Skip Navigation

Cardiovascular Research 2005 65(4):803-812; doi:10.1016/j.cardiores.2004.12.004
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 Xu, Z.
Right arrow Articles by Boysen, P. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Xu, Z.
Right arrow Articles by Boysen, P. G.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2004, European Society of Cardiology

Adenosine produces nitric oxide and prevents mitochondrial oxidant damage in rat cardiomyocytes

Zhelong Xua,*, Sung-Sik Parka, Robert A. Muellera,b, Robert C. Bagnellc, Cam Pattersond and Philip G. Boysena

aDepartment of Anesthesiology, CB#7010 University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7010, USA
bDepartment of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
cDepartment of Pathology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
dDepartment of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA

* Corresponding author. Tel.: +1 919 843 4174; fax: +1 919 843 3805. Email address: zxu{at}aims.unc.edu

Received 21 September 2004; revised 16 November 2004; accepted 7 December 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgment
 References
 
Objective: To examine if adenosine prevents oxidant-induced mitochondrial dysfunction by producing nitric oxide (NO) in cardiomyocytes.

Methods and results: Adenosine significantly enhanced the fluorescence of DAF-FM, a dye specific for NO, implying that adenosine induces synthesis of NO. Adenosine-induced NO production was blocked by both the nonspecific NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) and N5-(1-Iminoethyl)-L-ornithine dihydrochloride (L-NIO), an inhibitor of endothelial NOS (eNOS), but not by N6-(1-Iminoethyl)-L-lysine hydrochloride (L-NIL), an inhibitor of inducible NOS (iNOS), indicating that adenosine activates eNOS. Adenosine also enhances eNOS phosphorylation and its activity. The adenosine A2 receptor antagonist 8-(3-chlorostyryl)caffeine but not the A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine prevented the increase in NO production. CGS21680, an adenosine A2 receptor agonist, markedly increased NO, further supporting the involvement of A2 receptors. Adenosine-induced NO production was blocked by 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine (PP2), a selective Src tyrosine kinase inhibitor, suggesting that Src tyrosine kinase is crucial for adenosine-induced NO production. Adenosine-induced NO production was partially reversed by both wortmannin and Akt inhibitor indicating an involvement of PI3-kinase/Akt. Pretreatment of cells with adenosine prevented H2O2-induced depolarization of mitochondrial membrane potential ({Delta}{Psi}m). The protective effect was blocked by L-NAME and L-NIO but not by L-NIL, indicating that eNOS plays a role in the action of adenosine. The protective effect of adenosine was further suppressed by KT5823, a specific inhibitor of protein kinase G (PKG), indicating the PKG may serve as a downstream target of adenosine.

Conclusion: Adenosine protects mitochondria from oxidant damage through a pathway involving A2 receptors, eNOS, NO, PI3-kinase/Akt, and Src tyrosine kinase.

KEYWORDS Adenosine; Nitric oxide; Mitochondria; Signal transduction

This article is referred to in the Editorial by J.S. Karliner (pages 763–765) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Acknowledgment
 References
 
It has been well documented that adenosine protects the myocardium from ischemia–reperfusion injury [1,2]. Although PKC and mitochondrial KATP channels seem to be crucial, the mechanisms by which adenosine protects myocardium remain unclear [3]. It has been reported that eNOS plays a role in adenosine A1 receptor-induced delayed preconditioning in mouse hearts [4]. Recently, Yang et al. demonstrated that the protective effect of NECA, an agonist of adenosine, on reperfusion injury was abolished by L-NAME in intact rabbit hearts, suggesting a possible involvement of NO in the action of NECA [5]. Nonetheless, there has also been evidence that NO is not responsible for adenosine-induced early preconditioning [6] or adenosine-induced protective effect on reperfusion injury [7]. Although the reason for this discrepancy is not clear, the role of NO was determined by either suppressing NOS activity or detecting NOS expression, without direct evidence of whether adenosine verily altered intracellular NO concentration in cardiomyocytes or not. Accordingly, direct measurements of the change in cardiomyocyte NO concentration would greatly help ascertain the actual role of NO in the cardioprotective effect of adenosine. Moreover, given that adenosine enhances NO generation, it is tempting to determine the mechanisms underlying this phenomenon.

Reactive oxygen species (ROS) are involved in the pathogenesis of reperfusion injury [8], and have been reported to cause mitochondrial dysfunction in cardiomyocytes [9]. Recently mitochondrial dysfunctions such as {Delta}{Psi}m dissipation, matrix Ca2+ overload and mitochondrial permeability transition (MPT) pore opening have been proposed to be associated with the mechanism for ischemia–reperfusion injury and modulation of the mitochondrial dysfunctions may lead to cardioprotection [10]. It has been addressed that exogenous NO exerts cardioprotection by modulating mitochondrial function [11]. NO was also shown to reduce cytochrome c release by inhibiting MPT pore opening [12]. Thus, it is plausible that adenosine prevents mitochondrial oxidant damage by elevating availability of NO within cardiomyocytes.

In the present study, we first tested whether adenosine can increase NO production in isolated rat cardiomyocytes. We then sought to investigate the detailed mechanisms whereby adenosine produces NO. Lastly, we determined whether adenosine could modulate mitochondrial oxidant damage, and whether NO mediates the effect of adenosine.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Acknowledgment
 References
 
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1996).

