© 1997 American Heart Association, Inc.
Articles |
Selective Activation of A3 Adenosine Receptors With N6-(3-Iodobenzyl)Adenosine-5'-N-Methyluronamide Protects Against Myocardial Stunning and Infarction Without Hemodynamic Changes in Conscious Rabbits
From the Division of Cardiology, University of Louisville (Ky).
Correspondence to Roberto Bolli, MD, Division of Cardiology, 550 S Jackson St, ACB 3rd Floor, University of Louisville, Louisville, KY 40292. E-mail r0boll01{at}ulkyvm.louisville.edu
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Abstract |
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Abstract To examine the cardioprotective role of A3 adenosine receptors during myocardial ischemia/reperfusion injury, we tested the effect of N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA), a potent and selective A3 adenosine receptor agonist, in models of myocardial stunning and infarction in chronically instrumented conscious rabbits. In phase I (studies of myocardial stunning), rabbits were subjected to six 4-minute coronary occlusions, each separated by 4-minute reperfusion periods, after which the recovery of systolic wall thickening was measured (ultrasonic crystals). In phase II (studies of myocardial infarction), rabbits were subjected to a 30-minute coronary occlusion followed by 3 days of reperfusion. In both phases, IB-MECA was administered as an intravenous bolus (100 µg/kg) 10 minutes before the first coronary occlusion. This dose of IB-MECA was determined in pilot studies to have no effect on heart rate, arterial blood pressure, or plasma histamine concentration in rabbits. In phase I, IB-MECA markedly improved the recovery of wall thickening after the six occlusion/reperfusion cycles, and this effect was sustained throughout the 5-hour observation period; the total deficit of wall thickening (a measure of the overall severity of myocardial stunning) was reduced by 68% (control, 129±16 arbitrary units, n=7; IB-MECA, 41±6 arbitrary units, n=6; P<.01). The protective effects of IB-MECA against stunning were completely blocked by pretreatment with the nonselective adenosine receptor antagonist 8-p-sulfophenyl theophylline or the specific protein kinase C inhibitor chelerythrine. In phase II, IB-MECA reduced myocardial infarct size by 61%; infarct size (tetrazolium staining) was 41±4% of the risk region in control animals (n=8) and 16±6% in IB-MECA–treated animals (n=8, P<.01). These results demonstrate that in conscious rabbits the A3 adenosine receptor agonist IB-MECA confers a powerful protection against both reversible (stunning) and irreversible (infarction) injury during acute myocardial ischemia and reperfusion by a protein kinase C–mediated pathway, suggesting that selective activation of A3 receptors is an effective means of protecting the ischemic myocardium without hemodynamic changes.
Key Words: ischemia/reperfusion injury • myocardial stunning • myocardial infarction • adenosine receptor • protein kinase C
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Adenosine exerts numerous physiological effects, which were originally thought to be mediated by three receptors, A1, A2A, and A2B. In the early 1990s, however, a new adenosine receptor (termed A3) was cloned from rat tissues, first by Myerhof et al1 and then by Zhou et al.2 More recently, human,3 4 sheep,5 dog,6 and rabbit7 A3 receptors have been cloned. Functional expression of A3 receptors from various species in COS or CHO cells reveals that, like A1 receptors, A3 receptors bind to the radioligands N6-2-(4-amino-3-[125I]iodophenyl)ethyladenosine (125I-APNEA), N6-(4-amino-3-[125I]iodobenzyl)adenosine (125I-ABA), and N6-(4-amino-3-[125I]iodobenzyl)adenosine-5'-N-methyluronamide (125I-AB-MECA) and are negatively coupled to adenylyl cyclase.2 3 5 6 7 8 In RBL-2H3 cells and in rat brain tissue, A3 receptors have been shown to stimulate phospholipase C via pertussis toxin–sensitive G proteins.9 10 11 One unique feature of A3 receptors is that there are major differences among species in the binding profile of adenosine receptor ligands, particularly xanthine antagonists.12
A3 receptor transcripts have been detected in
testes,1 2 3 5 6 spleen,5 6
lung,2 3 4 5 6 liver,3 4 6 heart,2 4 6
kidney,2 4 5 6 inflammatory cells,6 9 and
various regions of the brain.2 3 4 5 Although A3
receptors are widely distributed, little is known regarding their
functions, primarily because of a lack of selective agonists and
antagonists. Recently, however, Gallo-Rodriguez et
al13 performed careful structure-activity relationship
studies with recombinant rat adenosine receptors and found that
5' substitution of
N6-benzyladenosine increases the
affinity and selectivity of this compound for A3 receptors.
