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1343
Existence and Role of Substrate Cycling
Between AMP and Adenosine in Isolated
Rabbit Cardiomyocytes Under Control
Conditions and in ATP Depletion
Daniel R. Wagner, MD; Frangoise Bontemps, PhD; Georges van den Berghe, MD
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Background Adenosine, a physiological coronary vasodilator, has been proposed to regulate coronary circulation according to myocardial oxygen demand. In the present study,
we investigated the mechanisms of adenosine formation and
utilization in isolated rabbit cardiomyocytes and, in particular,
the existence and the role of substrate cycling between AMP
and adenosine in the regulation of its concentration.
Methods and Results Rabbit cardiomyocytes were isolated
by collagenase perfusion and incubated in HEPES-buffered
Krebs-Henseleit solution at 37°C, pH 7.4, in control conditions
and in ATP depletion achieved by inhibiting glycolysis with 5
mmol/L iodoacetate. Under control conditions, adenosine
accumulated at a rate of 4 pmol . min'.*6 cells. The 13-fold
elevation of adenosine accumulation induced by iodotubercidin (ITu), an inhibitor of adenosine kinase, proves that
adenosine is normally recycled into AMP. This recycling
involves 95% of the adenosine formed. In ATP depletion,
adenosine accumulated at the rate of 335 pmol* minm . 106
In the heart, as well as in several other organs,
elevation of extracellular adenosine provoked by
hypoxia has been proposed to induce vasodilation,
aimed at restoration of the oxygen supply.' The physiological role of adenosine and its properties as a cardiovascular agent have been characterized in great
detail over the past decade.2 However, the exact mechanisms of adenosine formation and utilization in cardiac
myocytes are still poorly understood.
Adenosine can be formed by dephosphorylation of
AMP and by hydrolysis of S-adenosylhomocysteine
(SAH). Dephosphorylation of AMP (Fig 1), either
intracellularly by cytosolic 5'-nucleotidase(s) or extracellularly by ecto-5'-nucleotidase, is a major source of
adenosine under conditions that provoke an increase in
AMP, such as ischemia and hypoxia, although the
participation of the various 5'-nucleotidases remains
unclear.3-5 Besides dephosphorylation, catabolism of
AMP can also proceed by deamination into IMP by
AMP deaminase, followed by dephosphorylation of
Received January 17, 1994; revision accepted April 15, 1994.
From the Laboratory of Physiological Chemistry, International
Institute of Cellular and Molecular Pathology, and University of
Louvain Medical School, Brussels, Belgium.
Correspondence to Dr Georges van den Berghe, Laboratory of
Physiological Chemistry, International Institute of Cellular and
Molecular Pathology, UCL-ICP 75.39, Ave Hippocrate 75, B-1200
Brussels, Belgium.
C 1994 American Heart Association, Inc.
cells and was no longer rephosphorylated after 20 minutes, as
shown by the absence of effect of ITu after this time interval.
Moreover, adenosine was deaminated, as indicated by the
twofold increase of its accumulation induced by deoxycoformycin (dCF), an inhibitor of adenosine deaminase. Both in
control conditions and in ATP depletion, adenosine-dialdehyde, an inhibitor of S-adenosylhomocysteine (SAH) hydrolase, had no significant effect on adenosine formation, indicating that the transmethylation pathway is not an important
source of adenosine in rabbit cardiomyocytes.
Conclusions The results indicate that recycling of adenosine into AMP is essential for the maintenance of low,
nonvasodilatory concentrations of the nucleoside under control conditions and that interruption of recycling plays an
important role in elevating adenosine during ATP depletion.
(Circulation. 1994;90:1343-1349.)
Key Words * adenosine * myocardium
IMP into inosine by 5'-nucleotidase. In the guinea pig
heart, hydrolysis of SAH, a product of the transmethylation pathway, has been reported to contribute to the
production of adenosine in control conditions but not to
the hypoxia-induced elevation of adenosine.6
The concentration of adenosine is determined not
only by the rate of its formation but also by the rate of
its utilization. Two enzymes can utilize adenosine:
adenosine deaminase, which produces inosine, and
adenosine kinase, which rephosphorylates adenosine
into AMP, using ATP as the phosphate donor (Fig 1).
