1 Division of Gastroenterology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; and 2 Department of Physiology, The Chinese University of Hong Kong, Hong Kong, People's Republic of China
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ABSTRACT |
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The nucleoside transport systems in cultured epididymal epithelium were characterized and found to be similar between the proximal (caput and corpus) and distal (cauda) regions of the epididymis. Functional studies revealed that 70% of the total nucleoside uptake was Na+ dependent, while 30% was Na+ independent. The Na+-independent nucleoside transport was mediated by both the equilibrative nitrobenzylthioinosine (NBMPR)-sensitive system (40%) and the NBMPR-insensitive system (60%), which was supported by a biphasic dose response to NBMPR inhibition. The Na+-dependent [3H]uridine uptake was selectively inhibited 80% by purine nucleosides, indicating that the purine nucleoside-selective N1 system is predominant. Since Na+-dependent [3H]guanosine uptake was inhibited by thymidine by 20% and Na+-dependent [3H]thymidine uptake was broadly inhibited by purine and pyrimidine nucleosides, this suggested the presence of the broadly selective N3 system accounting for 20% of Na+-dependent nucleoside uptake. Results of RT-PCR confirmed the presence of mRNA for equilibrative nucleoside transporter (ENT) 1, ENT2, and concentrative nucleoside transporter (CNT) 2 and the absence of CNT1. It is suggested that the nucleoside transporters in epididymis may be important for sperm maturation by regulating the extracellular concentration of adenosine in epididymal plasma.
sperm maturation
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INTRODUCTION |
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NUCLEOSIDE TRANSPORTERS are involved in nucleotide biosynthesis by salvage pathways. This is especially important in tissues or cells that lack or have low capacity for de novo nucleotide biosynthesis (14, 25). These transporters also play key roles in many physiological processes such as coronary vasodilation (34), neurotransmission (36), and platelet aggregation (1). Pharmacologically, they serve as an entry for cellular uptake of many synthetic nucleoside analogs that are used in the treatment of cancer and viral diseases. The development of nucleoside transporter inhibitors also provides therapeutic benefits in cardiovascular disorders and parasitic infections (3).
Based on Na+ dependence, two major classes of nucleoside transport systems in mammalian cells have been described (15). The equilibrative transport systems are Na+ independent and are inhibited by coronary vasodilators such as nitrobenzylthioinosine (NBMPR). This class of nucleoside transport systems is broadly selective, accepting both purine and pyrimidine nucleosides. They are further subdivided into two types on the basis of their sensitivities to inhibition by NBMPR (7, 15). The equilibrative-sensitive system (ES) is potently inhibited by a nanomolar concentration of NBMPR. In contrast, the equilibrative-insensitive system (EI) is resistant to NBMPR up to 1 µM. Both the ES and EI transporters have been cloned and are named equilibrative nucleoside transporter (ENT) 1 and ENT2 (16, 17, 45), respectively. Kinetic characterization of cloned human ENT1 (hENT1) and ENT2 (hENT2) reveals that although both hENT1 and hENT2 are broadly selective, hENT2 exhibits a low affinity for guanosine and cytidine but a high affinity for inosine (40). Pharmacologically, hENT1 and hENT2 have a 7,000-fold difference in sensitivity to NBMPR and a 71-fold difference in sensitivity to dipyridamole (40). Interestingly, although rat ENT1 (rENT1) exhibits a similar IC50 to NBMPR inhibition as hENT1, rENT1 is resistant to inhibition by dipyridamole and dilazep. This difference is due to subtle differences in human and rat ENT1 proteins between the putative transmembrane domains 3-6 (35).