IB-MECA was one of the most potent analogues identified, with an
affinity of 1.1 nmol/L and 
50-fold selectivity over A1
and A2A receptors.13 Similar potency (2
nmol/L) and selectivity (15-fold) for IB-MECA were reported by Hill et
al7 for rabbit A3 receptors. Administration of
IB-MECA to mice was found to decrease locomotor activity, and this was
not blocked by selective A1 or A2A receptor
antagonists, demonstrating effectiveness and selectivity of
IB-MECA for A3 receptors in vivo.14 IB-MECA
has also been shown to decrease blood pressure in rodents, most likely
by activating mast cells.15 The hypotensive effects of
IB-MECA persist for at least 90 minutes, demonstrating that the
half-life of this compound is relatively long compared with
adenosine.15
Adenosine and synthetic adenosine receptor agonists have been shown to be protective in many different models of myocardial ischemia/reperfusion injury. In most cases, the cardioprotection has been attributed to activation of A1 and/or A2A receptors.16 17 18 19 20 21 22 23 24 25 26 Recently, however, evidence has emerged that A3 adenosine receptors may also be involved.27 28 29 Liu et al27 and Armstrong and Ganote28 were the first to suggest that the beneficial actions of adenosine on the ischemic/reperfused myocardium may be mediated in part by the A3 receptor. Strickler et al29 have subsequently reported results that are consistent with this notion. Although these studies support a cardioprotective action of A3 receptors in isolated hearts27 and isolated myocytes,28 29 the role of A3 receptors during myocardial ischemia in vivo has not been assessed. Furthermore, no data are available regarding whether activation of A3 receptors is cardioprotective in a conscious animal preparation and whether it attenuates myocardial stunning. With the recent availability of selective A3 receptor agonists, it has become possible to examine these issues using ligands that act more specifically on this receptor subtype. Accordingly, the goal of the present study was to determine whether selective activation of A3 receptors with IB-MECA protects against myocardial ischemia/reperfusion injury under conditions that are as physiological as possible. To eliminate the influence of variable levels of collateral perfusion, we used rabbits, a species that lacks a significant coronary collateral circulation.30 To avoid the confounding factors associated with open-chest preparations,31 32 33 34 35 36 we performed all studies in chronically instrumented conscious animals. To perform a comprehensive investigation of the effect of A3 receptor activation during ischemia, we used two different experimental settings. In phase I of the study, we examined the ability of IB-MECA to alleviate the reversible injury induced by brief ischemic episodes (myocardial stunning), whereas in phase II, we explored the ability of the drug to limit the irreversible injury induced by a sustained ischemic episode (myocardial infarction). As an initial step toward exploring the mechanism of action of IB-MECA, we tested whether the protective effects of this agent are blocked by the PKC inhibitor chelerythrine. The results demonstrate that IB-MECA protects against both stunning and infarction, that it acts via a PKC-mediated pathway, and that the protection occurs in the absence of any hemodynamic changes, suggesting that selective activation of A3 receptors may be a useful therapy to protect the myocardium during acute ischemia.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A total of 74 rabbits and 3 rats were used in this investigation (including pilot studies). The study was performed in accordance with the guidelines of the Animal Care and Use Committee of the University of Louisville School of Medicine and with the Guide for the Care and Use of Laboratory Animals (Department of Health and Human Services, publication No. [NIH] 86-23).
Conscious Rabbit Preparation
New Zealand White male rabbits (2.0 to 2.5 kg) were
anesthetized with an intravenous bolus of sodium
methohexital (10 mg/kg) to allow intubation with an orotracheal tube
using a pediatric laryngoscope. Anesthesia was maintained
with sodium pentobarbital (35 mg/kg IV); additional doses of
pentobarbital were given during surgery as needed. Rabbits were
ventilated with a positive-pressure respirator using room air
supplemented with 100% oxygen. Under sterile conditions, a thoracotomy
was performed through the fourth left intercostal space to expose the
heart, and an inflatable occluder was fastened around the left marginal
coronary artery. The occluder was inflated briefly to determine
the region at risk by observing regional cyanosis. At the center of the
region at risk, a 10-MHz pulsed Doppler ultrasonic
crystal37 was sutured to the epicardial surface to measure
systolic WTh. All wires and the occluder tubing were tunneled
under the skin and exteriorized through small incisions in the back. A
bipolar lead was sutured to the chest wall to record the ECG. The
chest was closed in layers. Antibiotics were administered
intramuscularly before surgery and for 2 days thereafter (gentamicin,
0.7 mg/kg once a day). All rabbits were allowed to recover for a
minimum of 10 days after surgery.
Experimental Protocols
Throughout the experiments, rabbits were kept in a cage in a
quiet, dimly lit room. In the studies of myocardial infarction (phase
II), diazepam was administered 20 minutes before the onset of
ischemia (1 mg/kg IV) to relieve the stress caused by the
coronary occlusion. No sedatives were used in the studies of
myocardial stunning (phase I). Left ventricular WTh, range
gate depth, and the ECG were recorded throughout the experiments on
a thermal array chart recorder (Gould TA6000).
Phase I: Studies of Myocardial Stunning
The experimental protocol for phase I is depicted in Fig 1
. All rabbits were subjected to a sequence of six
4-minute coronary occlusion/4-minute reperfusion cycles. In
previous studies in conscious rabbits,38 we found that
this protocol produces severe stunning but does not cause infarction.
Systolic WTh was measured 3 minutes into each occlusion and
reperfusion period and 5, 15, 30, 60, 120, 180, 240, and 300 minutes
after the sixth reperfusion. Rabbits were assigned to six groups. Group
I (control group) received 0.5 mL of vehicle (DMSO) 10 minutes before
the first coronary occlusion via a marginal ear vein. In group
II, IB-MECA (Research Biochemicals Intl) was given as a bolus (100
µg/kg in 0.5 mL DMSO) 10 minutes before the first coronary
occlusion. Four additional groups of animals were studied to determine
whether the adenosine receptor blocker SPT (Research
Biochemicals Intl) or the PKC inhibitor chelerythrine
(Research Biochemicals Intl) affects the cardioprotective action of
IB-MECA against myocardial stunning. Groups III and IV were treated 15
minutes before the first occlusion with SPT (10 mg/kg IV bolus followed
immediately by an intravenous infusion of 1 mg/kg per
minute, which was continued until the end of the sixth occlusion; total
dose, 70 mg/kg) and then received either IB-MECA (100 µg/kg 10
minutes before the first occlusion, group III) or no treatment (group
IV). Groups V and VI were given chelerythrine (5 mg/kg IV bolus 15
minutes before the first occlusion) followed either by IB-MECA (100
µg/kg 10 minutes before the first occlusion, group V) or no treatment
(group VI). SPT was dissolved in normal saline (8 mg/mL), and
chelerythrine was dissolved in DMSO (10 mg/mL in 50% DMSO). SPT is an
adenosine receptor antagonist that blocks all of
the subtypes of adenosine receptors, including the rabbit
A3 receptor,7 and chelerythrine is a highly
selective PKC inhibitor.39
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Phase II: Studies of Myocardial Infarction
Rabbits were subjected to a 30-minute coronary artery
occlusion followed by 3 days of reperfusion. IB-MECA or vehicle was
given as an intravenous bolus (100 µg/kg) 10 minutes
before the occlusion.
