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Physiology and Pharmacology  |   February 2013
Na+-Independent Nucleoside Transporters Regulate Adenosine and Hypoxanthine Levels in Müller Cells and the Inner Blood–Retinal Barrier
Author Affiliations & Notes
  • Shin-ichi Akanuma
    From the Department of Pharmaceutics, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan; and the
  • Tatsuya Soutome
    From the Department of Pharmaceutics, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan; and the
  • Eikichi Hisada
    From the Department of Pharmaceutics, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan; and the
  • Masanori Tachikawa
    From the Department of Pharmaceutics, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan; and the
    Division of Membrane Transport and Drug Targeting, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan.
  • Yoshiyuki Kubo
    From the Department of Pharmaceutics, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan; and the
  • Ken-ichi Hosoya
    From the Department of Pharmaceutics, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan; and the
  • Corresponding author: Ken-ichi Hosoya, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama, 930‐0194, Japan; hosoyak@pha.u-toyama.ac.jp
Investigative Ophthalmology & Visual Science February 2013, Vol.54, 1469-1477. doi:10.1167/iovs.12-10905
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      Shin-ichi Akanuma, Tatsuya Soutome, Eikichi Hisada, Masanori Tachikawa, Yoshiyuki Kubo, Ken-ichi Hosoya; Na+-Independent Nucleoside Transporters Regulate Adenosine and Hypoxanthine Levels in Müller Cells and the Inner Blood–Retinal Barrier. Invest. Ophthalmol. Vis. Sci. 2013;54(2):1469-1477. doi: 10.1167/iovs.12-10905.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: To elucidate the mechanism(s) of hypoxanthine production in Müller cells and the elimination of hypoxanthine across the inner blood–retinal barrier (BRB).

Methods.: The hypoxanthine biosynthesis and adenosine transport in Müller cells were investigated using a conditionally immortalized rat Müller cell line, TR-MUL5 cells. The elimination of hypoxanthine across the inner BRB was assessed by an in vivo microdialysis method and an in vitro transport study using a conditionally immortalized rat retinal capillary endothelial cell line, TR-iBRB2 cells.

Results.: [3H]Hypoxanthine was detected in TR-MUL5 cells and TR-MUL5 cell-cultured medium 3 hours after [3H]adenosine incubation, indicating that the hypoxanthine is produced in TR-MUL5 cells. [3H]Adenosine was taken up into TR-MUL5 cells, which express mRNAs of nucleoside transporters (ENT1-2 and CNT1-2), in an Na+-independent and concentration-dependent manner (K m = 20 μM). Moreover, 100 μM nitrobenzylmercaptopurine riboside (NBMPR) and azidothymidine, which are inhibitors of ENT2, inhibited [3H]adenosine uptake, suggesting that ENT2 is a major contributor to adenosine transport in Müller cells. [3H]Hypoxanthine was eliminated from the rat vitreous humor and this process was inhibited in the presence of NBMPR. [3H]Hypoxanthine uptake by TR-iBRB2 cells took place in an Na+-independent and concentration-dependent manner with Km values of 4.3 μM and 2.9 mM, and was inhibited by 100 μM NBMPR.

Conclusions.: Our findings suggest that hypoxanthine is produced from adenosine in Müller cells and ENT2 plays a major role in adenosine uptake in Müller cells. Hypoxanthine in the retina is eliminated via Na+-independent equilibrative nucleoside transporters.

