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INTRODUCTION |
It is well established that ATP acts as an extracellular signal in
many tissues and is involved in a variety of regulatory processes
including the control of vascular tone, muscle contraction, pain, or
neuronal communication (for review see Ref. 1). In particular, ATP
serves as a neurotransmitter or co-transmitter in central, as well as
in peripheral neurons (1, 2). For instance, ATP is co-released with
noradrenaline or acetylcholine from sympathetic or parasympathetic
nerve endings, respectively, and even from neuromuscular synapses (1).
Another source of extracellular ATP is that released from parenchymal
cells under hypoxic or ischemic conditions (3).
Many biological responses to ATP are mediated via P2
purinoceptors, which belong to either of two structurally and
functionally distinct families of receptors: ligand-gated, nonselective
cation channels (P2X-type), or G-protein-coupled receptors
linked to the phospholipase C signaling cascade (P2Y-type)
(1, 4). Activation of P2X receptors leads to sodium and
calcium influx and to cell depolarization and is therefore stimulatory
in nature (e.g. it causes smooth muscle cell contraction).
Although P2Y purinoceptor activation also results in an
increased cytosolic calcium concentration (at least in part via
inositoltrisphosphate-mediated calcium release), it induces both
stimulatory and inhibitory effects (the latter through the opening of
potassium channels or indirectly through nitric oxide production). In
the heart, extracellular ATP and ATP analogues were found to exert
pronounced although relatively complex effects on coronary tone and
mechanical activity, depending on the type of preparation studied and
on the purine concentrations used (5-8).
More recent studies using isolated cardiomyocytes have shown that
micromolar levels of extracellular ATP increase (i) plasma membrane
conductances for cations (9) and also for chloride (10, 11), (ii) the
cytosolic calcium concentration (9, 12-15), (iii) the rate of
phosphoinositide hydrolysis (14, 16, 17), and (iv) the contraction
amplitude (12, 14, 15). These results, along with pharmacological and
immunohistochemical data (10, 11, 13, 14, 18-20), suggest that members
of both families of P2 receptors are expressed in and
linked to the electrical and contractile function of cardiac muscle cells.
By contrast, it is not known whether ATP may also impinge on
cardiomyocyte metabolism, whose regulation often closely matches the
changes in energy demand and contractile status. The aim of the present
investigation was therefore to investigate possible effects of ATP and
other purinergic agents on the trans-sarcolemmal transport and
utilization of glucose that, besides fatty acids and lactate,
represents an essential substrate in cardiac myocytes. In these
studies, we observed a pronounced inhibitory action of purinergic
agonists on the rate of glucose transport and explored the underlying mechanisms.
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EXPERIMENTAL PROCEDURES |
Chemicals--
All chemicals were of the highest purity grade
available. Chemicals for media used for cell isolation and the glucose
transport assay were purchased from Merck (Darmstadt, Germany), except
for bovine serum albumin (fraction V, fatty acid-free), which was from
Boehringer Mannheim. All nucleotides (except
2MeSATP),1 adenosine, and
L-phenylephrine were from Sigma (Deisenhofen, Germany);
dichloroacetic acid was from Aldrich (Steinheim, Germany); 2-methylthioadenosine triphosphate,
pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid, suramin, and
Reactive Blue 2 were from RBI (Biotrend, Cologne, Germany); purified
bovine insulin was a kind gift from Prof. Axel Wollmer (Aachen,
Germany). 2-Deoxy-D-[3H]glucose (glucose
transport assay), [3-3H]-D-glucose
(glycolytic measurements), [1-14C]-pyruvic acid (pyruvate
oxidation assay), and ECL (Western blots) were from Amersham Pharmacia
Biotech (Braunschweig, Germany). The antiserum used to immunodetect
GLUT4 (OSCRX) was a kind gift from Prof. A. Zorzano (Barcelona, Spain),
and that against GLUT1 was purchased from Biogenesis Inc. (Quartett,
Berlin, Germany).
