Uridine triphosphate (UTP) is released during cardiac ischemia

International Journal of Cardiology 100 (2005) 427 – 433 www.elsevier.com/locate/ijcard

Uridine triphosphate (UTP) is released during cardiac ischemia David Erlingea,T,1, Jan Harnekb,1, Catharina van Heusdenc, Gfran Olivecronaa, Sverker Jernd, Eduardo Lazarowskic a Department of Cardiology, Lund University Hospital, S-221 85 Lund, Sweden Thoracic Radiology, Heart & Lung Division, Lund University Hospital, Sweden c Department of Medicine, University of North Carolina, School of Medicine, Chapel Hill, USA d ¨ stra, Go¨teborg University, Go¨teborg, Sweden Clinical Experimental Research Laboratory, Sahlgrenska University Hospital/O b

Received 11 March 2004; received in revised form 15 September 2004; accepted 2 October 2004 Available online 8 February 2005

Abstract Background: Extracellular uridine triphosphate (UTP) stimulates vasodilatation, automaticity in ventricular myocytes and release of tissueplasminogen activator (t-PA), indicating that UTP may be important in cardiac regulation. We took advantage of a recently developed quantitative assay for UTP to test the hypothesis that UTP is released in the circulation during cardiac ischemia. Methods: In ten pigs, a balloon catheter in the left anterior descending artery was introduced to induce ischemia. Samples were collected from the coronary sinus. Blood flow in the coronary sinus was assessed by a Doppler velocity transducer. Results: Plasma UTP levels increased early during ischemia and early after reperfusion (by 257F100 and 247F72%, pb0.05). Cardiac blood flow, ventricular arrhythmias and t-PA release were markedly increased at the same time points. In contrast, after 30 min, a second period of ischemia did not result in any significant increase of UTP or blood flow. Furthermore, ventricular arrhythmias were less frequent. UTP levels correlated with ventricular arrhythmia and blood flow. Similar results were found for ATP. Conclusion: For the first time we have shown that UTP is released during cardiac ischemia. UTP released during ischemia may stimulate blood flow, arrhythmia and t-PA release. D 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Uridine triphosphate; Nucleotide; Adenosine triphosphate; Coronary circulation; Arrhythmia; Heart

1. Introduction Uridine triphosphate (UTP) belongs to the family of extracellular nucleotide signalling molecules [together with adenosine triphosphate (ATP), adenosine diphosphate (ADP) and uridine diphosphate (UDP)], which regulate vascular tone and blood pressure by stimulating P2 receptors [1]. P2 receptors can be divided into two classes on the basis of their signal transmission mechanisms and their characteristic molecular structures; ligand-gated intrinsic ion channels, P2X receptors (seven subtypes), and G-

T Corresponding author. Tel.: +46 46 17 25 97; fax: +46 46 15 78 57. E-mail address: [email protected] (D. Erlinge). 1 David Erlinge and Jan Harnek contributed equally to the article. 0167-5273/$ - see front matter D 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2004.10.005

protein coupled P2Y receptors (eight subtypes) [1]. Various P2X and P2Y receptor subtypes have been detected in the heart. Nucleotides have been shown to have inotropic effects, and the capability to trigger automaticity and afterdepolarizations which indicates arrhythmogenic properties [2]. Extracellular UTP and ATP stimulate DAG, protein kinase C, p38 MAPK and ATP-sensitive K+ channels in cardiomyocytes, indicating that release of UTP during ischemia could trigger intracellular events known to be important for preconditioning [2]. UTP sensitive P2Y2 receptors on the endothelium induce vasodilatation by the release of prostaglandins, nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) [1,3–5]. Furthermore, UTP has been shown to release tissue-plasminogen activator (t-PA) from endothelial cells (EC), both in culture and in vivo in man [6,7]. ADP

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stimulates platelet aggregation and the P2Y12 ADP receptor antagonist (clopidogrel), is used in the clinic to reduce cardiac events [8]. Purines (ATP, ADP) are released in the heart during ischemia from cardiac myocytes, endothelial cells, red blood cells and sympathetic nerves [9]. However, little is known about the release of the pyrimidine UTP and no study of its release in the heart has been performed. We recently developed the first sensitive quantitative assay for UTP, making it possible to measure the release of UTP to the extracellular milieu [16]. The multiple actions of uridine triphosphate (UTP) including stimulation of vasodilatation, release of tissueplasminogen activator (t-PA) and automaticity in ventricular myocytes, indicate that UTP may be important during ischemia in the heart. We hypothesized that UTP is released during cardiac ischemia and that it correlates with coronary flow, arrhythmia and t-PA release. To test the hypothesis we used a pig cardiac ischemia in vivo model.