Introduction
Hypoxanthine, a nucleobase, is one of the sources of reactive oxygen species (ROS) via xanthine oxidase/xanthine dehydrogenase. 1 The involvement of oxidative stress induced by ROS is reported in the pathogenesis of retinal degenerative diseases such as age-related macular degeneration. 2 The concentrations of hypoxanthine and its precursor, adenosine, are elevated during reperfusion after retinal ischemic treatment in rats. 3 Thus, the modulation of the hypoxanthine concentration in the retina is important to avoid retinal oxidative stress in pathologic conditions such as ischemia–reperfusion. In the brain, astrocytes work as glial cells, and their involvement in hypoxanthine biosynthesis from adenosine has been reported. 4 Müller cells are retinal glial cells that play a role in the regulation of homeostasis of a number of compounds, such as glutamate, in the retinal interstitial fluid (ISF) as well as providing structural support to retinal cells. 5,6 Hypoxanthine is enzymatically synthesized from adenosine in the intracellular compartment. 7 Almost all the adenosine is distributed around the processes of Müller cells in the layers of ganglion, inner plexiform, and inner nuclear cells. 8 Thus, it is hypothesized that Müller cells participate in the production of hypoxanthine from adenosine, which is transported from the retinal ISF. 
As an elimination pathway of hypoxanthine from the retinal ISF, the metabolic conversion to xanthine and inosine nucleotide has been proposed. 9,10 However, the hypoxanthine concentration is not markedly increased by the intraperitoneal administration of inhibitors of xanthine oxidase/xanthine dehydrogenase to rats causing retinal ischemia–reperfusion. 11 This report indicates other clearance mechanism(s) of hypoxanthine in the retina. Previously, we have reported that the blood–retinal barrier (BRB), which is composed of the inner BRB and outer BRB, 12,13 is involved in the elimination of organic anionic compounds and amino acids, such as estradiol 17β-d-glucuronide, p-aminohippurate, and l-proline, from the retina to the circulating blood via a transporter-mediated process. 1416 The retinal blood vessels form the inner BRB, and are present in the ganglion cell layer, the inner and outer plexiform layers, and the inner nuclear layer, 17 exhibiting a similar localization to the retinal distribution of adenosine. 8 These pieces of evidence prompted us to hypothesize the involvement of transporters expressed at the inner BRB in hypoxanthine elimination from the retina. 
Na+-independent equilibrative nucleoside transporters (ENT/SLC29A) and Na+-dependent concentrative nucleoside transporters (CNT/SLC28A) have been studied in detail. In addition, rat Na+-dependent nucleobase transporter (SNBT1/SLC23A4) transports a number of nucleobases including hypoxanthine. 18 Thus, they are regarded as possibly being involved in adenosine transport in Müller cells and in hypoxanthine transport at the inner BRB. ENTs and CNTs have four groups (ENT1–4/SLC29A1–4) 19 and three subtypes (CNT1–3/SLC28A1–3), 20 respectively. Although dos Santos-Rodrigues et al. 21 have reported that adenosine is taken up into chick retinal glial cells via ENT1 and ENT2, their contributions to adenosine transport in Müller cells has not been fully evaluated. Regarding the transport of nucleosides and nucleobases at the inner BRB, we have reported the expression of ENT1, ENT2, and CNT2 mRNAs in a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells), and blood-to-retina adenosine transport across the inner BRB most likely takes place via ENT2. 22 Since ENT2 accepts hypoxanthine as a substrate, 23 it is suggested that hypoxanthine transport mechanism(s) are located in the inner BRB. To better understand the regulation of the hypoxanthine concentration in the retina, the molecular mechanism(s) of adenosine transport in Müller cells and hypoxanthine elimination from the retina across the BRB could provide useful information. 
The purpose of this study was to investigate the production of hypoxanthine in Müller cells and the elimination of hypoxanthine across the BRB. To evaluate the conversion of hypoxanthine from adenosine and the molecular mechanism(s) of adenosine transport, a conditionally immortalized rat Müller cell line (TR-MUL5 cells) was used as an in vitro model of rat Müller cells. 24 Moreover, the elimination of hypoxanthine across the inner BRB and molecular mechanism(s) of this process were assessed in an in vivo microdialysis study after intravitreous injection and an in vitro cellular uptake experiment using TR-iBRB2 cells as an in vitro model of inner BRB. 25  
Materials and Methods
Animals
Male Wistar rats, weighing 250 to 300 g, were purchased from a commercial supplier (Japan SLC Inc., Hamamatsu, Japan). The investigations using the rats described in this report conformed to the provisions of the Animal Care Committee, University of Toyama, and the ARVO Statement on the Use of Animals in Ophthalmic and Vision Research. 
Reagents
[2,8-3H]Adenosine ([3H]adenosine, 39.2 Ci/mmol) and [2,8-3H]hypoxanthine ([3H]hypoxanthine, 31.7 Ci/mmol) were purchased from a commercial provider (Moravek Biochemicals, Brea, CA). [1-14C]D-Mannitol ([14C]D-mannitol, 56 mCi/mmol) was purchased commercially (American Radiolabeled Chemicals, St. Louis, MO). All other chemicals were commercial reagent-grade products. 
Cell Culture
TR-MUL5 cells and TR-iBRB2 cells were cultured at 33°C in Dulbecco's modified Eagle's medium (Nissui Pharmaceuticals, Tokyo, Japan), supplemented with 20 mM sodium bicarbonate, 100 U/mL benzylpenicillin potassium, 100 μg/mL streptomycin sulfate, and 10% fetal bovine serum under 5% CO2/air. 24,25 Primary-cultured rat Müller cells were prepared as described previously, with minor modification. 26 (The detailed methods of isolation and cultivation of Müller cells are included in Supplementary Materials; see Supplementary Materials.) 
HPLC Analysis
The sample preparation and HPLC analysis were performed as described previously, with minor modifications (see Supplementary Materials).27,28 The efflux of 3H-labeled compound by TR-MUL5 cells and primary-cultured rat Müller cells preloaded with [3H]adenosine was expressed as follows:  The retention times of authentic compounds monitored at 260 nm were as follows: hypoxanthine, 4.2 minutes; inosine, 4.5 minutes; adenosine, 7.1 minutes.  
[3H]Adenosine and [3H]Hypoxanthine Uptake by TR-MUL5 Cells and TR-iBRB2 Cells
Uptake studies of [3H]adenosine (0.1 μCi/200 μL; 13 nM) by TR-MUL5 cells21 and primary-cultured rat Müller cells, and [3H]hypoxanthine (0.1 μCi/200 μL; 16 nM) by TR-iBRB2 cells22 were performed as described previously, with minor modifications. The detailed methods used in the uptake studies are included in the Supplementary Materials (see Supplementary Materials). The uptake of [3H]adenosine and [3H]hypoxanthine was expressed as the cell/medium ratio (μL/mg protein; Equation 2).    
Reverse Transcription–Polymerase Chain Reaction
Total RNA preparation and reverse transcription–polymerase chain reaction (RT-PCR) analysis were performed as described previously, with minor modifications. 22,27 The details of the methods are included in the Supplementary Materials. The oligonucleotide sequences of the specific primers for PCR analysis are shown in Supplementary Table S1 (see Supplementary Materials and Supplementary Table S1). 
Microdialysis Study
Microdialysis after intravitreal [3H]hypoxanthine administration was performed as described previously. 16 The details of the method are included in the Supplementary Materials (see Supplementary Materials). In brief, rats were anesthetized with an intraperitoneal injection of sodium pentobarbital and then mounted on a stereotaxic frame. [3H]Hypoxanthine (2 μCi) and [14C]D-mannitol (0.2 μCi) were intravitreally administered. The microdialysis probe was implanted into the vitreous chamber and Ringer's–HEPES solution (141 mM NaCl, 4.0 mM KCl, 2.8 mM CaCl2, 10 mM HEPES–NaOH, pH 7.4) at 37°C was delivered to the probe continuously at 2 μL/min and samples of the dialysate were collected. Each inhibitor was dissolved in Ringer's–HEPES solution and delivered to the probe. 
The probe recovery was assessed in the in vitro medium containing a constant level of compounds and calculated as follows:  where CT (dpm/mL) is the concentration in the dialysate solution and CV (dpm/mL) is the concentration in the test solution. The lag time for the passage through the polyethylene tubing connected to the distal end of the microdialysis probe was approximately 10 minutes. The recovery of [3H]hypoxanthine and [14C]D-mannitol from the solution was 11.3 ± 0.1% and 6.25 ± 0.09% (n = 3), respectively. Recovery values were constant over 180 minutes after the lag time.  
Data Analysis
The kinetic parameters of adenosine uptake by TR-MUL5 cells and hypoxanthine uptake by TR-iBRB2 cells were estimated from Equations 4 and 5, respectively:    where V is the uptake rate of adenosine or hypoxanthine, Vmax is the maximum uptake rate of adenosine, [S] is the concentration of adenosine or hypoxanthine in the medium, Km is the corresponding Michaelis–Menten constant, Vmax,1 is the maximum uptake rate for the high-affinity process, Km,1 is the corresponding Michaelis–Menten constant, Vmax,2 is the maximum uptake rate for the low-affinity process, and Km,2 is the corresponding Michaelis–Menten constant. To obtain the kinetic parameters, the equation was fitted using the nonlinear least-square regression analysis program.  
The vitreous concentrations normalized by the injected dose (Cp [% dose/mL]) were estimated from the radioactivities in the dialysate using Equation 6 and the Cp at time (Cp[t]) was fitted to a biexponential equation (Equation 7) by nonlinear least-squares regression analysis:   where CT is the concentration in the dialysate (dpm/mL) and Dosetracer (dpm) is the total radioactivity of the substance after intravitreal injection, The constants A and B are intercepts on the y-axis for each exponential segment of the curve in Equation 6. The constants α and β are the apparent first-order rate constants for the initial and terminal phases, respectively.  
The kinetic parameters are represented as the mean ± SD. Other data are given as the mean ± SEM. Statistical analysis of the data was performed by using an unpaired Student's t-test, and by one-way analysis of variance followed by Dunnett's test for single and multiple comparisons, as appropriate. 
Results
Hypoxanthine Biosynthesis from Adenosine in TR-MUL5 Cells
After preincubation of TR-MUL5 cells and primary-cultured rat Müller cells with [3H]adenosine, the 3H-label compounds were effluxed from TR-MUL5 cells and primary-cultured rat Müller cells for 3 hours with fractional release values of 48.6 ± 0.3% and 67.7 ± 1.8%, respectively (n = 3–4). To identify the 3H-label compounds in the cells and culture medium, we performed the HPLC analysis using TR-MUL5 cells 3 hours after [3H]adenosine incubation. After incubation in the efflux reaction medium, the peak of [3H]hypoxanthine at 3.5 to 5.0 minutes (Fig. 1A) was detected both in TR-MUL5 cells (Fig. 1B) and in culture medium (Fig. 1C), and this was distinct from the peak of [3H]adenosine (Fig. 1D). This result indicates that [3H]hypoxanthine was synthesized from [3H]adenosine in TR-MUL5 cells and transported from TR-MUL5 cells to the extracellular space. 
Figure 1
 
