Inhibition of Nitrobenzylthioinosine-Sensitive Adenosine

Transport by Elevated D-Glucose Involves Activation of P2Y2

Purinoceptors in Human Umbilical Vein Endothelial Cells
Jorge Parodi, Carlos Flores, Claudio Aguayo, M. Isolde Rudolph, Paola Casanello, Luis Sobrevia

Abstract—Chronic incubation with elevated D-glucose reduces adenosine transport in endothelial cells. In this study,
exposure of human umbilical vein endothelial cells to 25 mmol/L D-glucose or 100 ␮mol/L ATP, ATP-␥-S, or UTP, but
not ADP or ␣,␤-methylene ATP, reduced adenosine transport with no change in transport affinity. Inhibition of transport
by D-glucose, ATP, and ATP-␥-S was associated with reduced maximal binding, with no changes in the apparent
dissociation constant for nitrobenzylthioinosine (NBMPR). A significant reduction (⬇60⫾10%, P⬍0.05; n⫽6) in the
number of human equilibrative NBMPR-sensitive nucleoside transporters (hENT1s) per cell (1.8⫾0.1⫻106 in 5 mmol/L
D-glucose) and in hENT1 mRNA levels was observed in cells exposed to D-glucose or ATP-␥-S. Incubation with
elevated D-glucose, but not with D-mannitol, increased the ATP release by 3⫾0.2-fold . The effects of D-glucose and
nucleotides on the number and activity of hENT1 and hENT1 mRNA were blocked by reactive blue 2 (nonspecific P2Y
purinoceptor antagonist), suramin (G␣s protein inhibitor), or hexokinase but not by pyridoxal phosphate-6-azophenyl-
2⬘,4⬘-disulfonic acid (nonselective P2 purinoceptor antagonist). Our findings demonstrate that inhibition of adenosine
transport via hENT1 in endothelial cells cultured in 25 mmol/L D-glucose could be due to stimulation of P2Y2
purinoceptors by ATP, which is released from these cells in response to D-glucose. This could be a mechanism to explain
in part the vasodilatation observed in the early stages of diabetes mellitus or in response to D-glucose infusion. (Circ Res.
2002;90:570-577.)
Key Words: endothelium 䡲 adenosine 䡲 nitric oxide 䡲 glucose 䡲 purinoceptors

R emoval of extracellular adenosine is an essential step in

the modulation of several of the biological actions of this
endogenous nucleoside.1– 4 Plasma and tissue levels of aden-
osine are regulated by an efficient membrane transport
mediated by the Na⫹-independent, nitrobenzylthioinosine
(NBMPR)-sensitive equilibrative nucleoside transporter (sys-
tem es or ENT1)3,4 in human vascular endothelium5,6 and
smooth muscle.7,8 Human ENT1 (hENT1) expression in Raji
cells (a human B-lymphocyte cell line) is dependent on NO
levels and the activity of protein kinase C (PKC).9 Incubation
of human umbilical vein endothelial cells (HUVECs) with
25 mmol/L D-glucose for 24 hours has been reported to
reduce the NBMPR-sensitive adenosine transport associated
with increased protein levels and the activity of endothelial
NO synthase, intracellular Ca2⫹, PKC, and mitogen-activated
protein kinases p42/p44mapk.6,10 Thus, hENT1 adenosine trans-
porters could be expressed and modulated in HUVECs.
It has been reported that ATP inhibits dipyridamole-
sensitive adenosine transport in human pulmonary artery
endothelium.11 ATP also induces activation of PKC in endo-
thelium from human umbilical vein,12 bovine pulmonary
artery,13 and porcine aorta.14,15 Activation of P2Y1 and P2Y2
purinoceptors with ATP induced the phosphorylation of
p42mapk in the human endothelial cell line EAhy 92616 and
p42/p44mapk in bovine aortic endothelium.17 Therefore, the
cellular effects of elevated D-glucose and activation of P2Y
purinoceptors could involve common signal transduction
pathways in human endothelium.
We have investigated the involvement of P2Y purinoceptors in
the effect of elevated D-glucose on NBMPR-sensitive adenosine
transport in cultures of HUVECs. We established that endothe-
lial cells express the hENT1 isoform of nucleoside transporters
and that incubation with 25 mmol/L D-glucose leads to inhibition
of adenosine transport by a mechanism that involves the activa-
tion of P2Y2 purinoceptors. In addition, elevated D-glucose
diminished hENT1 mRNA levels, an effect mimicked by ATP
and blocked by P2Y antagonists. A preliminary account of the
present study has been reported.18

Original received July 27, 2001; revision received January 29, 2002; accepted January 29, 2002.
From the Cellular and Molecular Physiology Laboratory, Department of Physiology (J.P., C.F., C.A., M.I.R., P.C., L.S.), the Department of
Pharmacology (M.I.R.), Faculty of Biological Sciences, and the Department of Obstetrics and Gynecology (P.C.), Faculty of Medicine, University of
Concepción, Concepción, Chile.
Presented in part at The Physiological Society meeting, King’s College London, UK, December 18 –20, 2000, and published in abstract form [J Physiol
(Lond). 2001;531:36P].
Correspondence to Dr L. Sobrevia, Cellular and Molecular Physiology Laboratory (CMPL), Department of Physiology, Faculty of Biological Sciences,
University of Concepción, PO Box 160-C, Concepción, Chile. E-mail lsobrev@udec.cl
© 2002 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org DOI: 10.1161/01.RES.0000012582.11979.8B