2. Methods

ensure positioning of the catheter. The catheter was also used to place a 0.014-in. Doppler blood flow velocity transducer (Jometrics Flowire, Jomed NV) in the transition between the great cardiac vein and the coronary sinus, to measure the total change in blood flow from the heart (Fig. 1). By positioning a catheter in the heart’s left anterior descending artery (LAD) and inflating a balloon, an ischemic area was created. A 6 F introducer sheath (Onset, Cordis Co., Miami, FL, USA) was inserted into the surgically exposed left carotid artery. The side port of the introducer was connected to a pressure transducer and balanced to atmospheric pressure with zero reference at the mid-axillary level for continuously monitoring of the arterial pressure. Displaying a three-lead ECG on the same monitor we monitored cardiac rhythm and rate. An angiogram of the coronary arteries was obtained via a 6F 3.5-JL guidecatheter with perfusion-ports (Scimed Inc., Maple Grove, MN, USA) using 5–8 mL contrast medium. A 3.5 mm20 mm angioplasty balloon (Cobra 14, Scimed Europe, Vervieris, Belgium) was inflated with 3 atm in the LAD distally to the first diagonal branch. The balloon was oversized 0.5 mm in diameter to ensure occlusion.

2.1. Animals 2.2. Protocol Ten healthy domestic pigs of both sexes weighing 25 kg were fasted overnight with free access to water and were premedicated with azaperone (Stresnil Vet., Leo; Helsingborg, Sweden), 2 mg/kg intramuscularly 30 min before the procedure. After induction of anesthesia with thiopental 5– 25 mg/kg (Pentothal, Abbott, Stockholm, Sweden), the animals were orally intubated with cuffed endotracheal tubes. A slow infusion of 1.25 AL/mL Fentanyl (Fentanyl, Pharmalink AB, Stockholm, Sweden) in Ringer-acetate was started at a rate of 1.5 mL/min and adjusted as needed. Mechanical ventilation was then established with a Siemens-Elema 300B ventilator in the volume-controlled mode. Initial settings were respiratory rate of 15/min, tidal volume of 10 mL/kg, and positive end-expiratory pressure of 5 cm H2O. Minute volume was subsequently adjusted in order to obtain normocapnia (35–40 mm Hg). The animals were ventilated with a mixture of nitrous oxide (70%) and oxygen (30%). Anesthesia was complemented with small intermittent doses of 5 mg meprobamat (Mebumal, DAK, Copenhagen, Denmark), if needed. Throughout the experiment the perfusion pressure was stabilised to approximately 100 mm Hg in systolic blood pressure by a dopamine infusion. By positioning a catheter in the coronary sinus, samples could be taken from the venous outflow of the heart. An 8F introducer (Onset, Cordis Co., Miami, FL, USA) was inserted into the right internal jugular vein and an 8F AL 1.0 (Cordis Co., Miami, FL, USA) was inserted into the coronary sinus. 10,000 IU Heparin was given. A venography was obtained using 8–10 mL contrast medium Omnipaque 300 mg I /mL (Nycomed, Oslo, Norway) to

After baseline blood samples were collected, the balloon in LAD was inflated to cause ischemia for a duration of 15 min. Sampling after 1, 10 and 15 min was performed (see Fig. 2). After the 15-min period the balloon was deflated

Fig. 1. Normal anatomy of the coronary sinus. By positioning a catheter in the coronary sinus samples could be taken from the venous outflow of the heart. An introducer was inserted into the right internal jugular vein and a catheter was inserted into the coronary sinus. A venography was obtained to ensure positioning of the catheter. The catheter was also used to place a 0.014-in. Doppler blood flow velocity transducer in the transition between the great cardiac vein and the coronary sinus, to measure the total blood flow from the heart. SCV: superior caval vein; ICV: inferior caval vein; RA: right atrium; CS: coronary sinus; AV: azygos vein (drainage from the abdominal cavity); C: confluens (vein of the CS and AV); GCV: great cardiac vein.

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UTP (% increase)

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ischemia

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University of North Carolina, School of Medicine, Chapel Hill, USA for analysis.

