Skip Navigation

Cardiovascular Research 2006 71(4):764-773; doi:10.1016/j.cardiores.2006.06.011
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Gündüz, D.
Right arrow Articles by Schäfer, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gündüz, D.
Right arrow Articles by Schäfer, C.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2006, European Society of Cardiology

Accumulation of extracellular ATP protects against acute reperfusion injury in rat heart endothelial cells

Dursun Gündüz1, Sascha A. Kasseckert1, Frauke V. Härtel, Muhammad Aslam, Yaser Abdallah, Matthias Schäfer, Hans Michael Piper, Thomas Noll* and Claudia Schäfer

Physiologisches Institut, Justus-Liebig-Universität Giessen, Aulweg 129, D-35392 Giessen, Germany

* Corresponding author. Tel.: +49 641 99 47343; fax: +49 641 99 47219. Email address: thomas.noll{at}physiologie.med.uni-giessen.de

Received 6 June 2005; revised 1 June 2006; accepted 6 June 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Material and methods
 3. Results
 4. Discussion
 References
 
Objective: Ischemia–reperfusion provokes barrier failure of the coronary microvasculature, leading to myocardial edema development that jeopardizes functional recovery of the heart during reperfusion. Here, we tested whether adenosine 5'-triphosphate (ATP), either exogenously applied or spontaneously released during reperfusion, protects the endothelial barrier against an imminent reperfusion injury and whether interventions preventing ATP breakdown augment this protective ATP effect.

Methods Cultured microvascular coronary endothelial monolayers and isolated-perfused hearts of rat were used.

Results: After ischemic conditions were induced, reperfusion of endothelial monolayers activated the endothelial contractile machinery and caused intercellular gap formation. It also led to the release of ATP. When its breakdown was inhibited by 6-N,N-diethyl-β,{gamma}-dibromomethylene-D-ATP (ARL 67156; 100 µM), a selective ectonucleotidase inhibitor, contractile activation and gap formation were significantly reduced. Reperfusion in the presence of exogenously added ATP (10 µM) plus ARL caused an additional reduction of both aforementioned effects. In contrast, elevation of ATP degradation by apyrase (1 U/ml), a soluble ectonucleotidase, or addition of adenosine (10 µM) provoked an increase in gap formation during reperfusion that could be completely inhibited by 8-phenyltheophylline (8-PT; 10 µM), an adenosine receptor antagonist. In Langendorff-perfused rat hearts, the reperfusion-induced increase in water content was significantly reduced by ARL plus ATP. Under conditions favouring ATP degradation, an increase in myocardial edema was observed that could be blocked by 8-PT.

Conclusion ATP, either released from cells or exogenously applied, protects against reperfusion-induced failure of the coronary endothelial barrier. Inhibition of ATP degradation enhances the stabilizing effect of ATP on barrier function.

KEYWORDS ATP release; Endothelial barrier function; Endothelial contractile machinery; Ectonucleotidases; Reperfusion injury


   1. Introduction
Top
 Abstract
 1. Introduction
2. Material and methods
 3. Results
 4. Discussion
 References
 
The endothelial lining of blood vessels forms a selective permeability barrier between plasma and interstitial spaces. In the course of ischemia–reperfusion the endothelial barrier of the coronary microvasculature becomes disturbed. For that reason myocardial edema is a characteristic feature of the pathophysiology of reperfusion following coronary occlusion [1]. The resulting myocardial edema inhibits functional recovery of the heart during reperfusion [2,3] and may jeopardize survival of myocardial tissue. In a recent study on cultured microvascular coronary endothelial monolayers we showed that simulated reperfusion conditions trigger the opening of intercellular gaps between endothelial cells [4]. Cell retraction is due to a Ca2+-dependent activation of the endothelial contractile apparatus. Reperfusion-induced gap formation could be blocked by inhibitors of myosin light chain kinase (MLCK) or manoeuvres which prevented the rise in cytosolic Ca2+ concentration. This indicates that activation of the contractile machinery contributes to barrier failure under reperfusion.

Endothelial cells possess an actin–myosin based contractile machinery controlled by the phosphorylation state of the regulatory myosin light chains (MLC) [5]. The phosphorylation state of MLC is regulated by a Ca2+/calmodulin-dependent MLCK, which phosphorylates MLC and thus enhances development of force, and a MLC phosphatase complex, which acts antagonistically. Activation of MLC phosphatase may therefore reduce reperfusion-induced barrier failure. Exogenously applied ATP is a strong activator of endothelial MLC phosphatase. We showed that ATP stabilizes barrier function of endothelial monolayers through specific purine receptors [6,7] and effectively reduces hyperpermeability in endothelial monolayers stimulated by inflammatory mediators such as thrombin [8]. In the present study we investigated if exogenously applied ATP can prevent the destabilization of the endothelial barrier caused by reperfusion conditions.

ATP may act on endothelial cells not only when exogenously applied, but also when released spontaneously from endothelial cells themselves. This was the second point to be investigated in this study. It is well documented that ATP is released from endothelial as well as from other vascular cells under diverse (patho)physiological conditions, e.g. inflammation [9], thrombosis [10], shear stress [11,12], osmotic stress [13,14], and even a sole rise at cytosolic Ca2+ concentration [14]. ATP is also released from cells of the cardiovascular system under conditions of ischemia–reperfusion [15,16]. Release of endogenous ATP normally has a very brief biological half life, since it is rapidly degraded by ubiquitously distributed ectonucleotidases [16]. Quiescent endothelial cells express high levels of the membrane-associated CD39/nucleoside triphosphate diphosphohydrolase (NTPDase1) which catalyses degradation of ATP and ADP to AMP [17,18]. Further degradation to adenosine is catalysed by ecto-5'-nucleotidase (CD73) [10,17,18], also present on the surface of endothelial cells. In previous studies [19–21] we showed that microvascular coronary endothelial monolayers respond with a strong increase in macromolecule permeability to an activation of adenosine receptors, i.e. opposite to their response to ATP. The effect of adenosine is mediated by cAMP/PKA-dependent signalling towards the contractile machinery and cell adhesion structures [21].

