animal was ventilated with room air supplemented with 100% oxygen. The chest was opened by a median sternotomy, and the pericardium was opened to expose the heart. Heparin (300 U/kg) was given through the ear vein before removal of the heart.

The heart was removed from the chest and immediately immersed in cold KH buffer containing (in mM) 120 NaCl, 4.5 KCl, 1.2 KH2PO4, 1.3 MgSO4 · 7 H2O, 2.55 CaCl2 · 2 H2O, 11 glucose, and 25 NaHCO3. The heart was then quickly attached to the perfusion cannula and perfused with KH buffer at a constant pressure of 80 mmHg. Maximal flow of the perfusion apparatus at 80 mmHg perfusion pressure was >200 ml/min. Pacing leads were attached to the right atrium (185 beats/min), and a temperature probe was placed into the right ventricle. The left atrium was then removed, and the mitral valve was disrupted to allow a saline-filled latex balloon connected to a pressure transducer to be secured into the left ventricle with a suture around the valve annulus. Total coronary flow and left ventricular balloon and perfusion pressure measurements were monitored continuously and collected for analysis at baseline and 5, 15, 30, 45, and 60 min of reperfusion with use of Spectrum, a cardiovascular data collection and analysis program developed in our laboratory. From these data, developed pressure and maximum and minimum time derivatives of pressure (dP/dt) were derived from >= 25 beats.

After 30 min of stabilization, the perfusion line was clamped for 15 min to create normothermic ischemia, before the hearts were reperfused for 60 min. Each of the following groups was studied in the presence and absence of PMNs infused at reperfusion: 1) control, 2) 2 nM Cl-IB-MECA, and 3) 10 nM Cl-IB-MECA. Perfusion with Cl-IB-MECA, where appropriate, was started at the initiation of reperfusion and continued for the first 30 min of reperfusion. PMN-mediated damage was induced by the infusion of 28 × 106 canine PMNs per minute for 10 min beginning 5 min into the reperfusion period through a side port just above the perfusion cannula.

Pilot studies conducted in the laboratory demonstrated that, in buffer-perfused hearts that were not paced, 2 and 10 nM Cl-IB-MECA changed neither the basal coronary flow nor the heart rate, suggesting that there are no hemodynamic effects of A3 adenosine receptor activation. Additional pilot studies showed that coronary flow reserve (vasodilator response to nitroprusside challenge) was present after 15 min of global ischemia and reperfusion with PMNs.

Myocardial MPO activity. At the end of the protocol, tissue samples were taken from the left ventricle and the interventricular septum to assay for MPO activity. Tissue samples were cleaned of fatty tissue, weighed, and diluted with 0.5% hexadecyltrimethylammonium bromide to make a 10% (wt/vol) solution. The samples were then homogenized and sonicated to release all MPO from PMNs within the sample before pelleting of cellular debris. The supernatant was then analyzed spectrophotometrically for the maximal change in slope in the presence of hydrogen peroxide and o-dianisidine and expressed as relative units of MPO activity (change in absorbance per minute).

Immunohistochemistry. Samples from the left ventricle of hearts undergoing ischemia and reperfusion in the perfusion setup were prepared for immunohistochemical analysis for PMN accumulation. The samples were washed in cold saline and fixed in 4% paraformaldehyde buffered with 0.1 M Na2PO4 (pH 7.4) for 3 h at 4°C, cryoprotected in 15% sucrose-PBS overnight, embedded in optimal cutting temperature compound (Sakura Finetek, Torrence, CA), and frozen in liquid nitrogen. Sections (7-10 µm) were obtained using a cryostat and thaw-mounted onto slides and stored at -70°C until processed. Slides were fixed in acetone and air-dried, and endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide in methanol. Nonspecific binding was blocked with 1% gelatin before incubation with the primary antibody against CD18, R15.7/H4 (a gift from Dr. Robert Rothlein, Boehringer Ingelheim Pharmaceuticals, Ridgefield, CT). After primary antibody application, the slide was washed with PBS and incubated with biotinylated horse anti-mouse IgG (1:4,000 dilution; Vector Laboratories, Burlingame, CA) and stained with the ABC Elite peroxidase kit (Vector Laboratories). The slides were then dehydrated in graded alcohols, counterstained with hematoxylin, and prepared for viewing. PMNs were counted manually (24 fields/slide at ×200 magnification) and reported as the number of PMNs counted per slide. Control experiments were performed by eliminating the primary antibody from the labeling procedure.