In contrast, the concentrative transport systems are Na+ dependent. This class of nucleoside transport systems is able to concentrate nucleosides against a concentration gradient. There are five subclasses based on substrate selectivity. N1 is purine nucleoside selective but also accepts uridine (11, 38). N2 is pyrimidine nucleoside selective (21, 31). N3 has broad selectivity for both purine and pyrimidine nucleosides (43, 44). N4 is pyrimidine nucleoside selective, but it also transports adenosine and guanosine (18). N5 is NBMPR sensitive and appears to be guanosine specific (13). Recently, both N1 and N2 systems have been cloned by functional complementation in oocytes. The former is termed concentrative nucleoside transporter 2 (CNT2) and the latter is CNT1 (11, 21, 31, 38).
Acquisition of sperm fertility occurs during their transit through the epididymis. Adenosine, a substrate of nucleoside transporters, has been known to stimulate sperm motility (2, 37). However, little is known regarding the epithelial transport of nucleosides in the reproductive tissues. Therefore, the present study was carried out to characterize the nucleoside transport systems in the epithelium of proximal region (caput and corpus) and distal region (cauda) of the epididymis. This study may aid our understanding of the role of epididymis in providing a microenvironment for the maturation of spermatozoa.
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MATERIALS AND METHODS |
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Culture of rat epididymal epithelium.
The primary culture of rat epididymal epithelial cells has been
described previously (12, 42). Briefly, immature male Sprague-Dawley rats weighing 150-175 g were killed by cervical dislocation and the lower abdomen was opened. Proximal region (caput
and corpus) and distal region (cauda) of epididymis were dissected out.
The tissues were finely chopped with scissors and then digested with
0.25% (wt/vol) trypsin, followed by 0.1% (wt/vol) collagenase I. The
cells were suspended in Eagle's minimum essential medium supplemented
with nonessential amino acids (0.1 mM), glutamine (4 mM),
5-dihydrotestosterone (1 nM), sodium pyruvate (1 mM), 10% fetal
bovine serum, penicillin (100 IU/ml), and streptomycin (100 µg/ml)
and were incubated for 5-6 h at 32°C in 5% CO2.
During this period, fibroblasts and smooth muscle cells were removed as
these cells attached to the bottom of the cultured flask while the
epididymal epithelial cells remained suspended. The resulting epithelial cells in suspension were seeded into 24-well culture plates.
These epithelial cells became attached onto the plate after 12 h.
Four days after seeding, the cultures reached confluency and were used
for the experiments. These confluent cultures have been previously
characterized by us (8) and by Kierszenbaum and coworkers
(22). They are shown to resemble intact epididymal epithelia with respect to morphology, tight junction, and absorptive and secretory functions (6, 8, 22). These cells are
capable of secreting acidic epididymal glycoprotein, a spermatozoa
coating protein secreted by principal cells of rat epididymis
(22).
RNA isolation and RT-PCR.
Total RNA was isolated from cultured cells of the proximal and distal
regions of epididymis using TRIzol reagent (GIBCO BRL). Two micrograms
of total RNA was used for first-strand cDNA synthesis using random
hexamer primers and SuperScript II RNase H Reverse
Transcriptase (SuperScript Preamplification System, GIBCO BRL). The
resulting first-strand cDNA was directly used for PCR amplification.
Nucleoside uptake. All experiments were carried out in HEPES-buffered Ringer solution containing (in mM) 135 NaCl, 5 KCl, 3.33 NaH2PO4, 0.83 Na2HPO4, 1.0 CaCl2, 1.0 MgCl2, 10 glucose, and 5 HEPES (pH 7.4). Na+-free buffer contained (in mM) 140 N-methyl-D-glucamine, 5 HEPES, 5 KH2PO4, 1.0 CaCl2, 1.0 MgCl2, and 10 glucose (pH 7.4).