Measurement of Regional Myocardial Function
In both phases I and II, regional myocardial function was
assessed as systolic thickening fraction using the pulsed
Doppler probe, as described previously.37 Percent
systolic thickening fraction was calculated as the ratio of net
systolic thickening to end-diastolic wall
thickness, multiplied by 100.37 The total deficit of WTh
after reperfusion (an integrative measure of the severity of
postischemic dysfunction) was calculated by measuring the
area between the systolic WTh-versus-time line and the baseline
(100% line) during the 5-hour recovery period after the sixth
reperfusion.40 41 In all animals, measurements were
averaged from at least 10 beats at baseline and from at least 5 beats
at all subsequent time points.
Measurement of Region at Risk and Infarct Size
At the conclusion of the study, the rabbits were given heparin
(1000 U IV), after which they were anesthetized with sodium
pentobarbital (50 mg/kg IV) and then euthanized with KCl. The hearts
were excised, and the size of the ischemic/reperfused region
(region at risk) was determined by tying the coronary artery at
the site of the previous occlusion and by perfusing the aortic root for
2 minutes with a 2.0% solution of Monastral blue dye in normal saline
at a pressure of 70 mm Hg using a Langendorff
apparatus. With this technique, the nonischemic
portion of the left ventricle is stained dark blue, whereas the region
at risk remains unstained. The heart was frozen at -20°C for 15
minutes and then cut into six or seven transverse slices, which were
incubated for 10 minutes at 37°C in a 1% solution of
triphenyltetrazolium chloride in phosphate
buffer (pH 7.4). All atrial and right ventricular tissues
were then excised. In phase I, the region at risk was separated from
the rest of the left ventricle, and both components were weighed. In
phase II, the slices were weighed, fixed in 10% formaldehyde solution,
and photographed with a digital camera. For each slice, the image was
magnified 10 times, and the areas of the infarcted,
ischemic/reperfused, and nonischemic regions were
measured using a software program (Sigmascan); from these measurements,
infarct size was calculated as a percentage of the region at
risk.42
Measurement of Plasma Histamine Levels
Six noninstrumented rabbits were anesthetized with
an intramuscular injection of ketamine (35 mg/kg) plus xylazine
(5 mg/kg). Catheters were placed in the central ear artery and in the
marginal ear vein for collection of blood and for administration of
drugs, respectively. After a stabilization period (15 minutes), the
rabbits were randomly allocated to two groups (n=3 per group): group 1
received 100 µg/kg of IB-MECA, whereas group 2 received 20 mg/kg of
dextran sulfate (average molecular weight, 500 000; 10 mg/mL in normal
saline; Pharmacia Biotech). Baseline blood samples were taken
immediately before drug administration. Immediately after drug
administration, blood was withdrawn into an EDTA-coated syringe for 10
minutes at a rate of 0.2 mL/min with a Harvard infusion/withdrawal
pump. The blood samples were centrifuged (1000g for
10 minutes at 4°C), and the plasma was stored at -20°C until it
was analyzed by radioimmunoassay (Immunotech).
For purposes of comparison, the effect of IB-MECA on plasma histamine levels was also determined in rats. Three Sprague-Dawley rats were anesthetized with sodium pentobarbital (60 mg/kg IP), and catheters were placed in the carotid artery and jugular vein. IB-MECA (100 µg/kg in 100 µL DMSO) was administered intravenously, and blood samples were collected over 10 minutes at a rate of 0.1 mL/min and analyzed for histamine as described above.
Statistical Analysis
All data are reported as mean±SEM. Hemodynamic
variables and thickening fraction were analyzed by a
two-way repeated measures ANOVA (time and drug treatment) to determine
whether there was a main effect of time, a main effect of treatment, or
a time-treatment interaction. If global tests showed a main effect or
interaction, post hoc contrasts between time points or treatments were
performed with Student's t tests for unpaired or paired
data, as appropriate, with the Bonferroni correction.43
The total deficits of wall thickening, infarct sizes, and risk region
sizes were compared with Student's t tests for unpaired
data. Changes in plasma histamine concentrations were analyzed
with Student's t tests for paired data.
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Results |
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Hemodynamic Effects of IB-MECA
Pilot studies were performed to determine the hemodynamic effects of IB-MECA. Catheters were placed in the central ear artery of 9 conscious rabbits, and blood pressure was recorded continuously with a pressure analyzer (Micro-Med BP-99). The effects of IB-MECA were compared with those of the A1-selective agonist, CCPA,44 and the A2A-selective agonist, CGS 21680.45 All three agonists were used at the same dose (100 µg/kg; molar amounts: IB-MECA, 196 nmol/kg; CCPA, 270 nmol/kg; and CGS 21680, 187 nmol/kg). IB-MECA was dissolved in 100 µL of DMSO; CCPA and CGS 21680 were dissolved in 100 µL of normal saline. As shown in Fig 2
, administration of CCPA decreased heart rate and blood
pressure, an effect that lasted for at least 1 hour. CGS 21680 produced
a short-lived decrease in blood pressure but increased heart rate, most
likely via a reflex mechanism. These effects of CCPA and CGS 21680 are
characteristic of A1 and A2A receptor
activation. In contrast, IB-MECA had no significant effect on heart
rate or blood pressure (Fig 2). We were concerned that higher doses of
IB-MECA might be necessary to activate A3 receptors
in rabbits; however, we found in 2 rabbits that even 300 µg/kg of
IB-MECA produced no hemodynamic effects (average heart
rate: baseline, 240 bpm; 15 and 30 minutes after IB-MECA, 236 and 239
bpm, respectively; mean blood pressure: baseline, 70 mm Hg; 15
and 30 minutes after IB-MECA, 67 and 68 mm Hg). These results
showing lack of hemodynamic effects confirm that at
doses of 100 and 300 µg/kg, IB-MECA does not interact with
A1 or A2A receptors in rabbits.