Exclusion criteria and statistical analysis. Each of the in vitro assays was performed with positive (PAF or thrombin) and negative (unstimulated) controls. All data from entire experimental runs were excluded if the unstimulated controls exhibited elevated homotypic aggregation or significant adherence. Failure of PAF or thrombin to stimulate PMN adherence to endothelium resulted in exclusion of these experimental runs as well. Individual adherence slides were excluded for obvious damage to the coronary segments or improperly mounted slides.

Values are means ± SE. Group differences for the in vitro assays were analyzed using a one-way ANOVA. Where group differences were detected, post hoc Tukey's or Student-Newman-Keuls multiple comparison tests were performed to determine which groups differed. To determine the receptor selectivity of the adherence response, the Cl-IB-MECA groups were compared with the individual antagonists by t-test. Differences were considered significant when P < 0.05.

In the isolated, perfused hearts the hemodynamic data were compared using a one-way ANOVA. When group differences were detected by ANOVA, a Student-Newman-Keuls multiple comparison test was performed to determine the groups that were significantly different. Where the sample data were not normally distributed, a Kruskal-Wallis ANOVA on ranks was followed by a post hoc Dunn's test for multiple comparisons.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Competition radioligand binding assays. The results of competition binding assays are shown in Table 1. Cl-IB-MECA competitively inhibited the binding of [125I]ABA to canine A1 and A3 receptors. The Ki values for Cl-IB-MECA were calculated to be 30.3 ± 5.2 and 0.33 ± 0.1 nM for the A1 and A3 adenosine receptors, respectively. Thus Cl-IB-MECA is 91.8-fold selective for canine A3 receptors vs. canine A1 receptors. However, Cl-IB-MECA is much less selective for canine A3 receptors than has been reported previously for rat A3 receptors (2,500-fold A3 selective vs. A1) (14, 17).

                              
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  		Table 1.   Ki values of competitors for canine A1 and A3 adenosine receptors determined in radioligand binding assays

The antagonists KW-3902, KF-21326, and MRS-1220, which have previously been reported to be selective for A1, A2a, and A3 adenosine receptors (15, 28), were also tested in radioligand binding assays by use of recombinant canine A1 and A3 adenosine receptors. As shown in Table 1, KW-3902 was ~150-fold selective for the canine A1 adenosine receptor and MRS-1220 was ~100-fold selective for the A3 adenosine receptor. Thus 5 µM KW-3902 was used in subsequent functional assays with PMNs to selectively inhibit A1 receptors, and 100 nM MRS-1220 was used to target A3 receptors. The A2a receptor antagonist KF-21326 was found to bind poorly to canine A1 and A3 receptors and was subsequently used in assays at 5 µM.

Superoxide anion production. PMNs activated with 10 µM PAF increased their production of superoxide anion ~35-fold over the unstimulated controls (from 2.15 ± 0.31 to 74.07 ± 3.83 nmol · 10 × 106 PMNs-1 · 5 min-1). Treatment with 10 µM adenosine before activation decreased the response by approximately two-thirds (18.80 ± 6.83 nmol · 10 × 106 PMNs-1 · 5 min-1). These data are in agreement with previous studies that demonstrate an inhibitory effect on PMN-derived superoxide anion production (7, 42) and serve as a positive control for our assay. For 0.9-909 nM Cl-IB-MECA, there was no significant modulation of superoxide anion produced from PAF-stimulated (Fig. 1A) or unstimulated PMNs. These data demonstrate that activation of the A3 adenosine receptor does not directly modify PMN-derived superoxide anion production in our assay system.



