AJP - Heart AJP citation statistics
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 288: H2756-H2762, 2005. First published February 4, 2005; doi:10.1152/ajpheart.00921.2004
0363-6135/05 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
288/6/H2756    most recent
00921.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (8)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Leung, G. P. H.
Right arrow Articles by Tse, C.-M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Leung, G. P. H.
Right arrow Articles by Tse, C.-M.

D-Glucose upregulates adenosine transport in cultured human aortic smooth muscle cells

George P. H. Leung,1 Ricky Y. K. Man,1 and Chung-Ming Tse2

1Department of Pharmacology, University of Hong Kong, Hong Kong; and 2Division of Gastroenterology, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland

Submitted 3 September 2004 ; accepted in final form 29 January 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The etiology of the atherosclerosis that occurs in diabetes mellitus is unclear. Adenosine has been shown to inhibit growth of rat aortic smooth muscle cells. Nucleoside transporters play an integral role in adenosine function by regulating adenosine levels in the vicinity of adenosine receptors. Therefore, we studied the effect of 25 mM D-glucose, which mimics hyperglycemia of diabetes, on adenosine transport in cultured human aortic smooth muscle cells (HASMCs). Although RT-PCR demonstrated the presence of equilibrative nucleoside transporter-1 (ENT-1) and ENT-2 mRNA, functional studies revealed that adenosine transport in HASMCs was predominantly mediated by ENT-1 and inhibited by nitrobenzylmercaptopurine riboside (NBMPR, IC50 = 0.69 ± 0.05 nM). Adenosine transport in HASMCs was increased by >30% after treatment for 48 h with 25 mM D-glucose, but not with equimolar D-mannitol and L-glucose. Kinetic studies showed that D-glucose increased Vmax of adenosine transport without affecting Km. Similarly, D-glucose increased Bmax of high-affinity [3H]NBMPR binding, while the dissociation constant (Kd) was not changed. Consistent with these observations, 25 mM D-glucose increased mRNA and protein expression of ENT-1. Treatment of serum-starved cells with the selective inhibitors of MAPK/ERK, PD-98059 (40 µM) and U-0126 (10 µM), abolished the effect of D-glucose on ENT-1. We conclude that D-glucose upregulates the protein and message expression and functional activity of ENT-1 in HASMCs, possibly via MAPK/ERK-dependent pathways. Pathologically, the increase in ENT-1 activity in diabetes may affect the availability of adenosine in the vicinity of adenosine receptors and, thus, alter vascular functions in diabetes.

nucleoside transporter; diabetes

ADENOSINE MODULATES CELLULAR functions via G protein-coupled receptors to protect cells and organs and is released in response to cell injury and stress. For instance, adenosine levels in blood and interstitial fluid increase during hypoxia and ischemia. Increased extracellular adenosine causes vasodilation by acting through A2 adenosine receptors on vascular smooth muscle cells (SMCs) (5, 31) and, thus, increases blood flow and oxygenation. Adenosine inhibits platelet aggregation (33), decreases heart rate (14), and antagonizes the stimulatory effects of catecholamines (37). Because of these important functions of adenosine as a negative-feedback modulator of cell and organ energy demand and consumption and as a cell- and organ-protective agent, it is known as a "retaliatory metabolite" (25). Recent reports also showed that adenosine released during preconditioning by short periods of ischemia followed by reperfusion induces cardioprotection to a subsequent sustained ischemia (17). This effect is mediated in part by activation of A1 and A3 adenosine receptors in cardiomyocytes and involves PKC and mitochondrial ATP-sensitive K+ channels (24).

Nucleoside transporters fine-tune the local concentrations of adenosine in the vicinity of adenosine receptors. Two major classes of nucleoside transporters in mammalian cells have been characterized by Na+ dependence (16). The equilibrative nucleoside transporters (ENTs) are facilitated-diffusion systems and are Na+ independent. They are further subdivided into two types on the basis of their sensitivities to inhibition by nitrobenzylmercaptopurine riboside (NBMPR) (4, 43). ENT-1 is potently inhibited by nanomolar concentrations of NBMPR, but ENT-2 is resistant to NBMPR up to 1 µM. ENT-1 and ENT-2 are broadly selective, accepting purine and pyrimidine nucleosides, but ENT-2 also transports nucleobases such as hypoxanthine (6, 26, 46). In contrast, the concentrative nucleoside transporters (CNTs) are Na+ dependent. CNT-1 is pyrimidine selective (36), CNT-2 is purine selective (42), and CNT-3 is broadly selective (35, 41).

Diabetes mellitus is a major risk factor for cardiovascular diseases. Because adenosine inhibits growth of vascular SMCs (10, 11, 12, 13) and nucleoside transporters are involved in adenosine homeostasis, in the present study, we characterized the nucleoside transport systems in cultured human aortic SMCs (HASMCs). We also determined the effect on adenosine transport of long-term exposure of cells to 25 mM D-glucose, a condition that mimics hyperglycemia of diabetes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Culture of HASMCs. HASMCs were obtained from American Type Culture Collection (Manassas, VA) and cultured in DMEM (containing 5 mM D-glucose) supplemented with 10% (vol/vol) fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in 95% air-5% CO2. All experiments were carried out with cells from passages 6 to 14. At 48 h before uptake study and mRNA and protein isolation, cells were incubated in serum-free DMEM. To study the effect of D-glucose, 20 mM D-glucose was added to the medium for a final concentration of 25 mM. Under these conditions, there were no changes in cell number and morphology when the cells were grown in the presence of 5 mM (control) and 25 mM glucose (32).

