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1 Division of Cardiothoracic
Surgery, We tested the hypothesis that selective
adenosine A3-receptor stimulation
reduces postischemic contractile dysfunction through activation of
ATP-sensitive potassium (KATP)
channels. Isolated, buffer-perfused rat hearts
(n = 8/group) were not drug pretreated (control) or were pretreated with adenosine (20 µM),
2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide
(Cl-IB-MECA; A3 agonist, 100 nM),
Cl-IB-MECA + 8-(3-noradamantyl)-1,3-dipropylxanthine (KW-3902;
A1 antagonist, 5 µM), Cl-IB-MECA + glibenclamide (Glib; KATP-channel blocker, 0.3 µM),
or Glib alone for 12 min before 30 min of global normothermic
ischemia followed by 2 h of reperfusion. After 2 h of
reperfusion, left ventricular developed pressure (LVDP, %baseline) in
control hearts was depressed to 34 ± 2%. In hearts pretreated with
Cl-IB-MECA, there was a statistically significant increase in LVDP (50 ± 6%), which was reversed with coadministration of Glib (37 ± 1%). Control hearts also showed similar decreases in left ventricular
peak positive rate of change in pressure
(dP/dt). Therefore, the
A3 agonist significantly
attenuated postischemic cardiodynamic injury compared with the control,
which was reversed by Glib. Cumulative creatine kinase (CK in U/min) activity was most pronounced in the control group (10.4 ± 0.6) and
was significantly decreased by Cl-IB-MECA (7.5 ± 0.4), which was
reversed by coadministration of Glib (9.4 ± 0.2). Coronary flow was
increased during adenosine infusion (160% of baseline) but not during
Cl-IB-MECA infusion. Effects of Cl-IB-MECA were not reversed by the
specific A1 antagonist KW-3902. We
conclude that cardioprotection afforded by
A3-receptor stimulation may be
mediated in part by KATP channels.
Cl-IB-MECA may be an effective pretreatment agent that attenuates
postischemic cardiodynamic dysfunction and CK release without the
vasodilator liability of other adenosine agonists.
myocardial protection; purinergic receptors; rat; adenosine
5'-triphosphate-sensitive potassium channels
PROTECTIVE STRATEGIES to reduce myocardial
ischemia-reperfusion injury can potentially lead to better
recovery of the postischemic heart, decrease postoperative
complications, and lower patient mortality and morbidity. Postischemic
myocardial injury is mediated during both the ischemic interval and the
myocardial reperfusion and is mediated by ionic dyshomeostasis, edema
formation, neutrophils, and oxygen-free radical production. Adenosine,
a purine nucleoside, has been shown to delay the onset of ischemic
contracture (18), reduce the rates of ATP catabolism and intracellular
H+ and
Ca2+ accumulation during
ischemia (17, 18), attenuate myocardial stunning (17, 25, 40),
and reduce infarct size (31). Cardioprotective effects of adenosine are
exerted when it is administered as a pretreatment either before
ischemia (30, 31) or before reperfusion (23, 31). The
cardioprotective effects of pretreatment with adenosine administered
before ischemia may be mediated by adenosine A1-receptor activation (17, 18,
31), whereas protection during reperfusion may be mediated by adenosine
A2-receptor activation (23, 31).
Although adenosine is beneficial to myocardial preservation, its
untoward effects produced by A1-
(bradycardia) and A2a-mediated (vasodilation, hypotension) actions have impeded the routine clinical use of these agents. A recently described adenosine-receptor subtype, designated adenosine A3 (20, 43),
could potentially provide myocardial protection without systemic
untoward effects. The adenosine A3
receptor shares sequence similarity yet has specific pharmacological characteristics not typical of adenosine
A1 or
A2 receptors.
Activation of the adenosine A3
receptor has been proposed to enhance the release of inflammatory
mediators from mast cells (7, 10, 35), lower blood pressure (4, 6),
depress locomotor activity (13), either induce (16) or reduce apoptosis (38), and protect against cerebral ischemia (36). To date, only
a few studies have described the cardioprotective effects of adenosine
A3-receptor activation in models
of myocardial ischemia-reperfusion (3, 29), as well as its
ability to mimic or induce myocardial preconditioning (1, 19, 34, 37).