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  		Fig. 1.   A: effect of 2-chloro-N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (Cl-IB-MECA) on superoxide production by isolated canine neutrophils (PMNs) stimulated with platelet-activating factor (PAF). Superoxide production, measured by cytochrome c reduction, is expressed as nanomoles of superoxide anion produced in 5 min by 10 × 106 PMNs. Veh, vehicle. B: effect of Cl-IB-MECA on myeloperoxidase (MPO) release from isolated canine PMNs stimulated with PAF. Data are presented as a percentage of amount of MPO released from PAF-stimulated group. dagger  P < 0.05 vs. PAF-stimulated group.

PMN degranulation. MPO released from unstimulated controls was <30% of that released from PMNs stimulated with 10 µM PAF. Preincubation of PMNs with 0.1-100 nM Cl-IB-MECA did not change the amount of enzyme released by PAF-activated (Fig. 1B) or unstimulated PMNs. Adenosine again served as a positive control and, at 10 and 50 µM, significantly reduced PAF-stimulated MPO release (19.5 ± 1.7 and 19.3 ± 1.7% reduction, respectively). This modest inhibition of degranulation by adenosine is in agreement with previous studies (5, 7, 25).

PMN adherence to coronary artery segments. Figure 2A shows the effect of increasing concentrations of Cl-IB-MECA on PMN adherence to coronary artery endothelium. Costimulation of PMNs and endothelial segments with PAF caused a greater than eightfold increase in the number of PMNs adhered to the endothelial surface compared with basal levels (422 ± 14 vs. 50 ± 2 PMNs/mm2). There was a concentration-dependent decrease in adherence with 0.01-1 nM Cl-IB-MECA (lowest adherence value 45 ± 3 at 1 nM Cl-IB-MECA). To demonstrate that the inhibitory effect of Cl-IB-MECA at the most efficacious concentration (1 nM) was due to actions on the A3 adenosine receptor subtype, we employed the selective adenosine receptor antagonists KW-3902 (A1 antagonist) and KF-21326 (A2a antagonist). Neither blockade of A1 adenosine receptors with KW-3902 nor blockade of A2a receptors with KF-21326 reversed the effects of 1 nM Cl-IB-MECA (Fig. 2B), suggesting that the inhibitory actions of the agonist were due to activation of the A3 receptor subtype. Additionally, the inhibitory effect of 1 nM Cl-IB-MECA produced the same degree of inhibition previously observed for 100 µM adenosine (reduced adherence back to basal levels) (42). The A3 receptor antagonist MRS-1220 was not used to block the effect of Cl-IB-MECA in this protocol but was used to antagonize Cl-IB-MECA when the endothelium was selectively activated with thrombin (presented below).




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  		Fig. 2.   A: effect of Cl-IB-MECA on adherence of PMNs to coronary artery segments in vitro. Unstimulated, fluorescently labeled PMNs were added to coronary artery segments pretreated with increasing concentrations of Cl-IB-MECA. PAF was added to costimulate PMNs and endothelium. After samples were washed to remove loose PMNs, number of adherent PMNs was quantified using epifluorescence microscopy. Cl-IB-MECA produced a biphasic inhibition of PMN adherence. B: effect of 5 µM KW-3902 (KW, A1 antagonist) and 5 µM KF-21326 (KF, A2a antagonist) on inhibition of PAF-stimulated PMN adherence to coronary artery endothelium caused by 1 nM Cl-IB-MECA. Blockade of A1 or A2a receptors did not interfere with inhibition of adhesion by 1 nM Cl-IB-MECA. C: effect of KF-21326 (selective A2a receptor antagonist) on inhibition of PMN adherence to coronary artery endothelium. Unstimulated, labeled PMNs were added to coronary artery segments incubated with a high concentration Cl-IB-MECA (1,000 nM) and KF-21326 (5 µM). Blockade of adenosine A2a receptors reverses 2nd-phase inhibitory actions of high-dose (1,000 nM) Cl-IB-MECA. * P < 0.05 vs. nonactivated group; dagger  P < 0.05 vs. PAF-stimulated group.

