AJP - Heart Watch the video to see how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 293: H3325-H3332, 2007. First published October 5, 2007; doi:10.1152/ajpheart.01006.2007
0363-6135/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/6/H3325    most recent
01006.2007v1
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 Google Scholar
Google Scholar
Right arrow Articles by Bone, D. B. J.
Right arrow Articles by Hammond, J. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bone, D. B. J.
Right arrow Articles by Hammond, J. R.

Nucleoside and nucleobase transporters of primary human cardiac microvascular endothelial cells: characterization of a novel nucleobase transporter

Derek B. J. Bone and James R. Hammond

Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Canada

Submitted 30 August 2007 ; accepted in final form 3 October 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Levels of cardiovascular active metabolites, like adenosine, are regulated by nucleoside transporters of endothelial cells. We characterized the nucleoside and nucleobase transport capabilities of primary human cardiac microvascular endothelial cells (hMVECs). hMVECs accumulated 2-[3H]chloroadenosine via the nitrobenzylmercaptopurine riboside-sensitive equilibrative nucleoside transporter 1 (ENT1) at a Vmax of 3.4 ± 1 pmol·µl–1·s–1, with no contribution from the nitrobenzylmercaptopurine riboside-insensitive ENT2. Inhibition of 2-chloroadenosine uptake by ENT1 blockers produced monophasic inhibition curves, which are also compatible with minimal ENT2 expression. The nucleobase [3H]hypoxanthine was accumulated within hMVECs (Km = 96 ± 37 µM; Vmax = 1.6 ± 0.3 pmol·µl–1·s–1) despite the lack of a known nucleobase transport system. This novel transporter was dipyridamole-insensitive but could be inhibited by adenine (Ki = 19 ± 7 µM) and other purine nucleobases, including chemotherapeutic analogs. A variety of other cell types also expressed the nucleobase transporter, including the nucleoside transporter-deficient PK(15) cell line (PK15NTD). Further characterization of [3H]hypoxanthine uptake in the PK15NTD cells showed no dependence on Na+ or H+. PK15NTD cells expressing human ENT2 accumulated 4.5-fold more [3H]hypoxanthine in the presence of the ENT2 inhibitor dipyridamole than did PK15NTD cells or hMVECs, suggesting trapping of ENT2-permeable metabolites. Understanding the nucleoside and nucleobase transporter profiles in the vasculature will allow for further study into their roles in pathophysiological conditions such as hypoxia or ischemia.

adenosine; hypoxanthine; human; cardiovascular

THE TRANSMEMBRANE FLUX OF hydrophilic nucleosides and nucleobases is mediated by integral membrane proteins, including equilibrative nucleoside transporters (SLC29; ENTs) and concentrative nucleoside transporters (SLC28; CNTs) (3, 14). These nucleoside transporters (NTs) have roles in salvaging endogenous nucleosides, such as adenosine, and have been exploited for cancer chemotherapy and treatments for viral infection (17). The CNTs are Na+-dependent transporters that facilitate movement of nucleosides against their concentration gradient. Three members of the CNT family have been cloned, each with distinct substrate specificity. CNT1 transports adenosine and pyrimidine nucleosides, CNT2 transports purine nucleosides and uridine, and CNT3 is broadly selective for both purine and pyrimidine nucleosides (22). The ENTs, on the other hand, are Na+-independent facilitative diffusion transporters that rely on the concentration gradient of the substrate and thus are capable of mediating both influx and efflux of nucleosides. The two classical ENTs have been identified as being sensitive (ENT1) or insensitive (ENT2) to the nucleoside analog nitrobenzylmercaptopurine riboside (NBMPR) (28). ENT2 is also able to transport nucleobases like hypoxanthine (21). Recently, two more ENT family members have been identified: ENT3 is associated with intracellular acidic environments (15), and ENT4 was initially discovered as a monoamine transporter in the brain (12).

The purine nucleoside adenosine is an important compound in the vasculature. Acting through a variety of G-protein-coupled receptors, adenosine mediates physiological actions such as, among others, vasodilation, bradycardia, and inhibition of platelet aggregation (27). NTs play a role in regulating adenosine levels in the vasculature by facilitating its removal from the extracellular environment (19). In fact, cardiovascular drugs like dipyridamole inhibit NTs, thereby extending the lifespan of adenosine in the vasculature (25). Analogs of NBMPR (29) and dipyridamole (18) are now being designed and tested for the potential to act as cardioprotective agents, thus highlighting the importance of vascular NTs as potential therapeutic targets.

Investigations of NTs in the vasculature widely utilize models derived from the macrovasculature, with a particular focus on umbilical vein endothelial cells. However, the majority of exchange between the blood and peripheral tissues occurs in the microvasculature, in which microvascular endothelial cells (MVECs) are responsible for the regulation of this exchange. It has been shown that endothelial cells in the heart are responsible for the uptake of up to 65% of free adenosine (9). Therefore, changes in the ability of MVECs to transport or metabolize adenosine can have profound effects on the cardiovasculature and surrounding tissues.

