© 2001 American Heart Association, Inc.
Integrative Physiology |
A1 or A3 Adenosine Receptors Induce Late Preconditioning Against Infarction in Conscious Rabbits by Different Mechanisms
From the Division of Cardiology (H.T., R.B., R.G.B., E.K., X.-L.T., Z.Y., J.A.A.), University of Louisville and Jewish Heart and Lung Institute, Louisville, Ky; Department of Microbiology (S.B.), Columbia University, New York, NY.
Correspondence to John A. Auchampach, PhD, Department of Pharmacology, 8701 Watertown Plank Road, Medical College of Wisconsin, Milwaukee, WI 53226. E-mail jauchamp{at}mcw.edu
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Abstract |
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Abstract—We investigated whether activation of A1 or A3 adenosine receptors (ARs) induces late preconditioning (PC) against infarction in conscious rabbits using the selective AR agonists 2-chloro-N6-cyclopentyladenosine (CCPA) and N6-3-iodobenzyladenosine-5'-N-methylcarboxamide (IB-MECA). In vitro radioligand binding and cAMP assays demonstrated CCPA to be
200- to 400-fold selective for the rabbit
A1AR and IB-MECA to be 20-fold selective for
the rabbit A3AR. We observed that (1)
pretreatment of rabbits 24 hours earlier with CCPA (100 µg/kg IV
bolus) or IB-MECA (100 or 300 µg/kg) resulted in an 35% to 40%
reduction in the size of the infarct induced by 30 minutes of coronary
artery occlusion and 72 hours of reperfusion compared with
vehicle-treated rabbits, whereas pretreatment with the selective
A2AAR agonist CGS 21680 (100 µg/kg) had no
effect; (2) the delayed cardioprotective effect of CCPA, but not that
of IB-MECA, was completely blocked by coadministration of the highly
selective A1AR antagonist N-0861; (3) inhibition
of nitric oxide synthase (NOS) with
N
-nitro-L-arginine
during the 30-minute occlusion abrogated the infarct-sparing action of
CCPA but not that of IB-MECA; and (4) inhibition of ATP-sensitive
potassium (KATP) channels with sodium
5-hydroxydecanoate during the 30-minute occlusion blocked the
cardioprotective effects of both CCPA and IB-MECA. Taken together,
these results indicate that activation of either
A1ARs or A3ARs (but not
A2AARs) elicits delayed protection against
infarction in conscious rabbits and that both
A1AR- and A3AR-induced
cardioprotection involves opening of KATP
channels. However, A1AR-induced late PC uses an
NOS-dependent pathway whereas A3AR-induced late
PC is mediated by an NOS-independent
pathway.
Key Words: adenosine receptors • ischemia/reperfusion injury • myocardial infarction • ATP-dependent potassium channels • nitric oxide synthase
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Introduction |
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Although it is well established that stimulation of adenosine receptors (ARs) can induce early preconditioning (PC), it is controversial whether ARs can elicit the late phase of PC.1 2 Studies by Baxter et al1 first demonstrated in an open-chest, anesthetized rabbit model that administration of the A1AR agonist 2-chloro-N6-cyclopentyladenosine (CCPA) limited infarct size after a 30-minute coronary artery occlusion produced 24 to 72 hours later. On the other hand, we found in conscious rabbits that a 24-hour pretreatment with CCPA did not attenuate myocardial stunning.2 One interpretation of these results is that stimulation of ARs is capable of inducing late PC against infarction, but not late PC against myocardial stunning. This hypothesis is not implausible, because myocardial infarction and myocardial stunning are pathophysiologically distinct.3 4 However, since the studies by Baxter et al1 were performed in open-chest, anesthetized rabbits whereas our studies were performed in conscious rabbits, differences in the model systems that were used is another potential explanation for the disparate results. Given the lack of uniformity of the studies described above, the ability of ARs to induce late PC remains uncertain.
Of the four subtypes of ARs known to exist (A1, A2A, A2B, and A3), it is generally thought that the A1AR subtype is responsible for mediating the cardioprotective effects of adenosine.5 There is increasing evidence, however, that A3ARs may also exert cardioprotection.6 7 8 9 10 Evidence in support of this hypothesis includes the observations that early PC is not blocked by selective A1AR antagonists but is blocked by nonselective antagonists at high concentrations capable of blocking the A3AR, suggesting that the A3AR can also induce early PC.6 9 Furthermore, activation of A3ARs has been suggested to reduce injury in several different models of ischemia/reperfusion injury.7 8 10 Based on these results, it has been hypothesized that A3ARs are also expressed in ventricular cardiomyocytes and that they provide protection via a similar mechanism as A1ARs. The role of A3ARs in late PC, however, is unknown.