In the present study we tested the hypothesis (1) whether exogenously applied ATP can protect endothelial barrier against imminent failure provoked by reperfusion conditions, (2) whether endogenous ATP spontaneously released from endothelial cells during ischemia–reperfusion can provide such a protective effect, and (3) whether manoeuvres which prevent either ATP degradation to adenosine or inhibit activation of adenosine-receptors can augment or preserve the stabilization effect of ATP on the endothelial barrier. The effect of ATP to protect the endothelial barrier against reperfusion-induced failure was tested in a culture model of microvascular coronary endothelial monolayers and in the vascular system of isolated-perfused hearts. To inhibit degradation of extracellular ATP, 6-N,N-diethyl-β,{gamma}-dibromomethylene-D-ATP (ARL 67156; ARL), an ATP analogue and specific inhibitor of NTPDase1, was applied [22,23]. To exclude contractile effects of the applied substances in the whole heart preparation, cardioplegic conditions were chosen.


   2. Material and methods
Top
 Abstract
 1. Introduction
 2. Material and methods
3. Results
 4. Discussion
 References
 
2.1. Cell cultures
The investigation conforms to 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). Microvascular coronary endothelial cells were isolated from 200–250 g male Wistar rats and cultured as previously described [8].

2.2. Experimental protocols
Endothelial cells on glass cover slips were placed in a perfusion chamber (1 ml filling volume) and were superfused at a flow rate of 0.5 ml/min with a modified Hepes buffer (containing in mM: 140.0 NaCl, 2.6 KCl, 1.2 KH PO4, 1.2 MgSO4, 1.3 CaCl2, 2.5 glucose and 25.0 HEPES) adjusted to either pH 6.4 during ischemia (anoxic medium) or 7.4 during reperfusion (reoxygenation with normoxic medium) at 37 °C. Normoxic medium was equilibrated with air. The anoxic medium was glucose-free and equilibrated before and during experiments with 100% N2. Po2 in the anoxic medium was <1 mm Hg as determined at the chamber outlet with a polarographic oxygen sensor. Anoxic and normoxic media were serum-free. The medium was transferred into the perfusion chamber through gas-tight steel capillaries. Under standard conditions cells were exposed to anoxia at pH 6.4 for 40 min followed by 30 min reperfusion with normoxic medium. Only reperfusion conditions were varied: (a) continuing perfusion at pH 7.4, (b) with or without ATP (10 µM) or ARL 67156 (ARL; 100 µM), (c) with or without adenosine (ADO; 10 µM) or 8-phenyltheophylline (8-PT; 10 µM), (d) with soluble apyrase (1 U/ml). In pilot experiments the optimum effective concentrations of each agonist and inhibitor were determined in concentration–response experiments.

The basal medium used in incubations was modified Tyrode's solution (composition in mM: 150.0 NaCl, 2.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, 1.0 CaCl2, and 30.0 HEPES; pH 7.4, 37 °C) supplemented with 2% (vol/vol) heat-inactivated fetal calf serum (15 min, 65 °C).

Basal MLC phosphorylation was determined after an initial equilibration period of 30 min. Agents were added as indicated. Stock solution of ATP, ADO, apyrase and ARL were prepared immediately before use with basal medium. Stock solutions of 8-PT were prepared with dimethyl sulfoxide (DMSO). Appropriate volumes of these solutions were added to the cells yielding final solvent concentrations <0.1% (vol/vol). Same final concentrations of DMSO were also included in all respective control experiments. Appropriate volumes of these solutions were added to the cells. Identical additions of basal medium were included in all respective control experiments.

2.3 Free cytosolic Ca2+ concentration
Free cytosolic Ca2+ concentration (Cai) was determined using the fluorescent Ca2+ indicator Fura-2 as previously described [8,24].

2.4. Determination of intercellular gaps
Simultaneously to the measurement of the Fura-2 ratio, intercellular gap formation was quantified by planimetric analysis of the fluorescence images as previously described [24].

2.5. Myocardial water content
Hearts from 250-g male Wistar rats were mounted immediately after isolation on a Langendorff perfusion system in a temperature-controlled chamber (37 °C), as previously described [25]. Briefly, during normoxic perfusion (10 ml/min) with an oxygenated saline medium (composition in mM: 140.0 NaCl, 24.0 NaHCO3, 2.7 KCl, 0.4 KH2PO4, 1.0 MgSO4, 1.8 CaCl2, 5.0 glucose, pH 7.4; gassed with 95% O2 [vol/vol]/5% CO2[vol/vol]), the chamber was flushed with humidified air, and during anoxia (no-flow-perfusion), with humidified 100% N2. After no-flow ischemia, hearts were again supplied with oxygen by perfusion of a cardioplegic, hyperkalemic saline solution (composition in mM: 127.7 NaCl, 24.0 NaHCO3, 15.0 KCl, 0.4 KH2PO4, 1.0 MgCl2, 1.8 CaCl2, 5.0 glucose, pH 7.4; gassed with 95% O2 [vol/vol]/5% CO2 [vol/vol]). ARL, ATP, ADO, apyrase, 8-PT were added to the perfusion medium 1 min before the onset of cardioplegic reperfusion, respectively. It remained in the perfusion medium during the entire period of reperfusion. At the end of each experiment, wet weight and after 24 h, dry weight of the perfused rat hearts were measured.