Here, we describe the nucleoside and nucleobase transport capabilities of primary human cardiac MVECs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. 2-[8-3H]chloroadenosine (4-7 Ci/mmol), [2,8-3H]hypoxanthine (24-35 Ci/mmol), [2,8-3H]adenine (50 Ci/mmol), [3H] NBMPR (5.5 Ci/mmol), and [3H]water (1 mCi/g) were purchased from Moravek Biochemicals (Brea, CA). Nonradiolabeled 2-chloroadenosine, hypoxanthine, NBMPR, nitrobenzylthioguanine riboside, dipyridamole {2,6-bis(diethanolamino)-4,8-dipiperidinopyrimido-[5,4-d] pyrimidine}, and all other nucleosides and nucleobases were obtained from Sigma-Aldrich (St. Louis, MO). Dilazep {N,N'-bis[3-(3,4,5-trimethoxybenzoyloxy)propyl] homopiperazine} was from Asta Werke (Frankfurt, Germany), and draflazine {2-(aminocarbonyl)-4-amino-2,6-dichlorophenyl-4-[5,5-bis(4-fluorophenyl)-pentyl]-1-piperazineacetamide 2HCl} was provided by Janssen Research Foundation (Beerse, Belgium).

Cell culture. Primary human cardiac MVECs were purchased from Cambrex Bioscience or Lonza (Walkersville, MD) and were cultured as per manufacturer's instructions. Cells were supplied at passage 3 and used between passage 4 and 7. All assays were conducted with cells from the same lot (subject was a 4-year-old female). No morphological changes occurred during culture up to passage 7. Quality assurance from the manufacturer verified the isolation as pure endothelial cells using immunohistologic staining for the presence of acetylated LDL and von Willebrand's (factor VIII) antigen and the absence of smooth muscle {alpha}-actin. Some studies required the use of additional cell types, and all media and culture reagents were from Invitrogen (Burlington, ON). Primary rat MVECs were isolated as previously described (2) and cultured in M199 medium containing 10% FBS, 100 U/ml penicillin G, 100 µg/ml streptomycin sulfate, 0.25 µg/ml amphotericin B, 2 mM L-glutamine, 0.025 U/ml heparin, and 75 µg/ml endothelial cell growth supplement. Nucleoside transporter-deficient PK(15) cell line (PK15NTD) and PK15NTD-human ENT2 cells were a generous gift from Dr. Ming Tse (Johns Hopkins University) and were cultured in MEM containing 10% bovine growth serum with penicillin G (100 U/ml), streptomycin sulfate (100 mg/ml), nonessential amino acids (0.1 mM), and sodium pyruvate (1 mM). G418 (300 µg/ml) was added to the culture medium of the PK15NTD-human ENT2 cells to maintain a selection pressure on the stable transfection. UMR-108 and HEK-293 cells were a gift from Dr. Peter Chidiac (Department of Physiology and Pharmacology, University of Western Ontario). UMR-108 cells were cultured in {alpha}-MEM with 10% FBS, penicillin G (100 U/ml), and streptomycin sulfate (100 mg/ml). HEK-293 cells were cultured in MEM with 10% FBS and 10 µg/ml gentamicin. Madin-Darby canine kidney cells were cultured in MEM with 10% FBS and penicillin G (100 U/ml) and streptomycin sulfate (100 µg/ml). All cells were maintained in a humidified atmosphere at 37°C with 5% CO2 in T175 culture flasks. For experimental assays, cells were trypsinized (0.05% trypsin-0.53 mM EDTA) and then diluted fourfold in 500 µg/ml trypsin inhibitor in PBS (in mM: 137 NaCl, 6.3 Na2HPO4, 2.7 KCl, 1.5 KH2PO4, 0.9 CaCl2·2H2O, 0.5 MgCl2·6H2O; pH 7.4). Cells were collected by centrifugation, and pellets were washed in either PBS or a Na+-free buffer [NMG (in mM): 140 N-methyl-D-glucamine, 5 KCl, 4.2 KH2PO4, 0.36 K2HPO4, 10 HEPES, 1.3 CaCl2·2H2O, and 0.5 MgCl2·6H2O; pH 7.4] and resuspended in the same buffer as required for immediate use. In some cases, cells were depleted of ATP by sequential incubation at 37°C with rotenone and 2-deoxyglucose as described previously (2).

[3H]NBMPR binding. Cells (~75,000 cells/assay) were suspended in PBS and incubated with [3H]NBMPR for 45–60 min at room temperature (~22°C). Cells were collected on S&S Biopath (Riviera Beach, FL) glass fiber filters using a 24-port Brandel cell harvester and washed twice with Tris buffer (10 mM Tris, pH 7.4, 4°C) and analyzed for 3H content using standard liquid scintillation counting techniques. Specific binding was defined as total binding minus cell-associated [3H]NBMPR in the presence of 10 µM nitrobenzylthioguanine riboside (nonspecific binding).

