Skip Navigation

Cardiovascular Research 1999 44(2):333-343; doi:10.1016/S0008-6363(99)00207-2
© 1999 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
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
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Gustafson, L. A
Right arrow Articles by Kroll, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gustafson, L. A
Right arrow Articles by Kroll, K.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 1999, European Society of Cardiology

Increased hypoxic stress decreases AMP hydrolysis in rabbit heart

Lori A Gustafsona,b,*, Coert J Zuurbiera,c, John E Bassetta, Jan Paul F Barendsb, Johannes H.G.M van Beekb, James B Bassingthwaightea and Keith Kroll1,a

aCenter for Bioengineering, University of Washington, Seattle, WA 98195, USA
bLaboratory for Physiology, Institute of Cardiovascular Research, Vrije Universiteit, Amsterdam, The Netherlands
cDepartment of Experimental Anesthesiology, Universiteit van Amsterdam, Amsterdam, The Netherlands

* Corresponding author. Laboratory for Physiology, Institute for Cardiovascular Research, Vrije Universiteit, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands. Tel.: +31-20-444-8133; fax: +31-20-444-8255 lorig{at}physiol.med.vu.nl

Received 24 November 1998; accepted 16 June 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: AMP conversion to adenosine by cytosolic 5'-nucleotidase (5NT) or to IMP by AMP deaminase determines the degree of nucleotide degradation, and thus ATP resynthesis, during reoxygenation. To elucidate the regulation of AMP hydrolysis during ischemia, data from 31P NMR spectroscopy and biochemical analyses were integrated via a mathematical model. Since 5NT is downregulated during severe underperfusion (5% flow), we tested 5NT regulation during less severe underperfusion (10% flow) and then made the perfusate hypoxic to see if the greater stress reactivated 5NT. Methods: 31P NMR spectra and coronary venous effluents were obtained from Langendorff-perfused rabbit hearts subjected to two 30-min periods of underperfusion (10% flow); the second period with or without additional hypoxia (30% O2). Data were analyzed with a mathematical model describing the kinetics of myocardial energetics and metabolism. Results: A single 30-min period of 10% flow causes downregulation of AMP hydrolysis and the data from the second period of underperfusion are best described by lower 5NT activity, even in the presence of extra hypoxia. Thirty percent less purines appear in the venous effluent than predicted by the phosphoenergetics (PCr and ATP) when IMP is not allowed to accumulate by the model, however the model indicates that a constant accumulation of IMP via AMP deaminase could explain the discrepancy between expected and measured purines in the venous effluent. Conclusions: While AMP hydrolysis to adenosine is prominent in early ischemia and acts to preserve cellular energy potential, during a second ischemic period, nucleotides are conserved by the stable inhibition of AMP hydrolysis. Furthermore, during 10% flow conditions, nucleotides are conserved, possibly via an IMP-accumulatory pathway.

KEYWORDS Adenosine; Computer modeling; Energy metabolism; Hypoxia/anoxia; Ischemia


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
In the heart, an imbalance between oxygen supply and demand causes an immediate fall in PCr, followed by a net hydrolysis of ATP and an increase in the concentration of AMP. AMP can then be dephosphorylated to adenosine by the enzyme 5'-nucleotidase (5NT, E.C. 3.1.3.5 [EC] ), or deaminated to IMP by the enzyme AMP deaminase (AMPD, E.C. 3.5.4.6 [EC] ). Cytosolic ATP availability is essential for myocardial contraction and relaxation. Prolonged depletion of ATP during ischemia leads to irreversible damage during reperfusion [1], thus the treatment of myocardial ischemia or the provision of cardiac protection during coronary bypass will be aided by preventing ATP depletion. Adenosine is membrane permeable and its release evokes cardioprotective mechanisms such as coronary vasodilation, improving the tissue oxygen supply [2]. Ischemic preconditioning is mediated by adenosine receptor activation. Most importantly, AMP hydrolysis to adenosine provides a mechanism whereby the phosphorylation potential is preserved by mass balance during compromised energy supply (i.e. low energy nucleotides are removed) [3]. While AMP hydrolysis is beneficial during acute ischemia, the continued loss of nucleosides during prolonged ischemia will lead to the depletion of nucleotide pools [4]. The regulation of AMP hydrolysis is therefore important for myocardial survival during prolonged ischemia and cardioplegia.

AMP hydrolysis is downregulated during severe and prolonged underperfusion [5]. Purine efflux at similar cytosolic AMP concentrations was depressed during the second of two identical periods of underperfusion. Model analysis indicated that 5NT was downregulated late in the first period of underperfusion [5]. Our question is: ‘Does this downregulation of 5NT persist if the energy supply mechanisms are stressed further by reducing the pO2 during underperfusion?’ In order to test the hypothesis that 5NT is downregulated during prolonged underperfusion yet can become re-upregulated during greater hypoxic stress, we performed experiments designed to increase cytosolic AMP levels by using hypoxic perfusate (30% oxygen, compared to 95% in controls) during the second period of underperfusion (10% flow).