2.6. Determination of MLC phosphorylation
The phosphorylation of MLC was determined by glycerol–urea polyacrylamide gel electrophoresis and Western blot analysis as described [6]. This procedure allows separation of non-phosphorylated from phosphorylated MLC protein, the latter of which migrates more rapidly. Briefly, electrophoretically separated proteins were transblotted on PVDF membranes and incubated with an anti-MLC antibody followed by an alkaline phosphatase-conjugated anti-IgM antibody. Blots were scanned densitometrically, and the percentage of MLC phosphorylation (expressed as percentage of total MLC) was calculated from densitometric values of non- (MLC), mono- (MLC~P) and diphosphorylated MLC (MLC~PP) as follows:


Formula

2.7. Bioluminescence detection of ATP
The bioluminescence detection of ATP was determined according to [14], with minor modifications. Briefly, 50 µl medium of the tested endothelial monolayers and 50 µl of an ATP luciferin/luciferase assay reagent were mixed in a microcentrifuge tube and immediately placed into a TD-20/20 Luminometer (Turner Designs). All assays were performed at room temperature.

2.8. Materials
Falcon plastic tissue culture dishes were from Becton Dickinson (Heidelberg, Germany); ATP from Boehringer (Mannheim, Germany); ATP luciferin/luciferase assay reagent (ENLITEN®) was from Promega (Mannheim, Germany); Fura-2/AM was from Molecular Probes (Leiden, The Netherlands); polyvinylidene difluorid (PVDF) was from Millipore (Eschborn, Germany); anti-MLC antibody (clone MY-21) and peroxidase-coupled second antibody were from Sigma (Deisenhofen, Germany); ARL 67156 was from Tocris Bioscience (Bristol, UK). All other chemicals were of the best available quality, usually analytical grade.

2.9. Statistical analysis
Data are given as means±SEM in all optical measurements or S.D. of n experiments using independent cell preparations. The comparison of means between groups was performed by one way analysis of variance (ANOVA) followed by a Bonferroni post-hoc test. Changes of parameters within the same group were assessed by multiple ANOVA analysis. Probability (P) values of less than 0.05 were considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Material and methods
 3. Results
4. Discussion
 References
 
3.1 Effect of extracellular ATP on Cai, gap formation and MLC phosphorylation
Endothelial monolayers were exposed to 40 min anoxic medium (pH 6.4), to simulate in part ischemic conditions, and were then reoxygenated at physiological pH 7.4, to simulate reperfusion conditions. In the context of this paper these simulated conditions are addressed briefly as "ischemia" and "reperfusion". Superfusion of endothelial cells with ischemic perfusion medium induced a biphasic increase of Cai (Fig. 1A). Cai peaked at 8 min initially, dropped subsequently towards basal levels, and started to increase continuously after 20 min during the ongoing ischemic period. If the perfusion was changed to reperfusion medium, endothelial cells immediately responded with a steep further increase in Cai. With the onset of ischemia, interendothelial gaps started forming (Fig. 1B). Reperfusion provoked a further steep rise in gap formation, which increased to approximately threefold of the end-ischemic value during the 30 min period of reperfusion. In contrast, a 30 min continuation of ischemic perfusion led only to a slight further increase in gap formation.


Figure 1
View larger version (17K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Cytosolic Ca2+ concentration and gap formation of microvascular coronary endothelial monolayers during ischemia and reperfusion. Representative recordings of (A) cytosolic Ca2+ concentrations (Fura-2 ratio) and (B) formation of intercellular gaps are given.

 
To test whether extracellular ATP can affect reperfusion-induced gap formation, reperfusion medium was supplemented with 10 µM ATP. Although endothelial cells responded with an additional increase in Cai (Fig. 2A), presence of extracellular ATP reduced reperfusion-induced endothelial gap formation distinctly during reperfusion (Figs. 2B, 3Go). When 100 µM ARL, a NTPDase inhibitor, was added to reperfusion medium, the effects of ATP were enhanced, both with regard to Cai rise (Fig. 2C) and reduction of gap formation (Figs. 2D, 3Go). In the combined presence of ATP and ARL reperfusion-induced increase in gap formation was abolished.


Figure 2
View larger version (43K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Effect of extracellular ATP (10 µM), the NTPDase inhibitor ARL 67156 (ARL, 100 µM) or ATP plus ARL on cytosolic Ca2+ concentration and gap formation of endothelial monolayers during reperfusion. Representative recordings of (A, C) cytosolic Ca2+ concentrations (Fura-2 ratio) and (B, D) formation of intercellular gaps are given.

 

Figure 3
View larger version (15K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Gap formation after 40 min ischemia (I) followed by 30 min reperfusion in absence (R) or presence of 10 µM ATP, the NTPDase inhibitor ARL 67156 (ARL, 100 µM) or ARL plus ATP. Data are means±SD of 10 separate experiments with independent cell preparations. *P<0.05 vs. R; #P<0.05 as indicated; n.s., not significantly different to I.

 
To analyse the effect of extracellular ATP on activation of the contractile machinery, MLC phosphorylation was determined during ischemia and reperfusion in absence or presence of ATP (Fig. 4). At the end of ischemia, MLC phosphorylation was 63±3%. Reperfusion caused a distinct increase in MLC phosphorylation, i.e. to 78±4%. Reperfusion with medium containing 10 µM ATP prevented the reperfusion-induced increase in MLC phosphorylation, when ATP plus ARL were present during reperfusion, MLC phosphorylation was even reduced below the end-ischemic level, i.e. to 47±4%.


Figure 4
View larger version (30K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Effect of ATP, the NTPDase inhibitor ARL 67156 (ARL, 100 µM) or ATP plus ARL on reperfusion-induced increase in MLC phosphorylation of endothelial monolayers. Cells were incubated for 40 min under ischemia (I), or 40 min ischemia followed by 30 min reperfusion in the absence (R) or presence of 10 µM ATP, the NTPDase inhibitor ARL 67156 (ARL, 100 µM) or ARL plus ATP. Upper panel: Western blot analysis of MLC phosphorylation. A representative experiment is given. The bands represent, from top to bottom, non- (MLC), mono- (MLC~P), and di-phosphorylated MLC (MLC~PP), respectively. Lower panel: statistical analysis of MLC phosphorylation expressed as percentage of total MLC. Data are means±SD of 6 separate experiments with independent cell preparations. *P<0.05 vs. R.; #P<0.05 as indicated.