[3H]nucleoside/nucleobase uptake. Uptake was initiated by the addition of suspended cells (~750,000 cells/assay) to 2-[3H]chloroadenosine (nucleoside) or [3H]hypoxanthine (nucleobase) layered over 200 µl of silicon/mineral oil (21:4 vol/vol) in 1.5-ml microcentrifuge tubes. After a defined incubation time, uptake was terminated by centrifugation for 10 s (~12,000 g). Aqueous substrate and oil layers were removed by aspiration, and pelletted cells were digested in 1 M sodium hydroxide overnight (12–16 h). A sample of the digest was removed and analyzed for 3H content using standard liquid scintillation counting techniques. Uptake data are presented as picomoles per microliter of intracellular volume after correction for the amount of extracellular 3H in the cell pellet. Total volume was determined by incubating cells with 3H2O for 3 min and processed as above. Extracellular water space was estimated by extrapolation of the linear time course of nonmediated uptake to zero time.

Data analysis and statistics. Data are presented as means ± SE. Curves were fitted with the use of Graphpad Prism 4.03 software. Where appropriate, statistical analysis was performed using a two-way Student's t-test with P < 0.05 considered significant. Ki values were determined with the Cheng-Prusoff relationship (8).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
[3H]NBMPR binding. Specific [3H]NBMPR binding to human cardiac MVECs revealed 53,000 ± 6,000 ENT1 sites/cell with a Kd of 0.031 ± 0.003 nM (Fig. 1). A second population of low-affinity (Kd > 5 nM) binding sites was also present.


Figure 1
View larger version (21K):
[in this window]
[in a new window]

  		Fig. 1. 3H-labeled nitrobenzylmercaptopurine riboside (NBMPR) binding to human cardiac microvascular endothelial cells (MVECs). Human cardiac MVECs were incubated with increasing concentrations of [3H]NBMPR in the presence (nonspecific) or absence (total) of 10 µM nitrobenzylthioguanine riboside (NBTGR) for 45 min. Cells were collected on glass fiber filters, and cell-associated 3H content was determined. Specific binding was determined as total bound minus nonspecific bound (n = 5).

 
2-[3H]chloroadenosine uptake. A time course profile showed that human cardiac MVECs accumulated 10 µM 2-[3H]chloroadenosine with an initial rate of 1.6 ± 0.5 pmol·µl–1·s–1. Incubation of cells with 50 nM NBMPR to block ENT1-mediated uptake resulted in a rate of uptake that was not significantly different from that seen in the presence of 10 µM dipyridamole and NBMPR (Fig. 2A). Furthermore, when uptake buffer was changed from NMG (–Na+) to PBS (+Na+), there was no change in initial rate of uptake or maximal intracellular accumulation (data not shown). Uptake of increasing concentrations of 2-[3H]chloroadenosine demonstrated a Km of 42 ± 19 µM and a Vmax of 3.4 ± 1.0 pmol·µl–1·s–1 (Fig. 2B).


Figure 2
View larger version (21K):
[in this window]
[in a new window]

  		Fig. 2. 2-[3H]chloroadenosine uptake characteristics of human cardiac MVECs. A: time course of 2-[3H]chloroadenosine uptake. Human cardiac MVECs were incubated with 10 µM 2-[3H]chloroadenosine for the times indicated. Some cell lots were preincubated with 100 nM NBMPR to inhibit only equilibrative nucleoside transporter 1 (ENT1). Total uptake (no inhibitors) minus ENT1 inhibited uptake represents ENT2-mediated uptake. Nonmediated uptake was determined with cells preincubated with 10 µM NBMPR and 10 µM dipyridamole (n = 5). B: concentration-dependent uptake of 2-[3H]chloroadenosine. Cells were incubated with increasing concentrations of 2-[3H]chloroadenosine for 7 s. Transporter-mediated uptake is presented as pmol·µl–1·s–1 and was defined as total uptake minus nonmediated uptake (n = 4).

 
Inhibition of 10 µM 2-[3H]chloroadenosine was examined with the classical ENT inhibitors NBMPR, dipyridamole, draflazine, and dilazep. All four compounds (Fig. 3) displayed high-affinity inhibition with no indication of an inhibitor-insensitive (ENT2) component to the uptake. The lack of ENT2 was especially evident from the monophasic NBMPR inhibition curve. Ki values derived from these data are listed in Table 1.


