AJP - Endo Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Endocrinol Metab 290: E968-E975, 2006. First published December 20, 2005; doi:10.1152/ajpendo.00160.2005
0193-1849/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/5/E968    most recent
00160.2005v1
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 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 ISI Web of Science (6)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kato, R.
Right arrow Articles by Tamai, I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kato, R.
Right arrow Articles by Tamai, I.

Characterization of novel Na+-dependent nucleobase transport systems at the blood-testis barrier

Ryo Kato,1,2 Tomoji Maeda,1 Toshihiro Akaike,2 and Ikumi Tamai1

1Faculty of Pharmaceutical Sciences, Department of Molecular Biopharmaceutics, Tokyo University of Science, Noda, Chiba; and 2Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Kanagawa, Japan

Submitted 3 November 2005 ; accepted in final form 12 December 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In the testis, nucleosides and nucleobases are important substrates of the salvage pathway for nucleotide biosynthesis, and one of the roles of Sertoli cells is to provide nutrients and metabolic precursors to spermatogenic cells located within the blood-testis barrier (BTB). We have already shown that concentrative and equilibrative nucleoside transporters are expressed and are functional in primary-cultured rat Sertoli cells as a BTB model, but little is known about nucleobase transport at the BTB or about the genes encoding specific nucleobase transporters in mammalian cells. In the present study, we examined the uptake of purine ([3H]guanine) and pyrimidine ([3H]uracil) nucleobases by primary-cultured rat Sertoli cells. The uptake of both nucleobases was time and concentration dependent. Kinetic analysis showed the involvement of three different transport systems in guanine uptake. In contrast, uracil uptake was mediated by a single Na+-dependent high-affinity transport system. Guanine uptake was inhibited by other purine nucleobases but not by pyrimidine nucleobases, whereas uracil uptake was inhibited only by pyrimidine nucleobases. In conclusion, it was suggested that there might be purine- or pyrimidine-selective nucleobase transporters in rat Sertoli cells.

nucleoside; Sertoli cells; transporter

NUCLEOTIDES PLAY A KEY ROLE as precursors of DNA and RNA, as activated intermediates in many biosynthetic processes, and as metabolic regulators. In general, nucleotides are synthesized via two routes, i.e., the de novo pathway and the salvage pathway (6). In de novo biosynthesis of purine nucleotides, the heterocyclic ring is built up from beta-D-phosphoribosylamine using six different precursors to generate IMP, and then IMP is used for the production of AMP and GMP. The pyrimidine ring is preformed as orotate and then reacts with {alpha}-D-ribose 5-phosphoribosyl-1-pyrophosphate (PRPP) to give orotidylate. On the other hand, the salvage pathway is advantageous since degradation products of nucleic acids can be recycled rather than destroyed, which is much less costly than the energy-demanding reactions of the de novo pathway. Both nucleobases and nucleosides are used for salvage nucleotide biosynthesis. A part of the salvage route involves the reaction of purine and pyrimidine nucleosides with ribose 1-phosphate. The reaction is catalyzed by a nucleoside phosphorylase, and the resultant ribonucleosides are converted into the corresponding 5'-nucleotides by a cellular kinase. On the other hand, purine nucleobases, which are produced by hydrolytic degradation of nucleotides and nucleic acids, react with PRPP to provide the corresponding purine ribonucleotides. Adenine phosphoribosyltransferase is specific for the reaction with adenine, whereas hypoxanthine-guanine phosphoribosyltransferase catalyses the formation of IMP and GMP. Similarly, there are phosphoribosyltransferases for the pyrimidine nucleobases.

Plasma membrane transport processes for nucleosides and nucleobases play key roles as the first step of salvage nucleotide synthesis. In human and other mammalian cells and tissues, uptake of nucleosides is mediated by members of the concentrative nucleoside transporter (CNT) and equilibrative nucleoside transporter (ENT) families (3, 17). Human (h) and rat (r)CNT1 and CNT2 both transport uridine and also selectively transport pyrimidine (hCNT1 and rCNT1) or purine (hCNT2 and rCNT2) nucleosides (9, 25, 39, 40). In contrast, hCNT3 and mouse (m)CNT3 accept both purine and pyrimidine nucleosides as substrates (38). Human and rat ENT1 and ENT2, which exhibit broad selectivity for purine and pyrimidine nucleosides, are distinguished functionally by differential sensitivity to nitrobenzylthioinosine (NBMPR), which has IC50 values of 1.4 nM and >10 µM for ENT1 and ENT2, respectively (52, 57).

On the other hand, there are a few reports about nucleobase transport mediated by nucleoside transporters. Recombinant hCNT1, rCNT1, hCNT2, rCNT2, and hCNT3 do not transport hypoxanthine (58), and recombinant hCNT3 and mCNT3 do not transport uracil (38). Only the hENT2 and rENT2 isoform has the dual capability of transporting both nucleosides and nucleobases (58).

In addition to the shared mechanisms of transport of nucleobases and nucleosides, it was suggested that there are independent transport processes specific for nucleosides or nucleobases (12, 20). Nucleobase transport has been studied most extensively in microorganisms. The processes operating in mammalian cells are less well defined, although both equilibrative (Na+-independent) and concentrative (Na+-dependent) nucleobase-specific transport activities have been described in a variety of cells and tissues. Equilibrative nucleobase transport has been found in human erythrocytes, human T lymphoblastoid cells, LLC-PK1 cells, rabbit cornea, and S49 mouse-derived lymphoma cells (16, 18, 32, 50), whereas concentrative nucleobase transport occurs in kidney, intestine, placenta, and choroid plexus (4, 18, 43, 48, 53). However, little is known about the molecular basis of nucleobase transport in mammalian cells, and no cDNA encoding a functional mammalian nucleobase transporter has been cloned. Although human and rat Na+-dependent ascorbate transporter (SVCT)1 and SVCT2 have been defined as orthologs of bacterial nucleobase transporters in mammals (24), it was reported that human SVCT1 does not transport nucleobases or nucleosides (51).