The latter process results in recycling of adenosine in a
so-called "substrate cycle." We have reported previously that recycling of adenosine to AMP proceeds at a
high rate in normoxic isolated rat hepatocytes.7 Recently, interruption of this recycling has been shown to
play a major role in the elevation of adenosine induced
by anoxia in these cells.8
In the present study, we investigated the existence of
a substrate cycle between AMP and adenosine in isolated rabbit cardiomyocytes and its role in regulating
adenosine concentration in control conditions and in
ATP depletion induced by inhibition of glycolysis. The
contribution of the hydrolysis of SAH to adenosine
formation was also evaluated.
Methods
Isolation of Cardiomyocytes
Cardiac ventricular myocytes were isolated from male New
Zealand White rabbits weighing 1.7 to 2.3 kg by a modified
Circulation Vol 90, No 3 September 1994
1344
HCl04, followed by vigorous mixing. After standing on ice for
NH3
AMP
(5)
IMP
ITu
(2)
ATP
SAH
aD
(4)
(1)
\
Pi
Pi
NH3
Inosine
Homocysteine
FIG 1. Schematic of pathways of adenosine formation and
utilization in isolated cardiomyocytes and targets of the inhibitors: (1) 5T-nucleotidase(s), (2) adenosine kinase, (3) adenosine
deaminase, (4) SAH hydrolase, and (5) AMP deaminase. SAH
indicates S-adenosylhomocysteine; 1Tu, 5-iodotubercidin; dCF,
deoxycoformycin; ADD, adenosine-dialdehyde; and Pi, inor-
ganic phosphate.
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procedure of Altschuld et al.9 Hearts were perfused by the
Langendorff method and myocytes isolated by the addition of
collagenase (Wako). After isolation, the preparations were
enriched with viable myocytes by three sequential sedimentations in 4% bovine serum albumin (Serva). Before incubations,
the myocytes were counted in a Nageotte cell chamber, and
their viability was assessed by trypan blue exclusion. Myocytes
were adjusted to ±400 000 cells/mL. Contamination of the
preparation with endothelial cells was ruled out by measuring
hypoxanthine, a catabolite that is formed only in the latter cells
and not in myocytes.10 The protein content of cell suspensions
was determined according to Lowry et al,"1 220 000 cells being
equivalent to 1 mg of Lowry protein. Myocytes were incubated
in HEPES-buffered Krebs-Henseleit solution (pH 7.4) containing (in mmol/L): NaCI 118, KCl 4.8, KH2PO4 1.2, MgSO4
1.2, HEPES 25, and CaCi2 1, supplemented with 11 mmol/L
glucose and 1% bovine serum albumin (fatty acid free, Sigma
Chemical Co) and gassed with 100% 02. Incubations were
performed at 37°C in a gently shaking water bath. Cells were
preincubated for 20 minutes before the experiments were
started.
Inhibitors
lodotubercidin 2 ,mol/L (ITu) (Research Biochemicals)
and deoxycoformycin 2 ,umol/L (dCF) (Warner-Lambert)
were used to inhibit adenosine kinase and adenosine deami-
respectively.'12"3
Preliminary studies, performed as in Bontemps et al,14 had
shown that these concentrations inhibited by 94% and 99% the
phosphorylation and the deamination, respectively, of "4Clabeled adenosine by the cardiomyocytes. When used, dCF was
added at the beginning of the preincubation, owing to the time
required for this tight-binding inhibitor to penetrate the cells
and bind to its target. ITu was added at time 0. Adenosinedialdehyde 10 ,umol/L (Sigma), also added during preincubation, was used to inhibit SAH15; the concentration used was
verified to inhibit by 99% the formation of SAH from endogenous adenosine in the presence of a saturating, 1-mmol/L
concentration of L-homocysteine thiolactone. ATP depletion
was induced by addition at time 0 of 5 mmol/L iodoacetate, an
inhibitor of glycolysis acting at the level of glyceraldehyde
3-phosphate dehydrogenase. Whereas in control conditions, the
proportion of viable cells, measured by trypan blue exclusion,
decreased from -90% to 80% over the duration of the experiments, it decreased to 5% in the presence of iodoacetate.
