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The following is the abstract of the article discussed in the subsequent letter:
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ABSTRACT |
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Kilpatrick EL, P Narayan, RM Mentzer, and RD Lasley. Adenosine A3 agonist cardioprotection in isolate rat and
rabbit hearts is blocked by the A1 antagonist DPCPX.
Am J Physiol Heart Circ Physiol 281: H847-H853, 2001.
Adenosine A3 agonists have been shown to protect
ischemic rat and rabbit myocardium. However, these agonists
have been reported to exert A3 independent effects, and no
cardiac A3 receptor has yet been identified. We thus tested whether A3 agonist protection is due to A1
receptor activation. Isolated rat and rabbit hearts were subjected to
25 and 45 min of global ischemia, respectively. Rat hearts
pretreated with adenosine (100 µM), the A3 agonist
2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide
(Cl-IB-MECA, 50 nM), and vehicle recovered 73 ± 2%, 75 ± 4%, and 46 ± 4%, respectively, of preischemic left
ventricular developed pressure (LVDP) after 30 min of reperfusion. The
A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX,
100 nM) blocked the beneficial effects of Cl-IB-MECA (51 ± 5%)
and adenosine (47 ± 6%). In rabbit hearts, the beneficial effects of the A3 agonist
N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide
(50 nM) and the A1 agonist
2-chloro-N6-cyclopentyladenosine (100 nM) on
postischemic LVDP (75 ± 4 and 74 ± 5%,
respectively) were blocked by DPCPX (34 ± 4 and 36 ± 3%,
respectively). The reduction in infarct size with both agonists was
also completely blocked by DPCPX. These results suggest that these
A3 agonists protect ischemic myocardium via
A1 receptor activation.
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LETTER |
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To the Editor: In the August 2001 issue of the American Journal of Physiology-Heart Circulatory Physiology, Kilpatrick and colleagues (9) examined the ability of selective and nonselective adenosine receptor antagonists to abrogate cardioprotection with A3-selective agonists in the rat and rabbit. Cardioprotective functions of adenosine receptor subtypes remain a source of much debate, and various groups continue to probe the roles of A1, A2A, A2B, and A3 adenosine receptors in ischemic and reperfused myocardium. In terms of the cardiac effects of A3 receptors, the weight of evidence from the literature supports A3-mediated cardioprotection via pathways distinct from those for A1 receptors in multiple species (3, 8, 10, 17). Even discounting studies employing A3 agonists, expression of A3 receptors themselves confers tolerance to injury in cardiac myocytes (6). Nonetheless, the interesting study by Kilpatrick et al. does address an important issue: which receptor subtypes are activated by so-called "A3-selective" agonists employed in these varied studies?
Through comparing responses to A3 agonists [N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) and 2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IB-MECA)] in the absence or presence of A1-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) or nonselective antagonist 8-sulfophenyltheophylline (8-SPT), the authors conclude that the supposedly A3-selective agonists in fact protect via A1 receptors. However, the absence of appropriate control experiments renders this conclusion highly questionable. Specifically, effects of the antagonists themselves were not determined in ischemic-reperfused hearts.
To identify effects of exogenous receptor agonism in a tissue in which background endogenous responses exist, the appropriate "control" experiment is treatment of ischemic hearts with receptor antagonist alone. This is particularly important when endogenous agonist levels (and therefore responses) are enhanced, as during ischemia. If addition of agonist under these conditions no longer elicits a response, it can be concluded that the agonist acts via the targeted receptor. Importantly, a considerable literature reveals that A1- or nonselective adenosine receptor antagonism impairs ischemic or postischemic function in different species and models (7, 12, 14-16, 20). This demonstrates that endogenous adenosine serves a protective function in ischemic-reperfused myocardium. The recoveries observed by Kilpatrick et al. in hearts cotreated with agonist (adenosine, CCPA, Cl-IB-MECA, or IB-MECA) plus antagonist (DPCPX or 8-SPT), which in all instances equal recoveries for untreated hearts, are likely to exceed the recoveries for ischemic-reperfused hearts treated with the antagonists alone (the appropriate control group). This would lead to the equivocal conclusion that: 1) effects of agonists are not dependent on the targeted receptor, and/or 2) competitive antagonism fails to effectively counter responses to applied agonist. Stated another way, the apparent lack of protection during cotreatment with A3 agonist and A1 antagonist may reflect a balance between beneficial effects of A3 agonism and injurious effects of antagonism of an endogenous A1 response.