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Phase I: Studies of Myocardial Stunning
Of the 36 rabbits instrumented for phase I, 7 were assigned
to group I (control group), 6 to group II (IB-MECA), 5 to group III
(IB-MECA+SPT), 6 to group IV (SPT), 6 to group V
(IB-MECA+chelerythrine), and 6 to group VI (chelerythrine). One rabbit
each in groups IV and V died from ventricular fibrillation
during coronary occlusion; one rabbit in group VI was excluded
because of malfunction of the thickening probe. The remaining rabbits
(33 total) form the basis of this report. Postmortem tissue staining
with triphenyltetrazolium confirmed the
absence of infarction in all animals.
Heart rate did not differ appreciably among the six groups
(Table), except that it was slightly elevated at
baseline in group III (IB-MECA+SPT) and during the first occlusion in
group V (IB-MECA+chelerythrine). Furthermore, administration of the
various drugs, including IB-MECA, had no effect on systolic
thickening fraction (Figs 3, 4, and 5). These results are in agreement
with our pilot studies and confirm that IB-MECA has no
hemodynamic effects in rabbits.
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Figs 3
, 4, and 5 illustrate the serial measurements of thickening
fraction, expressed as percentages of baseline values, during the six
occlusion/reperfusion cycles and the subsequent recovery phase. In all
of the six groups, the extent of paradoxical systolic thinning
during ischemia did not change significantly with subsequent
occlusions, so that during the sixth occlusion it was similar to that
measured during the first occlusion.
In control rabbits (group I), the thickening fraction recovered to only
44.1±9.4% of baseline after the first coronary
occlusion/reperfusion cycle and did not exhibit further deterioration
with the subsequent five cycles (Fig 3). Five minutes
after the sixth reperfusion, the thickening fraction averaged
50.5±5.8% of baseline values. The thickening fraction remained
significantly depressed for 4 hours after the sixth reperfusion
(P<.01 versus baseline at all time points through 4 hours)
and returned to values not significantly different from baseline at 5
hours (Fig 3). Thus, the sequence of six 4-minute occlusions resulted
in severe myocardial stunning, which lasted, on average, 4 hours.
In group II, administration of IB-MECA markedly improved the
recovery of WTh (Fig 3). The measurements of thickening fraction in
IB-MECA–treated animals were significantly greater than those in
control animals throughout the first 4 hours of reperfusion
(79.7±4.2% of baseline versus 63.1±4.0% at 30 minutes
[P<.01]; 83.7±3.5% versus 66.8±3.9% at 1 hour [P<.01];
92.6±2.9% versus 66.1±4.7% at 2 hours [P<.01]; 99.6±0.3%
versus 80.5±3.0% at 3 hours [P<.01]; and 100.1±0.5% versus
84.9±3.3% at 4 hours [P<.01]). Whereas it took 5 hours for the
thickening fraction to return to 90% of baseline in control
rabbits, in the IB-MECA–treated group, the thickening fraction reached
100% of baseline after just 3 hours of reperfusion. The total deficit
of WTh after the sixth reperfusion (an integrative assessment of the
overall severity of myocardial dysfunction during the recovery
phase38 39 ) was 68% less in IB-MECA–treated rabbits
compared with control rabbits (P<.01 [Fig 3]). Thus,
administration of IB-MECA provided a powerful protection against
myocardial stunning, which became manifest early after reperfusion and
was sustained for the entire duration of the recovery phase.
Fig 4 illustrates systolic thickening
fraction in groups III and IV (groups I and II are also depicted for
comparison). In group III, rabbits were given SPT 15 minutes before the
occlusion/reperfusion cycles and IB-MECA 5 minutes after SPT (10
minutes before the first occlusion), whereas in group IV, rabbits were
given only SPT. In group III, the recovery of regional myocardial
function was similar to that observed in the control group (group I):
after the sequence of six coronary occlusions, thickening
fraction remained significantly depressed for 4 hours, averaging
59.4±11.5% of baseline at 1 hour, 65.0±11.8% at 2 hours,
83.0±3.0% at 3 hours, and 84.4±5.6% at 4 hours (P=NS
versus control group at all time points). As a result, the total
deficit of WTh after the sixth reperfusion was similar in groups I and
III (Fig 4). Thus, blockade of adenosine receptors with SPT
completely abrogated the cardioprotective effects of IB-MECA. In group
IV, SPT alone had no appreciable effect on the recovery of regional
myocardial function after the sequence of six occlusion/reperfusion
cycles (Fig 4), indicating that blockade of adenosine receptors
does not exacerbate myocardial stunning in this model.
Fig 5 illustrates systolic thickening
fraction in groups V and VI (groups I and II are also depicted for
comparison). In group V, chelerythrine was given 15 minutes before the
occlusion/reperfusion cycles, and IB-MECA was administered 5 minutes
after chelerythrine (10 minutes before the first occlusion), whereas in
group VI, rabbits were given only chelerythrine. In group V, the
recovery of WTh after the sixth reperfusion was similar to that
observed in group I (control group): systolic thickening
fraction was 64.9±6.4 at 1 hour, 69.9±5.7% at 2 hours, 76.7±5.7%
at 3 hours, and 77.9±3.7% at 4 hours (P=NS versus controls
at all time points). The total deficit of WTh was also
indistinguishable from that in the control group (Fig 5). Thus, the
protection against stunning afforded by IB-MECA was completely
abolished by the PKC inhibitor chelerythrine. In group VI,
chelerythrine alone had no appreciable effect on the severity of
myocardial stunning (Fig 5).
Phase II: Studies of Myocardial Infarction
Of the 23 rabbits instrumented for phase II, 5 died because of
ventricular fibrillation during the 30-minute
coronary occlusion (2 in the control group and 3 in the
IB-MECA–treated group). In addition, 2 rabbits (one in the control
group and one in the IB-MECA–treated group) were excluded because of
inadequate tissue staining during the postmortem perfusion. One treated
rabbit developed ventricular fibrillation after 5 minutes
of reperfusion but was resuscitated and included in data
analysis. Thus, 16 animals were used for data analysis:
8 in the control group and 8 in the IB-MECA–treated group.