Confluent monolayers of cells were washed three times in HEPES-buffered solution. Three hundred microliters of HEPES-buffered solution containing [3H]nucleoside (10 µM, 4 µCi/ml) was then added to each well. After incubation of 1.5-3 min, as described in the figures, the plates were washed three times rapidly with ice-cold phosphate-buffered saline containing (in mM) 137 NaCl, 2.68 KCl, 8.1 Na2PO4, and 1.47 KH2PO4 (pH 7.4). Cells were solubilized in 0.5 ml of 5% Triton X-100. The radioactivity was measured by aMaterials. All the nucleosides, NBMPR, collagenase I, and trypsin were purchased from Sigma Chemical (St. Louis, MO). [5-3H]uridine (0.81 TBq/mmol), [8-3H]guanosine (273 GBq/mmol), and [methyl-3H]thymidine (740 GBq/mmol) were from ICN Pharmaceuticals (Irvine, CA). Cell culture media and supplements were from GIBCO BRL (Grand Island, NY).
Statistical analysis. Data are expressed as means ± SE. Student's t-test and analysis of variance were used for paired and multiple variants, respectively. P < 0.05 was considered statistically significant.
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RESULTS |
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Time course of uridine uptake in proximal and distal regions of
epididymis.
[3H]uridine uptake (10 µM) was measured as a function
of time in the presence and in the absence of Na+
(Fig. 1). Cultured epithelial cells
isolated from both the proximal and distal regions of epididymis
demonstrated Na+-dependent [3H]uridine
uptake. The remaining Na+-independent
[3H]uridine uptake was completely inhibited by 0.5 mM
NBMPR (Fig. 1). This result suggested the presence of both
Na+-dependent and -independent nucleoside transport. At 3 min, Na+-dependent and -independent uridine uptake was
34.7 ± 1.2 pmol/mg protein and 16.2 ± 0.2 pmol/mg protein,
respectively, which represents 68 and 32% of total uridine uptake,
respectively, for the proximal region. Na+-dependent and
-independent uridine uptake was 26.4 ± 2.6 pmol/mg protein and
10.7 ± 3.5 pmol/mg protein, respectively, which represents 71 and
29% of total uridine uptake, respectively, for the distal region. The
Na+-dependent uridine uptake was not affected by 0.5 mM
NBMPR (data not shown).
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Na+-independent uridine uptake.
To test whether the Na+-independent nucleoside transport
consisted of both ES and EI, we pharmacologically defined the ES system as the nucleoside transport that was inhibited by 100-nM NBMPR, and the
NBMPR-resistant activity (resistant to 100 nM but sensitive to 0.5 mM)
as EI transport. As shown in Fig. 2, ES
and EI transport systems contributed ~40 and 60%, respectively, in
epithelial cells isolated from both the proximal and distal regions of
epididymis. To further confirm that the proximal region contained both
ES and EI systems, the dose response of NBMPR inhibition of
[3H]uridine transport was determined (Fig.
3A). This dose-response curve
was biphasic with a plateau between 10 nM and 1 µM, consistent with
the coexistence of ES and EI. If this dose-response curve was dissected
into ES (sensitive to 100 nM; Fig. 3B) and EI (resistant to
100 nM; Fig. 3C) transport components, the IC50
was 2.3 nM and 14 µM, respectively. These values are consistent with
studies on cloned rENT1 and rENT2 (45). Similar results
were obtained for epithelial cells isolated from the distal region of
epididymis (data not shown).
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Na+-dependent uridine uptake.
As shown in Fig. 1, Na+-dependent uridine uptake is present
in both the proximal and distal regions of epididymis. Since five Na+-dependent nucleoside transport systems have been
described based on substrate selectivity, we tested the effect of
purine and pyrimidine nucleosides on Na+-dependent
[3H]uridine uptake. All the subsequent experiments were
carried out in the presence of 0.5 mM NBMPR to avoid any contribution from Na+-independent transport systems. As shown in Fig.
