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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 SobreviaAbstract—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 purinoceptorsR emoval of extracellular adenosine is an essential step in the modulation of several of the biological actions of thisendogenous 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 P2Y2osine are regulated by an efficient membrane transport purinoceptors with ATP induced the phosphorylation ofmediated 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, thetem es or ENT1)3,4 in human vascular endothelium5,6 and cellular effects of elevated D-glucose and activation of P2Ysmooth muscle.7,8 Human ENT1 (hENT1) expression in Raji purinoceptors could involve common signal transductioncells (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 inof human umbilical vein endothelial cells (HUVECs) with the effect of elevated D-glucose on NBMPR-sensitive adenosine25 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 transporterswith increased protein levels and the activity of endothelial and that incubation with 25 mmol/L D-glucose leads to inhibitionNO 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-glucoseporters 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 thesensitive 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 ofPharmacology (M.I.R.), Faculty of Biological Sciences, and the Department of Obstetrics and Gynecology (P.C.), Faculty of Medicine, University ofConcepción, 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 firstname.lastname@example.org © 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 24Cell Culture hours were rinsed with PBS, and mRNA was extracted by using theHUVECs were isolated from full-term normal pregnancies. Informed Dynabeads technique (Dynal). The mRNA was reversed-transcribedwritten consent was given from the hospital for the use of the into cDNA by using oligo(dT18) plus random hexamers and Moloneyumbilical cords. Cells isolated by collagenase (0.25 mg/mL) diges- murine leukemia virus reverse transcriptase (Promega) for 1 hour attion were cultured (37°C, 5% CO2) in medium 199 (M199) contain- 37°C. Polymerase chain reactions (PCRs) were performed in a totaling 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/Land 100 U/mL penicillin-streptomycin as described.5 Twenty-four MgCl2, 2 U Taq DNA polymerase (GIBCO Life Technologies), andhours before an experiment, the incubation medium was changed to sequence-specific oligonucleotide primers (0.5 mol/L) for humanserum-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 . Expectedadenosine 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 and12, 18, or 24 hours to M199 containing 5 mmol/L D-glucose, Materials25 mmol/L D-glucose or L-glucose, or 5 mmol/L D-glucose plus Newborn and fetal calf serum and agarose were from GIBCO Life20 mmol/L D-mannitol as osmotic control.6,19 The kinetics of Technologies. Collagenase type II (Clostridium histolyticum) wasadenosine transport was measured in cells incubated with increasing from Boehringer-Mannheim. Bradford protein reagent was fromconcentrations of adenosine (0 to 500 mol/L, 5 seconds, 22°C) inKrebs 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 liquidscintillation counting.6,8 Adenosine transport was also determined in cells exposed to theP2Y antagonists reactive blue 2 (RB2, 0.1 to 100 nmol/L, 5 minutesor 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 sprotein inhibitor 8-(3-benzamido-4-methylbenzamido)-naphthalene-1,3,4-trisulfonic acid (suramin, 100 mol/L, 15 minutes or 24hours).22 Cells were then exposed to ATP (0.1 to 100 mol/L, 2minutes), which is a nucleotide hydrolyzed by ectonucleotidases inhuman endothelium,5 ATP- -S (0.1 to 100 mol/L, 2 minutes or 24hours), which is a nonhydrolyzable analogue of ATP,23 ADP (0.1 to100 mol/L, 2 minutes), UTP (0.1 to 100 mol/L, 2 minutes), or , -methylene ATP dilithium ( , -MeATP, 0.1 to 100 mol/L, 2minutes), which is a nonselective P2X purinoceptor agonist, in theabsence or presence of RB2, PPADS, or suramin. The effects ofD-glucose and ATP were also assayed in cells preincubated (10minutes or 24 hours) with 10 U/mL hexokinase.24NBMPR Binding[3H]NBMPR equilibrium binding studies were performed in cellspreincubated 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. Specificbinding was defined as the difference in the binding in the presenceand absence of 10 mol/L NBMPR.5,6Measurement of Extracellular ATPExtracellular ATP was determined in M199 from cells cultured in 5or 25 mmol/L D-glucose or in 5 mmol/L D-glucose plus 20 mmol/LD-mannitol for 2, 4, 10, or 60 minutes and 12, 18, or 24 hours byluminometry.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 cultured16 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 orluciferase reagent (pH 7.7), and the reaction was processed with the presence of RB2, PPADS, or suramin. B, Adenosine transportATP bioluminescence assay kit CLS II (Roche). Bioluminescence of was determined in cells cultured in 5 mmol/L D-glucose andsamples and standards was monitored at 562 nm (10 seconds, 22°C) incubated with ATP- -S (24 hours) or ATP (2 minutes), underin a luminometer (Lumat LB 9501, Berthold). Detection limit was 1 the same conditions as in panel A. Values are mean SEMfmol ATP per sample. (n 6). *P 0.05 vs all other values.
