1 Institute of Experimental Cardiology, National Cardiology Research Center, Moscow 121 552, Russia; and 2 Laboratoire de Physiopathologie Cardiovasculaire, Institut National de la Santé et de la Recherche Médicale U-390, F-34295 Montpellier Cédex 5, France
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ABSTRACT |
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With the aim of estimating interstitial levels and the breakdown process of ATP, cardiac microdialysis was performed in the left ventricular wall of in situ control and postinfarcted as well as of isolated, Langendorff-perfused rat hearts. With the use of a bioluminescence technique for dialysate ATP measurement, the baseline interstitial fluid ATP concentration in in situ hearts was estimated to be 38 ± 8 nM. Regional ischemia induced an early peak increase in interstitial fluid ATP to 373 ± 73 nM that correlates with the maximal incidence of ventricular arrhythmias. During continuous infusion of individual adenine nucleotides (50 µM ATP, ADP, or AMP), the dialysate samples were analyzed for adenine nucleotides, nucleosides, and bases using HPLC with ultraviolet detection. The patterns of catabolites were consistent with the major pathway of metabolism, that is, sequential dephosphorylation catalyzed by a chain of separate ecto-nucleotidases. In in situ postinfarcted hearts as well as in perfused hearts, a reduced catabolism rate of extracellular adenine nucleotides was observed. In conclusion, in in situ rat hearts, ATP can be released in substantial amounts in the interstitium where it readily undergoes enzymatic degradation. Dephosphorylation occurs sequentially and faster in in situ control hearts than in in situ postinfarcted or in perfused hearts.
cardiac microdialysis; adenine nucleotides; adenine nucleotide breakdown products; ecto-nucleotidases; ischemia-reperfusion
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INTRODUCTION |
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ENDOGENOUS ADENOSINE IS A potent regulator of coronary circulation. In hearts, adenosine is generally assumed to be generated by dephosphorylation of intracellular AMP and by hydrolysis of S-adenosyl-L-homocysteine (7, 13). However, in recent years, it has become evident that a chain of highly active ecto-nucleotidases capable of metabolizing adenine nucleotides present in the extracellular space is located on the surface of cardiac cells (9, 13, 18). Moreover, although basal ATP released from isolated, saline-perfused hearts has been shown to range below 1 nM (3, 31), the levels of AMP or total adenine nucleotides found in venous effluent (3, 12, 14), epicardial transudate (12, 14), or myocardial dialysate (11) were much higher and nearly in the same range as the release of adenosine, which varies from 180 to 420 nM in these studies. The possible sources of extracellular adenine nucleotides include leakage by damaged cardiomyocytes (31), release from endothelial cells (3), stimulated blood platelets (5, 9), and adrenergic and purinergic nerve endings (9, 11, 13, 14). It has also been shown that hypoxia (3, 31), ischemia (3), and catecholamine stimulation (3, 6, 11, 31) may produce a significant increase in adenine nucleotide levels in myocardial interstitium as well as in coronary effluent. In some pathophysiological situations, extracellular ATP concentrations may temporarily exceed 100 µM (5). Thus at least a portion of myocardial adenosine may be formed in the interstitium because of release of adenine nucleotides with subsequent degradation by a cascade of ecto-nucleotidases. Furthermore, if present in the extracellular space at sufficient concentrations, adenine nucleotides themselves may play a physiological role because of their vasodilator potency, which is nearly equal to that of adenosine, by influencing platelet activation or by modulating cardiac beating frequency (9). Although extracellular adenine nucleotide catabolism has been of undoubted physiological importance, it has been studied only in in vitro rat ventricular myocytes incubated with adenine nucleotides (18) or, indirectly, in isolated perfused hearts after a single coronary passage of exogenous nucleotides (3, 6, 8, 9).
In the present study, adenine nucleotide breakdown was examined in in situ rat hearts using cardiac microdialysis technique (11, 16, 17, 29). This technique allows local delivery of exogenous adenine nucleotide into the extracellular space and simultaneously allows sampling of intramyocardial interstitial fluid for subsequent analysis of adenine nucleotides and breakdown products. The standard conditions of microdialysis experiments permitted comparison of extracellular adenine nucleotide catabolism in in situ and in isolated perfused rat hearts. Using this microdialysis technique, we also estimated the level of interstitial ATP in cardiac dialysate samples by the highly sensitive detection luciferin-luciferase assay as well as its marked increase during regional ischemia/reperfusion. During these latter phases, increases in ATP levels were associated with electrical disturbances.
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METHODS |
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Animal preparation. All experiments performed in this study conformed to national guidelines regarding use and care of laboratory animals.