UTP

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2.5. Nucleotide measurements

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ischemia

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Time (min) Fig. 2. UTP levels measured in blood samples from the venous outflow of the heart were increased in a biphasic pattern, with an increase 1 min after onset of cardiac ischemia, and a second increase 1 min after reperfusion. During a second period of ischemia no significant increase of UTP was observed. Data are expressed as percentage increase from baseline and shown as meansFS.E.M., *pb0.05, **pb0.01, n=10.

and samples were collected during this reperfusion period at 1 and 10 min. After 30 min a second identical ischemic period was performed with identical blood sampling, except for t-PA. To reduce the total number of blood samples, t-PA was only collected during the first period of ischemia and only at baseline, at 1 min after ischemia, and 1 min after reperfusion. VT/VF episodes were terminated by a rapid DC shock. Each VT/VF episode was counted as one. 2.3. Measurement of cardiac venous outflow The diameter of the coronary vein was measured by venogram at baseline, early ischemia (1 min) and reperfusion (1 min). The increase in vessel area was calculated and multiplied to the change in Doppler flow in the coronary vein to get the total change in venous flow. However, the diameter never changed more than 10% from baseline, and in our experiments this compensation did not markedly influence the results. 2.4. Blood sampling Blood samples were taken from the catheter inserted in the coronary sinus (major vein), by gentle, slow aspiration to avoid activation of platelets or other cells. 5 mL blood was added to tubes containing citrate and immediately centrifugated at 1200g, 4 8C. Platelet contamination was excluded by Burker chamber examination. A similar method used to determine dinucleotides was validated against best practice conditions for limiting platelet activation, with no significant differences [15]. The plasma was aspirated and mixed with an equal amount of 10% trichloroacetic acid (TCA) to precipitate all proteins. After centrifugation, the protein-free supernatant was frozen at 808. Samples were sent by courier on dry ice, to the Department of Medicine,

Samples were thawed at room temperature and TCA was extracted three times with six volumes of ethyl ether. Ethyl ether was removed by gassing N2, and the resulting samples diluted in the corresponding nucleotide assay buffer as indicated below. 2.5.1. Measurement of UTP Sample extracts were diluted 1:4 with phosphate-free MEM–HEPES (pH 7.4) containing 2 mM MgCl2 and 2 mM CaCl2. UTP concentrations were determined using the UDP-glucose pyrophosphoryalse-based reaction described previously [16]. Briefly, samples (150 AL) were incubated with ~100,000 cpm, 1 AM [14C]glucose-1P, 0.5 U/mL UDPglucose pyrophosphorylase, and 0.5 U/mL inorganic pyrophosphatase for 1 h at 30 8C. Heating the samples at 95 8C for 2 min terminated reactions. Conversion of [14C]glucose1P to [14C]UTP was determined by HPLC (Shimadzu) via a Nova Pack C18 column and ion pairing mobile phase. Radioactivity was measured on-line with a Packard Flo-One detector [17]. 2.5.2. Derivatization of adenosine and adenine nucleotides We have adopted and slightly modified the derivatization protocol originally described by Levitt et al. [18], sample extracts were diluted 1:2 with water and incubated for 30 min at 72 8C in the presence of 1.0 M chloroacetaldehyde and 25 mM Na2HPO4 (pH 4.0) in a 200-AL final volume. Samples were transferred to ice, alkalinized with 50 AL 0.5 M NH4HCO3, and analysed by HPLC within 24 h. Identification and quantification of ethenylated species were performed with an automated Waters HPLC apparatus equipped with a fluorescence detector. Derivatized samples were transferred to 0.7 mL plastic shell vials and kept at 4 8C in the sample injector rack. A 100 AL sample aliquot was injected into a 250mm, 10 Am Hamilton PRP-X100 anion exchange column. The mobile phase (2 mL/min, 30% methanol) developed linearly from 0.250 to 0.275 M NH4HCO3 (pH 8.5) during the first 8 min, remaining isocratic at 0.275 M NH4HCO3 for an additional 4 min. The column was subsequently rinsed for 3 min with 0.425 M NH4HCO3 in 30% methanol, and re-equilibrated to the initial conditions for 15 min. Elution times (in min) were q-ADO, 3.2; q-AMP, 5.9; q-ADP, 7.6, and q-ATP, 9.4. 2.6. Reagents [14C]glucose-1P (300 mCi/mmol) and molecular biology grade ATP and UTP were purchased from Amersham Pharmacia Biotech (Piscataway, NJ). ADP, AMP, and adenosine were from Roche Molecular Biochemicals

