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(Circulation Research. 2002;90:570.)
© 2002 American Heart Association, Inc.

Cellular Biology

Inhibition of Nitrobenzylthioinosine-Sensitive Adenosine Transport by Elevated D-Glucose Involves Activation of P_2Y2 Purinoceptors in Human Umbilical Vein Endothelial Cells

Jorge Parodi, Carlos Flores, Claudio Aguayo, M. Isolde Rudolph, Paola Casanello, Luis Sobrevia

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.

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{at}udec.cl

	Abstract

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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 µmol/L ATP, ATP--S, or UTP, but not ADP or ,ß-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±0.1x10⁶ 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 P_2Y 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 P_2Y2 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.

Key Words: endothelium • adenosine • nitric oxide • glucose • purinoceptors

	Introduction

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Removal 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 adenosine are regulated by an efficient membrane transport mediated by the Na⁺-independent, nitrobenzylthioinosine (NBMPR)-sensitive equilibrative nucleoside transporter (system es or ENT1)^3,4 in human vascular endothelium^5,6 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/p44^mapk.^6,10 Thus, hENT1 adenosine transporters 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 endothelium from human umbilical vein,¹² bovine pulmonary artery,¹³ and porcine aorta.^14,15 Activation of P_2Y1 and P_2Y2 purinoceptors with ATP induced the phosphorylation of p42^mapk in the human endothelial cell line EAhy 926¹⁶ and p42/p44^mapk in bovine aortic endothelium.¹⁷ Therefore, the cellular effects of elevated D-glucose and activation of P_2Y purinoceptors could involve common signal transduction pathways in human endothelium.

We have investigated the involvement of P_2Y purinoceptors in the effect of elevated D-glucose on NBMPR-sensitive adenosine transport in cultures of HUVECs. We established that endothelial 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 activation of P_2Y2 purinoceptors. In addition, elevated D-glucose diminished hENT1 mRNA levels, an effect mimicked by ATP and blocked by P_2Y antagonists. A preliminary account of the present study has been reported.¹⁸

	Materials and Methods

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Cell Culture
HUVECs were isolated from full-term normal pregnancies. Informed written consent was given from the hospital for the use of the umbilical cords. Cells isolated by collagenase (0.25 mg/mL) digestion were cultured (37°C, 5% CO₂) in medium 199 (M199) containing 5 mmol/L D-glucose, 20% bovine sera, 3.2 mmol/L L-glutamine, and 100 U/mL penicillin-streptomycin as described.⁵ Twenty-four hours before an experiment, the incubation medium was changed to serum-free M199.

Adenosine Transport
Adenosine transport (4 µCi/mL) was measured as described.^5,6 Cells were rinsed with warmed (37°C) Krebs solution containing (mmol/L) NaCl 131, KCl 5.6, NaHCO₃ 25, NaH₂PO₄ 1, D-glucose 5, HEPES 20, CaCl₂ 2.5, and MgCl₂ 1 (pH 7.4), containing 100 µmol/L L-arginine. Triplicate monolayer wells were then preincubated (30 minutes, 22°C) in Krebs solution or in Krebs solution containing the adenosine transport inhibitor NBMPR (10 µmol/L).

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, 25 mmol/L D-glucose or L-glucose, or 5 mmol/L D-glucose plus 20 mmol/L D-mannitol as osmotic control.^6,19 The kinetics of adenosine transport was measured in cells incubated with increasing concentrations of adenosine (0 to 500 µmol/L, 5 seconds, 22°C) in Krebs solution. Tracer uptake was terminated by rinsing the monolayers (3 times) with 200 µL ice-cold Krebs solution containing 10 µmol/L NBMPR, and cell radioactivity was determined by liquid scintillation counting.^6,8

Adenosine transport was also determined in cells exposed to the P_2Y 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 µmol/L, 15 minutes or 24 hours).²² Cells were then exposed to ATP (0.1 to 100 µmol/L, 2 minutes), which is a nucleotide hydrolyzed by ectonucleotidases in human endothelium,⁵ ATP--S (0.1 to 100 µmol/L, 2 minutes or 24 hours), which is a nonhydrolyzable analogue of ATP,²³ ADP (0.1 to 100 µmol/L, 2 minutes), UTP (0.1 to 100 µmol/L, 2 minutes), or ,ß-methylene ATP dilithium (,ß-MeATP, 0.1 to 100 µmol/L, 2 minutes), which is a nonselective P_2X 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 µmol/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 µmol/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 µL 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 µL were mixed with 100 µL 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.