Throughout the mammalian spermatogenic pathway, differentiating spermatogenic cells move from the basal to the luminal compartment of the testis across the blood-testis barrier (BTB), which is formed mainly by Sertoli cells. Sertoli cells and spermatogenic cells are in close contact, which is essential for the proliferation and differentiation of spermatogenic cells (31, 42). Sertoli cells form tight junctions and act as a barrier to protect developing germ cells against harmful agents while allowing the passage of nutrients, such as nucleobases, nucleosides, amino acids, glucose, and lactic acid, from circulating blood for spermatogenesis (5, 26). For the transport of these nutrients, several membrane transporters are expressed in the BTB (2). Because mitosis and meiosis occur during the development of germ cells, an efficient route for nucleotide biosynthesis is required to promote spermatogenesis. In the rat testis, the specific activity of purine phosphoribosyltransferase is higher than that of amidophosphoribosyltransferase (1, 36). Rat testis also has a higher activity of uridine kinase, the rate-limiting enzyme of the pyrimidine salvage pathway, than other tissues (23). These reports suggest that the salvage pathway may be dominant and that nucleotides are likely to be synthesized from nucleosides and nucleobases supplied from circulating blood via transporters in the BTB.

We have already shown that primary-cultured rat Sertoli cells express ENT1, ENT2, ENT3, CNT1, CNT2, and CNT3 and exhibit nucleoside uptake activities mediated by ENT1-, ENT2-, and CNT-type transporters (27). However, there is no information about the transport of nucleobases, which are used for salvage biosynthesis of nucleotides, at the spermatogenic and Sertoli cells. In the present study, we investigated the mechanism of the supply of nucleobases from the bloodstream to seminiferous tubules and spermatogenic cells by measuring the transport of nucleobases in primary-cultured rat Sertoli cells as a model of the BTB.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. [8-3H]guanine (15 Ci/mmol) and [5,6-3H]uracil (37.3 Ci/mmol) were purchased from American Radiolabeled Chemicals (St. Louis, MO) and PerkinElmer Life Sciences (Boston, MA), respectively. [5-3H]uridine (16.2 Ci/mmol) was purchased from Moravek Biochemicals (Brea, CA). Collagenase and trypsin were obtained from Sigma-Aldrich (St. Louis, MO) and BD Microbiology Systems (Sparks, MD), respectively. All other reagents were purchased from Sigma-Aldrich and Wako Pure Chemical (Osaka, Japan).

Preparation and primary culture of rat Sertoli cells. Sertoli cells were isolated from 20-day-old Donryu rats (Saitama Experimental Animal Supply, Saitama, Japan) according to the method reported by Nagao (36) and Shiratsuchi et al. (45). Briefly, testes were decapsulated, and seminiferous tubules were gently expressed and then incubated in 35 ml of 0.25% collagenase in PBS for 20 min at 37°C with occasional stirring. The seminiferous tubules were washed with serum-free F12-L15 medium and then incubated with occasional gentle pipetting in 35 ml of 0.25% trypsin in PBS for 20 min at 37°C. F12-L15 medium was composed of a 1:1 mixture of Ham's F-12 medium (MP Biomedicals, Irvine, CA) and L-15 medium (MP Biomedicals), containing 15 mM HEPES, 10 U/ml penicillin, 0.1 mg/ml streptomycin, and 10% FBS (Invitrogen, Carlsbad, CA). Trypsin treatment was terminated by adding 5 ml of FBS and 10 ml of F12-L15 medium containing 10% FBS. The resultant cell suspension was filtered two times through four sheets of gauze to remove cell aggregates and tissue debris, after which the cells were collected by centrifugation (1,200 rpm x 10 min). The cells were suspended in 30 ml of F12-L15 medium containing 10% FBS and washed by centrifugation (900 rpm x 10 min). Finally, the cells were suspended in F12-L15 medium containing 10% FBS and passed once through nylon mesh (70 µm; BD Bioscience Discovery Labware, Bedford, MA). The isolated Sertoli cells thus obtained were cultured, and they adhered to the culture dish (353003; BD Bioscience Discovery Labware). Cells were grown in F12-L15 medium containing 1 µg/ml norepinephrine in a humidified incubator at 32.5°C for 3 days and at 37°C for 3 days. Sertoli cells in culture were isolated after removal with pipettes of the spermatogenic cells floating on the surface of the coculture of testicular cells. About 90% of the cells adhering to the culture dish were Sertoli cells, as judged from staining with Nile red (Molecular Probes, Eugene, OR), which is a marker for Sertoli cells (33). After seeding (6 days), the cultures reached confluence and were used for the transport experiments.

Nucleoside and nucleobase transport experiments. For the transport experiments using primary-cultured rat Sertoli cells, cells were harvested with a cell scraper and suspended in transport medium containing (in mM) 137 NaCl, 5 KCl, 0.39 NaHCO3, 0.44 KH2PO4, 0.95 CaCl2, 0.8 MgSO4, 25 D-glucose, and 10 HEPES, adjusted to pH 7.4. The cell suspension was preincubated at 37°C for 20 min in the transport medium and then centrifuged; the resultant cell pellets were resuspended in transport medium containing 3H-labeled nucleobase or 3H-labeled nucleoside to initiate uptake. After a designated time, the cell suspension (200 µl) was diluted with 800 µl of ice-cold transport medium and centrifuged immediately (10,000 rpm x 1 min) to terminate the uptake reaction. Next, the cells were resuspended in ice-cold transport medium and obtained as the pellet after centrifugation. The resultant cell pellets were solubilized in 1 M NaOH, and the cell-associated radioactivity was measured by means of a liquid scintillation counter (Aloka, Tokyo, Japan) with Cleasol-1 (Nacalai Tesque, Kyoto, Japan) as a liquid scintillation fluid. Na+-free transport medium was prepared by replacing 137 mM NaCl and 0.39 mM NaHCO3 of the standard transport medium with 137 mM N-methyl-D-glucamine (NMG) and 0.39 mM KHCO3, respectively, and was used to assess the uptake in the absence of Na+. NBMPR was dissolved in DMSO and was diluted to desired concentrations with transport medium such that the final DMSO concentration was 0.5% or less.