Isolation of Cardiomyocytes and Glucose Transport
Assay--
Cardiomyocytes from adult, female Sprague-Dawley rats
(220-250 g, fed ad libitum) were obtained as described
previously (21). Unless otherwise indicated, treatment of
cardiomyocytes (~1.5 mg protein/sample) was performed in medium A (6 mM KCl, 1 mM Na2HPO4, 0.2 mM NaH2PO4, 1.4 mM
MgSO4, 128 mM NaCl, 10 mM HEPES, 1 mM CaCl2, and 2% fatty acid-free bovine serum
albumin, pH 7.4) at 37 °C, equilibrated with 100% oxygen. The rates
of carrier-mediated 2-deoxy-D-glucose (dGlc) uptake were
determined over a period of 30 min as described (21).
Ionic Gradients--
In the experiments examining the relevance
of ionic gradients (see Fig. 3), cardiomyocytes were incubated in media
whose composition was identical to medium A, except that the
concentration of the ion to be tested was varied as indicated in the
figure. All the media used were isotonic; thus sodium was replaced by
an equimolar amount of choline (see Fig. 3A), potassium was
replaced by sodium (see Fig. 3C), or chloride was replaced
by HEPES (see Fig. 3D). The calcium concentration was
adjusted according to the method of Fabiato and Fabiato (22) (see Fig.
3B). The experiments in which the sodium concentration was
lowered (see Fig. 3A) were performed at a calcium
concentration of 10
7 M to avoid a calcium
overload of the cells due to impairment of the
sodium-dependent calcium extrusion via the sarcolemmal Na+/Ca2+ exchanger.
Glycolytic Flux--
The flux through the
6-phosphofructo-1-kinase reaction was estimated by measuring the rate
of 3H2O formation from
[3-3H]glucose with a protocol adapted from Ref. 23. In
brief, cardiomyocytes were treated (with or without ATP and
insulin) as described in the legend of Fig. 5 before
[3-3H]glucose (0.75 µCi/sample) was added for 30 min at
37 °C. The incubation was then stopped with perchloric acid (0.3 M). The extracts were neutralized with
KOH/KHCO3, and the tritiated water glycolytically formed
was separated on Dowex-borate columns (Bio-Rad, Munich, Germany) as
described (23). The amount of nonspecific 3H2O
(i.e. that which is not related to the glycolytic activity of the myocytes) was determined by adding perchloric acid to a parallel
set of cell samples prior to the incubation with
[3-3H]glucose.
Measurement of 14CO2 Production
from [1-14C]Pyruvate--
Flux through the pyruvate
dehydrogenase complex was determined by monitoring the production of
14CO2 from [1-14C]pyruvate as
described (24). In brief, cardiomyocytes were exposed to the
experimental conditions to be investigated (ATP and/or insulin
treatment) at 37 °C in 20-ml vials sealed with rubber stoppers
(under the same conditions as for the glucose transport assays,
i.e. in a total volume of 1.5 ml and with ~1.5 mg cell
protein/sample). The oxidation reaction was then started by injecting
0.1 µCi of [1-14C]pyruvate (final concentration, 0.1 mM) into the cell suspension. After another 10 min at
37 °C, the incubation was terminated by injecting 300 µl of 0.3 N perchloric acid. The released
14CO2 was trapped (over 18 h at 4 °C)
in 0.3 ml ethanolamine/ethylene glycol (1:1, v/v) contained in a
polypropylene center well (placed in the vials before the incubations).
Nonspecific values (determined by injecting perchloric acid in parallel
cell samples before adding [1-14C]pyruvate) were
subtracted from all samples. The quenching caused by the
ethanolamine/ethylene glycol mixture was determined in each experiment
and taken into account for the calculation of pyruvate oxidation rates.
Preparation of Purified Membrane Fractions from Cardiomyocytes
and Western Blot Analysis--
Cardiomyocytes were treated as detailed
in the legend of Fig. 4 and then rapidly washed once with TES buffer
(20 mM Tris, 1 mM EDTA, 250 mM
sucrose, 0.1 mM (phenylmethylsulfonyl fluoride, pH 7.4) and
immediately frozen in liquid nitrogen in a ratio of 107
cells/2.7 ml of TES. Preparation of purified plasma and intracellular membrane fractions (25) and semi-quantitative immunodetection and
quantitation of GLUT4 and GLUT1 (26) were performed as described.
Calculations--
Cell protein was measured by the biuret
method. The curves shown in Fig. 1, as well as the IC50
values of glucose transport inhibition, were obtained from the dGlc
transport data using a computer program (Prism) from GraphPad Software,
Inc. (San Diego, CA), according to the following equation.