Interestingly, at 100 nM Cl-IB-MECA (Fig. 2A), PMN adherence was increased (258 ± 23 PMNs/mm2), whereas at 1 µM Cl-IB-MECA, a second inhibitory phase became evident (128 ± 12 PMNs/mm2). We hypothesized that these effects of higher concentrations of Cl-IB-MECA were the result of actions on adenosine receptor subtypes other than the A3 receptor (i.e., A1 or A2a receptors). To test this hypothesis, we examined the effects of A1 or A2a receptor antagonists, KW-3902 and KF-21326, on the adherence patterns of the higher concentrations of Cl-IB-MECA on PMN adherence. The selective A1 adenosine antagonist KW-3902 did not block the increased level of adherence observed at 100 nM Cl-IB-MECA (data not shown). This result suggests a lack of involvement of A1 receptors in this response. In contrast, the A2a adenosine antagonist KF-21326 effectively reversed the second inhibitory phase of Cl-IB-MECA observed at 1 µM Cl-IB-MECA (Fig. 2C), suggesting that this concentration of Cl-IB-MECA may activate A2a receptors.

Selective activation of the endothelium by thrombin resulted in an increase in the number of adherent PMNs compared with the unstimulated controls (Fig. 3). Pretreatment of the endothelium for 5 min with 1 nM Cl-IB-MECA, washing, and then transfer to fresh KH buffer before incubation with unstimulated PMNs resulted in a significant reduction in adherence. This reduction in adherence was blocked by pretreatment with the selective A3 adenosine antagonist MRS-1220 before addition of Cl-IB-MECA.


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  		Fig. 3.   Effect of selective endothelial pretreatment with Cl-IB-MECA on PMN adherence to coronary artery endothelium activated with thrombin. Coronary artery segments were incubated with or without 1 nM Cl-IB-MECA. After 5 min of pretreatment, segments were transferred to fresh buffer and labeled, unstimulated PMNs were added. Endothelium was then activated with thrombin (2 U/ml). Cl-IB-MECA (1 nM) significantly reduced PMN adherence under these conditions. This effect of Cl-IB-MECA was completely blocked by addition of selective A3 agonist MRS-1220 (100 nM) during pretreatment period. * P < 0.05 vs. PMN group (control); dagger  P < 0.05 vs. thrombin-stimulated group; ddager  P < 0.05 vs. 1 nM Cl-IB-MECA group.

Studies in rabbit hearts. To confirm the interactions between canine PMNs and rabbit vascular endothelium, in vitro adherence of fluorescently labeled canine PMNs and isolated rabbit aortic segments exhibited a low basal level of adherence of PMNs in the unstimulated controls (40.8 ± 6.5 PMNs/mm2) that was significantly increased on activation with PAF (158.4 ± 16.9 PMNs/mm2). Increasing concentrations of Cl-IB-MECA (0.2-200 nM) progressively decreased the adherence of stimulated canine PMNs to rabbit aortic endothelium back to basal levels at concentrations of 10 and 20 nM (51.0 ± 3.8 and 31.3 ± 3.7 PMNs/mm2, respectively).

Hemodynamics. Table 2 summarizes the hemodynamic data from the isolated perfused hearts. Heart rate remained constant throughout the experiment with the use of a right atrial pacemaker in all groups. The hearts were paced at ~185 beats/min, which did not differ significantly between groups at any of the time points. The addition of PMNs caused a small but significant increase in perfusion pressure during and subsequent to their infusion (15 and 30 min of reperfusion). Although statistically significant increases in perfusion pressure were found, the maximum increase was only 6 mmHg and normalized by 45 min of reperfusion.

                              
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  		Table 2.   Hemodynamic results from isolated rabbit hearts

Postischemic infusion of PMNs caused a decrease in coronary flow throughout the reperfusion period compared with controls not receiving PMN infusion (Fig. 4). This PMN-mediated decrease in flow was attenuated by Cl-IB-MECA treatment at reperfusion.


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  		Fig. 4.   Total coronary flow, normalized for total heart weight, throughout experiment and expressed as a percentage of baseline flow (from Table 2). Infusion of PMNs is represented by shaded bar. Baseline, -15 min; reperfusion was initiated at time 0. * P < 0.05, PMN group vs. buffer group; dagger  P < 0.05, PMN + 10 nM Cl-IB-MECA group vs. PMN group; ddager  P < 0.05, PMN + 2 nM Cl-IB-MECA group vs. PMN group.