Typical HPLC chromatograms of samples of TR-MUL5 cells (B) and TR-MUL5 cell-cultured medium (C). TR-MUL5 cells were incubated in extracellular fluid (ECF) buffer containing 0.5 μCi/mL [3H]adenosine at 37°C for 5 minutes and the efflux of 3H-labeled compounds was initiated by applying 37°C-warmed ECF buffer (culture medium). After incubation for 3 hours, culture medium was collected and TR-MUL5 cells were lysed. These samples were deproteinized before being used as HPLC samples. (A, D) Typical chromatograms of [3H]hypoxanthine and [3H]adenosine, respectively. Closed and open arrowheads were indicated as the typical retention times of [3H]hypoxanthine and [3H]adenosine, respectively.
Figure 1
 
Typical HPLC chromatograms of samples of TR-MUL5 cells (B) and TR-MUL5 cell-cultured medium (C). TR-MUL5 cells were incubated in extracellular fluid (ECF) buffer containing 0.5 μCi/mL [3H]adenosine at 37°C for 5 minutes and the efflux of 3H-labeled compounds was initiated by applying 37°C-warmed ECF buffer (culture medium). After incubation for 3 hours, culture medium was collected and TR-MUL5 cells were lysed. These samples were deproteinized before being used as HPLC samples. (A, D) Typical chromatograms of [3H]hypoxanthine and [3H]adenosine, respectively. Closed and open arrowheads were indicated as the typical retention times of [3H]hypoxanthine and [3H]adenosine, respectively.
Adenosine Uptake by TR-MUL5 Cells and Primary-Cultured Rat Müller Cells
To investigate the transport of extracellular adenosine in Müller cells, [3H]adenosine uptake by TR-MUL5 cells and primary-cultured rat Müller cells was examined. In TR-MUL5 cells, [3H]adenosine uptake exhibited a time-dependent increase for at least 10 minutes (Fig. 2A) and saturable kinetics with a K m of 20.3 ± 1.1 μM and a V max of 1.22 ± 0.02 nmol/(minute·mg protein) (Fig. 2B). Moreover, the [3H]adenosine uptake by TR-MUL5 cells under Na+-free conditions was not significantly different from the control (Fig. 3). Similar to [3H]adenosine uptake by TR-MUL5 cells, [3H]adenosine uptake by primary-cultured rat Müller cells showed a time-dependent increase for at least 10 minutes (see Supplementary Material and Supplementary Fig. S1A). Moreover, [3H]adenosine uptake by primary-cultured rat Müller cells was significantly inhibited by 96% in the presence of 2 mM unlabeled adenosine, whereas Na+-replacement had little effect (see Supplementary Material and Supplementary Fig. S1B). 
Figure 2
 
Time-course (A) and concentration-dependence (B) of [3H]adenosine uptake by TR-MUL5 cells. (A) [3H]Adenosine uptake (13 nM) was measured at 37°C for the indicated times. The solid line was fitted using a nonlinear least-squares regression analysis program. Each point represents the mean ± SEM (n = 4). (B) Adenosine uptake was measured at 37°C for 3 minutes. [3H]Adenosine (13 nM) was present in all uptake measurements as a tracer, and the concentration of adenosine was varied using unlabeled adenosine. The solid line was fitted using a nonlinear least-squares regression analysis program. Each point represents the mean ± SEM (n = 4).
Figure 2
 
Time-course (A) and concentration-dependence (B) of [3H]adenosine uptake by TR-MUL5 cells. (A) [3H]Adenosine uptake (13 nM) was measured at 37°C for the indicated times. The solid line was fitted using a nonlinear least-squares regression analysis program. Each point represents the mean ± SEM (n = 4). (B) Adenosine uptake was measured at 37°C for 3 minutes. [3H]Adenosine (13 nM) was present in all uptake measurements as a tracer, and the concentration of adenosine was varied using unlabeled adenosine. The solid line was fitted using a nonlinear least-squares regression analysis program. Each point represents the mean ± SEM (n = 4).
Figure 3
 