Heart rates were similar in the two groups during the 30-minute
occlusion period and at 15 minutes after reperfusion (Table). There
were no significant differences between the two groups with respect to
left ventricular weight (control, 5.16±0.42 g; IB-MECA,
4.81±0.32 g) or weight of the risk region (control, 0.88±0.12 g
[17±2% of left ventricular weight]; IB-MECA, 0.92±0.11
g [19±1% of left ventricular weight]). However, infarct
size, expressed as a percentage of the region at risk, was smaller in
treated compared with control animals (16±6% versus 41±4%,
respectively; P<.01 [Fig 6]). Thus,
IB-MECA produced a marked (60%) reduction in myocardial infarct
size after 30 minutes of coronary occlusion and 3 days of
reperfusion.
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Effect of IB-MECA on Plasma Histamine Levels in Rabbits
To gain insights into the mechanism of action of IB-MECA and,
specifically, to examine the possibility that its protective effects
could be secondary to degranulation of resident cardiac mast cells, we
performed a series of studies in which the effect of IB-MECA on plasma
histamine levels was assessed in rabbits (n=3) and rats (n=3). The
results are shown in Figs 7 and 8.
Baseline levels taken before drug administration were 1203±461 nmol/L
(rabbits) and 143±9 nmol/L (rats). In rabbits, plasma histamine
concentrations were not altered by the administration of 100 µg/kg of
IB-MECA (972±565 nmol/L [Fig 7]). In contrast, in rats the same dose
of IB-MECA increased plasma histamine levels over 12-fold (to 1783±148
nmol/L [Fig 8]), similar to the data reported recently by Hannon et
al.46 As a positive control, injection of dextran sulfate
(20 mg/kg) produced a 10-fold increase in plasma levels in a separated
group of three rabbits (from 675±238 to 6800±757 nmol/L [Fig 7]).
Thus, the effect of A3 receptor activation on plasma
histamine levels differs between species.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The goal of the present study was to examine the potential cardioprotective effects of IB-MECA in two different models of myocardial ischemia/reperfusion injury in conscious rabbits. We found that administration of IB-MECA reduced both the severity of myocardial stunning after a sequence of six 4-minute coronary occlusion/4-minute reperfusion cycles (phase I) and the size of myocardial infarction after a 30-minute coronary occlusion and 3 days of reperfusion (phase II). The protective effects of IB-MECA were considerable, with a 68% decrease in the total deficit of WTh in phase I and a 61% reduction in infarct size in phase II. The protection against myocardial stunning was completely blocked by STP, demonstrating that the beneficial effects of IB-MECA are mediated by adenosine receptors. In contrast to CCPA and CGS 21680, IB-MECA had no effect on heart rate or blood pressure, demonstrating that the dose of IB-MECA used does not activate A1 or A2A receptors. The mitigation of myocardial stunning by IB-MECA was completely abolished by chelerythrine, indicating that IB-MECA exerts its salutary actions via a PKC-mediated signaling pathway. Because of the absence of hemodynamic effects and because rabbits lack a significant coronary collateral circulation,30 the beneficial effects of IB-MECA cannot be ascribed to favorable modifications of the myocardial oxygen supply-demand balance and therefore must reflect a direct cardioprotective action of this ligand. Taken together, the present results demonstrate that in the conscious rabbit, selective activation of A3 receptors protects the myocardium against both reversible and irreversible ischemia/reperfusion injury, acting via a direct cardioprotective mechanism that involves PKC. Previous work by Liu et al,27 Armstrong and Ganote,28 and Strickler et al29 suggested that activation of A3 receptors exerts beneficial effects on ischemic injury in isolated hearts and isolated myocytes. The present results expand these previous observations in several respects. To our knowledge, this is the first study to identify a cardioprotective role of A3 receptors during acute myocardial ischemia in vivo using a selective agonist for these receptors. This is also the first investigation to suggest that the cardioprotective actions of A3 receptors in vivo are mediated by PKC. Furthermore, this study demonstrates that A3 agonists mitigate not only myocardial infarction but also myocardial stunning, that they are protective in a conscious animal preparation, and that they ameliorate ischemic injury in rabbits without hemodynamic side effects.
One of the goals of the present investigation was to rigorously test the potential cardioprotective actions of IB-MECA under conditions that are as physiological as possible. Therefore, we sought to avoid the potentially confounding influence of anesthesia, surgical trauma, abnormal hemodynamics, elevated catecholamine levels, fluctuations in body temperature, exaggerated free radical formation, release of cytokines, and other factors associated with open-chest preparations.31 For example, previous studies have demonstrated significant differences between open-chest and conscious preparations with respect to the severity of myocardial stunning,32 magnitude of free radical generation,33 and duration of ischemic preconditioning.34 35 Furthermore, the size of a myocardial infarction is affected to a major extent by myocardial temperature,36 which is unstable in open-chest models.32 Accordingly, in the present study, all experiments were conducted in conscious rabbits.
The fact that in the present study IB-MECA did not produce any
measurable effects (ie, changes in heart rate, blood pressure, or
plasma histamine levels) could raise the concern that the protective
actions of this ligand resulted from some as-yet-unknown nonspecific
effect rather than from an interaction with adenosine
A3 receptors. Based on previous data demonstrating that
IB-MECA is a functional agonist of A3 receptors in vitro
and in vivo in rats and mice14 15 and based on previous in
vitro data supporting the concept that A3 adenosine
receptors are involved in preconditioning,27 28 29 this
possibility seems to be unlikely. Nevertheless, to positively exclude a
non–receptor-mediated mechanism of action for IB-MECA, we administered
this ligand together with the nonselective adenosine receptor
antagonist SPT. The finding that SPT completely abolished
the protective effects of IB-MECA against stunning (Fig 4) clearly
demonstrates that such effects are mediated by adenosine
receptors. Because of the lack of selectivity of SPT for any receptor
subtype, these results do not directly identify the A3
receptor as the effector. However, the complete absence of
hemodynamic effects of IB-MECA provides cogent evidence
that our dose of this ligand did not activate A1 or
A2 receptors and therefore indicates that the
cardioprotective effects of IB-MECA are mediated by A3
receptors.