Detection of hENT1
Cells cultured in M199 containing 5 or 25 mmol/L D-glucose for 24 hours were rinsed with PBS, and mRNA was extracted by using the Dynabeads technique (Dynal). The mRNA was reversed-transcribed into cDNA by using oligo(dT₁₈) plus random hexamers and Moloney murine leukemia virus reverse transcriptase (Promega) for 1 hour at 37°C. Polymerase chain reactions (PCRs) were performed in a total volume of 20 µL containing 2 µL of 10x PCR buffer, 2 mmol/L MgCl₂, 2 U Taq DNA polymerase (GIBCO Life Technologies), and sequence-specific oligonucleotide primers (0.5 µmol/L) for human 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, 6 minutes at 63°C, and a final extension for 7 minutes at 61°C. ß-Actin primers were used as housekeepers.

Oligonucleotide primers were for hENT1 (sense) 5'-CATGAT- CTGCGCTATTGCCAGTGG-3', hENT1 (antisense) 5'-AACCA- GGCATCGTGCTCGAAGACCA-3', ß-actin (sense) 5'-AACCGC-GAGAAGATGACCCAGATCATCTTT-3', and ß-actin (antisense) 5'-AGCAGCCGTGGCCATCTCTTGCTCGAAGTC-3'. Expected size products were 617 bp for hENT1 and 350 bp for ß-actin.

Materials
Newborn and fetal calf serum and agarose were from GIBCO Life Technologies. Collagenase type II (Clostridium histolyticum) was from Boehringer-Mannheim. Bradford protein reagent was from Bio-Rad Laboratories. D-Glucose, D-mannitol, hexokinase, and ethidium bromide were from Sigma Chemical Co. [2,8,5'-³H]Adenosine (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

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 µmol/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 K_i values (Table 1). Neither ADP nor ,ß-MeATP changed adenosine transport in HUVECs. Adenosine transport in 25 mmol/L D-glucose was unaltered by nucleotides (Figure 2B). Preincubation of the cells with hexokinase blocked (P<0.05, n=4) the inhibitory effect of 2-minute exposure (45±5 pmol/10⁶ cells per second) or 24-hour exposure (37±6 pmol/10⁶ cells per second) to 25 mmol/L D-glucose or 2-minute exposure to 100 µmol/L ATP (41±3 pmol/10⁶ cells per second) on 10 µmol/L adenosine transport.

View larger version (26K): [in this window] [in a new window]   Figure 1. Involvement of P2 purinoceptors in adenosine transport in HUVECs. A, Overall transport of adenosine (10 µmol/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.

View larger version (20K): [in this window] [in a new window]   Figure 2. Effect of different nucleotides on adenosine transport in HUVECs. Adenosine transport (10 µmol/L, 20 seconds, 22°C) was determined in cells cultured for 24 hours in M199 containing 5 mmol/L (A) or 25 mmol/L (B) D-glucose in the absence or presence of ATP--S or UTP. Cells were also exposed for 2 minutes to ATP, ADP, or ,ß-MeATP. Adenosine transport in the absence of nucleotides (100% transport) was 32±5 and 12±5 pmol/106 cells per second for 5 and 25 mmol/L D-glucose, respectively. Values are mean±SEM (n=8). Some error bars (<7.5% of measured transport) and connecting lines were deleted for clarity.

View this table: [in this window] [in a new window]   Table 1. Effect of d-Glucose and Nucleotides on the Kinetic Parameters of Adenosine Transport in HUVECs

Inhibition of adenosine transport by D-glucose, ATP--S, or UTP (24 hours) was associated with reduced V_max for saturable transport, with negligible changes in apparent K_m (Table 1). Cells incubated for 2 minutes with D-glucose or ATP exhibited a reduced adenosine transport that was also associated with lower V_max (245±56 or 225±34 pmol/10⁶ cells per second for D-glucose or ATP, respectively), with no significant changes in apparent K_m (112±34 or 109±13 µmol/L for D-glucose or ATP, respectively). Cell incubation with RB2, but not with PPADS (not shown), restored the reduced V_max for adenosine transport induced by 2-minute incubation with D-glucose (574±63 pmol/10⁶ cells per second, K_m 107±44 µmol/L) or ATP (633±76 pmol/10⁶ cells per second, K_m 118±51 µmol/L) or 24-hour incubation with elevated D-glucose (Figure 3A) or ATP--S (Figure 3B) to values in cells cultured in 5 mmol/L D-glucose (V_max 641±29 pmol/10⁶ cells per second, K_m 90±11 µmol/L). RB2 or PPADS had no significant effect on adenosine transport kinetics in cells in 5 mmol/L D-glucose (Table 1).

View larger version (19K): [in this window] [in a new window]   Figure 3. Involvement of P2 purinoceptors in the effect of elevated 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 µmol/L) or ATP--S and RB2 (100 nmol/L). Values are mean±SEM (n=6).