Effect of sodium replacement and several inhibitors on [3H]adenosine uptake by TR-MUL5 cells. [3H]Adenosine uptake (13 nM) was performed at 37°C for 3 minutes in the absence or presence (control) of Na+, or in the absence (control) or presence of inhibitors. Each column represents the mean ± SEM (n = 4–8). **P < 0.01, significantly different from the control. DMSO, dimethylsulfoxide.
Figure 3
 
Effect of sodium replacement and several inhibitors on [3H]adenosine uptake by TR-MUL5 cells. [3H]Adenosine uptake (13 nM) was performed at 37°C for 3 minutes in the absence or presence (control) of Na+, or in the absence (control) or presence of inhibitors. Each column represents the mean ± SEM (n = 4–8). **P < 0.01, significantly different from the control. DMSO, dimethylsulfoxide.
To characterize the adenosine uptake transport in rat Müller cells, [3H]adenosine uptake by TR-MUL5 cells was examined in the presence of various substrates and inhibitors of nucleoside transporters (Fig. 3). At a concentration of 2 mM, [3H]adenosine uptake by TR-MUL5 cells was inhibited by more than 54% in the presence of nucleosides, such as inosine, thymidine, uridine, guanosine, and cytidine. Nitrobenzylmercaptopurine riboside (NBMPR), which is an inhibitor of rat ENT1 and rat ENT2, 29 inhibited [3H]adenosine uptake by TR-MUL5 cells by 30% and 91% at a concentration of 0.1 and 100 μM, respectively. 3′-Azido-3′-deoxythymidine (AZT) is a selective substrate of rat ENT2, 30 and this inhibited [3H]adenosine uptake by 48% at a concentration of 2 mM. 
mRNA Expression of Nucleoside Transporters in TR-MUL5 Cells and Primary-Cultured Rat Müller Cells
To determine ENT and CNT expression in TR-MUL5 cells and primary-cultured rat Müller cells, RT-PCR analysis was performed with total RNA extracted from TR-MUL5 cells and primary-cultured rat Müller cells. We also performed this analysis using rat lung, brain, kidney, or liver as the corresponding positive controls (Fig. 4). The bands indicative of ENT1, ENT2, and ENT3 expression were detected from TR-MUL5 cells, primary-cultured rat Müller cells, and the respective positive control at the position of 157, 278, and 406 bp, respectively. In the case of CNTs, bands indicative with CNT1 and CNT2 expression were detected from TR-MUL5 cells and primary-cultured rat Müller cells at 479 and 298 bp, respectively, whereas there was no evidence of CNT3 expression in these cells. The nucleotide sequence of each amplified product was identical to that of the corresponding gene. 
Figure 4
 
mRNA expression of ENTs and CNTs in TR-MUL5 cells and primary-cultured rat Müller cells. RT-PCR analysis was performed in the presence (+) or absence (−) of RT using specific primers for rat ENT1–3 and CNT1–3. Rat lung, brain, kidney, and liver were used as respective positive controls.
Figure 4
 
mRNA expression of ENTs and CNTs in TR-MUL5 cells and primary-cultured rat Müller cells. RT-PCR analysis was performed in the presence (+) or absence (−) of RT using specific primers for rat ENT1–3 and CNT1–3. Rat lung, brain, kidney, and liver were used as respective positive controls.
In Vivo [3H]Hypoxanthine Elimination from Rat Vitreous Humor
The time profile of the remaining [3H]hypoxanthine in the vitreous humor after bolus injection is shown in Figure 5A. [14C]D-Mannitol, as a bulk flow marker, was simultaneously administrated. [3H]Hypoxanthine and [14C]D-mannitol were biexponentially eliminated from the vitreous humor and the decline in [3H]hypoxanthine was significantly greater than that of [14C]D-mannitol. The apparent elimination rate constant (β) during the terminal phase of [3H]hypoxanthine was 1.69 × 10−2 ± 0.14 × 10−2 min−1 and this was 1.46-fold greater than that of [14C]D-mannitol (1.16 × 10−2 ± 0.08 × 10−2 min−1) (Fig. 5B). Moreover, the difference between the β value of [3H]hypoxanthine and [14C]D-mannitol was significantly reduced by more than 30% in the presence of 20 mM uridine and 200 μM NBMPR (Fig. 6). This value was slightly reduced in the presence of 20 mM thymidine, but the difference was not significant. 
Figure 5
 
In vivo [3H]hypoxanthine elimination from rat vitreous humor after vitreous bolus injection. (A) Time profile of [3H]hypoxanthine and [14C]D-mannitol in the vitreous humor after vitreous bolus injection. Open circles and closed triangles represent the concentration in the dialysate of [3H]hypoxanthine and [14C]D-mannitol, respectively. Each point represents the mean ± SEM (n = 5). (B) Elimination rate constants (β) of [3H]hypoxanthine (open column) and [14C]D-mannitol (closed column) during the terminal phase. Each column represents the mean ± SEM (n = 5). **P < 0.01, significant difference.
Figure 5
 
In vivo [3H]hypoxanthine elimination from rat vitreous humor after vitreous bolus injection. (A) Time profile of [3H]hypoxanthine and [14C]D-mannitol in the vitreous humor after vitreous bolus injection. Open circles and closed triangles represent the concentration in the dialysate of [3H]hypoxanthine and [14C]D-mannitol, respectively. Each point represents the mean ± SEM (n = 5). (B) Elimination rate constants (β) of [3H]hypoxanthine (open column) and [14C]D-mannitol (closed column) during the terminal phase. Each column represents the mean ± SEM (n = 5). **P < 0.01, significant difference.
Figure 6
 
Effect of inhibitors on the elimination rate constant difference between [3H]hypoxanthine and [14C]D-mannitol during the terminal phase. Each inhibitor was perfused in the microdialysis probe. Each column represents the mean ± SEM (n = 3–6). *P < 0.05, significantly different from the control. Percentage of control was calculated as follows: (β value of [3H]hypoxanthine − β value of [14C]D-mannitol in the presence of inhibitor)/(β value of [3H]hypoxanthine − β value of [14C]D-mannitol in the absence of inhibitor) × 100.
Figure 6
 