In previous cloning studies, A3 receptor transcripts have been identified in heart tissue by Northern analysis and reverse transcription–polymerase chain reaction.2 4 6 However, since the specific cell types that express A3 receptors in the heart have not been fully elucidated, the exact mechanism by which IB-MECA exerted its protective effects in the present study remains to be determined. One possibility is that IB-MECA may have influenced resident cardiac mast cells.12 Ramkumar et al10 have recently shown that RBL-2H3 cells (a rat tumor mast cell line) express A3 receptors and that stimulation of these receptors causes the release of stored mediators. Duling's group47 48 found that the transient constriction induced by adenosine in arterioles isolated from the hamster cheek pouch is the result of mediators released from mast cells via the A3 receptor. Furthermore, it has been reported that in rats, plasma histamine levels increase after the administration of Cl-IB-MECA,49 an adenosine agonist with even greater A3 selectivity than IB-MECA,50 or APNEA, a potent agonist of A1 and A3 receptors.46 Since many mediators released by mast cells (such as histamine, leukotrienes, thromboxanes, platelet-activating factor, various cytokines, and proteases51 ) could be noxious during ischemia and/or could act to stress the myocardium,52 53 it could be hypothesized that the protection observed in the present study was secondary to depletion of mast cell mediators before ischemia, which would be beneficial by attenuating mast cell degranulation during ischemia and/or by inducing a preconditioned state. However, our data demonstrating the lack of any rise in plasma histamine levels after IB-MECA administration to rabbits strongly argue against this hypothesis.
Our data also demonstrate important species differences in the responsiveness to A3 receptor activation, namely, a marked release of histamine in the rat versus no release in the rabbit. These results are consonant with the recent findings that the secretion of inflammatory mediators from canine BR mastocytoma cells54 and human HMC-1 cells55 is stimulated by A2B receptors and not A3 receptors. Our histamine measurements also help to explain the lack of hemodynamic effects of IB-MECA in our study. In rats, activation of A3 receptors causes hypotension, which is blunted by the mast cell inhibitor sodium cromoglycate or by mast cell depletion with repeated treatment with compound 48/80.46 These results suggest that at least some of the hemodynamic effects observed in this species are secondary to mast cell degranulation, which may be the reason we did not observe hypotension in our study. Given the above species differences, characterization of the effects of A3 receptor activation on hemodynamics and histamine release in additional species, particularly in humans, is warranted.
In view of the lack of hemodynamic changes and
histamine release, the most plausible mechanism for the beneficial
effects observed with IB-MECA in the present study is activation of
A3 receptors in cardiac myocytes, resulting in a direct
cardioprotective action. In this regard, two recent studies have
demonstrated that A3 receptors are expressed in
cardiomyocytes and that these receptors are able to induce
preconditioning in isolated cell preparations.28 29 The
signal transduction mechanisms triggered by A3 receptors in
myocytes are unclear. In an effort to gain insight into the mechanism
of action of IB-MECA in our conscious rabbit model, we performed
additional experiments in which this agonist was given in conjunction
with chelerythrine, a highly selective inhibitor of
PKC.39 Our results demonstrate that the attenuation of
myocardial stunning by IB-MECA was completely lost in the presence of
chelerythrine, despite the fact that chelerythrine in itself had no
significant effect (Fig 5). These findings support the concept that
activation of A3 receptors alleviates ischemic
injury in vivo via a PKC-mediated pathway, a mechanism analogous to
that of A1 receptors.56 In support of this
proposal is the finding that in brain tissue11 and RBL 2H3
cells,9 the A3 receptor couples to
phospholipase C, suggesting that this subtype of adenosine
receptor is capable of activating PKC. Furthermore, Armstrong and
Ganote28 found that the preconditioning effect elicited by
activation of A3 receptors in isolated
cardiomyocytes was abolished by the PKC
inhibitor calphostin C.
Since in the present study IB-MECA was administered 10 minutes before ischemia, its protective effects may have been due to the induction of early preconditioning. Our results do not enable us to distinguish between a preconditioning effect of IB-MECA (which would require activation of A3 receptors before ischemia) and an anti-ischemic effect (which would require activation of A3 receptors only during ischemia), particularly since IB-MECA most likely has a long circulating half-life.15 Although it is well established that activation of adenosine receptors increases the tolerance of the myocardium to a subsequent ischemic insult (Downey's hypothesis of preconditioning56 ), the exact receptor subtypes involved have not been definitely ascertained. Early studies suggested that A1 receptors mediate preconditioning.57 58 Subsequent work involving isolated buffer-perfused rabbit hearts (Liu et al27 ) and isolated adult rabbit cardiomyocytes (Armstrong and Ganote28 ) led to the hypothesis that A3 receptors also mediate the response, based on three pieces of evidence: (1) pretreatment with the A1-selective antagonist CPX did not block ischemic preconditioning; (2) administration of APNEA (an A1/A3 agonist) induced preconditioning during A1 receptor blockade with CPX; and (3) 1,3-dipropyl-8-(4-acrylate)phenylxanthine (BWA-1433), a potent but nonselective antagonist for sheep A3 receptors,5 effectively blocked preconditioning induced by ischemia. More recently, Strickler et al29 have demonstrated in isolated neonatal chick myocytes that Cl-IB-MECA inhibits cAMP accumulation in response to isoproterenol during A1 receptor blockade with CPX (indicating the presence of functional A3 receptors in this myocyte preparation) and induces a CPX-insensitive preconditioning effect against hypoxia-induced cell death. These studies, taken together with a number of investigations demonstrating that activation of A1 receptors with selective agonists can induce preconditioning,28 29 57 58 support the hypothesis that both A3 and A1 receptors are involved in the preconditioning response. This concept is further supported by studies by Ganote's group demonstrating that the concentration-response curves for the cardioprotective effects of adenosine are biphasic, suggesting the involvement of two populations of adenosine receptors in preconditioning.59
In conclusion, although adenosine and adenosine receptor agonists are cardioprotective, a major limiting factor preventing their use in the clinical setting is the well-known potential of these agents to induce undesirable hemodynamic effects, such as sinus bradycardia, atrioventricular block, and hypotension, via A1 and A2 receptors. The present study demonstrates that in the conscious rabbit, activation of A3 adenosine receptors confers powerful protection against both mild reversible injury associated with brief ischemia (myocardial stunning) and severe irreversible injury associated with sustained ischemia (myocardial infarction) without causing any hemodynamic effects. The magnitude of these beneficial actions is comparable to that previously observed with A1 agonists.18 19 Accordingly, the present results suggest that therapeutic strategies targeting A3 receptors could be a novel and useful approach to the protection of the ischemic myocardium.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported in part by National Institutes of Health grants RO1 HL-43151 and HL-55757 (Dr Bolli); by American Heart Association, National Center, grant 9630083N (Dr Auchampach); and by American Heart Association, Kentucky Affiliate, Inc, grants KY-96-GB-31 (Dr Tang) and KY-96-GB-32 (Dr Qiu). This research was made possible through a grant of the Jewish Hospital Foundation, Louisville, KY (960915-02, Auchampach). We gratefully acknowledge Christiane A. Trauss, Wen-Jian Wu, and Ai-Ru Yang for their expert technical assistance.