The rate of adenosine formation from AMP in the cytosol is influenced by at least four factors: the AMP concentration, and the activities of three enzymes which influence its concentration: adenosine kinase (AK), which rephosphorylates adenosine to AMP, AMP deaminase, which deaminates AMP to IMP, and cytosolic, AMP-preferring 5NT (cN-I), which dephosphorylates AMP to adenosine [6–8]. Our previous mathematical model [5] did not include AMP deaminase, so this pathway, and the IMP->inosine pathway catalyzed by the IMP-preferring isoform of 5NT (cN-II), have been included in the present model metabolically depicted in Fig. 1. The results show that a single 30-min period of underperfusion at 10% flow downregulates AMP hydrolysis and that additional hypoxic stress elevates cytosolic AMP levels further but purine efflux increases only a little. The data are best described by the mathematical model with a lowered activity of 5NT (cN-I) and a constant AMP deaminase activity which allows IMP accumulation. The conclusion is that 5NT is an important regulator of AMP hydrolysis.


Figure 1
View larger version (16K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Model description of myocardial phosphoenergetics and nucleoside metabolism. Abbreviations: Cr, creatine; PCr, phosphocreatine; ATP, adenosine triphosphate; ADP, adenosine diphosphate, AMP, adenosine monophosphate, Cr kinase, creatine kinase; {Delta}rATP, rate of ATP synthesis minus rate of ATP hydrolysis; 5NT, AMP-preferring isoform of 5'-nucleotidase (cN-I); 5NT-II, IMP-preferring isoform of 5'-nucleotidase (cN-II); Ado kinase, adenosine kinase; Ado deaminase, adenosine deaminase; Pi, inorganic phosphate; PS, permeability surface area products for membrane transport.

 

   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Isolated heart preparation
All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996) and approved by the institutional animal experimental ethics committee.

New Zealand White rabbits (2.2–3.0 kg) were sedated with acetylpromazine (0.8 mg/kg, s.c.), and anesthetized with ketamine (45 mg/kg, i.m.) plus xylazine (4 mg/kg, i.m.). The rabbits were tracheotomized and ventilated with room air supplemented with oxygen. After opening the thorax and administration of heparin (200 U, i.v.), the aorta was cannulated in situ and perfusion was started, followed by excision of the heart. In situ cannulation was implemented to prevent any cardioplegic or preconditioning effects upon the myocardial metabolism. The hearts were Langendorff-perfused at 37°C at a constant flow (perfusion pressure=80–100 mmHg) with non-recirculating, modified Krebs—Henseleit bicarbonate buffer containing (in mM) 118 NaCl, 3.8 KCl, 1.2 KH2PO4, 0.7 MgSO4, 2.1 CaCl2 0.1 EDTA, 25 NaHCO3, 11 glucose, 5 pyruvate, and 0.1% bovine serum albumin, equilibrated with 95% O2/5% CO2 using a membrane oxygenator, resulting in a pH of 7.35–7.45. A fluid-filled latex balloon was inserted in the left ventricle, connected to a pressure transducer and inflated, yielding a systolic pressure between 70–90 mmHg, with an end-diastolic pressure less than 5 mmHg. The hearts were electrically paced at 180 beats per min.

Coronary venous effluent samples were collected as described previously [5]. The hearts were submerged in 37°C perfusate in a cylinder encircled with a solenoid style radiofrequency NMR coil. After the set-up was placed inside the magnet, the radiofrequency coil was tuned (81 MHz) and matched, the gradient coils were shimmed, and a fully relaxed 31P NMR spectrum was acquired. Wet ventricular weight was determined after each experiment.

2.2 Experimental protocol
Twelve rabbits were used to investigate AMP hydrolysis during two sequential and identical periods of underperfusion. The control group of hearts (n=6) were subjected to two 30-min periods of underperfusion with perfusate equilibrated with 95% O2/5% CO2 at a flow of 10% of the baseline flow (approximately 3 ml/min, or 0.5 ml/min/g). The two periods of underperfusion were separated by a 20-min period of reperfusion at baseline flow. The hypoxia group (n=6) underwent an identical procedure, except that during the second period of underperfusion the perfusate was equilibrated via the membrane oxygenator with 30% O2, 5% CO2 and 65% N2.

2.3 NMR spectroscopy
Phosphorus NMR measurements were obtained using a 4.7-Tesla superconducting magnet (Bruker) and a CSI spectrometer (GE-Omega) and analyzed using an automated fitting routine [9] as previously described [3]. Intracellular Pi was determined and intracellular pH and the free intracellular Mg2+ concentration were calculated as described [3]. Due to interference by extracellular Pi in the perfusion medium, baseline and reperfusion intracellular pH were assumed to equal 7.1 [10]. Cytosolic free [AMP] was calculated using creatine kinase and adenylate kinase equilibrium expressions, adjusted to the calculated H+ and Mg2+ concentrations, and it was assumed that the total concentration of PCr+Cr decreased linearly by 5% during each of the two periods of underperfusion [5].

2.4 Determination of venous purines and lactate
Coronary venous effluent samples were collected and analyzed by HPLC as previously described [5]. Total purine release was calculated by summing the purines (adenosine+inosine+hypoxanthine) released during each 2- or 5-min period for the entire 30-min period of underperfusion. Effluent lactate concentration was measured with a YSI glucose analyzer equipped with a lactate membrane.