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(Indianapolis, IN). Etheno-adenyl standards were from Sigma (St. Louis, MO). Other chemicals were of the highest purity available. Unless otherwise stated drugs were purchased from Sigma Co., USA. 2.7. Measurement of t-PA Plasma levels of total t-PA antigen were determined by an enzyme-linked immunoassay (TintElize t-PA, catalogue No. 1105, Biopool AB) that detects free and complexed t-PA with equal efficiency. Calibration was performed with porcine t-PA diluted in t-PA-depleted porcine plasma as earlier described [19]. Active t-PA was determined with a spectrophotometric parabolic rate assay (SpectrolyseTM/fibrin t-PA, catalogue No. 101101, Biopool AB). By quenching with polyclonal goat antiporcine t-PA IgG (catalogue No. 105301, Biopool AB), we have earlier shown that this assay is specific for t-PA [19]. Human single-chain t-PA calibrated against the International Standard for t-PA (World Health Organization’s First International Standard for t-PA coded 86/670 from the National Institute for Biological Standards and Control, Hertfordshire, England) was used as standard in this assay. Thus, in the following, t-PA activity is expressed in units, with 1 unit of porcine t-PA being equivalent in the employed assay to 1 international unit of human t-PA. Samples from each experimental animal were analysed on 1 single microtiter plate. All samples were analysed in duplicate, and mean intra-assay coefficients of variation were 2.5% and 3.5% for total and active t-PA, respectively.

(before second period of ischemia) were similar with only a 10–15% reduction compared to the first baseline values. UTP was released in a biphasic pattern, with an increase 1 min after onset of ischemia (increase by 257F100%, pb0.01) and a second increase directly after reperfusion (247F72%, pb0.05; Fig. 2). ATP was also released in a biphasic pattern, with an increase 1 min after onset of ischemia, and a second increase directly after reperfusion (Fig. 3a). The relative increase of ATP was lower in comparison to UTP (increase by 76F16% and 73F23%, pb0.05; Fig. 3a). ADP increased in a similar pattern as UTP (Fig. 3b). The second phase of ischemia did not result in any significant increases in either UTP, ATP or ADP. There was a tendency for a late increase of the nucleotides after ischemia but this did not reach significance (Figs. 2 and 3). It cannot be excluded that a further increase would have been revealed if subsequent samples had been collected.

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ATP (% increase)

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2.8. Ethics

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The Ethics Committee of Lund University approved the project.

Time (min)

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2.9. Calculation and statistics

ADP ADP (% increase)

Calculations and statistics were performed using the GraphPad Prism 3.02 software. Values are presented as meanFS.E.M., n=10. Statistical significance was accepted when pb0.05 (two-tailed test). One-way analysis of variance (ANOVA) test followed by the Dunnett multiple comparisons test was used. Pearson’s test was used for regression analysis.

ATP ischemia

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3. Results 3.1. Nucleotide release Nucleotide baseline values were determined prior to induction of ischemia. Initial baseline values were UTP 187F61, ATP 2292F726, ADP 1398F423, AMP 239F47, and adenosine 14F6 nmol/L. The second baseline values

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75

Time (min) Fig. 3. ATP (a) and ADP levels (b) measured in blood samples from the venous outflow of the heart were increased in a biphasic pattern, with an increase 1 min after onset of cardiac ischemia, and a second increase 1 min after reperfusion. During a second period of ischemia no significant increase of ATP or ADP was observed. Data are expressed as percentage increase from baseline and shown as meansFS.E.M., *pb0.05, n=10.

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t-PA (% increase)

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Time (min)

Time (min) Fig. 4. The total venous blood flow from the heart increased 1 min after onset of cardiac ischemia, with a second increase directly after reperfusion. During the second period of ischemia no increase of blood flow was observed. Data are expressed as percentage increase from baseline and shown as meansFS.E.M., *pb0.05, n=10.

Fig. 6. Tissue-plasminogen activator (t-PA) levels measured in blood samples from the venous outflow of the heart at baseline, 1 min after cardiac ischemia and 1 min after reperfusion. t-PA levels were significantly increased both at early ischemia and reperfusion. Data are expressed as percentage increase from baseline and shown as meansFS.E.M., *pb0.05, n=10.

3.2. Blood flow

with fewer episodes during the second period (after preconditioning) of ischemia (Fig. 5). Regression analysis revealed a positive correlation between UTP release and ventricular arrhythmias (r 2=0.38, pb0.05), and for ATP and ventricular arrhythmias (r 2=0.50, pb0.01).