Extracellular LDH, which increased from about 5% to 10%
under control conditions, reached =12% after iodoacetate.
nase,
Determination of Nucleotides and Nucleosides
Nucleotides and nucleosides were extracted by transferring
500-,L aliquots of cell suspension into 150 ,L of ice-cold 10%
15 minutes, the extracts were centrifuged with an Eppendorf
microfuge, and the resultant supernatants were neutralized
with 3 mol/L KOH and 3 mol/L KHCO3. Nucleotides were
quantified by anion-exchange high-performance liquid chromatography (HPLC) on a 12.5xO.46-cm PartiSphere 5 SAX
column (Whatman) according to the method described by
Hartwick and Brown16 and nucleosides by reversed-phase
HPLC on a 12.5x0.46-cm PartiSphere C-18 column
(Whatman).8
Statistics
Results are expressed as mean+± SEM, in nanomoles per
milligram total Lowry protein for nucleotides and in nanomoles per milliliter of cell suspension for nucleosides. Data
obtained at the different time points and accumulation rates
were subjected to factorial ANOVA (one-way ANOVA,
Fisher test), and a value of P<.05 was considered to be
statistically significant.
Results
Concentrations of Adenine Nucleotides in
Control Myocytes
After isolation, >90% of the cardiac myocytes were
viable as assessed by trypan blue exclusion, and 75% to
90% of the cells were rod-shaped. In the control conditions, ATP content was 28.9±1.2 nmol/mg Lowry protein (n= 12) at the beginning of the experiments and did
not change significantly over the ensuing 60 minutes of
incubation. The ADP content was 5.4±0.3 nmol/mg
Lowry protein at time 0 and decreased to 4.3±0.3 after
60 minutes of incubation. AMP content was 1.1±0.1
nmol/mg Lowry protein initially and remained stable
over the whole duration of the incubation. IMP was
undetectable. These results are in accordance with
observations by others in cardiomyocytes isolated from
rabbits and rats.9'17'8 The sum of adenine nucleotides
and the individual contents of ATP, ADP, and AMP
were not significantly affected by the addition of either
2 ,umol/L ITu, 2 ,umol/L dCF, a combination of both
ITu and dCF, or the addition of 10 ,umol/L adenosinedialdehyde (results not shown).
Influence of lodoacetate on Adenine
Nucleotide Contents
Addition of 5 mmol/L iodoacetate to the myocyte
suspensions provoked a biphasic decrease of the ATP
content (Fig 2A). A rapid, -15% initial diminution was
followed by a 10-minute plateau, which was succeeded
by a linear decline, up to complete depletion after 60
minutes. The ADP content increased slightly during 40
minutes and decreased progressively thereafter (Fig
2B). The AMP content increased nearly 10-fold within
5 minutes of the addition of iodoacetate (Fig 2C) and
remained at that level over the whole duration of the
experiment. IMP accumulated to 7 nmol/mg Lowry
protein within 5 minutes and to 10 nmol/mg Lowry
protein after 60 minutes of incubation (Fig 2D). These
results accord with published studies.9"18
Inhibition of adenosine deaminase by dCF and of
adenosine kinase by ITu, as well as addition of both
inhibitors, had no significant effect on the degradation
of ATP (Fig 2A). The inhibitors also did not influence
ADP (Fig 2B) and IMP (Fig 2D) content. Addition of
ITu or dCF alone had no effect on AMP content (Fig
2C), but the combination of both inhibitors decreased
Wagner et al Recycling of Adenosine in Cardiomyocytes
A
*
30
*
A
-c
6
No addition
flu
dCF
dCF + ITu
C
4~
CL
C
A. 520
0
No addition
dCF
a0.
0
rru
Eb 10
E 2
E
E
0
rru
FIG 2. Graphs showing time course of ATP
(A), ADP (B), AMP (C), and IMP (D) content
in isolated rabbit cardiomyocytes incubated
in HEPES-buffered Krebs-Henseleit solution
after addition of 5 mmol/L iodoacetate.
Deoxycoformycin (dCF), an inhibitor of
adenosine deaminase, was added during a
prior 20-minute incubation; iodotubercidin
(ITu), an inhibitor of adenosine kinase, and
iodoacetate, an inhibitor of glycolysis, were
added at time 0. Values are mean±SEM of
No addition
three to six experiments.
dCF
+
rru
0
c
OL
20r
8
B
D
-E
p
.,
4-6
0.
dCF
dCF
C
010
c,
1345
+
ITu
01
,E
0
0
E
E 2
c
c
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01
oj
0
20
40
40
Time (min)
60
Time (min)
the latter by 50% after 40 minutes. However, the sum of
adenine nucleotides was not significantly affected.