It is plausible that the cardioprotective effects afforded by the
activation of the adenosine A3
receptor may be mediated via activation of ATP-sensitive potassium
(KATP) channels, because the
adenosine A3 receptor is similar
to the adenosine A1 receptor in
receptor transduction through an inhibition in adenylate cyclase (43)
and stimulation in protein kinase C (1). The potential involvement of
KATP channels by the activation of
the adenosine A3 receptors remains undefined.
Therefore, we tested the hypothesis that stimulation of the adenosine
A3 receptor with the highly
selective A3 agonist
2-chloro-N 6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide
(Cl-IB-MECA) would reduce postischemic dysfunction and
morphological injury in a neutrophil-free isolated perfused rat heart
model via activation of KATP channels.
Experimental preparation. The animals
in the investigation were handled in full accordance with the
Guide for the Care and Use of Laboratory
Animals published by the National Research Council in
1996, and the protocol was approved by the Institutional Animal Care
and Use Committee of Emory University. Sprague-Dawley rats weighing
~200-250 g were anesthetized with pentobarbital sodium (30 mg/kg) intraperitoneally. After adequate anesthesia was given, each rat
was heparinized intravenously with 1,000 U porcine sodium heparin.
After 3 min, the chest was opened rapidly, and the heart was excised.
Immediately after excision, the aorta was mounted on a Langendorff
perfusion apparatus (Radnoti Glass Technology, Monrovia, CA), and
retrograde aortic perfusion was initiated in a nonrecirculating fashion
with modified Krebs-Henseleit buffer (KHB) containing (in mM) 4.7 KCl,
1.2 KH2PO4,
1.2 MgSO4, 118 NaCl, 2.04 CaCl2, 0.05 EDTA tetrasodium salt,
25 NaHCO3, and 11 dextrose. The
perfusate was pH balanced to 7.4 after full aeration and was filtered
through a 5-µm magna nylon membrane (Fisher Scientific, Pittsburgh,
PA). The perfusate temperature was maintained at 37°C with a
circulating water-bath heater (Precision Scientific, Chicago, IL) and
was continuously oxygenated with 95%
O2-5%
CO2. The heart was suspended in a
water-jacketed chamber to maintain the temperature at 37°C.
All hearts underwent a 25-min stabilization period. During this period,
the left atrium was excised and a latex fluid-filled balloon on the end
of a PE-100 catheter was inserted into the left ventricle across the
mitral annulus. The balloon was inflated to achieve an end-diastolic
pressure between 7 and 10 mmHg, after which no further alterations in
balloon volume were made. During the stabilization period, two
electrodes were placed in the right ventricle, the sinoatrial node was
crushed, and the heart was paced at 250 beats/min with a Grass S9
stimulator (Grass Medical Instruments, Kings Park, NY).
To measure coronary perfusion pressure, a solid-state blood pressure
transducer (Kent Scientific, Litchfield, CT) was connected to a
vertical column of perfusate, mounted in line with and proximal to the
aortic cannula. The pressure transducer was positioned at the level of
the heart, and coronary perfusion pressure was maintained at >70
mmHg. Coronary flow was continuously measured with an in-line
ultrasonic flow probe and T101 ultrasonic blood flow meter (Transonic
Systems, Ithaca, NY).
The left ventricular balloon pressure and the aortic perfusion pressure
were monitored with fluid-filled pressure transducers (TRN050 blood
pressure transducer) and connected to a TRN005 base instrument
amplifier (Kent Scientific, Litchfield, CT). The pressure and flow
signals were sampled by a personal computer with an analog-to-digital converter (Data Translation, Marlboro, MA). The data were captured, stored, and analyzed with SPECTRUM cardiovascular acquisition and
analysis software (Wake Forest University, Winston-Salem, NC).