Analytical methods. Cellular protein content was determined according to the method of Lowry et al. (31) with BSA as the standard. Cellular uptake was usually expressed as cell-to-medium ratio (µl/mg protein), which was an indicator of transport activity independent of the concentration of substrate. The cell-to-medium ratio was obtained by dividing the uptake amount by the concentration of test compound in the transport medium. The apparent kinetic parameters Km (Michaelis constant) and Vmax (maximal transport rate) of nucleobase uptake by Sertoli cells were calculated by nonlinear least squares regression curve fitting according to the following Michaelis-Menten- type equations, where v and [s] are the transport rate and substrate concentration, respectively. The value of Kd was obtained from the 3H-labeled nucleobase uptake in the presence of excess nonlabeled nucleobase (100 µM guanine or 200 µM uracil).

Formula 1(1)

Formula 2(2)

When the data were fitted to Eq. 2 with two saturable transport components, the indexes 1 and 2 indicate the high- and low-affinity components, respectively. Nonlinear regression analysis was performed using the MULTI program (56).

All data were expressed as means ± SE, and statistical analysis was performed with Student's t-test. The criterion of significance was taken to be P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Nucleobase uptake by nucleoside transporters in Sertoli cells. We evaluated the involvement of nucleoside transporters in the transport of nucleobases in Sertoli cells by using uridine as the substrate (Fig. 1). [3H]uridine uptake was decreased to 77% in the absence of Na+, suggesting that primary-cultured rat Sertoli cells contain both Na+-dependent and -independent nucleoside transport systems. NBMPR, an ENT-specific inhibitor, reduced [3H]uridine uptake in a concentration-dependent manner under Na+-free conditions, indicating contributions of ENT1 and ENT2 (Fig. 1). To examine whether nucleobases share transporters with nucleosides, the inhibitory effects of various nucleobases on [3H]uridine uptake were measured (Fig. 1). The [3H]uridine uptake was decreased significantly in the presence of 0.33–1 mM adenine, cytosine, guanine, thymine, and uracil to ~71–79% of control uptake in the presence of Na+, although various nucleosides reduced [3H]uridine uptake to 20–40% of control uptake in the same concentration range (0.33–1 mM; see Ref. 27). These results indicated that Sertoli cells contain ENT1, ENT2, and CNTs and that these nucleoside transporters might contribute to nucleobase transport.


Figure 1
View larger version (11K):
[in this window]
[in a new window]
  		Fig. 1. Inhibitory effect of nitrobenzylthioinosine (NBMPR) and nucleobases on [3H]uridine uptake by primary-cultured rat Sertoli cells. Uptake of [3H]uridine was measured for 3 min in primary-cultured rat Sertoli cells in the absence or presence of Na+. The concentration of uridine was 70 nM. The results are shown as a percentage of control uptake in the presence of Na+. In the Na+-free condition, all Na+ was replaced with N-methyl-D-glucamine (NMG+). NBMPR was used at 0.1 and 500 µM in the absence of Na+, and guanine (G) was used at 500 µM. The concentration of the other nucleobases [adenine (A), cytosine (C), thymine (T), and uridine (U)] was 1 mM. Each column represents the mean ± SE (n = 3 or 4 experiments). Significant differences from the control uptake in the presence (*) and absence ({dagger}) of Na+ (P < 0.05).

 
Nucleobase uptake by Sertoli cells. To characterize the mechanisms of uptake of nucleobases by primary-cultured rat Sertoli cells, [3H]guanine and [3H]uracil were used as model purine and pyrimidine nucleobases, respectively. Figure 2 shows the uptake of [3H]guanine and [3H]uracil by primary-cultured rat Sertoli cells in the presence of Na+. Because the uptakes of both guanine and uracil by primary-cultured rat Sertoli cells increased linearly up to 5 and 10 min, respectively, initial uptake was analyzed at 3 min in the subsequent studies. The uptakes of guanine and uracil exhibited saturation as the concentration was increased from 40 nM to 5 µM in Na+ buffer (Figs. 3A and 4A). To confirm that there are Na+-independent guanine transport processes in Sertoli cells, we analyzed transport both in the presence and absence of Na+. Those results indicated the involvement of two or three saturable transport systems (Fig. 3). An Eadie-Hofstee plot for guanine uptake by the cells, after correction for nonsaturable uptake (4.91 ± 0.44 µl/mg protein) estimated as the first-order rate constant in the presence of nonlabeled 100 µM guanine (Fig. 3A), indicated the involvement of three saturable transport systems (Fig. 3, B and D). In addition, the kinetic parameters obtained by nonlinear least squares regression analysis were as follows: high-affinity transport system (Km1 = 0.08 ± 0.04 µM, Vmax1 = 2.02 ± 0.76 pmol·3 min–1·mg protein–1), low-affinity transport system (Km2 = 3.15 ± 1.29 µM, Vmax2 = 18.1 ± 2.93 pmol·3 min–1·mg protein–1) in the presence of Na+. The kinetic parameters Km and Vmax in the absence of Na+ were 0.079 ± 0.032 µM and 1.00 ± 0.11 pmol·3 min–1·mg protein–1, respectively. Because the uptake of guanine was mediated by three different components, two concentrations of guanine (40 nM and 1 µM) were used in the following studies. The low-affinity guanine transport systems made a greater contribution at 1 µM (40%) than at 40 nM (16%) in Na+ buffer condition. The uptake of guanine increased linearly up to 5 min and then attained a steady state at both concentrations (Fig. 2A). In contrast, the uptake of the pyrimidine nucleobase uracil appeared to be mediated by one saturable component (Fig. 4B), and the kinetic parameters obtained after correction for nonsaturable uptake (2.03 ± 0.10 µl/mg protein, estimated from the first-order rate constant in the presence of nonlabeled 200 µM uracil) were Km = 0.29 ± 0.09 µM and Vmax = 5.00 ± 0.59 pmol·3 min–1·mg protein–1 using Eq. 1. These results indicate that both high- and low-affinity guanine transport systems and a high-affinity uracil transport system operate in Sertoli cells.