The present study was undertaken to address these issues and to further characterize the cardioprotective actions of AR stimulation. The results demonstrate, for the first time, that activation of A1 and A3ARs induces late PC against infarction via different mechanisms.
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Materials and Methods |
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Radioligand Binding Assays and cAMP Assays
Binding assays were performed with membranes prepared from HEK 293 cells expressing recombinant rabbit A1ARs or A3ARs using N6-(4-amino-3-[125I]iodobenzyl)adenosine-5'-N-methylcarboxamide ([125I]AB-MECA) as the radioligand. The rabbit A1AR cDNA used for transfections was as described by Bhattacharya et al,11 and the rabbit A3AR cDNA was cloned from a rabbit brain cDNA library. Additional binding assays with [125I]AB-MECA were performed with membranes prepared from rabbit brain and spleen. Inhibition of isoproterenol-induced cAMP accumulation by adenosine agonists was performed on HEK 293 cells expressing rabbit A1ARs or A3ARs using radioimmunoassay.
Reverse Transcriptase–Polymerase Chain
Reaction (RT-PCR) Analysis
mRNA expression of A1ARs and
A3ARs was determined by RT-PCR of total RNA
obtained from rabbit heart or 100 to 200 ventricular rabbit
cardiomyocytes isolated by enzymatic digestion.
Studies in Conscious Rabbits
New Zealand White rabbits (2.2 to 2.9 kg;
Myrtle’s Rabbitry, Thompson Station, Tenn) were instrumented with a
balloon occluder around a major branch of the left coronary artery for
occlusion and reperfusion and bipolar ECG leads on the chest wall. In
some studies (groups I through IV; see below), a Doppler thickening
crystal was sutured to the epicardial surface of the region at risk to
measure wall thickening (WTh). The rabbits were allowed to recover for
a minimum of 10 days after surgery. All animal experiments conformed to
the guidelines established by the University of Louisville.
The experimental protocol is depicted in
Figure 1
. All rabbits were subjected to 30 minutes of
coronary artery occlusion and 3 days of reperfusion. At the end of the
experiments, infarct size and the area-at-risk size were measured by
dual staining with phthalo blue dye and triphenyltetrazolium
hydrochloride. Rabbits were randomly assigned to 12 treatment groups.
Group I (control group) received 1 mL of vehicle as an IV bolus 24
hours before the occlusion. In groups II, III, and IV, CCPA (100
µg/kg), CGS-21680 (100 µg/kg; A2A receptor
agonist), or IB-MECA (100 µg/kg or 300 µg/kg) were given as boluses
24 hours before the coronary occlusion. Groups V and VI were treated 24
hours before the occlusion with N-0861 (7.5 mg/kg IV bolus followed
immediately by an intravenous infusion of 0.3 mg ·
kg–1 · min–1,
which was maintained for 3 hours; total dose, 55.5 mg/kg); 5 minutes
after the bolus injection of N-0861, the rabbits were treated with
either CCPA (100 µg/kg IV bolus) or IB-MECA (300 µg/kg IV bolus).
Groups VII, VIII, and IX were given
N-nitro-L-arginine
(L-NA) (13 mg/kg as an IV bolus) and groups X, XI, and XII were given
5-hydroxydecanoate (5-HD) (5 mg/kg IV bolus) 5 minutes before
the 30-minute occlusion in rabbits pretreated 24 hours earlier with
either CCPA (100 µg/kg IV bolus; groups VII and X), IB-MECA (300
µg/kg IV bolus; groups VIII and XI), or vehicle (groups IX and
XII).
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An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.
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Results |
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Pharmacological Characterization of Rabbit A1ARs and A3ARs
Because the pharmacology of A3ARs differs markedly among species,12 preliminary studies were performed to characterize rabbit A1ARs and A3ARs using radioligand binding analysis and functional assays.
Binding Studies
Specific binding of
[125I]AB-MECA to HEK 293 cell membranes
expressing either rabbit A1ARs or
A3ARs fit best to a one-site binding model. The
Kd and
Bmax values for the rabbit
A1AR were 6.18±1.92 nmol/L and 2675±569
fmol/mg membrane protein (n=3), respectively, and for the rabbit
A3AR were 0.39±0.11 nmol/L and 1033±433
fmol/mg membrane protein (n=3), respectively. No specific binding of
[125I]AB-MECA was observed to
nontransfected cells (data not shown).