 
3.2. Effect of released endogenous ATP on gap formation
To verify whether ATP is released spontaneously from endothelial cells during reperfusion, ATP was determined in samples of the effluate (Fig. 5). It is expressed in relative terms, since in this experimental setup the actual concentration of ATP within the unstirred layer just above the endothelial surfaces is diluted into a large stream of fluid passing through the incubation chamber, and samples from the effluate of the perfusion chamber therefore reflect only on a relative scale the changes at the endothelial cell surface. Reperfusion after 40 min ischemia caused a transient rise of ATP in the medium. When ARL was added to the reperfusion medium, the rise of ATP in the medium was enlarged. Comparing the integrals underneath the curves, the amount of extracellular ATP cleared into the passing medium in absence of ARL amounts to 28% of that in presence of ARL. Under normoxic control conditions no significant ATP release was detected. These data show that reperfusion indeed causes an ATP release from the endothelial cells into the surrounding medium. They also indicate that the extracellular concentration resulting from that spontaneous release is distinctly higher when the NTPDase inhibitor ARL is applied.


Figure 5
View larger version (25K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 ATP in the effluate of superfused endothelial monolayers during reoxygenation. Cells were incubated for 40 min under ischemia followed by 30 min reperfusion in the absence ({blacktriangleup}) or presence of the NTPDase inhibitor ARL 67156 ({blacksquare}; ARL, 100 µM) or for 70 min under ischemia (Continued Isch.) in absence ({bigtriangleup}) or presence of ARL 67156 ({square}). Data are given as percentage of maximum. Mean±SD of 6 separate experiments with independent cell preparations. *P<0.05 vs. onset of reperfusion.

 
To verify if the ARL-induced accumulation of ATP during reperfusion can stabilize the endothelial barrier, gap formation was estimated (Fig. 3). Compared to the end-ischemic value, gaps were threefold increased after subsequent 40 min reperfusion. Reperfusion of EC in presence of ARL reduced the reperfusion-induced gap formation significantly. It was then tested whether conditions with different results with respect to gap formation in reperfusion, also differ with respect to the activation state of the endothelial contractile machinery. Reperfusion of endothelial cells with medium containing ARL prevented the reperfusion-induced increase in MLC phosphorylation almost entirely (Fig. 4).

To test whether degradation products of ATP can influence gap formation during reperfusion, exogenous adenosine was applied (Fig. 6). Adenosine (10 µM) alone augmented gap formation during reperfusion, up to 4.6-fold of the end-ischemic value. This adenosine effect was completely blocked by simultaneous addition of the adenosine receptor antagonist, 8-phenyltheophylline (8-PT, 10 µM). Similar effects could be achieved by addition of a different adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (50 nM; data not shown). Interestingly, presence of 8-PT alone in reperfusion had no effect, indicating that the amount of adenosine generated by spontaneous breakdown and staying at the cells' surface is too small to stimulate adenosine receptors.


Figure 6
View larger version (21K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 Effects on gap formation in endothelial monolayers by adenosine (ADO; 10 µM), the adenosine receptor antagonist 8-phenyltheophylline (8-PT; 10 µM) or simultaneous addition of ADO plus 8-PT, the soluble ectonucleotidase apyrase (Apyr; 1 U/ml) or simultaneous addition of Apyr plus ATP (10 µM) after 40 min ischemia followed by 30 min reperfusion. (I) Endothelial cells were exposed for 40 min to ischemia without further additions. Data are means±SD of 10 separate experiments with independent cell preparations. *P<0.05 vs. R; n.s., not significantly different.

 
When the soluble ectonucleotidase apyrase (1 U/ml) was added at reperfusion, a significant increase in gaps was observed. This indicates that in abundant presence of ectonucleotidase activity in the external medium, the amount of adenosine generated from the spontaneously released ATP is sufficient to stimulate adenosine receptors on the endothelial cells. When apyrase was applied together with 10 µM ATP, reperfusion-induced gap formation was enhanced to the same level as in presence of an equivalent concentration of 10 µM adenosine. Together these data show that gap formation during reperfusion is influenced by adenosine that can be rapidly generated from ATP.

3.3. Effect of ATP and its degradation product adenosine on myocardial water content
To verify whether ATP can stabilize the vascular barrier in situ, experiments were performed on Langendorff-perfused hearts under cardioplegic arrest (Fig. 7). Myocardial water content was determined as parameter for tissue edema. Cardioplegic conditions were chosen to exclude indirect effects on myocardial water content through changes in contractile function. Hearts were exposed for 40 min to no-flow ischemia and then reperfused for 30 min. Effluate samples were taken at the 5th min of reperfusion to determine ATP concentrations (Table 1). These results show that, in presence of the NTPDase inhibitor ARL, 100 µM, about 79% of the ATP added to the perfusate is preserved after passage through the coronary circulation. In absence of ARL, only 18% of the added ATP is preserved. When hearts are perfused without exogenous addition of ATP to the perfusate, a small release of ATP was detectable. In presence of ARL this amounted to 0.11 µM which would correspond to an initially released concentration of 0.14 µM, if the same 79% preservation is assumed. The concentration found in the absence of ARL in the effluate was 0.02 µM, from which a preservation of 14% of the initially released concentration can be calculated. This figure is comparable to the degradation determined for exogenously applied ATP. Compared to the exogenously applied ATP concentration, that produced by endogenous release from reperfused endothelial cells is only 1.4%.


