0022-3565/97/2801-0301$03.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics
JPET 280:301–309, 1997
Vol. 280, No. 1
Printed in U.S.A.
Adenosine A2 Receptor Activation Attenuates Reperfusion
Injury by Inhibiting Neutrophil Accumulation, Superoxide
Generation and Coronary Endothelial Adherence1
JAMES E. JORDAN, ZHI-QING ZHAO, HIROKI SATO, SPENCER TAFT and JAKOB VINTEN-JOHANSEN
Bowman Gray School of Medicine, Department of Physiology and Pharmacology, Department of Cardiothoracic Surgery, Research Laboratory,
Winston-Salem, North Carolina
Accepted for publication September 25, 1996
Reperfusion, although a necessary event for the preservation of reversibly injured myocardium, is associated with
additional injury mediated, in large part, by neutrophils
(Dreyer et al., 1991; Lefer et al., 1991). The restoration of
blood flow to the ischemic myocardium initiates a cascade of
inflammatory-like processes that may contribute to postischemic injury. Within minutes of the initiation of reperfusion,
endothelial dysfunction occurs (Tsao and Lefer, 1990). This
endothelial dysfunction results from the close proximity of
the neutrophil to the coronary endothelium during the adhesion process and the subsequent release of cytotoxic agents
into this microenvironment. These agents, including PAF,
superoxide anion radical, hydrogen peroxide and elastase,
Received for publication February 8, 1996.
1
This study was supported by grants from the National Institutes of Heath
(Grant HL46179) and from the American Heart Association, North Carolina
Affiliate.
P , .05 vs. vehicle-treated group). In in vitro studies, CGS21680 reduced platelet-activating factor (PAF)-activated canine
neutrophil adherence to the endothelial surface of normal homologous coronary artery segments. Compared with PAFstimulated neutrophils (188.4 6 9.4 adhered neutrophils/mm2),
CGS-21680 reduced adherence close to base-line levels
(46.6 6 5.8 adhered neutrophils/mm2) at concentrations of 10
mM (65.6 6 8.2 adhered neutrophils/mm2, P , .05 vs. PAFstimulated group) and 50 mM (56.6 6 4.6 adhered neutrophils/
mm2, P , .05 vs. PAF-stimulated group). Superoxide anion
production (cytochrome c reduction) by activated neutrophils
was reduced by CGS-21680 from 33.8 6 5.0 to 8.9 6 3.6
nmol/5 min/5 3 106 cells (P , .05 vs. PAF-stimulated group).
We conclude that specific A2 receptor stimulation with CGS21680 at reflow reduces reperfusion injury by inhibiting neutrophil-related processes.
attract and activate more neutrophils to the area (amplification stage) and damage the endothelium. Although the migration of neutrophils across the endothelium into the myocardium occurs much later after reperfusion is initiated
(approximately 3 hr after reperfusion), the early neutrophilendothelial events produce microvascular injury and ultimately myocellular necrosis (Entman et al., 1991; Lefer et al.,
1994; Lefer, 1995).
Adenosine is an endogenous autacoid that has multiple
effects on the progression of ischemia and reperfusion injury
(Ely and Berne, 1992; Vinten-Johansen et al., 1995a). Adenosine works primarily through two distinct membrane receptors, with A1 receptor-mediated effects predominating during
ischemia and A2 receptor effects being exerted primarily
during reperfusion in models of lethal injury (i.e., infarction)
(Zhao et al., 1993). The role of the A3 receptor in cardioprotection has yet to be precisely determined. Activation of aden-
ABBREVIATIONS: AAR, area at risk; CK, creatine kinase; EDSL, end-diastolic segment length; ESSL, end-systolic segment length; LAD, left
anterior descending coronary artery; LV, left ventricle; MAP, mean aortic pressure; MPO, myeloperoxidase; PAF, platelet-activating factor; PBS,
phosphate-buffered saline; PMN, polymorphonuclear leukocyte; PRP, pressure-rate product; SOD, superoxide dismutase.
301
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ABSTRACT
This study tests the hypothesis that adenosine A2 receptor
activation reduces reperfusion injury by inhibiting neutrophils in
a canine model of ischemia and reperfusion. In 16 anesthetized,
open-chest dogs, the left anterior descending coronary artery
was ligated for 60 min and reperfused for 3 hr. An intracoronary
infusion of either the selective adenosine A2 agonist CGS21680 at 0.2 mg/kg/min (n 5 8) or vehicle (n 5 8) was started 5
min before reperfusion and discontinued after 60 min. The area
at risk was comparable between vehicle-treated and CGS21680-treated groups (39.6 6 4.1 vs. 37.1 6 2.5% of left
ventricle). Infarction size, determined with triphenyltetrazolium
chloride, was smaller in the CGS-21680-treated group than in
the vehicle-treated group (15.4 6 2.9 vs. 29.8 6 2.3% of area
at risk, P , .05 vs. vehicle-treated group). CGS-21680 significantly reduced neutrophil accumulation (myeloperoxidase activity) in the nonnecrotic area at risk tissue, compared with the
vehicle-treated group (2.12 6 0.5 vs. 6.47 6 0.6 U/g of tissue,
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Jordan et al.