570
 Parodi et al Inhibition of Adenosine Transport by Glucose 571

Materials and Methods Detection of hENT1

Cells cultured in M199 containing 5 or 25 mmol/L D-glucose for 24
Cell Culture hours were rinsed with PBS, and mRNA was extracted by using the
HUVECs were isolated from full-term normal pregnancies. Informed Dynabeads technique (Dynal). The mRNA was reversed-transcribed
written consent was given from the hospital for the use of the into cDNA by using oligo(dT18) plus random hexamers and Moloney
umbilical cords. Cells isolated by collagenase (0.25 mg/mL) diges- murine leukemia virus reverse transcriptase (Promega) for 1 hour at
tion were cultured (37°C, 5% CO2) in medium 199 (M199) contain- 37°C. Polymerase chain reactions (PCRs) were performed in a total
ing 5 mmol/L D-glucose, 20% bovine sera, 3.2 mmol/L L-glutamine, volume of 20 ␮L containing 2 ␮L of 10⫻ PCR buffer, 2 mmol/L
and 100 U/mL penicillin-streptomycin as described.5 Twenty-four MgCl2, 2 U Taq DNA polymerase (GIBCO Life Technologies), and
hours before an experiment, the incubation medium was changed to sequence-specific oligonucleotide primers (0.5 ␮mol/L) for human
serum-free M199. ENT1. Samples were incubated for 3 minutes at 97°C, followed by
5 cycles of 30 seconds at 94°C, 4 minutes at 67°C, 5 cycles of 30
seconds at 94°C, 4 minutes at 65°C, 35 cycles of 45 seconds at 94°C,
Adenosine Transport
6 minutes at 63°C, and a final extension for 7 minutes at 61°C.
Adenosine transport (4 ␮Ci/mL) was measured as described.5,6 Cells ␤-Actin primers were used as housekeepers.
were rinsed with warmed (37°C) Krebs solution containing Oligonucleotide primers were for hENT1 (sense) 5⬘-CATGAT-
(mmol/L) NaCl 131, KCl 5.6, NaHCO3 25, NaH2PO4 1, D-glucose 5, CTGCGCTATTGCCAGTGG-3⬘, hENT1 (antisense) 5⬘-AACCA-
HEPES 20, CaCl2 2.5, and MgCl2 1 (pH 7.4), containing 100 ␮mol/L GGCATCGTGCTCGAAGACCA-3⬘, ␤-actin (sense) 5⬘-AACCGC-
L-arginine. Triplicate monolayer wells were then preincubated (30 GAGAAGATGACCCAGATCATCTTT-3⬘, and ␤-actin (antisense)
minutes, 22°C) in Krebs solution or in Krebs solution containing the 5⬘-AGCAGCCGTGGCCATCTCTTGCTCGAAGTC-3⬘. Expected
adenosine transport inhibitor NBMPR (10 ␮mol/L). size products were 617 bp for hENT1 and 350 bp for ␤-actin.
Endothelial cells were preexposed for 2, 4, 10, or 60 minutes and
12, 18, or 24 hours to M199 containing 5 mmol/L D-glucose, Materials
25 mmol/L D-glucose or L-glucose, or 5 mmol/L D-glucose plus Newborn and fetal calf serum and agarose were from GIBCO Life
20 mmol/L D-mannitol as osmotic control.6,19 The kinetics of Technologies. Collagenase type II (Clostridium histolyticum) was
adenosine transport was measured in cells incubated with increasing from Boehringer-Mannheim. Bradford protein reagent was from
concentrations of adenosine (0 to 500 ␮mol/L, 5 seconds, 22°C) in
Krebs solution. Tracer uptake was terminated by rinsing the mono-
layers (3 times) with 200 ␮L ice-cold Krebs solution containing 10
␮mol/L NBMPR, and cell radioactivity was determined by liquid
scintillation counting.6,8
Adenosine transport was also determined in cells exposed to the
P2Y antagonists reactive blue 2 (RB2, 0.1 to 100 nmol/L, 5 minutes
or 24 hours), pyridoxal phosphate-6-azophenyl-2⬘,4⬘-disulfonic acid
(PPADS, 0.1 to 100 nmol/L, 5 minutes or 24 hours),20,21 or the G␣s
protein inhibitor 8-(3-benzamido-4-methylbenzamido)-naphthalene-
1,3,4-trisulfonic acid (suramin, 100 ␮mol/L, 15 minutes or 24
hours).22 Cells were then exposed to ATP (0.1 to 100 ␮mol/L, 2
minutes), which is a nucleotide hydrolyzed by ectonucleotidases in
human endothelium,5 ATP-␥-S (0.1 to 100 ␮mol/L, 2 minutes or 24
hours), which is a nonhydrolyzable analogue of ATP,23 ADP (0.1 to
100 ␮mol/L, 2 minutes), UTP (0.1 to 100 ␮mol/L, 2 minutes), or
␣,␤-methylene ATP dilithium (␣,␤-MeATP, 0.1 to 100 ␮mol/L, 2
minutes), which is a nonselective P2X purinoceptor agonist, in the
absence or presence of RB2, PPADS, or suramin. The effects of
D-glucose and ATP were also assayed in cells preincubated (10
minutes or 24 hours) with 10 U/mL hexokinase.24

NBMPR Binding
[3H]NBMPR equilibrium binding studies were performed in cells
preincubated in Krebs solution or in Krebs solution containing 10
␮mol/L NBMPR. Cells were then exposed (30 minutes, 22°C) to
[3H]NBMPR in the presence of 5 or 25 mmol/L D-glucose. Specific
binding was defined as the difference in the binding in the presence
and absence of 10 ␮mol/L NBMPR.5,6