Figure 7
View larger version (25K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 7 Myocardial water content after ischemia–reperfusion of saline-perfused rat hearts. Hearts were exposed for 40 min to ischemia (I) or for 40 min to ischemia followed by 30 min reperfusion (R). The following agents were added 1 min before onset of reperfusion: ATP (10 µM), ATP plus the NTPDase inhibitor ARL 67156 (ARL; 100 µM), ATP plus the adenosine receptor antagonist 8-phenyltheophylline (8-PT; 10 µM), ATP plus the soluble ectonucleotidase apyrase (Apyr; 1 U/ml), adenosine (ADO; 10 µM) plus the adenosine receptor antagonist 8-phenyltheophylline (8-PT; 10 µM) or ADO alone. Data are means±SD of 5 separate experiments with independent cell preparations. *P<0.05 vs. R; #P<0.05 as indicated; n.s., not significantly different.

 

View this table:
[in this window]
[in a new window]

  		Table 1 ATP concentration in the effluate during reperfusion of the isolated-perfused rat heart

 
Reperfusion of ischemic hearts caused an increase in myocardial water content from 402±29 to 543±23 ml H2O/100 g dry weight. If hearts were reperfused with a medium containing 10 µM ATP plus 100 µM ARL, i.e. conditions which prevent 79% of ATP breakdown, the reperfusion-induced increase in myocardial water content was markedly reduced, i.e. to 458±17 ml H2O/100 g dry weight. When 10 µM ATP was applied without ARL, i.e. conditions which prevent only 18% ATP breakdown, it caused an increase in myocardial water content during reperfusion to 656±36 ml H2O/100 g dry weight. When ARL alone was applied, i.e. conditions with only 1.4% of the exogenously applied ATP present, no significant effect was observed. Insignificant was also the effect of exogenously applied ATP (10 µM) in presence of the adenosine receptor antagonist 8-PT, indicating that the edema augmenting effect of ATP alone is due to its degradation product adenosine.

To verify this last mentioned hypothesis, it was tested whether acceleration of ATP degradation by apyrase (1 U/ml) can enhance myocardial edema formation. When 10 µM ATP was applied in presence of myocardial water content increased to 824±20 ml H2O/100 g dry weight. A comparable increase in myocardial water content was obtained when hearts were reperfused with 10 µM adenosine, and this affect could be blocked by 8-PT. Addition of apyrase alone to the reperfusion medium induced a small increase in myocardial water content, i.e. to 609±39 ml H2O/100 g dry weight, suggesting that adenosine concentrations as low as 0.1 µM already affect vascular permeability. In general the results on perfused hearts confirm those obtained on endothelial monolayers, that ATP when degraded by ectonucleotidases acts through its metabolite adenosine and that this effect is opposite to that of ATP.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Material and methods
 3. Results
 4. Discussion
References
 
Ischemia–reperfusion leads to myocardial edema which jeopardizes functional recovery of the heart during reperfusion [2,3] as a consequence of endothelial barrier failure of the coronary microvasculature. Here we demonstrate that extracellular ATP either released from the cells or exogenously applied reduces reperfusion-induced endothelial barrier failure. However, when ATP is degraded, the barrier stabilizing effect of ATP is overruled by the permeability promoting effect of its metabolite adenosine. Manoeuvres preventing ATP degradation or inhibiting adenosine receptor activation preserve the stabilizing effect of ATP on the endothelial barrier.

Previously, we showed that reperfusion of microvascular coronary endothelial cells leads to an increase in Cai accompanied by a marked increase in interendothelial gap formation. The effect of reperfusion on Cai and gaps exceeds markedly the effect of continued ischemic conditions, demonstrating a kind of acute reperfusion injury of the endothelial barrier [24]. This reperfusion-induced disturbance of endothelial barrier function is due to an activation of the endothelial contractile machinery. To date, strategies to interfere with this form of reperfusion-induced endothelial barrier failure have not been identified.

In the present study we showed that extracellular ATP protects against reperfusion-induced gap formation even though it provokes a further increase in Cai. The mechanistic explanation for this apparently paradoxical effect comes from our recent studies [6–8] which have shown that extracellular ATP is not only able to reduce endothelial permeability despite increasing cytosolic Ca2+ concentration but also to antagonize endothelial hyperpermeability caused by inflammatory mediators such as thrombin [8]. The detailed analysis has demonstrated that extracellular ATP reduces mechanical force development of the endothelial contractile machinery by interfering at the level of myosin light chains, by activation of MLC phosphatase in a Ca2+-independent manner [6]. As shown by us in another study [26], extracellular ATP may also activate MLC-kinase in an Ca2+-dependent step. However, this ATP effect on the contractile machinery is completely overruled by the strong activation of MLC phosphatase.

Consistent with this explanation is the finding of the present study that addition of ATP to the reperfusion medium can completely prevent the rise of MLC phosphorylation during reperfusion and may even lower the phosphorylation of MLC below the value found otherwise as a result of ischemic conditions. The reduction of MLC phosphorylation correlates with the suppression of reperfusion-induced gap formation. These results indicate that the protective effect of ATP during reperfusion on the endothelial barrier is indeed due to an inhibition of the endothelial contractile machinery.

It is well documented that ATP is released from vascular cells and under a variety of (patho)physiological conditions [9,11,15,16,27]. The nature of the ATP release mechanism is not yet fully understood. We found here that reperfusion conditions cause a significant release of ATP from endothelial cells which is neither found under normoxic control conditions nor during simulated ischemia. The accumulation of ATP in the extracellular medium could be enhanced when the ectonucleotidase inhibitor ARL was added to the reperfusion medium. These results show that reperfusion conditions cause release of ATP from endothelial cells which normally is rapidly degraded by ectonucleotidases. ARL alone did not lead to a significant ATP accumulation in normoxic-perfused endothelial monolayers without preceding ischemia, indicating that there is indeed no or very little ATP release under normoxic control conditions.