Figure 3
View larger version (23K):
[in this window]
[in a new window]

  		Fig. 3. Inhibition of 2-[3H]chloroadenosine uptake. Cells were incubated with increasing concentrations of inhibitor for 15 min before uptake of 10 µM 2-[3H]chloroadenosine for 15 s. Uptake in the absence of any inhibitor was defined as 100%, and totally inhibited cells (10 µM NBMPR + 10 µM dipyridamole) was defined as 0% (n ≥ 4). Ki values are reported in Table 1. DY, dipyridamole.

 

View this table:
[in this window]
[in a new window]

  		Table 1. Ki values for inhibition of uptake by hMVECs 2-chloroadenosine and hypoxanthine

 
[3H]hypoxanthine uptake. Human cardiac MVECs were able to accumulate [3H]hypoxanthine under Na+-free conditions despite an apparent lack of ENT2 activity. Uptake was insensitive to dipyridamole; however, the addition of 1 mM adenine was able to block uptake (Fig. 4A). As with 2-[3H]chloroadenosine, Na+ had no effect on the uptake of [3H]hypoxanthine (data not shown). ATP depletion of the cells before exposure to 5 µM [3H]hypoxanthine reduced the extrapolated maximum intracellular accumulation from 39 ± 18 to 4.1 ± 0.2 pmol/µl (Fig. 4B). Consequently, subsequent experiments were performed under ATP-depleted conditions to minimize the effects of intracellular metabolism. With the use of a 15-s time point to estimate initial rate, the Vmax of [3H]hypoxanthine influx was determined as 1.6 ± 0.3 pmol·µl–1·s–1 with a Km of 96 ± 37 µM (Fig. 4C).


Figure 4
View larger version (21K):
[in this window]
[in a new window]

  		Fig. 4. [3H]hypoxanthine uptake characteristics of human cardiac MVECs (hMVECs). A: [3H]hypoxanthine time course. Cells were incubated with 5 µM [3H]hypoxanthine for the times indicated. Some cells were preincubated with 10 µM DY to inhibit any ENT2 that may be present in the cells. To completely block uptake, cells were coincubated with 1 mM adenine (n = 5). B: ATP-depleted [3H]hypoxanthine time course. Uptake was performed identical to A, except that cells were depleted of ATP before assay (n = 6). C: concentration-dependent [3H]hypoxanthine uptake. ATP-depleted cells were incubated with increasing concentrations of [3H]hypoxanthine for 15 s. Transporter-mediated uptake is presented and was defined as uptake in the absence of any inhibitor minus uptake in the presence of 1 mM adenine (n = 4).

 
Additional nucleobases were tested for the ability to inhibit 5 µM [3H]hypoxanthine uptake. Adenine was the most effective, with a Ki of 19 ± 7 µM. Guanine produced significant inhibition of 17 ± 4% at a concentration of 1 µM (Fig. 5A). Higher concentrations of guanine could not be achieved because of low solubility in assay buffers at physiological pH. Pyrimidines were much less effective, with thymine producing inhibition only at 1 mM (59 ± 6%) (Fig. 5A), and cytosine and uracil had no effect at 1 mM concentrations (data not shown).


Figure 5
View larger version (21K):
[in this window]
[in a new window]

  		Fig. 5. Inhibition of [3H]hypoxanthine uptake. A: inhibition of [3H]hypoxanthine uptake by nucleobases and nucleosides. Cells were coincubated with 5 µM [3H]hypoxanthine and indicated purine for 60 s. Uptake in the absence of inhibitor was defined as 100%, and totally inhibited uptake (1 mM adenine) was defined as 0% (n ≥ 3). B: inhibition of [3H]hypoxanthine uptake by purine nucleobase analogs. 6-MP, 6-mercaptopurine; 6-TG, 6-thioguanine. Assays were conducted in the same manner as A (n ≥ 3). Ki values are reported in Table 1.

 
The chemotherapeutic thiopurine analogs 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) and the pyrimidine analog 5-fluorouracil (5-FU) were also examined. Both 6-MP and 6-TG were able to inhibit 5 µM [3H]hypoxanthine uptake with an IC50 of 37 ± 11 µM and 20 ± 2 µM, respectively (Fig. 5B). As with other pyrimidines, 5-FU had no effect on [3H]hypoxanthine uptake (data not shown).

To confirm that the dipyridamole-insensitive hypoxanthine uptake mechanism was nucleobase selective, the nucleosides adenosine and uridine were tested. Adenosine had an extrapolated IC50 value of 1.3 ± 0.9 mM (Fig. 5A), and uridine had no effect (data not shown).

Nucleobase transport in other cell types. The identification of Na+-independent [3H]hypoxanthine transport in human cardiac MVECs, by a mechanism other than ENT2, led to the examination of other cell types for evidence of a similar dipyridamole-insensitive nucleobase transport system. Established cell lines from human (U2OS and HEK-293), rat (UMR-108), pig (PK15NTD), and dog (Madin-Darby canine kidney) were examined along with primary rat skeletal muscle MVECs. Figure 6 shows the time course profiles for dipyridamole-insensitive hypoxanthine uptake by these cells. All cells tested displayed evidence of dipyridamole-insensitive nucleobase transport with the exception of HEK-293 cells.


