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

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

3. 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.

4. 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 (106
Km, mol/L Cells) 1 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 RB2 and PPADS alone did not significantly alter hENT1
Transport and ATP Release mRNA in cells in 5 mmol/L D-glucose. Similarly, when cells
ATP release from cells cultured in M199 containing were incubated with ATP- -S, hENT1 mRNA was signifi-
5 mmol/L D-glucose was increased by 25 mmol/L D-glucose cantly reduced, an effect blocked by RB2 but not by PPADS
for different time periods (Figure 4A). The effect of (Figure 6). The hENT1 mRNA level was unchanged in cells
D-glucose was not due to osmotic changes, inasmuch as cells exposed for 2 to 60 minutes to elevated D-glucose, ATP, or
incubated with equimolar concentrations of D-mannitol (ie, ATP- -S (not shown).
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
Discussion
The present study has established that HUVECs express the
hours was marginal. D-Glucose–induced ATP release was
paralleled by reduced adenosine transport, an effect blocked hENT1 transporter isoform and that inhibition of adenosine
by hexokinase (Figure 4B) and RB2 but not by PPADS (not transport and of NBMPR binding by elevated D-glucose is
shown). 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 UTP, but not ADP or , -MeATP, mimicked the inhibitory
mRNA Levels effects of D-glucose on adenosine transport and NBMPR
Compared with incubation of the cells in 5 mmol/L binding. D-Glucose and ATP- -S also reduced the number of
D-glucose, incubation of the cells in 25 mmol/L D-glucose for NBMPR-sensitive adenosine transporters and hENT1 mRNA
24 hours reduced the hENT1 mRNA level (Figure 5). The levels; this effect was blocked by P 2Y purinoceptor
effect of D-glucose was inhibited by RB2 but not by PPADS. antagonists.

5. 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
stress condition associated with metabolic alterations in
Figure 3. Involvement of P2 purinoceptors in the effect of ele- vascular endothelium,2,32,33 which could explain our findings
vated D-glucose on kinetics of adenosine transport in HUVECs.
A, Initial rates of adenosine transport (20 seconds, 22°C) were
of a higher extracellular ATP level.
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 Involvement of P2Y2 Purinoceptors in the Effect of
in M199 containing 5 mmol/L D-glucose in the absence (control) D-Glucose on Adenosine Transport
or presence of ATP- -S (100 mol/L) or ATP- -S and RB2 (100
nmol/L). Values are mean SEM (n 6). 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
Adenosine transport was inhibited after the incubation of
mediate several cellular responses.20,21,36 P2Y2 and P2Y4 puri-
endothelial cells with 25 mmol/L D-glucose, confirming our
noceptors are stimulated by ATP and UTP but are insensitive
previous observations in this cell type.6 The inhibition of
adenosine transport induced by D-glucose was blocked by the to ADP; P2Y1 purinoceptors are stimulated by ATP and ADP
noncompetitive nonspecific P2Y purinoceptor antagonist but not by UTP; and P2Y6 purinoceptors are stimulated by
RB227,28 and by the G s protein inhibitor suramin29,30 but was ADP but are insensitive to ATP or UTP.21,36 Thus, the
unaffected by the nonselective P2 purinoceptor antagonist inhibition of adenosine transport by high D-glucose, ATP,
PPADS, suggesting the involvement of P2 purinoceptors in ATP- -S, or UTP could result from the activation of P2Y2 or
the effects of D-glucose. This could be due to ATP released P2Y4 purinoceptors in HUVECs. In addition, P2Y2, but not P2Y1,
from HUVECs in response to D-glucose, inasmuch as hex- purinoceptors are stimulated by UTP; both purinoceptors are
okinase, an ATP-degrading enzyme,24 blocked the effect of inhibited by RB220; and P2Y4 purinoceptors are insensitive to
D-glucose, and a 3-fold increase in the extracellular ATP level inhibition by suramin.22 Thus, P2Y2 purinoceptors (the former
was detected in cells cultured in 25 mmol/L D-glucose P2U receptors)37 could be responsible for the inhibitory effect
compared with 5 mmol/L D-glucose ( 35 pmol/mL). Basal of D-glucose on adenosine transport in human endothelium.
ATP release from HUVECs is within the range of concen- Because , -MeATP, a general P2X purinoceptor agonist,20,21
trations reported for this cell type ( 40 pmol/mL).25 In- does not alter adenosine transport, it is suggested that these
creased extracellular ATP derived from freshly dissociated or purinoceptors are not involved in the effect of elevated
cultured endothelial cells has been shown to be a rapid D-glucose on adenosine transport.