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(Eq. 1)
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where X is the logarithm of agonist concentration,
Ymin and Ymax are the
minimum and maximum dGlc transport rates, IC50 is the
agonist concentration producing a half-maximal inhibition, and
S the Hill slope of the curves. For the statistical tests used to assess the difference of data sets, see the figure legends.
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RESULTS |
Effects of Purinergic Agonists and Antagonists on Glucose Transport
in Cardiomyocytes--
Glucose transport is the first step and under
many physiological conditions the rate-limiting step of myocardial
glucose utilization and is known to be the target of a host of
regulatory factors in heart muscle cells, such as insulin,
catecholamines, contraction, anoxia, ischemia, or alternative
substrates (24, 27-30).
In an initial series of experiments, we therefore investigated the
action of purinergic agonists on cardiomyocyte glucose transport. As
documented in Table I, ATP produced a
substantial inhibition of glucose transport in basal (nonstimulated)
myocytes, as well as in cells stimulated with insulin or with the
1-adrenergic agent phenylephrine. The effect of ATP was
rapid; thus the extent of inhibition of insulin-stimulated glucose
transport was the same, regardless of whether ATP was added to the
cells 30, 20, 10, or 0 min before or even 2 min after insulin addition
(not shown).
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Table I
Effects of adenine nucleotides on dGlc uptake in cardiomyocytes
Cardiomyocytes were incubated for 10 min (at 37 °C in medium A) with
or without nucleotides at the indicated concentrations before insulin
(12 nM) or phenylephrine (120 µM) were added
for another 30 min. In another series of samples, cells were treated
for 60 min with oligomycin B (0.6 µM) in the presence or
absence of ATP. At the end of these incubations, the rate of dGlc
uptake was determined as described under "Experimental Procedures."
Data are the mean values expressed in pmol/h/mg protein ± S.E.
The numbers of independent experiments are indicated in parentheses.
The percentage values given in the third column in relation to the
control values in the corresponding experiments. The p
values given are in relation to the corresponding uptake rates measured
in the absence of nucleotide (analysis of variance, except for the
comparison done with basal and phenylephrine-stimulated values that
were paired in each experiment and were therefore analyzed with a
paired Student's t test).
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Similarly, other purine nucleotides known to mainly interact with
P2 purinoceptors (2, 31) efficiently depressed glucose transport; thus addition of ATP and nonhydrolyzable ATP analogues (ATP
S1, 2MeSATP, and
,
meATP) at micromolar
concentrations resulted in a decrease in insulin-dependent
glucose transport by up to ~50%. Inhibitory effects were also
observed with other purine nucleotides such as ADP, NADH, and
NAD+ (Table I); NADPH and NADP+ also tended to
inhibit glucose transport, although these effects did not quite reach
statistical significance (Table I). By contrast, adenosine (100 µM), which preferentially activates members of the
P1 purinoceptor family, had no effect on the basal or on
the insulin-stimulated rate of glucose transport (Table I).
In an attempt to discriminate the potential P2 receptor
type involved, we examined the concentration dependence of the effects of some of the above agonists. As illustrated in Fig.
1, the P2-subtype nonselective agonists ATP, ATP
S, and ADP were effective in the low
micromolar range (IC50 = 4.0 ± 0.7, 5.4 ± 1.5, and 7.8 ± 3.4 µM, respectively). The
P2Y-selective analogue 2MeSATP (31) also produced a
significant inhibition (p < 0.001) at 1-10
µM, but higher concentrations than with ATP or ATP
S
were required to reach a maximal degree of inhibition (Fig. 1); in this
case the dose-response relationship could not be fitted to a simple
sigmoidal function. The selective P2X purinoceptor agonist
,
meATP (31) was clearly less potent (with an apparent
IC50 of ~100 µM) (Fig. 1). UTP, which
specifically activates so-called P2U purinoceptors (which
belong to the P2Y family and are also expressed in the myocardium; Ref. 32), was without effect at a concentration as high as
100 µM (Table I).

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Fig. 1.