Experimental design. Animals were
randomized to 6 groups (n = 8 in each
group) according to pretreatment drug administration: 1) KHB (control),
2) adenosine (20 µM),
3) Cl-IB-MECA (100 nM), 4) Cl-IB-MECA + 8-(3-noradamantyl)-1,3-dipropylxanthine (KW-3902; a selective
A1 antagonist, 5 µM),
5) Cl-IB-MECA + glibenclamide (a
KATP-channel blocker, 0.3 µM),
and 6) glibenclamide alone (0.3 µM). Hearts received treatment with adenosine or
Cl-IB-MECA for 12 min without a washout period before the onset of
ischemia via a syringe infusion pump 22 (Harvard Apparatus,
South Natick, MA) in a side port located just proximal to the aortic
cannulation site. In groups receiving the
A1 antagonist KW-3902 and the
KATP-channel blocker
glibenclamide, the antagonists were administered 2 min before the
infusion of Cl-IB-MECA, after which both Cl-IB-MECA and the respective
antagonist were infused for the 12 min of drug treatment. This assured
that the antagonist was present at the time of
A3-receptor agonist infusion.
After stabilization and drug administration, all hearts underwent 30 min of normothermic (37°C) global ischemia by the ceasing
of all buffer perfusion to the heart. Global myocardial
ischemia was initiated immediately after drug administration
without a "washout" interval. Pacing was stopped at the onset of
ischemia and was resumed 5 min after initiation of reperfusion.
Static intraventricular balloon pressure was monitored via the left
ventricular balloon pressure transducer throughout the ischemic
interval to assess contracture. Reperfusion was initiated by the
resumption of buffer perfusion to the heart. Hemodynamic and
cardiodynamic measurements were obtained before and after drug
administration and after 15, 30, 60, 90, and 120 min of reperfusion.
Coronary effluent was collected before and after drug administration
and at 15, 30, 60, 90, and 120 min of reperfusion. Samples were stored
in a CK determination. CK (Sigma
Diagnostics, St. Louis, MO) activity was calculated
spectrophotometrically from the effluent collected at the
aforementioned time points. Briefly, effluent samples were mixed in a
cuvette with 1 ml of freshly prepared reagent. The solutions were
maintained at a constant temperature (37°C) and incubated for 3 min. The Statistical analysis. The data were
analyzed by a repeated-measures two-way ANOVA for group and time
(within group) differences. Tukey's or Student-Newman-Keuls methods
were used to determine pairwise differences between groups. A
P value <0.05 was considered statistically significant. Data are expressed as means ± SE.
Materials. Adenosine (Calbiochem, La
Jolla, CA) was dissolved with KHB to obtain a final perfusate
concentration of 20 µM. The selective
A3 agonist Cl-IB-MECA was
reconstituted in DMSO to a concentration of 10 mM and diluted to
achieve a final heart concentration of 100 nM. The final heart
perfusate concentration of DMSO was 0.01 nM. The
A1-selective adenosine antagonist
KW-3902 (Kyowa Hakko Kogyo) was dissolved in 100 µl ethyl alcohol and 50 µl of 1.0 N NaOH first and then diluted with 0.9% saline to achieve a final heart concentration of 5 µM. This concentration for
KW-3902 has previously been shown to be a specific adenosine A1-receptor antagonist (41). The
specific KATP-channel antagonist glibenclamide (Sigma, St. Louis, MO) was dissolved in 0.5 ml of ethanol, 0.5 ml of 1 N NaOH, and 0.5 ml of polyethylene glycol 400 to
achieve a final heart concentration of 0.3 µM. This concentration has
previously been shown to be specific for
KATP-channel blockade (9).
Left ventricular developed pressure.
The contractile state of the isolated rat heart was assessed by
measuring the left ventricular developed pressure (LVDP), as measured
by a fixed-volume balloon located within the left ventricle. The LVDP
was defined as the peak systolic pressure minus the peak end-diastolic
pressure. All baseline LVDPs were comparable among groups before and
after drug administration (Fig. 1,
A and
B). After 2 h of reperfusion, control hearts exhibited a 34 ± 2% recovery in LVDP from baseline values. In contrast, LVDP was significantly greater after pretreatment with adenosine and Cl-IB-MECA (54 ± 3 and 50 ± 6% recovery
from baseline, respectively; Fig.