2.1. Isolation of adult rat cardiomyocytes
Male Wistar rats weighing 250–350 g were anesthetized with thiobutabarbital sodium (100 mg/kg, i.P.). A midline thoracotomy was performed and the heart was removed and rapidly mounted on a Langendorff apparatus. The heart was perfused in a non-recirculating mode with Krebs–Henseleit buffer (37 °C) containing (in mM) NaCl 118, NaHCO3 25, KCl 4.7, KH2PO4 1.2, MgSO4 1.2, CaCl2 1.25, and glucose 10 for 5 min to wash out blood. The buffer was bubbled with 95% O2/5% CO2. Then the heart was perfused with calcium-free buffer that contained all of the above components except CaCl2. After 5 min of perfusion, collagenase (type II) was added to the buffer (0.1%) and the heart was perfused in a recirculating mode for ~15 min. The heart was removed from the apparatus and the ventricles were placed into a beaker containing the calcium-free buffer. The ventricles were agitated in a shaking bath (37 °C) at a rate of 50 cycles/min until individual cells were released. The released cells were suspended in an incubation buffer containing all the components of the calcium-free buffer, 1% bovine serum albumin, 30 mM HEPES, 60 mM taurine, 20 mM creatine, and amino acid supplements at 37 °C. Calcium was gradually added to the buffer containing the cells to a final concentration of 1.2 mM. The cells were filtered through nylon mesh and centrifuged briefly. The cell viability was determined using trypan blue. Finally the cells were suspended in culture medium M199 for 4 h before experiments.

2.2. Confocal imaging of DAF-FM
To measure intracellular NO concentration, cardiomyocytes (1 x 105 cells) cultured in a specific temperature-controlled culture dish were incubated with 2 µM DAF-FM diacetate in standard Tyrode solution containing (mM) NaCl 140, KCl 6, MgCl2 1, CaCl2 1, HEPES 5, and glucose 5.8 (pH 7.4) for 20 min. The cells were then mounted on the stage of an Olympus FV 500 laser scanning confocal microscope. The cells were randomly selected and imaged. The green fluorescence was excited at 488 nm and imaged through a 525 nm long-path filter. Temperature was maintained at 37 °C with Delta T Open Dish Systems (Bioptechs, Butler, PA). The images recorded on a computer were quantified using Image J.

2.3. Confocal imaging of {Delta}{Psi}m
{Delta}{Psi}m was measured by loading cardiomyocytes with tetramethylrhodamine ethyl ester (TMRE, 100 nM). The red fluorescence was excited with a 543 nm line of argon–krypton laser line and imaged through a 560 nm long-path filter.

2.4. Western blotting analysis of phosphorylated eNOS activity
After exposure to adenosine (100 µM) for 10 min, cells were homogenized in ice-cold lysis buffer. Equal amounts of protein were loaded and electrophoresed on 4% SDS–polyacrylamide gel and transferred to a PVDF membrane. The membrane was probed with the primary antibody that recognizes phospho-eNOS (Ser 1177). The primary antibody binding was detected with a secondary anti-rabbit antibody and visualized by the ECL method.

2.5. NOS activity assay
NOS activity was determined by measuring the conversion of L-[3H]arginine to L-[3H]citrulline with the NOS activity assay kit from Calbiochem. Briefly, cardiomyocytes were homogenized in homogenization buffer (25 mM Tris–HCl, 1 mM EDTA, and 1 mM EGTA) and pelleted at 14,000 x g for 5 min at 4 °C. To determine eNOS activity, the supernatant (40 µg of protein per assay) was incubated for 30 min at 30 °C in 40 µl of reaction mix containing 25 µl of 2 x reaction buffer (50 mM Tris–HCl, 6 µM tetrahydrobiopterin, 2 µM FAD, and 2 µM FMN), 5 µl of 10 mM NADPH, 1 µl of 3H-arginine (1 µCi/µl), 5 µl of 6 mM CaCl2, and 4 µl of H2O. To determine iNOS activity (Ca2+-independent), the assay was conducted in the presence of 2 mM EGTA without CaCl2. The reaction was stopped by adding 400 µl of stop buffer containing 50 mM HEPES and 5 mM EDTA, and the reaction mixture containing 100 µl of resin was transferred to a spin cup. The mixture was centrifuged for 30 s, and the eluate was added to a scintillation vial containing 5 ml of scintillation fluid. Finally, the radioactivity was quantified in a liquid scintillation counter.

2.6. Experimental protocol
In the experiments measuring NO generation, adenosine (or CGS21680) was given immediately after baseline (time 0') measurements, whereas the inhibitors were given 10 min prior to the application of adenosine (Fig. 1A). The measurements were terminated 10 min after the baseline recordings. In the study evaluating the effect of exogenous zinc on {Delta}{Psi}m (Fig. 1B), cardiomyocytes were exposed to 100 µM H2O2 for 20 min to cause mitochondrial oxidant damage. Adenosine (or SNAP) was given 10 min before exposure to H2O2.


Figure 1
View larger version (23K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Experimental protocol.

 
2.7. Statistical analysis
Data are expressed as mean ± S.E.M. and were obtained from 5 to 10 separate experiments. Statistical significance was determined using Student's t-test or one way ANOVA followed by Tukey's test. A value of P<0.05 was considered as statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Acknowledgment
 References
 