Similar to the release of UTP, ATP and ADP, the total venous blood flow from the heart increased during early ischemia and reperfusion (Fig. 4). Furthermore, as for UTP release, these increases in flow during early ischemia and reperfusion were reduced after preconditioning. Regression analysis revealed a positive correlation between UTP release and cardiac blood flow (r 2=0.36, pb0.05), ATP showed a tendency to correlate with blood flow, but did not reach significance (r 2=0.19, p=0.15). 3.3. Ventricular arrhythmias Episodes of ventricular fibrillation or tachycardia peaked at time points when the UTP and ATP release was high, and 20

UTP

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To reduce the total number of blood samples t-PA was only collected during the first period of ischemia and at baseline, at 1 min after ischemia, and 1 min after reperfusion. Significantly increased t-PA release was observed at 1 min after ischemia and following reperfusion (Fig. 6). At the corresponding time points both UTP and ATP levels were markedly increased.

4. Discussion

VF/VT

VT/VF episodes

UTP (% increase)

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3.4. t-PA release

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Time (min) Fig. 5. Episodes of ventricular fibrillation/tachycardia (VF/VT) peaked at times where the UTP levels were high, 1 min after onset of cardiac ischemia, and 1 min after reperfusion. The episodes of ventricular fibrillation/tachycardia were fewer during the second period of ischemia. UTP data are expressed as percentage increase from baseline and shown as meansFS.E.M., n=10.

For the first time we have shown that extracellular UTP levels in the blood increase during heart ischemia. UTP was released in a biphasic pattern: early during ischemia and early after reperfusion. The increase in cardiac blood flow and the incidence of ventricular arrhythmias peaked when extracellular UTP levels were maximal. This release pattern of UTP was lost at a second period of ischemia, as was the increase in blood flow and the incidence of ventricular arrhythmia. The biphasic release pattern for UTP and ATP seen in the venous blood from the heart, with an increase 1 min after onset of ischemia and a second increase directly after reperfusion, has been observed previously for ATP with interstitial microdialysis in rat [12]. Furthermore, ADP and AMP had a similar accumulation pattern although not as marked as ATP and UTP. The baseline ATP levels were in the micromolar range, which is similar to previously

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reported levels of circulating ATP and diadenosine polyphosphates in man [15,20]. Baseline levels of adenosine in the pig plasma were markedly lower than those of its nucleotides, most likely due to both rapid uptake into the red blood cells and degradation. Because we have not included adenosine deaminase and nucleoside transport inhibitors in the perfused solution, the magnitude of changes in adenosine levels in blood have not been fully appreciated in our study. However, the role of adenosine has been studied extensively before and was not the focus of the present study [21]. Unlike the large number of studies measuring extracellular ATP (or ATP release), detection of UTP in biological samples has been scarce due to the lack of sensitive methodology. The first demonstration of UTP release used [ 3H]uridine-labelled endothelial cells. In that study, [3H]UTP release occurred in response to increased flow [22]. Recently, a newly developed sensitive assay for UTP has made it possible to quantify extracellular UTP levels in different cell types [16,18]. We used a new approach for the measurement of the total coronary blood flow. Doppler flow measurements are traditionally used on the arterial side. The limitation of this approach is that the artery selected for measurement, may not represent the whole cardiac circulation. To the best of our knowledge this is the first time the Doppler flow method has been used in the main cardiac vein. The flow should represent the total coronary blood flow, resulting from the ischemic area, non-ischemic area, and the penumbra zone. This revealed distinct peak increases in flow during early ischemia and reperfusion that were reduced by preconditioning. Samples for t-PA analysis were collected at 1 min after ischemia and directly after reperfusion. t-PA was released on both occasions. At these times UTP and ATP release was high. Previously, we showed that both UTP and ATP release t-PA in vivo in man [7]. It is possible that nucleotides contribute to the release of t-PA during cardiac ischemia. The resulting concentrations of released nucleotides are indeed sufficient to release t-PA. In our previous study, we infused both UTP and ATP in the human forearm and found that they induced the released t-PA at sub-micromolar concentrations [7]. UTP and ATP induced t-PA release may dissolve the thrombus that is the most common cause for myocardial infarctions in patients. The increase of nucleotide levels may be a protective mechanism to limit thrombus formation. Both UTP and ATP infused in the human forearm cause vasodilatation and increase blood flow and t-PA release in lower than micromolar concentrations [7]. Thus, the release levels of UTP and ATP found in the present study are physiologically relevant. The UTP levels are lower than the ATP levels, but since UTP or its degradation product UDP acts on several P2 receptors, some of which are not activated by ATP (human P2Y2 and P2Y6), UTP release may have an importance of its own. Furthermore, many of the effects of