Measurement of Extracellular ATP

Extracellular ATP was determined in M199 from cells cultured in 5
or 25 mmol/L D-glucose or in 5 mmol/L D-glucose plus 20 mmol/L
D-mannitol for 2, 4, 10, or 60 minutes and 12, 18, or 24 hours by
luminometry.25 Aliquots of 200 ␮L were collected at the beginning
(time 0) and after indicated periods of time and stored at ⫺20°C for
16 to 17 hours. Aliquots of 100 ␮L were mixed with 100 ␮L
luciferase reagent (pH 7.7), and the reaction was processed with the
ATP bioluminescence assay kit CLS II (Roche). Bioluminescence of
samples and standards was monitored at 562 nm (10 seconds, 22°C)
in a luminometer (Lumat LB 9501, Berthold). Detection limit was 1
fmol ATP per sample.
Figure 1. Involvement of P2 purinoceptors in adenosine trans-
port in HUVECs. A, Overall transport of adenosine (10 ␮mol/L,
20 seconds, 22°C) was determined in passage-2 cells cultured
for 24 hours in 5 or 25 mmol/L D-glucose in the absence or
presence of RB2, PPADS, or suramin. B, Adenosine transport
was determined in cells cultured in 5 mmol/L D-glucose and
incubated with ATP-␥-S (24 hours) or ATP (2 minutes), under
the same conditions as in panel A. Values are mean⫾SEM
(n⫽6). *P⬍0.05 vs all other values.
572 Circulation Research March 22, 2002

Bio-Rad Laboratories. D-Glucose, D-mannitol, hexokinase, and

ethidium bromide were from Sigma Chemical Co. [2,8,5⬘-3H]Aden-
osine (60 Ci/mmol) and D-[1-14C]mannitol (49.3 mCi/mmol) were
from NEN. [3H]NBMPR (80 mCi/mmol) was from Moraveck
Biochemicals. Agonists and antagonists were from RBI Research
Biochemical International.

Statistical Analysis
Values are mean⫾SEM, and n indicates different umbilical vein
endothelial cell cultures with 3 to 6 replicate measurements per
experiment. Statistical analyses were carried out on raw data by
using the Peritz F multiple means comparison test.26 A Student t test
was applied for unpaired data, and a value of P⬍0.05 was considered
statistically significant.

Results
Effect of D-Glucose on Adenosine Transport
We have reported that adenosine transport is inhibited by 10
nmol/L NBMPR or after incubation with 25 mmol/L
D-glucose.5,6 In the present study, inhibition of NBMPR-
sensitive adenosine (10 ␮mol/L) transport induced by
25 mmol/L D-glucose was blocked after incubation of the
cells with RB2 or suramin but not PPADS (Figure 1A).
Adenosine transport was also inhibited by ATP-␥-S or UTP;
this effect was blocked by RB2 and suramin (Figure 1B).
Inhibition of adenosine transport by ATP, ATP-␥-S, or UTP
in cells cultured in 5 mmol/L D-glucose was concentration
dependent (Figure 2A), with similar apparent Ki values (Table
1). Neither ADP nor ␣,␤-MeATP changed adenosine trans-
port in HUVECs. Adenosine transport in 25 mmol/L
D-glucose was unaltered by nucleotides (Figure 2B). Prein-
cubation of the cells with hexokinase blocked (P⬍0.05, n⫽4)
the inhibitory effect of 2-minute exposure (45⫾5 pmol/106 Figure 2. Effect of different nucleotides on adenosine transport
in HUVECs. Adenosine transport (10 ␮mol/L, 20 seconds, 22°C)
cells per second) or 24-hour exposure (37⫾6 pmol/106 cells
was determined in cells cultured for 24 hours in M199 contain-
per second) to 25 mmol/L D-glucose or 2-minute exposure to ing 5 mmol/L (A) or 25 mmol/L (B) D-glucose in the absence or
100 ␮mol/L ATP (41⫾3 pmol/106 cells per second) on 10 presence of ATP-␥-S or UTP. Cells were also exposed for 2
␮mol/L adenosine transport. minutes to ATP, ADP, or ␣,␤-MeATP. Adenosine transport in the
absence of nucleotides (100% transport) was 32⫾5 and 12⫾5
Inhibition of adenosine transport by D-glucose, ATP-␥-S, pmol/106 cells per second for 5 and 25 mmol/L D-glucose,
or UTP (24 hours) was associated with reduced Vmax for respectively. Values are mean⫾SEM (n⫽8). Some error bars
saturable transport, with negligible changes in apparent Km (⬍7.5% of measured transport) and connecting lines were
(Table 1). Cells incubated for 2 minutes with D-glucose or deleted for clarity.
ATP exhibited a reduced adenosine transport that was also
associated with lower Vmax (245⫾56 or 225⫾34 pmol/106
cells per second for D-glucose or ATP, respectively), with no equilibrium binding was determined.5 Table 2 shows that
significant changes in apparent Km (112⫾34 or 109⫾13 D-glucose or ATP-␥-S (24 hours) reduced the maximal
␮mol/L for D-glucose or ATP, respectively). Cell incubation binding (Bmax) of [3H]NBMPR by 58⫾12%, with no signifi-
with RB2, but not with PPADS (not shown), restored the cant changes in the Kd. The effects of D-glucose and ATP-␥-S
reduced Vmax for adenosine transport induced by 2-minute on Bmax were blocked by RB2 but not by PPADS. Scatchard
incubation with D-glucose (574⫾63 pmol/106 cells per sec-
plots of specific binding data were lineal (not shown),
ond, Km 107⫾44 ␮mol/L) or ATP (633⫾76 pmol/106 cells
indicating a single population of high-affinity NBMPR bind-
per second, Km 118⫾51 ␮mol/L) or 24-hour incubation with
ing sites in cells cultured in 5 or 25 mmol/L D-glucose, in the
elevated D-glucose (Figure 3A) or ATP-␥-S (Figure 3B) to
absence or presence of ATP-␥-S and/or RB2. Similar results
values in cells cultured in 5 mmol/L D-glucose (Vmax 641⫾29
pmol/106 cells per second, Km 90⫾11 ␮mol/L). RB2 or were obtained in cells exposed for 2 minutes to elevated
6
D-glucose (Bmax 1.1⫾0.2 pmol/10 cells, Kd 0.17⫾0.02
PPADS had no significant effect on adenosine transport
kinetics in cells in 5 mmol/L D-glucose (Table 1). nmol/L) or ATP (Bmax 0.9⫾0.3 pmol/106 cells, Kd 0.22⫾0.03
nmol/L) compared with values in 5 mmol/L D-glucose (Bmax
Effect of D-Glucose on NBMPR Binding 3.1⫾0.2 pmol/106 cells, Kd 0.21⫾0.02 nmol/L). RB2 blocked
To determine whether the effects of D-glucose or ATP-␥-S on the effect of 2 minutes of D-glucose (Bmax 2.9⫾0.4 pmol/106
Vmax for adenosine transport were due to changes in the cells, Kd 0.18⫾0.02 nmol/L) or ATP (Bmax 3.3⫾0.6 pmol/106
number of available adenosine transport sites, [3H]NBMPR cells, Kd 0.20⫾0.02 nmol/L) on NBMPR binding.
 Parodi et al Inhibition of Adenosine Transport by Glucose 573