To determine the exact role of NO in the cardioprotective effect of adenosine, we tested whether adenosine could induce NO production by loading cardiomyocytes with the NO-specific fluorescence dye DAF-FM diacetate. This membrane permeant probe is converted by intracellular esterases to DAF-FM, which reacts with NO to form a green-fluorescent product. DAF-FM has been reported to be useful for detection of NO in living cells [13,14]. Fig. 2A shows confocal images of cells loaded with DAF-FM. Compared to the control cells, cells treated with adenosine revealed a dramatic increase in DAF-FM fluorescence, indicating that adenosine can trigger NO production. Fig. 2B shows that adenosine increases NO production in a dose-dependent manner. The increase peaked with 100 µM adenosine. Fig. 3 shows that adenosine (100 µM)-induced increase in DAF-FM fluorescence was reversed by the NOS inhibitor L-NAME (200 µM), implying that adenosine increased NO production via NOS. To verify the specific NOS subtype that is responsible for the action of adenosine, we then treated cells with adenosine in the presence of either the selective iNOS inhibitor L-NIL (100 µM) or the potent eNOS inhibitor L-NIO (10 µM). As shown in Fig. 3, the increased DAF-FM signal was abolished by L-NIO but not by L-NIL, suggesting that eNOS but not iNOS accounts for adenosine-induced NO production. To confirm that eNOS is responsible for adenosine-induced NO production, we next determined if adenosine can activate eNOS. As shown in Fig. 4, cells treated with adenosine (100 µM) for 10 min indeed had a significant increase in phosphorylation of eNOS at Ser1177. We then determined if adenosine could alter NOS activity by measuring the conversion of L-[3H]arginine to L-[3H]citrulline. As shown in Fig. 5A, adenosine (100 µM) significantly increased eNOS activity, confirming the role of eNOS in NO generation. In contrast, adenosine had no effect on iNOS activity (Fig. 5B), supporting the above observation that eNOS but not iNOS is responsible for the action of adenosine on NO generation.


Figure 2
View larger version (21K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 (A) Confocal fluorescence images of cardiomyocytes loaded with DAF-FM. Adenosine (100 µM) dramatically increased fluorescence of DAF-FM. (B) Summarized data for DAF-FM fluorescence intensity 10 min after exposure to adenosine expressed as percentage of baseline. Six cells were imaged in each group. Adenosine enhanced NO production in a dose-dependent manner with the peak at 100 µM. *P<0.05 vs. control.

 

Figure 3
View larger version (23K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Summarized data for DAF-FM fluorescence intensity 10 min after exposure to adenosine (100 µM). The effect of adenosine (n=9) on NO production was reversed by both L-NAME (200 µM, n=6) and L-NIO (10 µM, n=7) but not by L-NIL (100 µM, n=6). Ado, adenosine. *P<0.05 vs. control; #P<0.05 vs. adenosine.

 

Figure 4
View larger version (26K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Western blotting analysis of phospho-eNOS (Ser 1177) activity in cardiomyocytes. Adenosine (100 µM) significantly increased eNOS phosphorylation (n=6). *P<0.05 vs. control.

 

Figure 5
View larger version (16K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 NOS activity in cardiomyocytes. (A) Adenosine (100 µM, n=6) significantly increases eNOS activity, which was blocked by both AKTI (partially, n=6) and PP2 (completely, n=6). Cells were exposed to adenosine for 10 min. The inhibitors were given 10 min prior to the treatment with adenosine. (B) Adenosine (100 µM, n=6) does not alter iNOS activity. *P<0.05 vs. control; #P<0.05 vs. adenosine.

 
Adenosine receptors are divided into three major subtypes (A1, A2 and A3), and adenosine primarily activates A1 and A2 receptors [15]. To clarify the specific receptor that is responsible for the action of adenosine, we tested if specific subtype adenosine receptor antagonists can alter the effect of adenosine on NO production. As shown in Fig. 6, the adenosine-induced increase in the fluorescence was abolished by the adenosine A2 receptor antagonist CSC (10 µM) but not by the A1 receptor antagonist DPCPX (1 µM). Furthermore, the adenosine A2 receptor agonist CGS21680 (100 nM) mimicked the effect of adenosine to increase NO production, confirming that the activation of A2 receptor accounts for the action of adenosine.


Figure 6
View larger version (23K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 DAF-FM fluorescence intensity 10 min after exposure to adenosine (100 µM). The effect of adenosine on NO production was abolished by the adenosine A2 receptor antagonist CSC (10 µM, n=8) but not by A1 receptor antagonist DPCPX (1 µM, n=7). The adenosine A2 receptor agonist CGS21680 (100 nM, n=6) mimicked the effect of adenosine to increase NO generation. Ado, adenosine; CGS, CGS21680. *P<0.05 vs. control; #P<0.05 vs. adenosine.

 
Akt/PKB mediates NO production in endothelial cells [16] and adenosine activates PI3-kinase in myocardium [17]. Therefore, it is possible that adenosine produces NO through activation of the PI3-kinase/Akt signal pathway. Fig. 7 indicates that adenosine-induced NO production was abolished partially but significantly by both the PI3-kinase inhibitors (wortmannin and LY294002) and the Akt inhibitor (10 µM). The involvement of Akt signal in the action of adenosine was also evidenced by the observation that adenosine-induced eNOS phosphorylation was blocked by the Akt inhibitor (Fig. 4). Since Src tyrosine kinase has been reported as a critical upstream signal of PI3-kinase/Akt induced eNOS activation [18,19], we tested if it is involved in the action of adenosine on NO production. Fig. 8 shows that the adenosine-induced increase in the green fluorescence was suppressed by both the nonspecific tyrosine kinase inhibitor genistein (100 µM) and the specific Src tyrosine kinase inhibitor PP2 (1 µM), indicating that Src tyrosine kinase may also play a role in the mechanism by which adenosine induces NO production. The importance of Src tyrosine kinase in NO production by adenosine can also be found in another experiment (Fig. 5) in which PP2 completely reversed the effect of adenosine on eNOS activity.


Figure 7
View larger version (23K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 7 DAF-FM fluorescence intensity 10 min after exposure to adenosine (100 µM). The effect of adenosine on NO production was partially but significantly suppressed by the PI3-kinase inhibitors wortmannin (100 nM, n=10) and LY294002 (10 µM, n=6). The AKT inhibitor (10 µM, n=9) also prevented the increase in NO generation by adenosine. Ado, adenosine; Wort, wortmannin; LY, LY294002; AKTI, Akt inhibitor. *P<0.05 vs. control; #P<0.05 vs. adenosine.