With respect to the selectivity of the antagonist employed, DPCPX is selective for A1 receptors but also inhibits A2B and A2A receptors with a inhibitory constant (Ki) from 50 to 150 nM (11). It can therefore be argued that effects of DPCPX are complicated by A2B antagonism. However, this is not a likely explanation for the observations of Kilpatrick et al., because there is little evidence of A2-mediated protection in isolated asanguinous hearts, and A2-mediated protection from ischemia in vivo is resistant to DPCPX (18).
A final point relates to their experiments employing 100 µM exogenous adenosine. In these studies it is predicted that protection should occur via receptor-mediated (A1, potentially A2 and A3) and nonreceptor-mediated mechanisms. We and others (4, 13) have demonstrated a metabolic component to adenosine-mediated protection involving purine salvage. Nonetheless, selective A1 blockade apparently abolished protection in their study, implicating a single protective mechanism for adenosine (A1 activation). This unexpected observation might again reflect combined effects of inhibition of A1-mediated protection and protection via A1-independent pathways.
These uncertainties cannot be resolved in the absence of data on effects of receptor antagonism in ischemic-reperfused hearts. Studies examining adenosine antagonism reveal protection by endogenous adenosine in human (14), canine (19), rabbit (16, 20), rat (7, 15), and mouse myocardium (12). Interestingly, failure to identify effects of adenosine antagonism alone is not uncommon. For example, investigators often apply antagonists to test adenosines role in models of preconditioning, yet they fail to identify effects of antagonism in nonpreconditioned tissue (e.g., 1, 2, 5, 17). Impaired recovery in these cases may not reflect adenosine-mediated preconditioning per se, but cardioprotection by endogenous adenosine independent of a preconditioning response.
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Adenosine A3 agonist cardioprotection in isolated rat and rabbit hearts is blocked by the A1 antagonist DPCPX.
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John P. Headrick, Heart Foundation Research Centre School of Health Science Griffith University Southport, QLD 4217 Australia (E-mail: j.headrick{at}mailbox.gu.edu.au) |
To the Editor: We thank Dr. Headrick for his interest
in our recent report that the adenosine A1 receptor
antagonist DPCPX blocked the protection of the A3 agonists
IB-MECA and Cl-IBMECA (8). As he pointed out, despite the
acknowledged cardioprotective effects of exogenous and endogenous
adenosine, there remain several controversial and unresolved aspects to
this phenomenon. One of our primary concerns has been the numerous
reports implicating the involvement of adenosine A3
receptors, based primarily on results obtained with agonists such as
IB-MECA and Cl-IBMECA and the nonselective adenosine antagonist
8-sulfophenyltheophylline (8-SPT). This hypothesis continues to receive
support despite the fact there has been no documented physical evidence
of the existence of functional A3 receptors in the adult
mammalian ventricular myocardium. Furthermore, the results of
radioligand binding (17, 19) and functional studies
(10, 21) indicate that adenosine A3 agonists
can exert their effects via activation of A1 and
A2a receptors.