Vol. 280
Methods
All animals were handled in accordance with the Guide for the
Care and Use of Laboratory Animals published by the National
Institutes of Health (National Institutes of Health Publication 85–
23, revised 1985). The protocol was also approved by the Animal
Care and Use Committee of the Bowman Gray School of Medicine of
Wake Forest University.
Twenty-two mongrel dogs of either gender were initially anesthetized with sodium thiamylal (20 mg/kg i.v.). After endotracheal intubation and cannulation of the right femoral vein, deep anesthesia
was maintained by continuous infusion of 0.3 mg/kg/min fentanyl
citrate and 0.03 mg/kg/min diazepam. The dog was ventilated with
oxygen-enriched room air using a Harvard volume-cycled respirator
to maintain arterial pO2 greater than 100 mm Hg. Normal blood gas
levels and acid-base status were maintained by adjustment of the
rate and volume of respiration or by i.v. administration of sodium
bicarbonate, as appropriate. A left thoracotomy was performed at the
fifth intercostal space, and the heart was suspended in a pericardial
cradle. Millar MPC-500 solid-state pressure transducers (Millar Instruments, Houston, TX) were inserted into the aortic root via the
right internal mammary artery and into the LV through a puncture
in the apex of the heart, to measure MAP and instantaneous LV
blood pressure, respectively. An umbilical snare was placed around
the inferior vena cava for transient preload reduction. A pair of
piezoelectric crystals were placed into the subendocardium of the
area of the LV perfused by the LAD in the direction of circumferential fiber shortening, to measure segmental systolic and diastolic
length. Anticoagulation was achieved with bolus injections of 300 U
of sodium heparin given every 90 min, to maintain activated clotting
times of .400 sec. The left carotid artery was cannulated and connected to a large-bore silastic catheter in which a Transonic ultrasonic cannulating-type flow probe (Transonic Systems, Ithaca, NY)
was interposed. This circuit was capable of accommodating up to
95-ml/min flow at 100 mm Hg mean pressure. A proximal segment of
the LAD was isolated and cannulated with a 14-gauge angiocatheter
and immediately perfused via the left carotid artery. Ischemia encountered during the cannulation process lasted no longer than 40
sec and did not alter segmental function, relative to the precannulation period.
Experimental protocol. Hemodynamic and segmental function
data were collected before ischemia for baseline, at the end of
ischemia and at 15, 60, 120 and 180 min of reperfusion. After baseline measurements were recorded, 60 min of collateral-deficient ischemia was created by clamping the carotid-LAD perfusion circuit,
disconnecting the perfusion circuit from the coronary catheter and
venting the collateral blood flow into a beaker. Arteriotomy and
collateral diversion reduce the variability in infarction size observed
in the canine model of ischemia-reperfusion by reducing the nutritive
collateral blood flow (Eng et al., 1987). After 55 min of collateral
diverted ischemia, an intracoronary infusion of either CGS-21680 at
0.2 mg/kg/min (n 5 8) or vehicle (n 5 8) was begun by reconnecting
the extracorporeal circuit to the cannula in the LAD, while the
circuit remained clamped, and inserting a 25-gauge needle into the
silastic tubing just proximal to the cannula and distal to the clamp.
At the end of 60 min of ischemia, reperfusion was initiated by
removing the clamp, and the intracoronary infusion of CGS-21680 or
vehicle was continued for the next 55 min. The AAR was reperfused
for a total of 3 hr.
Drug preparation. CGS-21680 was prepared from a dry powder
and dissolved in 0.5 ml of dimethylsulfoxide. When completely dissolved, the drug was diluted with normal saline to achieve a dose of
0.2 mg/kg/min, to be delivered by 60-min intracoronary infusion.
Fresh drug was prepared for each experiment. The saline vehicle was
prepared with dimethylsulfoxide and saline in the same concentration as used in the CGS-21680-treated group. The dose of drug was
calculated to deliver a concentration of drug of about 180 nM, which
was a compromise between maximal neutrophil inhibition determined by in vitro tests and systemic vasodilation.
Data collection and analysis. LV and aortic pressures, segment
length data and LAD blood flow measurements were collected in
duplicate or triplicate during 12-sec intervals of respiratory apnea.
Data were digitized at 200 Hz using a 12-bit analog-to-digital converter (model DT2821; Data Translation Devices, Marlboro, MA) and
an IBM-PC computer. Data were stored both on removable media
and on the hard disk for archival and subsequent analysis. Two
measurements were taken during steady state and three measurements were taken during caval occlusion (for determination of segmental diastolic stiffness). The data were processed using SPECTRUM (Triton Technology, San Diego, CA), a video-graphical
interface computer algorithm program developed in our laboratory.