Effect of inhibitors on the elimination rate constant difference between [3H]hypoxanthine and [14C]D-mannitol during the terminal phase. Each inhibitor was perfused in the microdialysis probe. Each column represents the mean ± SEM (n = 3–6). *P < 0.05, significantly different from the control. Percentage of control was calculated as follows: (β value of [3H]hypoxanthine − β value of [14C]D-mannitol in the presence of inhibitor)/(β value of [3H]hypoxanthine − β value of [14C]D-mannitol in the absence of inhibitor) × 100.
Hypoxanthine Uptake by TR-iBRB2 Cells
[3H]Hypoxanthine uptake increased linearly for at least 10 minutes (Fig. 7A) and this uptake by TR-iBRB2 cells was concentration dependent and composed of high- and low-affinity saturable processes (Fig. 7B). The apparent K m,1 and V max,1 of the high-affinity process were found to be 4.34 ± 1.43 μM and 73.5 ± 18.1 pmol/(minute·mg protein), respectively, and the apparent K m,2 and V max,2 of the low-affinity process were 2.93 ± 1.63 mM and 5.90 ± 2.57 nmol/(minute·mg protein) (mean ± SD), respectively. Na+-free conditions had little effect on [3H]hypoxanthine uptake by TR-iBRB2 cells (Fig. 8). 
Figure 7
 
Time-course (A) and concentration-dependence (B) of [3H]hypoxanthine uptake by TR-iBRB2 cells. (A) [3H]Hypoxanthine uptake (16 nM) was measured at 37°C for the indicated times. The solid line was fitted using a nonlinear least-squares regression analysis program. Each point represents the mean ± SEM (n = 3). (B) Hypoxanthine uptake was measured at 37°C for 5 minutes. [3H]Hypoxanthine (16 nM) was present in all uptake measurements as a tracer, and the concentration of hypoxanthine was varied using unlabeled hypoxanthine. The solid line was fitted using a nonlinear least-squares regression analysis program. Each point represents the mean ± SEM (n = 3).
Figure 7
 
Time-course (A) and concentration-dependence (B) of [3H]hypoxanthine uptake by TR-iBRB2 cells. (A) [3H]Hypoxanthine uptake (16 nM) was measured at 37°C for the indicated times. The solid line was fitted using a nonlinear least-squares regression analysis program. Each point represents the mean ± SEM (n = 3). (B) Hypoxanthine uptake was measured at 37°C for 5 minutes. [3H]Hypoxanthine (16 nM) was present in all uptake measurements as a tracer, and the concentration of hypoxanthine was varied using unlabeled hypoxanthine. The solid line was fitted using a nonlinear least-squares regression analysis program. Each point represents the mean ± SEM (n = 3).
Figure 8
 
Effect of sodium replacement and several inhibitors on [3H]hypoxanthine uptake by TR-iBRB2 cells. [3H]Hypoxanthine uptake (16 nM) was performed at 37°C for 5 minutes in the absence or presence (control) of Na+, or in the absence (control) or presence of inhibitors. Each column represents the mean ± SEM (n = 3–6). *P < 0.05, **P < 0.01, significantly different from the control.
Figure 8
 