In situ studies. Male Wistar rats weighing 300-400 g were used in these experiments. Some rats underwent experimental myocardial infarction under ketamine anesthesia according to Pfeffer et al. (22). These rats were used in dialysis experiments 3 wk after coronary artery ligation. After anesthesia and tracheostomy, rats were ventilated with room air, and body temperature was maintained at 37°C. Blood pressure, heart rate, and a lead II electrocardiogram were continuously monitored throughout the experiment. Analog signals from blood pressure and the electrocardiogram were sampled by the physiological data acquisition system BioShell (National Cardiology Research Center, Moscow, Russia) on a personal computer. The electrocardiogram as well as blood pressure signals were also analyzed for the appearance of rhythm disturbances: ventricular premature beats, ventricular tachycardia, and fibrillation. Ventricular tachycardia was defined as four or more consecutive ventricular premature beats. Ventricular fibrillation was defined as a signal in which individual QRS complexes could not be distinguished from one another and for which a rate could not be determined. Ventricular fibrillation was also seen as a nearly complete absence of blood pressure.
In vitro studies. Hearts were rapidly excised from anesthetized (1 g/kg urethane, ip) and heparinized (600 units, ip) male Wistar rats (300-400 g). Hearts were subjected to retrograde perfusion with Krebs-Henseleit solution (in mM: 118.5 NaCl, 25 NaHCO3, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, 2.5 CaCl2, and 11 glucose) equilibrated with a mixture of 95% O2-5% CO2 at 37°C. An initial resting tension of 1.0 g was preloaded with a string connected to a hook at the apex of the heart. Cardiac contractile force was estimated using a force-displacement transducer (Gould Statham UC2, Ballainvilliers, France). Changes in the developed heart force were continuously monitored and recorded on a tape recorder (DTR-1203, Biologic) and a thermal pen recorder (DASH-IV, Astro-Med).
Cardiac microdialysis. Two or three microdialysis probes (10 mm long) were pulled through the left ventricular wall. Each end of the probes was inserted into inflow and outflow silicone tubes and sealed in place with cyanoacrylic glue (16, 17). The microdialysis technique was similar in isolated and in situ rat hearts. After implantation, the microdialysis probes were perfused with a Ringer solution (in mM: 147 NaCl, 4.0 KCl, and 2.3 CaCl2) at a rate of 3.0 µl/min (CMA/100 microinjection pump, Carnegie Medicine, Stockholm, Sweden). Dialysate sampling (10-min duration or, most generally, 20-min duration) was started 60 min after the last probe implantation.
Three series of experiments were performed. In the first series, two microdialysis probes were inserted in the myocardial left ventricular wall in the control (nonischemic) region and in the perfusion area to be occluded. After control dialysate samples were collected, the left main coronary artery was ligated ~2 mm from its origin, and 10-min dialysate samples were collected during the 20-min ischemia followed by a 30-min reperfusion period. Dialysates obtained from both ischemic and control zones immediately after collection were analyzed for ATP using the bioluminescence technique with luciferin-luciferase reagent. The in vivo recovery in heart tissue for the poorly hydrolyzable ADP analogAnalytical procedures. For endogenous interstitial ATP level determination, a 30-µl dialysate sample was mixed with 30 µl of luciferin-luciferase reagent (ATP bioluminescence assay kit CLS II, Boehringer Mannheim). The ATP-induced bioluminescence was detected in a luminometer (Luminometer 1250, LKB-WALLAC). The limit of ATP detection was 0.2 pmol/ml. Because recovery of the microdialysis probe is proportional to its length, all results of analysis were recalculated for a standard probe length of 10 mm.
In the experimental series dealing with dialysis probe perfusion of adenine nucleotide, the dialysate samples were analyzed for adenine nucleotides, nucleosides, and bases by HPLC with ultraviolet detection at 254 nm. A two-step separation was performed on an 80 × 4.6 mm (ID) HR-80 column packed with C-18 3-µm particles (ESA, Bedford, MA). Immediately after collection, a portion of the dialysate was used for the determination of adenosine, AMP, AOPCP, ADP, and ATP levels with a mobile phase of 50 mM KH2PO4, 3.2 mM tetrabutylammonium chloride, and 7% (vol) acetonitrile, pH 5.70, at a flow rate of 1 ml/min. The detection limits (signal-to-noise ratio of 3:1) in these conditions were calculated as 25 pmol/ml for adenosine, 50 pmol/ml for AMP, 65 pmol/ml for ADP and AOPCP, and 150 pmol/ml for ATP. The remainder of the dialysate sample stored at ![]() |
RESULTS |
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Interstitial ATP levels in control and ischemia-reperfusion hearts. In the first experimental series with dialysis probe collection, the dialysate levels of adenine nucleotides, nucleosides, and bases were detected by HPLC. In in situ rat hearts, baseline dialysate adenosine concentration was estimated to be 0.23 ± 0.09 µM and total cardiac purine (inosine + hypoxanthine + xanthine) concentration was estimated to be 0.9 ± 0.3 µM (n = 6). These values increased to 0.93 ± 0.20 and 1.36 ± 0.3 µM for adenosine and to 10.5 ± 2.4 and 15.7 ± 3.2 µM for total cardiac purines during the 0- to 10-min and 10- to 20-min sampling periods of ischemia, respectively. Then, concentrations recovered to initial values within 1 h. These values and their time courses during and after ischemia compare well with previously published data using the microdialysis technique in various species (16, 17, 28, 29).