The beginning of systole was marked when instantaneous LV dP/dt
exceeded 250 mm Hg/sec, and the end-systolic point was marked at
peak negative dP/dt. These points were checked visually and adjusted manually as necessary. Segmental shortening was calculated
by the formula [(EDSL 2 ESSL)/EDSL] 3 100 and expressed as a
percentage. Segmental work was calculated using a point-by-point
integration of the pressure-segment length loop over the entire cardiac cycle. Segmental stiffness was calculated by fitting the enddiastolic pressure-segment length data of the variably preloaded
pressure-segment length loops obtained during caval occlusion to the
equation PED 5 a(ebL), where PED is the end-diastolic pressure, a and
b coefficients are the end-diastolic pressure axis intercept and the
degree of curvature, respectively, and L is the EDSL. The b coefficient is a unitless measure of the curvature of the end-diastolic
relation (modulus of stiffness).
Determination of AAR and infarction size. At the end of the
experiment, the heart was rapidly arrested with a bolus injection of
sodium pentobarbital and excised. The proximal aorta was cannulated, and the heart was stained using a gentian violet infusion
delivered via the ascending aorta (infusion pressure was maintained
at 100 mm Hg). The atria and the right ventricle were removed, and
the LV was cut into 3- to 4-mm-thick sections. The unstained region
(AAR) was then separated from the blue-stained nonischemic area
and incubated for 10 min in a 1% solution of triphenyltetrazolium
chloride solution in phosphate buffer, warmed to 37°C. After incubation, all tissues were placed in a 10% formalin solution overnight.
The AAR was then subdivided into pale necrotic and brick-red nonnecrotic areas. The AAR and area of necrosis were determined gravimetrically and expressed as a percentage of the total LV mass. The
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osine A1 receptors during the ischemic phase has been shown
to reduce infarction size and contractile dysfunction, possibly
through inhibition of ATP-sensitive K1 channels (Gross and
Auchampach, 1992; Toombs et al., 1993) and the subsequent
effects of hyperpolarization and reduced calcium overload.
Adenosine A2 receptor activation during the reperfusion
phase has been shown to reduce infarction size (Norton et al.,
1992; Schlack et al., 1993). Selective A2 receptor activation
inhibits neutrophil adherence to endothelium and decreases
the production of superoxide anion (Cronstein et al., 1992;
Zhao et al., 1995) independent of ATP-sensitive K1 channels.
These inhibitory effects may be related to A2-mediated reductions in infarction size. Recently, a specific adenosine A2
agonist, CGS-21680, was reported to reduce infarction size in
both rabbit and canine models (Norton et al., 1992; Schlack et
al., 1993). However, those studies did not determine whether
the mechanism of adenosine A2 receptor-mediated cardioprotection conferred by CGS-21680 was related to the inhibition
of neutrophil actions. In the present study, we tested the
hypothesis that adenosine A2 receptor activation, with the
A2-selective agonist CGS-21680 given only during reperfusion, is cardioprotective through inhibition of neutrophil activities.
1997
303
CA). Superoxide radical production was calculated using an extinction coefficient of 21 mM21 cm21 for cytochrome c. Results are
reported as nanomoles of SOD-inhibitable O22 produced by a suspension of 5 3 106 neutrophils during the 5-min measurement period.
Statistical analysis. Data are expressed as mean 6 S.E.M. Differences were considered significant when P , .05. Differences in
end-point variables between groups were analyzed by Student’s t
test. Time-related differences within groups and group differences
were analyzed by two-way analysis of variance for repeated measures, with post hoc t tests to determine the points that differed at
each time. In vitro data were analyzed with one-way analysis of
variance followed by Duncan’s multiple comparisons analysis.
Results
Twenty-two dogs were entered into the study, of which 16
(eight in each group) are represented in the final analysis of
the results. Of the six dogs that were excluded from the data
analysis, five were from the CGS-21680-treated group and
one from the vehicle-treated group. Animals were fully randomized to either group. Two dogs did not finish the protocol
(one from each group) because of malignant arrhythmias,
three were not reperfused (all from the CGS-21680-treated
group and characterized by the absence of reactive hyperemia and the persistence of cyanosis due to thrombus material in the extracorporeal circuit) and one broke protocol
(from the CGS-21680-treated group) because of persistent
and long-lasting respiratory acidosis.