Adenosine uptake. All experiments were carried out in HEPES-buffered Ringer solution containing (in mM) 135 NaCl, 5 KCl, 3.33 NaH2PO4, 0.83 Na2HPO4, 1.0 CaCl2, 1.0 MgCl2, 5 HEPES, and 10 D-glucose (pH 7.4). Na+-free buffer contained (in mM) 140 N-methyl-D-glucamine, 5 HEPES, 5 KH2PO4, 1.0 CaCl2, 1.0 MgCl2, and 10 D-glucose (pH 7.4).

Confluent monolayers of cells in 24-well plates were washed three times in HEPES-buffered solution. HEPES-buffered solution (300 µl) containing [3H]adenosine (10 µM, 2 µCi/ml) was added to each well. The plates were washed three times rapidly with ice-cold phosphate-buffered saline (PBS) containing (in mM) 137 NaCl, 2.68 KCl, and 1.47 KH2PO4 (pH 7.4). Cells were solubilized in 0.5 ml of 5% (vol/vol) Triton X-100. The radioactivity was measured by a {beta}-scintillation counter. The protein content was determined spectrophotometrically using a commercial bicinchoninic acid assay (Pierce Biochemicals, Rockford, IL).

To determine passive adenosine uptake, monolayers of cells were incubated in the Na+-free buffer containing [3H]adenosine in the presence of 0.5 mM NBMPR. NBMPR (0.5 mM) was dissolved in DMSO [in a final concentration of 0.5% (vol/vol)] with gentle heating. In a control experiment, DMSO [0.5% (vol/vol)] did not alter adenosine uptake.

High-affinity [3H]NBMPR binding. Microsomal membranes of HASMCs (300 µg) were incubated with various concentrations of [3H]NBMPR (0.03–10 nM with and without 10 µM nonradioactive NBMPR) for 20 min at room temperature. Bound and free [3H]NBMPR were then separated by rapid filtration with a Whatman GF/B filter (43). The radioactivity that was retained on filters (bound [3H]NBMPR) was counted by a {beta}-scintillation counter after dissolving in Liquiscint (Amersham Biosciences, Piscataway, NJ).

RNA isolation and RT-PCR. Total RNA was isolated from HASMCs using TRIzol reagent (Invitrogen, Grand Island, NY). Two micrograms of total RNA were used for first-strand cDNA synthesis using random hexamer primers and Superscript II RNase H– reverse transcriptase (SuperScript Preamplification System, Invitrogen). The resulting first-strand cDNA was directly used for PCR amplification.

Sets of primers were designed and synthesized for PCR analysis. The two primers used for amplifying ENT-1 (accession no. AF079117) were 5'-GCAGCACCCTTGCCTGAG-3' (sense) and 5'-GAAGGCACTTTCTGATAG-3' (antisense), which generated a 420-bp PCR product. The two primers for ENT-2 (accession no. AF029358) were 5'-ACAGCCAGGATCCTGAGC-3' (sense) and 5'-CATGGACAGGAGCATGGC-3' (antisense), which generated a 399-bp product. The two primers for CNT-1 (accession no. NM_004212) were 5'-ACCCCTCGAGACGAAGAG-3' (sense) and 5'-AAACAGAGCCAGGGCCCT-3' (antisense), which yielded a 329-bp PCR product. The two primers for CNT-2 (accession no. NM_004213) were 5'-GGGCTGGAGCTCATGGAA-3' (sense) and 5'-GGAGACTCCTGCAAACAC-3' (antisense), which yielded a 399-bp PCR product. The two primers for CNT-3 (accession no. AF305210) were 5'-GCAGCCCCCAGAGCTGAG-3' (sense) and 5'-ACAAAAAGAGGAAGGGCT-3' (antisense), which yielded a 380-bp PCR product. Reactions were carried out for 30 cycles with the following parameters: denaturation at 94°C for 30 s, annealing at 55°C for 1 min, and extension at 72°C for 1.5 min. PCR products were analyzed by agarose gel electrophoresis and visualized by staining with ethidium bromide. The human ileum cDNA was used as a positive control, because all the nucleoside transporters are expressed in this tissue (41, 44). To semiquantify the PCR products of nucleoside transporters, optical density of nucleoside transporter bands was normalized to that of the ribosomal protein S16 (accession no. BC004324). The two primers used to amplify S16 were 5'-CCCGCTGCAGTCTGTGCAGGT-3' (sense) and 5'-CCAAACTTTTTGGACTCGCAG-3' (antisense), which yielded a 384-bp PCR product.