2.5 Model analysis
A mathematical model of myocardial phosphoenergetics and nucleotide metabolism, adapted from [3] was used to analyze the data. The metabolic processes described by the model are depicted in Fig. 1. A full description of the rate equations, differential equations and the model parameter values can be found in the appendix of Ref. [3]. The model describes the intracellular concentrations of PCr, Cr, ATP, ADP, AMP, Pi, adenosine, and inosine, the enzymes creatine kinase, myokinase, AMP-preferring and IMP-preferring isoforms of cytosolic 5NT, AMP deaminase, adenosine kinase and adenosine deaminase, the membrane transport of adenosine and inosine, and Pi and Cr in exchange with an interstitial region. The processes whereby ATP is synthesized (oxidative phosphorylation, glycolysis) and hydrolyzed (e.g. Ca2+ and ion pumps, myofibril contraction, ATPases, homeostasis, etc.) were described as a continuous function, {Delta}rATP ({Delta}rATP=rate of ATP synthesis–rate of ATP hydrolysis), providing a flexible means for describing the time-course and extent of the net energy imbalance experienced during underperfusion and reperfusion [3]. The parameters describing {Delta}rATP were estimated empirically using optimization procedures. The baseline data showed a low, continuous outflow of adenosine indicating a slightly greater hydrolysis than synthesis of ATP during baseline conditions in the buffer perfused rabbit hearts. The current model allows for this small net ATP breakdown, differing thereby from the original model [3], which assumed baseline {Delta}rATP=0.

Permeability surface area (PS) products described transmembrane exchange of adenosine, inosine, Pi and creatine. Enzyme dissociation constants and PS products were taken from literature values [3], with the exception of the Vmax of 5'-nucleotidase and AMP deaminase, which were determined by the model. The present model incorporates an alternative pathway of AMP hydrolysis to IMP by AMP deaminase. Literature values from the rabbit for AMP deaminase [11] were used as starting values (i.e. Vmax=60 µmol/min/g, Km=1.7 mM). The Km and Vmax of AMP deaminase were then optimized prior to each simulation run. The product of this reaction, IMP, was further allowed to be hydrolyzed to inosine by an IMP-preferring isoform of 5'-nucleotidase (cN-II) [12].

To fit the model to the NMR and purine data, an automated least squares optimization routine (SIMPLEX) was used to simultaneously fit the PCr, ATP, adenosine and inosine curves by adjusting the Vmax of 5NT (predictions of the Km were found to vary only slightly, see results) and the parameters of the {Delta}rATP function. All other model parameters were either held constant during fitting or were changed according to direct measurements (flow, intracellular pH, Mg+2). Because the model does not include hypoxanthine, the coronary venous hypoxanthine data were added to the inosine data for fitting with the model inosine data. ‘Best fits’ were derived from the SIMPLEX routine using various starting values to ensure avoidance of local minima. Means and standard errors of the parameter estimates were obtained by fitting the data from each individual experiment. Initial concentrations of ATP (6.0 mM), PCr (10.5 mM) and Cr (13.2 mM) used in the modeling were taken from biochemical measurements of freeze-clamped hearts performed previously in this laboratory [3].

2.6 Statistical analysis
All data are presented as mean values±standard error of the mean (S.E.M. for n=6). Mean values of the model results (i.e. Vmax of 5NT and the {Delta}rATP parameters) were determined from the separate model solutions for each experiment. Statistical comparisons between the first period of underperfusion and the second period of underperfusion, with and without hypoxia, were made using a repeated measures ANOVA with Tukey's post-hoc test for individual comparisons. A value of P<0.05 was considered to be indicative of statistical significance.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Purine efflux during increased hypoxic stress
The NMR, purine and metabolic data for the two groups are presented in Fig. 2. The first period of underperfusion (10% of basal flow, approximately 0.5 ml/min/g) yielded very similar results for each group, therefore this data has been grouped (n=12). The second periods of underperfusion, one under normoxic (95% oxygen) conditions, and one under hypoxic (30% oxygen) conditions are, thus, directly comparable. During the first period of underperfusion, PCr fell from 10.5 to 4.6 mM and then slightly recovered toward baseline, while ATP slowly fell from 6 to 4.3 mM by the end of the first underperfusion period. During the second period of underperfusion, PCr fell to the same level (4.6 mM) and showed a similar recovery, while ATP only slightly decreased (from 4.3 to 3.9 mM). When hypoxia, however, was applied during the second period of underperfusion, PCr fell to 2.7 mM, after a delay, and ATP fell from 4.3 to 3.3 mM. The calculated concentrations of ADP and AMP indicate that hypoxia during the second period of underperfusion indeed resulted in an elevation in the cytosolic concentrations of ADP and AMP. Peak ADP for the first, second and second+hypoxia groups, respectively, were: 145, 94, and 130 µM. Peak AMP concentrations for the first, second and second+hypoxia groups, respectively, were: 3.4, 1.8, and 4.5 µM. This elevation of 5NT substrate concentration (i.e. AMP) due to hypoxia resulted in a higher purine efflux: 244.6 (34.3) nmol/g total purines as compared to 179 (15.0) nmol/g during the second period of underperfusion with normoxia. Both levels of purine efflux, however, were significantly attenuated as compared to the purine efflux from the first period of underperfusion (i.e. 404.7 (24.8) nmol/g). Lactate efflux and the intracellular Pi concentration were elevated during the second period of underperfusion with hypoxia as compared to normoxic underperfusion (P<0.05), while the cytosolic hydrogen ion concentration was similar for the two periods. Diastolic pressure did not rise during the periods of underperfusion, but developed left ventricular pressure fell quickly to 10–12 mmHg after the onset of underperfusion and was similar for the three different underperfusions.