Figure 2
View larger version (8K):
[in this window]
[in a new window]
  		Fig. 2. Time course of 3H-labeled nucleobase uptake by primary-cultured rat Sertoli cells. Uptake of 40 nM (bullet) and 1 µM ({circ}) [3H]guanine (A) and 8 nM [3H]uracil (B) by primary-cultured rat Sertoli cells was measured at 37°C. Cellular uptake is expressed as cell-to-medium ratio. Each result represents the mean ± SE (n = 3 or 4). If the SE. is not shown, it is smaller than the symbol.

 

Figure 3
View larger version (16K):
[in this window]
[in a new window]
  		Fig. 3. Concentration dependence of guanine uptake by primary-cultured rat Sertoli cells. A: total (bullet), saturable (dotted line), and nonsaturable (broken line) uptakes of guanine by primary-cultured rat Sertoli cells were measured at 37°C for 3 min. B: guanine uptake at 37°C for 3 min shown as an Eadie-Hofstee plot after correction for nonsaturable uptake evaluated from the first-order rate constant obtained in the presence of 100 µM unlabeled guanine, as described in RESULTS. V, transport rate; S, substrate concentration. respectively C: total (bullet), Na+-independent (dotted line), and nonsaturable (broken line) uptake of guanine measured at 37°C for 3 min in Na+-free buffer. D: Eadie-Hofstee plots of guanine uptake at 37°C for 3 min after correction for nonsaturable uptake evaluated from the first-order rate constant obtained in the presence of 100 µM unlabeled guanine. Each result represents the mean ± SE (n = 3 or 4). If the SE is not shown, it is smaller than the symbol.

 

Figure 4
View larger version (10K):
[in this window]
[in a new window]
  		Fig. 4. Concentration dependence of uracil uptake by primary-cultured rat Sertoli cells. A: total (bullet), carrier-mediated (dotted line), and nonsaturable (broken line) uptake of uracil by primary-cultured rat Sertoli cells at 37°C for 3 min. B: Eadie-Hofstee plot of uracil uptake at 37°C for 3 min after correction for nonsaturable uptake evaluated from the first-order rate constant obtained in the presence of 200 µM unlabeled uracil, as described in RESULTS. Each result represents the mean ± SE (n = 3 or 4). If the SE is not shown, it is smaller than the symbol.

 
Na+ dependence of nucleobase uptake by Sertoli cells. Na+-dependent and -independent nucleobase transport systems have been reported in various mammalian cells (20, 12), and the uptake of 3H-labeled nucleobases was measured in the presence and absence of Na+ in the transport medium (Fig. 5). When extracellular Na+ was replaced with NMG+ at equimolar concentration, the uptake of [3H]guanine (40 nM) was reduced to 30% of control (Fig. 5A). In addition, at a high concentration of guanine (1 µM), the uptake of [3H]guanine (1 µM) was reduced to 50% when extracellular Na+ was replaced with NMG+ at equimolar concentration (Fig. 5A). These results further indicated that the uptake of guanine was mediated by both Na+-dependent and -independent nucleobase transport systems in primary-cultured rat Sertoli cells. [3H]uracil uptake was also reduced to 3.9% in the absence of Na+ (Fig. 5B), suggesting that there is one Na+-dependent transport system for uracil in these cells.


Figure 5
View larger version (10K):
[in this window]
[in a new window]
  		Fig. 5. Na+ dependence of [3H]nucleobase uptake by primary-cultured rat Sertoli cells. Uptake of 40 nM and 1 µM [3H]guanine (A) and 8 nM [3H]uracil (B) was measured at 37°C for 3 min in primary-cultured rat Sertoli cells. The results are shown as percentage of control uptake in the presence of Na+. In the Na+-free condition, all Na+ was replaced with NMG+. Each column represents the mean ± SE (n = 3 or 4). and *Significant difference from the uptake in the presence of Na+ (P < 0.05).

 
Inhibitory effects of several compounds on nucleobase uptake by Sertoli cells. Transporter molecules involved in the transport of nucleobases have not yet been cloned, except for ENT2. To analyze whether guanine and uracil transport is mediated by known transporters, the inhibitory effects of substrates for these transporters on nucleobase uptake were measured (Table 1). p-Aminohippuric acid, estrone 3-sulfate (E13S), and tetraethylammonium are typical substrates of organic anion transporters (OATs; see Ref. 28), organic anion transporting peptides (OATPs; see Refs. 22 and 47), and organic cation transporters (OCTs; see Refs. 45 and 46), respectively. Arginine, dopamine, glucose, norepinephrine, and thiamine were used as substrates for cationic amino acid transporters (13), dopamine transporter (41), glucose transporters (55, 49), neurotransmitter transporters (7), and thiamine transporter (15). Because SVCTs are mammalian orthologs of bacterial nucleobase transporters (24), ascorbate was used as a substrate of SVCTs. Concentrations of inhibitors were fixed at 1 mM, which should be high enough to inhibit the respective transporters, considering the respective affinities. As shown in Table 1, no compounds inhibited [3H]guanine uptake at the low guanine concentration (40 nM), and only E13S was inhibitory at the high concentration (1 µM). [3H]uracil uptake was not reduced by any of the compounds examined in this assay (Table 1). These results suggested that the transport systems for nucleobases are distinct from previously known transporters.