Figure 6
View larger version (33K):
[in this window]
[in a new window]

  		Fig. 6. Dipyridamole-insensitive [3H]hypoxanthine uptake in various cell types. Cells were incubated with 5 µM [3H]hypoxanthine for the times indicated. Cell lots were preincubated with 10 µM DY to inhibit any ENT2. To completely block uptake, cells were coincubated with 1 mM adenine (n ≥ 2). UMR, UMR-108 cells; NTD, nucleoside transporter-deficient PK(15) cells (PK15NTD); MDCK, Madin-Darby canine kidney cells; rMVEC, rat skeletal muscle MVECs; HEK, HEK-293 cells.

 
PK15NTD cells were chosen for further characterization of this nucleobase transporter. These cells do not have ENT1 or ENT2 (26), thereby eliminating any contribution from other nucleoside/nucleobase transporters to the results obtained. [3H]hypoxanthine uptake by PK15NTD cells was concentration dependent with a similar Km (122 ± 31 pmol/µl) to the human cardiac MVEC in ATP-depleted conditions and a Vmax of 7.0 ± 0.6 pmol·µl–1·s–1 (Fig. 7A). There was no effect of pH on [3H]hypoxanthine uptake, and the thiopurines 6-MP and 6-TG displayed Ki values (21 ± 5 and 19 ± 1 µM, respectively) similar to those for human cardiac MVECs (data not shown).


Figure 7
View larger version (22K):
[in this window]
[in a new window]

  		Fig. 7. PK15NTD cells as a model for nucleobase transport. A: concentration-dependent uptake of [3H]hypoxanthine under ATP-depleted conditions. Cells were incubated with increasing concentrations of [3H]hypoxanthine for 15 s. Transporter-mediated uptake is presented and was defined as uptake in the absence of any inhibitor minus uptake in the presence of 1 mM unlabeled adenine (n = 3). B: [3H]adenine uptake time course. ATP-depleted cells were incubated with 500 nM [3H]adenine in the presence or absence of 150 µM hypoxanthine or 1 mM adenine (n = 2).

 
[3H]adenine was tested as a transport substrate with unlabeled hypoxanthine as the inhibitor (Fig. 7B). Hypoxanthine at 150 µM was able to produce ~20% inhibition of 500 nM [3H]adenine uptake under ATP-depleted conditions. The use of unlabeled 1 mM adenine was required for total inhibition.

[3H]hypoxanthine uptake in the presence or absence of dipyridamole was also examined in PK15NTD cells that had been previously transfected with human ENT2. Accumulation of 5 µM [3H]hypoxanthine in ATP-depleted conditions reached a maximum intracellular concentration of 3.8 ± 0.03 pmol/µl; however, in the presence of dipyridamole, the amount of [3H]hypoxanthine that accumulated in the cell rose to 18 ± 4 pmol/µl. Blocking ENT1 with NBMPR in the human cardiac MVECs (no ENT2) did not have an effect on [3H]hypoxanthine accumulation (Fig. 8).


Figure 8
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 8. Complex effects of nucleoside and nucleobase transporter coexpression and inhibition. A: PK15NTD [3H]hypoxanthine uptake ± dipyridamole. ATP-depleted PK15NTD cells were incubated with 5 µM [3H]hypoxanthine for the times indicated in the presence or absence of 5 µM DY (n = 3). B: PK15NTD-human ENT2 (hENT2) [3H]hypoxanthine uptake ± DY. ATP-depleted PK15NTD-human ENT2 cells were incubated with 5 µM [3H]hypoxanthine for the times indicated in the presence or absence of 5 µM DY (n = 2). C: hMVEC [3H]hypoxanthine uptake ± NBMPR. Cells were incubated in 100 µM [3H]hypoxanthine in the presence or absence of 50 nM NBMPR (n = 4).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The microvasculature forms a barrier between the circulation and peripheral tissues and is responsible for mediating nutrient exchange between these two compartments. MVEC regulation of nucleosides via NTs can have profound effects on the cardiovascular actions of adenosine, warranting further investigation of NTs as potential therapeutic targets (19).

Human cardiac MVECs have both high- and low-affinity binding sites for the ENT1-selective probe NBMPR. The Kd of the high-affinity site is consistent with reported values for ENT1 (15). Our group, in rat skeletal muscle MVECs (2), and others (6) have reported the presence of the low-affinity population of NBMPR binding sites, which may be a result of [3H]NBMPR binding to intracellular ENT1 proteins. The high-affinity maximum binding capacity of ~53,000 ENT1 sites/cell likely represents the transporters associated with the plasma membrane.