Figure 2
View larger version (40K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Nuclear magnetic resonance, purine and metabolic data from two successive periods of underperfusion (10% baseline flow) with and without hypoxia. PCr (bullet) and ATP ({diamond}). Calculated cytosolic ADP (bullet) and AMP ({diamondsuit}); AMP was multiplied by a factor of 10 to aid viewing. Intracellular Pi (bullet). Intracellular pH (bullet). Lactate concentration in venous effluent (bullet). Purine concentration in venous effluent, adenosine ({blacksquare}), hypoxanthine+inosine ({blacktriangleup}). Total purine release for the first 30-min period was 405±25 nmol/g; for the second period (normoxic), 179±15 nmol/g and for the hypoxic second period, 245±34 nmol/g. Left ventricular developed pressure (bullet).

 
3.2 Model analysis
A second, consecutive period of underperfusion, under normoxic conditions, caused an attenuation of purine release in the venous effluent. While this could indicate a downregulation of the activity of 5NT, under these conditions, the cytosolic concentration of AMP was also attenuated, thus, substrate limitation could be the cause of such attenuation. During a second period of underperfusion under hypoxic conditions, purine efflux increased, suggesting a possible upregulation of 5NT. However, the simultaneous elevation of cytosolic AMP concentration could have increased purine efflux by a simple mass action effect, without 5NT activation. We, therefore, analyzed the simultaneously-obtained NMR and purine data with a mathematical model which is able to differentiate between the mass action effects of the AMP concentration and the enzymatic effects of 5NT (or AMPD). Simultaneous model fits to the PCr, ATP, adenosine and inosine (inosine+hypoxanthine) data, using a four-region mathematical model of myocardial energetics and enzyme kinetics, are given in Fig. 3. Separate fits were obtained from each of the six individual experiments of the three different periods of underperfusion. The only parameters allowed to vary during the fitting optimization were the Vmax of 5NT and the parameters describing the degree of energy imbalance ({Delta}rATP). The means and standard errors of these analyses are given in Table 1. The model analysis indicates that a 30-min period of 90% flow reduction induces a downregulation of 5NT activity. Furthermore, the analysis indicates that, while the elevated AMP levels during increased hypoxic stress do lead to an increase in purine efflux, the data are best described by a lower 5NT activity. The high-energy phosphate and purine data were best fit for the first period of underperfusion with an average Vmax of 164 nmol/min/g for 5NT. The second period of underperfusion, under normoxic conditions, was best fit with a significantly lower (P<0.05) Vmax of 129 nmol/min/g. The Vmax for the hypoxic second period of underperfusion was significantly lower (P<0.05) than for the first period of underperfusion, as well as for the second (normoxic) period, and was best fit using a value of 85 nmol/min/g. Analysis of the initial part of the purine efflux curves indicated a high affinity of 5NT for its substrate during all three conditions, thus the Km for 5NT was set to 0.8 µM during all model optimizations. Qualitatively similar results were achieved when the Vmax was set to a literature value of 290 nmol/min/g [13], i.e. Km was the lowest under control conditions (3.0 µM), and highest for the hypoxic group (10.9 µM) (Fig. 4). The fits obtained by varying the Km, however, were poorer than the Vmax variations, due to a delay of the purine efflux curves. The model, however, best describes AMP hydrolysis, as a whole, and has difficulty in discerning between Km or Vmax effects. It remains unclear at this stage whether downregulation of 5NT during underperfusion is due to a change in affinity for AMP or the Vmax.


Figure 3
View larger version (15K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Simultaneous model fits to the PCr (bullet), ATP ({diamond}), adenosine ({square}) and hypoxanthine+inosine ({triangleup}) data using the four region model of myocardial energetics and enzymatic kinetics (Fig. 1). Data are indicated by the symbols, model fits by the lines.

 

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

  		Table 1 Model predictions of Vmax for 5NT and {Delta}rATPa

 

Figure 4
View larger version (19K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Exploratory model fits to the PCr (bullet), ATP ({diamond}), adenosine ({square}) and hypoxanthine+inosine ({triangleup}) data using the four-region model of myocardial energetics and enzymatic kinetics with (left) and without (right) AMP deaminase activity. Data are indicated by the symbols and are identical for (A) and (B); optimized model fits indicated by the lines. Note the comparable fits to the high energy phosphate curves, but an over-estimation of purine efflux in the fits without AMP deaminase activity.

 
A surprising result of this study was that the measured purine efflux at 10% flow was considerably lower (30%) than that predicted by initial runs with the model which had been validated at 5% flow levels. This suggested that another pathway of AMP hydrolysis and accumulation was active at 10% flow and not at 5% flow. A post hoc aim of the model analysis, therefore, was to test the possibility that another enzyme, for example AMP deaminase, plays a regulatory role during ischemia-induced purine efflux. It has been shown, for example, that AMP degradation in de-energized heart cells can occur through either deamination (AMPD) or dephosphorylation (5NT) [14]. Thus, the relative rates of the IMP and adenosine pathways reflect the competition between AMPD and 5NT for AMP (Fig. 1). Since a decrease in AMPD activity during a second period of underperfusion would also result in a decreased purine efflux, this pathway was added to the mathematical model.