572 Circulation Research March 22, 2002Bio-Rad Laboratories. D-Glucose, D-mannitol, hexokinase, andethidium bromide were from Sigma Chemical Co. [2,8,5 -3H]Aden-osine (60 Ci/mmol) and D-[1-14C]mannitol (49.3 mCi/mmol) werefrom NEN. [3H]NBMPR (80 mCi/mmol) was from MoraveckBiochemicals. Agonists and antagonists were from RBI ResearchBiochemical International.Statistical AnalysisValues are mean SEM, and n indicates different umbilical veinendothelial cell cultures with 3 to 6 replicate measurements perexperiment. Statistical analyses were carried out on raw data byusing the Peritz F multiple means comparison test.26 A Student t testwas applied for unpaired data, and a value of P 0.05 was consideredstatistically significant. ResultsEffect of D-Glucose on Adenosine TransportWe have reported that adenosine transport is inhibited by 10nmol/L NBMPR or after incubation with 25 mmol/LD-glucose.5,6 In the present study, inhibition of NBMPR-sensitive adenosine (10 mol/L) transport induced by25 mmol/L D-glucose was blocked after incubation of thecells 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 UTPin cells cultured in 5 mmol/L D-glucose was concentrationdependent (Figure 2A), with similar apparent Ki values (Table1). Neither ADP nor , -MeATP changed adenosine trans-port in HUVECs. Adenosine transport in 25 mmol/LD-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 or100 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 barssaturable 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 alsoassociated with lower Vmax (245 56 or 225 34 pmol/106cells per second for D-glucose or ATP, respectively), with no equilibrium binding was determined.5 Table 2 shows thatsignificant 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- -Sreduced Vmax for adenosine transport induced by 2-minute on Bmax were blocked by RB2 but not by PPADS. Scatchardincubation 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 theelevated D-glucose (Figure 3A) or ATP- -S (Figure 3B) to absence or presence of ATP- -S and/or RB2. Similar resultsvalues in cells cultured in 5 mmol/L D-glucose (Vmax 641 29pmol/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 0.02PPADS had no significant effect on adenosine transportkinetics 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 (BmaxEffect of D-Glucose on NBMPR Binding 3.1 0.2 pmol/106 cells, Kd 0.21 0.02 nmol/L). RB2 blockedTo 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/106Vmax 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/106number 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 (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 hENT1Transport and ATP Release mRNA in cells in 5 mmol/L D-glucose. Similarly, when cellsATP 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 PPADSfor different time periods (Figure 4A). The effect of (Figure 6). The hENT1 mRNA level was unchanged in cellsD-glucose was not due to osmotic changes, inasmuch as cells exposed for 2 to 60 minutes to elevated D-glucose, ATP, orincubated with equimolar concentrations of D-mannitol (ie, ATP- -S (not shown).5 mmol/L D-glucose 20 mmol/L D-mannitol) exhibited ATPrelease similar to that of cells in 5 mmol/L D-glucose. ATPrelease in cells exposed to hexokinase for 2 minutes or 24 Discussion The present study has established that HUVECs express thehours was marginal. D-Glucose–induced ATP release wasparalleled by reduced adenosine transport, an effect blocked hENT1 transporter isoform and that inhibition of adenosineby hexokinase (Figure 4B) and RB2 but not by PPADS (not transport and of NBMPR binding by elevated D-glucose isshown). associated with the activation of P2Y2 purinoceptors. D-Glucose increased ATP release, and ATP, ATP- -S, orEffect of D-Glucose and ATP- -S on hENT1 UTP, but not ADP or , -MeATP, mimicked the inhibitorymRNA Levels effects of D-glucose on adenosine transport and NBMPRCompared with incubation of the cells in 5 mmol/L binding. D-Glucose and ATP- -S also reduced the number ofD-glucose, incubation of the cells in 25 mmol/L D-glucose for NBMPR-sensitive adenosine transporters and hENT1 mRNA24 hours reduced the hENT1 mRNA level (Figure 5). The levels; this effect was blocked by P 2Y purinoceptoreffect of D-glucose was inhibited by RB2 but not by PPADS. 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 stress condition associated with metabolic alterations inFigure 3. Involvement of P2 purinoceptors in the effect of ele- vascular endothelium,2,32,33 which could explain our findingsvated 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 or25 mmol/L D-glucose in the absence or presence of RB2 (100nmol/L). B, Adenosine transport was measured in cells cultured Involvement of P2Y2 Purinoceptors in the Effect ofin M199 containing 5 mmol/L D-glucose in the absence (control) D-Glucose on Adenosine Transportor presence of ATP- -S (100 mol/L) or ATP- -S and RB2 (100nmol/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 insensitiveprevious observations in this cell type.6 The inhibition ofadenosine transport induced by D-glucose was blocked by the to ADP; P2Y1 purinoceptors are stimulated by ATP and ADPnoncompetitive nonspecific P2Y purinoceptor antagonist but not by UTP; and P2Y6 purinoceptors are stimulated byRB227,28 and by the G s protein inhibitor suramin29,30 but was ADP but are insensitive to ATP or UTP.21,36 Thus, theunaffected 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 orthe 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 areokinase, an ATP-degrading enzyme,24 blocked the effect of inhibited by RB220; and P2Y4 purinoceptors are insensitive toD-glucose, and a 3-fold increase in the extracellular ATP level inhibition by suramin.22 Thus, P2Y2 purinoceptors (the formerwas detected in cells cultured in 25 mmol/L D-glucose P2U receptors)37 could be responsible for the inhibitory effectcompared 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,20,21trations reported for this cell type ( 40 pmol/mL).25 In- does not alter adenosine transport, it is suggested that thesecreased extracellular ATP derived from freshly dissociated or purinoceptors are not involved in the effect of elevatedcultured endothelial cells has been shown to be a rapid D-glucose on adenosine transport.