Received November 12, 1996; accepted March 27, 1997.
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References |
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R. J. Cerniway, Z. Yang, M. A. Jacobson, J. Linden, and G. P. Matherne Targeted deletion of A3 adenosine receptors improves tolerance to ischemia-reperfusion injury in mouse myocardium Am J Physiol Heart Circ Physiol, October 1, 2001; 281(4): H1751 - H1758. [Abstract] [Full Text] [PDF]
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K. Mubagwa and W. Flameng Adenosine, adenosine receptors and myocardial protection: An updated overview Cardiovasc Res, October 1, 2001; 52(1): 25 - 39. [Abstract] [Full Text] [PDF]
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 J. E. LEE, G. BOKOCH, and B. T. LIANG A novel cardioprotective role of RhoA: new signaling mechanism for adenosine FASEB J, September 1, 2001; 15(11): 1886 - 1894. [Abstract] [Full Text] [PDF]
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D. A Liem, M. A van den Doel, S. de Zeeuw, P. D Verdouw, and D. J Duncker Role of adenosine in ischemic preconditioning in rats depends critically on the duration of the stimulus and involves both A1 and A3 receptors Cardiovasc Res, September 1, 2001; 51(4): 701 - 708. [Abstract] [Full Text] [PDF]
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E. L. Kilpatrick, P. Narayan, R. M. Mentzer Jr., and R. D. Lasley Adenosine A3 agonist cardioprotection in isolated rat and rabbit hearts is blocked by the A1 antagonist DPCPX Am J Physiol Heart Circ Physiol, August 1, 2001; 281(2): H847 - H853. [Abstract] [Full Text] [PDF]
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E. Kodani, K. Shinmura, Y.-T. Xuan, H. Takano, J. A. Auchampach, X.-L. Tang, and R. Bolli Cyclooxygenase-2 does not mediate late preconditioning induced by activation of adenosine A1 or A3 receptors Am J Physiol Heart Circ Physiol, August 1, 2001; 281(2): H959 - H968. [Abstract] [Full Text] [PDF]
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R. D. Lasley, M. S. Jahania, and R. M. Mentzer Jr. Beneficial effects of adenosine A2a agonist CGS-21680 in infarcted and stunned porcine myocardium Am J Physiol Heart Circ Physiol, April 1, 2001; 280(4): H1660 - H1666. [Abstract] [Full Text] [PDF]
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R. Zucchi, G. Yu, S. Ghelardoni, F. Ronca, and S. Ronca-Testoni A3 adenosine receptor stimulation modulates sarcoplasmic reticulum Ca2+ release in rat heart Cardiovasc Res, April 1, 2001; 50(1): 56 - 64. [Abstract] [Full Text] [PDF]
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H. Takano, R. Bolli, R. G. Black Jr, E. Kodani, X.-L. Tang, Z. Yang, S. Bhattacharya, and J. A. Auchampach A1 or A3 Adenosine Receptors Induce Late Preconditioning Against Infarction in Conscious Rabbits by Different Mechanisms Circ. Res., March 16, 2001; 88(5): 520 - 528. [Abstract] [Full Text] [PDF]
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J. D. McCully, Y. Toyoda, M. Uematsu, R. D. Stewart, and S. Levitsky Adenosine-enhanced ischemic preconditioning: adenosine receptor involvement during ischemia and reperfusion Am J Physiol Heart Circ Physiol, February 1, 2001; 280(2): H591 - H602. [Abstract] [Full Text] [PDF]
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 D. Belhomme, J. Peynet, E. Florens, O. Tibourtine, M. Kitakaze, and P. Menasche Is adenosine preconditioning truly cardioprotective in coronary artery bypass surgery? Ann. Thorac. Surg., August 1, 2000; 70(2): 590 - 594. [Abstract] [Full Text] [PDF]
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M. PARSONS, L. YOUNG, J. E. LEE, K. A. JACOBSON, and B. T. LIANG Distinct cardioprotective effects of adenosine mediated by differential coupling of receptor subtypes to phospholipases C and D FASEB J, July 1, 2000; 14(10): 1423 - 1431. [Abstract] [Full Text]
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Z. Yang, B. Zingarelli, and C. Szabo Crucial Role of Endogenous Interleukin-10 Production in Myocardial Ischemia/Reperfusion Injury Circulation, March 7, 2000; 101(9): 1019 - 1026. [Abstract] [Full Text] [PDF]
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C. E. Ganote and S. C. Armstrong Adenosine and preconditioning in the rat heart Cardiovasc Res, January 1, 2000; 45(1): 134 - 140. [Full Text] [PDF]
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J. E. Jordan, V. H. Thourani, J. A. Auchampach, J. A. Robinson, N.-P. Wang, and J. Vinten-Johansen A3 adenosine receptor activation attenuates neutrophil function and neutrophil-mediated reperfusion injury Am J Physiol Heart Circ Physiol, November 1, 1999; 277(5): H1895 - H1905. [Abstract] [Full Text] [PDF]
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V. H. Thourani, M. Nakamura, R. S. Ronson, J. E. Jordan, Z.-Q. Zhao, J. H. Levy, F. Szlam, R. A. Guyton, and J. Vinten-Johansen Adenosine A3-receptor stimulation attenuates postischemic dysfunction through KATP channels Am J Physiol Heart Circ Physiol, July 1, 1999; 277(1): H228 - H235. [Abstract] [Full Text] [PDF]
