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Cardiovascular Research 2002 55(1):161-170; doi:10.1016/S0008-6363(02)00329-2
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Copyright © 2002, European Society of Cardiology

Coronary function and adenosine receptor-mediated responses in ischemic-reperfused mouse heart

Amanda J Flood, Laura Willems and John P Headrick*

Heart Foundation Research Centre, School of Health Science, Griffith University Gold Coast Campus, Southport, Qld 4217, Australia

* Corresponding author. Tel.: +61-7-5552-8292; fax: +61-7-5552-8802 j.headrick{at}mailbox.gu.edu.au

Received 6 November 2001; accepted 14 February 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objectives: To assess the impact of ischemia-reperfusion (I/R) on coronary function, and the role of endogenous adenosine in modifying post-ischemic vascular function in asanguinous hearts. Methods: Vascular function was studied in Langendorff perfused mouse hearts subjected to 20–25-min ischemia and 30-min reperfusion. Results: Ischemia altered the dependence of flow on work-rate observed in normoxic hearts, and inhibited reflow by mechanisms additional to diastolic compression. Coronary responses were selectively reduced: 2-chloroadenosine and ADP dilated with pEC50s of 8.4±0.1 and 7.4±0.1 in non-ischemic hearts versus 7.7±0.1 and 7.1±0.1 after 20-min ischemia (P<0.05). Sensitivity was further reduced after 25-min ischemia. Responses to nitroprusside were unaltered. NO-synthase antagonism (50 µM nitro-L-arginine methylester) reduced sensitivities to 2-chloroadenosine and ADP up to 10-fold, and eliminated inhibitory effects of I/R. KATP blockade with 5 µM glibenclamide impaired sensitivity pre- and post-ischemia, not eliminating the inhibitory effects of I/R. A1 adenosine receptor antagonism with 100 nM 8-cyclopentyl-1,3-dipropylxanthine worsened effects of ischemia on sensitivity. A2A adenosine receptor antagonism with 100 nM 8-(3-chlorostyryl)caffeine reduced post-ischemic flow by 50%, yet paradoxically enhanced post-ischemic contractile recovery. Conclusions: Ischemia modifies vascular control and impairs NO- versus KATP-dependent coronary dilation in an asanguinous model. Endogenous adenosine protects against vascular dysfunction via A1 receptors, and determines coronary reflow via A2A receptors. However, intrinsic A2A activation apparently worsens contractile dysfunction.

KEYWORDS Adenosine; Coronary circulation; Ischemia; KATP channel; Reperfusion; Vasoconstriction/dilation; Ventricular function


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
While research into the pathogenesis of ischemic injury has focused on cardiac myocytes, increasing evidence supports a role for coronary injury [1–3]. Ku first documented impaired endothelium-dependent responses in post-ischemic coronaries [4]. Subsequent studies verify vascular dysfunction in a variety of models and species [5–11], including humans [12]. Dysfunction is an early event, evident after brief ischemia [6,13], and endothelial cells appear the primary site of injury.

While the pathogenesis of vascular injury remains incompletely understood, oxidant-mediated NO inactivation is implicated [14,15]. Radical generation reduces NO-synthase activity [16] and basal and agonist-stimulated NO release [17]. Proximity of endothelial cells to sites of radical generation may render them susceptible. Moreover, they appear less tolerant of oxidant stress compared with other cells, likely due to low anti-oxidant content [18,19]. Compounding this, endothelial cells generate radicals [20]. Leukocytes are also thought to play a major role through radical release and capillary obstruction [3,21–23]. However, impaired reflow and coronary dysfunction occur in asanguinous hearts [9,10,24,25], implicating non-leukocyte dependent mechanisms. The impact of ischemia-reperfusion on coronary function in the absence of blood has been the focus of few studies [25]. The first goal of the present study was to examine the impact of ischemia-reperfusion on coronary function in an asanguinous model.