Effect of sodium replacement and several inhibitors on [3H]hypoxanthine uptake by TR-iBRB2 cells. [3H]Hypoxanthine uptake (16 nM) was performed at 37°C for 5 minutes in the absence or presence (control) of Na+, or in the absence (control) or presence of inhibitors. Each column represents the mean ± SEM (n = 3–6). *P < 0.05, **P < 0.01, significantly different from the control.
The inhibition study was performed to characterize the [3H]hypoxanthine transport mechanism(s) in TR-iBRB2 cells (Fig. 8). At a concentration of 250 μM, adenosine and guanosine inhibited [3H]hypoxanthine uptake by more than 90%, whereas uridine partially inhibited it by 35% and cytidine had little effect. Regarding the nucleobases, uracil and adenine at a concentration of 250 μM also significantly inhibited [3H]hypoxanthine uptake by more than 20%. NBMPR inhibited [3H]hypoxanthine uptake by TR-iBRB2 cells by 74% at a concentration of 100 μM, whereas 0.1 μM NBMPR had little effect. Dipyridamole, which has been reported previously to inhibit ENT1 and ENT2 in other rat cell lines, 22,31,32 also inhibited [3H]hypoxanthine uptake by TR-iBRB2 cells by 79% at a concentration of 10 μM. 
Discussion
The results of our studies provide evidence of hypoxanthine biosynthesis from adenosine in Müller cells. [3H]Hypoxanthine was identified in TR-MUL5 cells and TR-MUL5 cell-culture medium 3 hours after [3H]adenosine incubation (Fig. 1), suggesting that extracellular adenosine is taken up and converted to hypoxanthine in Müller cells, and then released into the extracellular compartment as hypoxanthine. Although the expression of ENT1-3 and CNT1-2 mRNAs was detected in TR-MUL5 cells and primary-cultured rat Müller cells (Fig. 4), adenosine uptake by these cells occurred in an Na+-independent manner (Fig. 3; also see Supplementary Material and Supplementary Fig. S1). These results indicate the role of an ENT-mediated process in adenosine transport in Müller cells. 
Adenosine was taken up into TR-MUL5 cells in a saturable manner (Km = 20 μM; Fig. 2), with a Km value similar to that of rat ENT1- and ENT2-mediated adenosine uptake (ENT1, Km = 6.1 μM; ENT2, K m = 26 μM). 29 Because of the dominant localization of ENT3 in intracellular membranes, 33 it would appear that ENT1 and ENT2 are responsible for adenosine uptake transport into TR-MUL5 cells. ENT1 and ENT2 accept various nucleosides as substrates, with a lower affinity of ENT2 for guanosine and cytidine than that of ENT1. 34,35 The inhibition degree of [3H]adenosine uptake into TR-MUL5 cells by adenosine, inosine, thymidine, and uridine was greater than that by guanosine and cytidine (Fig. 3). Nishimura et al. 29 reported that 100 μM NBMPR inhibited adenosine uptake by both rat ENT1- and rat ENT2-expressing Xenopus laevis oocytes, whereas 0.1 μM NBMPR inhibited only rat ENT1-mediated adenosine transport. AZT is a substrate of rat ENT2 but not rat ENT1. 30 [3H]Adenosine uptake by TR-MUL5 cells was strongly inhibited by 100 μM NBMPR and 2 mM AZT (Fig. 3), suggesting a greater contribution of ENT2 than that of ENT1. The adenosine concentration in the rat retina is reported to be approximately 0.153 μM under normal conditions, and was increased to 4.56 μM by inducing retinal ischemia–reperfusion. 3 The K m value of adenosine uptake by TR-MUL5 cells is higher than these values. Although the retinal ISF concentration of adenosine under these conditions has not been measured, the adenosine influx transport in Müller cells appears not to be saturated. Hypoxanthine, which is produced in Müller cells, is also a substrate for ENT2. 23 The affinity of adenosine for rat ENT2 is reported to be 38-fold greater than that of hypoxanthine, 23,29 suggesting that ENT2 in Müller cells is mainly involved in the uptake of adenosine rather than that of hypoxanthine in the retinal ISF. Considering these findings, it is proposed that ENT2-mediated adenosine uptake plays a role in the modulation of the adenosine concentration in retinal ISF and hypoxanthine production in Müller cells. 
Some efflux transport mechanisms of hypoxanthine were considered to be present in Müller cells because [3H]hypoxanthine was detected in TR-MUL5 cell-cultured medium (Fig. 1C). In rat C6 glioma cells, a putative hypoxanthine-preferring nucleobase transporter, NBT2, contributes to hypoxanthine efflux transport, although its responsible molecule has not been identified. 7 In addition, ENT2 is an equilibrative transporter with no driving force and the direction of transport is dependent on the gradient of the substrate concentration. 19 ENT2 is suggested to be involved in the efflux of hypoxanthine in rat muscle microvascular endothelial cells. 32 Although the contribution of these transporters to hypoxanthine efflux transport in Müller cells will need to be assessed in further studies such as transport experiments under ENT2-specific cells (Fig. 4), the contribution also takes a part in hypoxanthine efflux transport. 
The dialysate concentration of [3H]hypoxanthine and [14C]D-mannitol, a bulk flow marker for passage from the vitreous humor to Schlemm's canal and/or the uveoscleral outflow route, in the vitreous humor, is reduced in a biexponential manner (Fig. 5A). The initial slope of the drug concentration–time profile was steeper than that of the later slope. This result supports the hypothesis that the first and second declines reflect diffusion into the vitreous humor, including the microdialysis tube, after vitreous bolus injection and elimination of both [3H]hypoxanthine and [14C]D-mannitol from the vitreous humor, respectively. 16 The β value of [3H]hypoxanthine was significantly higher than that of [14C]D-mannitol (Fig. 5B), indicating that [3H]hypoxanthine undergoes retina-to-blood transport across the BRB in addition to elimination from the vitreous humor via bulk flow and passive diffusion. Moreover, the β-value difference between [3H]hypoxanthine and [14C]D-mannitol was reduced in the presence of NBMPR and uridine (Fig. 6), suggesting the involvement of NBMPR-sensitive nucleoside transporters in the retina-to-blood transport of hypoxanthine at the BRB. 
[3H]Hypoxanthine uptake by TR-iBRB2 cells exhibited time dependence and Na+ independence (Figs. 7A, 8), indicating that Na+-independent transporter(s), such as ENTs but not CNTs and SNBT1, are involved in the hypoxanthine transport in TR-iBRB2 cells. It has been reported that mRNAs of ENT1 and ENT2 are expressed in TR-iBRB2 cells 22 and rat ENT1 does not accept hypoxanthine. 23 The hypoxanthine uptake exhibited high- and low-affinity transport processes (Fig. 7B). The low-affinity K m value (K m,2 = 2.9 mM) is in good agreement with that obtained from hypoxanthine transport in rat ENT2-expressing Xenopus laevis oocytes (K m = 1.0 mM), 23 suggesting that a putative high-affinity hypoxanthine transporter is present in the inner BRB in addition to ENT2. The high-affinity K m value (K m,1 = 4.3 μM) is similar to that of hypoxanthine uptake by mouse ENT2-expressing COS-7 cells (K m = 2.8 μM). 36 The inhibition properties of NBMPR and dipyridamole to [3H]hypoxanthine uptake by TR-iBRB2 cells (Fig. 8) is similar to that of [3H]adenosine uptake by TR-iBRB2 cells, which is mainly involved in ENT2-mediated processes. 22 On the other hand, [3H]hypoxanthine uptake by TR-iBRB2 cells was strongly inhibited by 250 μM guanosine, which has a high affinity for mouse ENT2 (Km = 7.1 μM) and a low affinity for human (Km = 2.7 mM) and rat ENT2. 22,35,36 From these findings, it is suggested that hypoxanthine transport at the inner BRB consists of two processes involving both rat ENT2 and a putative hypoxanthine transporter, which shows mouse ENT2-like characteristics. The localization of rat ENT2 protein at the inner BRB has not been reported yet. NBMPR and uridine inhibited both in vivo hypoxanthine elimination from the rat vitreous humor and in vitro hypoxanthine transport in TR-iBRB2 cells (Figs. 6, 8), suggesting that rat ENT2 and/or a putative hypoxanthine transporter at the inner BRB is involved in hypoxanthine elimination from the retina. 
Adenosine, the precursor of hypoxanthine, possibly contributes to the retinal physiologic responses because adenosine receptors are reported to be expressed in the retina. 37 As one of the adenosine clearance mechanisms in the retina, our studies proposed that adenosine in the retinal ISF is taken up into Müller cells and is converted to hypoxanthine, and this hypoxanthine is eliminated from the retina (Fig. 9). Because ROS are produced via hypoxanthine metabolism to xanthine, the hypoxanthine efflux transport across the inner BRB is important for the prevention of retinal oxidative stress. Moreover, these processes are, at least in part, mediated via an Na+-independent nucleoside transporter such as ENT2. ENT2 is also involved in blood-to-retina adenosine transport at the inner BRB. 22 Thus, it is considered that ENT2 regulates adenosine-mediated retinal physiologic responses and the oxidative stress induced by hypoxanthine metabolism. Nucleoside drugs, such as AZT, which inhibit adenosine uptake by TR-MUL5 cells (Fig. 3), is given to treat patients who are positive for human immunodeficiency virus, 38,39 and it is reported to induce maculopathy as one of its adverse effects. 38 Therefore, the inhibition of ENT2-mediated transport of hypoxanthine and adenosine should be considered in the development of antiviral drugs to maintain adenosine and hypoxanthine concentrations in the retina and to avoid any resulting retinal adverse effects. 
Figure 9
 