4, [3H]uridine uptake (10 µM) in epididymal epithelial cells was inhibited by ~70 and 80% by
the purine nucleosides guanosine and inosine (both at 100 µM),
respectively. In contrast, the pyrimidine nucleosides cytidine and
thymidine (both at 100 µM) inhibited [3H]uridine uptake
in epididymal epithelial cells by ~20%. This suggested that the
purine nucleoside-selective N1 transport system is the dominant system
expressed in the proximal and distal regions of epididymis. The
pyrimidine nucleoside-sensitive component of [3H]uridine
uptake would represent either the pyrimidine nucleoside-selective N2
system or the broadly selective N3 system. To distinguish between these
two systems, we tested the ability of thymidine and inosine (both at
100 µM) to inhibit [3H]guanosine uptake (10 µM; Fig.
5). As expected, inosine inhibited >80%
of [3H]guanosine uptake. However, thymidine (100 µM)
also inhibited 20% of [3H]guanosine uptake, suggesting
the presence of the broadly selective N3 transport system.
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Identification of nucleoside transporters in proximal and distal
regions of rat epididymis by RT-PCR.
Since the Na+-dependent pyrimidine nucleoside-selective N2
(CNT1), purine nucleoside-selective N1 (CNT2), and
Na+-independent ES (ENT1) and EI (ENT2) nucleoside
transport systems have been cloned, RT-PCR was used to confirm the
expression of ENT1, ENT2, and CNT2, and the absence of CNT1, in the
cultured epithelial cells isolated from the proximal and distal regions of epididymis (Fig. 7). The rat ileum
cDNA was used as a positive control because all four of these
nucleoside transporters were expressed in this tissue
(39). As predicted, PCR products of ENT1 (416 bp), ENT2
(399 bp), and CNT2 (399 bp) were amplified by RT-PCR from RNA isolated
from cultured epithelial cells in the proximal and distal regions of
epididymis. In contrast, there was no amplified CNT1 PCR product from
cultured epithelial cells in the proximal and distal regions of
epididymis. Therefore, these results complemented our functional
studies that the rat epididymal epithelial cells expressed ES (ENT1),
EI (ENT2), and N1 (CNT2) systems.
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DISCUSSION |
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Both Na+-independent equilibrative and Na+-dependent concentrative nucleoside transport systems are involved in the transport of nucleosides across mammalian cell membranes. The housekeeping equilibrative nucleoside transporters, in particular the ES system, are found in almost all cell types, whereas the concentrative transporters are limited to specialized cells such as liver, intestinal and renal epithelia, and lymphoma cells (4, 5, 24, 29). The present study demonstrated the presence of ES (ENT1; 12.8% in the proximal region and 12.5% in the distal region), EI (ENT2; 19.2% in the proximal region and 16.5% in the distal region), N1 (CNT2; 54.4% in the proximal region and 56.8% in the distal region), and N3 systems (13.6% in the proximal region and 14.2% in the distal region). To our knowledge, this is the first description of nucleoside transport in epididymis. The presence of the N3 system in epididymis, previously described in choroids plexus and intestine (20, 43, 44), further suggests the existence of this yet to be cloned broadly selective Na+-dependent nucleoside transporter.
The Na+-dependent transport was predominant and contributed 68 and 71% of the total nucleoside uptake in epithelial cells isolated from the proximal and distal regions of epididymis, respectively (Fig. 1). The presence of the N1 system was supported by 1) [3H]uridine uptake preferentially inhibited by guanosine and inosine (Fig. 4), and 2) [3H]guanosine uptake preferentially inhibited by inosine (Fig. 5). In both cases, the pyrimidine nucleosides thymidine and cytidine inhibited the [3H]nucleoside uptake by ~20%, with such inhibition of uridine and guanosine uptake likely reflecting the presence of the N3 system. The existence of the N3 system was supported by 1) 20% of [3H]guanosine uptake was inhibited by thymidine (Fig. 5), and 2) [3H]thymidine uptake was inhibited by both purine and pyrimidine nucleosides (Fig. 6). It has been described that the N4 system has similar substrate selectivity as the pyrimidine nucleoside-selective N2 system except that N4 is able to transport guanosine but not inosine. Our study showed that inosine inhibited [3H]thymidine uptake in epididymal epithelial cells by 80%, eliminating the possibility of the N4 system. Additionally, Na+-dependent uridine transport was not affected by NBMPR (data not shown), eliminating the possibility of the N5 system. The presence of the N1 system was further confirmed by RT-PCR by which CNT2 message was found in the proximal and distal regions of epididymal epithelial cells (Fig. 7). The absence of the N2 system was confirmed by the absence of CNT1 message in epididymal epithelial cells.