 

Figure 8
View larger version (18K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 8 DAF-FM fluorescence intensity 10 min after exposure to adenosine (100 µM). The effect of adenosine on NO production was blocked by both the tyrosine kinase inhibitor genistein (100 µM, n=7) and the selective Src tyrosine kinase inhibitor PP2 (1 µM, n=7). Ado, adenosine; Gen, genistein. *P<0.05 vs. control; #P<0.05 vs. adenosine.

 
To determine the physiological significance of the effect of adenosine on NO production, we tested if adenosine can modulate mitochondrial dysfunction caused by oxidant stress. Fig. 9A shows confocal images of cardiomyocytes loaded with the {Delta}{Psi}m indicator TMRE. The control cell exposed to 200 µM H2O2 for 20 min revealed a marked decrease in TMRE intensity, indicating that oxidant stress induces a loss of {Delta}{Psi}m. Compared to the control, the cell treated with adenosine (100 µM) showed a much smaller change in TMRE fluorescence, implying that adenosine prevents the {Delta}{Psi}m loss. To test if NO is involved in the signaling mechanism by which adenosine prevents {Delta}{Psi}m loss, cells were pretreated with the NOS inhibitor L-NAME 10 min before the treatment with adenosine. As shown in Fig. 9A, L-NAME reversed the effect of adenosine on {Delta}{Psi}m, suggesting that NO may serve as a down stream signal of adenosine receptor activation. The effect of adenosine was also blocked by the eNOS inhibitor L-NIO but not by the iNOS inhibitor L-NIL, confirming that eNOS is essential in the action of adenosine. To further investigate the downstream signal that mediates the protective effect of adenosine, we tested if KT5323, a specific inhibitor of PKG, could alter the action of adenosine. As shown in the bottom of Fig. 9A, the effect of adenosine was blocked by KT5823, indicating that PKG plays a role in the action of adenosine. TMRE intensity at the end of experiments (20 min after exposure to H2O2) was expressed as % of baseline and summarized in Fig. 9B.


Figure 9
View larger version (20K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 9 (A) Confocal fluorescence images of TMRE at baseline and 20 min after exposure to H2O2 (200 µM) in rat cardiomyocytes. (B) Summarized data for TMRE fluorescence intensity 20 min after exposure to H2O2 expressed as percentage of baseline. The protective effect of adenosine (100 µM, n=6) was partially but significantly blocked by both L-NAME (200 µM, n=6), L-NIO (10 µM, n=6) and KT5823 (1 µM, n=6) but not by L-NIL (100 µM, n=6). Ado, adenosine. *P<0.05 vs. control; #P<0.05 vs. adenosine.

 
Having demonstrated that NO may play a role in the protective effect of adenosine, we were interested to test if exogenous NO can mimic the effect of adenosine. As shown in Fig. 10A, compared to control, cells treated with SNAP revealed much less decrease in TMRE fluorescence, indicating that exogenous NO can mimic the effect of adenosine to prevent oxidant-induced dissipation of {Delta}{Psi}m. This result further confirms the essential role of NO in the protective effect of adenosine.


Figure 10
View larger version (25K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 10 (A) Confocal fluorescence images of TMRE at baseline and 20 min after exposure to 200 µM H2O2 in rat cardiomyocytes. (B) Summarized data for TMRE fluorescence intensity 20 min after exposure to H2O2 showing that SNAP (20 µM, n=6) prevented dissipation of {Delta}{Psi}m caused by oxidant stress. *P<0.05 vs. control.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Acknowledgment
 References
 
Adenosine can induce NO production in endothelial cells [20–23] and smooth muscle cells [24,25]. Although Ikeda et al. have reported an indirect effect of adenosine on NO production by chronically augmenting interleukin-1β-induced NO synthesis in neonatal rat cardiomyocytes [26], there has been no data describing a direct and acute effect of adenosine on NO production in cardiomyocytes. In the present study, we have demonstrated for the first time that adenosine is able to directly produce NO in cardiomyocytes. NO is synthesized from L-arginine by three isoenzymes: nNOS, iNOS, and eNOS. Among these enzymes, the existence of iNOS and eNOS in cardiomyocytes has been well documented in the literature [27]. In the present study, adenosine-induced NO production was blocked by the potent eNOS inhibitor L-NIO [28,29] but not by the selective iNOS inhibitor L-NIL [30], suggesting eNOS but not iNOS is responsible the effect of adenosine on NO production. The importance of eNOS in NO production by adenosine is further supported by the observation that adenosine enhances phosphorylation of eNOS at its activation site (Ser 1177). Moreover, adenosine significantly increased eNOS but not iNOS activity, corroborating the above observation. It has been suggested that a large amount of NO produced by iNOS is toxic, whereas eNOS is a protective enzyme [31]. Therefore, it is highly likely that adenosine-induced NO generation through eNOS can result in cardioprotection, which may explain the cardioprotective effect of adenosine.

It has been demonstrated that adenosine enhances interleukin-1β-induced NO production through activation of adenosine A2 receptor [24,26]. Activation of adenosine A2 receptors could increase NO production in porcine coronary endothelial cells [21] and in human umbilical vein endothelial cells [23]. In addition, 5'-(N-cyclopropyl)-carboxam-idoadenosine (CPCA), a selective adenosine A2 receptor agonist, increases NO release in isolated perfused rat hearts [32]. The present results support these observations confirming that adenosine A2 receptors are responsible for adenosine-induced NO production. Since we have detected the changes in NO production by imaging rod-shaped single cardiomyocytes, the current data also suggest that adenosine directly increased NO production by stimulating A2 receptors on cardiomyocytes but not on endothelial cells.