Hemodynamic Data
The hemodynamic data for both groups are represented in
table 1. There were no differences between groups for any of
the hemodynamic parameters at the control time point. In
both groups the heart rate increased over the course of the
experiment; however, none of the increases were significant,
compared with the previous time point. CGS-21680 treatment after reperfusion showed a statistically significant reduction in heart rate, from that with the vehicle, only at 180
min of reperfusion. The vehicle-treated group displayed a
small but significant decrease in LV peak-systolic pressure
during ischemia, which was maintained over the remainder
of the experiment. The CGS-21680-treated group showed a
similar time-related decrease in LV peak-systolic pressure,
which did not reach significance. The LV end-diastolic pressure increased slightly in both groups during ischemia but
showed no significant group- or time-related differences during the entire experiment. The MAP in the vehicle-treated
group remained constant throughout the procedure. However, CGS-21680 treatment at reperfusion caused 13 and
20% decreases in MAP at 15 and 60 min of reperfusion,
respectively. There was a group difference in MAP only at 60
min of reperfusion, when the vehicle-treated group displayed
a significantly higher pressure than the CGS-21680-treated
group. Maximum and minimum dP/dt decreased during ischemia and remained at similar lower levels throughout the
procedure in both groups. Coronary blood flow data showed a
marked hyperemic response in both groups beginning upon
restoration of flow and lasting through the first 1 hr of
reperfusion. At 15 and 60 min of reperfusion, the CGS-21680treated group exhibited a significantly greater blood flow,
compared with vehicle. By 120 min of reperfusion, blood flow
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AAR was calculated as 100 3 [(mass of necrotic tissue 1 mass of
nonnecrotic ischemic-reperfused tissue)/total mass of LV]. This
gravimetric method of infarction sizing compares favorably with the
planimetric method (Vinten-Johansen et al., 1992).
Plasma CK activity. Blood samples for measuring plasma CK
activity were taken from the left femoral artery at the same time
points at which hemodynamic data were collected. The plasma was
analyzed spectrophotometrically for total CK activity (CK-10 kit;
Sigma Diagnostics, St. Louis, MO) and protein content. CK levels
were expressed as international units per gram of protein.
Cardiac MPO activity. Tissue samples were taken from the
nonischemic zone and the nonnecrotic and necrotic zones of the AAR
for spectrophotometric analysis of MPO activity as a measure of
neutrophil accumulation in the myocardium, using a method described previously (Mullane et al., 1985; Sato et al., 1995). MPO
activity (units per gram of tissue) was calculated as micromoles per
minute 5 DA460/11.3 mmol/(CM 3 milliliters of reactant 3 D)/sample
weight, where DA460 is equal to the absorbance change over time at
460 nm, 11.3 is the molar extinction coefficient for H2O2 at 460 nm,
CM represents the cuvette path length and D is the volume of
supernatant used. One unit of MPO activity is defined as the enzyme
activity degrading 1 mmol of H2O2/min at 25°C.
In vitro PMN adherence assay. Adherence of canine neutrophils to the endothelial cell surface of normal, homologous, coronary
arteries (from naive animals not subjected to ischemia and reperfusion) was assessed using neutrophils labeled with Zynaxis PKH26
(Zynaxis Cell Science, Malvern, PA) vital fluorescent dye. One milliliter each of diluent and PKH26 dye (4 mM) was added to a neutrophil suspension of 5 3 106 cells/ml and incubated for 5 min. Two
milliliters of PBS containing 10% plasma were subsequently added
to stop the labeling reaction, and another 5 ml of PBS were added to
the cell suspension. The cells were then centrifuged at 600 3 g for 10
min at 4°C. The pellet was resuspended in PBS, and the cells were
counted by an automated Coulter counter to determine the population per milliliter of cell suspension. This labeling procedure yields
cells with normal function and morphology. In validation studies
performed with the labeling procedure, unlabeled and labeled neutrophils demonstrated 95% and 97% viability, respectively, using
trypan blue exclusion.
Canine coronary arteries were carefully isolated in cold oxygenated Krebs-Henseleit buffer, so as not to disturb the endothelium.
After removal of the fat and connective tissue, the arteries were cut
into segments 2 to 3 mm in length. These segments were carefully
opened and placed in plastic dishes containing 3 ml of Krebs-Henseleit buffer at 37°C. Labeled neutrophils (4 3 106 cells) were added to
the bath alone or in combination with different concentrations of
CGS-21680 (1–50 mM). PAF (100 nM) was added to the dishes 5 min
after incubation with CGS-21680, and the incubation was continued
for an additional 15 min. After incubation, the segments were removed, washed three times with Krebs-Henseleit buffer, placed on
glass slides (endothelial side up) and covered with immersion oil and
a glass coverslip. Adherence was measured by counting the number
of neutrophils adhering to the endothelial surface in six separate
microscopic fields under epifluorescence microscopy (490-nm excitation, 504-nm emission).
In vitro superoxide production by PMNs. Adherence-independent superoxide radical production by activated neutrophils was
determined by measuring the SOD-inhibitable reduction of ferricytochrome c to ferrocytochrome c. Neutrophils (5 3 106 cells/ml) were
prewarmed to 37°C in the presence of 160 mM cytochrome c and
incubated with cytochalasin B (5 mg/ml) and CGS-21680 (1–50 mM)
for 5 min. The neutrophils were then stimulated with PAF (100 nM)
in a final reaction volume of 0.5 ml. The tests were run in two groups,
with and without SOD, to correct for nonspecific activity or color
generation. The tubes were centrifuged at 500 3 g for 10 min at 4°C.