Concentration dependence of the inhibiting
effects of P2 purinoceptor agonists on insulin-stimulated
glucose transport. Cardiomyocytes were exposed for 10 min to ATP,
ATP S, ADP (P2-subtype-nonselective agonists),
, meATP (P2X-selective), or 2MeSATP
(P2Y-selective) at the indicated concentrations before
insulin (12 nM) was added for another 30 min. Following
this incubation the rate of dGlc transport was measured as described
under "Experimental Procedures." Each point represents the mean
from three to eight independent experiments; S.E. values were less than
10% in all cases. The curves corresponding to the effects of ATP,
ATP S, ADP, and , meATP were calculated using Equation 1;
R2 was >0.99 in all cases (with the 2MeSATP
data, the fit did not converge; the points were therefore connected by
a simple line). Significant statistical difference (p < 0.05) versus the corresponding control (in the absence of
agonist) was reached at 1 µM 2MeSATP, 3 µM
ATP, and 10 µM ATP S, ADP, or , meATP (paired
Student's t test).
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A second approach aimed to address the question of the
P2-type mediating the inhibiting effect of ATP was to use
purinoceptor antagonists. Surprisingly, neither did the
P2Y-specific antagonist Reactive Blue (31) nor suramin,
which is known to antagonize the binding of agonists to and the
activation of both P2X and P2Y purinoceptors
(31), affect the decrease in glucose transport brought about by ATP
(Fig. 2). By contrast, the poorly
subtype-selective antagonist
pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, 100 µM; Ref. 33) suppressed the ATP-dependent
inhibition of glucose transport (Fig. 2). It should also be mentioned
that the effect of ATP (30 µM) was not influenced by the
P1 receptor antagonists 1,3-diethyl-8-phenylxanthine, or
3,7-dimethyl-1-propargylxanthine (100 µM each) (not
shown).

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Fig. 2.
Influence of P2 purinoceptor
antagonists on the inhibitory action of ATP on glucose transport.
Cardiomyocytes were incubated for 10 min in the presence of the
antagonists Reactive Blue (300 µM;
P2Y-selective), PPADS (100 or 300 µM; poorly
P2X-selective), or suramin (300 µM;
nonselective P2X and P2Y antagonist), before
ATP (50 µM) or , meATP (100 µM) was
added for 20 min. Subsequently, the cells were exposed to insulin (12 nM) for another 30 min, and the rate of dGlc transport was
measured as described. Values are means from three to five independent
experiments ± S.E. * denotes statistical significance at the
p < 0.05, and ** denotes statistical significance at
the p < 0.01 level (analysis of variance).
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Influence of Ionic Gradients on the Inhibitory Effect of ATP on
Glucose Transport--
Because the pharmacological data presented
above were not sufficient to clearly define or exclude either
P2X or P2Y receptors as the mediator of the
effect of ATP on glucose transport, we considered the possible
involvement of the former purinoceptor type by using a functional
approach. P2X purinoceptors are nonselective cation
channels whose activation produces a depolarizing inward sodium and
calcium current (1, 2, 4). We therefore examined the influence of
trans-sarcolemmal ionic gradients on the inhibition of glucose
transport by ATP. To this end, we modified the composition of the
incubation medium with respect to sodium, calcium, and potassium over a
wide range of concentrations (while keeping the osmolarity constant).
The results of this series of experiments are summarized in Fig.
3. Even a reduction of the extracellular
sodium concentration to nominally 0 mM (Fig. 3A)
or of the free calcium concentration down to 10
9
M (Fig. 3B) did not suppress the ATP-induced
glucose transport inhibition. It is noteworthy that the basal rate of
glucose transport (in the absence of ATP) significantly rose when the
calcium level was lowered from the physiological value of
10
3 M (Fig. 3); conversely, we had previously
found that an increase in calcium from 1 to 5 mM depresses
the rate of glucose transport in these cells (34). Hence it appears
that calcium exerts an inhibiting influence of the glucose transport
system of cardiomyocytes. However, our present data (Fig. 3,
A and B) show that the action of ATP on glucose
transport is not dependent on extracellular calcium and sodium, thus
ruling out that a depolarizing influx of these ions could mediate the
action of the nucleotide. In addition, depolarization with 30 mM potassium chloride (in the absence of ATP) did not
produce an inhibition of glucose transport (Fig. 3C,
open bars).

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Fig. 3.
Influence of the extracellular concentrations
of sodium, calcium, potassium, and chloride on the inhibition of
insulin-dependent glucose transport by ATP. Cardiac
myocytes were incubated in isotonic media corresponding to medium A,
except that the concentration of one of the ions to be tested was
varied as indicated (for details see "Experimental Procedures").