Because adenosine A1 and A3 receptors but not A2 receptors are involved in ischemic preconditioning [33], it is unlikely that NO produced by adenosine (through activation of A2 receptors) plays a role in the mechanism underlying preconditioning. It has been proposed that activation of A2 receptors at reperfusion plays a role in protecting myocardium from reperfusion injury [34]. In canine hearts, CGS-23680 given at reperfusion significantly reduced infarct size [35]. In addition, Zhao et al. reported that adenosine at reperfusion can block ischemia–reperfusion-induced apoptosis by activating A2 receptors [36]. Recently, Xu et al. have demonstrated that AMP579, a novel adenosine A1/A2 receptor agonist, given at reperfusion significantly reduces infarct size in rabbit hearts [37,38], and that the activation of adenosine A2 receptors at reperfusion accounts for the protective effects of AMP579 [37]. Interestingly, Yang et al. have documented that the NECA-induced cardioprotection against infarction at reperfusion was blocked by both the adenosine A2 receptor antagonist CSC and the NOS inhibitor L-NAME [5]. Thus, it is tenable to propose that NO produced by the activation of adenosine A2 receptor may be involved in the mechanism by which adenosine or its analogues at reperfusion prevents reperfusion injury.

Akt is a downstream target of PI3-kinase [39], and the PI3-kinase/AKT signal pathway may mediate cardioprotection at reperfusion [40]. PI3-kinase/Akt is also important in phosphorylation of eNOS at Ser 1177 [16,41]. Therefore, it is possible that adenosine activates eNOS and thus generates NO through a pathway involving PI3-kinase and Akt. In the present study, adenosine-induced NO production was suppressed by the PI3-kinase inhibitors and Akt inhibitor, indicating an involvement of this pathway in NO generation. In addition, we have demonstrated that Src tyrosine kinase is also crucial for adenosine-induced NO production. It has been documented that Src tyrosine kinase serves as an essential upstream regulator of the estrogen-stimulated PI3-kinase/Akt/eNOS pathway [18]. Similarly, Src tyrosine kinase is an upstream stimulator of the PI3-kinase/Akt pathway that mediates high density lipoprotein (HDL)-induced eNOS activation [19]. It has been shown by others that acetylcholine and adenosine can activate PI3-kinase in an Src-tyrosine kinase dependent manner in rabbit hearts [17]. Therefore, it is conceivable that adenosine activates Src tyrosine kinase, which in turn activates PI3-kinase/Akt pathway leading to the activation of eNOS, although we could not determine the exact signaling sequence in the present study. In this study, we have also noted that adenosine-induced NO production was partially blocked by the inhibition of PI3-kinase/Akt pathway, making us speculate that there may be some other parallel signaling pathways contributing to Src tyrosine kinase-mediated eNOS activation by adenosine. Indeed, several different signal components such as HSP90 and PKA have been implicated in the activation of eNOS [42]. Interestingly, it was also proposed that MAP kinase serves as the downstream signal of Src tyrosine kinase leading to the activation of eNOS [19].

Mitochondria play an important role in both necrosis and apoptosis caused by ischemia–reperfusion. In isolated cardiomyocytes subjected to ischemia–reperfusion, the mitochondrial electron transport chain is the main source of ROS upon reperfusion [43]. The generated ROS not only cause cellular membrane damage but also induce mitochondrial dysfunction by causing mitochondrial membrane protein oxidation and lipid peroxidation [44,45]. In neonatal rat cardiomyocytes, H2O2 causes dissipation of {Delta}{Psi}m and accumulation of matrix Ca2+, which are crucial for cell death [9]. The dissipation of {Delta}{Psi}m causes uncoupling of oxidative phosphorylation resulting in a decrease in ATP synthesis. Therefore the loss of {Delta}{Psi}m may lead to necrosis, since cells die from necrosis when they cannot maintain adequate ATP [46]. It has been reported that Ca2+ is accumulated in the mitochondrial matrix during ischemia–reperfusion and the accumulation of Ca2+ can damage mitochondria either by inhibiting oxidative phosphorylation or by inducing MPT [10]. MPTP is characterized by a progressive permeabilization of the inner mitochondrial membrane, which gradually becomes permeable to protons, ions [47]. MPTP not only causes necrosis by accelerating cellular energy depletion but also induces apoptosis by releasing proapoptotic molecules [46]. Since mitochondrial dysfunctions (the loss of {Delta}{Psi}m, Ca2+ overload, and MPT) are so closely associated with the cell death pathways (both necrosis and apoptosis), interventions that prevent mitochondrial dysfunction could be cardioprotective. In the present study, adenosine attenuated oxidant-induced {Delta}{Psi}m dissipation. Since oxidant stress has been proposed to be an important mechanism of reperfusion injury [8], this finding may suggests that adenosine or its analogues is able to protect the heart from reperfusion injury by modulating mitochondrial functions. Our data have also shown that NO generated by eNOS but not by iNOS accounts for the protective effect of adenosine on mitochondrial oxidant damage. This finding is consistent with the observation that eNOS but not iNOS is responsible for adenosine-induced NO generation. We further found that the PKG inhibitor KT5823 can block the protective effect of adenosine, suggesting that PKG may play an important role in the action of adenosine on mitochondrial damage. PKG is an important downstream target of NO, and a recent study showed that cGMP attenuates oxidant-induced reduction of {Delta}{Psi}m, cytochrome c release, MPTP opening through PKG [48]. Thus, it is likely that adenosine protects mitochondria by activating PKG via NO. Although PKC and mitochondrial KATP channels are important in the protective effect of adenosine A1 receptor activation, their roles in A2 receptor activation remain unknown. In this study, the essential role of NO in the action of adenosine was further supported by the observation that the exogenous NO donor SNAP mimicked the effect of adenosine to prevent dissipation of {Delta}{Psi}m (Fig. 10). Thus, these findings together support the above-mentioned notion that NO produced by adenosine may be involved in the cardioprotection at reperfusion. It has been reported that the loss in NO release is responsible for the decreased adenosine protection against reperfusion injury of aging rat hearts [32]. In addition, NO has been implicated in the adenosine A1/A2 receptor agonist NECA-induced cardioprotection against reperfusion injury. Recently, Hausenloy and Yellon have proposed that NO produced from eNOS may play a role in the protection of reperfusion injury by blocking mitochondrial damage [40]. Therefore, it is reasonable to propose that NO mediates the protective effect of adenosine on mitochondrial oxidant damage, which may serve as a critical mechanism whereby reperfusion injury is reduced. Although NO is essential for the protective effect of adenosine, it should be mentioned that there may be some other signaling elements participating in the mechanism by which adenosine protects mitochondria, since the inhibitors of NOS could partially but not completely block the effect of adenosine. The observation that SNAP (75.3 ± 12.6 % of baseline) was less effective than adenosine (91.3 ± 4.9 % of baseline) in preventing dissipation of {Delta}{Psi}m further confirmed that some unknown signaling elements other than NO are also involved in the action of adenosine.