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 wasFigure 4. Time-course effect of elevated D-glucose on ATPrelease and adenosine transport in HUVECs. A, Cells were cul- blocked by RB2 but was unaltered by PPADS, indicating thattured for different periods of time in M199 containing 5 or activation of P2Y purinoceptors leads to a lower uptake of25 mmol/L D-glucose, 5 mmol/L D-glucose 20 mmol/L adenosine by reducing hENT1 expression. hENT1 has beenD-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 centralindicated 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 hENT1sine (20 seconds, 22°C) was measured in M199 containing5 mmol/L D-glucose (time 0) or M199 containing 25 mmol/L transporters could be crucial in human pathological tissues inD-glucose in the absence or presence of hexokinase (10 U/mL) which high levels of D-glucose or adenosine nucleotidesfor 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-glucoseEffect 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). ATPD-glucose was associated with a reduced Vmax.6 The effect of may mediate the effect of elevated D-glucose, inasmuch asD-glucose was mimicked by ATP, ATP- -S, and UTP and extracellular levels of this nucleotide are elevated inblocked by RB2. These results were similar to changes 25 mmol/L D-glucose, and ATP (and ATP- -S) mimicked theinduced by D-glucose, ATP, and ATP- -S in NBMPR- effects of D-glucose on adenosine transport and expression ofbinding kinetics. The adenosine transport inhibitor NBMPR hENT1. Thus, ATP could be playing an autocrine role inbinds specifically to ENT1 (system es) transporters but is not response to elevated D-glucose in HUVECs. The presenttransported itself; therefore, it can be used to estimate the study is the first report to demonstrate modulation of hENT1surface density of ENT1 transporters in intact cells.5,38,39 expression and activity in human endothelium since theThus, the inhibition of adenosine transport by elevated cloning of this transporter from human tissue.3,39,42 RemovalD-glucose and adenine or uridine nucleotides could be due to of extracellular adenosine is a key mechanism in the reduc-
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 inshown to mediate vasodilatation via adenosine receptors by patients affected by this syndrome.2,32,33,45increasing NO synthesis from endothelial cells.43,44 Thus, areduced removal of extracellular adenosine by the endotheli- Acknowledgmentsum 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. 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.Figure 6. Effect of ATP- -S on hENT1 mRNA levels in HUVECs. 6. Montecinos VP, Aguayo C, Flores C, Wyatt AW, Pearson JD, Mann GE,RT-PCR was performed for mRNA extracted from cells cultured Sobrevia L. Regulation of adenosine transport by D-glucose in humanfor 24 hours in M199 containing 5 mmol/L D-glucose and ATP- fetal endothelial cells: involvement of nitric oxide, protein kinase C and -S in the absence or presence of RB2 or PPADS. Reverse mitogen-activated protein kinase. J Physiol (Lond). 2000;529:777–790.transcription and PCR for hENT1 (size product 617 bp) were 7. Aguayo C, Sobrevia L. Nitric oxide, cGMP and cAMP modulate nitro-performed as described in the Figure 5 legend. -Actin (size benzylthioinosine sensitive adenosine transport in human umbilical arteryproduct 350 bp) was used as housekeeper. Data are represen- smooth muscle cells from gestational diabetes. Exp Physiol. 2000;85:tative of 5 different cell cultures. 399 – 409.
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