Left ventricular contractile function. Left ventricular developed pressure for all groups was equivalent at baseline (Table 3). Ischemia caused a severe reduction in developed pressure that recovered only slightly by 5 min of reperfusion. Buffer-treated hearts recovered >40% of their baseline developed pressure by 30 min of reperfusion and maintained this level of recovery to the end of the experiment (60 min of reperfusion; Fig. 5A). Infusion of PMNs (PMN group) at reperfusion resulted in significantly less recovery of developed pressure throughout the reperfusion period (30, 45, and 60 min of reperfusion). Although 2 nM Cl-IB-MECA in the presence of PMNs tended to improve the recovery of developed pressure (36 ± 4% of baseline), this did not reach statistical significance in comparison to buffer-perfused hearts. However, treatment of PMN-perfused hearts with 10 nM Cl-IB-MECA (PMN + 10 nM Cl-IB-MECA) significantly improved recovery of developed pressure (40 ± 3% of baseline) compared with the untreated (PMN) group (23 ± 3% of baseline). Treatment with Cl-IB-MECA in the absence of PMNs did not significantly affect the recovery of developed pressure compared with the buffer group.

                              
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  		Table 3.   Left ventricular function from isolated rabbit hearts





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  		Fig. 5.   A: left ventricular (LV) developed pressure as a percentage of baseline value (from Table 3) for hearts subjected to 15 min of global ischemia followed by 60 min of reperfusion. Infusion of PMNs (where appropriate) is represented by shaded bar. B and C: peak positive and peak negative time derivatives of pressure (dP/dt) as a percentage of their baseline values (from Table 2) for hearts subjected to 15 min of global ischemia followed by 60 min of reperfusion. Infusion of PMNs (where appropriate) is represented by shaded bar. Baseline, -15 min; reperfusion was initiated at time 0. * P < 0.05, PMN group vs. buffer group; dagger  P < 0.05, PMN + 10 nM Cl-IB-MECA group vs. PMN group; ddager  P < 0.05, PMN + 2 nM Cl-IB-MECA group vs. PMN group.

The addition of PMNs reduced the recovery of peak positive dP/dt at 45 and 60 min of reperfusion (Fig. 5B) compared with the buffer control. As with developed pressure, Cl-IB-MECA (PMN + 10 nM Cl-IB-MECA) significantly improved recovery of peak positive dP/dt at 45 and 60 min of reperfusion. Recovery of peak negative dP/dt was attenuated in the PMN group beginning at 15 min of reperfusion and persisting through the end of the experiment (Fig. 5C). Both concentrations of Cl-IB-MECA significantly improved recovery of peak negative dP/dt at 45 min of reperfusion, whereas only the PMN + 10 nM Cl-IB-MECA group remained significantly elevated at 60 min of reperfusion compared with PMN alone.

MPO activity. PMN accumulation was assessed by MPO activity in the ischemic-reperfused tissue of the left ventricle and interventricular septum. All samples taken from hearts not exposed to PMNs during the experiment showed equivalent, low levels of MPO activity (Fig. 6). In the left ventricle and the septum, MPO activity was elevated in the PMN group. Cl-IB-MECA caused a concentration-dependent decrease in MPO activity in both areas of the myocardium. MPO levels in the left ventricular samples with 10 nM Cl-IB-MECA were significantly lower than in the samples with 2 nM Cl-IB-MECA.


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  		Fig. 6.   PMN accumulation in LV from hearts subjected to 15 min of global ischemia followed by 60 min of reperfusion. Tissue MPO activity (relative units of absorbance change/min) is displayed for LV samples at end of protocol in groups infused with and without PMNs. * P < 0.05 vs. PMN + buffer group; dagger  P < 0.05 vs. PMN + 2 nM Cl-IB-MECA group.

