Cardiovascular Research 44 (1999) 333–343
www.elsevier.com / locate / cardiores
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Increased hypoxic stress decreases AMP hydrolysis in rabbit heart
a,b ,
a,c
a
b
Lori A. Gustafson *, Coert J. Zuurbier , John E. Bassett , Jan Paul F. Barends ,
Johannes H.G.M. van Beek b , James B. Bassingthwaighte a , Keith Kroll a ,1
a
b
Center for Bioengineering, University of Washington, Seattle, WA 98195, USA
Laboratory for Physiology, Institute of Cardiovascular Research, Vrije Universiteit, Amsterdam, The Netherlands
c
Department of Experimental Anesthesiology, Universiteit van Amsterdam, Amsterdam, The Netherlands
Received 24 November 1998; accepted 16 June 1999
Abstract
Objective: AMP conversion to adenosine by cytosolic 59-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 31 P 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: 31 P 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% O 2 ). 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.  1999 Elsevier
Science B.V. All rights reserved.
Keywords: Adenosine; Computer modeling; Energy metabolism; Hypoxia / anoxia; Ischemia
1. Introduction
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 59-nucleotidase (5NT, E.C. 3.1.3.5), or deaminated to IMP by the enzyme AMP deaminase (AMPD,
E.C. 3.5.4.6). Cytosolic ATP availability is essential for
*Corresponding author. Laboratory for Physiology, Institute for Cardiovascular Research, Vrije Universiteit, Van der Boechorststraat 7, 1081
BT Amsterdam, The Netherlands. Tel.: 131-20-444-8133; fax: 131-20444-8255.
E-mail address: [email protected] (L.A. Gustafson)
1
Deceased 15 July, 1997.
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
Time for primary review 28 days.
0008-6363 / 99 / $ – see front matter  1999 Elsevier Science B.V. All rights reserved.
PII: S0008-6363( 99 )00207-2
334
L. A. Gustafson et al. / Cardiovascular Research 44 (1999) 333 – 343
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 pO 2
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, AMPpreferring 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.
2. Methods
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
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; DrATP, rate of ATP synthesis minus rate of ATP
hydrolysis; 5NT, AMP-preferring isoform of 59-nucleotidase (cN-I); 5NT-II, IMP-preferring isoform of 59-nucleotidase (cN-II); Ado kinase, adenosine
kinase; Ado deaminase, adenosine deaminase; Pi , inorganic phosphate; PS, permeability surface area products for membrane transport.
L. A. Gustafson et al. / Cardiovascular Research 44 (1999) 333 – 343
Langendorff-perfused at 378C at a constant flow (perfusion
pressure580–100 mmHg) with non-recirculating, modified
Krebs–Henseleit bicarbonate buffer containing (in mM)
118 NaCl, 3.8 KCl, 1.2 KH 2 PO 4 , 0.7 MgSO 4 , 2.1 CaCl 2
0.1 EDTA, 25 NaHCO 3 , 11 glucose, 5 pyruvate, and 0.1%
bovine serum albumin, equilibrated with 95% O 2 / 5% CO 2
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
378C 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 31 P 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 (n56) were subjected to
two 30-min periods of underperfusion with perfusate
equilibrated with 95% O 2 / 5% CO 2 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 (n56) underwent an identical procedure, except that
during the second period of underperfusion the perfusate
was equilibrated via the membrane oxygenator with 30%
O 2 , 5% CO 2 and 65% N 2 .
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 Mg 21 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 1 and Mg 21 concentrations, and it was assumed that the
total concentration of PCr1Cr 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
335
analyzed by HPLC as previously described [5]. Total
purine release was calculated by summing the purines
(adenosine1inosine1hypoxanthine) 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. Ca 21 and ion pumps, myofibril contraction, ATPases, homeostasis, etc.) were described
as a continuous function, DrATP (DrATP5rate of ATP
synthesis2rate 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 DrATP 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 DrATP50.