In summary (Fig. 11), we have demonstrated that adenosine increases NO production through activation of adenosine A2 receptor, Src tyrosine kinase, PI3-kinase and eNOS, and that NO may play a crucial role in the protective effect of adenosine on mitochondrial oxidant damage in adult rat cardiomyocytes. These findings will provide new insights into the mechanisms by which adenosine (or its analogue) protects myocardium at reperfusion.


Figure 11
View larger version (21K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 11 Signaling mechanism by which adenosine produces NO and prevents mitochondrial oxidant damage in cardiomyocytes. Src TK, Src tyrosine kinase; Gen, genistein; AKTI, Akt inhibitor.

 


   Acknowledgment
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgment
References
 
This study was supported by a grant from the AHA Mid-Atlantic Affiliate (0365534U) to Z.X. We thank Dr. James M. Downey at the University of South Alabama for critical reading of the manuscript and helpful discussions.


   Notes
 
Time for primary review 16 days


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

  1. Thornton J.D., Liu G.S., Olsson R.A., Downey J.M. Intravenous pretreatment with A1-selective adenosine analogues protects the heart against infarction. Circulation (1992) 85:659–665.[Abstract/Free Full Text]
  2. Toombs C.F., McGee D.S., Johnston W.E., Vinten-Johansen J. Myocardial protective effects of adenosine: infarct size reduction with pretreatment and continued receptor stimulation during ischemia. Circulation (1992) 86:986–994.[Abstract/Free Full Text]
  3. Mubagwa K., Flameng W. Adenosine, adenosine receptors and myocardial protection: an updated overview. Cardiovasc. Res. (2001) 52:25–39.[Abstract/Free Full Text]
  4. Bell R.M., Smith C.C., Yellon D.M. Nitric oxide as a mediator of delayed pharmacological (A(1) receptor triggered) preconditioning; is eNOS masquerading as iNOS? Cardiovasc. Res. (2002) 53:405–413.[Abstract/Free Full Text]
  5. Yang X.M., Krieg T., Cui L., Downey J.M., Cohen M.V. NECA and bradykinin at reperfusion reduce infarction in rabbit hearts by signaling through PI3K, ERK, and NO. J. Mol. Cell Cardiol. (2004) 36:411–421.[CrossRef][ISI][Medline]
  6. Peart J., Hedrick J.P. Adenosine-mediated early preconditioning in mouse: protective signaling and concentration dependent effects. Cardiovasc. Res. (2003) 58:589–601.[Abstract/Free Full Text]
  7. Cargnoni A., Ceconi C., Boraso A., Bernocchi P., Monopoli A., Curello S., et al. Role of A2A receptor in the modulation of myocardial reperfusion damage. J. Cardiovasc. Phamacol. (1999) 33:883–893.[CrossRef][ISI][Medline]
  8. Park J.L., Lucchesi B.R. Mechanisms of myocardial reperfusion injury. Ann. Thorac. Surg. (1999) 68:1905–1912.[Abstract/Free Full Text]
  9. Akao M., O'Rourke B., Teshima Y., Seharaseyon J., Marban E. Mechanistically distinct steps in the mitochondrial death pathway triggered by oxidative stress in cardiac myocytes. Circ. Res. (2003) 92:186–194.[Abstract/Free Full Text]
  10. Suleiman M.S., Halestrap A.P., Griffiths E.J. Mitochondria: a target for myocardial protection. Pharmacol. Ther. (2001) 89:29–46.[CrossRef][ISI][Medline]
  11. Rakhit R.D., Mojet M.H., Marber M.S., Duchen M.R. Mitochondria as targets for nitric oxide-induced protection during simulated ischemia and reoxygenation in isolated neonatal cardiomyocytes. Circulation (2001) 103:2617–2623.[Abstract/Free Full Text]
  12. Brookes P.S., Salinas E.P., Darley-Usmar K., Eiserich J.P., Freeman B.A., Darley-Usmar V.M., et al. Concentration-dependent effects of nitric oxide on mitochondrial permeability transition and cytochrome c release. J. Biol. Chem. (2000) 275:20474–20479.[Abstract/Free Full Text]
  13. Itoh Y., Ma F.H., Hoshi H., Oka M., Noda K., Ukai Y., et al. Determination and bioimaging method for nitric oxide in biological specimens by diaminofluorescein fluorometry. Anal. Biochem. (2000) 287:203–209.[CrossRef][ISI][Medline]
  14. Strijdom H., Muller C., Lochner A. Direct intracellular nitric oxide detection in isolated adult cardiomyocytes: flow cytometric analysis using the fluorescent probe, diaminofluorescein. J. Mol. Cell. Cardiol. (2004) 37:897–902.[CrossRef][ISI][Medline]
  15. Peralta C., Hotter G., Closa D., Prats N., Xaus C., Gelpi E., et al. The protective role of adenosine in inducing nitric oxide synthesis in rat liver ischemia preconditioning is mediated by activation of adenosine A2 receptors. Hepatology (1999) 29:126–132.[CrossRef][ISI][Medline]
  16. Fulton D., Gratton J.P., McCabe T.J., Fontana J., Fujio Y., Walsh K., et al. Regulation of endothelium-derived nitric oxide production by the protein kinase Akt. Nature (1999) 399:597–601.[CrossRef][Medline]
  17. Krieg T., Qin Q., McIntosh E.C., Cohen M.V., J.M.D. ACh and adenosine activate PI3-kinase in rabbit hearts through transactivation of receptor tyrosine kinases. Am. J. Physiol. Heart Circ. Physiol. (2002) 283:H2322–H2330.[Abstract/Free Full Text]
  18. Haynes M.P., Li L., Sinha D., Russell K.S., Hisamoto K., Baron R., et al. Src kinase mediates phosphatidylinositol 3-kinase/Akt-dependent rapid endothelial nitric-oxide synthase activation by estrogen. J. Biol. Chem. (2003) 278:2118–2123.[Abstract/Free Full Text]
  19. Mineo C., Yuhanna I.S., Quon M.J., Shaul P.W. High density lipoprotein-induced endothelial nitric-oxide synthase activation is mediated by Akt and MAP kinases. J. Biol. Chem. (2003) 278:9142–9149.[Abstract/Free Full Text]
  20. Li J.M., Fenton R.A., Cutler B.S. Jr., Dobson J.G. Adenosine enhances nitric oxide production by vascular endothelial cells. Am. J. Physiol. (1995) 269.
  21. Olanrewaju H.A., Mustafa S.J. Adenosine A(2A) and A(2B) receptors mediated nitric oxide production in coronary artery endothelial cells. Gen. Pharmacol. (2000) 35:171–177.[Medline]