Western blotting. Polyclonal anti-human ENT-1 antibody was raised in rabbit as previously described (18). HASMCs were grown to confluence on 10-cm petri dishes. All subsequent steps were conducted at 4°C with ice-cold solutions. The cells were washed three times with PBS, scraped in 2 ml of 5 mM sodium phosphate, pH 8, with protease inhibitor cocktail [Sigma, St. Louis, MO; 1:100 (vol/vol)]. Cells were sonicated briefly and centrifuged at 3,000 g for 10 min to remove nuclei and unbroken cells. The resulting supernatant was centrifuged at 30,000 g for 30 min to pellet the crude microsomal membranes, which was resuspended in 5 mM sodium phosphate. The crude membranes were then resolved on 9% (wt/vol) SDS-polyacrylamide gels and electrotransferred onto nitrocellulose membranes. After blocking with 5% (wt/vol) nonfat dry milk in PBS overnight at 4°C, nitrocellulose membranes were incubated with the anti-human ENT-1 antibody [1:100 (vol/vol) dilution in blocking solution] at room temperature for 2 h. Nitrocellulose membranes were then washed extensively with 0.02% (vol/vol) Triton X-100 in PBS. After they were washed, the membranes were incubated with the horseradish peroxidase-conjugated goat anti-rabbit secondary antibody [1:5,000 (vol/vol) dilution in blocking solution] at room temperature for 2 h. Excess secondary antibody was again washed, and the bound secondary antibody was detected by enhanced chemiluminescence (Western Blot Chemiluminescence Reagent Plus, NEN Life Science Products, Boston, MA). Protein expression of {beta}-actin was similarly detected with the monoclonal mouse antiactin antibody (Chemicon, Temecula, CA). The molecular sizes of ENT-1 and {beta}-actin are 40 and 43 kDa, respectively. Optical density of the ENT-1 band was normalized to that of {beta}-actin.

Materials. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise noted. [2-3H]adenosine was obtained from Amersham Biosciences (Piscataway, NJ), [3H]NBMPR from Moravek Biochemicals (Brea, CA), and cell culture media and supplements from Invitrogen.

Statistical analysis. Adenosine uptake data are expressed as means ± SE of three experiments performed in triplicate. Apparent Km and Vmax values were calculated by nonlinear regression analysis of the v vs. v/s plots and by the Hill equation { where v is the rate of [3H]adenosine uptake, [S] is substrate concentration, n is the Hill coefficient, and K' is affinity for substrate} using Origin software. Student's t-test and analysis of variance were used for paired and multiple variants, respectively. P < 0.05 was considered statistically significant. Equilibrium binding of [3H]NBMPR was transformed for Scatchard analysis to calculate the dissociation constant (Kd) and Bmax.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Characterization of adenosine transport in HASMCs. The time course of [3H]adenosine uptake (10 µM) in HASMCs is shown in Fig. 1A. There was no difference between Na+-dependent and Na+-independent [3H]adenosine uptake. Na+-independent [3H]adenosine uptake was linear for up to 5 min and was completely inhibited by 0.5 mM NBMPR. We also compared the [3H]adenosine uptake rate between control and ATP-depleted cells, in which adenosine phosphorylation was inhibited (34). ATP depletion was achieved by incubating cells with oligomycin (2 µg/ml) and 2-deoxy-D-glucose (5 mM) for 1 h at 37°C (22, 34, 41). The initial rate of [3H]adenosine uptake measured at 1 min was similar in control and ATP-depleted cells: 3.7 ± 0.5 and 3.6 ± 0.4 pmol/mg protein (n = 3, P > 0.05). Similarly, erythro-9-(2-hydroxy-3-nonyl)-adenine hydrochloride (1 µM), an adenosine deaminase inhibitor, had no effect on the 1-min uptake of [3H]adenosine (data not shown). Therefore, subsequent initial rate studies were carried out with 1 min of incubation.



View larger version (18K):
[in this window]
[in a new window]
  		Fig. 1. Functional characterization of adenosine transport in human aortic smooth muscle cells (HASMCs). A: [3H]adenosine uptake (10 µM, 2 µCi/ml) in HASMCs in the presence ({blacksquare}) and absence ({square}) of Na+ and in the absence of Na+ with the addition of 0.5 mM nitrobenzylmercaptopurine riboside (NBMPR, {circ}). B: initial rate (1 min) of [3H]adenosine uptake (10 µM, 2 µCi/ml) in the absence of Na+ and the presence of 0–300 µM NBMPR. Values are means ± SE of 3 experiments performed in triplicate.

 
To test whether the adenosine transport was mediated by ENT-1 and/or ENT-2, the dose response of NBMPR inhibition of [3H]adenosine transport was determined. Adenosine uptake was inhibited by NBMPR in a dose-dependent manner (IC50 = 0.69 ± 0.05 nM) and was completely abolished by 10 nM NBMPR (Fig. 1B).

RT-PCR was used to confirm the expression of ENT-1 in HASMCs (Fig. 2). As predicted, the PCR product of ENT-1 was amplified from RNA isolated from HASMCs. The PCR product of ENT-2 was also amplified, although ENT-2 activity was not functionally detected. There was no amplification of CNT-1, CNT-2, or CNT-3 (Fig. 2). These results complemented the functional studies, in which Na+-dependent adenosine transport was absent in HASMCs.



View larger version (34K):
[in this window]
[in a new window]
  		Fig. 2. RT-PCR analysis of nucleoside transporter mRNA in HASMCs. PCR products are seen only in reactions using oligonucleotide primer pairs for equilibrative nucleoside transporter (ENT)-1 and ENT-2 but not for concentrative nucleoside transporter (CNT)-1, CNT-2, and CNT-3. Positive controls with human ileum cDNA indicate expected sizes of amplified fragments: 420-bp (ENT-1), 399-bp (ENT-2), 329-bp (CNT-1), 399-bp (CNT-2), and 380-bp (CNT-3). DNA size markers are shown at left.

 
Effect of D-glucose on adenosine uptake in HASMCs. Diabetes is characterized by high blood D-glucose, and it has recently been shown that expression of nucleoside transporters during diabetes is differentially regulated in different tissues and cell types (28, 29). Therefore, we studied the effect of 25 mM D-glucose on HASMCs. Adenosine uptake was unaffected after incubation of the cultured cells with 25 mM D-glucose for up to 12 h. However, adenosine uptake was increased by 30.7 and 38.3% after incubation with 25 mM D-glucose for 24 and 48 h, respectively. D-Mannitol or L-glucose did not affect adenosine transport (data not shown), suggesting that the effect was due to the change in D-glucose level, rather than the change in osmolarity.