Note that physiological concentrations of sodium, potassium, chloride,
and calcium are ~145, 4, 120, and 1.5 mM, respectively.
After a 10-min preincubation, ATP (50 µM) was added for
20 min. The myocytes were then treated for another 30 min with insulin
(12 nM) before the rate of dGlc was measured as described.
Data are means of two to five independent experiments (± S.E. or
range). In the data sets shown in panels A, C,
and D, all values obtained in the presence of ATP were
significantly lower than those measured in the corresponding ATP-free
controls at the same ion concentration (p < 0.05 or
less; paired Student's t test). Statistical analysis of
data in panel C: *, p < 0.05; **,
p < 0.01; ***, p < 0.001 versus rate of insulin-stimulated glucose transport in
ATP-free samples at the corresponding Ca2+ level (paired
t test); ¶, p < 0.05; ,
p < 0.01 versus basal rate of glucose
transport measured at [Ca2+] = 10 3
M (analysis of variance).
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Since purinoceptor activation was also shown to activate cardiac
potassium channels (18, 35), we also examined the possible influence of
the potassium gradient across the plasma membrane. As shown in Fig.
3C (closed bars), ATP significantly reduced the rate of glucose transport at all tested potassium concentrations, although the percentage of inhibition tended to be smaller at 0.6 mM potassium. However, such a low potassium concentration may limit the function of the Na+/K+-ATPase,
i.e. of Na+ extrusion, thus impairing
Ca2+ efflux or even promoting Ca2+ influx via
the sarcolemmal Na+/Ca2+ exchanger, which would
eventually result in an increased cytosolic Ca2+
concentration. We therefore also studied the effect of ATP at a very
low extracellular Ca2+ concentration (10
7
M) (at which the Na+/Ca2+ exchanger
is not required to maintain a low cytosolic calcium concentration). We
found that ATP inhibited glucose transport by 57 ± 7% even in
the complete absence of extracellular potassium (two independent
experiments; not shown). Moreover, the effect of ATP (as tested at 6 mM potassium and 1 mM Ca2+) was not
affected by the potassium channel blockers Tedisamil, Apamin, or
Glibenclamide (3-10 µM): thus, in the presence of these inhibitors, the percentage of inhibition by ATP was 48 ± 1%,
37 ± 16%, and 49 ± 2%, respectively (n = 2-3). In conclusion, we found no indication that the trans-sarcolemmal
potassium gradient or potassium channels may be relevant to the action
of ATP on glucose uptake.
Finally, it was reported that in cardiomyocytes ATP activates
trans-sarcolemmal bicarbonate/chloride exchange (10, 11). We therefore
also investigated the influence of the extracellular chloride
concentration on the effect of the nucleotide on glucose transport. As
illustrated in Fig. 3D, this parameter did not affect the
action of ATP; neither did the inhibitor of bicarbonate/chloride exchange DIDS (50 µM) (not shown).
Effect of ATP on the Subcellular Distribution of the Glucose
Transporters GLUT4 and GLUT1--
We next addressed the mechanism by
which ATP decreases the rate of glucose transport. In particular, we
investigated the possibility that this effect might be explained by a
decrease in the amount of the glucose transporters GLUT4 and GLUT1
(which mediate the uptake of glucose in cardiomyocytes; Ref. 26) in the
plasma membrane of these cells. As previously reported (25, 26), insulin (in the absence of ATP) produced an increase in the GLUT4 and
GLUT1 content in the plasma membrane fraction, which was paralleled by
a decreased of these transporters in the intracellular membrane fraction (LDM) (Fig. 4). In the presence
of ATP (30 µM) the insulin-dependent effect
on plasma membrane GLUT4 and GLUT1 was largely suppressed (Fig. 4).
Concomitantly, ATP treatment resulted in a significant increase in the
amount of GLUT4 and GLUT1 detected in LDM (Fig. 4). In other words, ATP
caused a redistribution of GLUT1 and GLUT4 from the plasma membrane to
an intracellular site, thus offsetting the insulin-induced recruitment
of these transporters to the cell surface.

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Fig. 4.