TABLE 1. Effect of D-Glucose and Nucleotides on the Kinetic Parameters of

Adenosine Transport in HUVECs
Vmax, pmol 䡠 (106
Km, ␮mol/L Cells)⫺1 䡠 s⫺1 Ki, ␮mol/L
5 mmol/L D-glucose
Control 90⫾11 641⫾29 ND
ATP 98⫾45 156⫾21* 0.35⫾0.06
ATP-␥-S 128⫾41 211⫾26* 0.42⫾0.09
UTP 127⫾39 198⫾64* 0.41⫾0.05
ADP 101⫾29 598⫾54 No inhibition
RB2 102⫾34 598⫾45 ND
PPADS 95⫾45 624⫾47 ND
ATP-␥-S⫹RB2 108⫾30 660⫾70† ND
ATP-␥-S⫹PPADS 118⫾50 271⫾31* ND
25 mmol/L D-glucose
Control 127⫾44 227⫾30* ND
ATP 131⫾26 254⫾49* No inhibition
ATP-␥-S 145⫾41 237⫾61* No inhibition
UTP 112⫾21 199⫾32* No inhibition
ADP 125⫾19 187⫾44* No inhibition
RB2 86⫾26 554⫾59‡ ND
PPADS 132⫾31 199⫾58* ND
ATP-␥-S⫹RB2 95⫾32 559⫾61‡ ND
ATP-␥-S⫹PPADS 112⫾14 199⫾34* ND
ND indicates not determined. Values are mean⫾SEM (n⫽8). Saturable adenosine transport was
determined in cells cultured for 24 hours in M199 containing 5 or 25 mmol/L D-glucose in the
absence or presence of 100 ␮mol/L ATP-␥-S, 100 ␮mol/L UTP, 100 nmol/L RB2, or 100 nmol/L
PPADS. The effect of 100 ␮mol/L ATP or 100 ␮mol/L ADP on transport was assayed by incubation
of cells for 2 minutes with these nucleotides. For inhibition studies, adenosine transport was
determined in cells exposed to increasing concentrations (0 to 100 ␮mol/L) of nucleotides. The
apparent inhibition constants (Ki) were calculated by using the expression Ki⫽IC50/(1⫹[Ado]/Km),
where Km is the apparent Km value for adenosine transport, [Ado] is adenosine concentration
(10 ␮mol/L), and IC50 is the half-maximal inhibitory concentration of the inhibitors.5
*P⬍0.05 vs control in 5 mmol/L D-glucose; †P⬍0.05 vs ATP-␥-S in 5 mmol/L D-glucose; and
‡P⬍0.05 vs control in 25 mmol/L D-glucose.

Time-Course Effect of D-Glucose on Adenosine
Transport and ATP Release
ATP release from cells cultured in M199 containing
5 mmol/L D-glucose was increased by 25 mmol/L D-glucose
for different time periods (Figure 4A). The effect of
D-glucose was not due to osmotic changes, inasmuch as cells
incubated with equimolar concentrations of D-mannitol (ie,
5 mmol/L D-glucose⫹20 mmol/L D-mannitol) exhibited ATP
release similar to that of cells in 5 mmol/L D-glucose. ATP
release in cells exposed to hexokinase for 2 minutes or 24
hours was marginal. D-Glucose–induced ATP release was
paralleled by reduced adenosine transport, an effect blocked
by hexokinase (Figure 4B) and RB2 but not by PPADS (not
shown).
RB2 and PPADS alone did not significantly alter hENT1
mRNA in cells in 5 mmol/L D-glucose. Similarly, when cells
were incubated with ATP-␥-S, hENT1 mRNA was signifi-
cantly reduced, an effect blocked by RB2 but not by PPADS
(Figure 6). The hENT1 mRNA level was unchanged in cells
exposed for 2 to 60 minutes to elevated D-glucose, ATP, or
ATP-␥-S (not shown).
Discussion
The present study has established that HUVECs express the
hENT1 transporter isoform and that inhibition of adenosine
transport and of NBMPR binding by elevated D-glucose is
associated with the activation of P2Y2 purinoceptors.
D-Glucose increased ATP release, and ATP, ATP-␥-S, or

Effect of D-Glucose and ATP-␥-S on hENT1
mRNA Levels
Compared with incubation of the cells in 5 mmol/L
D-glucose, incubation of the cells in 25 mmol/L D-glucose for
24 hours reduced the hENT1 mRNA level (Figure 5). The
effect of D-glucose was inhibited by RB2 but not by PPADS.
UTP, but not ADP or ␣,␤-MeATP, mimicked the inhibitory
effects of D-glucose on adenosine transport and NBMPR
binding. D-Glucose and ATP-␥-S also reduced the number of
NBMPR-sensitive adenosine transporters and hENT1 mRNA
levels; this effect was blocked by P 2Y purinoceptor
antagonists.
574 Circulation Research March 22, 2002