View this table:
[in this window]
[in a new window]
  		Table 1. Inhibitory effects of various compounds on [3H]nucleobase uptake by primary-cultured rat Sertoli cells

 
Structural selectivity of nucleobase transport systems in Sertoli cells. In the case of nucleoside transporters, rCNT1 and rCNT2 preferentially transport pyrimidine and purine nucleosides, respectively (9, 25), whereas CNT3 and ENTs transport both purine and pyrimidine nucleosides (3, 38). To examine whether nucleobase transport systems show selectivity for purine or pyrimidine nucleobase structure, the inhibitory effects of several nucleobases on [3H]guanine and [3H]uracil uptake were measured (Table 2). Purine nucleobases adenine, guanine, and hypoxanthine, significantly inhibited uptake of 40 nM [3H]guanine to 17.1, 19, and 52.7% of control uptake, respectively, although cytosine, thymine, and uracil exhibited no inhibition (Table 2). In addition, only purine nucleobases showed similar inhibition of the uptake of guanine at higher concentration (Table 2). In contrast, [3H]uracil uptake was inhibited by pyrimidine nucleobases but not by purine nucleobases adenine, guanine, and hypoxanthine (Table 2).


View this table:
[in this window]
[in a new window]
  		Table 2. Inhibitory effect of various nucleobases on 3H-labeled nucleobase uptake by primary-cultured rat Sertoli cells

 
A similar inhibition assay was performed using various nucleosides (Table 3). When purine and pyrimidine nucleosides were used as inhibitors, uptake of 40 nM [3H]guanine was significantly inhibited by adenosine, guanosine, and inosine to 20, 46.7, and 48.2% of the control (Table 3). However, pyrimidine nucleosides failed to inhibit [3H]guanine uptake (Table 3). Similar inhibitory patterns were observed at the higher concentration (1 µM) of guanine (Table 3). On the other hand, [3H]uracil uptake was inhibited by cytidine, thymidine, and uridine, although purine nucleosides exhibited no inhibition (Table 3).


View this table:
[in this window]
[in a new window]
  		Table 3. Inhibitory effect of various nucleosides on 3H-labeled nucleobase uptake by primary-cultured rat Sertoli cells

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the mammalian spermatogenic pathway, one of the roles of Sertoli cells is to provide nutrients and metabolic precursors to spermatogenic cells located within the BTB (25, 31). Because large amounts of nucleotides are required for spermatogenesis, salvage biosynthesis of nucleotides using nucleobases and nucleosides transported across the BTB is expected to play a key role. We have already demonstrated molecular and functional expression of nucleoside transporters ENT1, ENT2, ENT3, CNT1, CNT2, and CNT3 in primary-cultured rat Sertoli cells (27). However, nucleobases are also important materials for the salvage pathway (6), although little is known about nucleobase transport at the molecular level in mammalian cells (20, 12). Accordingly, we investigated the transport mechanism of both purine and pyrimidine nucleobases at the BTB using primary-cultured rat Sertoli cells.

Concentration-dependent inhibition of the uptake of uridine by NBMPR suggested that rENT1 and rENT2 contributed 51 and 24% of total uridine uptake by primary-cultured rat Sertoli cells, respectively (Fig. 1). We have reported that transport activity of uridine was inhibited by NBMPR in the presence and absence of Na+ (27). Accordingly, it was thought that NBMPR could work in the presence and absence of Na+. Uridine uptake was inhibited by several nucleobases, although the extents of inhibition were less than those by nucleosides (27). One possible reason for this observation is that uptake of uridine is mainly (~70–0% of total uptake) mediated by nucleoside-specific transporters such as CNTs and ENT1, which cannot transport nucleobases (38, 58). As shown in Fig. 1, various nucleobases inhibited uridine uptake by ~20–30%, and the inhibitory pattern was consistent with the characteristics of rENT2, which transports both purine and pyrimidine nucleobases (58). However, because the affinity of nucleobases for ENT2 is low, and it was suggested that there are higher-affinity nucleobase transport systems than ENT2 in other epithelial tissues (12), it is likely that there are distinct transport systems for nucleosides and nucleobases in primary-cultured rat Sertoli cells.

As shown in Fig. 2, uptake of both guanine and uracil by rat Sertoli cells increased linearly. Uptake of guanine was saturable, and an Eadie-Hofstee plot showed that three different saturable components were involved, with kinetic parameters of Km1 = 0.08 ± 0.04 µM, Km2 = 3.15 ± 1.29 µM, and Km3 = 0.079 ± 0.032 µM (Fig. 3). Here, transports shown by Km1 and Km2 are Na+-dependent transport systems, whereas that of Km3 is a Na+-independent transport system. Because the physiological blood concentration of guanine is 0.4 µM in rats (54), the Na+-dependent high- and low-affinity and the Na+-independent guanine transport systems can be estimated by the kinetic parameters to mediate ~28, 34, and 14% of total uptake, respectively. Thus it is suggested that these two Na+-dependent guanine transport systems are mainly functional in vivo. At the lower concentration (40 nM), the uptake of guanine was mainly mediated by the Na+-dependent high-affinity transport system (49%). In addition, such Na+ dependence was observed at 1 µM guanine (Fig. 5A). At the higher concentration (1 µM), the uptake of guanine was mainly mediated by the Na+-dependent low-affinity transport system (40%), and the contributions of the Na+-dependent high-affinity and Na+-independent transport systems were 15 and 9.2%, respectively.

Uptake of uracil appeared to be mediated by a single saturable component, with Km = 0.29 µM (Fig. 4). This uptake was abrogated in the absence of extracellular Na+ (Fig. 5B). Because ENT2 is a Na+-independent nucleobase transporter (58), it appears that there is a Na+-dependent nucleobase transport system for uracil in primary-cultured rat Sertoli cells. There are no published kinetic parameters for guanine and uracil transport, although Ki values of guanine (0.6–6.4 µM) and uracil (2.9–64.6 µM) for hypoxanthine uptake were calculated (18, 19, 22, 43, 48, 53). These Ki values are ~10 times higher than the Km values of the high-affinity transport system for guanine and uracil obtained in the present study. Therefore, the transport systems in this study have higher affinities for guanine and uracil than previously known transporters. However, further transport studies to evaluate directly the transport of hypoxanthine are needed to compare with the previously reported nucleobase transport systems.