1A). To assess if the preservation of LVDP after pretreatment with Cl-IB-MECA was attributable to activation of adenosine A1
receptors, the A1 antagonist
KW-3902 was coadministered with Cl-IB-MECA. Rat hearts pretreated with Cl-IB-MECA + KW-3902 had a 55 ± 2% recovery from baseline LVDP values after 2 h of reperfusion (Fig.
1B), which was not statistically different from treatment with Cl-IB-MECA alone. To determine whether recovery of function with Cl-IB-MECA involved activation of
KATP channels, the
KATP-channel blocker glibenclamide
was coadministered with Cl-IB-MECA. The protective effect of Cl-IB-MECA
on LVDP after 2 h of reperfusion was completely reversed by coinfusion
of glibenclamide (37 ± 1% recovery from baseline, Fig.
1B) to values not different from
those for control and glibenclamide alone. The administration of
glibenclamide alone did not significantly alter LVDP compared with
control (36 ± 4%, Fig. 1B).
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
70°C freezer until used for creatine kinase (CK) analysis.
-absorbances at 0- and 30-s intervals for a period of 120 s
were used to calculate CK activity (expressed as U/l). In accordance
with the coronary flow at each specific time point of collection, CK
activity per minute of flow was determined. The sum of the individual
CK values per minute at each time point is represented as the
cumulative CK activity.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
A and
B: left ventricular developed pressure
(in mmHg) as determined from a fixed-volume fluid-filled balloon within
left ventricle. Values are means ± SE. Drug
concentrations include adenosine (20 µM),
2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide
(Cl-IB-MECA; 100 nM), 8-(3-noradamantyl)-1,3-dipropylxanthine (KW-3902;
5 µM), and glibenclamide (Glib; 0.3 µM).
* P < 0.05, control vs. all
other groups; + P < 0.05, Cl-IB-MECA vs. control and Glib alone;
@ P < 0.05, Cl-IB-MECA vs.
all other groups. r, Reperfusion; m, minutes.
Peak positive rate of change in pressure. The
changes in developed pressure over time were accompanied by similar
changes in peak positive rate of change in pressure
(dP/dtmax; Fig.
2, A and
B). All baseline
dP/dtmax were
comparable among groups before and after drug administration. After 2 h
of reperfusion, control hearts recovered to only 34 ± 2% of
baseline dP/dtmax
values. In hearts pretreated with adenosine and Cl-IB-MECA,
dP/dtmax recovery compared with baseline values was significantly greater (56 ± 3 and
52 ± 6% recovery, respectively) than that in control hearts (Fig.
2A). The cardioprotection associated
with administration of Cl-IB-MECA was not reversed with the addition of
the A1 antagonist KW-3902 (56 ± 2% recovery after 2 h of reperfusion, Fig.
2A). In contrast, the
coadministration of the KATP
antagonist glibenclamide with Cl-IB-MECA reduced recovery of
dP/dtmax at the
end of reperfusion to 37 ± 2% of baseline value (Fig.
2B), which was comparable with that
for control hearts. The administration of glibenclamide alone did not
significantly alter
dP/dtmax compared
with control (36 ± 4%, Fig.
2B). With glibenclamide
alone, dP/dtmax
after 2 h of reperfusion was not significantly different from control
(34 ± 2%).
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Left ventricular end diastolic
pressure. The diastolic state of the isolated rat heart
was assessed by measuring the left ventricular end diastolic pressure
(LVEDP) in a fixed-volume balloon located within the left ventricle
(Fig. 3, A
and B). Baseline LVEDPs were
comparable among groups before and after drug administration. After 2 h
of reperfusion, LVEDP in control hearts increased by 687 ± 30%
from baseline values. Increases in LVEDP after 2 h of reperfusion were
significantly less in hearts pretreated with adenosine and Cl-IB-MECA
(347 ± 49 and 359 ± 47% increase, respectively) compared with
control hearts (Fig. 3A). The lower
LVEDP with Cl-IB-MECA was not reversed by the
A1 antagonist KW-3902 (384 ± 33% increase from baseline, Fig.