  22. Li J., Fenton R.A., Wheeler H.B., Powell C.C., Peyton B.D., Cutler B.S., et al. Adenosine A2a receptors increase arterial endothelial cell nitric oxide. J. Surg. Res. (1998) 80:357–364.[CrossRef][ISI][Medline]
  23. Wyatt A.W., Steinert J.R., Wheeler-Jones C.P., Morgan A.J., Sugden D., Pearson J.D., et al. Early activation of the p42/p44MAPK pathway mediates adenosine-induced nitric oxide production in human endothelial cells: a novel calcium-insensitive mechanism. FASEB J. (2002) 16:1584–1594.[Abstract/Free Full Text]
  24. Ikeda U., Kurosaki K., Ohya K., Shimada K. Adenosine stimulates nitric oxide synthesis in vascular smooth muscle cells. Cardiovasc. Res. (1997) 35:168–174.[Abstract/Free Full Text]
  25. Dubey R.K., Gillespie D.G., Jackson E.K. Cyclic AMP-adenosine pathway induces nitric oxide synthesis in aortic smooth muscle cells. Hypertension (1998) 31:296–302.[Abstract/Free Full Text]
  26. Ikeda U., Kurosaki K., Shimpo M., Okada K., Saito T., Shimada K. Adenosine stimulates nitric oxide synthesis in rat cardiac myocytes. Am. J. Physiol. (1997) 273:H59–H65.[ISI][Medline]
  27. Kelly R.A., Balligand J.L., Smith T.W. Nitric oxide and cardiac function. Circ. Res. (1996) 79:363–380.[Free Full Text]
  28. Jiang M.H., Kaku T., Hada J., Hayashi Y. Different effects of eNOS and nNOS inhibition on transient forebrain ischemia. Brain Res. (2002) 946:139–147.[CrossRef][ISI][Medline]
  29. Benamar K., Yondorf M.Z., Kon D., Geller E.B., Adler M.W. Role of the nitric-oxide synthase isoforms during morphine-induced hyperthermia in rats. J. Pharmacol. Exp. Ther. (2003) 307:219–222.[Abstract/Free Full Text]
  30. Connor J.R., Manning P.T., Settle S.L., Moore W.M., Jerome G.M., Webber R.K., et al. Suppression of adjuvant-induced arthritis by selective inhibition of inducible nitric oxide synthase. Eur. J. Pharmacol. (1995) 273:15–24.[CrossRef][ISI][Medline]
  31. Albrecht E.W., Stegeman C.A., Heeringa P., Henning R.H., H. vG. Protective role of endothelial nitric oxide synthase. J. Pathol. (2003) 199:8–17.[CrossRef][ISI][Medline]
  32. Gao F., Christopher T.A., Lopez B.L., Friedman E., Cai G., Ma X.L. Mechanism of decreased adenosine protection in reperfusion injury of aging rats. Am. J. Physiol. Heart Circ. Physiol. (2000) 279:H329–H338.[Abstract/Free Full Text]
  33. Cohen M.V., Baines C.P., Downey J.M. Ischemic preconditioning: from adenosine receptor to KATP channel. Annu. Rev. Physiol. (2000) 62:79–109.[CrossRef][ISI][Medline]
  34. Vinten-Johansen J., Zhao Z.Q., Corvera J.S., Morris C.D., Budde J.M., Thourani V.H., et al. Adenosine in myocardial protection in on-pump and off-pump cardiac surgery. Ann. Thorac. Surg. (2003) 75:S691–S699.[Abstract/Free Full Text]
  35. Jordan J.E., Zhao Z.-Q., Sato H., Taft S., Vinten-Johansen J. Adenosine A2 receptor activation attenuates reperfusion injury by inhibiting neutrophil accumulation, superoxide generation and coronary endothelial adherence. J. Pharmacol. Exp. Ther. (1997) 280:301–309.[Abstract/Free Full Text]
  36. Zhao Z.-Q., Budde J.M., Morris C., Wang N.-P., Velez D.A., Muraki S., et al. Adenosine attenuates reperfusion-induced apoptotic cell death by modulating expression of Bcl-2 and Bax proteins. J. Mol. Cell. Cardiol. (2001) 33:57–68.[CrossRef][ISI][Medline]
  37. Xu Z., Downey J.M., Cohen M.V. AMP 579 reduces contracture and limits infarction in rabbit heart by activating adenosine A2 receptors. J. Cardiovasc. Pharmacol. (2001) 38:474–481.[CrossRef][ISI][Medline]
  38. Xu Z., Jiao Z., Cohen M.V., Downey J.M. Protection from AMP 579 can be added to that from either cariporide or ischemic preconditioning in ischemic rabbit heart. J. Cardiovasc. Pharmacol. (2002) 40:510–518.[CrossRef][ISI][Medline]
  39. Burgering B.M., Coffer P.J. Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction. Nature (1995) 376:599–602.[CrossRef][Medline]
  40. Hausenloy D.J., Yellon D.M. New directions for protecting the heart against ischaemia–reperfusion injury: targeting the Reperfusion Injury Salvage Kinase (RISK)-pathway. Cardiovasc. Res. (2004) 61:448–460.[Abstract/Free Full Text]
  41. Dimmeler S., Fleming I., Fisslthaler B., Hermann C., Busse R., Zeiher A.M. Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature (1999) 399:601–605.[CrossRef][Medline]
  42. Boo Y.C., Jo H. Flow-dependent regulation of endothelial nitric oxide synthase: role of protein kinases. Am. J. Physiol. Cell Physiol. (2003) 285:C499–C508.[Abstract/Free Full Text]
  43. Vanden Hoek T.L., Shao Z., Li C., Schumacker P., Becker L.B. Mitochondrial electron transport can become a significant source of oxidative injury in cardiomyocytes. J. Mol. Cell. Cardiol. (1997) 29:2441–2450.[CrossRef][ISI][Medline]
  44. Valle V.G., Fagian M.M., Parentoni L.S., Meinicke A.R., Vercesi A.E. The participation of reactive oxygen species and protein thiols in the mechanism of mitochondrial inner membrane permeabilization by calcium plus prooxidants. Arch. Biochem. Biophys. (1993) 307:1–7.[CrossRef][ISI][Medline]
  45. Vladimirov Y.A., Olenev V.I., Suslova T.B., Cheremisina Z.P. Lipid peroxidation in mitochondrial membrane. Adv. Lipid Res. (1980) 17:173–249.[ISI][Medline]
  46. Weiss J.N., Korge P., Honda H.M., Ping P. Role of the mitochondrial permeability transition in myocardial disease. Circ. Res. (2003) 93:292–301.[Abstract/Free Full Text]
  47. Zoratti M., Szabo I. The mitochondrial permeability transition. Biochim. Biophys. Acta (1995) 1241:139–176.[Medline]
  48. Takuma K., Phuagphong P., Lee E., Mori K., Baba A., Matsuda T. Anti-apoptotic effect of cGMP in cultured astrocytes: inhibition by cGMP-dependent protein kinase of mitochondrial permeable transition pore. J. Biol. Chem. (2001) 276:48093–48099.[Abstract/Free Full Text]