The temporal profiles of ATP levels in dialysate collected from control and ischemic zones of the in situ control rat heart are shown in Fig. 1. The baseline dialysate ATP levels were similar in both control and ischemic zones, respectively: 5.3 ± 1.2 nM and 7.9 ± 1.2 nM (n = 6). In the ischemic zone, the dialysate ATP level transiently increased 10-fold (range of 15-103 nM) during the first 10 min of the ischemic period; it fell back nearly to control levels during the second 10- to 20-min ischemic sampling period. On reperfusion, ATP levels increased transiently again before decreasing toward control values. In the control zone, no significant change in the dialysate ATP level occurred during both the ischemia and reperfusion periods. When a probe recovery of 14% (see below) was taken into account, the intramyocardial interstitial ATP concentration was in control conditions and increased 38 ± 8 nM to 373 ± 73 nM during the early phase of ischemia.
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Patterns of degradation of adenine nucleotides. Infusion of ATP by the dialysis probe was followed by its extensive catabolism as assayed by analysis of breakdown products in the dialysate effluent. Under conditions of steady-state delivery and breakdown, as indicated by the similarity of metabolite contents in two successive 20-min dialysate samples, ADP was the predominant metabolite. The pattern of catabolites formed was consistent with sequential dephosphorylation of ATP to ADP to AMP to adenosine (Fig. 2). The inhibition of ecto-5'-nucleotidase (ecto-5'-AMPase) by AOPCP before and during extracellular ATP infusion (50 µM) in in situ control hearts resulted in a reduced ATP degradation mainly as a consequence of a reduced formation of purine nucleosides and bases in the interstitium (Fig. 2). Furthermore, these experiments allowed us to estimate the in vivo recovery of the dialysis probe. The averaged dialysis probe in vivo recovery factor of AOPCP, an exogenous nondegradable compound, was 14.1 ± 2.6% under these experimental conditions.
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DISCUSSION |
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The present work using microdialysis and bioluminescence techniques demonstrates that the interstitial heart fluid contains a significant amount of ATP under resting conditions. During ischemia, the interstitial ATP concentration can increase 10-fold or more. In addition, as suggested by infusion of ATP or its derivatives, the adenine nucleotides are sequentially dephosphorylated in the interstitial space by a chain of separate ecto-enzymes.
On the basis of kinetic analysis of hydrolysis of adenine nucleotides at the surface of isolated rat ventricular myocytes, it has been suggested that three different ecto-enzymes, ecto-ATPase, ecto-ADPase, and ecto-5'-nucleotidase, are responsible for sequential degradation of ATP to ADP to AMP to adenosine. Moreover, ecto-ATPase and ecto-ADPase were supposed to be clustered together, providing preferential delivery of ADP from ATPase to ADPase (18, 19). The concept that ecto-ATPase and ecto-ADPase are separate enzymes has been recently questioned by the finding of an ectoenzyme, termed (Ca2+- Mg2+)-ATP-diphosphohydrolase (ATPDase), in many organs and tissues including blood vessels (23, 33). Besides ecto-nucleotidases, nonspecific phosphatases and ATP pyrohydrolase could be responsible for myocardial extracellular adenine nucleotide hydrolysis (8, 9, 14, 18). However, it has been recently demonstrated that nonspecific phosphatases do not contribute to adenine nucleotide metabolism at the surface of cardiomyocytes and endothelial cells (18, 19).
In each series of experiments in the present study, under conditions of steady-state delivery of adenine nucleotides, the predominant metabolite was the product of the first step of adenine nucleotide hydrolysis. Metabolite accumulation in the interstitium suggests that the rate of conversion of the initial substrate by appropriate ecto-nucleotidase was significantly greater than the rate of delivery of a newly formed metabolite by this enzyme to the subsequent one in the degradation pathway. Regardless of whether the initial substrate was ATP, ADP, or AMP, the amounts and relative contents of the major metabolite as well as total amounts of metabolites formed were nearly similar. Furthermore, during local infusion of 50 µM ATP, the kinetics of its extracellular degradation was likely to be far from saturation. Thus it is suggested that the activities of ecto-ATPase, ecto-ADPase, and ecto-5'-nucleotidase did not differ greatly from each other and showed no preferential delivery of locally produced adenine nucleotides in intact hearts. The results of the present study also show that direct conversion of extracellular ATP into AMP by ATP pyrohydrolase did not occur to any significant extent. Thus these adenine nucleotide degradation pathways are of minor importance when compared with hydrolysis by ecto-nucleotidases.