Our second goal was to examine the role of adenosine in modifying post-ischemic coronary function. There is evidence for reduced vascular injury through adenosine-mediated inhibition of neutrophil activation and radical formation [26–28], and also for impaired vascular injury with adenosinergic therapy in humans [29,30]. While it is generally thought these responses are dependent on blood elements and A2/A3 adenosine receptors [26–28], there is some evidence for A1-mediated vascular protection [31] and reduced vascular injury with interventions involving A1 receptors (e.g. preconditioning) [32]. We therefore wished to identify roles of endogenous adenosine in controlling post-ischemic coronary function.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Langendorff heart model
Investigations conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Hearts were isolated and prepared as described previously [33–35]. Specifically, 196 adult male C57/Bl6 mice (7–12 weeks age, 26.0±0.3 g body weight, 105±2 mg blotted heart weight) were anesthetized with 50 mg kg–1 sodium pentobarbitone, thoracotomy was performed and hearts excised into ice-cold perfusion fluid. The aorta was cannulated and perfused at 80 mmHg with Krebs bicarbonate buffer containing (in mM): NaCl, 120; NaHCO3, 25; KCl, 4.7; KH2PO4, 1.2; CaCl2, 2.5; Mg2SO4 1.2; glucose, 15; and EDTA, 0.6. Perfusate was equilibrated with 95% O2, 5% CO2 at 37 °C, giving a pH of 7.4. Perfusate temperature was maintained at 37 °C and hearts were bathed in a chamber maintained at 37 °C. The left ventricle was vented with a polyethylene drain to prevent Thebesian accumulation. Coronary flow was monitored via ultrasonic flow-probe (Transonic Systems, Ithaca, NY, USA). Perfusion pressure was monitored using a P23XL pressure transducer (Viggo-Spectramed, Oxnard, CA, USA) connected to a MacLab (ADInstruments, Castle Hill, Australia).

To assess contractile function, fluid-filled polyvinyl chloride balloons were inserted into the left ventricle via the mitral [33,34]. Balloons were connected to a second P23XL pressure transducer by fluid-filled polyethylene tube. The balloons have an unstressed volume of ~60 µl as described previously [34]. Balloon volume was increased to give an end-diastolic pressure of 5 mmHg. Functional data were recorded at 1 KHz on a 4/s MacLab unit connected to an Apple computer. The ventricular pressure signal was digitally processed to yield systolic and diastolic pressures, and heart rate. After 20-min stabilization hearts were switched to pacing via silver electrodes, using a Grass S9 stimulator (Grass, Quincy, MA, USA). Hearts were paced at 400 beats min–1 via silver left ventricular electrodes (0.5 ms square pulses, 20% above threshold, typically 2–5 V) and stabilized for a further 10 min.

2.2 Functional responses to ischemia
To assess functional responses to ischemia, hearts were subjected to 20- (n = 8) or 25-min (n = 11) global normothermic ischemia followed by 30-min reperfusion. To identify effects of coronary dilation, and receptor-mediated effects of endogenous adenosine, hearts subjected to 20-min ischemia were treated with 5 nM of the dilatory A2A agonist 2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosine (CGS21680, n = 7) or 150 nM of A1 adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, n = 7) prior to and for 15 min following ischemia. Hearts subjected to 25-min ischemia were treated with 5 nM of A2A agonist CGS21680 (n = 7) or 100 nM of A2A antagonist 8-(3-chlorostyryl)caffeine (n = 7), prior to and following ischemia.

2.3 Relationships between diastolic pressure, work-rate and coronary flow
To assess the impact of diastolic vascular compression on coronary flow, normoxic hearts were perfused with an intra-ventricular balloon in place (n = 6). Balloon volume was increased using a calibrated Hamilton 500-µl threaded syringe (Hamilton, Reno, NV, USA) to give end-diastolic pressures of 5–60 mmHg [34]. After 2-min stabilization at each volume, diastolic pressure and coronary flow were measured. This was first performed under control conditions, after which balloon volume was reduced to baseline levels and hearts stabilized for 15 min prior to maximal coronary dilation with 5 nM CGS21680. After 10 min the protocol was repeated. The relationship between diastolic pressure and coronary flow was then determined. Data for flow and diastolic pressure during the initial 5-min reperfusion for the ischemic hearts described in Section 2.2 were also plotted with these data. Early reflow occurs in maximally or near maximally dilated vessels and is therefore largely unrelated to metabolic rate. There are also no differences in systolic compression between groups at this time. Thus, early reflow is determined primarily by mechanical effects of diastolic compression.