Accumulation of Adenosine and Inosine in
Control Conditions
Under control conditions, adenosine accumulated
slowly and linearly in the cardiomyocyte suspensions (Fig
3A), at a rate (Table) reaching 4±1 pmol/min per 106 cells
c
c
dCF +
&
-*
O
c
flu
0
0
C=
ITu
dCF
No addition
B
10
c
8
0
_C
o
0
.E0
**
6
/Tu
-E
s0.
4
E
0
E
c
2
No addition
a
0
20
40
dCF
+
ITu
dCF
-
60
Time (min)
FIG 3. Graphs showing time course of adenosine (A) and
inosine (B) accumulation in rabbit cardiomyocyte suspensions
incubated under normoxic conditions in HEPES-buffered KrebsHenseleit solution. Inhibitors were as in Fig 2. Values are
mean±+-SEM of three to six experiments. **P<.01 and
***P<.005 vs control.
(n=6). This indicates that the production of adenosine by
these cells slightly surpasses its utilization. Inosine is the
terminal adenine nucleotide catabolite in cardiomyocytes,
owing to the absence or very low activity of purine
nucleoside phosphorylase in these cells.'0,19 20 Inosine production in normoxic myocytes (Fig 3B) occurred at the
rate of 6±4 pmol/min per 106 cells (n=6).
The addition of dCF completely suppressed the production of inosine. This indicates that under normoxic
conditions, the very slow production of inosine proceeds
exclusively by dephosphorylation of AMP into adenosine, followed by deamination of adenosine into inosine.
Indeed, if catabolism of AMP had occurred in part via
the alternative pathway, deamination into IMP by AMP
deaminase followed by dephosphorylation of IMP into
inosine (Fig 1), a residual production of inosine would
have been observed in the presence of an inhibitor of
adenosine deaminase.
The rate of accumulation of adenosine was not significantly increased by the addition of dCF. However,
the addition of ITu increased the rate of accumulation
of adenosine more than 10 -fold, to 53 18 pmol/min per
106 cells (n=6) (P<.05). This observation demonstrates
that adenosine is rephosphorylated into AMP by adenosine kinase in the absence of ITu. The preferential
utilization of adenosine by adenosine kinase can be
explained by its higher affinity for this enzyme compared with adenosine deaminase.21 In the presence of
ITu, the rate of production of inosine increased more
than 20-fold, to 138±7 pmol/min per 106 cells (n=6)
(P<.005). This is explained by the fact that under this
condition, adenosine can no longer be rephosphorylated into AMP and is consequently further converted
into inosine by adenosine deaminase.
In the presence of both inhibitors, the rate of accumulation of adenosine increased more than 50-fold, to
210±32 pmol/min per 106 cells (n=6) (P<.005). This
high rate reflects the total rate of formation of adenosine by the cells. That this rate was severalfold higher
1346
Circulation Vol 90, No 3 September 1994
Accumulation Rates of Adenosine, Inosine, and the Sum of Both in Isolated
Rabbit Cardiomyocytes
dCF, 2
No Addition
Adenosine
Control
lodoacetate
Inosine
Control
lodoacetate
Sum
Control
lodoacetate
4-+i
335±63
gmol/L
5±5
743±123t
6+4
1936+214
0
1611±311
10
2271
5
2354
1Tu, 2 ,mol/L
dCF+ITu
53+18*
504±66
210+32*
814±16t
138±7$
0
1125±266
1771±+213
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191
210
2275
1939
in
were
incubated
HEPES-buffered
in
Krebs-Henseleit
solution
Cardiomyocytes
control conditions
and in ATP depletion achieved with the glycolysis inhibitor iodoacetate 5 mmol/L. Values are
expressed in pmol/min per 106 cells. Deoxycoformycin (dCF), an inhibitor of adenosine deaminase,
was added during a prior 20-minute incubation; iodotubercidin (ITu), an inhibitor of adenosine kinase,
and iodoacetate were added at time 0. Values are mean±SEM of three to six experiments. *P<.05,
tP<.01, and tP<.005 vs no addition.
than upon addition of ITu alone confirmed that in the
latter condition, a substantial quantity of adenosine was
deaminated, owing to the increase of its concentration.
In the presence of both inhibitors, the production of
inosine was completely suppressed.
Effect of lodoacetate on Adenosine and
Inosine Accumulation
Fig 4A shows that the biphasic depletion of ATP
induced by iodoacetate was reflected in a similarly
A
30
dCF ITu
dCF
+
0
c
1CL
o 0
- -5
rru
0 0.