Immunohistochemical analysis of PMN accumulation. CD18-positive cells (PMNs) were rarely detected (5 ± 1 PMNs/slide, n = 9) from hearts not exposed to PMNs during reperfusion (buffer and 2 and 10 nM Cl-IB-MECA). However, the PMN group exhibited significant accumulation of CD18-positive cells (183 ± 69, n = 4) that was reduced by 2 nM (42 ± 9, n = 3) and 10 nM (54 ± 9, n = 6) Cl-IB-MECA. These results are consistent with the MPO data, although the reduction in CD18-positive cells did not reach statistical significance (P = 0.09 for PMN vs. PMN + 10 nM Cl-IB-MECA).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We examined the in vitro actions of the selective A3 adenosine receptor agonist Cl-IB-MECA on PMN superoxide anion production, release of MPO from azurophilic granules, and PMN-endothelial cell interactions resulting in adherence to coronary artery segments. For these studies, we selected PAF as the stimulus. This choice was made on the basis of earlier studies that demonstrated the inability of N-formyl-methionyl-leucyl-phenylalanine and leukotriene B4 to stimulate superoxide anion production and PMN adherence in canine cells, whereas PAF is a potent stimulus of both processes (42). Our results demonstrated that Cl-IB-MECA does not regulate superoxide anion production or the release of MPO (degranulation) from isolated canine PMNs. However, Cl-IB-MECA potently inhibited PAF-induced PMN adherence to coronary artery endothelium. The inhibitory action of Cl-IB-MECA was concentration dependent and appears to involve interaction with A3 receptors at low concentration (0.1-10 nM) and A2a receptors at high concentrations (>100 nM). When the endothelium was treated independently, low concentrations of Cl-IB-MECA still inhibited PMN adherence and could be inhibited by the A3 receptor antagonist MRS-1220, suggesting an A3-selective action directly on the endothelium. These data suggest that the selective A3 adenosine receptor agonist Cl-IB-MECA has no direct inhibitory effect on PMNs, in contrast to adenosine (but in agreement with the lack of A3 receptors on PMNs), but exerts potent inhibition of PMN-endothelial interaction involving direct actions on the endothelium. We also examined the effect of reperfusing the heart with Cl-IB-MECA in a model of PMN-mediated reperfusion injury after 15 min of global ischemia. Our results suggest that Cl-IB-MECA reduces PMN reperfusion (MPO and immunohistochemical staining) accumulation after ischemia and in association with improved functional recovery and postischemic perfusate flow.

Radioligand binding studies were performed to determine the potency and selectivity of Cl-IB-MECA for canine A3 adenosine receptors. These studies are important, since it is well known that the pharmacological properties of A3 receptors vary markedly among species (21). We found that Cl-IB-MECA binds to canine A3 receptors with a Ki of 0.33 nM and is ~100-fold selective vs. the A1 receptor, its closest pharmacological relative. These results suggest that Cl-IB-MECA can act on other adenosine receptors at concentrations >10 nM, which was confirmed with our adherence data. Cl-IB-MECA has previously been reported to be >2,500-fold selective for rat A3 receptors vs. A1 receptors, owing primarily to lower A1 receptor affinity (17). Cl-IB-MECA appears to be a much more A3-selective agonist for rats than for dogs.

A potential role for A3 receptors in attenuating PMN superoxide production has not been addressed previously, and our data do not support their involvement in regulation of this process. The ability of A3 adenosine receptors to influence PMN degranulation, however, is controversial. It has previously been suggested by Bouma et al. (5) that activation of A3 adenosine receptors inhibits PMN degranulation. These investigators demonstrated that >1 µM IB-MECA inhibits the release of bacterial permeability-increasing protein, elastase, and defensins from human PMNs in whole blood samples in response to cytokine and endotoxin stimulation (5). However, the results of the present study demonstrate that 0.1-100 nM Cl-IB-MECA does not inhibit degranulation of isolated canine PMNs. We speculate that in the studies of Bouma et al. the higher concentrations of IB-MECA may have inhibited degranulation by influencing other adenosine receptor subtypes, notably the A2a receptor. Other possible explanations for the disparate observations may be related to species differences, measurement of different degranulation proteins, or differences in the stimulators used to activate PMNs.