An additional limitation of studies examining adenosine A3
receptors is the species-dependent effects of currently available A3 antagonists and the lack of information on the effects
of these agents in the heart. Despite these limitations, it is well
known that rodent adenosine A3 receptors are relatively
insensitive to methylxanthine-based antagonists. It has been reported
that cloned rat and rabbit A3 receptors exhibit
Ki values for DPCPX > 5 µM and 1 µM,
respectively (6, 7). In addition, although high doses (100 µM) of 8-SPT have been used to implicate A3
cardioprotection in the rabbit myocardium [Ki
for cloned rabbit A3 receptors Although we recognize the concerns of Dr. Headrick regarding the
potential effects of DPCPX alone, we disagree with his conclusion that
our omission of such a group "renders [our] conclusion highly questionable." As Dr. Headrick pointed out, there are reports on the
modulation of ischemia-reperfusion injury by various adenosine antagonists; however, we did not include this group based on several reports (in multiple species) documenting the lack of effect of DPCPX
alone on ischemia-reperfusion injury when administered only before ischemia (3, 4, 12, 13, 18, 20). Hearts in our study were exposed to DPCPX (100 nM) only for 10 min immediately before ischemia. Two reports (14, 16) indicate
that low doses of DPCPX (100-200 nM), even when administered only
before ischemia, did exacerbate early recovery of ventricular
function in isolated perfused mouse hearts. However, these hearts were
only reperfused for 30 min, and in the latter study (16),
during the final 10 min of the 30-min reperfusion period, both DPCPX
groups exhibited a more rapid rate of recovery of preischemic
function than control hearts. Whether this effect would have persisted
for the duration of reperfusion remains unknown. In addition to
potential differences between murine myocardium and that of other
species, these conflicting reports indicate that the effects of
adenosine receptor antagonists in ischemic-reperfused
myocardium may be dependent on the time of administration, as has been
widely documented for adenosine agonists.
Dr. Headrick's final point related to the "unexpected observation"
that the beneficial effects of 100 µM adenosine in our study were
essentially completely blocked by DPCPX. He apparently interpreted this
to indicate that we were excluding the metabolic effects of adenosine,
such as purine salvage. In fact our initial studies (2,
22) on adenosine cardioprotection were based on the hypothesis
that adenosine would be protective via this mechanism. However, purine
salvage is an oxygen-dependent, reperfusion-related process of ATP
resynthesis, and we observed beneficial metabolic effects of exogenous
adenosine during ischemia. We subsequently reported that the
ability of adenosine to retard the rate of ATP depletion during
ischemia could be mimicked by an A1 agonist
(9, 11), and there have been numerous reports of metabolic
effects of adenosine agonists during ischemia. Given that we
infused adenosine for only 5 min before ischemia, it is
unlikely that purine salvage would play a role in this protection.
Although we cannot discount potential metabolic effects of DPCPX during
ischemia, there is substantial evidence that
preischemic treatment with DPCPX does not exacerbate
ischemia-reperfusion injury (3, 4, 12, 13, 18, 20)
in several species. As we discussed in the paper, this does not appear
to be the case with another antagonist BWA1433 (15), which
has been used to implicate A3 receptors in adenosine cardioprotection.
As we concluded in our final statement in the paper "until the
A3 receptor is identified in cardiac myocytes and more
selective A3 agonists and antagonists become available, the
exact role, if any, that A3 receptors play in adenosine
cardioprotection remains to be determined." This statement is further
reinforced by the results of two studies indicating that knockout of
the A3 receptor in the mouse confers resistance to
myocardial ischemia-reperfusion (1, 5).
REPLY
38 µM
(6)], we were not able to document any published reports of the use of low doses of this methylxanthine in the presence of
an A3 agonist. These deficiencies in the literature
provided the basis for our study. The methylxanthine DPCPX, at doses
(100 nM) similar to what we used in our study, has been used
extensively in A1 receptor radioligand binding studies and
to study the adenosine A1 anti-adrenergic effect. Although
this dose of DPCPX may have exerted some effects on A2a, we
have previously reported that preischemic treatment with an
A2 agonist in this same model is not cardioprotective. At
the present time there is no evidence supporting a role for
A2b receptors in adenosine cardioprotection. Thus it is
likely the effects associated with low-dose DPCPX and 8-SPT in our
study were due to A1 receptor antagonism.
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Robert Lasley, Eric Kilpatrick, Prakash Narayan, Robert Mentzer, Jr., Dept. of Surgery University of Kentucky College of Medicine Lexington, KY 40536-0298 |
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