To examine dependence of flow on myocardial work-rate, coronary flow rate and cardiac work (indicated by the rate–pressure product), were determined in a group of normoxic hearts (n = 19) beating at varying rates. The rate–pressure product (heart ratexventricular developed pressure) was employed as this is linearly related to oxygen consumption under varying conditions [36], and accounts for contributions of heart rate and pressure work to work-rate. Hearts were stabilized at intrinsic rate (381±11 beats min–1), then subjected to 5-min periods of pacing at 420, 480, and 540 beats min–1. Flow and function were measured at the end of each period. A similar experiment was undertaken in hearts subjected to 20-min ischemia and 30-min reperfusion (n = 8). After ischemia-reperfusion function and flow were assessed at 400, 420, 480 and 540 beats min–1. Data for flow and function (between 10- and 30-min reperfusion) for the experimental groups described in Section 2.2 were also plotted to contrast relationships in the different groups. We examined later reperfusion times since post-ischemic hyperemia has abated and hearts recovered substantial cardiac function at these times. While the latter hearts were not subjected to changes in pacing rate, the data permit assessment of the qualitative nature of the relationships between flow and work-rate (if any) in the varied experimental groups.

2.4 Vascular responses to 2-chloroadenosine, ADP and nitroprusside
To assess coronary sensitivity, empty hearts were switched to constant flow perfusion 10 min prior to acquisition of concentration–response curves, as described previously [35]. Flow was controlled by a Gilson MiniPuls 2 peristaltic pump (Gilson, Middleton, WI, USA) and set to a value yielding 100-mmHg aortic pressure. Hearts were treated with dilatory agonist (2-chloroadenosine, ADP or sodium nitroprusside), infused incrementally for 1–3 min at each concentration (during which vascular responses stabilized). One drug was applied per heart. Changes in aortic pressure were measured. For NO-synthase or KATP channel inhibition, infusion of 50 µM nitro-L-arginine methylester (L-NAME) or 5 µM glibenclamide was initiated 10 min prior to concentration–response curves. To assess the role of A1 adenosine receptors in modifying dysfunction, responses to 2-chloroadenosine were studied in hearts subjected to 20-min ischemia in the presence of 150 nM DPCPX (applied prior to ischemia and for the initial 15 min of reperfusion).

2.5 Chemicals
2-Chloroadenosine, CGS21680, 8-(3-chlorostyryl)caffeine, ADP, sodium nitroprusside, L-NAME, DPCPX, and glibenclamide were purchased from Sigma/RBI (Sigma, Castle Hill, Australia). All other chemicals were of analytical grade or better.

2.6 Data analysis
EC50 values were obtained from concentration–response data by fitting the following equation to data from individual experiments:


Formula

where A is the pre-infusion value and B the response at infinite dose. Dilatory responses were scaled as % of maximal dilation and data fit to the equation using Statistica (Statsoft, Tulsa, OK, USA). Individual EC50 values were derived and pEC50s (–log of EC50) compared by analysis of variance with Tukeys H.S.D. post-hoc test. Effects of ischemia and drug treatments were tested via multi-way analysis of variance for repeated measures, followed by Tukeys H.S.D. post-hoc test. In all tests P<0.05 was indicative of statistical significance. All values are reported as mean±S.E.M.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Functional effects of ischemia-reperfusion
Coronary flow initially recovered to above pre-ischemic levels during reperfusion following 20-min ischemia (Fig. 1A). This hyperemia was absent after 25-min ischemia (Fig. 2A). Flow through reperfusion was lower in the 25- versus 20-min ischemic group (Fig. 2A). Recoveries for diastolic (Figs. 1B and 2BGo) and systolic pressures (Figs. 1C and 2CGo) were incomplete following ischemia. Diastolic pressure was significantly higher throughout much of the reperfusion period in the 25- versus 20-min group (Fig. 2B), whereas systolic pressure only differed at the final time point (Fig. 2C). Coronary dilation with CGS21680 failed to alter early reflow but modestly increased flow during late reperfusion (Figs. 1A and 2AGo). The dilator failed to modify contractile recovery from 20- (Fig. 1) or 25-min ischemia (Fig. 2).