Effect of ATP on the subcellular distribution
of the glucose transporters GLUT4 and GLUT1. Cardiomyocytes were
treated for 10 min with or without ATP (30 µM) and then
without (Bas) or with 12 nM insulin
(Ins) for another 30 min. Subsequently, the cells were
fractionated to obtain plasma membranes and intracellular membranes
(LDM), and quantitation of GLUT4 and GLUT1 content was
performed by Western blot analysis. Upper panels,
representative autoradiograms. Lower panels, quantitative
analysis of data from three to four independent experiments (means ± S.E.). **, p < 0.01; ***, p < 0.001 versus insulin alone (paired t test).
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Effects of ATP on the Rates of Glycolysis, Pyruvate, and Palmitate
Oxidation--
In view of the large effects produced by purinergic
agents on the uptake of glucose, the question arose as to whether
extracellular ATP might regulate other steps of glucose metabolism such
as glycolysis and pyruvate oxidation. The glycolytic flux in
cardiomyocytes was estimated from the rate of detritiation of
[3-3H]glucose, which is a measure of the flux through the
6-phosphofructo-1-kinase reaction (23, 36). When the measurements were
performed at a glucose concentration at which glucose transport
represents the limiting step in the utilization of the sugar in these
cells (i.e. 0.5 mM; Ref. 37), ATP caused a
significant decrease in the rate of basal and insulin-stimulated
[3-3H]glucose detritiation, as expected (Fig.
5A). On the other hand, when
the cells were incubated at a glucose concentration of 5 mM, a condition where the transport step no longer limits
the uptake and metabolism of glucose (at least in the presence of insulin; Ref. 37), ATP failed to decrease the rate of glycolysis (Fig.
5B), which indicates that 6-phosphofructo-1-kinase reaction is not a direct target of purinergic action in these cells.

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Fig. 5.
Effect of ATP on the flux through
6-phosphofructo-1-kinase (A and B) and on the
rate of 14CO2 formation from
[1-14C]pyruvate (C). A and
B, glycolysis. After a 10-min preincubation at 37 °C,
cardiomyocytes were treated with or without ATP (30 µM)
for 20 min and subsequently with or without insulin (20 nM)
for 30 min in medium A supplemented with either 0.5 or 5 mM
D-glucose, as indicated. [3-3H]Glucose was
then added, and its rate of detritiation was determined over a period
of 30 min, as described under "Experimental Procedures." Values are
means from three to four independent experiments ± S.E. *,
denotes a significant difference at the p < 0.05 level
(paired Student's t test). C, pyruvate
decarboxylation. Cardiomyocytes were incubated for 10 min with or
without ATP (50 µM) and for an additional 20 min with or
without dichloroacetate (2 mM) before the rate of
14CO2 formation from
[1-14C]pyruvate was measured over a period of 10 min as
described under "Experimental Procedures." Shown are means ± S.E. from three to four independent experiments.
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The rate of 14CO2 formation from
[1-14C]pyruvate by cardiac myocytes was used to monitor
the activity of the pyruvate dehydrogenase complex of the cells in the
presence and in the absence of ATP. To investigate the pyruvate
dehydrogenase complex reaction in two different states of the enzyme
complex, cells were either used in basal, nonstimulated conditions, as
well as in the presence of the pyruvate dehydrogenase complex
stimulator dichloroacetate (2 mM). As shown in Fig.
5C, extracellular ATP had no inhibitory action on the basal
and dichloroacetate-stimulated rate of [1-14C]pyruvate
oxidation. On the contrary, ATP even tended to increase pyruvate
oxidation in dichloroacetate-stimulated cells (Fig. 5C).
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DISCUSSION |
Extracellular ATP acting through purinergic receptors has been
shown to exert pronounced effects on ionic currents and contractility in hearts and isolated cardiomyocytes (5-14, 17). In this report, we
present first evidence that in heart muscle cells ATP is also endowed
with metabolic properties, in terms of a marked inhibition of glucose
transport, independently of the electrical or mechanical activity of
these cells. Thus we found that in isolated, noncontracting cardiac
myocytes, addition of micromolar concentrations of ATP and other
purinergic agents brings about a large inhibition of glucose transport
(Table I), which is independent of trans-sarcolemmal ionic gradients
(Fig. 3; see also below).