3B). However, the coadministration
of glibenclamide reversed the decrease in LVEDP seen with Cl-IB-MECA
alone to 585 ± 26% of baseline value (Fig.
3B). The administration of
glibenclamide alone resulted in no change in LVEDP (668 ± 32%,
Fig. 3B) compared with control.
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Coronary perfusate flow. Coronary
perfusate flow as measured by an in-line flow probe is summarized in
Table 1. All baseline coronary flow
measurements (before drug infusion) were comparable among groups.
Administration of 20 µM of adenosine significantly increased flow to
160% of its predrug value. However, pretreatment with Cl-IB-MECA (100 nM) did not produce significant coronary vasodilation (9% increase in
flow from baseline values). When a greater concentration of Cl-IB-MECA
(1 µM) was administered, there was a significant increase (142%) in
coronary flow (17 ± 1.2 ml/min) compared with its predrug baseline
value (12 ± 0.4 ml/min). After 2 h of reperfusion, coronary
perfusate flow (%baseline) was depressed in control hearts to 28 ± 3% of baseline. At the end of reperfusion, coronary
perfusate flow as compared with control was increased after
pretreatment with Cl-IB-MECA (41 ± 3%), but only in the
adenosine-treated group (47 ± 3%) was the flow significantly greater than the control group at the end of reperfusion.
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Cumulative effluent CK. CK activity
was calculated from coronary effluent, which was collected at the
aforementioned time points (Fig. 4). The
sum of the individual CK values per minute at each time point is
represented as the cumulative CK. In control hearts, cumulative CK
concentration (U/min) averaged 10.4 ± 0.6. Adenosine pretreatment
decreased cumulative effluent CK activity by 26% relative to the
control group (Fig. 4). Cl-IB-MECA was also associated with a similar
decrease in CK activity (28%) relative to control. The coinfusion of
KW-3902 did not alter the effects of Cl-IB-MECA alone compared with
control hearts. However, when glibenclamide was coadministered with
Cl-IB-MECA, CK values were significantly greater than those from
Cl-IB-MECA administration alone and were not significantly different
from values in control hearts (Fig. 4). Therefore, the coadministration
of glibenclamide reversed the effect of Cl-IB-MECA on CK release. There
was no significant reduction in cumulative CK values in hearts treated with glibenclamide alone.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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In this study, we examined the cardiodynamic effects of a selective adenosine A3 agonist, Cl-IB-MECA, in cell-free perfused rat hearts. Our model is devoid of cellular and other humoral components (e.g., neutrophils, complement, histamine), and our study specifically tests the neutrophil-independent cardioprotection afforded by Cl-IB-MECA. We found that pretreatment with a highly selective A3 agonist attenuated postischemic systolic and diastolic dysfunction and CK release. Pretreatment with Cl-IB-MECA for 12 min before 30 min of normothermic global ischemia without an intervening washout period resulted in a significant improvement in recovery of LVDP and dP/dtmax compared with control hearts after 2 h of reperfusion. In addition, LVEDP and effluent cumulative CK activity over the course of ischemia and reperfusion were significantly reduced in Cl-IB-MECA-treated hearts compared with control hearts. We have also shown that the cardioprotection afforded by pretreatment with A3 agonists were blocked by the KATP-channel antagonist glibenclamide, suggesting involvement of KATP-channel activation. Incomplete reversal of reductions in CK release may suggest that mechanisms other than KATP-channel activation may be the primary mechanism involved in protection against reversible injury. Furthermore, these cardioprotective effects of Cl-IB-MECA did not result in coronary vasodilation. Our study suggests that selective stimulation of the adenosine A3 receptor attenuates postischemic dysfunction after myocardial ischemia involving the activation of KATP channels.