The
Ki
values of competing ligands are shown in the
Table.
The salient findings of these studies can be summarized as follows: (1)
CCPA is a potent and selective agonist for recombinant rabbit
A1ARs (377-fold); (2) IB-MECA is a potent
A3AR agonist; however, it is only 21-fold
selective versus the A1AR; (3) like rodent
A3ARs,13
the rabbit A3AR is resistant to blockade by
xanthine antagonists and no selective antagonists were identified; (4)
the xanthine and adenine antagonists had high affinity for the
A1AR, therefore useful A1
selective antagonists were identified including CPX (7750-fold) and
N-0861 (1400-fold); and (5) the A2AAR agonist
CGS 21680 and the A2AAR antagonist ZM 241385 had
low affinity for both A1ARs and
A3ARs.
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We next measured [125I]AB-MECA
binding to membranes prepared from rabbit lung and spleen to determine
whether we could detect endogenous expression of
A1ARs and A3ARs and to
confirm that endogenous rabbit ARs expressed in tissues are
pharmacologically similar to recombinant rabbit receptors expressed
heterologously in HEK 293 cells. Previous studies have demonstrated
that high levels of A3AR transcript exist in
spleen,12 and that the brain
is a rich source of A1ARs. These studies could
not be performed in heart, because the level of expression of all of
the adenosine receptor subtypes is too low to be detected accurately in
heart tissue with agonist radioligands. For these studies, binding to
A1ARs was defined by specific binding displaced
by 500 nmol/L CPX, A3AR binding was defined by
the difference in specific binding displaced by 500 nmol/L CPX and 500
nmol/L MRS 1220, and A2AAR binding was defined
by specific binding displaced by 100 nmol/L ZM 241385. As shown in
Figure 2, incubation of rabbit brain or spleen membranes
with 0.3 nmol/L [125I]AB-MECA resulted
in 90±1% and 74±1% specific binding, respectively. In brain tissue,
75% of the specific binding sites were A1,
12% were A3, and 13% were
A2A. In contrast, 25% of the specific
binding sites in spleen were A1, 70% were
A3, and <5% were A2A.
These data demonstrate that (1)
[125I]AB-MECA labels multiple AR subtypes
in rabbit brain and spleen, and (2) the majority of
[125I]AB-MECA binding in rabbit brain is
to A1ARs whereas the majority of binding in
rabbit spleen is to A3ARs.
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Using rabbit brain membranes as a source of
A1ARs and rabbit spleen membranes as a source of
A3ARs, we next performed competition binding
assays to compare the affinity of CCPA and IB-MECA for endogenously
expressed rabbit ARs. [125I]AB-MECA was
included at a concentration of 6 nmol/L for assays of brain membranes
and 0.3 nmol/L for assays of spleen membranes, ie, concentrations
equivalent to its predicted
Kd value
for A1ARs and A3ARs,
respectively. In addition, 100 nmol/L ZM 241385 was included in all of
the assays to inhibit binding to A2AARs, and 500
nmol/L CPX was included in assays of spleen tissue to selectively block
A1ARs. Both CCPA and IB-MECA concentration
dependently competed for [125I]AB-MECA
binding in rabbit brain and spleen tissues
(Figure 2
). In brain tissue, the CCPA and IB-MECA competition
binding data fit best to a two-site binding model; the high-capacity
binding site reflected binding to the A1AR. In
spleen tissue when 500 nmol/L CPX and 100 nmol/L ZM 241825 were added
to the assays, the data fit best to a single-site binding model
reflecting binding to the A3AR. The
IC50 values of CCPA were calculated to be
0.32±0.06 nmol/L for the high-capacity binding site in brain
(A1AR) and 50.1±18.1 nmol/L for the binding
site in spleen (A3AR). The
IC50 values for IB-MECA were calculated to be
0.78±0.26 nmol/L for the single binding site in spleen
(A3AR) and 10.1±2.2 nmol/L for the
high-capacity binding site in brain (A1AR).