In this study, the involvement of a Na+-dependent low-affinity guanine transport system has been shown (Fig. 3). In addition, Km of the Na+-dependent low-affinity guanine transport system (3.15 µM) found in the present study is similar to that reported in LLC-PK1 cells (18).

rOATs, especially rOAT1 and rOAT3, transport nucleobase-related compounds such as 6-mercaptopurine and adefovir (11, 34). In addition, Chen and Nelson (10) suggested that rOCTs contribute to renal secretion of nucleoside analogs cytosine arabinoside and azidethymidine. To investigate the role of known transporters, including rOATs and rOCTs, we measured the inhibitory effects of several substrates of known transporters on guanine and uracil uptake (Table 1). However, none of the compounds examined showed significant inhibition on either guanine or uracil uptake at 1 mM, which is a higher concentration than the Km values of the respective transporters. Accordingly, it appears that there are specific transporters for nucleobases. Uptake of guanine at the higher concentration was slightly reduced by E13S. Several E13S transporters, OATP2 (37), OATP3 (8), and OATP4 (8), are expressed in rat Sertoli cells (2); but at present there is no information about nucleobase transport mediated by OATPs, so the contribution of these transporters to low-affinity guanine transport remains to be established.

It was reported previously that both purine and pyrimidine nucleobases equally inhibited the Na+-dependent uptake of hypoxanthine in mammals (18, 21). On the other hand, rat nucleoside transporters CNT1 and CNT2 preferentially transport pyrimidine and purine nucleosides, respectively (9, 25, 39, 40). In the present study, uptake of the purine nucleobase guanine was decreased only by purine nucleobases adenine, guanine, and hypoxanthine, whereas pyrimidine nucleobases caused no inhibition (Table 2). Accordingly, both the high- and low-affinity guanine transport systems are purine nucleobase selective. In contrast, only pyrimidine nucleobases cytosine, thymine, and uracil inhibited the uptake of uracil (Table 2). In conclusion, it was suggested that there might be purine- or pyrimidine-selective nucleobase transporters in rat Sertoli cells. These results suggest that there are multiple nucleobase transport systems that preferentially transport pyrimidine or purine nucleobases. However, further transport studies measuring the transport of each nucleobase, such as hypoxanthine, adenine, thiamine, and cytosine, are needed.

Guanine uptake was decreased only by purine nucleosides, and pyrimidine nucleosides did not inhibit the uptake of guanine at low or high concentration (Table 3). On the other hand, uptake of the pyrimidine nucleobase uracil was significantly inhibited only by pyrimidine nucleosides cytidine, thymidine, and uridine (Table 3). Because purine and pyrimidine nucleosides have a common nucleobase structure, these results suggest that nucleobase transport systems found in this study distinguish purine and pyrimidine nucleobases by recognizing nucleobase structure. Furthermore, these transporters may have the dual capability of transporting both nucleobases and the corresponding nucleosides, like ENT2 (58).

Analogs of purine and pyrimidine nucleobases are widely used in medicine, e.g., as antimetabolites to treat tumors, anti-viral compounds, immunosuppressants to prevent organ transplantation rejection, and agents to treat gout (14, 29). An understanding of nucleobase transport systems should be helpful to increase the effectiveness and specificity of such drugs and to reduce adverse effects.

In conclusion, this study has clarified the presence in primary-cultured rat Sertoli cells of nucleobase transport systems that are likely to be involved in trans-BTB substrate delivery for salvage nucleotide biosynthesis in spermatogenic cells and Sertoli cells. Because the nucleobase transport activities cannot be ascribed to known transporter molecules, further molecular identification is needed.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This investigation was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and by a grant from AstraZeneca.


   FOOTNOTES
 

Address for reprint requests and other correspondence: I. Tamai, Faculty of Pharmaceutical Sciences, Dept. of Molecular Biopharmaceutics, Tokyo Univ. of Science, 2641 Yamasaki, Noda, Chiba, 278-8510, Japan (e-mail: tamai{at}rs.noda.tus.ac.jp)