TABLE 2. Effect of D-Glucose and ATP-␥-S on the Kinetic

Parameters of NBMPR Binding in HUVECs
Kd, nmol/L Bmax, pmol/106 Cells
5 mmol/L D-glucose
Control 0.21⫾0.02 3.1⫾0.2
RB2 0.19⫾0.03 2.9⫾0.3
PPADS 0.22⫾0.01 2.9⫾0.4
ATP-␥-S 0.28⫾0.04 0.8⫾0.2*
ATP-␥-S⫹RB2 0.18⫾0.03 2.7⫾0.2†
ATP-␥-S⫹PPADS 0.19⫾0.04 1.1⫾0.3*
25 mmol/L D-glucose
Control 0.19⫾0.04 1.3⫾0.3*
RB2 0.18⫾0.02 3.5⫾0.5‡
PPADS 0.21⫾0.01 0.9⫾0.3*
ATP-␥-S 0.16⫾0.04 1.4⫾0.1*
ATP-␥-S⫹RB2 0.19⫾0.03 3.6⫾0.5‡
ATP-␥-S⫹PPADS 0.22⫾0.01 1.1⫾0.3*
Values are mean⫾SEM (n⫽6). Endothelial cells were cultured for 24 hours
in M199 containing 5 or 25 mmol/L D-glucose in the absence or presence of
100 ␮mol/L ATP-␥-S, 100 nmol/L RB2, or 100 nmol/L PPADS. Cells were then
washed and preincubated in Krebs buffer for 15 minutes in the absence or
presence of 10 ␮mol/L NBMPR. Monolayers were then incubated with
[3H]NBMPR for 30 minutes at 22°C in Krebs buffer. Specific cell-associated
radioactivity was defined as the difference between total binding and binding
in the presence of 10 ␮mol/L NBMPR.
*P⬍0.05 vs control in 5 mmol/L D-glucose; †P⬍0.05 vs ATP-␥-S in
5 mmol/L D-glucose; and ‡P⬍0.05 vs control in 25 mmol/L D-glucose.