The lack of an effect by Cl-IB-MECA on PAF-induced superoxide production and degranulation suggests that A3 receptors may not be functionally expressed on canine PMNs. In support of this hypothesis, we were unable to detect the expression of A3 adenosine receptors in isolated canine PMNs by Northern blot or radioligand binding analysis (data not shown). Additionally, our observations of a lack of effect on superoxide production and degranulation by Cl-IB-MECA are consistent with the hypothesis that PMNs do not express A3 adenosine receptors. A3 receptors may, however, be present in canine PMNs at levels too low to be detected by these methods. For instance, A3 adenosine receptor expression has been identified in human PMNs (5) and in cardiomyocytes (38) with use of the sensitive technique of RT-PCR. Therefore, it is possible that A3 receptors are expressed in canine PMNs but that they regulate functions other than superoxide anion production and degranulation of azurophilic granules.

Although we observed no direct effect of Cl-IB-MECA on PMN function, it strongly inhibited PMN adherence to coronary artery endothelium, which appeared to be mediated by the A3 receptor at low concentrations. This conclusion is based on the inability of KW-3902 and KF-21326 to block the inhibitory effect of 1 nM Cl-IB-MECA. Because KF-21326 blocked the antiadhesive effects of 1 µM Cl-IB-MECA, this higher concentration of Cl-IB-MECA appeared to be acting on A2a receptors. The use of these antagonists as selective agents was based on the results of the binding assays (Table 1), as well as previous experiments. KW-3902 has been used by our laboratory to examine adenosine receptor subtype involvement in the production of superoxide anion and PMN adherence. Zhao et al. (41) showed that 50 µM KW-3902 completely blocks the A1 adenosine receptor-mediated inhibition of catecholamine-stimulated positive inotropy induced by (+)-N6-phenylisopropyl adenosine in an isolated papillary muscle preparation. Furthermore, 1-50 µM KW-3902 did not attenuate the adenosine-mediated reduction in superoxide anion generation in isolated canine PMNs or the adherence of labeled PMNs to endothelial segments (42). Taken together, these data suggest that KW-3902, at the concentration used in this study (5 µM), acts in a manner consistent with a selective A1 adenosine antagonist. Similarly, Fernandez et al. (10) blocked A2a receptor-mediated inhibition of PMN adherence with KF-21326 in a concentration-dependent (0.5-5 µM) manner, suggesting that this drug selectively blocks A2a receptors.

Our data using selective treatment of coronary artery endothelium would support inhibition of PMN adhesion by the activation of A3 receptors expressed on endothelial cells by low concentrations of Cl-IB-MECA. Cl-IB-MECA at 1 nM inhibited PMN adhesion when thrombin-activated endothelium was selectively pretreated before incubation with PMNs. This inhibitory effect is likely mediated by the A3 receptor, since Cl-IB-MECA was effective at nanomolar concentrations, consistent with A3 selectivity, and since it was blocked by the A3-selective antagonist MRS-1220. We speculate that A3 receptors may regulate mechanisms leading to the surface expression of adhesion molecules. Bouma et al. (6) demonstrated that, in isolated human endothelial cell cultures, adenosine reduces the surface expression of two adhesion molecules, E-selectin and vascular cell adhesion molecule-1, when the culture is stimulated with tumor necrosis factor-alpha . It is possible that a similar mechanism plays a role in the decreased PMN adherence observed in this study.

In isolated hearts, contractile function (left ventricular developed pressure and positive and negative dP/dt) was substantially reduced by 15 min of global, normothermic ischemia and 60 min of reperfusion in the buffer controls. These hearts regained >40% of their baseline performance. However, when PMNs were added during reperfusion, the recovery of contractile performance was further reduced, regaining only ~25% of baseline performance. This loss of contractile function was associated with a significant increase in the accumulation of PMNs. Together, these data suggest that our model of contractile dysfunction is dependent on the presence of PMNs. These data are in agreement with those of others who have demonstrated a decrease in contractile performance induced by an infusion of PMNs (19, 29, 32). Initial studies were performed to ensure that there were no cross-species problems pertaining to the adherence of canine PMNs to rabbit endothelium, since PMN-mediated damage to the endothelium and myocardium is dependent on PMN-endothelial interaction. In these preliminary adherence studies, activation of canine PMNs and rabbit aortic endothelium with PAF led to an increase in PMN adherence that was reduced by Cl-IB-MECA.