2-Chloroadenosine is an established substrate of NTs and was chosen to limit the effects of intracellular metabolism on the measurement of transport kinetics (5, 16). Na+-independent uptake of 2-chloroadenosine in the presence or absence of a low concentration of NBMPR (50 nM) to inhibit ENT1 revealed no significant contribution of the NBMPR-insensitive ENT2 to the overall uptake. In addition, NBMPR, dilazep, draflazine, and dipyridamole all produced monophasic inhibition curves for inhibiting 2-chloroadenosine uptake (pseudo Hill coefficients were approximately –1) with Ki values typical of ENT1 inhibition (28). Numerous endothelial cell models express a combination of ENT1 and ENT2, including primary rat MVECs (2), human umbilical vein endothelial cells (1), primary mouse MVECs (unpublished data), and EVC 304 cells (21). In this regard, human cardiac MVECs, which express only ENT1, are an exception.

Although human cardiac MVECs had no functional ENT2, they were still able to accumulate hypoxanthine under Na+-free conditions. Uptake was insensitive to dipyridamole; however, other purine nucleobases were capable of blocking influx. There was no significant change in uptake in the presence of Na+, thus ruling out the ascorbic acid transport system. An increase in H+ had no effect, and uptake was not inhibited by monoamines (data not shown); thus this hypoxanthine transporter is not the recently identified ENT4 (11). The apparent Km of hypoxanthine uptake (96 ± 37 µM) was threefold lower, and the Ki of adenine inhibition of hypoxanthine uptake (19 ± 7 µM) reported in this study was ~140 times lower than previously reported for human ENT2 (21). Together, these data support that hypoxanthine uptake in human cardiac MVECs was mediated by a transport system distinct from ENT2. Further characterization identified the transport system as purine nucleobase selective. The only pyrimidine that could inhibit hypoxanthine uptake was thymidine at nonphysiological concentrations (>1 mM). The nucleoside adenosine also inhibited uptake at high concentrations (IC50 > 1 mM), but this may be the result of adenosine metabolism releasing adenine. The chemotherapeutic purine nucleobase analogs 6-MP and 6-TG demonstrated an affinity for the nucleobase transport system, whereas the pyrimidine analog 5-FU did not. The Ki results of 6-MP and 6-TG inhibition of hypoxanthine uptake (~20–30 µM) are similar to reported values of therapeutic concentrations found within cells (10). This has relevance in the role that the vasculature plays in delivering chemotherapeutic agents to the intended target such that if this nucleobase transport system is blocked or downregulated the chemotherapeutic purine nucleobase analogs would be less able to gain access to their intracellular site of cytotoxic action.

The ability of a cell to mediate the transmembrane flux of nucleobases is important, as evidenced by the existence of at least two distinct transport systems for nucleobases in many cell types. Regulation of intracellular levels of nucleobases, such as hypoxanthine, may be critical during periods of physiological stress, including hypoxia or ischemia. During hypoxia or ischemia in the heart, the breakdown of ATP in cardiomyocytes results in an increase in adenosine, which is released to the interstitial space via NTs (13). The surrounding endothelium then accumulates this extracellular adenosine, where it is metabolized to inosine via adenosine deaminase. Subsequent metabolism by purine nucleoside phosphorylase produces hypoxanthine as an intermediate to the final product of uric acid (27). It is in the production of hypoxanthine that the role of MVEC nucleobase transporters may hold importance. Because hypoxanthine is metabolized to xanthine by xanthine oxidase, reactive oxygen species (ROS) are produced (20). Links have been established between xanthine oxidase-derived ROS and endothelial dysfunction (4, 24). Therefore, having a system capable of regulating hypoxanthine levels, before xanthine oxidase-mediated metabolism of hypoxanthine were to occur, may prevent or reduce ROS-induced endothelial dysfunction.

The molecular identity of this distinctive dipyridamole-insensitive, purine-selective nucleobase transporter is presently not known. Despite this lack of identity, we have named the transporter equilibrative nucleobase transporter 1 (ENBT1). There is some evidence in the literature that similar transporters may exist (23, 26), and we have been able to identify its presence through function in several cell types from various species. Preliminary data from our laboratory have suggested that mouse MVECs are more similar to human cardiac MVECs with respect to their nucleoside and nucleobase transport properties. However, rat MVECs appear to have less of the dipyridamole-insensitive [3H]hypoxanthine transporter and instead express more ENT2 as the primary nucleobase transport system. This needs to be considered when using rat as a model for human extrapolation studies.

Of the cells tested, only HEK-293 cells appeared to be deficient in ENBT1. Thus HEK-293 cells may prove useful to the heterologous expression of candidate genes for ENBT1. In contrast, PK15NTD cells, which lack ENTs, have very high levels of ENBT1. Thus PK15NTD cells may provide a useful model to study dipyridamole-insensitive hypoxanthine transport. The lack of ENTs in these cells allows for the transfection of a NT of interest for the study of interactions between nucleoside and nucleobase transporters, as we have shown with the PK15NTD-human ENT2 cell line.