Purinergic Receptors Involved--
Our pharmacological data
clearly point to the involvement of P2 purinoceptors in the
inhibitory action of ATP on glucose transport: (i) absence of effect of
the P1 purinoceptor agonist adenosine (Table I), (ii)
failure of P1 antagonists to influence the effect of ATP
(not shown), (iii) inhibition of glucose transport by
P2-selective agonists in the micromolar range (Fig. 1), and
(iv) suppression of this inhibitory effect by the P2
antagonist PPADS (Fig. 2).
Furthermore, P2Y receptors seem more likely to mediate the
action of ATP than receptors of the P2X-type. Thus, the
selective P2X agonist
,
meATP was clearly less potent
that the P2Y agonist 2MeSATP (Fig. 1). Furthermore, ADP
produced a marked inhibition of glucose transport (Table I), whereas
this nucleotide is known to have only very weak agonist properties or
even to be completely inefficient at P2X sites (2, 38-41).
More importantly, P2X receptors are ligand-gated cation
channels, and their effects are mediated by a depolarizing influx of
sodium and especially of calcium (1, 2, 4). Therefore the insensitivity
of the effect of ATP on glucose transport toward drastic changes in
transmembrane gradients for sodium and calcium (Fig. 3) would be
inconsistent with a receptor of the P2X family being
involved here.
The characteristics of the purinergic effect observed in this study,
however, do not match the profile of known purinoceptors. In
particular, this applies to the recently cloned P2Y
subtypes that were shown to be expressed in cardiac tissue and more
specifically in adult cardiomyocytes of the rat, i.e. the
P2Y1, P2Y2, and P2Y6 receptors
(32). Thus, in contrast to the glucose transport response to ATP (Table
I and Figs. 1 and 2), activation of P2Y1 purinoceptors was
shown to be completely blocked by the P2 antagonists
suramin and Reactive Blue (42), and activation of P2Y2 and
P2Y6 receptors was shown to be potently induced by UTP (43,
44). Moreover, the agonist profiles of these receptors
(P2Y1, P2Y2, and P2Y6), as well as
of P2X receptors present in the adult heart
(P2X1, P2X4, and P2X5; Refs. 20,
40, and 45), are distinct from the rank of potencies found in the
present study (ATP
ATP
S
2MeSATP > ADP >
,
meATP; see Fig. 1) (38, 40, 42-45). On the other hand, it
cannot be ruled out that the depression of cardiomyocyte glucose
transport may involve more than one purinergic receptor.
Another point worth being highlighted is that purinergic inhibition of
glucose transport is likely to be mediated by receptors and/or signals
differing from those underlying the previously described effects of ATP
on ionic processes and contractility in rat cardiomyocytes. In
particular, many of these latter effects were shown to be dependent on
extracellular calcium (9, 12-14) or chloride ions (9, 11), in contrast
to the response of the glucose transport system (Fig. 3). Moreover,
ATP-dependent calcium influx was reportedly blocked by
suramin (19), whereas the effect of the nucleotide on glucose transport
was not influenced by this antagonist (Fig. 2). The same is true with
respect to the efficiency of agonists. Thus, ADP was ineffective in
inducing the cytosolic increase in calcium in cardiac cells (9, 12), although it clearly depressed glucose transport (Table I and Fig. 1).
Conversely, UTP was reported to increase the contraction amplitude as
potently as ATP (14), whereas it had no effect on glucose transport
(Table I).
Mechanism of Glucose Transport Inhibition--
The rate of glucose
transport is largely determined by the amount of glucose transporters
(GLUTs) present in the plasma membrane, and this amount is in turn
dependent on the trafficking of these transporters between the cell
surface and intracellular membrane vesicles (46, 47). As shown in Fig.
4, ATP causes a redistribution of GLUT4 and GLUT1 from the plasma
membrane to intracellular vesicles (LDM) and thus influences the
trafficking of the transporters between these compartments. There
appears to be a discrepancy between the observation that ATP nearly
abrogates the effect of insulin on the subcellular partition of GLUTs
(Fig. 4) and the finding that the nucleotide only depresses
insulin-dependent glucose transport by half (Table I). This
may be explained by the fact that the type of methodology used,
i.e. to fractionate different membrane compartments (plasma
membrane and LDM), only allows a semi-quantitative evaluation of
changes in the amount of transporters in these fractions. This is true,
in particular, for the plasma membrane fraction, which is contaminated
to some extend by intracellular membranes (26), so that the relative
content of transporters is overestimated in samples from basal cells.