response of cells to shear stress.25,31 Elevated D-glucose is a

Figure 3. Involvement of P2 purinoceptors in the effect of ele-
vated D-glucose on kinetics of adenosine transport in HUVECs.
A, Initial rates of adenosine transport (20 seconds, 22°C) were
measured in cells cultured for 24 hours in M199 containing 5 or
25 mmol/L D-glucose in the absence or presence of RB2 (100
nmol/L). B, Adenosine transport was measured in cells cultured
in M199 containing 5 mmol/L D-glucose in the absence (control)
or presence of ATP-␥-S (100 ␮mol/L) or ATP-␥-S and RB2 (100
nmol/L). Values are mean⫾SEM (n⫽6).
Adenosine transport was inhibited after the incubation of
endothelial cells with 25 mmol/L D-glucose, confirming our
previous observations in this cell type.6 The inhibition of
adenosine transport induced by D-glucose was blocked by the
noncompetitive nonspecific P2Y purinoceptor antagonist
RB227,28 and by the G␣s protein inhibitor suramin29,30 but was
unaffected by the nonselective P2 purinoceptor antagonist
PPADS, suggesting the involvement of P2 purinoceptors in
the effects of D-glucose. This could be due to ATP released
from HUVECs in response to D-glucose, inasmuch as hex-
okinase, an ATP-degrading enzyme,24 blocked the effect of
D-glucose, and a 3-fold increase in the extracellular ATP level
was detected in cells cultured in 25 mmol/L D-glucose
compared with 5 mmol/L D-glucose (⬇35 pmol/mL). Basal
ATP release from HUVECs is within the range of concen-
trations reported for this cell type (⬇40 pmol/mL).25 In-
creased extracellular ATP derived from freshly dissociated or
cultured endothelial cells has been shown to be a rapid
stress condition associated with metabolic alterations in
vascular endothelium,2,32,33 which could explain our findings
of a higher extracellular ATP level.
Involvement of P2Y2 Purinoceptors in the Effect of
D-Glucose on Adenosine Transport
HUVECs express at least 4 isoforms of P2Y purinergic
receptors, ie, P2Y1, P2Y2, P2Y4, and P2Y6,34,35 which exhibit
different sensitivities for nucleotides and have been shown to
mediate several cellular responses.20,21,36 P2Y2 and P2Y4 puri-
noceptors are stimulated by ATP and UTP but are insensitive
to ADP; P2Y1 purinoceptors are stimulated by ATP and ADP
but not by UTP; and P2Y6 purinoceptors are stimulated by
ADP but are insensitive to ATP or UTP.21,36 Thus, the
inhibition of adenosine transport by high D-glucose, ATP,
ATP-␥-S, or UTP could result from the activation of P2Y2 or
P2Y4 purinoceptors in HUVECs. In addition, P2Y2, but not P2Y1,
purinoceptors are stimulated by UTP; both purinoceptors are
inhibited by RB220; and P2Y4 purinoceptors are insensitive to
inhibition by suramin.22 Thus, P2Y2 purinoceptors (the former
P2U receptors)37 could be responsible for the inhibitory effect
of D-glucose on adenosine transport in human endothelium.
Because ␣,␤-MeATP, a general P2X purinoceptor agonist,20,21
does not alter adenosine transport, it is suggested that these
purinoceptors are not involved in the effect of elevated
D-glucose on adenosine transport.
 Parodi et al
Figure 4. Time-course effect of elevated D-glucose on ATP
release and adenosine transport in HUVECs. A, Cells were cul-
tured for different periods of time in M199 containing 5 or
25 mmol/L D-glucose, 5 mmol/L D-glucose⫹20 mmol/L
D-mannitol, or 25 mmol/L D-glucose⫹10 U/mL hexokinase. Ali-
quots (100 ␮L) of M199 collected at the beginning (time 0) or at
indicated incubation periods were mixed with 100 ␮L luciferase
reagent, and ATP bioluminescence was monitored at 562 nm for
10 seconds at 22°C. B, Overall transport of 10 ␮mol/L adeno-
sine (20 seconds, 22°C) was measured in M199 containing
5 mmol/L D-glucose (time 0) or M199 containing 25 mmol/L
D-glucose in the absence or presence of hexokinase (10 U/mL)
for the indicated incubation periods. Values are mean⫾SEM
(n⫽12).
Effect of D-Glucose on the Number of
Adenosine Transporters
As reported, inhibition of adenosine transport by elevated
D-glucose was associated with a reduced Vmax.6 The effect of
D-glucose was mimicked by ATP, ATP-␥-S, and UTP and
blocked by RB2. These results were similar to changes
induced by D-glucose, ATP, and ATP-␥-S in NBMPR-
binding kinetics. The adenosine transport inhibitor NBMPR
binds specifically to ENT1 (system es) transporters but is not
transported itself; therefore, it can be used to estimate the
surface density of ENT1 transporters in intact cells.5,38,39
Thus, the inhibition of adenosine transport by elevated
D-glucose and adenine or uridine nucleotides could be due to
Inhibition of Adenosine Transport by Glucose 575
the reduced number rather than the activity of an existing
pool of NBMPR-sensitive nucleoside transporters in the
plasma membrane of HUVECs. This conjecture is supported
by the finding that the number of adenosine transporters per
cell (1.8⫾0.1⫻06 transporters/cell) was significantly reduced
by 25 mmol/L D-glucose (0.7⫾0.2⫻06 transporters/cell,
P⬍0.05; n⫽8) or 100 ␮mol/L ATP-␥-S (0.5⫾0.1⫻06 trans-
porters/cell, P⬍0.04; n⫽12). However, the D-glucose– or
ATP-␥-S–induced reduction in adenosine transport is not due
to changes in the turnover number (ie, Vmax/number of
transporters per cell)5,8 for adenosine (356⫾30 versus
324⫾45 or 439⫾75 adenosine molecules/transporter per
second for 5 mmol/L versus 25 mmol/L D-glucose or 100
␮mol/L ATP-␥-S, respectively). These results are similar to
previous reports showing a reduced number of adenosine
membrane transporters without altering its turnover rate in
human vascular endothelium5 or smooth muscle cells7 ob-
tained from gestational diabetic pregnancies or in vascular
smooth muscle cells exposed to human insulin.8
Parallel experiments demonstrated a reduced hENT1
mRNA level in cells incubated with elevated D-glucose or
ATP-␥-S for 24 hours. However, as expected, acute incuba-
tion of cells with elevated D-glucose or ATP (2 minutes) did
not change hENT1 mRNA levels. Thus, possible explana-
tions for a reduced number of hENT1 transporters are a lower
transcription due to long exposure to D-glucose or an in-
creased turnover rate of hENT1 transporters as described in
other cell types.1–3 The latter is supported by the finding of a
reduced number of hENT1 transporters available at the