The 4.5-fold enhancement of hypoxanthine accumulation in the presence of dipyridamole in PK15NTD-human ENT2 cells (see Fig. 8) may be the result of intracellular trapping of hypoxanthine metabolites, or perhaps hypoxanthine itself, that normally are substrates for ENT2. It is possible for ENT2-preferring nucleosides, like inosine (28), to be formed from hypoxanthine by purine nucleoside phosphorylase when normal physiological conditions are changed to allow the enzyme to catalyze the preferred reaction of nucleoside synthesis (7). Thus nucleobases can be used to replenish depleted nucleoside and nucleotide stores, using ENBT1 as a salvage pathway. Cells that lack ENT2, like human MVECs, or ones that have their existing ENT2 transporters blocked will see an enhancement of this salvage pathway, possibly leading to improved cellular function during periods of physiological stress.

In summary, we have characterized the nucleoside and nucleobase transporters of primary human cardiac MVECs. Human cardiac MVECs rely solely on ENT1 for nucleoside transport, and we have identified a novel nucleobase transport system, which we have named ENBT1. ENBT1 is present in a variety of different cell types in combination with ENT2; however, this combination varies between species and cell types. We recommend that future studies involving nucleoside or nucleobase regulation in the microvasculature be conducted in mouse models if human models are unavailable. The role of nucleoside and nucleobase transporters in the vasculature is important and complex. Regulation of extracellular adenosine levels, either through ENT expression or pharmacological intervention, will impact cardiovascular function. Considering the vasodilatory effects of adenosine, ENT regulation of purine levels can impact perfusion and thus nutrient exchange between the microvasculature and surrounding tissues (19). Control of nucleobase levels by ENT2 or ENBT1 will have implications in nutrient salvage, chemotherapy, and ROS production during hypoxia or ischemia.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by a grant to J. R. Hammond from the Heart and Stroke Foundation of Ontario (no. T-5606). D. B. J. Bone acknowledges the financial support of the Schulich School of Medicine and Dentistry and is also the recipient of an Ontario Graduate Scholarship in Science and Technology.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. R. Hammond, Dept. of Physiology and Pharmacology, MSB 266, Medical Sciences Bldg., Univ. of Western Ontario, London, ON, Canada N6A 5C1 (e-mail: james.hammond{at}schulich.uwo.ca)