Kinetic studies were performed to provide insight into mechanisms by which the nucleoside transporters were modulated by D-glucose (Fig. 3A). Incubation of HASMCs with 25 mM D-glucose was associated with an increase in Vmax (28.93 ± 2.54 and 42.93 ± 4.25 pmol·mg protein–1·min–1 for control and D-glucose-treated cells, respectively, P < 0.05) with no change in apparent Km of adenosine (0.022 ± 0.003 and 0.024 ± 0.004 mM for control and D-glucose-treated cells, respectively, P > 0.05). The maximum capacity of adenosine transport (Vmax/Km) was 42.6% higher in D-glucose-treated than in control cells. This increase in adenosine transport by D-glucose was completely attenuated by treatment with cycloheximide (10 nM), an inhibitor of protein synthesis (data not shown), suggesting that protein synthesis was required for D-glucose-induced adenosine transport.



View larger version (23K):
[in this window]
[in a new window]
  		Fig. 3. Kinetic analysis of [3H]adenosine uptake and high-affinity [3H]NBMPR binding. A: concentration dependence of adenosine (0.1 µM–1 mM) uptake determined by measuring [3H]adenosine uptake at room temperature for 1 min. Inset: Eadie-Hoftsee plot (v vs. v/s), which was used for estimation of Km and Vmax. B: concentration dependence of [3H]NBMPR binding (0.03–10 nM, without and with 10 µM nonradioactive NBMPR). Inset: Scatchard analysis (B/F vs. B, where B is bound and F is free), which was used to estimate Bmax and Kd. Data represent a typical experiment performed in triplicate. {bullet}, Control cells; {circ}, cells treated with 25 mM D-glucose.

 
[3H]NBMPR binding has been used as an assay for the number of functional ENT-1 nucleoside transporters (43), so the affinity and the density of NBMPR-binding sites in HASMCs were determined. Incubation of HASMCs with 25 mM D-glucose was associated with an increase in Bmax (72.01 ± 1.91 and 103.05 ± 2.02 fmol/mg protein for control and D-glucose-treated cells, respectively, P < 0.05) without a significant change in Kd (0.343 ± 0.026 and 0.341 ± 0.013 nM for control and D-glucose-treated cells, respectively, P > 0.05; Fig. 3B).

To further explore whether the effect of D-glucose on adenosine transport in HASMCs was due to changes in ENT-1 and/or ENT-2 mRNA and protein expressions, semiquantitative RT-PCR and Western blotting were performed. ENT-1 mRNA levels in HASMCs were increased by 45.1 and 52.7% after incubation with 25 mM D-glucose for 24 and 48 h, respectively, whereas ENT-2 mRNA levels were not affected (Figs. 4, A and B). Similarly, ENT-1 protein levels in HASMCs were increased by 48.3 and 59.1% after incubation with 25 mM D-glucose for 24 and 48 h, respectively (Fig. 4, C and D).



View larger version (37K):
[in this window]
[in a new window]
  		Fig. 4. Effects of D-glucose on mRNA and protein expression of ENT-1 and ENT-2 in HASMCs. HASMCs were treated with 25 mM D-glucose for 0–48 h. A: semiquantitative RT-PCR analysis of ENT-1 and ENT-2 mRNA expression in HASMCs with reference to ribosomal protein S16. B: amount of ENT-1 (solid bars) and ENT-2 (open bars) mRNA products normalized to S16. C: Western blot analysis of ENT-1 protein expression in HASMCs with reference to {beta}-actin. D: amount of ENT-1 protein normalized to {beta}-actin. Values are means ± SE of 3 separate experiments. *P < 0.05 vs. control.

 
Lysophosphatidic acid is a known activator of MAP/ERK (7). Lysophosphatidic acid (25 µM) increased the amount of phosphorylated MAP/ERK in HASMCs (Fig. 5A), and this increase was abolished in the presence of the MAP/ERK inhibitors PD-98059 (40 µM) and U-0126 (10 µM). Interestingly, the upregulation of adenosine transport by glucose was abolished with the addition of PD-98059 (40 µM) and U-0126 (10 µM), suggesting the involvement of MAPK/ERK (Fig. 5B). Similarly, in the presence of PD-98059 and U-0126, D-glucose failed to increase ENT-1 mRNA (Fig. 6, A and B) and protein expression (Fig. 6, C and D) in HASMCs.



View larger version (25K):
[in this window]
[in a new window]
  		Fig. 5. Effect of PD-98059 and U-0126 on [3H]adenosine transport in HASMCs. A: lysophosphatidic acid (25 µM), a known activator of MAP/ERK (7), increased the amount of phospho-MAP/ERK proteins (42 and 44 kDa) as shown by Western blotting with an antiphospho-MAP/ERK antibody. Increase was abolished with MAP/ERK inhibitors PD-98059 and U-0126. B: HASMCs were treated with 25 mM D-glucose in the absence or presence of 40 µM PD-98059 and 10 µM U-0126 for 48 h. [3H]adenosine uptake (10 µM, 2 µCi/ml) was measured at room temperature for 1 min. Values are means ± SE of 3 experiments performed in triplicate. *P < 0.05 vs. control.