K-=
No addition
0
E
10
c
80r
c
o
B
No addition
60
rru
._
dCF
c
CL
0.
40
o
dCF +
0@
c
flu
20
o
W
0
20
40
60
Time (min)
FIG 4. Graphs showing time course of adenosine (A) and
inosine (B) accumulation in rabbit cardiomyocyte suspensions
incubated in HEPES-buffered Krebs-Henseleit solution after addition of 5 mmol/L iodoacetate. Inhibitors were as in Fig 2. Inset
shows the first 10 minutes of the experiments at a higher
magnification. *P<.05, **P<.01, ***P<.005 vs control.
biphasic accumulation of adenosine. In the absence of
inhibitors, adenosine increased fourfold within 10 minutes (Fig 4A, inset), to a plateau that was maintained
during the ensuing 10 minutes; thereafter, adenosine
accumulated linearly up to the end of the experiment.
The rate of accumulation of adenosine, which was
461±92 pmol/min per 106 cells over the first 10 minutes,
reached an overall value of 335±63 pmol/min per 106
cells (n=3) over 60 minutes, which is 85-fold the rate of
accumulation recorded in control conditions in the
absence of inhibitors. The effect of ITu was also timedependent. During the first 10 minutes, addition of ITu
significantly increased the rate of accumulation of adenosine by 598 pmol/min per 106 cells, to 1059 pmol/min
per 106 cells. This indicates that during this time
interval, rephosphorylation of adenosine by adenosine
kinase continued. Thereafter, the rate of elevation of
adenosine in the presence of ITu evolved in parallel
with that recorded in the absence of inhibitors, indicating that adenosine was no longer recycled. Addition of
dCF enhanced the rate of accumulation of adenosine
approximately twofold during the first 40 minutes of
incubation; thereafter, the rate increased about fourfold. Addition of both inhibitors resulted in an even
higher rate of accumulation of adenosine, reflecting the
total formation of adenosine and reaching 814±16
pmol/min per 106 cells (n=3). This is a nearly fourfold
increase compared with the total rate of production of
adenosine in the presence of both inhibitors in control
conditions.
In the absence of inhibitors, iodoacetate induced a
biphasic accumulation of inosine, resembling that of
adenosine (Fig 4B). Over the 20- to 60-minute time
interval, the rate of production of inosine reached 1484
pmol/min per 106 cells (n=3). As in control cardiomyocytes, addition of dCF decreased the rate of accumulation of inosine, but only during the first 20 minutes.
The only marginal inhibition of accumulation of inosine
by dCF thereafter indicates that the accumulation of
AMP induced by iodoacetate provokes catabolism of
this nucleotide not only by way of dephosphorylation
but also, and to an important extent, by way of AMP
Wagner et al Recycling of Adenosine in Cardiomyocytes
15
r
A
c
0
10i
CL
a
c
5
0
L
B
50
c
0
40
Control
c
0
to
cCL
o C*
30
C =
20
It-0
c
10
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0
0
20
40
60
Time (min)
FIG 5. Graphs showing time course of total adenosine accumulation in normoxic conditions (A) and in ATP depletion (B) in rabbit
cardiomyocyte suspensions incubated in HEPES-buffered KrebsHenseleit solution after addition of adenosine-dialdehyde (Adodialdehyde) 10 gmol/L, an inhibitor of S-adenosylhomocysteine
hydrolase. ATP depletion was achieved by adding 5 mmol/L
iodoacetate, an inhibitor of glycolysis. Total adenosine production
was assessed by addition of deoxycoformycin, an inhibitor of
adenosine deaminase, and iodotubercidin, an inhibitor of adenosine kinase. Values are mean±SEM of three experiments.
deaminase. This is in accordance with the elevation of
IMP under this condition (Fig 2D). There was little or
no effect of ITu, alone or in combination with dCF, on
the rate of accumulation of inosine, also in accordance
with the role of IMP as a source of inosine.
Role of SAH as a Source of Adenosine
In control conditions, the total rate of adenosine
formation, as assessed by addition of dCF and ITu, was
not significantly decreased by the supplemental addition
of adenosine-dialdehyde, an inhibitor of SAH hydrolase
(Fig SA). This indicates that under control conditions,
adenosine is nearly exclusively derived from AMP dephosphorylation without significant participation of
SAH hydrolysis. After inhibition of glycolysis with iodoacetate, total adenosine production, as assessed with
dCF and ITu, was also not decreased by addition of
adenosine-dialdehyde, indicating that adenosine was
formed exclusively from AMP (Fig 5B).