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. Adams A and Harkness RA. Developmental changes in purine phosphoribosyltransferases in human and rat tissues. Biochem J 160: 565–576, 1976.[ISI][Medline]
  2. Augustine LM, Markelewicz RJ, Boekelheide K, and Cherrington N. Xenobiotic and endobiotic transporter mRNA expression in the blood-testis barrier. Drug Metab Dispos 33: 182–189, 2005.[Abstract/Free Full Text]
  3. Baldwin SA, Beal PR, Yao SY, King AE, Cass CE, and Young JD. The equilibrative nucleoside transporter family, SLC29. Pflügers Arch 447: 735–743, 2004.[CrossRef][ISI][Medline]
  4. Barros LF. Hypoxanthine transport in the guinea pig and human placenta is a carrier-mediated process that does not involve nucleoside transporters. Am J Obset Gynecol 171: 111–117, 1994.[ISI][Medline]
  5. Bart J, Groen HJ, van der Graaf WT, Hollema H, Hendrikse NH, Vaalburg W, Sleijfer DT, and de Vries EG. An oncological view on the blood-testis barrier. Lancet Oncol 3: 357–363, 2002.[CrossRef][ISI][Medline]
  6. Blackburn GM and Gait MJ. Biosynthesis of nucleotides. In: Nucleic Acids in Chemistry and Biology. London, UK: IRL, 1990, p. 137–163.
  7. Blakely RD, De Felice LJ, and Hartzell HC. Molecular physiology of norepinephrine and serotonin transporters. J Exp Biol 196: 263–281, 1994.[Abstract/Free Full Text]
  8. Cattori V, van Montfoort JE, Stieger B, Landmann L, Meijer DK, Winterhalter KH, Meier PJ, and Hagenbuch B. Localization of organic anion transporting polypeptide 4 (Oatp4) in rat liver and comparison of its substrate specificity with Oatp1, Oatp2 and Oatp3. Pflügers Arch 443: 188–195, 2001.[CrossRef][ISI][Medline]
  9. Che M, Ortiz DF, and Arias IM. Primary structure and functional expression of a cDNA encoding the bile canalicular, purine-specific Na(+)-nucleoside cotransporter. J Biol Chem 270: 13596–13599, 1995.[Abstract/Free Full Text]
  10. Chen R and Nelson JA. Role of organic cation transporters in the renal secretion of nucleosides. Biochem Pharmacol 60: 215–219, 2000.[CrossRef][ISI][Medline]
  11. Cihlar T, Lin DC, Pritchard JB, Fuller MD, Mendel DB, and Sweet DH. The antiviral nucleotide analogs cidofovir and adefovir are novel substrates for human and rat renal organic anion transporter 1. Mol Pharmacol 56: 570–580, 1999.[Abstract/Free Full Text]
  12. de Koning H and Diallinas G. Nucleobase transporters. Mol Membr Biol 75: 75–94, 2000.
  13. Deves R and Boyd CA. Transporters for cationic amino acids in animal cells: discovery, structure, and function. Physiol Rev 78: 487–545, 1998.[Abstract/Free Full Text]
  14. Elion B. The purine path to chemotherapy. Science 244: 41–47, 1989.[Abstract/Free Full Text]
  15. Ganapathy V, Smith SB, and Prasad PD. SLC19: the folate/thiamine transporter family. Pflügers Arch 447: 641–646, 2004.[CrossRef][ISI][Medline]
  16. Gati WP, Paterson AR, Tyrrell DL, Cass CE, Moravek J, and Robins MJ. Nucleobase transporter-mediated permeation of 2',3'-dideoxyguanosine in human erythrocytes and human T-lymphoblastoid CCRF-CEM cells. J Biol Chem 267: 22272–22276, 1992.[Abstract/Free Full Text]
  17. Gray JH, Owen RP, and Giacomini KM. The concentrative nucleoside transporter family, SLC28. Pflügers Arch 447: 728–34, 2004.[CrossRef][ISI][Medline]
  18. Griffith DA, Doherty AJ, and Jarvis SM. Nucleoside transport in cultured LLC-PK1 epithelia. Biochim Biophys Acta 1106: 303–310, 1992.[Medline]
  19. Griffith DA and Jarvis SM. High affinity sodium-dependent nucleobase transport in cultured renal epithelial cells (LLC-PK1). J Biol Chem 268: 20085–20090, 1993.[Abstract/Free Full Text]
  20. Griffith DA and Jarvis SM. Characterization of a sodium-dependent concentrative nucleobase-transport system in guinea-pig kidney cortex brush-border membrane vesicles. Biochem J 303: 901–905, 1994.[Medline]
  21. Griffith DA and Jarvis SM. Nucleoside and nucleobase transport systems of mammalian cells. Biochim Biophys Acta 1286: 153–181, 1996.[Medline]
  22. Haddock PS. Evidence for sodium-dependent hypoxanthine uptake in isolated guinea pig ventricular myocytes: stimulation by extracellular Ni2+. Cardiovasc Res 30: 130–137, 1995.[CrossRef][ISI][Medline]
  23. Hagenbuch B and Meier PJ. Organic anion transporting polypeptides of the OATP/ SLC21 family: phylogenetic classification as OATP/SLCO superfamily, new nomenclature and molecular/functional properties. Pflügers Arch 447: 653–665, 2004.[CrossRef][ISI][Medline]
  24. Haugen TB, Hansson V, and Fritzson P. Cellular distribution, developmental changes and effects of cryptorchidism on uridine kinase in rat testis. J Reprod Fertil 83: 655–661, 1988.[Abstract]
  25. Hogue DL and Ling V. A human nucleobase transporter-like cDNA (SLC23A1): member of a transporter family conserved from bacteria to mammals. Genomics 59: 18–23, 1999.[CrossRef][ISI][Medline]
  26. Huang QQ, Yao SY, Ritzel MW, Paterson AR, Cass CE, and Young JD. Cloning and functional expression of a complementary DNA encoding a mammalian nucleoside transport protein. J Biol Chem 269: 17757–17760, 1994.[Abstract/Free Full Text]
  27. Jutte NH, Jansen R, Grootegoed JA, Rommerts FF, Clausen OP, and van der Molen HJ. Regulation of survival of rat pachytene spermatocytes by lactate supply from Sertoli cells. J Reprod Fertil 65: 431–438, 1982.[Abstract]
  28. Kato R, Maeda T, Akaike T, and Tamai I. Nucleoside transport at the blood-testis barrier studied with primary-cultured Sertoli cells. J Pharmacol Exp Ther 312: 601–608, 2005.[Abstract/Free Full Text]
  29. Koepsell H and Endou H. The SLC22 drug transporter family. Pflügers Arch 447: 666–676, 2004.[CrossRef][ISI][Medline]
  30. Kolb M. Novel and unusual nucleosides as drugs. Prog Drug Res 48: 195–232, 1997.[Medline]
  31. Lowry OH, Rosebrough NJ, Farr AL, and Randall RJ. Protein measurement with the Folin-phenol reagent. J Biol Chem 193: 265–275, 1951.[Free Full Text]
  32. Lui WY, Mruk D, Lee WM, and Cheng CY. Sertoli cell tight junction dynamics: their regulation during spermatogenesis. Biol Reprod 68: 1087–1097, 2003.[Abstract/Free Full Text]