Cytochrome c reduction was measured spectrophotometrically by
determining the optical density of the supernatant at 500 nm, using
a Vmax kinetic microtiter plate reader (Molecular Devices, Palo Alto,
A2 Activation Reduces Reperfusion Injury
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Vol. 280
TABLE 1
Hemodynamic parameters measured throughout the course of the experiment
Reperfusion
Parametera
Group
Control
Ischemia
92 6 17
94 6 12
124 6 12
118 6 12
133 6 7
139 6 11
126 6 7
139 6 12
136 6 8
116 6 7
142 6 10
115 6 7*
15 min
60 min
120 min
180 min
Vehicle
CGS-21680
LVSP (mm
Hg)
Vehicle
CGS-21680
111 6 6
115 6 6
104 6 4†
114 6 8
105 6 6
103 6 4
107 6 6
99 6 3
103 6 7
96 6 3
98 6 4
97 6 2
LVEDP (mm
Hg)
Vehicle
CGS-21680
8.5 6 1.4
11.9 6 1.5
13.7 6 3.6
14.1 6 1.5
12.1 6 1.3
12.9 6 2.3
9.9 6 1.3
9.8 6 1.9
9.3 6 1.3
10.4 6 1.8
9.1 6 1.3
10.8 6 1.7
MAP (mm Hg)
Vehicle
CGS-21680
95 6 5
99 6 7
95 6 4
99 6 9
95 6 4
86 6 7
96 6 6
79 6 5*
92 6 7
78 6 5
88 6 4
80 6 5
PRP z 1000
(mm Hg sec)
Vehicle
CGS-21680
9.8 6 1.6
11.0 6 1.7
12.8 6 1.3
13.7 6 2.2
13.9 6 0.7
14.4 6 1.5
13.4 6 0.9
13.7 6 1.2
14.0 6 1.2
11.1 6 0.8
14.1 6 1.5
11.2 6 0.8
dP/dtmax (mm
Hg/sec)
Vehicle
CGS-21680
1716 6 89
1700 6 134
1417 6 92
1506 6 100
1342 6 124
1441 6 75
1445 6 123
1555 6 88
1423 6 112
1310 6 76
1337 6 95
1292 6 99
dP/dtmin (mm
Hg/sec)
Vehicle
CGS-21680
21788 6 101
21789 6 94
21452 6 104
21625 6 138
21479 6 135
21437 6 21
21573 6 115
21452 6 38
21519 6 118
21372 6 48
21417 6 117
21349 6 103
CBF (ml/min)
Vehicle
CGS-21680
20.6 6 1.8
25.6 6 1.5
32.3 6 2.4
51.6 6 2.7*
22.4 6 2.8†
23.7 6 2.0†
19.8 6 1.6
21.9 6 2.3
35.6 6 2.7†
56.3 6 3.5†*
HR, heart rate; LVSP, LV peak systolic pressure; LVEDP, LV end-diastolic pressure; CBF, mean coronary (LAD) blood flow. Values are expressed as mean 6 S.E.M.
* P , .05 vs. vehicle-treated group.
† P , .05 vs. prior time point.
a
values returned to base-line levels and were comparable between groups.
The calculated PRP for the two groups is summarized in
table 1. Even though there were individual group differences
at various times for the variables used to calculate the PRP,
there were no significant differences in this variable during
ischemia and early reperfusion. Although there was a trend
toward a lower PRP in the CGS-21680-treated group at the
later stages of reperfusion, these differences never reached
significance.
Contractile Function Data
Segmental shortening. The vehicle-treated and CGS21680-treated groups were comparable with respect to EDSL
and ESSL at the control time point (table 2). Both groups
exhibited lengthened EDSL and ESSL values during ischemia and throughout the entire reperfusion period. However,
there were no group-related differences. During ischemia
both groups exhibited a rightward shift in the pressurelength relationship and displayed a significant decline in
segmental shortening, changing from active shortening to
dyskinesis (fig. 1; table 2). Both groups demonstrated a shift
back toward the left in the pressure-segment length relation
during the reperfusion phase, but neither group returned to
base-line levels of ESSL or segmental shortening. Wall motion remained dyskinetic throughout the entire reperfusion
period in the vehicle-treated group, whereas the CGS-21680treated group showed a slight improvement in shortening
during the first 1 hr of reperfusion before becoming dyskinetic for the remainder of the experiment. There were no
significant group differences at any of the time points.
Segment work. The calculated segment work for both
groups showed a significant decline from control values during ischemia (table 2). There was a transient slight recovery
of segment work in both groups just after 15 min of reperfusion, which dissipated with time. However, there were no
significant differences between the vehicle- and CGS-21680treated groups at any time during reperfusion.
Segment stiffness. Segmental stiffness (table 2), calculated as the b-coefficient of the end-diastolic pressure-segment length relationship, was slightly higher in the CGS21680-treated group at the control time point, compared with
the vehicle-treated group (0.36 6 0.05 vs. 0.21 6 0.03).
Throughout ischemia and reperfusion, the stiffness in both
groups increased. However, these changes did not reach significant levels at any other time points.