Figure 1
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  		Fig. 1 Recoveries for (A) coronary flow, (B) diastolic pressure, and (C) systolic pressure after 20-min ischemia. Data are shown for control hearts (n = 8), and hearts treated with dilatory A2A adenosine agonist CGS21680 (dilated, n = 7) or the A1 adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, n = 7). Values are means±S.E.M. *P<0.05 versus untreated. Note that pre-ischemic data points for some groups are obscured by symbols for other groups.

 

Figure 2
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  		Fig. 2 Recoveries for (A) coronary flow, (B) diastolic pressure, and (C) systolic pressure after 25-min ischemia. Data are shown for control hearts (n = 11), and hearts treated with dilatory A2A adenosine agonist CGS21680 (dilated, n = 7) or the A2A adenosine receptor antagonist 8-(3-chlorostyryl)caffeine (8-CSC, n = 7). Values are means±S.E.M. *P<0.05 versus untreated; {dagger}P<0.05 versus 20-min control. Note that pre-ischemic data points for some groups are obscured by symbols for other groups.

 
The dependence of flow on diastolic pressure (vascular compression) is depicted in Fig. 3A. Coronary flow displayed an inverse linear dependence on diastolic pressure in non-ischemic hearts (Fig. 3A). The relation predicts a 1.5 ml min–1 g–1 decline in sub-maximally or maximally dilated flow for every 10-mmHg increase in diastolic pressure. Coronary flow during the initial 5 min of reperfusion after 20-min ischemia falls within the range of flows and pressures for maximally dilated non-ischemic hearts (Fig. 3A). Supporting maximal dilation, CGS21680 increased pre-ischemic flow (Table 1) but failed to increase early reflow relative to diastolic pressure in the 20-min group (Fig. 3A). In contrast, early reflow following 25-min reperfusion was much lower than predicted from data for flow and diastolic pressure in non-ischemic hearts (and hearts subjected to 20-min ischemia) (Fig. 3A).


Figure 3
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  		Fig. 3 (A) Dependence of coronary flow on diastolic pressure in normoxic hearts (n = 6) untreated or treated with a maximal dilatory concentration of CGS21680 (5 nM), and during reperfusion following 20- or 25-min ischemia. (B) Relationship between coronary flow and work-rate (reflected by the rate–pressure product) in normoxic hearts subjected to electrical pacing (n = 19), post-ischemic hearts subjected to 20-min ischemia and 30-min reperfusion before assessing effects of pacing (n = 8), and in post-ischemic hearts paced at 400 beats min–1 and subjected to 20- (n = 8) or 25-min (n = 11) ischemia, 20-min ischemia with CGS21680 (dilated, n = 7) or DPCPX (n = 7), or 25-min ischemia with CGS21680 (dilated, n = 7) or 8-(3-chlorostyryl)caffeine (8-CSC, n = 7). All values are means±S.E.M.

 

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  		Table 1 Pre-ischemic functional parameters

 
Dependence of flow on cardiac work is depicted in Fig. 3B. These data reveal that coronary flow increases in a linear manner with the rate–pressure product when work-rate is increased by varying heart rate. Regression analysis of data for non-ischemic hearts yielded the relation: flow=–1.71+0.54xrate–pressure product (r = 0.71). In contrast, in post-ischemic hearts the pacing protocol elevated work-rate but failed to substantially alter coronary flow (flow=23.91+0.10xrate–pressure product, r = 0.49). A similar ‘flat’ relation was observed for post-ischemic hearts not subjected to the pacing protocol. While data for intrinsic function in these hearts was not acquired during pacing, the findings demonstrate qualitatively different relationships between cardiac function and coronary flow post-ischemia. Indeed, there was a paradoxical trend towards higher flows at lower work-rates post-ischemia (Fig. 3B). Flow relative to work-rate was also higher than predicted from non-ischemic data, and lower in 25- versus 20-min ischemic hearts.