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V. H. Thourani, R. S. Ronson, J. E. Jordan, R. A. Guyton, and J. Vinten-Johansen Adenosine A3 pretreatment before cardioplegic arrest attenuates postischemic cardiac dysfunction Ann. Thorac. Surg., June 1, 1999; 67(6): 1732 - 1737. [Abstract] [Full Text] [PDF]
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R. D. Lasley, P. Narayan, M. S. Jahania, E. L. Partin, K. R. Kraft, and R. M. Mentzer Jr. Species-dependent hemodynamic effects of adenosine A3-receptor agonists IB-MECA and Cl-IB-MECA Am J Physiol Heart Circ Physiol, June 1, 1999; 276(6): H2076 - H2084. [Abstract] [Full Text] [PDF]
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P. Ping, J. Zhang, X. Cao, R. C. X. Li, D. Kong, X.-L. Tang, Y. Qiu, S. Manchikalapudi, J. A. Auchampach, R. G. Black, et al. PKC-dependent activation of p44/p42 MAPKs during myocardial ischemia-reperfusion in conscious rabbits Am J Physiol Heart Circ Physiol, May 1, 1999; 276(5): H1468 - H1481. [Abstract] [Full Text] [PDF]
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A. M. Feldman, D. R. Wagner, and D. M. McNamara AMPD1 Gene Mutation in Congestive Heart Failure : New Insights Into the Pathobiology of Disease Progression Circulation, March 23, 1999; 99(11): 1397 - 1399. [Full Text] [PDF]
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P. Ping, H. Takano, J. Zhang, X.-L. Tang, Y. Qiu, R. C. X. Li, S. Banerjee, B. Dawn, Z. Balafonova, and R. Bolli Isoform-Selective Activation of Protein Kinase C by Nitric Oxide in the Heart of Conscious Rabbits : A Signaling Mechanism for Both Nitric Oxide–Induced and Ischemia-Induced Preconditioning Circ. Res., March 19, 1999; 84(5): 587 - 604. [Abstract] [Full Text] [PDF]
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J. A. Auchampach and R. Bolli Adenosine receptor subtypes in the heart: therapeutic opportunities and challenges Am J Physiol Heart Circ Physiol, March 1, 1999; 276(3): H1113 - H1116. [Full Text] [PDF]
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Y. Guo, W.-J. Wu, Y. Qiu, X.-L. Tang, Z. Yang, and R. Bolli Demonstration of an early and a late phase of ischemic preconditioning in mice Am J Physiol Heart Circ Physiol, October 1, 1998; 275(4): H1375 - H1387. [Abstract] [Full Text] [PDF]
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W.R. Tracey, W. Magee, H. Masamune, J. J. Oleynek, and R. J. Hill Selective activation of adenosine A3 receptors with N6-(3-chlorobenzyl)-5'-N-methylcarboxamidoadenosine (CB-MECA) provides cardioprotection via KATP channel activation Cardiovasc Res, October 1, 1998; 40(1): 138 - 145. [Abstract] [Full Text] [PDF]
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 V. Ralevic and G. Burnstock Receptors for Purines and Pyrimidines Pharmacol. Rev., September 1, 1998; 50(3): 413 - 492. [Abstract] [Full Text] [PDF]
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R. D. Lasley and R. M. Mentzer Jr. Dose-Dependent Effects of Adenosine on Interstitial Fluid Adenosine and Postischemic Function in the Isolated Rat Heart J. Pharmacol. Exp. Ther., August 1, 1998; 286(2): 806 - 811. [Abstract] [Full Text]
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J. A. Ismail and K. H. McDonough The role of coronary flow and adenosine in postischemic recovery of septic rat hearts Am J Physiol Heart Circ Physiol, July 1, 1998; 275(1): H8 - H14. [Abstract] [Full Text] [PDF]
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 B. T. Liang and K. A. Jacobson A physiological role of the adenosine A3 receptor: Sustained cardioprotection PNAS, June 9, 1998; 95(12): 6995 - 6999. [Abstract] [Full Text] [PDF]
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P. Ping, J. Zhang, Y. Qiu, X.-L. Tang, S. Manchikalapudi, X. Cao, and R. Bolli Ischemic Preconditioning Induces Selective Translocation of Protein Kinase C Isoforms {epsilon} and {eta} in the Heart of Conscious Rabbits Without Subcellular Redistribution of Total Protein Kinase C Activity Circ. Res., September 19, 1997; 81(3): 404 - 414. [Abstract] [Full Text]
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 K. A. Jacobson, R. Xie, L. Young, L. Chang, and B. T. Liang A Novel Pharmacological Approach to Treating Cardiac Ischemia. BINARY CONJUGATES OF A1 AND A3 ADENOSINE RECEPTOR AGONISTS J. Biol. Chem., September 22, 2000; 275(39): 30272 - 30279. [Abstract] [Full Text] [PDF]
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 M. Taira, J. Tamaoki, K. Nishimura, J. Nakata, M. Kondo, H. Takemura, and A. Nagai Adenosine A3 receptor-mediated potentiation of mucociliary transport and epithelial ciliary motility Am J Physiol Lung Cell Mol Physiol, March 1, 2002; 282(3): L556 - L562. [Abstract] [Full Text] [PDF]
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