 


View larger version (39K):
[in this window]
[in a new window]
  		Fig. 6. Effects of PD-98059 and U-0126 on mRNA and protein expression of ENT-1 and ENT-2 in HASMCs. HASMCs were treated with 25 mM D-glucose in the absence or presence of 40 µM PD-98059 and 10 µM U-0126 for 48 h. A: semiquantitative RT-PCR analysis of ENT-1 mRNA expression in HASMCs, with S16 used as a reference protein. B: ENT-1 mRNA products normalized to S16. C: Western blot analysis of ENT-1 protein expression in HASMC with {beta}-actin used as a reference. D: amount of ENT-1 protein normalized to {beta}-actin. Values are means ± SE of 3 separate experiments. *P < 0.05 vs. control.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
An association between circulating D-glucose levels and the development of cardiovascular complications has been well established. This notion is supported by the observation that hyperglycemia per se stimulates the proliferation of cultured vascular SMCs (15). Generation of reactive oxygen species, activation of PKC and MAPK signaling pathways, and increased intracellular Ca2+ are suggested mechanisms (38, 47). In addition, D-glucose has been shown to affect the regulation of angiotensin II and arginine vasopressin receptors in vascular SMCs (45). These peptide hormones are important modulators of vascular tone and proliferation. In the present study, we characterized the nucleoside transport systems in cultured human HASMCs and showed that adenosine transport in HASMCs was predominantly mediated by ENT-1. The existence of ENT-1 in HASMCs was supported by our findings as follows: 1) [3H]adenosine uptake was Na+ independent (Fig. 1A), 2) [3H]adenosine uptake was abolished by nanomolar NBMPR (Fig. 1B), and 3) ENT-1 mRNA was found in HASMCs (Fig. 2). Although ENT-2 mRNA was revealed by RT-PCR, the adenosine uptake study did not detect a functional ENT-2 in HASMCs. This discrepancy might be due to a very low ENT-2 expression level that was far below the detection threshold of the functional study. We also demonstrated that 25 mM D-glucose exerted a stimulating effect on adenosine transport in HASMCs (Fig. 3A). This effect was due to an increase in ENT-1 abundance in HASMCs and was supported by the increased mRNA and protein levels of ENT-1 (Fig. 4), as well as the increased number of high-affinity [3H]NBMPR binding sites in HASMCs (Fig. 3B). Our study is in agreement with the recent study by Aguayo and Sobrevia (1), which showed that the Vmax of adenosine transport in the freshly isolated human umbilical cord arterial SMCs from diabetic pregnancies is threefold higher than that in normal arterial SMCs. We also examined the possible involvement of the MAP/ERK signaling pathway, by which D-glucose increased ENT-1 expression. The MAPK/ERK pathway is activated by D-glucose and is important in the upregulation of plasminogen activator inhibitor-1 and platelet-derived growth factor-{beta} receptor genes in vascular SMCs (3, 39). We demonstrated that inhibition of MAPK/ERK by the selective inhibitors PD-98059 and U-0126 abolished the effect of D-glucose on ENT-1 (Figs. 5 and 6). Our study provides the first evidence that transcription of ENT-1 may be regulated by MAPK/ERK. However, the mechanisms by which MAPK/ERK increases ENT-1 gene expression remain to be elucidated.

Diabetes-induced changes in ENT-1 expression are cell specific. In contrast to vascular SMCs, ENT-1 expression in heart, liver, and kidney of streptozotocin-induced diabetic rats are decreased (29). Approximately 80% of adenosine release from heart is derived from cardiomyocytes (8), and ENT-1 is a major contributor to this adenosine efflux (29). Pawelczyk et al. (29) suggested that downregulation of ENT-1 in the heart may decrease adenosine release and, thus, impair the regulation of vascular functions by adenosine and its ability to exert a cardioprotective effect during ischemia and reperfusion. However, it is not clear whether the source of adenosine is intracellular or extracellular under stress conditions. Deussen et al. (9) showed that, in guinea pig hearts under well-oxygenated conditions, addition of NBMPR increased interstitial adenosine concentrations and the source of adenosine was extracellular. An example of such a source is the hydrolysis of AMP by ecto-5'-nucleotidase (8). In contrast to the heart, endothelial cells and SMCs act as sinks in adenosine homeostasis by transporting extracellular adenosine into cells. Interestingly, diabetes induces opposite changes in ENT-1 activity and message in endothelial cells and SMCs. ENT-1 expression is reduced in endothelial cells (28) but is increased in SMCs freshly isolated from human diabetic umbilical vein (1). In the present study, we also demonstrated that ENT-1 in aortic SMCs was increased when the cells were grown under elevated glucose concentration. Taken together, the results suggest a coordinated regulation of ENT-1 in these cells.