Exploratory modeling, performed to investigate the role of AMP deamination to IMP by AMP deaminase, indicated that the data were best described by allowing a constant flux through AMPD, which was allowed to accumulate as IMP (Fig. 4). The analysis, which used as determinants the ratio of inosine+hypoxanthine to adenosine, and the total amount of purines that appeared in the effluent during underperfusion, resulted in a predicted Vmax for AMPD of 90 nmol/min/g and a Km of 30 µM for AMP. The model predicted that IMP accumulated in the micromolar to low millimolar range during underperfusion. Separate freeze-clamp experiments indicated, indeed, an accumulation of 7–12 µmol/g (1–2 mM) IMP after a 30-min period of 10% flow (unpublished observations). These values are in agreement with literature values. It has been shown, for example, that rat hearts accumulated 200 µM IMP after 45 min of ischemia, in the presence of pyruvate and glucose, which increased to more than 600 µM in the presence of glucose alone [15]. During the final optimization runs in this study, the curve fits were not improved by allowing the parameters describing the deamination of AMP to IMP to vary. In other words, the data were best described by a constant AMPD activity, therefore, AMPD probably does not play a regulatory role under the conditions tested in these experiments. Furthermore, that the data were best fit when purines were allowed to accumulate suggests a very low activity of the IMP-preferring isoform of 5NT (cN-II) during underperfusion or hypoxia.

The present study was performed at a lesser degree of ischemic stress (i.e. 10% of baseline flow) than the previous study (i.e. 5% flow during underperfusion) and thus allows a comparison of the degree of hypoxic/ischemic stress necessary to induce the downregulation of 5NT. An interesting and surprising result, however, of the higher flow was the considerable degree of PCr recovery within the first 5 min of the initiation of underperfusion. Exploration of this phenomenon with the mathematical model indicated such steep recovery could not be predicted by a high, but constant, 5NT activity, as predicted by the open-adenylate hypothesis [3]. One could postulate that 5NT activity varies strongly during this period and that such rapid changes in the 5NT activity would cause such strong PCr recovery by a high degree of AMP hydrolysis, however, the purine efflux patterns were reasonably fit without such rapid changes. Based upon the lactate efflux data (Fig. 2), which suggest an early burst of glycolytic activity shortly after the initiation of underperfusion, we explored the possibility of a short period of positive energy balance occurring during this period. This was achieved by the implementation of a second {Delta}rATP function. It was found, empirically, that the data were best fit when a small positive burst of energy was allowed, measuring only 10–15% of the total negative energy balance which occurred after the onset of underperfusion (Fig. 5). Hard conclusions cannot be drawn from such empirical modeling, it does suggest the possibility of a short period of positive energy imbalance shortly after the onset of underperfusion. Possibly, the energy is glycolytic in nature, because Janier et al. [16] showed a burst of lactate release in glucose-perfused rabbit hearts within 10 min after the onset of 10% flow. Or it may be that mechanical downregulation precedes the metabolic downregulation and occurs within the first few minutes of underperfusion (i.e. developed left ventricular pressure falls to lowest level within 2 min), whereby the energy demands are actually even less than the energy supply. This is supported by the finding that the model predicts a very slight, yet positive energy imbalance (0.3%) during the prolonged phase of underperfusion (i.e. 10–30 min).


Figure 5
View larger version (19K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Exploratory model fits to the PCr (bullet), ATP ({diamond}), adenosine ({square}) and hypoxanthine+inosine ({triangleup}) data using the four-region model of myocardial energetics and enzymatic kinetics with (left) and without (right) extra glycolytic activity. Data are indicated by the symbols and are identical for (A) and (B); optimized model fits indicated by the lines. Note the inability to fit the high energy phosphate curves, with an over-estimation of purine efflux in the fits without the extra postulated glycolytic activity.

 
3.3 Relationship between cytosolic AMP and purine release
The relation between the calculated cytosolic AMP concentration and the measured purine efflux for each separate period of underperfusion is given in Fig. 6. The data points are from the venous purine data (bottom), while the lines are derived from the model fits (top). The figure indicates a lower purine efflux during the second period of normoxic underperfusion with similar levels of AMP concentrations, as compared to the first period of underperfusion. For example, during the first period of underperfusion, a capillary concentration of 40 µM is achieved at a cytosolic AMP concentration of 2 µM, while during the second period of underperfusion only 20 µM is achieved at a cytosolic AMP concentration of 2 µM. During the hypoxic period of underperfusion, the differences between the relationships become even more extreme. During the hypoxic second period of underperfusion, cytosolic AMP levels reach 4.5 µM, yet the purine efflux does not go above a capillary concentration of 20 µM. It is interesting to note that the hysteresis of these relationships, caused by transport delays in the system and by the phasic nature of the AMP concentration, disappears during the hypoxic situation. This is probably due to the fact that the AMP concentration is no longer phasic during continuous hypoxia (see Fig. 2).