Effect of D-Glucose on NBMPR Binding
To determine whether the effects of D-glucose or ATP--S on V_max for adenosine transport were due to changes in the number of available adenosine transport sites, [³H]NBMPR equilibrium binding was determined.⁵ Table 2 shows that D-glucose or ATP--S (24 hours) reduced the maximal binding (B_max) of [³H]NBMPR by 58±12%, with no significant changes in the K_d. The effects of D-glucose and ATP--S on B_max were blocked by RB2 but not by PPADS. Scatchard plots of specific binding data were lineal (not shown), indicating a single population of high-affinity NBMPR binding sites in cells cultured in 5 or 25 mmol/L D-glucose, in the absence or presence of ATP--S and/or RB2. Similar results were obtained in cells exposed for 2 minutes to elevated D-glucose (B_max 1.1±0.2 pmol/10⁶ cells, K_d 0.17±0.02 nmol/L) or ATP (B_max 0.9±0.3 pmol/10⁶ cells, K_d 0.22±0.03 nmol/L) compared with values in 5 mmol/L D-glucose (B_max 3.1±0.2 pmol/10⁶ cells, K_d 0.21±0.02 nmol/L). RB2 blocked the effect of 2 minutes of D-glucose (B_max 2.9±0.4 pmol/10⁶ cells, K_d 0.18±0.02 nmol/L) or ATP (B_max 3.3±0.6 pmol/10⁶ cells, K_d 0.20±0.02 nmol/L) on NBMPR binding.

View this table: [in this window] [in a new window]   Table 2. Effect of d-Glucose and ATP--S on the Kinetic Parameters of NBMPR Binding in HUVECs

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).

View larger version (20K): [in this window] [in a new window]   Figure 4. Time-course effect of elevated D-glucose on ATP release and adenosine transport in HUVECs. A, Cells were cultured 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. Aliquots (100 µL) of M199 collected at the beginning (time 0) or at indicated incubation periods were mixed with 100 µL luciferase reagent, and ATP bioluminescence was monitored at 562 nm for 10 seconds at 22°C. B, Overall transport of 10 µmol/L adenosine (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 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. 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 significantly 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).

View larger version (29K): [in this window] [in a new window]   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 oligonucleotide primers (0.5 µmol/L) for hENT1 (size product 617 bp), with ß-actin (size product 350 bp) used as housekeeper. Data are representative of 5 different cell cultures.

View larger version (33K): [in this window] [in a new window]   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. ß-Actin (size product 350 bp) was used as housekeeper. Data are representative of 5 different cell cultures.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 P_2Y2 purinoceptors. D-Glucose increased ATP release, and ATP, ATP--S, or UTP, but not ADP or ,ß-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.

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 P_2Y purinoceptor antagonist RB2^27,28 and by the G_s protein inhibitor suramin^29,30 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 hexokinase, 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 concentrations reported for this cell type (40 pmol/mL).²⁵ Increased extracellular ATP derived from freshly dissociated or cultured endothelial cells has been shown to be a rapid response of cells to shear stress.^25,31 Elevated D-glucose is a 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 P_2Y2 Purinoceptors in the Effect of D-Glucose on Adenosine Transport
HUVECs express at least 4 isoforms of P_2Y purinergic receptors, ie, P_2Y1, P_2Y2, P_2Y4, and P_2Y6,^34,35 which exhibit different sensitivities for nucleotides and have been shown to mediate several cellular responses.^20,21,36 P_2Y2 and P_2Y4 purinoceptors are stimulated by ATP and UTP but are insensitive to ADP; P_2Y1 purinoceptors are stimulated by ATP and ADP but not by UTP; and P_2Y6 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 P_2Y2 or P_2Y4 purinoceptors in HUVECs. In addition, P_2Y2, but not P_2Y1, purinoceptors are stimulated by UTP; both purinoceptors are inhibited by RB2²⁰; and P_2Y4 purinoceptors are insensitive to inhibition by suramin.²² Thus, P_2Y2 purinoceptors (the former P_2U receptors)³⁷ could be responsible for the inhibitory effect of D-glucose on adenosine transport in human endothelium. Because ,ß-MeATP, a general P_2X 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.

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 V_max.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 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±0.1x0⁶ transporters/cell) was significantly reduced by 25 mmol/L D-glucose (0.7±0.2x0⁶ transporters/cell, P<0.05; n=8) or 100 µmol/L ATP--S (0.5±0.1x0⁶ transporters/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, V_max/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 µmol/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 obtained 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 incubation of cells with elevated D-glucose or ATP (2 minutes) did not change hENT1 mRNA levels. Thus, possible explanations for a reduced number of hENT1 transporters are a lower transcription due to long exposure to D-glucose or an increased 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 elevated D-glucose (0.7±0.1x10⁶ transporters/cell, P<0.05; n=6) or ATP (0.5±0.2x10⁶ 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 P_2Y 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 umbilical vein endothelium by a mechanism that involves activation of P_2Y purinoceptors (possibly the P_2Y2 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 reduction 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 endothelium under pathological conditions in which plasma D-glucose 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 Tecnología (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 CONICYT (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.

	Footnotes

 
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].

Received July 27, 2001; revision received January 29, 2002; accepted January 29, 2002.

	References

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