The physiological consequence of increased ENT-1 expression of diabetes in vascular functions remains to be determined. It has been reported that patients with diabetes mellitus suffer greater morbidity from ischemia (2). Therefore, increased ENT-1 in vascular SMCs may result in decreased adenosine availability to the adenosine receptors in vascular SMCs and weaken the protective mechanism against ischemic injury. In addition, adenosine and selective adenosine A2 receptor agonists have been shown to inhibit the growth of aortic SMCs in normal and streptozotocin-induced diabetic rats (10, 19, 27, 40). Recent studies have provided evidence that dipyridamole, which is a putative ENT-1 inhibitor and increases extracellular adenosine concentration, can inhibit vascular SMC proliferation (20, 23). These studies suggest the relevance of adenosine in vascular functions and the role of nucleoside transporters in adenosine homeostasis. In addition to nucleoside transporters, other membrane-bound and cytosolic adenosine-metabolizing enzymes also contribute to adenosine homeostasis. Enzymes including adenosine kinase, adenosine deaminase, S-adenosylhomocysteine hydrolase, cytosolic 5'-nucleotidase, and the membrane ecto-5'-nucleotidase might also be affected by diabetes. For instance, it has been demonstrated that SMCs of mesenteric arteries of diabetic rats express more ecto-5'-nucleotidase than those of nondiabetic rats (21). Adenosine kinase expression is decreased in heart, kidney, and liver in rats with streptozotocin-induced diabetes mellitus, whereas cytosolic 5'-nucleotidase and adenosine deaminase activities are not changed (30). Nevertheless, these observations might not be extrapolated to the aortic SMCs, because the effects of diabetes on adenosine-metabolizing enzymes might be tissue specific, as in the case of diabetes on ENT-1 expression in various cell types and tissues.