An alternative explanation would be that insulin may, in addition to
recruiting intracellular GLUTs to the cell surface, also increase the
intrinsic activity of transporters and that this latter process is
insensitive to ATP. Be that as it may, the results illustrated in Fig.
4 show that activation of P2 purinergic receptors affects
the GLUT trafficking machinery, and this is likely to represent the
basis for the observed changes in the rate of glucose uptake.
Importantly, ATP influences the distribution of both GLUT4 and GLUT1.
Because these transporter isoforms are confined to partly different
intracellular locations (26), and display different trafficking
kinetics (47), the target of the purinergic mechanism may be a general
step of trafficking that is common to both GLUT4 and GLUT1, such as
endocytosis, or alternatively, the recruitment of an intracellular pool
containing both GLUT isoforms.
Likewise, it is of interest that the inhibitory effect of ATP on
glucose transport was observed both in basal, nonstimulated cells, as
well as in the presence of different glucose transport stimuli such as
insulin, and the
-adrenergic agent phenylephrine (Table I),
i.e. under a variety of conditions that are likely to
differently influence the GLUT trafficking machinery. Thus, the
trafficking kinetics of glucose transporters differ between the basal
and the insulin-stimulated state (48); moreover, other studies indicate
that phenylephrine probably affects another step of the trafficking
cycle or another pool of transporters than insulin (28). Hence, the
property of ATP to depress glucose transport regardless of the
experimental conditions suggests that the nucleotide acts on a basic
process involved in glucose transporter trafficking in these cells.
In addition to the ATP-dependent GLUT redistribution, one
may also consider the possibility of a direct interaction of adenine derivatives with glucose transporters, as it was described for ATP or
adenosine in erythrocyte membranes (49, 50). However, this mechanism is
unlikely to contribute to the effect of micromolar concentrations of
ATP in cardiomyocytes, because binding of ATP or adenosine to
erythrocyte glucose transporters only decreased the rate of glucose
transport when the adenine compounds were used at millimolar
concentrations (49, 50). In cardiomyocytes, the ATP concentration had
to be raised above the millimolar range (i.e. well beyond
the level required to saturate the effect described in Fig. 1) to
produce an additional depressing effect on glucose transport (down to
10% of control values; not shown).
Physiological Significance--
At this stage, one can only
speculate on the physiological significance of the purinergic effect on
glucose transport. A finding of interest in this context is that ATP
does not depress the glycolytic utilization of exogenous
[3-3H]glucose when the transmembrane transport no longer
limits the overall rate of uptake (i.e. in the presence of
insulin at 5 mM glucose; (37, 51) and Fig. 5). This
observation suggests that the inhibitory purinergic mechanism would
only be relevant in situations where the transport step does determine
the utilization of the sugar, i.e. for instance under basal
conditions or under the influence of stimuli other than insulin, such
as an increased myocardial workload or ischemia (37, 51, 52).
It is noteworthy that the range of ATP concentrations (~1-10
µM) found to be active with respect to glucose transport
inhibition corresponds to reported blood levels in rats and humans
(53). Hypoxia or ischemia cause a substantial release of ATP from heart cells (3). Although cardiac tissue contains high ectonucleotidase activities, locally released ATP (and ADP consequently formed) may
restrict the uptake of glucose in the affected areas. Whether partial
inhibition of glucose transport (and thus utilization) is deleterious
or not under these pathological conditions remains to be determined.
Alternatively, ATP that is co-released with noradrenaline from
sympathetic nerve endings may modulate the utilization of exogenous glucose in the (normoxic) heart, e.g. in terms of preventing
an excessive uptake of the sugar when the contractile activity is increased by the catecholamine. In whatever physiological condition the
purinergic inhibition of glucose transport may be effective, its very
large extent (Table I) makes it worth being investigated in further
detail in future studies.
Concluding Remarks--
The present study describes a novel,
metabolic action of extracellular ATP and related purine nucleotides,
namely a pronounced inhibition of glucose transport in isolated
cardiomyocytes. The decrease in glucose transport is brought about by a
redistribution of glucose transporters (GLUT1 and GLUT4) from the cell
surface to an intracellular location. This purinergic effect is
mediated by P2 purinoceptors, presumably of the
P2Y family. However, the purinoceptor type involved appears
to differ from previously characterized members of the P2
class and in particular from those mediating ionic and contractile
effects of ATP in these cells.