The physiological role of A3 receptors may be very different from A1 and A2a subtypes (12). Activation of A3 receptors by native adenosine requires relatively high concentrations of the purine; the inhibitory constant (Ki) value of adenosine at the rat A3 receptor is ~1 µM vs. 10-30 nM at rat A1 and A2a receptors, respectively (12). At first, the nonselective adenosine-receptor agonist N6-2-(4-aminophenyl)ethyladenosine (APNEA) had been used to activate A3 receptors, in combination with a xanthine such as 8-(p-sulfophenyl)theophylline to eliminate non-A3 receptor-mediated effects. Consequently, more specific A3 agonists like IB-MECA have been developed. IB-MECA has an affinity of 1.1 nM and is ~50-fold selective over adenosine A1 and A2a receptors in vitro (8). The chlorinated derivative, Cl-IB-MECA, displays a Ki value of 0.33 nM and is highly selective for A3 vs. A1 and A2a receptors by 2,500- and 1400-fold, respectively (12).
In the present study, Cl-IB-MECA selectivity was confirmed in its cardioprotective effect by the coadministration of a selective adenosine A1 antagonist, KW-3902, to rule out spillover into the A1 receptor. Furthermore, KW-3902 did not reverse the decreased release of CK after pretreatment with Cl-IB-MECA. Thus the cardioprotective effects of Cl-IB-MECA did not appear to involve A1-mediated effects resulting from a spillover effect mediated by the adenosine A1 receptor. This selectivity data are consistent with reports by Nonaka et al. (22), Nomura et al. (21), and Zhao et al. (41) showing selectivity of KW-3902 for adenosine A1 receptor. Coronary vasodilation was apparent when a higher concentration (1 µM) of Cl-IB-MECA was administered. However, because we were interested in subvasodilator effects of Cl-IB-MECA, we did not show these functional data or determine its involvement of adenosine A2-receptor activation.
The role of activation of the specific adenosine A3 receptor in mediating cardioprotection during ischemia per se (as opposed to effects during reperfusion) has not been clearly defined. Stambaugh et al (29), with chick embyro ventricular myocytes in ovo, have suggested that activation of adenosine A3 receptors with IB-MECA and Cl-IB-MECA during hypoxia can attenuate myocyte injury. In agreement, the present study found that stimulation of the adenosine A3 receptor with Cl-IB-MECA in the cell-free isolated rat heart model preserved cardiodynamics and reduced CK enzyme release. Although Cl-IB-MECA has been observed to inhibit selected neutrophil functions relevant to neutrophil-mediated postischemic injury (14), the cardioprotection observed in the present study was independent of any putative antineutrophil effects.
Studies evaluating the mechanisms or affectors leading to A3-mediated cardioprotection remain scarce. However, several studies have investigated the involvement of the A3 receptor with the cardioprotection mimicking preconditioning (1, 3, 19). With the use of a series of cell culture techniques in isolated rabbit myocytes, Armstrong and Ganote (1) suggest that the protection afforded by preconditioning may be secondary to stimulation of A3 receptors by a combination of APNEA (an A1/A3 agonist) and 8-cyclopentyl-1,3-dipropylxanthine (a selective A1-antagonist) to unmask the A3 effects. Liu et al. (19), in isolated rabbit hearts, and Auchampach et al. (3), in a rabbit chronic in vivo model, have suggested that stimulation of the adenosine A3 receptor with APNEA and 8-cyclopentyl-1,3-dipropylxanthine or IB-MECA, respectively, may mediate cardioprotection afforded by preconditioning. In the current study, we did not mimic chemical preconditioning, which incorporates a drug washout period. Instead, global myocardial ischemia was initiated immediately at the end of drug administration, without a washout interval so that the drug of interest remained in the myocardium during ischemia. In agreement with the aforementioned studies, the activation of adenosine A3 receptors before myocardial ischemia in our model was cardioprotective.