3.2 Effects of A1 and A2A adenosine receptor antagonism
Effects of A1 adenosine receptor antagonism are shown in Fig. 1. Antagonism failed to alter pre-ischemic function (Table 1), but modestly reduced post-ischemic reflow (Fig. 1A), elevated post-ischemic diastolic pressure (Fig. 1B), and reduced contractile recovery (Fig. 1C). DPCPX shifted relationships between reflow and diastolic pressure (Fig. 3A), and post-ischemic flow and contractile function (Fig. 3B). Early reflow was lower than predicted based on diastolic contracture, and the relation between flow and work-rate was almost identical to that for a more severe 25-min ischemic insult (Fig. 3B).

The A2A-selective antagonist 8-(3-chlorostyryl)caffeine reduced pre-ischemic coronary flow by ~7 ml min–1 g–1 and produced a modest decline in +dP/dt (Table 1, Fig. 2A). No other functional parameters were altered. Antagonism markedly reduced post-ischemic coronary flow by up to 10 ml min–1 g–1 (Fig. 2A). Reduced reflow occurred despite significantly reduced post-ischemic diastolic pressure (Fig. 2B) and enhanced contractile recovery (Fig. 2C).

3.3 Effects of ischemia-reperfusion on coronary vascular responses
Ischemia-reperfusion impaired sensitivity to 2-chloroadenosine (Fig. 4, Table 2) and endothelial-dependent ADP (Fig. 5, Table 2). Longer 25-min ischemia further reduced sensitivity. Sensitivity to endothelial-independent SNP was unaltered by ischemia (Fig. 5). Responses to 2-chloroadenosine were significantly inhibited by 50 µM L-NAME pre-ischemia (Fig. 4, Table 2). Similarly, ADP responses were significantly inhibited by L-NAME (Fig. 5, Table 2). In post-ischemic hearts, L-NAME was less effective in modifying responses to 2-chloroadenosine and ADP (Figs. 4 and 5Go, Table 2). Moreover, ischemia-reperfusion failed to alter sensitivity to 2-chloroadenosine and ADP in the presence of L-NAME. L-NAME failed to modify responses to SNP pre- or post-ischemia (Figs. 4 and 5Go).


Figure 4
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  		Fig. 4 Concentration-response curves for 2-chloroadenosine in non-ischemic and post-ischemic hearts. (A) Effects of NO-synthase inhibition with L-NAME, and (B) effects of KATP channel inhibition. Responses were determined for untreated non-ischemic hearts (n = 7), hearts subjected to 20- (n = 9) or 25-min of ischemia (n = 7), non-ischemic (n = 8) and post-ischemic hearts (n = 12) treated with 50 µM L-NAME, and non-ischemic (n = 9) and post-ischemic hearts (n = 6) treated with 5 µM glibenclamide. Values are means±S.E.M. *P<0.05 versus non-ischemic; {dagger}P<0.05 versus untreated.

 

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  		Table 2 Coronary concentration–response data for 2-chloroadenosine, ADP and sodium nitroprusside

 

Figure 5
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  		Fig. 5 Concentration response curves for (A) ADP, and (B) sodium nitroprusside in non-ischemic and post-ischemic hearts. Responses were determined for ADP in the absence (n = 7 non-ischemic, n = 8 post-ischemic) and presence of 50 µM L-NAME (n = 9 non-ischemic, n = 8 post-ischemic), and for sodium nitroprusside in the absence (n = 6 non-ischemic, n = 6 post-ischemic) and presence of 50 µM L-NAME (n = 6 non-ischemic, n = 8 post-ischemic). Values are means±S.E.M. *P<0.05 versus non-ischemic; {dagger}P<0.05 versus untreated.

 
In contrast to reduced effects of L-NAME post-ischemia, KATP channel blockade with glibenclamide significantly impaired sensitivity to 2-chloroadenosine post-ischemia (Fig. 4, Table 2). Reflecting roles for NO release and KATP channel activation in control of resting coronary tone, L-NAME increased resting coronary vascular resistance by 2.0±0.4 and 2.1±0.5 mmHg ml–1 min–1 g–1 in non-ischemic and post-ischemic hearts, respectively, and glibenclamide increased resistance by 2.0±0.4 and 2.0±0.4 mmHg ml–1 min–1 g–1 in non-ischemic and post-ischemic hearts, respectively. In the final series of experiments it was found that selective A1 antagonism with DPCPX during ischemia and early reperfusion worsened effects of ischemia-reperfusion on sensitivity to 2-chloroadenosine (Fig. 6, Table 2).