Proposed mechanism for the production of hypoxanthine in Müller cells and its elimination across the inner BRB, and the involvement of nucleoside transporters. ADO, adenosine; AMP, adenosine monophosphate; ATP, adenosine triphosphate; HX, hypoxanthine; 5′N, 5′-nucleotidase; NBT2, hypoxanthine-preferring nucleobase transporter; NTPDase, ectonucleoside triphosphate diphosphohydrolase; (a) Nagase et al., 22 (b) Iandiev et al., 40 (c) Lutty and McLeod, 37 (d) Robillard et al., 32 (e) Sinclair et al., 7 (f) Berry and Hare. 1
Figure 9
 
Proposed mechanism for the production of hypoxanthine in Müller cells and its elimination across the inner BRB, and the involvement of nucleoside transporters. ADO, adenosine; AMP, adenosine monophosphate; ATP, adenosine triphosphate; HX, hypoxanthine; 5′N, 5′-nucleotidase; NBT2, hypoxanthine-preferring nucleobase transporter; NTPDase, ectonucleoside triphosphate diphosphohydrolase; (a) Nagase et al., 22 (b) Iandiev et al., 40 (c) Lutty and McLeod, 37 (d) Robillard et al., 32 (e) Sinclair et al., 7 (f) Berry and Hare. 1
In conclusion, hypoxanthine is produced in Müller cells from adenosine, and ENT2 mainly contributes to the uptake from the extracellular compartment (Fig. 9). Moreover, hypoxanthine in the retina is eliminated via ENT2 and/or an Na+-independent putative hypoxanthine transporter at the inner BRB (Fig. 9). Our findings provide valuable information to help modulate the hypoxanthine concentration in the retina and the retinal pathophysiologic effect induced by oxidative stress. 
Supplementary Materials
References
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Footnotes
 Supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Sciences.
Footnotes
3  These authors contributed equally to the work presented here and should therefore be regarded as equivalent authors.
Footnotes
 Disclosure: S. Akanuma, None; T. Soutome, None; E. Hisada, None; M. Tachikawa, None; Y. Kubo, None; K. Hosoya, None
Figure 1
 
Typical HPLC chromatograms of samples of TR-MUL5 cells (B) and TR-MUL5 cell-cultured medium (C). TR-MUL5 cells were incubated in extracellular fluid (ECF) buffer containing 0.5 μCi/mL [3H]adenosine at 37°C for 5 minutes and the efflux of 3H-labeled compounds was initiated by applying 37°C-warmed ECF buffer (culture medium). After incubation for 3 hours, culture medium was collected and TR-MUL5 cells were lysed. These samples were deproteinized before being used as HPLC samples. (A, D) Typical chromatograms of [3H]hypoxanthine and [3H]adenosine, respectively. Closed and open arrowheads were indicated as the typical retention times of [3H]hypoxanthine and [3H]adenosine, respectively.
Figure 1
 
Typical HPLC chromatograms of samples of TR-MUL5 cells (B) and TR-MUL5 cell-cultured medium (C). TR-MUL5 cells were incubated in extracellular fluid (ECF) buffer containing 0.5 μCi/mL [3H]adenosine at 37°C for 5 minutes and the efflux of 3H-labeled compounds was initiated by applying 37°C-warmed ECF buffer (culture medium). After incubation for 3 hours, culture medium was collected and TR-MUL5 cells were lysed. These samples were deproteinized before being used as HPLC samples. (A, D) Typical chromatograms of [3H]hypoxanthine and [3H]adenosine, respectively. Closed and open arrowheads were indicated as the typical retention times of [3H]hypoxanthine and [3H]adenosine, respectively.
Figure 2
 
Time-course (A) and concentration-dependence (B) of [3H]adenosine uptake by TR-MUL5 cells. (A) [3H]Adenosine uptake (13 nM) was measured at 37°C for the indicated times. The solid line was fitted using a nonlinear least-squares regression analysis program. Each point represents the mean ± SEM (n = 4). (B) Adenosine uptake was measured at 37°C for 3 minutes. [3H]Adenosine (13 nM) was present in all uptake measurements as a tracer, and the concentration of adenosine was varied using unlabeled adenosine. The solid line was fitted using a nonlinear least-squares regression analysis program. Each point represents the mean ± SEM (n = 4).
Figure 2
 
Time-course (A) and concentration-dependence (B) of [3H]adenosine uptake by TR-MUL5 cells. (A) [3H]Adenosine uptake (13 nM) was measured at 37°C for the indicated times. The solid line was fitted using a nonlinear least-squares regression analysis program. Each point represents the mean ± SEM (n = 4). (B) Adenosine uptake was measured at 37°C for 3 minutes. [3H]Adenosine (13 nM) was present in all uptake measurements as a tracer, and the concentration of adenosine was varied using unlabeled adenosine. The solid line was fitted using a nonlinear least-squares regression analysis program. Each point represents the mean ± SEM (n = 4).
Figure 3
 
Effect of sodium replacement and several inhibitors on [3H]adenosine uptake by TR-MUL5 cells. [3H]Adenosine uptake (13 nM) was performed at 37°C for 3 minutes in the absence or presence (control) of Na+, or in the absence (control) or presence of inhibitors. Each column represents the mean ± SEM (n = 4–8). **P < 0.01, significantly different from the control. DMSO, dimethylsulfoxide.
Figure 3
 
Effect of sodium replacement and several inhibitors on [3H]adenosine uptake by TR-MUL5 cells. [3H]Adenosine uptake (13 nM) was performed at 37°C for 3 minutes in the absence or presence (control) of Na+, or in the absence (control) or presence of inhibitors. Each column represents the mean ± SEM (n = 4–8). **P < 0.01, significantly different from the control. DMSO, dimethylsulfoxide.
Figure 4
 
mRNA expression of ENTs and CNTs in TR-MUL5 cells and primary-cultured rat Müller cells. RT-PCR analysis was performed in the presence (+) or absence (−) of RT using specific primers for rat ENT1–3 and CNT1–3. Rat lung, brain, kidney, and liver were used as respective positive controls.
Figure 4
 
mRNA expression of ENTs and CNTs in TR-MUL5 cells and primary-cultured rat Müller cells. RT-PCR analysis was performed in the presence (+) or absence (−) of RT using specific primers for rat ENT1–3 and CNT1–3. Rat lung, brain, kidney, and liver were used as respective positive controls.
Figure 5
 