Significant improvement in the recovery of postischemic contractile performance was obtained with the addition of Cl-IB-MECA to the reperfusion buffer in the PMN-perfused groups without effect in groups not infused with PMNs. With 10 nM Cl-IB-MECA treatment, developed pressure and positive and negative dP/dt returned to the same values (at 60 min of reperfusion) as hearts not exposed to PMNs, suggesting a complete amelioration of PMN-mediated injury to postischemic contractile function. Additionally, there was a significant reduction in PMN accumulation, further supporting the idea that the improved contractile function was due to anti-PMN actions of Cl-IB-MECA.

Previous studies pertaining to A3 adenosine cardioprotection have been performed using agonist treatment before ischemia (13, 35, 36). In this modality of agonist administration, A3 receptor activation was associated with improved postischemic contractile function. Furthermore, Tracey et al. (36) demonstrated that protection afforded by A3 agonist pretreatment was dependent on KATP channel activation. However, all these studies were performed in isolated, perfused hearts completely devoid of blood formed elements (i.e., PMNs, macrophages, and monocytes), and the adenosine analog was present before the time of ischemia. Auchampach et al. (4) demonstrated protection from lethal and sublethal ischemia-reperfusion injury in vivo with pretreatment with an A3 agonist. Thus our study extended the understanding of A3 receptor-mediated cardioprotection by demonstrating that Cl-IB-MECA attenuates PMN-mediated postischemic dysfunction when given only at reperfusion. Because A3 receptor activation does not decrease PMN degranulation or superoxide production but does decrease the adherence of PMNs to the endothelium (16), a likely mechanism for the observed cardioprotection is related to the attenuated PMN-endothelial interaction and reduction in adherence-dependent vascular and contractile injury. The reduced adherence and attenuated PMN accumulation (MPO) support this conclusion. Lefer et al. (20) observed that endothelial injury and PMN accumulation were causally related and reduction in PMN adherence preserved endothelial function. Similarly, preservation of endothelial function is observed when adherence of PMNs is blocked with antibodies to endothelial adhesion molecules (24). Furthermore, Lefer et al. (19) showed complete functional recovery in hearts exposed to PMNs by blocking the adhesion of PMNs with sialyl Lewisx oligosaccharide. Indeed, data from the present study demonstrate that the preservation of contractile function was concomitant with a decrease in PMN accumulation, suggesting that decreased adherence and subsequent accumulation of PMNs in the ischemic-reperfused myocardium may be the mechanism underlying the improved contractile function.

Because there is substantial evidence for benefit from pretreatment with A3 agonists and new evidence that reperfusion injury may also be attenuated, agonists of this adenosine receptor subtype may provide clinicians with a new therapeutic strategy for treating ischemic heart disease. In view of the potent anti-PMN actions on adherence to endothelium at low concentrations and the lack of vasodilatory actions (4), A3 receptor agonists may be an effective therapeutic strategy in attenuating in vivo reperfusion injury with minimal hemodynamic or cardiodynamic effects.


    ACKNOWLEDGEMENTS

The authors are grateful to the Carlyle Fraser Heart Center of Crawford Long Hospital, Emory University, for their continued support of the research activities within the Cardiothoracic Research Laboratory. Cl-IB-MECA was provided by Research Biochemicals International as part of the Chemical Synthesis Program of the National Institute of Mental Health (Contract N01 MH-30003). [125I]ABA was provided by Dr. Joel Linden [Dept. of Medicine (Cardiology), University of Virginia]. The selective A1 and A2a adenosine antagonists KW-3902 and KF-21326 were provided as a gift from the Laboratory of Cardiovascular Pharmacology, Kyowa Hakko Kogyo (Shizuoka 411, Japan), through the efforts of Akira Karasawa.


    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: J. E. Jordan, Center for Experimental Therapeutics and Reperfusion Injury, Dept. of Anesthesia, Brigham and Women's Hospital, Harvard Medical School, 75 Francis St., Boston, MA 02115 (E-mail: jjordan{at}zeus.bwh.harvard.edu).

Received 18 August 1998; accepted in final form 10 June 1999.


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DISCUSSION
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