6. Parodi et al 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
Figure 4. Time-course effect of elevated D-glucose on ATP
release and adenosine transport in HUVECs. A, Cells were cul- blocked by RB2 but was unaltered by PPADS, indicating that
tured for different periods of time in M199 containing 5 or activation of P2Y purinoceptors leads to a lower uptake of
25 mmol/L D-glucose, 5 mmol/L D-glucose 20 mmol/L adenosine by reducing hENT1 expression. hENT1 has been
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
colocalized with A1 nucleoside receptors in the human central
indicated incubation periods were mixed with 100 L luciferase nervous system,4,40,41 suggesting a role of the hENT1-
reagent, and ATP bioluminescence was monitored at 562 nm for mediated transport process in the control of adenosine-
10 seconds at 22°C. B, Overall transport of 10 mol/L adeno- mediated biological actions.2,42,43 Thus, expression of hENT1
sine (20 seconds, 22°C) was measured in M199 containing
5 mmol/L D-glucose (time 0) or M199 containing 25 mmol/L transporters could be crucial in human pathological tissues in
D-glucose in the absence or presence of hexokinase (10 U/mL) which high levels of D-glucose or adenosine nucleotides
for the indicated incubation periods. Values are mean SEM could modulate endothelial cell function, such as in diabetes
(n 12).
mellitus.2
The present results demonstrate that elevated D-glucose
Effect of D-Glucose on the Number of induced a reduction in adenosine transport in human umbil-
Adenosine Transporters ical vein endothelium by a mechanism that involves activa-
As reported, inhibition of adenosine transport by elevated tion of P2Y purinoceptors (possibly the P2Y2 subtype). ATP
D-glucose was associated with a reduced Vmax.6 The effect of may mediate the effect of elevated D-glucose, inasmuch as
D-glucose was mimicked by ATP, ATP- -S, and UTP and extracellular levels of this nucleotide are elevated in
blocked by RB2. These results were similar to changes 25 mmol/L D-glucose, and ATP (and ATP- -S) mimicked the
induced by D-glucose, ATP, and ATP- -S in NBMPR- effects of D-glucose on adenosine transport and expression of
binding kinetics. The adenosine transport inhibitor NBMPR hENT1. Thus, ATP could be playing an autocrine role in
binds specifically to ENT1 (system es) transporters but is not response to elevated D-glucose in HUVECs. The present
transported itself; therefore, it can be used to estimate the study is the first report to demonstrate modulation of hENT1
surface density of ENT1 transporters in intact cells.5,38,39 expression and activity in human endothelium since the
Thus, the inhibition of adenosine transport by elevated cloning of this transporter from human tissue.3,39,42 Removal
D-glucose and adenine or uridine nucleotides could be due to of extracellular adenosine is a key mechanism in the reduc-

7. 576 Circulation Research March 22, 2002
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.
tion of extracellular levels of this nucleoside, modulating its is increased (such as in uncontrolled diabetes) could, in part,
biological actions on vascular cells.1– 4 Adenosine has been explain the early generalized vasodilatation observed in
shown to mediate vasodilatation via adenosine receptors by patients affected by this syndrome.2,32,33,45
increasing NO synthesis from endothelial cells.43,44 Thus, a
reduced removal of extracellular adenosine by the endotheli- Acknowledgments
um under pathological conditions in which plasma D-glucose 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.
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