On the other hand, Na+-independent transport contributed 32 and 29% of the total nucleoside uptake in epithelial cells isolated from the proximal and distal regions of the epididymis. Using differential sensitivity to NBMPR, we demonstrated that the NBMPR-sensitive and -insensitive components were present in epithelial cells isolated from both the proximal and distal regions of the epididymis (Fig. 2), which accounted for 40 and 60% of the Na+-independent nucleoside transport, respectively. The coexistence of the ES and EI transport systems in epithelial cells isolated from the proximal and distal regions of epididymis was further strengthened by the biphasic dose-response curve for NBMPR inhibition of [3H]uridine uptake. Furthermore, the result of RT-PCR confirmed the presence of ENT1 and ENT2 messages in these epithelial cells (Fig. 7).
The physiological role of nucleoside transporters in epididymis is not
well understood. In this study, we demonstrated that the epididymis has
both Na+-dependent and -independent nucleoside
transporters. Unfortunately, we do not yet have the antibodies against
these transporters to localize the transporters in epididymal
epithelial cells. Nevertheless, in a scenario similar to other
polarized epithelial cells, it is likely that the
Na+-dependent nucleoside transporters are localized to the
luminal membranes, whereas the Na+-independent transporters
are on the basolateral membranes (9, 30). It has been
shown that the physiologically important nucleoside adenosine regulates
Cl secretion via an apical adenosine receptor in
epididymal epithelial cells (41). Adenosine also modulates
sperm motility (2, 37), which involves an adenosine
receptor, possibly the A2 subtype, on the spermatozoa (32,
33). The adenosine is likely derived from the hydrolysis of
nucleotides by 5'-ectonucleotidase, which has been found on the plasma
membranes of spermatozoa (26-28) and epididymal
epithelial cells (23). The Na+-dependent
nucleoside transporters are therefore probably involved in fine tuning
the physiological effect of adenosine on spermatozoa and on apical
Cl
channels of epididymal epithelial cells by salvaging
adenosine from the epididymal fluid. This is consistent with our
observation that the purine nucleoside-selective N1 system is
predominantly expressed in epididymis and also with the observation by
others that spermatozoa are incapable of adenosine transport
(37). As in other epithelial cells, the basolateral
Na+-independent nucleoside transporters are likely to be
important for nucleoside salvage from interstitial fluid.
In conclusion, we have demonstrated the presence of Na+-dependent (N1 and N3) and -independent (ES and EI) nucleoside transport systems in epididymal epithelial cells. These transporters might be important for sperm maturation as spermatozoa transit along the epididymis.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institutes of Health (NIH) Grant R01-CA-85428 and American Heart Association, Maryland Affiliate, Grant-in-Aid S98645M (to C.-M. Tse), NIH Award K08-DK-02737 (to J. L. Ward), and by the Research Grant Council of Hong Kong (to P. Y. D. Wong).
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FOOTNOTES |
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Present address of G. P. H. Leung: Department of Physiology, The Chinese University of Hong Kong, Hong Kong, People's Republic of China.
Address for reprint requests and other correspondence: C.-M. Tse, Division of Gastroenterology, Dept. of Medicine, Johns Hopkins Univ. School of Medicine, 918 Ross Research Bldg., 720 Rutland Ave., Baltimore, MD 21205 (E-mail: mtse{at}welch.jhu.edu).
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.
Received 17 April 2000; accepted in final form 22 November 2000.
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