The activation of KATP channels has been well documented as a physiological effector of adenosine-receptor stimulation. In 1990, Kirsch et al. (15) reported that cardiac adenosine receptors activate the KATP in rat ventricular myocytes via adenosine A1 receptor-coupled activation of a Gi protein. Furthermore, KATP channels have been proposed to be involved in A1 receptor-mediated protective effects in ischemic-reperfused myocardium (5, 11, 28). Glibenclamide, a KATP-channel antagonist, abolishes the cardioprotection afforded by stimulation of the adenosine A1 receptor in many animal species (2, 26, 27, 32, 33, 39). Because the adenosine A3 receptor is similar to the adenosine A1 receptor in homology and receptor transduction through an inhibition in adenylate cyclase (43) and cAMP and a stimulation of protein kinase C translocation (1), it is plausible that activation of the adenosine A3 receptor may mediate cardioprotection via activation of KATP channels. In the present study, we have shown that the cardioprotection mediated by the specific activation of the adenosine A3 receptor with Cl-IB-MECA was reversed by the addition of the KATP-channel antagonist glibenclamide. Because the current study utilized pacing of the isolated perfused rat heart, conclusions regarding the arrhythmogenicity of stimulation of the KATP channels by A3-receptor activation cannot be made. In our model, the responses to the administration of glibenclamide alone did not significantly differ from those in control hearts. This observation suggests that A3-mediated cardioprotection is, in part, mediated via the activation of KATP channels. From the present study, we cannot delineate which KATP isoform is affected by the activation of the adenosine A3 receptor because glibenclamide blocks both the mitochondrial and sarcolemmal isoforms of the KATP channel.
Study limitations. The limitations in our study are inherent to the crystalloid perfused Langendorff model. Because our model is crystalloid perfused, adequate oxygen delivery to meet the demands of a heart contracting isovolumetrically against a filled balloon was a problem. However, we demonstrated a vasodilator reserve in our preparation with vasodilatory response to adenosine that increased perfusate flow by 160%. Because our perfusate lacked formed elements, the effects of the activation of the adenosine A3 receptor on other cells including neutrophils cannot be assessed. Furthermore, it has previously been demonstrated that KATP-channel activation may effect polymorphonuclear neutrophil function (24, 42). It was the intent of this study to investigate cardioprotective mechanisms independent of neutrophils. Other systemic inflammatory mediators (i.e., complement fragments) that may have direct effects on vascular endothelium or myocytes are absent. In the present in vitro study, we did not quantify the release of histamine from mast cells. Stimulation of the adenosine A3 receptor by some agonists has been shown to produce hypotension as a consequence of the release of histamine from mast cells in rats and mice (7, 10, 35). If this response occurs in humans, therapies targeting the stimulation of A3 receptors may be severely limited by side effects. We conclude that pretreatment with a specific adenosine A3-receptor agonist, via activation of KATP channels, may be a clinically relevant chemical preconditioning stimulus that attenuates postischemic cardiodynamic dysfunction and CK release without the vasodilator liability of nonspecific adenosine agonists.
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ACKNOWLEDGEMENTS |
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We are grateful to Gail Nechtman for formatting the manuscript for publication and to Dr. Bakthavachalam for the generous gift of Cl-IB-MECA. KW-3902 was a gift from Kyowa Hakko Kogyo through the efforts of Dr. Akira Karasawa. Furthermore, we thank the Carlyle Fraser Heart Center of Crawford W. Long Hospital-Emory University School of Medicine for their continued support of the research effort.
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FOOTNOTES |
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Funded in part by the Thoracic Surgery Research Foundation Fellowship Award (V. Thourani). Cl-IB-MECA was synthesized by Research Biochemicals (Natick, MA) as part of the Chemical Synthesis Program of the National Institute of Mental Health.
Presented at the Seventieth Scientific Sessions, American Heart Association, November 9-12, 1997, Orlando, Florida.
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. Vinten-Johansen, Cardiothoracic Research Laboratory, Crawford Long Hospital, 550 Peachtree St., NE, Atlanta, GA 30365-2225 (E-mail: jvinten{at}emory.edu).
Received 18 September 1998; accepted in final form 22 March 1999.
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REFERENCES |
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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