Infarction Size
The LV weight was significantly greater in the vehicletreated group, compared with the CGS-21680-treated group
(table 3). However, the AAR, when expressed as either mass
(table 3) or percentage of LV (fig. 2), showed no significant
group differences. CGS-21680 treatment at reperfusion significantly reduced the mass of necrotic tissue by 53% of the
average vehicle size (table 3). Infarction size was reduced
approximately 45% by CGS-21680, compared with vehicle,
when expressed either as a percentage of LV or as a percentage of the AAR (fig. 2).
Plasma CK Activity
Total CK activity was comparable between groups at the
control time point (fig. 3). There was a small increase in
plasma CK in both vehicle-treated and CGS-21680-treated
groups during ischemia. However, after reperfusion, there
was a marked increase in CK levels within each group (fig. 3).
Starting at 60 min of reperfusion, there was a strong ten-
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HR (beats/
min)
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305
0.53 6 0.11
0.55 6 0.14
0.49 6 0.12
0.57 6 0.18
0.54 6 0.12
0.66 6 0.20
0.53 6 0.13
0.49 6 0.14
72.8 6 34.1
67.1 6 24.1
36.4 6 26.1†
39.1 6 25.8
20.2 6 2.4
1.8 6 2.2
21.1 6 1.9†
21.0 6 1.6
23.2 6 0.9
23.3 6 1.3
25.5 6 1.8
24.5 6 1.4
13.8 6 1.6†
13.4 6 1.4
14.3 6 1.2
14.3 6 1.2
83.4 6 14.9
67.1 6 17.9
42.1 6 16.6
44.3 6 13.9
186.2 6 21.0
67.1 6 24.1†
16.5 6 1.6
22.4 6 1.7†
19.6 6 2.0
23.6 6 0.7†
11.3 6 1.3
15.9 6 1.7†
257.2 6 41.0
67.2 6 26.8†
mm Hg z mm
VEH
CGS
VEH
CGS
%
Segmental Shortening
17.4 6 1.7†
17.6 6 1.7
17.4 6 1.4
17.3 6 1.4
13.9 6 1.7
13.7 6 1.5
14.3 6 1.3
14.3 6 1.3
16.9 6 1.6
17.1 6 1.6
16.6 6 1.4
16.6 6 1.4
a
VEH, vehicle-treated; CGS, CGS-21680-treated. Values expressed as mean 6 S.E.M.
* P , .05 vs. vehicle-treated.
† P , .05 vs. prior time point.
12.6 6 1.1
19.8 6 1.7†
13.4 6 1.4
15.7 6 1.9†
Control
Ischemia
Reperfusion
15 min
60 min
120 min
180 min
VEHa
mm
15.7 6 1.3
19.2 6 1.7†
CGS
VEH
mm
ESSL
EDSL
Time
TABLE 2
Regional segmental length and function for the AAR throughout the experiment
Fig. 1. Instantaneous LV pressure-segment length loops for a representative experiment in vehicle-treated (n 5 8) (top) and CGS-21680treated (n 5 8) (bottom) groups. Segmental work was calculated as the
integral of each pressure-length loop. CNTL, control time point; ISCH,
end of ischemia; REP, time of reperfusion.
TABLE 3
Mass of the areas of the heart (entire LV mass, AAR and area of
necrosis) used for the determination of infarction size
AAR Weight
Area of Necrosis
Weight
Group
LV Weight
g
g
g
Vehicle
CGS-21680
140.7 6 7.8a
118.8 6 4.7*
39.6 6 4.1
37.1 6 2.5
12.2 6 1.9
5.7 6 1.3*
a
Values are expressed as mean 6 S.E.M.
* P , .05 vs. vehicle.
dency for the CGS-21680-treated group to have a lower
plasma CK activity than the vehicle-treated group. However,
these differences in total CK activity did not reach significance (P 5 .21 at 180 min of reperfusion).
Myocardial MPO Activity
MPO activity was similar between the two groups in the
normally perfused, nonischemic area of the LV (fig. 4). In the
nonnecrotic AAR, the CGS-21680-treated group showed significantly lower levels of MPO activity, compared with the
vehicle-treated group. In the necrotic AAR, CGS-21680 treatment decreased the MPO activity by 45%, compared with the
vehicle (P 5 .07). These data suggest that CGS-21680 re-
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Segmental Work
CGS
VEH
0.21 6 0.03
0.51 6 0.18
b
CGS
0.36 6 0.05*
0.69 6 0.18
A2 Activation Reduces Reperfusion Injury
306
Jordan et al.
Vol. 280
Fig. 4. MPO activity in three areas of the myocardium. NI, nonischemic, normally perfused tissue; Isch, ischemic nonnecrotic tissue; Nec,
necrotic tissue. MPO activity is expressed as units per gram of protein.
*P , .05 vs. vehicle-treated group.
Fig. 3. Plasma creatine kinase (CK) levels for the two groups, shown
for the entire experiment. CK levels are expressed as international units
per gram of protein. †P , .05 vs. prior time point. Cntl, control; Isch,
end of ischemia; R15, reperfusion for 15 min; R60, reperfusion for 60
min; R120, reperfusion for 120 min; R180, reperfusion for 180 min.
duced neutrophil accumulation in the ischemic-reperfused
myocardium.