ATP are counterbalanced by the effect of its degradation product adenosine. This is not the case for UTP, making a higher total effect possible. It is not clear from what cell types UTP is released but it is likely that cardiac myocytes, endothelial cells and platelets are the main sources. As mentioned above, increased flow or ischemia stimulates nucleotide release from endothelial cells [22]. Thus, increased flow could have stimulated nucleotide release in our experiments. Previous studies have often been performed in the Langendorff preparation perfused with buffer solutions, demonstrating that at least ATP can be released from solid tissues (mainly cardiac myocytes and endothelial cells). However, red blood cells have recently been shown to act as oxygen sensors, releasing ATP in response to hypoxia and low pH, causing vascular relaxation [23,24]. In fact, this has been shown to be an explanation for the previous enigma of the rapid increase of the skeletal muscle blood flow in response to exercise (hypoxic vasodilatation), that have been the focus of more than a century of research [20]. Our present findings demonstrate that not only ATP but also of UTP is released during ischemia and peaks at times of increased coronary blood flow. This observation gives support to the possibility that nucleotides may contribute to hypoxia induced increases in blood flow not only in skeletal muscle but also in the heart. Both UTP and ATP levels correlated with the occurrence of ventricular arrhythmias. A similar correlation has previously been shown for ATP in rat heart [12]. UTP and ATP trigger automaticity and afterdepolarization in cardiac myocytes, which indicates that they have arrhythmogenic properties [2]. Thus, it is possible that nucleotides released during ischemia and reperfusion may cause arrhythmias, and that antagonists against P2 receptors could have antiarrhythmic properties. Preconditioning is a brief ischemic event that protects cardiac tissue from damage by subsequent ischemic episodes [10,11]. The mechanisms are not clear but a role for adenosine-release has been suggested. ATP has been paid less attention to because of the assumption that it should be rapidly degraded to adenosine through ectonucleotidases. However, recent studies using microdialysis have found high levels of ATP in the myocardial interstitium [12–14]. Furthermore, using P2 receptor antagonists Ninomiya et al., found that extracellular ATP and adenosine play an equal and complementary role in ischemic preconditioning [14]. Several preconditioning time protocols have been used before. We used two identical ischemic episodes of 15 min separated by 30 min reperfusion. Preconditioning is well known to have several beneficial effects in the heart, one of which is the reduction of ventricular arrhythmias. We confirmed that preconditioning reduced the rate of ventricular arrhythmias. Surprisingly, we found that preconditioning also reduced both the peak increases in coronary flow and nucleotide release during early ischemia and reperfusion. Reduced nucleotide release after preconditioning was observed in a recent study using microdialysis in the

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rat heart [13]. Here we confirm their findings in a larger animal model and add evidence that UTP and ATP release is reduced after preconditioning. Furthermore, the reduction in the release of nucleotides coincides with a reduction in coronary flow after preconditioning. Extracellular UTP and ATP stimulate DAG, protein kinase C, p38 MAPK and ATP-sensitive K+ channels in cardiomyocytes [2]. These intracellular events are necessary mediators for preconditioning. Thus, our finding of a prominent release of UTP and ATP during ischemia could represent an important trigger for the previously established mediators of preconditioning. Consistent with this idea, it has been shown that the P2 receptor antagonist suramin, infused during the preconditioning period, abolishes approximately half of the beneficial effects of preconditioning on cardiac function (rate pressure product), and total coronary blood flow [14]. In summary, for the first time we have shown that UTP is released during cardiac ischemia. UTP was released concurrent with increases in blood flow, t-PA release and ventricular arrhythmia. Preconditioning reduced UTP release, blood flow and ventricular arrhythmias. Thus, UTP is a factor that is released together with previously known substances such as adenosine, ATP and bradykinin. UTP released during ischemia may stimulate blood flow, arrhythmia and t-PA release.

5. Uncited reference [17]

Acknowledgements The study was supported by the Swedish Heart and Lung Foundation, the Franke and Margareta Bergqvist Foundation, the Wiberg Foundation, the Bergwall Foundation, the Zoegas Foundation, the Westergren Foundation, the Swedish Medical Society, and the Swedish Medical Research Council, Grant 13130.

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