The most effective manoeuvre to stabilize the endothelial barrier was to apply extracellular ATP in presence of ARL, which prevents ATP degradation. A reduction of gap formation was also found, if endothelial cells were reperfused in presence of ARL alone, i.e. under conditions favouring accumulation of released endogenous ATP. These data demonstrate that ATP released from endothelial cells under this pathophysiological cell stress can contribute to a stabilization of barrier function.

It is well documented that the ATP metabolite adenosine can induce a loss of barrier function in certain monolayer models of microvascular endothelial cells [20,21,28] and in various intact microvessels [29–31]. As we showed before for coronary endothelial cells, this is due to an effect on cell adhesion structures, not on the contractile machinery [21]. These results led us to analyse the effects of adenosine during simulated ischemia–reperfusion in the same model of cultured coronary microvascular endothelial cells. Addition of adenosine enhanced the reperfusion-induced increase in gap formation. This effect could be blocked by simultaneous addition of the adenosine receptor antagonist 8-PT indicating that this gap-forming effect of adenosine is due to activation of adenosine receptors. Reperfusion-induced gap formation was also increased when the soluble ectonucleotidase apyrase was added to the reperfusion medium to enhance degradation of ATP to adenosine. Apyrase sequentially catalyses the hydrolysis of ATP to AMP, which is then converted to adenosine by action of the endothelial ecto-5'-nucleotidase (CD73). These data show that in endothelial monolayers derived from the coronary microvasculature reperfusion-induced gap forming is enhanced by adenosine, whereas ATP itself has an attenuating effect.

The effect of ATP on barrier function was also examined in the coronary system of the isolated, saline-perfused whole heart. In this model ATP caused a strong reduction of the reperfusion-induced increase in myocardial water content if applied in combination with the NTPDase inhibitor ARL during reperfusion. These data indicate that extracellular ATP is also a potent stabilizer of the vascular permeability barrier in the intact coronary system and that its application during reperfusion can protect the heart against an imminent reperfusion-induced edema. In the intact coronary system, however, this beneficial effect of ATP was only seen when it was applied in combination with ARL, the NTPDase inhibitor. Otherwise, ATP caused an increase in myocardial tissue edema. Measurements of ATP in the effluate during reperfusion of the isolated rat hearts showed a 79% disappearance of the ATP infused on the arterial side. This points towards a fast ATP degradation in the vascular system most probably via NTPDase1 which is the dominant ectonucleotidase in microvascular coronary endothelial cells [32,33]. In contrast to the situation in the cell culture model, in the perfused hearts a protective effect of the NTPDase inhibitor ARL, stabilizing the released endogenous ATP, was not observed. This seems due to the small value of medium concentration of ATP generated by the spontaneous release in the microvascular lumen at the given high flow rates of the saline-perfused heart model.

Not ATP itself but its metabolite adenosine induced an increase in myocardial water content acting via adenosine receptors. The data of this study show that the edema increasing effect of added ATP is completely blocked in presence of the adenosine receptor antagonist 8-PT. Myocardial edema formation could be additionally increased if ATP was applied in combination with soluble apyrase to accelerate its degradation. Direct application of adenosine caused a similar oedematous effect. This oedematous effect could also be blocked by simultaneous addition of 8-PT. It was postulated in the literature that adenosine receptor antagonists block ischemia–reperfusion injury in the lung [34] and in the heart [35] and have protective effects on postischemic damage to the coronary microcirculation [36]. In other vascular provinces this is apparently different. In ischemic-reperfused intestine, generation of adenosine is protective, and absence of NTPDase1 favours capillary leakage and tissue damage [37]. In a general pathophysiological context effects of adenosine other than the direct actions on the endothelium must be considered too, e.g. its vasodilatory effect and the helpful adverse effects on platelet aggregation and leukocyte adhesion.

The comparison of gap formation in the cell culture model and edema development in the intact, but saline-perfused heart should not be over-stretched. First, cultured coronary cells are not the same as coronary cells in situ. Second, the perfusion conditions are quite different, with a much higher ratio of volume flux to cell mass in the culture system. Third, tissue water uptake is only a coarse parameter for endothelial permeability which does not differentiate between the extravascular subcompartments filling up with water. But in spite of these limitations and the relative differences seen between these two experimental models, the results from both speak the same general language in respect to the ATP mediated protection.

In conclusion, extracellular ATP effectively protects the endothelial barrier against reperfusion-induced failure, in cultured endothelial monolayers as well as in the coronary system in situ, whereas its degradation product adenosine increases reperfusion-induced hyperpermeability. Therefore, additional inhibition of ATP degradation, released by endothelial cells in effective amounts during reperfusion, increases this protective effect. While these results identify a novel protective principle, it remains unclear how far this can be directly exploited for therapeutic use. First, in microcirculatory beds other than the coronary system adenosine improves endothelial barrier function, as shown for example for intestinal microvessels [38]. This however, is not of relevance if ATP or ectonucleotidase inhibitors are applied selectively to the coronaries. Second, ATP has adverse effects on vascular tone and platelet function which may be protective during reperfusion. ATP released from endothelial cells in response to shear stress or hypoxia induces endothelium-dependent vasodilatation [39]. Application of ATP plus ectonucleotidase inhibition can counteract the vasoconstriction induced during ischemia–reperfusion and may enhance reperfusion of the ischemic tissue. Furthermore, ATP can act as a week antagonist of the ADP-induced platelet activation [40]. However, this effect may not be so relevant, since patients receiving reperfusion therapy are mostly under antiaggregatory therapy anyway. Finally, even if enhancing ATP effects in the reperfused coronary circulation in vivo would overall not be advisable, the results of the present study are important. They identify for the first time principle of protecting the endothelium directly against reperfusion-induced barrier failure, and it may be parts of the ATP signalling directed towards myosin-light chain phosphatase that are exploitable for future clinical use.