Fig. 5. Production of superoxide anion by neutrophils, measured
spectrophotometrically as the SOD-inhibitable reduction of ferricytochrome c in vitro. The ordinate represents the amount of superoxide
produced in 5 min by a suspension of 5 3 106 neutrophils. *P , .05 vs.
PAF-treated group.
Superoxide Anion Production
Neutrophil Adherence to Normal Coronary Endothelium
As an index of the effect of CGS-21680 on adherenceindependent (directly stimulated) neutrophil activity, an in
vitro assay of superoxide production by isolated neutrophils
was performed. PAF stimulation of neutrophils caused a
.5-fold increase in superoxide anion production, compared
with unstimulated neutrophils (fig. 5). Preincubation with
CGS-21680 exhibited a concentration-dependent decrease in
superoxide anion production. At a concentration of 50 mM
CGS-21680, superoxide radical production was reduced by
nearly 75% of PAF-activated levels, which was not different
from the unstimulated value. Therefore, CGS-21680 had a
direct inhibitory effect on neutrophil superoxide anion generation (fig. 5) triggered by factors other than adherence to
the endothelium (fig. 6).
To further examine the effect of CGS-21680 on neutrophil
activity, adherence of activated neutrophils to normal coronary artery endothelium was determined in vitro. PAF stimulation of neutrophils caused a 4-fold increase in the number
of neutrophils adhered, compared with that of unstimulated
PMNs (fig. 6). Pretreatment of the endothelium and neutrophils with CGS-21680 before activation with PAF significantly reduced adherence, in a dose-dependent manner. At
10 mM, adherence was reduced to approximately one-third of
stimulated levels. CGS-21680, we conclude, had a profound
inhibitory effect on neutrophil adherence to coronary artery
endothelium. CGS-21680 therefore reduced adherence-independent superoxide generation and adherence to the coronary artery endothelium.
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Fig. 2. Size of the AAR expressed as a percentage of the LV (AAR/LV)
and infarction size (area of necrosis) expressed as a percentage of the
LV (AN/LV) or AAR (AN/AAR). Vehicle-treated, n 5 8; CGS-21680treated, n 5 8. *P , .05 vs. vehicle-treated group.
1997
A2 Activation Reduces Reperfusion Injury
Discussion
Reperfusion accounts for a substantial portion of the injury
that occurs as the result of reversible coronary occlusion
(Forman et al., 1990). This concept of reperfusion injury has
been cultivated from numerous studies in which pharmacological interventions were applied only during the reperfusion phase and resulted in a reduction in infarction size.
Neutrophils may contribute significantly to the pathogenesis
of injury occurring during reperfusion. Dreyer et al. (1991)
demonstrated that neutrophils are localized into the previously ischemic myocardium predominantly during the first 1
hr of reperfusion. The present study shows that intracoronary infusion of a specific adenosine A2 agonist, CGS-21680,
started during the first 1 hr of reperfusion, reduced infarction
size significantly, without marked effects on the determinants of myocardial oxygen demand. The infusion of CGS21680 was, however, associated with greater LAD blood flow
during the period of infusion. In addition, these data support
the hypothesis that this reduction in infarction size is associated with reduced neutrophil accumulation (tissue MPO
activity) in the ischemic-reperfused myocardium. Additionally, it was shown in vitro that neutrophil-derived superoxide
radical production was attenuated by A2 receptor stimulation, as was the adherence of neutrophils to coronary endothelium. These data are consistent with the hypothesis that
activation of adenosine A2 receptors during reperfusion reduces myocardial infarction by inhibiting neutrophil activation and associated myocardial injury after reperfusion has
been initiated.
The receptor agonist used in this study, CGS-21680, has
been used extensively as a specific adenosine A2 agonist
(Balwierczak et al., 1991; Norton et al., 1992; Schlack et al.,
1993). This compound is more potent at the A2 receptor than
at the A1 receptor (EC50 values of 22 nM vs. 760 nM) and is
140-fold more selective for the A2 receptor than the A1 receptor (Hutchison et al., 1989; Webb et al., 1992). CGS-21680
also has well-characterized vasodilator effects that are attributed to its selectivity for the adenosine A2 receptor
(Abebe et al., 1994). The half-life of CGS-21680, as measured
in rats, is approximately 19 min (Webb et al., 1992). Our
hemodynamic data (table 1) indicate a small decrease in
MAP during the infusion period. However, the decrease in
pressure did not differ from that in the vehicle-treated group
after the infusion was discontinued, arguing against a systemic buildup of the drug.
The chronotropic data do not suggest any A1-mediated
effects on the heart, although in an in vivo model with intact
cardiovascular reflexes these effects may be difficult to observe. The dose of CGS-21680 used in the present study was
greater than that used by some other investigators (Bullough
et al., 1995). However, as reported by Balwierczak et al.