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
 Am. J. Physiol. Heart Circ. Physiol.Home page
R. Merla, Y. Ye, Y. Lin, S. Manickavasagam, M.-H. Huang, R. J. Perez-Polo, B. F. Uretsky, and Y. Birnbaum
The central role of adenosine in statin-induced ERK1/2, Akt, and eNOS phosphorylation
Am J Physiol Heart Circ Physiol, September 1, 2007; 293(3): H1918 - H1928.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
X.-l. Moore, S.-l. Tan, C.-y. Lo, L. Fang, Y.-D. Su, X.-M. Gao, E. A. Woodcock, R. J. Summers, G. W. Tregear, R. A. D. Bathgate, et al.
Relaxin Antagonizes Hypertrophy and Apoptosis in Neonatal Rat Cardiomyocytes
Endocrinology, April 1, 2007; 148(4): 1582 - 1589.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
S.-S. Park, H. Zhao, Y. Jang, R. A. Mueller, and Z. Xu
N6-(3-Iodobenzyl)-adenosine-5'-N-methylcarboxamide Confers Cardioprotection at Reperfusion by Inhibiting Mitochondrial Permeability Transition Pore Opening via Glycogen Synthase Kinase 3beta
J. Pharmacol. Exp. Ther., July 1, 2006; 318(1): 124 - 131.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
K. Akassoglou
Nerve Growth Factor-Independent Neuronal Survival: A Role for NO Donors
Mol. Pharmacol., October 1, 2005; 68(4): 952 - 955.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
T. Okada, H. Otani, Y. Wu, T. Uchiyama, S. Kyoi, R. Hattori, T. Sumida, H. Fujiwara, and H. Imamura
Integrated pharmacological preconditioning and memory of cardioprotection: role of protein kinase C and phosphatidylinositol 3-kinase
Am J Physiol Heart Circ Physiol, August 1, 2005; 289(2): H761 - H767.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
J. S. Karliner
Symbols within words: Have we neglected the AdeNOsine A2 receptor?
Cardiovasc Res, March 1, 2005; 65(4): 763 - 765.
[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 Xu, Z.
Right arrow Articles by Boysen, P. G.