In conclusion, we have demonstrated that adenosine transport in HASMCs is predominantly mediated by ENT-1. Elevated D-glucose increases adenosine transport by increasing ENT-1 abundance and functional activity, possibly via an MAPK/ERK-dependent signaling pathway. Upregulation of ENT-1 may influence the availability of adenosine in the vicinity of adenosine receptors and, subsequently, alter vascular functions. However, it remains to be determined whether our observations in cultured HASMCs will be similar to observations in animal models.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Seed Funding for Basic Research Program of the University of Hong Kong and National Cancer Institute Grants CA-85428 and CA-94012.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. P. H. Leung, Dept. of Pharmacology, The Univ. of Hong Kong, Hong Kong (E-mail: gphleung{at}hkucc.hku.hk)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Aguayo C and Sobrevia L. Nitric oxide, cGMP and cAMP modulate nitrobenzylthioinosine-sensitive adenosine transport in human umbilical artery smooth muscle cells from subjects with gestational diabetes. Exp Physiol 85: 399–409, 2000.[Abstract]
  2. Blendea MC, McFarlane SI, Isenovic ER, Gick G, and Sowers JR. Heart disease in diabetic patients. Curr Diab Rep 3: 223–229, 2003.[Medline]
  3. Campbell M, Allen WE, Silversides JA, and Trimble ER. D-Glucose-induced phosphatidylinositol 3-kinase and mitogen-activated protein kinase-dependent upregulation of the platelet-derived growth factor-{beta} receptor potentiates vascular smooth muscle cell chemotaxis. Diabetes 52: 519–526, 2003.[Abstract/Free Full Text]
  4. Cass CE, Young JD, and Baldwin SA. Recent advances in the molecular biology of nucleoside transporters of mammalian cells. Biochem Cell Biol 76: 761–770, 1998.[CrossRef][Web of Science][Medline]
  5. Collis MG. The vasodilator role of adenosine. Pharmacol Ther 41: 143–162, 1989.[CrossRef][Web of Science][Medline]
  6. Crawford CR, Patel DH, Naeve C, and Belt JA. Cloning of the human equilibrative, nitrobenzylmercaptopurine riboside (NBMPR)-insensitive nucleoside transporter ei by functional expression in a transport-deficient cell line. J Biol Chem 273: 5288–5293, 1998.[Abstract/Free Full Text]
  7. Cui MZ, Zhao G, Winokur AL, Laag E, Bydash JR, Penn MS, Chisolm GM, and Xu X. Lysophosphatidic acid induction of tissue factor expression in aortic smooth muscle cells. Arterioscler Thromb Vasc Biol 23: 224–230, 2003.[Abstract/Free Full Text]
  8. Deussen A. Metabolic flux rates of adenosine in the heart. Naunyn Schmiedebergs Arch Pharmacol 362: 351–363, 2000.[CrossRef][Web of Science][Medline]
  9. Deussen A, Stappert M, Schafer S, and Kelm M. Quantification of extracellular and intracellular adenosine production: understanding the transmembranous concentration gradients. Circulation 99: 2041–2047, 1999.[Abstract/Free Full Text]
  10. Dubey RK, Gillespie DG, Mi Z, and Jackson EK. Adenosine inhibits growth of human aortic smooth muscle cells via A2B receptors. Hypertension 31: 516–521, 1998.[Abstract/Free Full Text]
  11. Dubey RK, Gillespie DG, Mi Z, Suzuki F, and Jackson EK. Smooth muscle cell-derived adenosine inhibits cell growth. Hypertension 27: 766–773, 1996.[Abstract/Free Full Text]
  12. Dubey RK, Gillespie DG, Shue H, and Jackson EK. A2B receptors mediate antimitogenesis in vascular smooth muscle cells. Hypertension 35: 267–272, 2000.[Abstract/Free Full Text]
  13. Feoktistov I, Ryzhov S, Zhong H, Goldstein AE, Matafonov A, Zeng D, and Biaggioni I. Hypoxia modulates adenosine receptors in human endothelial and smooth muscle cells toward an A2B angiogenic phenotype. Hypertension 44: 649–654, 2004.[Abstract/Free Full Text]
  14. Froldi G and Belardinelli L. Species-dependent effects of adenosine on heart rate and atrioventricular nodal conduction. Mechanism and physiological implication. Circ Res 67: 960–978, 1990.[Abstract/Free Full Text]
  15. Graier WF, Grubenthal I, Dittrich P, Wascher TC, and Kostner GM. Intracellular mechanism of high D-glucose-induced modulation of vascular cell proliferation. Eur J Pharmacol 290: 221–229, 1995.[CrossRef][Web of Science][Medline]
  16. Griffith DA and Jarvis SM. Nucleoside and nucleobase transport systems of mammalian cells. Biochim Biophys Acta 1286: 153–181, 1996.[Medline]
  17. Headrick JP, Hack B, and Ashton KJ. Acute adenosinergic cardioprotection in ischemic-reperfused hearts. Am J Physiol Heart Circ Physiol 285: H1797–H1818, 2003.[Abstract/Free Full Text]
  18. Jobbagy Z, Ward JL, Toan SV, Leung GP, and Tse CM. One-step unidirectional cloning of tandem repeats of DNA fragment: an application for fusion protein production. Anal Biochem 303: 104–107, 2001.
  19. Jonzon B, Nilsson J, and Fredholm BB. Adenosine receptor-mediated changes in cyclic AMP production and DNA synthesis in cultured arterial smooth muscle cells. J Cell Physiol 124: 451–456, 1985.[CrossRef][Web of Science][Medline]
  20. Kim SJ, Masaki T, Leypoldt JK, Kamerath CD, Mohammad SF, and Cheung AK. Arterial and venous smooth-muscle cells differ in their responses to antiproliferative drugs. J Lab Clin Med 144: 156–162, 2000.
  21. Kwan CY. Membrane abnormalities of vascular smooth muscle of mesenteric arteries of spontaneous diabetic BB rats. J Smooth Muscle Res 35: 77–86, 1999.[Medline]
  22. Little PJ, Neylon CB, Farrelly CA, Weissberg PL, Cragoe EJ, and Bobik A. Intracellular pH in vascular smooth muscle: regulation by sodium-hydrogen exchange and multiple sodium-dependent HCO3– mechanisms. Cardiovasc Res 29: 239–246, 1995.[CrossRef][Web of Science][Medline]
  23. Masaki T, Kamerath CD, Kim SJ, Leypoldt JK, Mohammad SF, and Cheung AK. In vitro pharmacological inhibition of human vascular smooth muscle cell proliferation for the prevention of hemodialysis vascular access stenosis. Blood Purif 22: 307–312, 2004.[CrossRef][Web of Science][Medline]
  24. Mubagwa K and Flameng W. Adenosine, adenosine receptors and myocardial protection: an updated overview. Cardiovasc Res 52: 25–39, 2001.[Abstract/Free Full Text]
  25. Newby AC. Adenosine and the concept of "retaliatory metabolites." Trends Biochem Sci 9: 42–44, 1984.[CrossRef][Web of Science]
  26. Osses N, Pearson JD, Yudilevich DL, and Jarvis SM. Hypoxanthine enters human vascular endothelial cells (ECV 304) via the nitrobenzylthioinosine-insensitive equilibrative nucleoside transporter. Biochem J 317: 843–848, 1996.[Web of Science][Medline]
  27. Pares-Herbute N, Hillaire-Buys D, Etienne P, Gross R, Loubatieres-Mariani MM, and Monnier L. Adenosine inhibitory effect on enhanced growth of aortic smooth muscle cells from streptozotocin-induced diabetic rats. Br J Pharmacol 118: 783–789, 1996.[Web of Science][Medline]
  28. Parodi J, Flores C, Aguayo C, Rudolph MI, Casanello P, and Sobrevia L. Inhibition of nitrobenzylthioinosine-sensitive adenosine transport by elevated D-glucose involves activation of P2Y2 purinoceptors in human umbilical vein endothelial cells. Circ Res 90: 570–577, 2002.[Abstract/Free Full Text]
  29. Pawelczyk T, Podgorska M, and Sakowicz M. The effect of insulin on expression level of nucleoside transporters in diabetic rats. Mol Pharmacol 63: 81–88, 2003.[Abstract/Free Full Text]
  30. Pawelczyk T, Sakowicz M, Szczepanska-Konkel M, and Angielski S. Decreased expression of adenosine kinase in streptozotocin-induced diabetes mellitus rats. Arch Biochem Biophys 375: 1–6, 2000.[CrossRef][Web of Science][Medline]
  31. Pearce WJ. Mechanisms of hypoxic cerebral vasodilation. Pharmacol Ther 65: 75–91, 1995.[CrossRef][Web of Science][Medline]
  32. Peiro C, Lafuente N, Matesanz N, Cercas E, Llergo JL, Vallejo S, Rodriguez-Manas L, and Sanchez-Ferrer CF. High glucose induces cell death of cultured human aortic smooth muscle cells through the formation of hydrogen peroxide. Br J Pharmacol 133: 967–974, 2001.[CrossRef][Web of Science][Medline]
  33. Picano E and Abbracchio MP. European Stroke Prevention Study-2 results: serendipitous demonstration of neuroprotection induced by endogenous adenosine accumulation? Trends Pharmacol Sci 19: 14–16, 1998.[CrossRef][Medline]
  34. Plagemann PG, Wohlhueter RM, and Woffendin C. Nucleoside and nucleobase transport in animal cells. Biochim Biophys Acta 947: 405–443, 1988.[Medline]
  35. Ritzel MW, Ng AM, Yao SY, Graham K, Loewen SK, Smith KM, Ritzel RG, Mowles DA, Carpenter P, Chen X, Karpinski E, Hyde RJ, Baldwin SA, Cass CE, and Young JD. Molecular identification and characterization of novel human and mouse concentrative Na+-nucleoside cotransporter proteins (hCNT3 and mCNT3) broadly selective for purine and pyrimidine nucleosides (system cib). J Biol Chem 276: 2914–2927, 2001.[Abstract/Free Full Text]
  36. Ritzel MW, Yao SY, Huang MY, Elliott JF, Cass CE, and Young JD. Molecular cloning and functional expression of cDNAs encoding a human Na+-nucleoside co-transporter (hCNT1). Am J Physiol Cell Physiol 272: C707–C714, 1997.[Abstract/Free Full Text]
  37. Rongen GA, Lenders JW, Lambrou J, Willemsen JJ, Van Bella H, Thien T, and Smits P. Presynaptic inhibition of norepinephrine release from sympathetic nerve endings by endogenous adenosine. Hypertension 27: 933–938, 1996.[Abstract/Free Full Text]
  38. Srivastava AK. High D-glucose-induced activation of protein kinase signaling pathways in vascular smooth muscle cells: potential role in the pathogenesis of vascular dysfunction in diabetes. Int J Mol Med 9: 85–89, 2002.[Web of Science][Medline]
  39. Suzuki M, Akimoto K, and Hattori Y. D-Glucose upregulates plasminogen activator inhibitor-1 gene expression in vascular smooth muscle cells. Life Sci 72: 59–66, 2002.[CrossRef][Web of Science][Medline]
  40. Takiguchi Y, Nagano M, Ikeda Y, and Nakashima M. Early administration of YT-146, an adenosine A2 receptor agonist, inhibits neointimal thickening after rat femoral artery endothelium injury. Eur J Pharmacol 281: 205–207, 1995.[CrossRef][Web of Science][Medline]
  41. Toan SV, To KK, Leung GP, de Souza MO, Ward JL, and Tse CM. Genomic organization and functional characterization of the human concentrative nucleoside transporter-3 isoform (hCNT3) expressed in mammalian cells. Pflügers Arch 447: 195–204, 2003.[CrossRef][Web of Science][Medline]
  42. Wang J, Su SF, Dresser MJ, Schaner ME, Washington CB, and Giacomini KM. Na+-dependent purine nucleoside transporter from human kidney: cloning and functional characterization. Am J Physiol Renal Physiol 273: F1058–F1065, 1997.[Abstract/Free Full Text]
  43. Ward JL, Sherali A, Mo ZP, and Tse CM. Kinetic and pharmacological properties of cloned human equilibrative nucleoside transporters, ENT1 and ENT2, stably expressed in nucleoside transporter-deficient PK15 cells. ENT2 exhibits a low affinity for guanosine and cytidine but a high affinity for inosine. J Biol Chem 275: 8375–8381, 2000.[Abstract/Free Full Text]
  44. Ward JL and Tse CM. Identification of the nucleoside transporters in human small intestine and colonic epithelial cell lines (Abstract). Gastroenterology 114: A431, 1998.[Web of Science]
  45. Williams B, Tsai P, and Schrier RW. D-Glucose-induced down-regulation of angiotensin II and arginine vasopressin receptors in cultured rat aortic vascular smooth muscle cells. Role of protein kinase C. J Clin Invest 90: 1992–1999, 1992.[Web of Science][Medline]
  46. Yao SY, Ng AM, Vickers MF, Sunderam M, Cass CE, Baldwin SA, and Young JD. Functional and molecular characterization of nucleobase transport by recombinant human and rat equilibrative nucleoside transporters 1 and 2. Chimeric construct reveals a role for the ENT2 helix 5–6 region in nucleobase translocation. J Biol Chem 277: 24938–24948, 2002.[Abstract/Free Full Text]
  47. Yoo HJ, Kozaki K, Akishita M, Watanabe M, Eyo M, Nagano K, Sudo N, Hashimoto M, Kim S, Yoshizumi M, Toba K, and Ouchi Y. Augmented Ca2+ influx is involved in the mechanism of enhanced proliferation of cultured vascular smooth muscle cells from spontaneously diabetic Goto-Kakizaki rats. Atherosclerosis 131: 167–175, 1997.[CrossRef][Web of Science][Medline]