(1991), dogs are much less sensitive to CGS-21680 than other
species. Previously, our group showed that adenosine, working through the A1 receptor, had very little effect on infarction size when given specifically during reperfusion (Zhao et
al., 1994a). In that study, A1 receptor activity could account
for only about 25% of the total protective effect of adenosine,
and this protection was exerted during the ischemic phase
(Zhao et al., 1994a). Therefore, it is unlikely that any A1
receptor activation by the dose of CGS-21680 used in the
present study would exert cardioprotection when administered only during reperfusion.
Our results are in apparent contrast to those of Lasley and
Mentzer (1992), who concluded that postischemic functional
recovery in an isolated rat heart model of myocardial stunning was achieved only by an A1 agonist and not by an A2
agonist. However, in that study the receptor-selective analogs were administered only before the onset of ischemia,
followed by reperfusion with drug-free buffer. Therefore, the
A2 agonist was not present during the reperfusion phase,
when it may have exerted its action. In addition, the buffer
solution was free of neutrophils, one of the primary targets of
adenosine A2 analog actions. Moreover, neutrophils may play
little role in ischemic-reperfusion injury producing myocardial “stunning” in the absence of infarction (Becker, 1991).
Therefore, the data reported by Lasley and Mentzer (1992)
are not inconsistent with the observations in the present
study and are not inconsistent with a role for A2-mediated
cardioprotection in models of lethal injury (infarction) where
neutrophils are present.
Consistent with the vasodilatory effects of an A2-selective
agonist, CGS-21680 exhibited a significant decrease in MAP
and an increase in LAD blood flow, relative to the vehicletreated group, for the duration of its infusion during reperfusion. The decrease in MAP occurred only during reperfusion. Although a correlation has been drawn between
reduced afterload (MAP) during ischemia and reduced infarction size, no such correlation has been reported for afterload
reduction specifically during reperfusion. In addition, the
reduction in aortic blood pressure was counterbalanced by an
increase in heart rate, resulting in no change in the PRP as
an index of myocardial oxygen demand. The increase in coronary blood flow to the ischemic-reperfused myocardium may
have played a role in the cardioprotection elicited by CGS21680. Stahl et al. (1986) observed an improvement in postischemic function when blood flow was increased to the isch-
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Fig. 6. Adherence of neutrophils to normal coronary artery endothelium. Labeled neutrophils were quantified using epifluorescent microscopy and are represented as the sum of neutrophils adhering to the
endothelial surface per square millimeter of endothelium. *P , .05 vs.
PAF-treated group. †P , .05 vs. PMN-only group.
307
308
Jordan et al.
endothelium. Therefore, adenosine inhibits the early neutrophil actions leading to necrosis and reduces neutrophil-mediated damage to both vascular endothelium and myocytes.
The data from the present study are consistent with the
A2-mediated antineutrophil actions of adenosine, because the
A2-selective analog CGS-21680 reduced superoxide generation and endothelial cell adherence of PAF-stimulated neutrophils in vitro. Accordingly, the MPO levels in the myocardium at risk, used as an index of neutrophils accumulated in
the tissue, were reduced in the group treated with CGS21680. Chen et al. (1994) demonstrated a correlation between
neutrophil accumulation in vivo, as assessed by histological
examination, and tissue MPO activity. In the present study,
the neutrophils accumulated in the ischemic-reperfused myocardium may have been either adhering to the vascular endothelium or present as microemboli in the microvasculature. It is unlikely that the accumulated neutrophils had
migrated into the parenchyma in proximity to the myocytes,
because this process takes several hours to accomplish
(Dreyer et al., 1991). MPO levels in the normally perfused
myocardium were not altered in the CGS-21680-treated
group, arguing against a significant contribution of passively
resident neutrophils trapped after tissue sampling to the
overall tissue MPO levels and arguing against a systemic
antineutrophil effect of CGS-21680. In contrast to the in vitro
studies using coincubated neutrophils and endothelium, we
cannot conclude that there is a direct cause-and-effect relationship between fewer neutrophils and decreases in infarction size. However, data from the present study, as well as
those reported by others (Tsao et al., 1990; Lefer et al., 1991;
Ma et al., 1992), strongly suggest that the inhibition of neutrophils reduces consequent injury to vascular endothelium
and myocytes. In addition, there is a strong correlation between neutrophil inhibition and infarction reduction (Lucchesi and Mullane, 1986; Lucchesi, 1990; Lefer et al., 1993).
In summary, the present study supports the cardioprotective effects of adenosine and adenosine analogs. Specifically,
a significant reduction in infarction may by accomplished by
selective A2 receptor activation during reperfusion. The period of reperfusion coincides with the initiation of neutrophil
activities culminating in vascular and myocyte injury. Reduction of in vitro superoxide generation and adherence to
the coronary vascular endothelium, with a concomitant reduction of MPO levels in the AAR, strongly suggest that
selective activation of adenosine A2 receptors during reperfusion reduces infarction size by inhibiting neutrophil-mediated damage.
Acknowledgments
We are grateful to Ciba-Geigy Pharmaceuticals (Summit,
NJ) for the gift of CGS-21680 and to Sharon Ireland for
preparation of the manuscript.
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A2 Activation Reduces Reperfusion Injury