Discussion
In accordance with experiments by others using isolated rat cardiomyocytes,918 we found that isolated
rabbit cardiomyocytes can degrade AMP both by dephosphorylation into adenosine followed by deamination into inosine and by deamination into IMP followed
by dephosphorylation into inosine (Fig 1). Also in
accordance with others, inosine was the terminal purine
catabolite in the cells under investigation, a finding
explained by their absent or very low purine nucleoside
phosphorylase activity.10"9'20 In addition, our results
1347
demonstrate that in isolated rabbit cardiomyocytes, as
in isolated rat hepatocytes,7,8 a substrate cycle operates
between AMP and adenosine under control conditions
and is impaired on depletion of ATP. These two conditions will be discussed separately.
Recycling of Adenosine in Control Conditions
Under control conditions, the catabolism of the adenine nucleotide pool of the isolated myocytes was very
slow, as evidenced by the minimal rate of accumulation
of adenosine and inosine in the cell suspension (Fig 3
and Table). The complete suppression of the accumulation of inosine by the adenosine deaminase inhibitor
dCF indicates, as already concluded,9"18 that this catabolism proceeds only by dephosphorylation of AMP
followed by deamination of adenosine.
Substrate cycling between AMP and adenosine, involving reutilization by adenosine kinase of adenosine
formed by 5'-nucleotidase(s), was postulated by Arch
and Newsholme22 as a mechanism that would permit a
rapid regulation of the concentration of adenosine. The
first demonstration of an active AMP/adenosine cycle,
achieved in normoxic isolated rat hepatocytes, was
based primarily on the effect of the adenosine kinase
inhibitor ITu to progressively decrease the concentration of ATP, to elevate the concentration of adenosine,
and to increase the rate of production of allantoin, the
terminal purine catabolite in rat liver.7 The marked,
approximately 10-fold increase of the rate of accumulation of adenosine (Fig 3A) and inosine (Fig 3B)
induced by ITu in control cardiomyocytes shows that in
these cells also, AMP is continuously dephosphorylated
and that the resulting adenosine normally is nearly not
deaminated but rather rephosphorylated into AMP.
Measurement of the absolute rate of adenosine production, achieved by addition of both ITu and dCF, revealed that it reaches 210 pmol/min per 106 cells
(Table), which is more than 20-fold the rate of accumulation of adenosine and inosine together recorded in the
absence of inhibitors (10 pmol/min per 106 cells). This
shows that 95% of adenosine produced is recycled into
AMP under control conditions. The absence of a decrease of ATP on inhibition of adenosine kinase can be
explained by the fact that adenosine accumulation after
60 minutes in the presence of ITu and dCF accounted
for <10% of the initial adenine nucleotide pool in the
cardiomyocytes, compared with 40% in hepatocytes.7,8
Studies in isolated rat hearts have either concluded
against the existence of an AMP/adenosine cycle23 or
claimed that recycling of adenosine occurs only at a very
low rate.24 The latter conclusion was based on a less
than twofold increase of total purine nucleoside release
on addition of ITu in normoxic hearts. Whether the
marked effect of ITu we observed is due to species
differences, to the use of isolated cells, or to the great
care taken to ensure that the concentration of ITu used
was nearly totally inhibitory of adenosine kinase remains to be determined. After submission of this article,
rapid recycling of adenosine has, nevertheless, been
reported in well-oxygenated isolated guinea pig heart.25
Our observation that the production of adenosine in
control conditions was only minimally decreased by
adenosine-dialdehyde, an inhibitor of SAH hydrolase,
indicates that adenosine was derived nearly exclusively
from AMP, with almost no participation of the trans-
1348
Circulation Vol 90, No 3 September 1994
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methylation pathway. This is in accordance with the
latest studies in normoxic perfused guinea pig heart.25
Impairment of Adenosine Recycling on
ATP Depletion
When ATP depletion was induced by iodoacetate,
degradation of the adenine nucleotides proceeded at
high rates by both pathways, as evidenced by the
accumulation of IMP (Fig 2D) and by the low inhibitory
effect of dCF on the huge accumulation of inosine,
particularly during the second half of the experiment
(Fig 4B and Table). In the absence of inhibitors of
adenosine metabolism, addition of iodoacetate increased the rate of accumulation of adenosine to 335
pmol/min per 106 cells (Fig 4A), compared with 4