These data demonstrate that CCPA binds selectively to endogenously
expressed rabbit A1ARs (160-fold selective)
and that IB-MECA binds potently and with moderate selectivity
(13-fold selective) to endogenously expressed rabbit
A3ARs. These results are similar to those
obtained with CCPA and IB-MECA in binding studies using recombinant
rabbit ARs expressed in HEK 293 cells (see
Table).
cAMP Assays With HEK 293 Cells
CCPA and IB-MECA were found to inhibit
isoproterenol-stimulated cAMP accumulation in HEK 293 cells transfected
with recombinant rabbit A1ARs and
A3ARs
(Figure 3). The EC50 values of CCPA
and IB-MECA were calculated to be 0.05±0.03 and 59.0±12.2 nmol/L,
respectively, for HEK 293 cells expressing rabbit
A1ARs and 10.3±4.11 and 0.14±0.08 nmol/L,
respectively, for HEK 293 cells expressing rabbit
A3ARs. CGS 21680 weakly acted on rabbit
A1ARs (EC50=821±98.2
nmol/L) and A3ARs
(EC50=116±24.6 nmol/L). These results
demonstrate that CCPA and IB-MECA are functional agonists of rabbit
A1ARs and A3ARs. None of
the agonists influenced isoproterenol-induced increases in cAMP levels
in nontransfected HEK 293 cells (data not shown).
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The cAMP-lowering effects of CCPA in HEK 293 cells
transfected with the A1AR were surmountably
blocked by the addition of 10 or 50 µmol/L N-0861
(Figure 3;
Kd=45±19
nmol/L by Schild regression analysis;
Figure 3). N-0861 did not antagonize the inhibitory effects
of IB-MECA in HEK 293 cells transfected with the
A3AR. Thus, N-0861 acts as a competitive and
selective antagonist of
A1ARs.
Detection of A1AR and
A3AR Message by RT-PCR
No bands corresponding to the
A1AR or A3AR were
detected in heart tissue or isolated ventricular cardiomyocytes after
RT-PCR, electrophoretic separation of the reactions through agarose
gels, and staining with ethidium bromide. However, as shown in
Figure 4, distinct bands corresponding to the
A1AR (310 bp) and A3AR
(415 bp) were detected in both heart tissue and isolated cardiomyocytes
after Southern blotting of the RT-PCR reactions and probing with
A1AR- or A3AR-specific
cDNA radioprobes. These results suggest that both
A1AR and A3AR mRNAs are
expressed in whole heart tissue as well as ventricular cardiomyocytes.
Note that no bands were detected in negative control samples in which
water was included in the RT-PCR reactions instead of RNA, excluding
the possibility that the reactions were contaminated with foreign
DNA.
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Delayed Cardioprotection by CCPA and IB-MECA in
Conscious Rabbits
Exclusions
Of the 129 rabbits instrumented for the studies of
myocardial infarction, 26 were excluded because of ventricular
fibrillation, technical problems, or a small risk region (<10% of
left ventricular weight; see Table 1 in the online data supplement,
available at http://www.circresaha.org).
Hemodynamic Variables on Day 1
Hemodynamic data for groups II, III, IVa, IVb, V, and
VI on day 1 are presented in
Figure 5. At baseline, heart rate and mean arterial blood
pressure were similar in all of the treatment groups (which ranged from
241 to 255 bpm and 74 to 82 mm Hg, respectively). In group II,
administration of CCPA (100 µg/kg) produced a transient reduction in
heart rate and mean arterial blood pressure (maximal decreases of 24%
and 14%, respectively). In group III, administration of 100 µg/kg
CGS 21680 increased heart rate by 29% and reduced mean arterial blood
pressure by 17%. In group IVb, administration of IB-MECA at 300
µg/kg caused an 11% decrease in heart rate but did not cause any
appreciable changes in mean arterial blood pressure. However, IB-MECA
at a dose of 100 µg/kg (group IVb) did not influence either
hemodynamic parameter. In group V and VI in which 100 µg/kg CCPA and
300 µg/kg IB-MECA were administered in animals treated concurrently
with the A1AR antagonist N-0861, there were no
appreciable changes in heart rate or blood pressure at any time,
indicating that N-0861 completely blocked the hemodynamic actions of
CCPA and IB-MECA.
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Heart Rate on Day 2
There were no significant differences in heart rate
among any of the groups during the 30-minute coronary occlusion or
during the 72-hour reperfusion period, except for an 20% decrease
in heart rate in all of the groups of rabbits treated with L-NA (see
Table 2 in the online data supplement).