   Acknowledgements
 
The study was supported by the Deutsche Forschungsgesellschaft, Project A3, A4 of SFB 547 and the Graduiertenkolleg 534. The technical support by H. Holzträger, A. Reis, and A. Seipp is gratefully acknowledged. This work is a part of the thesis submitted by S. Kasseckert.


   Notes
 
1 Contributed equally. Back

* Hiroshi Watanabe of Hamamatsu University School of Medicine (Hamamatsu, Japan) served as Guest Editor for this article.

Time for primary review 22 days


   References
Top
 Abstract
 1. Introduction
 2. Material and methods
 3. Results
 4. Discussion
 References
 

  1. Tranum-Jensen J., Janse M.J., Fiolet W.T., Krieger W.J., D'Alnoncourt C.N., Durrer D. Tissue osmolality, cell swelling, and reperfusion in acute regional myocardial ischemia in the isolated porcine heart. Circ Res (1981) 49:364–381.[Free Full Text]
  2. Garcia-Dorado D., Oliveras J. Myocardial oedema: a preventable cause of reperfusion injury? Cardiovasc Res (1993) 27:1555–1563.[Free Full Text]
  3. Mehlhorn U., Geissler H.J., Laine G.A., Allen S.J. Myocardial fluid balance. Eur J Cardiothorac Surg (2001) 20:1220–1230.[Abstract/Free Full Text]
  4. Schäfer C., Walther S., Schäfer M., Dieterich L., Kasseckert S., Abdallah Y., et al. Inhibition of contractile activation reduces reoxygenation-induced endothelial gap formation. Cardiovasc Res (2003) 58:149–155.[Abstract/Free Full Text]
  5. Goeckeler Z.M., Wysolmerski R.B. Myosin light chain kinase-regulated endothelial cell contraction: the relationship between isometric tension, actin polymerization, and myosin phosphorylation. J Cell Biol (1995) 130:613–627.[Abstract/Free Full Text]
  6. Noll T., Schäfer M., Schavier-Schmitz U., Piper H.M. ATP induces dephosphorylation of myosin light chain in endothelial cells. Am J Physiol Cell Physiol (2000) 279:C717–C723.[Abstract/Free Full Text]
  7. Noll T., Hölschermann H., Koprek K., Gündüz D., Haberbosch W., Tillmanns H., et al. ATP reduces macromolecule permeability of endothelial monolayers despite increasing [Ca2+]i. Am J Physiol (1999) 276:H1892–H1901.[ISI][Medline]
  8. Gündüz D., Hirche F., Härtel F.V., Rodewald C.W., Schäfer M., Pfitzer G., et al. ATP antagonism of thrombin-induced endothelial barrier permeability. Cardiovasc Res (2003) 59:470–478.[Abstract/Free Full Text]
  9. Bodin P., Burnstock G. Increased release of ATP from endothelial cells during acute inflammation. Inflamm Res (1998) 47:351–354.[CrossRef][ISI][Medline]
  10. Gordon E.L., Pearson J.D., Slakey L.L. The hydrolysis of extracellular adenine nucleotides by cultured endothelial cells from pig aorta. Feed-forward inhibition of adenosine production at the cell surface. J Biol Chem (1986) 261:15496–15507.[Abstract/Free Full Text]
  11. Bodin P., Burnstock G. Evidence that release of adenosine triphosphate from endothelial cells during increased shear stress is vesicular. J Cardiovasc Pharmacol (2001) 38:900–908.[CrossRef][ISI][Medline]
  12. Yegutkin G., Bodin P., Burnstock G. Effect of shear stress on the release of soluble ecto-enzymes ATPase and 5'-nucleotidase along with endogenous ATP from vascular endothelial cells. Br J Pharmacol (2000) 129:921–926.[CrossRef][ISI][Medline]
  13. Taylor A.L., Kudlow B.A., Marrs K.L., Gruenert D.C., Guggino W.B., Schwiebert E.M. Bioluminescence detection of ATP release mechanisms in epithelia. Am J Physiol (1998) 275:C1391–C1406.[ISI][Medline]
  14. Schwiebert L.M., Rice W.C., Kudlow B.A., Taylor A.L., Schwiebert E.M. Extracellular ATP signaling and P2X nucleotide receptors in monolayers of primary human vascular endothelial cells. Am J Physiol Cell Physiol (2002) 282:C289–C301.[Abstract/Free Full Text]
  15. Bergfeld G.R., Forrester T. Release of ATP from human erythrocytes in response to a brief period of hypoxia and hypercapnia. Cardiovasc Res (1992) 26:40–47.[Abstract/Free Full Text]
  16. Clemens M.G., Forrester T. Appearance of adenosine triphosphate in the coronary sinus effluent from isolated working rat heart in response to hypoxia. J Physiol (1981) 312:143–158.[Abstract/Free Full Text]
  17. Kaczmarek E., Koziak K., Sevigny J., Siegel J.B., Anrather J., Beaudoin A.R., et al. Identification and characterization of CD39/vascular ATP diphosphohydrolase. J Biol Chem (1996) 271:33116–33122.[Abstract/Free Full Text]
  18. Zimmermann H. Extracellular metabolism of ATP and other nucleotides. Naunyn-Schmiedeberg's Arch Pharmacol (2000) 362:299–309.[CrossRef][ISI][Medline]
  19. Watanabe H., Kuhne W., Schwartz P., Piper H.M. A2-adenosine receptor stimulation increases macromolecule permeability of coronary endothelial cells. Am J Physiol (1992) 262:H1174–H1181.[ISI][Medline]
  20. Hempel A., Noll T., Muhs A., Piper H.M. Functional antagonism between cAMP and cGMP on permeability of coronary endothelial monolayers. Am J Physiol (1996) 270:H1264–H1271.[Medline]