Figure 6
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  		Fig. 6 Effect of A1 adenosine receptor antagonism on post-ischemic sensitivity to 2-chloroadenosine. Data are shown for hearts subjected to 20-min ischemia and 30-min reperfusion in the absence (n = 9) or presence of antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, n = 6). Values are means±S.E.M. *P<0.05 versus untreated hearts.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The aims of this study were to identify effects of ischemia on coronary function in the absence of blood, and to identify roles of adenosine receptors in modifying post-ischemic vascular function. Data indicate that 20–25-min ischemia results in vascular dysfunction in an asanguinous model, implicating non-leukocyte dependent pathways. Furthermore, findings support vascular protection by A1 adenosine receptors, and control of reflow via A2A receptors.

4.1 Post-ischemic coronary vascular function in asanguinous hearts
As noted by Nevalainen et al. [37] and recently by Gavin et al. [2], capillary compression by edema and contracture may be primary determinants of reflow, with vascular dysfunction a secondary factor. Supporting this, we recently showed early reflow correlates inversely with diastolic contracture [38]. We verify here that reflow after 20-min ischemia is consistent with maximally dilated flow for non-ischemic hearts, when corrected for elevated diastolic pressure and compression. Maximal dilation is additionally verified by lack of dilation with CGS21680 (Figs. 1A and 2AGo). However, more prolonged ischemia results in early reflow not predicted from the relation between flow and diastolic compression for normoxic hearts (Fig. 3A). Thus, we conclude that peak reflow after 20-min ischemia is not limited by early vascular dysfunction, while there is evidence for an abnormality after 25-min ischemia.

In contrast to predicted links between work-rate and flow in normoxic hearts, post-ischemic coronary flow appeared largely insensitive to work-rate (Fig. 3B). Furthermore, coronary flow was lower, relative to work-rate, after more prolonged ischemia. This further supports vascular dysfunction, although we find no direct evidence of impaired reflow (relative to work-rate) after the relatively severe insults, previously shown to induce substantial injury and necrosis [34]. Indeed, post-ischemic coronary flow during late reperfusion (10–30 min) appears higher than necessary for the level of work performed (Fig. 3B). Significantly impaired reflow may therefore require the presence of blood components and related pathological processes.

4.2 Post-ischemic coronary dysfunction
In addition to dissociation of flow from work-rate, coronary dysfunction is directly reflected in impaired sensitivity to 2-chloroadenosine and ADP (Figs. 4 and 5Go, Table 2). 2-Chloroadenosine dilates via mixed NO-synthase and KATP-dependent paths [35], whereas ADP is endothelial-dependent, acting through release of NO, EDHF and possibly prostaglandins [39,40]. Since ischemia-reperfusion failed to modify sensitivity to endothelial independent SNP, dysfunction is specific for endothelial responses. A role for impaired NO bio-availability is supported by the fact that ischemia-reperfusion failed to alter sensitivity to 2-chloroadenosine or ADP when NO-synthase was inhibited with L-NAME (Figs. 4 and 5Go). Moreover, when KATP channel dependent dilation was inhibited, sensitivity to 2-chloroadenosine was still reduced by ischemia (Fig. 4). Thus, when the contribution of NO synthase dependent dilation is reduced (with L-NAME), ischemia has little additional effect, yet when the contribution of KATP channel-dependent dilation is reduced (with glibenclamide), ischemia still impairs vascular function.

Quite opposing data exist regarding the genesis of endothelial dysfunction. There is evidence for [41] and against [42] impaired NO-synthase activity, for [10,41,43] and against [5,8,9] impairment of receptor versus non-receptor mediated responses, and for [11] and against [7] inhibition of muscarinic responses. We exclude post-ischemic down-regulation of receptors, or impaired receptor-effector coupling, since this would require comparable reductions in receptor number and/or coupling for two distinct receptor groups with different transduction mechanisms. Irrespective of mechanism, our data indicate that endothelial dysfunction occurs independently of leukocyte-mediated injury. Endothelium itself may be a source of oxidants [20], just as underlying cells may generate radicals during ischemia-reperfusion. Post-ischemic dysfunction in the absence of blood observed here (Figs. 4 and 5Go), and previously [9,11,24,25], indicates that the ‘classical’ path, involving oxidant release and injury via leukocyte activation and adherence, is not the sole source of dysfunction.