Region at Risk and Infarct Size
There were no significant differences among the groups
with respect to the weight of the region at risk (which ranged from
15.8% to 20.1% of the left ventricle; see Table 3 in the online data
supplement), indicating that this important determinant of infarct size
is similar among the treatment groups. However, the average infarct
size was 33% smaller in group II (CCPA 100 µg/kg group), 44%
smaller in the group IVa (IB-MECA 100 µg/kg group), and 40% smaller
in group IVb (IB-MECA 300 µg/kg group) compared with the control
group (38.3±4.1%, 33.7±4.9%, and 31.8±2.9% versus 56.4±4.0% of
the risk region, respectively), indicating that both CCPA and IB-MECA
(the latter even at a hemodynamically inert dose of 100 µg/kg)
elicited protection against infarction 24 hours later
(Figure 6). The average infarct size in group III (CGS 21680
100 µg/kg group; 61.4±5.5%) did not differ from the control group,
indicating that activation of A2AARs does not
produce late PC against infarction. In group V (CCPA 100 µg/kg+N-0861
group), infarct size (59.8±3.8%) was indistinguishable from that
measured in the control group and significantly larger than that
measured in the CCPA group
(Figure 6), indicating that N-0681 completely blocked the
infarct-sparing effect of CCPA pretreatment. In group VI (IB-MECA 300
µg/kg+N-0861), infarct size (41.8±1.9%) was significantly smaller
than that of the control group and was not significantly larger than
that observed in the IB-MECA–pretreated group
(Figure 6). Thus, the same dose of N-0861 that blocked the
infarct-sparing effect of CCPA failed to block the infarct-sparing
effect of IB-MECA, implying that activation of
A3ARs alone is sufficient to produce late PC
against infarction.
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, Individual rabbits. •, Mean±SEM.
Having found that both CCPA and IB-MECA induced late PC
against infarction, we examined the effect of a nitric oxide synthase
(NOS) inhibitor and an ATP-sensitive potassium
(KATP) channel blocker on the cardioprotective
responses. In group VII (CCPA 100 µg/kg+L-NA), infarct size
(57.7±4.1%) was significantly greater than in group II (CCPA 100
µg/kg) and essentially indistinguishable from controls
(Figure 7), indicating that L-NA abrogated the protective
effect of CCPA. However, in group VIII (IB-MECA 300 µg/kg+L-NA)
infarct size (36.6±3.4%) was significantly smaller than the control
group and similar to that in group IVb (IB-MECA 300 µg/kg),
indicating that L-NA failed to block the protective effect of IB-MECA
pretreatment. In groups X (CCPA 100 µg/kg+5-HD) and XI (IB-MECA 300
µg/kg+5-HD), infarct size (54.8±5.8% and 55.0±4.4%) was greater
than in groups II (CCPA 100 µg/kg) and IVb (IB-MECA 300 µg/kg),
respectively, and essentially indistinguishable from the control group
(Figure 7). Thus, administration of 5-HD on day 2 completely
blocked the protection provided by pretreatment with either CCPA or
IB-MECA. In groups IX (L-NA) and XII (5-HD), infarct size did not
differ significantly from that in the control group
(Figure 7), indicating that administration of L-NA or 5-HD
did not affect infarct size in vehicle-treated myocardium (55.0±4.4%
and 52.0±5.5%, respectively).
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Functional Recovery
The recovery of regional contractile function in the
ischemic-reperfused region was measured in groups I through IV. In
keeping with the infarct size data, the recovery of systolic WTh was
improved in rabbits pretreated with 100 µg/kg CCPA, 100 µg/kg
IB-MECA, or 300 µg/kg IB-MECA after 72 hours of reperfusion compared
with that measured in the control group (3.5±3.9%, 5.5±9.4%, and
7.3±8.3% of baseline, respectively, versus -16.2±6.4% of
baseline; P<0.05). In the CGS
21680-treated group, the recovery of systolic WTh at 72 hours was not
significantly improved (-15.4±4.2% of
baseline).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The results presented herein demonstrate that, in conscious rabbits, activation of either A1ARs or A3ARs (but not A2AARs) can induce late PC against infarction acting via distinct mechanisms. To the best of our knowledge, this is the first investigation to demonstrate that ARs can induce late PC in a conscious rabbit model. This is also the first investigation to demonstrate that the A3AR subtype, in addition to the A1AR subtype, is capable of triggering the development of late PC, that this effect of the A3AR is mediated by KATP channels, and that NOS plays a differential role in A1AR- versus A3AR-induced late PC.