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, Casado J, Gonzalez M, Pearson JD, Martin RS, Casanello P, Pastor-Anglada M, Sobrevia L. Equilibrative nucleoside transporter 2 is expressed in human umbilical vein endothelium, but is not involved in the inhibition of adenosine transport induced by hyperglycaemia. Placenta 26: 641–653, 2005.[CrossRef][Web of Science][Medline]
  2. Archer RG, Pitelka V, Hammond JR. Nucleoside transporter subtype expression and function in rat skeletal muscle microvascular endothelial cells. Br J Pharmacol 143: 202–214, 2004.[CrossRef][Web of Science][Medline]
  3. Baldwin SA, Beal PR, Yao SY, King AE, Cass CE, Young JD. The equilibrative nucleoside transporter family, SLC29. Pflügers Arch 447: 735–743, 2004.[CrossRef][Web of Science][Medline]
  4. Berry CE, Hare JM. Xanthine oxidoreductase and cardiovascular disease: molecular mechanisms and pathophysiological implications. J Physiol 555: 589–606, 2004.[Abstract/Free Full Text]
  5. Bone DB, Robillard KR, Stolk M, Hammond JR. Differential regulation of mouse equilibrative nucleoside transporter 1 (mENT1) splice variants by protein kinase CK2. Mol Membr Biol 24: 294–303, 2007.[CrossRef][Web of Science][Medline]
  6. Boumah CE, Hogue DL, Cass CE. Expression of high levels of nitrobenzylthioinosine-sensitive nucleoside transport in cultured human choriocarcinoma (BeWo) cells. Biochem J 288: 987–996, 1992.[Web of Science][Medline]
  7. Bzowska A, Kulikowska E, Shugar D. Purine nucleoside phosphorylases: properties, functions, and clinical aspects. Pharmacol Ther 88: 349–425, 2000.[CrossRef][Web of Science][Medline]
  8. Cheng Y, Prusoff WH. Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol 22: 3099–3108, 1973.[CrossRef][Web of Science][Medline]
  9. Deussen A, Stappert M, Schafer S, Kelm M. Quantification of extracellular and intracellular adenosine production: understanding the transmembranous concentration gradient. Circulation 99: 2041–2047, 1999.[Abstract/Free Full Text]
  10. Duley JA, Florin TH. Thiopurine therapies: problems, complexities, and progress with monitoring thioguanine nucleotides. Ther Drug Monit 27: 647–654, 2005.[CrossRef][Medline]
  11. Engel K, Wang J. Interaction of organic cations with a newly identified plasma membrane monoamine transporter. Mol Pharmacol 68: 1397–1407, 2005.[Abstract/Free Full Text]
  12. Engel K, Zhou M, Wang J. Identification and characterization of a novel monoamine transporter in the human brain. J Biol Chem 279: 50042–50049, 2004.[Abstract/Free Full Text]
  13. Gorlach A. Control of adenosine transport by hypoxia. Circ Res 97: 1–3, 2005.[Free Full Text]
  14. Gray JH, Owen RP, Giacomini KM. The concentrative nucleoside transporter family, SLC28. Pflügers Arch 447: 728–734, 2004.[CrossRef][Web of Science][Medline]
  15. Hyde RJ, Cass CE, Young JD, Baldwin SA. The ENT family of eukaryote nucleoside and nucleobase transporters: recent advances in the investigation of structure/function relationships and the identification of novel isoforms. Mol Membr Biol 18: 53–63, 2001.[Web of Science][Medline]
  16. Jarvis SM, Martin BW, Ng AS. 2-Chloroadenosine, a permeant for the nucleoside transporter. Biochem Pharmacol 34: 3237–3241, 1985.[CrossRef][Web of Science][Medline]
  17. King AE, Ackley MA, Cass CE, Young JD, Baldwin SA. Nucleoside transporters: from scavengers to novel therapeutic targets. Trends Pharmacol Sci 27: 416–425, 2006.[CrossRef][Medline]
  18. Lin W, Buolamwini JK. Synthesis, flow cytometric evaluation, and identification of highly potent dipyridamole analogs as equilibrative nucleoside transporter 1 inhibitors. J Med Chem 50: 3906–3920, 2007.[CrossRef][Web of Science][Medline]
  19. Loffler M, Morote-Garcia JC, Eltzschig SA, Coe IR, Eltzschig HK. Physiological roles of vascular nucleoside transporters. Arterioscler Thromb Vasc Biol 27: 1004–1013, 2007.[Abstract/Free Full Text]
  20. Nemeth I, Talosi G, Papp A, Boda D. Xanthine oxidase activation in mild gestational hypertension. Hypertens Pregnancy 21: 1–11, 2002.[CrossRef][Web of Science][Medline]
  21. Osses N, Pearson JD, Yudilevich DL, 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]
  22. Podgorska M, Kocbuch K, Pawelczyk T. Recent advances in studies on biochemical and structural properties of equilibrative and concentrative nucleoside transporters. Acta Biochim Pol 52: 749–758, 2005.[Web of Science][Medline]
  23. Redzic ZB, Gasic JM, Segal MB, Markovic ID, Isakovic AJ, Rakic ML, Thomas SA, Rakic LM. The kinetics of hypoxanthine transport across the perfused choroid plexus of the sheep. Brain Res 925: 169–175, 2002.[Web of Science][Medline]
  24. Rieger JM, Shah AR, Gidday JM. Ischemia-reperfusion injury of retinal endothelium by cyclooxygenase- and xanthine oxidase-derived superoxide. Exp Eye Res 74: 493–501, 2002.[CrossRef][Web of Science][Medline]
  25. Schaper W. Dipyridamole, an underestimated vascular protective drug. Cardiovasc Drugs Ther 19: 357–363, 2005.[CrossRef][Web of Science][Medline]
  26. Shayeghi M, Akerman R, Jarvis SM. Nucleobase transport in opossum kidney epithelial cells and Xenopus laevis oocytes: the characterisation, structure-activity relationship of uracil analogues and oocyte expression studies of sodium-dependent and -independent hypoxanthine uptake. Biochim Biophys Acta 1416: 109–118, 1999.[Medline]
  27. Shryock JC, Belardinelli L. Adenosine and adenosine receptors in the cardiovascular system: biochemistry, physiology, and pharmacology. Am J Cardiol 79: 2–10, 1997.[Web of Science][Medline]
  28. Ward JL, Sherali A, Mo ZP, 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]
  29. Zhu Z, Hofmann PA, Buolamwini JK. Cardioprotective effects of novel tetrahydroisoquinoline analogs of nitrobenzylmercaptopurine riboside in an isolated perfused rat heart model of acute myocardial infarction. Am J Physiol Heart Circ Physiol 292: H2921–H2926, 2007.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
K. M. Hoque, L. Chen, G. P. H. Leung, and C.-M. Tse
A purine-selective nucleobase/nucleoside transporter in PK15NTD cells
Am J Physiol Regulatory Integrative Comp Physiol, June 1, 2008; 294(6): R1988 - R1995.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/6/H3325    most recent
01006.2007v1
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 Google Scholar
Google Scholar
Right arrow Articles by Bone, D. B. J.
Right arrow Articles by Hammond, J. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bone, D. B. J.
Right arrow Articles by Hammond, J. R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.