pmol/min per 106 cells in control conditions (Fig 3A). In
theory, this 85-fold increase could result from the
increase of AMP (Fig 2C), from a decreased recycling
into AMP, or from both. During the first 10 minutes
after the addition of iodoacetate, ITu enhanced the rate
of accumulation of adenosine by 598 pmol/min per 106
cells. This indicates that rephosphorylation of adenosine into AMP was still active, even more so than in
control conditions. During these 10 minutes, total adenosine production, determined by addition of dCF and
ITu, was 1250 pmol/min per 106 cells. It can thus be
concluded that, in the absence of inhibitors, 50% of the
adenosine produced was recycled into AMP during this
time interval. The accumulation of adenosine thus
results from a large production that exceeds the maximal recycling capacity. During later time intervals,
however, no more recycling of adenosine took place, as
evidenced by the observation that the concentrations of
adenosine evolved in parallel in the presence of ITu and
in the absence of inhibitor. The impairment of adenosine recycling at this stage can be explained by depletion
of the phosphate donor, ATP, and/or inhibition of
adenosine kinase by excess of its substrate, adenosine,
or of its product, AMP.2627 Since the Km of ATP for
adenosine kinase is approximately 0.8 mmol/L in rat
heart,28 one would expect recycling of adenosine to be
maintained until depletion of ATP to values below 5 to
7 nmol/mg Lowry protein. However, in our experiments, recycling of adenosine was already impaired at
ATP levels above 20 nmol/mg Lowry protein. The ATP
level is thus not the only determinant of the interruption
of the AMP/adenosine cycle. That the Km of adenosine
for adenosine kinase is < 1.0 ,umol/L in rat heart led to
the proposition that adenosine kinase might be nearly
saturated under basal conditions.24,28 Nevertheless,
Newby et a123 concluded that adenosine kinase is far
from saturated in neonatal rat heart cells, because the
basal rate of adenosine formation was only 9% of the
maximal activity of adenosine kinase measured in these
cells. In our studies, the activity of adenosine kinase in
control conditions, calculated from the rate of accumulation of adenosine and inosine on inhibition with ITu,
reached 181 pmol/min per 106 cells. It was increased
threefold, to 598 pmol/min per 106 cells, during the first
10 minutes of ATP depletion. This indicates that the
enzyme was not maximally active under basal conditions. After 10 minutes, however, the activity of adenosine kinase rapidly declined, probably because of substrate inhibition, provoking a large accumulation of
adenosine. Flux toward inosine then became possible
owing to the 50-fold higher Km of adenosine for adenosine deaminase.21
Physiological Significance of the
AMP/Adenosine Cycle
The magnitude of the rate of accumulation of adenosine measured in isolated cardiomyocytes under control conditions in the presence of dCF and ITu (Table)
demonstrates that the dephosphorylation of AMP proceeds at a substantial rate. This indicates that the
enzymes responsible for this process, the cardiac cytosolic IMP-GMP 5'-nucleotidase29 and/or the cytosolic
AMPase,30 display appreciable activity under physiological conditions. This finding is somewhat surprising in
view of the low affinity for AMP, with Km in the
millimolar range, that has been reported for both enzymes. In addition, the IMP-GMP 5'-nucleotidase is
subjected to inhibition by the concentration of inorganic
phosphate prevailing intracellularly. Because of this
substantial rate of adenosine formation, utilization of
adenosine becomes mandatory to avoid its accumulation to vasodilatory concentrations. For maintenance of
low concentrations of adenosine, utilization by adenosine kinase will be much more efficient than by adenosine deaminase owing to the severalfold lower Km for
adenosine of adenosine kinase. In addition, the marked
dependence of the activity of this enzyme on a narrow
range of ATP, AMP, and adenosine concentrations
evidenced in our work results in rapid saturation of the
cycle with adenosine. This provides a sensitive and rapid
mechanism for the elevation of the concentration of
adenosine under conditions of ATP depletion such as
hypoxia.
Acknowledgments
This work was supported by grants 3.4539.87 and 3.4557.93
from the Fund for Medical Scientific Research (Belgium) and
by the Belgian State-Prime Minister's Office for Science Policy
Programming. Dr van den Berghe is Director of Research of
the Belgian National Fund for Scientific Research.
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Circulation. 1994;90:1343-1349
doi: 10.1161/01.CIR.90.3.1343
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