Figure 6
View larger version (14K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 Top panel: Model predictions of the relationship between cytosolic free AMP and capillary purine efflux for the three different periods of underperfusion. (1) First period of underperfusion, (2C) second period of underperfusion (normoxic), (2H) second period of underperfusion (hypoxic). Bottom three panels: Data points depicting the measured relations between the cytosolic AMP concentration and the total purine release for the three different groups.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
4.1 Downregulation of 5'-nucleotidase
The relationship between the cytosolic AMP concentration and adenosine release has been the topic of much research. A linear relationship between free AMP and adenosine formation has generally been assumed and that the cytosolic concentration of AMP drives the reaction by mass action at the enzyme, 5NT. However, many conditions have been observed where there has been a dissociation observed between free AMP and adenosine release [17]. It has only recently been recognized that adenosine kinase inhibition amplifies the release of adenosine during hypoxic stress, thereby explaining the enhancement of adenosine release at only small or no increases in cytosolic AMP concentrations [17,18]. The role that 5NT may play during ischemic preconditioning has also been controversial. It has been proposed, for example, that 5NT becomes activated during ischemic preconditioning [19], yet others have shown purine release attenuation after brief exposures to global ischemia [20,21]. Possible confounding factors in these studies, however, are mass action effect of AMP and the degree of energetic imbalance.

Previous work from this laboratory, which implemented an integrative mathematical model to account for varying degrees of energetic imbalance, has shown that 5NT, indeed, becomes downregulated during prolonged underperfusion (45 min, 5% flow) [5]. This can be considered to be a protective mechanism whereby the cardiomyocyte attempts to prevent depletion of the nucleotide/nucleoside pool during prolonged oxygen deprivation. The question remained, however, whether 5NT remains downregulated in the face of additional hypoxic stress, or does it become re-activated in order to provide adenosine-derived benefits. The data presented here show clearly lower purine efflux at similar cytosolic AMP levels during a normoxic second period of underperfusion, which remains attenuated, even when cytosolic AMP levels are increased two-fold by hypoxia. The model-based analysis shows that the observed decrease in purine efflux can be ascribed to a lower 5NT activity. We conclude that 5NT does become downregulated during a 30-min period of 10% flow (Vmax=164 vs. 129 nmol/min/g; this paper), albeit to a lesser degree than after a 45-min period of 5% flow (Vmax=140 vs. 67 nmol/min/g; [5]).

Since we have shown 5NT activity to be downregulated after a 20-min period of 5% flow [5], we assume here that both second periods of underperfusion described in this paper (i.e. under normoxic and under hypoxic conditions), began with similarly reduced activities of 5NT. That the second period of underperfusion with hypoxia is best fit with an even lower 5NT activity indicates modulation during the second underperfusion period. The systematic over-prediction of the purine efflux during the last 20 min of the second period of underperfusion with hypoxia is suggestive that 5NT becomes further downregulated during this phase. The peak is reasonably fit by an activity of 88 nmol/min/g, yet the purine efflux decreases during the last 10 min of the underperfusion period – even while the cytosolic concentration of AMP is elevated during this period. A limitation of the current model is the inability to allow parameter values to vary during one period of underperfusion. These findings indicate the desirability to apply mathematical models in future work which have the ability to adapt 5NT levels during the underperfusion period.

4.2 In vivo regulation of AMP hydrolysis
Results presented here show a downregulation of AMP hydrolysis during a period of underperfusion. The primary enzymes known to regulate the net hydrolysis of AMP are 5'-nucleotidase, AMP deaminase and adenosine kinase. One should also not forget to take into account that Fredholm et al. in 1982 [22] showed that AMP itself is released from hearts by sympathetic nerve stimulation. Regulation of AMP levels and adenosine efflux is, thus, complex and finely tuned, as befitting an important regulator of myocardial energetics and survival. Indeed, the greater tolerance of neonatal myocardium to ischemia-reperfusion injury has been attributed to lesser AMP hydrolysis due to lower concentrations of 5NT [23]. In vitro studies on highly purified 5NT indicate H+ and Pi to be inhibitory, while AMP, ADP and Mg2+ are activators. A recent study by Bak and Ingwall [24] using hyperthyroid hearts to manipulate intracellular pH adds evidence that acidosis decreases the activity of 5NT and thus enhances the resynthesis of ATP during reperfusion. These data obtained during global ischemia, however, are difficult to relate to less severe ischemic episodes, since our preparations have never shown a resynthesis of ATP during the reperfusion period, even after a 45 min period of 5% flow [5]. Evidently a different accumulatory pathway is active during global ischemia than during low flow ischemia, even though convincing data exist for both preparations that AMP hydrolysis is downregulated during these periods. Our results tend to support the hypothesis of Pi as a regulator of 5NT, since pHi was similar during both second periods of underperfusion, while Pi levels were higher during hypoxia, thus possibly resulting in the lowered 5NT activity. Indeed, as shown by Itoh et al. [25], 5NT activity is more sensitive to changes in Pi concentrations at lower energy charges.

In vitro data, for 5NT isolated from dog [26], rat and human [12] heart obtain a Km of 1.5 mM under maximally activated conditions. This value, however, is quite different from the model-based predicted Km for 5NT obtained in this study. This model prediction of a high affinity of 5NT for AMP (approximately 1µM) results from the fact that AMP hydrolysis to adenosine is quickly achieved at AMP concentrations in the low micromolar range early in the underperfusion period. Since free cytosolic AMP levels rarely exceed low micromolar concentrations, it would appear the in vitro indications that the Km of 5NT for AMP is in the millimolar range are not physiologically realistic. Furthermore, exploratory modeling showed clearly that the measured purine efflux rates could not be achieved when in vitro values (~3mM) obtained from the literature were used for the Km of 5NT for AMP.