This article has been cited by other articles:


Home page
 Toxicol SciHome page
S. W. Smith
Chiral Toxicology: It's the Same Thing...Only Different
Toxicol. Sci., July 1, 2009; 110(1): 4 - 30.
[Abstract] [Full Text] [PDF]

Home page
 Exp. Biol. Med.Home page
S. B. Bender, J. D. Tune, L. Borbouse, X. Long, M. Sturek, and M. H. Laughlin
Altered Mechanism of Adenosine-Induced Coronary Arteriolar Dilation in Early-Stage Metabolic Syndrome
Experimental Biology and Medicine, June 1, 2009; 234(6): 683 - 692.
[Abstract] [Full Text] [PDF]

Home page
 PhysiologyHome page
J. B. Rose and I. R. Coe
Physiology of Nucleoside Transporters: Back to the Future. . . .
Physiology, February 1, 2008; 23(1): 41 - 48.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
O. Frobert, G. Haink, U. Simonsen, C. H. Gravholt, M. Levin, and A. Deussen
Adenosine concentration in the porcine coronary artery wall and A2A receptor involvement in hypoxia-induced vasodilatation
J. Physiol., January 15, 2006; 570(2): 375 - 384.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
288/6/H2756    most recent
00921.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (8)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Leung, G. P. H.
Right arrow Articles by Tse, C.-M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Leung, G. P. H.
Right arrow Articles by Tse, C.-M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2005 by the American Physiological Society.