The ability of AR agonists to induce late PC is
controversial. Studies in barbital-anesthetized, open-chest rabbits
support the concept that the A1AR agonist CCPA
can induce late PC against myocardial
infarction,1 whereas studies
in conscious rabbits suggest that CCPA cannot induce late PC against
myocardial stunning.2 One of
the goals of the present investigation was to gain insights into this
apparent discrepancy. Specifically, we sought to distinguish between
two basic possibilities: (1) ARs can induce late PC against infarction
but not late PC against myocardial stunning, or (2) the ability of AR
agonists to induce late PC is dependent on the system in which they are
studied. Our results provide evidence that the former hypothesis is
correct. Using the same conscious rabbit model in which we previously
observed that CCPA did not induce late PC against myocardial
stunning,2 we found that the
same dose of CCPA induced late PC against myocardial infarction
(Figure 6). To the best of our knowledge, this is the first
identification of a late PC stimulus that provides selective protection
against a specific type of ischemic injury. Our results imply that
there are important differences between the mechanism of late PC
against myocardial stunning and late PC against infarction.
A second major goal of the present investigation was to
determine whether the A3AR is capable of
inducing late PC against infarction. Our approach was to compare the
effects of CCPA to those of the recently characterized
A3AR agonist
IB-MECA.14 We found that
IB-MECA, at a dose of 300 µg/kg, produced reductions in infarct size
that were equivalent in magnitude to those elicited by CCPA
(Figure 6), suggesting that the A3AR
is also capable of inducing late PC against infarction. These results,
however, must be interpreted with caution, because IB-MECA at a dose of
300 µg/kg produced a modest decrease in heart rate
(Figure 5). This finding, coupled with the results of our in
vitro radioligand binding studies showing that IB-MECA is only 13- to
21-fold more potent at binding to rabbit A3ARs
compared with rabbit A1ARs
(Table
and
Figure 2), raises the possibility that IB-MECA may have
induced late PC not by interacting with A3ARs,
but rather through nonspecific interactions with the
A1AR. This possibility cannot be addressed using
an A3AR-selective inhibitor because no
antagonist is currently available that selectively inhibits rabbit
A3ARs
(Table).
Therefore, we performed two additional sets of experiments. First, we
administered IB-MECA or CCPA in the presence of the
A1AR antagonist N-0861 given at a dose predicted
to produce blood levels (20 to 50
µmol/L)15 that do not
block the A3AR. The finding that N-0861 blocked
the actions of CCPA against infarction but not those of IB-MECA
(Figure 6) demonstrates that IB-MECA is capable of eliciting
delayed cardioprotection through a mechanism independent of the
A1AR, thereby implicating the involvement of the
A3AR. Second, we administered IB-MECA at a lower
dose (100 µg/kg), which had no effect on heart rate
(Figure 5), indicating that it did not activate
A1ARs. The finding that this lower dose of
IB-MECA exerted a robust delayed cardioprotective action
(Figure 6) further corroborates the conclusion that
activation of A3ARs in itself induces late PC,
independent of A1AR activation. The possibility
that IB-MECA produced late PC by acting through
A2AARs can be excluded because the selective
A2AAR agonist CGS 21680 did not reduce infarct
size
(Figure 6). We can also exclude the possibility that IB-MECA
acted through A2BARs, because it has extremely
low affinity for this receptor
subtype.16 Thus, our results
indicate that, similar to early PC, the late phase of PC can also be
induced by activation of A3ARs.
Although current data indicate that A3ARs can induce both phases of PC, in recent in vivo studies we have observed that mice with genetic disruption of A3ARs exhibit infarcts that are smaller than those in wild-type mice,17 raising the interesting possibility that A3ARs may actually play an injurious role during acute myocardial ischemia in this species. These observations are not in conflict with the idea that A3ARs can trigger PC, since the role of A3ARs in modulating injury during acute myocardial ischemia is distinct from their role in eliciting PC before ischemia. It is also important to keep in mind that the findings obtained with A3AR knockout mice need to be confirmed with the use of A3AR-selective antagonists, because chronic disruption of these receptors may produce compensatory changes in other genes and/or signaling pathways resulting in protection from ischemic injury. In addition, there are marked differences in the properties and tissue expression of A3ARs among species,12 such that observations in mice should not be extrapolated to rabbits.