4.3 Vascular protection via intrinsic activation of A1 adenosine receptors
Since A1 receptor activation protects murine hearts from ischemia [33], we tested whether they might protect against vascular injury. DPCPX worsened diastolic contracture and contractile recovery (Fig. 1), consistent with prior observations [33]. Post-ischemic flow was also impaired modestly, and flow relative to workload was reduced to levels observed after more prolonged ischemia (Fig. 3B), suggesting vascular effects of the antagonist. In support of a vascular effect, DPCPX worsened effects of ischemia-reperfusion on coronary sensitivity to 2-cloroadenosine (Fig. 6). These findings implicate A1-mediated vascular protection by endogenous adenosine, consistent with the study of Maczewski and Beresewicz [31]. These findings may be of relevance to the clinically beneficial effects of adenosinergic therapies. For example, adenosine has been employed successfully as an adjunctive therapy in myocardial infarction and angioplasty [29,30]. While experimental data indicate vascular effects are due to A2 receptor-mediated changes in neutrophil/leukocyte function [26–28], whereas myocardial effects are A1 and/or A3 mediated, our data and the findings of Maczewski and Beresewicz [31] support the existence of vascular protection via A1 receptors independent of blood cells.

4.4 Role of A2A adenosine receptors in reflow and injury
The data support a key role for A2A adenosine receptors in controlling coronary flow pre- and post-ischemia (Fig. 2A, Table 1). There is support for vasoregulation by endogenous adenosine in vivo and in vitro under pathophysiological conditions [44–46]. Although there is evidence for vasoregulation under physiological conditions in animals [47] and humans [48], adenosine's physiological role remains controversial [49]. We targeted A2A receptors since murine coronary responses are A2A-mediated [34]. 8-(3-Chlorostyryl)caffeine is selective for A2A receptors, with poor antagonistic properties at A1 receptors. Lack of effect on A1 or A3 receptors is reflected in the fact that antagonism enhanced post-ischemic contractile and reduced diastolic pressure (Fig. 2), whereas A1 and A3 antagonists produce the opposite effect [33,38]. Reduced flow is therefore not the result of enhanced vascular compression or reduced workload. Since A2A agonism increases late but not early reflow, A2A-mediated dilatation by endogenous adenosine appears maximal initially, declining as reperfusion continues.

Improved recovery from ischemia with 8-(3-chlorostyryl)caffeine was unexpected, as there is little evidence for A2A receptor modulation of contractile dysfunction. We recently showed A2A agonism is ineffective in altering contractile recovery in the absence of blood [38], consistent with lack of effect of CGS21680 here (Figs. 1 and 2Go). However, Strickler et al. [50] have observed that A2A antagonism enhances protection with preconditioning in cultured myocytes. Whilst from a different model, the data of Strickler and colleagues together with our observations support a detrimental A2A-mediated effect in ischemic myocardium. Since A2A agonism did not worsen recovery whereas A2A antagonism enhanced recovery (Figs. 1 and 2Go), this response appears maximally activated by endogenous adenosine during ischemia and early reperfusion, consistent with the high sensitivity of A2A receptors.

4.5 Conclusions
In summary, graded coronary dysfunction is observed following 20–25-min ischemia in the absence of blood-borne elements. Dysfunction appears specific for NO-dependent responses. Additionally, we find that post-ischemic reflow is determined significantly by A2A receptor activation by endogenous adenosine, and that A1 receptor activation by endogenous adenosine protects against vascular injury. A vascular component may therefore exist in A1-mediated cardioprotection. Finally, we also observe improved functional recovery with A2A antagonism (despite reduced reflow), supporting a detrimental A2A mediated response to endogenous adenosine. The latter warrants further investigation.

Time for primary review 23 days.


   Acknowledgements
 
This work was supported by grants from the National Institutes of Health RO1 grant (HL 59419) and the National Heart Foundation of Australia (G 98B 0080 and G 99B 0246).


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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