In skeletal muscle, the IMP pathway is very active and remains dominant, even at high Pi concentrations. In the heart, however, AMPD activity is considerably lower, thus it has been often ignored. Data exist, however which show that the IMP pathway does play a regulatory role during energetic perturbations of the heart. It has been found, for example, that energy-depleted human cardiomyocytes dephosphorylate 70% of the AMP via 5NT, and deaminate 30% via AMPD [8]. In rat heart, the regulation of AMPD activity is complex, with allosteric modulation by multiple factors including ATP, GTP and Pi [27]. Perfusion with 2-deoxyglucose, which causes a fall in ATP levels without a concomitant rise in Pi, induces predominantly inosine release, while hypoxia induces a release of a combination of adenosine and inosine [27]. Thus, the IMP pathway dominates in the 2-deoxyglucose perfused heart, where ATP and Pi levels are low. Under such conditions, AMPD accounted for 97% of the AMP catabolites. In contrast, in the anoxic heart, where AMPD is inhibited by high Pi, the IMP pathway accounted for only 23% of the AMP flux. The previously described model [5], which lacked a purine-accumulating pathway, was able to quantitatively describe the purine efflux during 5% flow conditions, yet was unable to adequately describe purine efflux under 10% flow conditions. Since AMPD activity is regulated by Pi [27–29], one may speculate that the IMP pathway is active under 10% flow conditions, where Pi levels reach 5 mM, yet is inactivated by Pi during 5% flow conditions, where Pi levels reached 10–15 mM. The regulation of the hydrolysis of AMP to adenosine, or the deamination of AMP to IMP is emerging as a complex and finely tuned system. Both H+ and Pi may be primary regulators of these paths. Further work is needed to fully elucidate the underlying mechanisms of regulation since the path that is chosen, one of vasodilation and nucleotide depletion or one of accumulation has far-reaching ramifications for the bioenergetics of the heart.

In summary, our results give strong evidence for the persistent regulation of AMP hydrolysis during prolonged ischemic conditions. Previous data show that 5NT becomes downregulated 20 min after the onset of severe ischemia. The current results point out clearly that such stable downregulation is also achieved at a less severe level of ischemia, but that also a nucleoside/nucleotide accumulatory pathway also is active

4.3 Clinical implications
The two general schools of thought regarding adenosine release by metabolically-perturbed myocardium have been: (1) adenosine release is good, therefore more will be better, and (2) adenosine release results in nucleotide depletion, therefore less is better.

However, a more refined picture is now emerging. Severe underperfusion results in a stable downregulation of AMP hydrolysis during severe ischemia, which persists through a short period of reperfusion [5]. The data presented here show that the decreased AMP hydrolysis also persists during a period of even greater metabolic stress. Thus, a strategy is chosen whereby an initial, beneficial period of high purine efflux is followed by a nucleotide-saving strategy whereby AMP hydrolysis to adenosine is decreased and nucleotides are allowed to accumulate. Cardioprotective strategies need to be developed which will enhance and not antagonize these processes. One can envision, for example, pharmacological interventions which aid in the accumulation of nucleotides (i.e. IMP) without adversely influencing the phosphorylation potential, while at the same time the importance of adenosine receptor activation should not be forgotten during the early phases of ischemia.

Time for primary review 28 days.


   Acknowledgements
 
The authors thank Rodney Gronka for his invaluable expertise in conducting the NMR experiments. This study was supported by National Institutes of Health grants HL51152 and RR01243.