In vivo [3H]hypoxanthine elimination from rat vitreous humor after vitreous bolus injection. (A) Time profile of [3H]hypoxanthine and [14C]D-mannitol in the vitreous humor after vitreous bolus injection. Open circles and closed triangles represent the concentration in the dialysate of [3H]hypoxanthine and [14C]D-mannitol, respectively. Each point represents the mean ± SEM (n = 5). (B) Elimination rate constants (β) of [3H]hypoxanthine (open column) and [14C]D-mannitol (closed column) during the terminal phase. Each column represents the mean ± SEM (n = 5). **P < 0.01, significant difference.
Figure 5
 
In vivo [3H]hypoxanthine elimination from rat vitreous humor after vitreous bolus injection. (A) Time profile of [3H]hypoxanthine and [14C]D-mannitol in the vitreous humor after vitreous bolus injection. Open circles and closed triangles represent the concentration in the dialysate of [3H]hypoxanthine and [14C]D-mannitol, respectively. Each point represents the mean ± SEM (n = 5). (B) Elimination rate constants (β) of [3H]hypoxanthine (open column) and [14C]D-mannitol (closed column) during the terminal phase. Each column represents the mean ± SEM (n = 5). **P < 0.01, significant difference.
Figure 6
 
Effect of inhibitors on the elimination rate constant difference between [3H]hypoxanthine and [14C]D-mannitol during the terminal phase. Each inhibitor was perfused in the microdialysis probe. Each column represents the mean ± SEM (n = 3–6). *P < 0.05, significantly different from the control. Percentage of control was calculated as follows: (β value of [3H]hypoxanthine − β value of [14C]D-mannitol in the presence of inhibitor)/(β value of [3H]hypoxanthine − β value of [14C]D-mannitol in the absence of inhibitor) × 100.
Figure 6
 
Effect of inhibitors on the elimination rate constant difference between [3H]hypoxanthine and [14C]D-mannitol during the terminal phase. Each inhibitor was perfused in the microdialysis probe. Each column represents the mean ± SEM (n = 3–6). *P < 0.05, significantly different from the control. Percentage of control was calculated as follows: (β value of [3H]hypoxanthine − β value of [14C]D-mannitol in the presence of inhibitor)/(β value of [3H]hypoxanthine − β value of [14C]D-mannitol in the absence of inhibitor) × 100.
Figure 7
 
Time-course (A) and concentration-dependence (B) of [3H]hypoxanthine uptake by TR-iBRB2 cells. (A) [3H]Hypoxanthine uptake (16 nM) was measured at 37°C for the indicated times. The solid line was fitted using a nonlinear least-squares regression analysis program. Each point represents the mean ± SEM (n = 3). (B) Hypoxanthine uptake was measured at 37°C for 5 minutes. [3H]Hypoxanthine (16 nM) was present in all uptake measurements as a tracer, and the concentration of hypoxanthine was varied using unlabeled hypoxanthine. The solid line was fitted using a nonlinear least-squares regression analysis program. Each point represents the mean ± SEM (n = 3).
Figure 7
 
Time-course (A) and concentration-dependence (B) of [3H]hypoxanthine uptake by TR-iBRB2 cells. (A) [3H]Hypoxanthine uptake (16 nM) was measured at 37°C for the indicated times. The solid line was fitted using a nonlinear least-squares regression analysis program. Each point represents the mean ± SEM (n = 3). (B) Hypoxanthine uptake was measured at 37°C for 5 minutes. [3H]Hypoxanthine (16 nM) was present in all uptake measurements as a tracer, and the concentration of hypoxanthine was varied using unlabeled hypoxanthine. The solid line was fitted using a nonlinear least-squares regression analysis program. Each point represents the mean ± SEM (n = 3).
Figure 8
 
Effect of sodium replacement and several inhibitors on [3H]hypoxanthine uptake by TR-iBRB2 cells. [3H]Hypoxanthine uptake (16 nM) was performed at 37°C for 5 minutes in the absence or presence (control) of Na+, or in the absence (control) or presence of inhibitors. Each column represents the mean ± SEM (n = 3–6). *P < 0.05, **P < 0.01, significantly different from the control.
Figure 8
 
Effect of sodium replacement and several inhibitors on [3H]hypoxanthine uptake by TR-iBRB2 cells. [3H]Hypoxanthine uptake (16 nM) was performed at 37°C for 5 minutes in the absence or presence (control) of Na+, or in the absence (control) or presence of inhibitors. Each column represents the mean ± SEM (n = 3–6). *P < 0.05, **P < 0.01, significantly different from the control.
Figure 9
 
Proposed mechanism for the production of hypoxanthine in Müller cells and its elimination across the inner BRB, and the involvement of nucleoside transporters. ADO, adenosine; AMP, adenosine monophosphate; ATP, adenosine triphosphate; HX, hypoxanthine; 5′N, 5′-nucleotidase; NBT2, hypoxanthine-preferring nucleobase transporter; NTPDase, ectonucleoside triphosphate diphosphohydrolase; (a) Nagase et al., 22 (b) Iandiev et al., 40 (c) Lutty and McLeod, 37 (d) Robillard et al., 32 (e) Sinclair et al., 7 (f) Berry and Hare. 1
Figure 9
 
Proposed mechanism for the production of hypoxanthine in Müller cells and its elimination across the inner BRB, and the involvement of nucleoside transporters. ADO, adenosine; AMP, adenosine monophosphate; ATP, adenosine triphosphate; HX, hypoxanthine; 5′N, 5′-nucleotidase; NBT2, hypoxanthine-preferring nucleobase transporter; NTPDase, ectonucleoside triphosphate diphosphohydrolase; (a) Nagase et al., 22 (b) Iandiev et al., 40 (c) Lutty and McLeod, 37 (d) Robillard et al., 32 (e) Sinclair et al., 7 (f) Berry and Hare. 1
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