plasma membrane after a brief (2-minute) exposure to ele-
vated D-glucose (0.7⫾0.1⫻106 transporters/cell, P⬍0.05;
n⫽6) or ATP (0.5⫾0.2⫻106 transporters/cell, P⬍0.05; n⫽6).
Reduction in the number of adenosine transporters and
hENT1 mRNA by D-glucose, ATP, and ATP-␥-S was
blocked by RB2 but was unaltered by PPADS, indicating that
activation of P2Y purinoceptors leads to a lower uptake of
adenosine by reducing hENT1 expression. hENT1 has been
colocalized with A1 nucleoside receptors in the human central
nervous system,4,40,41 suggesting a role of the hENT1-
mediated transport process in the control of adenosine-
mediated biological actions.2,42,43 Thus, expression of hENT1
transporters could be crucial in human pathological tissues in
which high levels of D-glucose or adenosine nucleotides
could modulate endothelial cell function, such as in diabetes
mellitus.2
The present results demonstrate that elevated D-glucose
induced a reduction in adenosine transport in human umbil-
ical vein endothelium by a mechanism that involves activa-
tion of P2Y purinoceptors (possibly the P2Y2 subtype). ATP
may mediate the effect of elevated D-glucose, inasmuch as
extracellular levels of this nucleotide are elevated in
25 mmol/L D-glucose, and ATP (and ATP-␥-S) mimicked the
effects of D-glucose on adenosine transport and expression of
hENT1. Thus, ATP could be playing an autocrine role in
response to elevated D-glucose in HUVECs. The present
study is the first report to demonstrate modulation of hENT1
expression and activity in human endothelium since the
cloning of this transporter from human tissue.3,39,42 Removal
of extracellular adenosine is a key mechanism in the reduc-
576 Circulation Research March 22, 2002
tion of extracellular levels of this nucleoside, modulating its
biological actions on vascular cells.1– 4 Adenosine has been
shown to mediate vasodilatation via adenosine receptors by
increasing NO synthesis from endothelial cells.43,44 Thus, a
reduced removal of extracellular adenosine by the endotheli-
um under pathological conditions in which plasma D-glucose
Figure 6. Effect of ATP-␥-S on hENT1 mRNA levels in HUVECs.
RT-PCR was performed for mRNA extracted from cells cultured
for 24 hours in M199 containing 5 mmol/L D-glucose and ATP-
␥-S in the absence or presence of RB2 or PPADS. Reverse
transcription and PCR for hENT1 (size product 617 bp) were
performed as described in the Figure 5 legend. ␤-Actin (size
product 350 bp) was used as housekeeper. Data are represen-
tative of 5 different cell cultures.
Figure 5. Effect of elevated D-glucose on
hENT1 mRNA levels in HUVECs.
RT-PCR was performed for mRNA
extracted from cells cultured for 24
hours in M199 containing 5 or
25 mmol/L D-glucose in the absence or
presence of RB2 or PPADS. The mRNA
was reversed-transcribed into cDNA (1
hour, 37°C), and PCRs were performed
by using sequence-specific oligonucleo-
tide primers (0.5 ␮mol/L) for hENT1 (size
product 617 bp), with ␤-actin (size prod-
uct 350 bp) used as housekeeper. Data
are representative of 5 different cell
cultures.
is increased (such as in uncontrolled diabetes) could, in part,
explain the early generalized vasodilatation observed in
patients affected by this syndrome.2,32,33,45
Acknowledgments
This study was supported by Fondo Nacional de Ciencia y Tec-
nología (FONDECYT 1000354 and 7000354) and Dirección de
Investigación, University of Concepción (DIUC 201.084.003-1.0),
Concepción, Chile, and The Wellcome Trust (UK). J. Parodi holds
an MSc fellowship and P. Casanello holds a PhD fellowship from
Beca Docente University of Concepción. C. Aguayo holds a CONI-
CYT (Chile) PhD fellowship. We thank Dr J. Villegas (Universidad
La Frontera, Chile) for contributing the ATP measurements. We also
thank the midwives of Hospital Regional, Concepción, Chile, labor
wards for the supply of umbilical cords, Susana Rojas for technical
assistance, and Isabel Jara for secretarial assistance.
References
1. Griffith DA, Jarvis SM. Nucleoside and nucleobase transport systems of
mammalian cells. Biochim Biophys Acta. 1996;1286:153–181.
2. Sobrevia L, Mann GE. Dysfunction of the nitric oxide signalling pathway
in diabetes and hyperglycaemia. Exp Physiol. 1997;82:1–30.
3. Baldwin S, Mackey J, Cass C, Young J. Nucleoside transporters:
molecular biology and implications for therapeutic development. Mol
Med Today. 1999;53:216 –224.
4. Jennings LL, Hao C, Cabrita MA, Vickers MF, Baldwin SA, Young JD,
Cass CE. Distinct regional distribution of human equilibrative nucleoside
transporter proteins 1 and 2 (hENT1 and hENT2) in the central nervous
system. Neuropharmacology. 2001;40:722–731.
5. Sobrevia L, Jarvis SM, Yudilevich DL. Adenosine transport in cultured
human umbilical vein endothelial cells is reduced in diabetes. Am J
Physiol. 1994;267:C39 –C47.
6. Montecinos VP, Aguayo C, Flores C, Wyatt AW, Pearson JD, Mann GE,
Sobrevia L. Regulation of adenosine transport by D-glucose in human
fetal endothelial cells: involvement of nitric oxide, protein kinase C and
mitogen-activated protein kinase. J Physiol (Lond). 2000;529:777–790.
7. Aguayo C, Sobrevia L. Nitric oxide, cGMP and cAMP modulate nitro-
benzylthioinosine sensitive adenosine transport in human umbilical artery
smooth muscle cells from gestational diabetes. Exp Physiol. 2000;85:
399 – 409.
 Parodi et al
8. Aguayo C, Flores C, Parodi J, Rojas R, Mann GE, Pearson JD, Sobrevia
L. Modulation of adenosine transport by insulin in human umbilical artery
smooth muscle cells from normal or gestational diabetic pregnancies.
J Physiol (Lond). 2001;534:243–254.
9. Soler C, Felipe A, Mata JF, Casado FJ, Celada A, Pastor-Anglada M.
Regulation of nucleoside transport by lipopolysaccharide, phorbol esters,
and tumor necrosis factor-␣ in human B-lymphocytes. J Biol Chem.
1998;273:26939 –26945.
10. Sobrevia L, Nadal A, Yudilevich DL, Mann GE. Activation of L-arginine
transport (system y⫹) and nitric oxide synthase by elevated glucose and
insulin in human endothelial cells. J Physiol (Lond). 1996;490:775–781.
11. Dawicki DD, Chatterjee D, Wyche J, Rounds S. Extracellular ATP and
adenosine cause apoptosis of pulmonary artery endothelial cells. Am J
Physiol. 1997;273:L485–L494.