  33. Majumdar S, Tirucherai GS, Pal D, and Mitra AK. Functional differences in nucleoside and nucleobase transporters expressed on the rabbit corneal epithelial cell line (SIRC) and isolated rabbit cornea (Abstract). AAPS Pharm Sci 5: E15, 2003.[CrossRef]
  34. Mather JP, Attie KM, Woodruff TK, Rice GC, and Phillips DM. Activin stimulates spermatogonial proliferation in germ-Sertoli cell cocultures from immature rat testis. Endocrinology 127: 3206–3214, 1990.[Abstract]
  35. Mori S, Ohtsuki S, Takanaga H, Kikkawa T, Kang YS, and Terasaki T. Organic anion transporter 3 is involved in the brain-to-blood efflux transport of thiopurine nucleobase analogs. J Neurochem 90: 931–941, 2004.[CrossRef][ISI][Medline]
  36. Nagao Y. Viability of meiotic prophase spermatocytes of rats is facilitated in primary culture of dispersed testicular cells on collagen gel by supplementing epinephrine or norepinephrine: evidence that meiotic prophase spermatocytes complete meiotic divisions in vitro. In Vitro Cell Dev Biol 25: 1088–1098, 1989.[ISI][Medline]
  37. Natsumeda Y, Prajda N, Donohue JP, Glover JL, and Weber G. Enzymic capacities of purine de novo and salvage pathways for nucleotide synthesis in normal and neoplastic tissues. Cancer Res 44: 2475–2479, 1984.[Abstract/Free Full Text]
  38. Noe B, Hagenbuch B, Stieger B, and Meier PJ. Isolation of a multispecific organic anion and cardiac glycoside transporter from rat brain. Proc Natl Acad Sci USA 94: 10346–10350, 1997.[Abstract/Free Full Text]
  39. Ritzel MW, Ng AM, Yao SY, Graham K, Loewen SK, Smith KM, Ritzel RG, Mowles DA, Carpenter P, Chen XZ, 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]
  40. 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 cotransporter (hCNT1). Am J Physiol Cell Physiol 272: C707–C714, 1997.[Abstract/Free Full Text]
  41. Ritzel MW, Yao SY, Ng AM, Mackey JR, Cass CE, and Young JD. Molecular cloning, functional expression and chromosomal localization of a cDNA encoding a human Na+/nucleoside cotransporter (hCNT2) selective for purine nucleosides and uridine. Mol Membr Biol 15: 203–211, 1998.[ISI][Medline]
  42. Rothman RB and Baumann MH. Monoamine transporters and psychostimulant drugs. Eur J Pharmacol 479: 23–40, 2003.[CrossRef][ISI][Medline]
  43. Russel LD, Ettlin RA, Hikim AP, and Clegg ED. The Sertoli cell. In: Histological and Histopathological Evaluation of the Testis. Clearwater, FL: Cache River, 1990, p. 29–36.
  44. Shayeghi M, Akerman R, and 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]
  45. Shiratsuchi A, Umeda M, Ohba Y, and Nakanishi Y. Recognition of phosphatidylserine on the surface of apoptotic spermatogenic cells and subsequent phagocytosis by Sertoli cells of the rat. J Biol Chem 272: 2354–2358, 1997.[Abstract/Free Full Text]
  46. Tamai I, Yabuuchi H, Nezu J, Sai Y, Oku A, Shimane M, and Tsuji A. Cloning and characterization of a novel human pH-dependent organic cation transporter, OCTN1. FEBS Lett 419: 107–111, 1997.[CrossRef][ISI][Medline]
  47. Tamai I, Ohashi R, Nezu J, Yabuuchi H, Oku A, Shimane M, Sai Y, and Tsuji A. Molecular and functional identification of sodium ion-dependent, high affinity human carnitine transporter OCTN2. J Biol Chem 273: 20378–20382, 1998.[Abstract/Free Full Text]
  48. Tamai I, Nezu J, Uchino H, Sai Y, Oku A, Shimane M, and Tsuji A. Molecular identification and characterization of novel members of the human organic anion transporter (OATP) family. Biochem Biophys Res Commun 273: 251–260, 2000.[CrossRef][ISI][Medline]
  49. Theisinger A, Grenacher B, and Scharrer E. Na+ gradient-dependent transport of hypoxanthine by calf intestinal brush border membrane vesicles. J Comp Physiol [B] 173: 165–170, 2003.[Medline]
  50. Uldry M and Thorens B. The SLC2 family of facilitated hexose and polyol transporters. Pflügers Arch 447: 480–489, 2004.[CrossRef][ISI][Medline]
  51. Ullman B, Patrick J, and McCartan K. Expression of the high-affinity purine nucleobase transporter in mutant mouse S49 cells does not require a functional wild-type nucleoside-nucleobase transporter. Mol Cell Biol 7: 97–103, 1987.[Abstract/Free Full Text]
  52. Wang Y, Mackenzie B, Tsukaguchi H, Weremowicz S, Morton CC, and Hediger MA. Human vitamin C (L-ascorbic acid) transporter SVCT1. Biochem Biophys Res Commun 267: 488–494, 2000.[CrossRef][ISI][Medline]
  53. 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]
  54. Washington CB and Giacomini KM. Mechanisms of nucleobase transport in rabbit choroid plexus. Evidence for a Na(+)-dependent nucleobase transporter with broad substrate selectivity. J Biol Chem 270: 22816–22819, 1995.[Abstract/Free Full Text]
  55. Weber G, Jayaram HN, Pillwein K, Natsumeda Y, Reardon MA, and Zhen YS. Salvage pathways as targets of chemotherapy. Adv Enzyme Regul 26: 335–352, 1987.[CrossRef][ISI][Medline]
  56. Wright EM and Turk E. The sodium/glucose cotransport family SLC5. Pflügers Arch 447: 510–518, 2004.[CrossRef][ISI][Medline]
  57. Yamaoka K, Tanigawara Y, Nakagawa T, and Uno T. A pharmacokinetic analysis program (MULTI) for microcomputer. J Pharmacobiodyn 4: 879–885, 1981.[Medline]
  58. Yao SY, Ng AM, Muzyka WR, Griffiths M, Cass CE, Baldwin SA, and Young JD. Molecular cloning and functional characterization of nitrobenzylthioinosine (NBMPR)-sensitive (es) and NBMPR-insensitive (ei) equilibrative nucleoside transporter proteins (rENT1 and rENT2) from rat tissues. J Biol Chem 272: 28423–28430, 1997.[Abstract/Free Full Text]
  59. Yao SY, Ng AM, Vickers MF, Sundaram 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 constructs reveal a role for the ENT2 helix 5–6 region in nucleobase translocation. J Biol Chem 277:24938–24948, 2002.[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]

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]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/5/E968    most recent
00160.2005v1
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 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 ISI Web of Science (6)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kato, R.
Right arrow Articles by Tamai, I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kato, R.
Right arrow Articles by Tamai, I.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2006 by the American Physiological Society.