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
59-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 560
mmol / min / g, Km 51.7 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 59-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
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L. A. Gustafson et al. / Cardiovascular Research 44 (1999) 333 – 343
the Km were found to vary only slightly, see results) and
the parameters of the DrATP function. All other model
parameters were either held constant during fitting or were
changed according to direct measurements (flow, intracellular pH, Mg 12 ). 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 values6standard error of
the mean (S.E.M. for n56). Mean values of the model
results (i.e. Vmax of 5NT and the DrATP 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
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 (n512). 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 second1hypoxia groups, respec-
tively, were: 145, 94, and 130 mM. Peak AMP concentrations for the first, second and second1hypoxia
groups, respectively, were: 3.4, 1.8, and 4.5 mM. 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.
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 (inosine1
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
(DrATP). 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
L. A. Gustafson et al. / Cardiovascular Research 44 (1999) 333 – 343
Fig. 2. Nuclear magnetic resonance, purine and metabolic data from two successive periods of underperfusion (10% baseline flow) with and without hypoxia. PCr (d) and ATP
(앳). Calculated cytosolic ADP (d) and AMP (♦); AMP was multiplied by a factor of 10 to aid viewing. Intracellular Pi (d). Intracellular pH (d). Lactate concentration in
venous effluent (d). Purine concentration in venous effluent, adenosine (j), hypoxanthine1inosine (m). Total purine release for the first 30-min period was 405625 nmol / g;
for the second period (normoxic), 179615 nmol / g and for the hypoxic second period, 245634 nmol / g. Left ventricular developed pressure (d).
337
338
L. A. Gustafson et al. / Cardiovascular Research 44 (1999) 333 – 343
Fig. 3. Simultaneous model fits to the PCr (d), ATP (앳), adenosine (h) and hypoxanthine1inosine (^) 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.
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 mM
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 mM), and highest for the
hypoxic group (10.9 mM) (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 .
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 inosine1hypoxanthine to adeno-
Table 1
Model predictions of Vmax for 5NT and DrATP a
Parameter
1st period of
underperfusion
2nd period of
underperfusion
2nd period of
underperfusion1hypoxia
Vmax 5NT (nmol / min / g)
Integral of DrATP (mmol / l)
16466
210.3
129610*
28.8
8565*
210.6
a
Values (means6S.E.) were obtained from optimized model solutions of individual experiments during the entire 30-min period of underperfusion
(n56). Vmax , maximal reaction velocity. 5NT, 59-nucleotidase; Integral of DrATP, estimation of total net high energy phosphate breakdown during
underperfusion, a negative value indicates a net breakdown of energy. * Significantly different from first period of underperfusion (P#0.05).
L. A. Gustafson et al. / Cardiovascular Research 44 (1999) 333 – 343
339
Fig. 4. Exploratory model fits to the PCr (d), ATP (앳), adenosine (h) and hypoxanthine1inosine (^) 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.
sine, 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 mM 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 mmol / 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
mM IMP after 45 min of ischemia, in the presence of
pyruvate and glucose, which increased to more than 600
mM 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 DrATP 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
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L. A. Gustafson et al. / Cardiovascular Research 44 (1999) 333 – 343
Fig. 5. Exploratory model fits to the PCr (d), ATP (앳), adenosine (h) and hypoxanthine1inosine (^) 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.
(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).
mM. 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).
3.3. Relationship between cytosolic AMP and purine
release
4. Discussion
4.1. Downregulation of 59 -nucleotidase
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 mM is achieved
at a cytosolic AMP concentration of 2 mM, while during
the second period of underperfusion only 20 mM is
achieved at a cytosolic AMP concentration of 2 mM.
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 mM, yet the purine
efflux does not go above a capillary concentration of 20
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
L. A. Gustafson et al. / Cardiovascular Research 44 (1999) 333 – 343
341
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 5164
vs. 129 nmol / min / g; this paper), albeit to a lesser degree
than after a 45-min period of 5% flow (Vmax 5140 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
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.
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
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
59-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 ischemiareperfusion injury has been attributed to lesser AMP
hydrolysis due to lower concentrations of 5NT [23]. In
vitro studies on highly purified 5NT indicate H 1 and Pi to
be inhibitory, while AMP, ADP and Mg 21 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
342
L. A. Gustafson et al. / Cardiovascular Research 44 (1999) 333 – 343
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 pH i 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 1mM) 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 1 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.
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.
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