We also investigated the mechanisms by which CCPA and
IB-MECA induce the delayed cardioprotection against infarction. We have
previously found that both the anti-stunning and anti-infarct effects
of ischemia-induced late PC are mediated by increased activity of NOS,
specifically, the inducible isoform of NOS
(iNOS).18 Additional studies
have demonstrated that the cardioprotective effects of CCPA-induced
late PC can be blocked by inhibitors of the KATP
channel and are absent in iNOS knockout
mice.19 20 Based
on these observations, we hypothesized that late PC induced by both
CCPA and IB-MECA is also the result of increased NOS activity and
increased function of KATP channels. We found
that the nonselective NOS inhibitor L-NA and the
KATP channel antagonist 5-HD completely
abrogated the protective effects of CCPA when they were administered
immediately before the 30-minute coronary occlusion on day 2
(Figure 7). On the other hand, IB-MECA–induced late PC was
not blocked by L-NA but was completely abrogated by 5-HD
(Figure 7). It appears, therefore, that CCPA-induced late PC
against infarction involves a mechanism similar to that of
ischemia-induced late PC18 ;
that is, cardioprotection is due to enhanced production of NO via
induction of NOS and enhanced function of KATP
channels. IB-MECA–induced late PC also appears to utilize a mechanism
requiring KATP channels; however, upregulation
of NOS is not a necessary component. Thus, the results suggest that the
mechanisms by which A1ARs and
A3ARs induce late PC against infarction involve
different pathways that ultimately converge on the
KATP channel.
Elucidation of the mechanisms by which ARs modulate PC
requires knowledge of the cell types within the heart that express
A1ARs and A3ARs. It is
well established that A1ARs coupled to
inhibition of adenylyl cyclase via Gi/o proteins
are expressed in cardiomyocytes in both atria and ventricles. These
receptors classically are known to counteract the positive inotropic
actions of catecholamines.21
Recent data indicate that cardiac A1ARs induce
delayed PC via signaling pathways involving protein kinase C, tyrosine
kinases, and mitogen-activated/stress-activated protein
kinases.22 With regard to
the A3AR, however, little is known regarding the
specific cell types within the heart that express this receptor
subtype. Strickler et al10
recently demonstrated that A3ARs are expressed
in cultured embryonic chicken cardiomyocytes and that activation of
these receptors produces protection against cell death induced by
simulated ischemia and reperfusion. Thus, it is possible that
cardiomyocytes also express A3ARs, which may
induce PC via similar signaling pathways as the
A1AR. This hypothesis is supported by the
results of the present investigation in which we were able to detect
A3AR mRNA in adult rabbit cardiomyocytes by
RT-PCR analysis
(Figure 4). The cell types within the heart that express
A3ARs may not be limited to cardiomyocytes,
however. For example, in other tissues, the A3AR
is known to be expressed in resident leukocytes such as macrophages and
mast cells.12
A3ARs have also been suggested to be expressed
in vascular smooth muscle cells and endothelial
cells.23 Based on these
observations, it remains possible that A3AR
agonists may elicit PC through the release of mediators from
nonmyocytic cells. Clearly, additional studies of
A3ARs in the heart are warranted.
In conclusion, the present study demonstrates that activation of ARs induces a long-lasting cardioprotective effect in conscious rabbits, suggesting that AR agonists could be used to maintain patients in a protracted preconditioned state. Our results further demonstrate that selective agonists of either A1ARs or A3ARs can induce a late PC effect. Because A3AR agonists can provide protection with minimal hemodynamic effects, these results imply that targeting the A3AR could be a novel and useful approach to the protection of the ischemic myocardium. Finally, the present study elucidates the molecular mechanisms of A1AR- and A3AR-induced late PC by identifying common and differential roles for the KATP channel and NOS, respectively, in theses two forms of delayed cardioprotection.
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Acknowledgments |
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This study was supported in part by National Institutes of Health Grants R01 HL60051 (J.A.A.), HL-43151 (R.B.), and HL-55757 (R.B.); by American Heart Association (AHA) National Center Grant 9630083N (J.A.A.); by AHA Kentucky Affiliate Grant KY 9804558 (H.T.), and by the Jewish Hospital Research Foundation, Louisville, Ky.
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Footnotes |
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Original received March 3, 2000; resubmission received October 12, 2000; revised resubmission received January 17, 2001; accepted January 17, 2001.
This manuscript was sent to James T. Willerson, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
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References |
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