   Notes
 
1 Deceased 15 July, 1997. Back


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

  1. Vary T.C., Angelakos E.T., Schaeffer S.W. Relationship between adenine nucleotide metabolism and irreversible ischemic damage in isolated perfused rat heart. Circ Res (1979) 45:218–225.[Abstract]
  2. Matherne G.P., Headrick J.P., Berr S., Berne R.M. Metabolic and functional responses of immature and mature rabbit hearts to hypoperfusion, ischemia and reperfusion. Am J Physiol (1993) 264:H2141–H2153.[Web of Science][Medline]
  3. Kroll K., Kinzie D.J., Gustafson L.A. Open system kinetics of myocardial phosphoenergetics during coronary underperfusion. Am J Physiol (1997) 272:H2563–H2576.[Web of Science][Medline]
  4. Reimer K.A., Murry C.E., Yamasawa I., Hill M.L., Jennings R.B. Four brief periods of myocardial ischemia cause no cumulative ATP loss or necrosis. Am J Physiol (1986) 251:H1306–H1315.[Web of Science][Medline]
  5. Gustafson L.A., Kroll K. Downregulation of 5'-nucleotidase in rabbit heart during coronary underperfusion. Am J Physiol (1998) 274:H529–H538.[Web of Science][Medline]
  6. Kroll K., Decking U.K.M., Dreikorn K., Schrader J. Rapid turnover of the AMP-adenosine metabolic cycle in the guinea pig heart. Circ Res (1993) 73:846–856.[Abstract/Free Full Text]
  7. Newby A.C. The pigeon heart 5'-nucleotidase responsible for ischaemia-induced adenosine formation. Biochem J (1988) 253:123–130.[Web of Science][Medline]
  8. Smolenski R.T., Suitters A., Yacoub M.H. Adenine nucleotide catabolism and adenosine formation in isolated human cardiomyocytes. J Mol Cell Cardiol (1992) 24:91–96.[Web of Science][Medline]
  9. Heineman F.W., Eng J., Berkowitz B.A., Balaban R.S. NMR spectral analysis of kinetic data using natural lineshapes. Magn Reson Med (1990) 13:490–497.[Web of Science][Medline]
  10. Mallet R.T., Kang Y.H., Mukohara N., Bunger R. Use of cytosolic metabolite patterns to estimate free magnesium in normoxic myocardium. Biochim Biophys Acta (1992) 1139:239–247.[Medline]
  11. Thakkar J.K., Janero D.R., Yarwood C., Hreniuk D. Isolation and characterization of AMP deaminase from mammalian (rabbit) myocardium. Biochem J (1993) 290:335–341.[Web of Science][Medline]
  12. Skladanowski A.C., Hoffmann C., Krass J., Jastorff B., Makarewicz W. Structure—activity relationship of cytoplasmic 5'-nucleotidase substrate sites. Biochem J (1996) 314:1001–1007.[Web of Science][Medline]
  13. Darvish A., Pomerantz R.W., Zografides P.G., Metting P.J. Contribution of cytosolic and membrane-bound 5'-nucleotidases to cardiac adenosine production. Am J Physiol (1996) 271:H2162–H2167.[Web of Science][Medline]
  14. Hohl C.M., Wimsatt D.K., Brierley G.P., Altschuld R.A. IMP production by ATP-depleted adult rat heart cells. Circ Res (1989) 65:754–760.[Abstract/Free Full Text]
  15. De Groot M.J.M., Coumans W.A., van der Vusse G.J. The nucleotide metabolism in lactate perfused hearts under ischaemic and reperfused conditions. Mol Cell Biochem (1992) 118:1–14.[CrossRef][Web of Science][Medline]
  16. Janier M.F., Vanoverschelde J.L., Bergmann S.R. Ischemic preconditioning stimulates anaerobic glycolysis in the isolated rabbit heart. Am J Physiol (1994) 26:H1353–H1360.
  17. Decking U.K., Schlieper G., Kroll K., Schrader J. Hypoxia-induced inhibition of adenosine kinase potentiates adenosine release. Circ Res (1997) 81:154–164.[Abstract/Free Full Text]
  18. Wagner D.R., Bontemps F., van den Berghe G. The AMP-adenosine cycle is active during normoxia and impaired in ATP depletion in isolated rabbit cardiomyocytes. Adv Exp Med Biol (1994) 370:323–326.[Medline]
  19. Kitakaze M., Hori M., Takashima S., Sato H., Inoue M., Kamada T. Ischemic preconditioning increases adenosine release and 5'-nucleotidase activity during myocardial ischemia and reperfusion in dogs. Implications for myocardial salvage. Circulation (1993) 87:208–215.[Abstract/Free Full Text]
  20. Bradamante S., Piccinini F., Delu C., Janssen M., de Jong J.W. NMR evaluation of changes in myocardial high energy metabolism produced by repeated short periods of ischemia. Biochim Biophys Acta (1995) 1243:1–8.[Medline]
  21. Harrison G.J., Willis R.J., Headrick J.P. Extracellular adenosine levels and cellular energy metabolism in ischemically preconditioned rat heart. Cardiovasc Res (1998) 40:74–87.[Abstract/Free Full Text]
  22. Fredholm B.B., Hedqvist P., Lindstrom K., Wennmalm M. Release of nucleosides and nucleotides from the rabbit heart by sympathetic nerve stimulation. Acta Physiol Scand (1982) 116:285–295.[Web of Science][Medline]
  23. Grosso M.A., Banjeree A., St. Cyr J.A., et al. Cardiac 5'-nucleotidase activity increases with age and inversely relates to recovery from ischemia. J Thorac Cardiovasc Surg (1992) 103:206–209.[Abstract]
  24. Bak M.I., Ingwall J.S. Regulation of cardiac AMP-specific 5'-nucleotidase during ischemia mediates ATP resynthesis on reflow. Am J Physiol (1998) 274:C992–C1001.[Web of Science][Medline]
  25. Itoh R., Oka J., Ozasa H. Regulation of rat heart cytosol 5'-nucleotidase by adenylate charge. Biochem J (1986) 235:847–851.[Web of Science][Medline]
  26. Darvish A., Metting P.J. Purification and regulation of an AMP-specific cytosolic 5'-nucleotidase from dog heart. Am J Physiol (1993) 264:H1528–H1534.[Web of Science][Medline]
  27. Chen W., Gueron M. AMP degradation in the perfused rat heart during 2-deoxy-D-glucose perfusion and anoxia. J Mol Cell Cardiol (1996) 28:2175–2182.[CrossRef][Web of Science][Medline]
  28. Hu B., Altschuld R.A., Hohl C.M. Adenosine stimulation of AMP deaminase activity in adult rat cardiac myocytes. Am J Physiol (1993) 264:C48–C53.[Web of Science][Medline]
  29. Spychala J., Marszalek J. Regulatory properties of AMP deaminase from rat tissues. Int J Biochem (1991) 23:1155–1159.[CrossRef][Web of Science][Medline]

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


This article has been cited by other articles:


Home page
 J. Physiol.Home page
Z. Papp, A. Szabo, J. P. Barends, and G J M Stienen
The mechanism of the force enhancement by MgADP under simulated ischaemic conditions in rat cardiac myocytes
J. Physiol., August 15, 2002; 543(1): 177 - 189.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
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
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Gustafson, L. A
Right arrow Articles by Kroll, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gustafson, L. A
Right arrow Articles by Kroll, K.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?