12. Ethier MF, Dobson JG Jr. Adenosine stimulation of DNA synthesis in
human endothelial cells. Am J Physiol. 1997;272:H1470 –H1479.
13. Chen BC, Lin WW. PKC␤I mediates the inhibition of P2Y receptor-
induced inositol phosphate formation in endothelial cells. Br J
Pharmacol. 1999;27:1908 –1914.
14. Castro AF, Amorena C, Müller A, Ottaviano G, Tellez-Inon MT, Taquini
AC. Extracellular ATP and bradykinin increase cGMP in vascular endo-
thelial cells via activation of PKC. Am J Physiol. 1998;275:C113–C119.
15. Noll T, Holschermann H, Koprek K, Gunduz D, Haberbosch W,
Tillmanns H, Piper HM. ATP reduces macromolecule permeability of
endothelial monolayers despite increasing [Ca2⫹]i. Am J Physiol. 1999;
276:H1892–H18901.
16. Graham A, McLees A, Kennedy C, Gould GW, Plevin R. Stimulation by
the nucleotides, ATP and UTP of mitogen-activated protein kinase in
EAhy 926 endothelial cells. Br J Pharmacol. 1996;117:1341–1347.
17. Patel J, Abeles A, Block ER. Nitric oxide exposure and sulfhydryl
modulation alter L-arginine transport in cultured pulmonary artery endo-
thelial cells. Free Radic Biol Med. 1996;20:629 – 637.
18. Parodi J, Roa J, Rudolph I, Flores C, Aguayo C, Guijarro MV, Sobrevia
L. Modulation of L-arginine and adenosine transport by elevated
D-glucose is mediated by activation of P2Y purinoceptors involving acti-
vation of p44/p42mapkin human fetal vein endothelium. J Physiol (Lond).
2001;531:36P. Abstract.
19. Sobrevia L, Cesare P, Yudilevich DL, Mann GE. Diabetes-induced acti-
vation of system y⫹ and nitric oxide synthase in human endothelial cells:
association with membrane hyperpolarization. J Physiol (Lond). 1995;
489:183–192.
20. Ralevic V, Burnstock G. Receptors for purines and pyrimidines
Pharmacol Rev. 1998;50:413– 492.
21. Boarder M, Hourani S. The regulation of vascular function by P2
receptors: multiple sites and multiple receptors. Trends Physiol Sci. 1998;
19:99 –107.
22. Freissmuth M, Boehm S, Beindl W, Nickel P, Ijzerman AP, Hohenegger
M, Nanoff C. Suramin analogues as subtype selective G protein inhibi-
tors. Mol Pharmacol. 996;49:602– 611.
23. Han J, Kim N, Kim E, Ho WK, Earm YE. Modulation of ATP-sensitive
potassium channels by cGMP-dependent protein kinase in rabbit ventric-
ular myocytes. J Biol Chem. 2001;276:22140 –22147.
24. Nicholas RA, Watt WC, Lazarowski ER, Li Q, Harden TK. Uridine
nucleotide selectivity of three phospholipase C-activating P2 receptors:
identification of a UDP-selective, a UTP-selective, and an ATP- and
UTP-specific receptor. Mol Pharmacol. 1996;50:224 –229.
25. Bodin P, Burnstock G. Increased release of ATP from endothelial cells
during acute inflammation. Inflamm Res. 1998;47:351–354.
Inhibition of Adenosine Transport by Glucose 577
26. Harper JF. Peritz’ F test: BASIC program of a robust multiple comparison
test for statistical analysis of all differences among group means. Comput
Biol Med. 1984;14:437– 445.
27. Burnstock G, Warland JJ. P2-purinoceptors of two subtypes in the rabbit
mesenteric artery: reactive blue 2 selectively inhibits responses mediated
via the P2Y- but not the P2X-purinoceptor. Br J Pharmacol. 1987;90:
383–391.
28. Chen BC, Lee C-M, Lin WW. Inhibition of ecto-ATPase by PPADS,
suramin and reactive blue 2 in endothelial cells, C6 glioma cells and RAW
264.7 macrophages. Br J Pharmacol. 1996;119:1628 –1634.
29. Dunn PM, Blakeley AG. Suramin. a reversible P2-purinoceptor antagonist
in the mouse vas deferens. Br J Pharmacol. 1988;93:243–245.
30. Usune S, Katsuragi T, Furukawa T. Effects of PPADS and suramin on
contractions and cytoplasmic Ca2⫹changes evoked by AP4A, ATP and
␣,␤-methylene ATP in guinea-pig urinary bladder. Br J Pharmacol.
1996;117:698 –702.
31. Bodin P, Bailey D, Burnstock G. Increased flow-induced ATP release
from isolated vascular endothelial cells but not smooth muscle cells. Br J
Pharmacol. 1991;103:1203–1205.
32. Pieper GM. Review of alterations in endothelial nitric oxide production in
diabetes. Hypertension. 1998;31:1047–1060.
33. De Vriese AS, Verbeuren TJ, Van de Voorde J, Lameire NH, Vanhoutte PM.
Endothelial dysfunction in diabetes. Br J Pharmacol. 2000;130:963–974.
34. Jin J, Dasari R, Sistare FD, Kunapuli S. Distribution of P2Y receptor
subtypes on haematopoietic cells. Br J Pharmacol. 1998;123:789 –794.
35. Kunapuli SP, Daniel JL. P2 receptor subtypes in the cardiovascular
system. Biochem J. 1998;336:513–523.
36. Vassort G. Adenosine 5⬘-triphosphate: a P2-purinergic agonist in the
myocardium. Physiol Rev. 2001;81:767– 806.
37. Lustig KD, Shiau AK, Brake AJ, Julius D. Expression cloning of an ATP
receptor from mouse neuroblastoma cells. Proc Natl Acad Sci U S A.
1993;90:5113–5117.
38. Paterson AR, Kolassa N, Cass CE. Transport of nucleoside drugs in
animal cells. Pharmacol Ther. 1981;12:515–536.
39. Hyde RJ, Cass CE, Young JD, Baldwin SA. The ENT family of eukaryote
nucleoside and nucleobase transporters: recent advances in the investi-
gation of structure/function relationships and the identification of novel
isoforms. Mol Membr Biol. 2001;18:53– 63.
40. Pennycooke M, Chaudary N, Shuralyova I, Zhang Y, Coe IR. Differential
expression of human nucleoside transporters in normal and tumor tissue.
Biochem Biophys Res Commun. 2001;280:951–959.
41. Anderson CM, Xiong W, Geiger JD, Young JD, Cass CE, Baldwin SA,
Parkinson FE. Distribution of equilibrative, nitrobenzylthioinosine-
sensitive nucleoside transporters (ENT1) in brain. J Neurochem. 1999;
73:867– 873.
42. Griffiths M, Beaumont N, Yao SY, Sundaram M, Boumah CE, Davies A,
Kwong FY, Coe I, Cass CE, Young JD, Baldwin SA. Cloning of a human
nucleoside transporter implicated in the cellular uptake of adenosine and
chemotherapeutic drugs. Nat Med. 1997;3:89 –93.
43. Sobrevia L, Yudilevich DL, Mann GE. Activation of A2-purinoceptors by
adenosine stimulates L-arginine transport (system y⫹) and nitric oxide
synthesis in human fetal endothelial cells. J Physiol (Lond). 1997;499:
135–140.
44. Sexl V, Mancusi G, Höller C, Gloria-Maercker E, Schutz W, Freissmuth
M. Stimulation of the mitogen-activated protein kinase via the A2a-aden-
osine receptor in primary human endothelial cells. J Biol Chem. 1997;
272:5792–5799.
45. Hayoz D, Ziegler T, Brunner HR, Ruiz J. Diabetes mellitus and vascular
lesions. Metabolism. 1998;47:16 –19.