  21. Bindewald K., Gündüz D., Härtel F., Peters S.C., Rodewald C., Nau S., et al. Opposite effect of cAMP signaling in endothelial barriers of different origin. Am J Physiol Cell Physiol (2004) 287:C1246–C1255.[Abstract/Free Full Text]
  22. Crack B.E., Pollard C.E., Beukers M.W., Roberts S.M., Hunt S.F., Ingall A.H., et al. Pharmacological and biochemical analysis of FPL 67156, a novel, selective inhibitor of ecto-ATPase. Br J Pharmacol (1995) 114:475–481.[ISI][Medline]
  23. Westfall T.D., Kennedy C., Sneddon P. Enhancement of sympathetic purinergic neurotransmission in the guinea-pig isolated vas deferens by the novel ecto-ATPase inhibitor ARL 67156. Br J Pharmacol (1996) 117:867–872.[ISI][Medline]
  24. Schäfer C., Walther S., Schäfer M., Dieterich L., Kasseckert S., Abdallah Y., et al. Inhibition of contractile activation reduces reoxygenation-induced endothelial gap formation. Cardiovasc Res (2003) 58:149–155.[Abstract/Free Full Text]
  25. Noll T., Wozniak G., McCarson K., Hajimohammad A., Metzner H.J., Inserte J., et al. Effect of factor XIII on endothelial barrier function. J Exp Med (1999) 189:1373–1382.[Abstract/Free Full Text]
  26. Klingenberg D., Gündüz D., Härtel F., Bindewald K., Schäfer M., Piper H.M., et al. MEK/MAPK as a signaling element in ATP control of endothelial myosin light chain. Am J Physiol Cell Physiol (2004) 286:C807–C812.[Abstract/Free Full Text]
  27. Bodin P., Burnstock G. Purinergic signalling: ATP release. Neurochem Res (2001) 26:959–969.[CrossRef][ISI][Medline]
  28. Watanabe H., Kuhne W., Spahr R., Schwartz P., Piper H.M. Macromolecule permeability of coronary and aortic endothelial monolayers under energy depletion. Am J Physiol (1991) 260:H1344–H1352.[ISI][Medline]
  29. Gawlowski D.M., Duran W.N. Dose-related effects of adenosine and bradykinin on microvascular permselectivity to macromolecules in the hamster cheek pouch. Circ Res (1986) 58:348–355.[Abstract/Free Full Text]
  30. Rubinstein I., Chandilawa R., Dagar S., Hong D., Gao X.P. Adenosine A(1) receptors mediate plasma exudation from the oral mucosa. J Appl Physiol (2001) 91:552–560.[Abstract/Free Full Text]
  31. Murray M.A., Heistad D.D., Mayhan W.G. Role of protein kinase C in bradykinin-induced increases in microvascular permeability. Circ Res (1991) 68:1340–1348.[Abstract/Free Full Text]
  32. Enjyoji K., Sevigny J., Lin Y., Frenette P.S., Christie P.D., Esch J.S., et al. Targeted disruption of cd39/ATP diphosphohydrolase results in disordered hemostasis and thromboregulation. Nat Med (1999) 5:1010–1017.[CrossRef][ISI][Medline]
  33. Sevigny J., Sundberg C., Braun N., Guckelberger O., Csizmadia E., Qawi I., et al. Differential catalytic properties and vascular topography of murine nucleoside triphosphate diphosphohydrolase 1 (NTPDase1) and NTPDase2 have implications for thromboregulation. Blood (2002) 99:2801–2809.[Abstract/Free Full Text]
  34. Neely C.F., Keith I.M. A1 adenosine receptor antagonists block ischemia–reperfusion injury of the lung. Am J Physiol (1995) 268:L1036–L1046.[ISI][Medline]
  35. Neely C.F., DiPierro F.V., Kong M., Greelish J.P., Gardner T.J. A1 adenosine receptor antagonists block ischemia–reperfusion injury of the heart. Circulation (1996) 94:II376–II380.[Medline]
  36. Di Napoli P., Di Crecchio A., Taccardi A.A., Di Muzio M., Statile D., Maggi A., et al. Effect of A1 adenosine receptor blockade on postischemic damage to the coronary microcirculation. Cardiologia (1998) 43:387–393.[Medline]
  37. Guckelberger O., Sun X.F., Sevigny J., Imai M., Kaczmarek E., Enjyoji K., et al. Beneficial effects of CD39/ecto-nucleoside triphosphate diphosphohydrolase-1 in murine intestinal ischemia–reperfusion injury. Thromb Haemost (2004) 91:576–586.[ISI][Medline]
  38. Michel C.C., Curry F.E. Microvascular permeability. Physiol Rev (1999) 79:703–761.[Abstract/Free Full Text]
  39. Burnstock G., Knight G.E. Cellular distribution and functions of P2 receptor subtypes in different systems. Int Rev Cytol (2004) 240:31–304.[ISI][Medline]
  40. Park H.S., Hourani S.M. Differential effects of adenine nucleotide analogues on shape change and aggregation induced by adnosine 5-diphosphate (ADP) in human platelets. Br J Pharmacol (1999) 127:1359–1366.[CrossRef][ISI][Medline]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 CirculationHome page
A. L. Moens, H. C. Champion, M. J. Claeys, B. Tavazzi, P. M. Kaminski, M. S. Wolin, D. J. Borgonjon, L. Van Nassauw, A. Haile, M. Zviman, et al.
High-Dose Folic Acid Pretreatment Blunts Cardiac Dysfunction During Ischemia Coupled to Maintenance of High-Energy Phosphates and Reduces Postreperfusion Injury
Circulation, April 8, 2008; 117(14): 1810 - 1819.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Gündüz, D.
Right arrow Articles by Schäfer, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gündüz, D.
Right arrow Articles by Schäfer, C.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?