In in situ postinfarcted rat hearts, a slower degradation of each individual adenine nucleotide was observed when compared with in situ control hearts, suggesting that the activities of ecto-ATPase, ecto-ADPase, and ecto-5'-nucleotidase were reduced nearly by a similar extent (Fig. 3). Surprisingly, a similar pattern was observed in isolated perfused hearts. Furthermore, it has been recently reported that intracellular 5'-nucleotidase is downregulated during coronary underperfusion (10). Myocardial infarction is known to induce a prolonged Mg2+ loss in infarcted as well as in noninfarcted areas of the heart (4, 25). We suggest that the decreased Mg2+ plasma levels in the latter condition and the standard perfusate Mg2+ concentrations are low to reduce the activity of the ecto-nucleotidases, which are Ca2+- and Mg2+-dependent enzymes, as shown in vitro (2, 20).
The baseline interstitial fluid concentrations of endogenous ATP estimated in in situ hearts (38 ± 8 nM) greatly exceeded the concentrations of ATP (<1 nM) detected in the venous effluent of isolated, saline-perfused hearts previously reported (3, 31). Numerous studies have demonstrated that, in isolated perfused hearts, ATP is metabolized by more than 97% during a single coronary passage (3, 6, 9). In accordance with this finding, in some preliminary studies, no ATP could be detected in the myocardial dialysate after 50 µM ATP was added to the coronary perfusing solution of the isolated rat heart. When infused extracellularly via the dialysis probe, ATP degradation was reduced to one-third or less. This suggests that the activity of ecto-nucleotidases on smooth muscle and endothelial cells of cardiac vasculature is markedly higher than that on cardiomyocytes. The coronary endothelium might thus be a highly effective barrier for the movement of purines between the interstitial and vascular spaces (21).
During ischemia, dialysate ATP demonstrates only an initial burst of ATP, as previously reported by Vial et al. (31); at this time, the intramyocardial interstitial ATP concentration increased up to 373 ± 73 nM (Fig. 1). This profile differs from the continuous accumulation of adenine nucleotide breakdown products with time in the interstitium of the ischemic zone shown in rats (Ref. 29; these results) as well as dogs (16, 17). A reduction in extracellular space during ischemia is known to occur (15); it might perturb our ATP estimation but in any case would account for its large variations. Source(s) and mechanism(s) of ATP release during ischemia are poorly understood. Interstitial norepinephrine accumulation during ischemia (1) might be responsible for the peak ATP release, since catecholamine stimulation transiently increases coronary venous release of ATP in isolated perfused rat hearts (3, 6). In addition, as in nerves, it should be considered that the release of ATP might be accompanied by release of soluble nucleotidases that inactivate the neurotransmitter (27). Finally, the present findings that ventricular rhythm disturbances occurred transiently during the early period of ischemia when interstitial ATP peaked in the ischemic zone are in accordance with the deleterious arrhythmogenic effects of ATP previously hypothesized at the cellular level (30). In postinfarcted hearts in which adenosine nucleotide breakdown products appeared more slowly, the high-ATP concentration occurring at the early ischemic phase would be prolonged and further increase the susceptibility to arrhythmia.
In conclusion, the patterns of catabolites formed during extracellular infusion of adenine nucleotide in in situ and isolated rat hearts were consistent with the major pathway of metabolism being sequential dephosphorylation catalyzed by the chain of separate ecto-nucleotidases located on the surface of cardiomyocytes. In pathological situations (in situ postinfarcted hearts) as well as in in vitro experiments (isolated perfused hearts), a reduced catabolism rate of extracellular adenine nucleotides was observed. The present study also demonstrated that, in rat heart, interstitial fluid concentration of endogenous ATP is ~40 nM and it may increase 10-fold during early regional ischemia, a period when major electrical disturbances are known to occur.
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ACKNOWLEDGEMENTS |
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This study was supported by Institut National de la Santé et de la Recherche Médicale (INSERM) Grant Réseau Est-Ouest 93 EO 07 and by Action Concertée Coordonnée-Science du Vivant N-9 to INSERM U-390.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: G. Vassort, INSERM U-390, CHU Arnaud de Villeneuve, F-34295 Montpellier Cédex 5, France.
Received 23 February 1998; accepted in final form 29 May 1998.
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