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Regular Article |
a Department of Physiology, Faculty of Medicine, Chinese University of Hong Kong, Shatin, N.T., Hong Kong
b Department of Medicine, Division of Gastroenterology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
ABSTRACT
The effect of nucleoside on Na+ reabsorption via Na+/nucleoside cotransporter in cultured rat epididymal epithelia was studied by short-circuit current (Isc) technique. Guanosine added apically stimulated Isc in a dose-dependent manner, with a median effective concentration (EC50) of 7 ± 2 µM (mean ± SEM). Removal of Na+ from the apical bathing solution or pretreatment with a nonspecific Na+/nucleoside cotransporter inhibitor, phloridzin, completely blocked the Isc response to guanosine. Moreover, the guanosine response was abolished by pretreatment of the tissue with ouabain, a Na+/K+-ATPase inhibitor, suggesting the involvement of Na+/nucleoside cotransporter on the apical side and Na+/K+-ATPase on the basolateral side in Na+ reabsorption. In contrast, the Isc response to guanosine was not affected after desensitization of purinoceptors by ATP. Addition of the Na+/K+/2Cl- symport inhibitor bumetanide to the basolateral side or the nonspecific Cl- channel blocker diphenylamine-2-carboxylate to the apical side showed no effect on the Isc response to guanosine, excluding stimulation of Cl- secretion by guanosine as the cause of the guanosine-induced Isc. The Isc response to purine nucleoside (guanosine and inosine) was much higher than that to pyrimidine nucleoside (thymidine and cytidine). Consistent with substrate specificity, results of reverse transcription-polymerase chain reaction revealed mRNA for concentrative nucleoside transporter (CNT2), which is a purine nucleoside-selective Na+/nucleoside cotransporter in the epididymis, but not for CNT1. It is suggested that the Na+/nucleoside cotransporter (i.e., CNT2) may be one of the elements involved in Na+ and fluid reabsorption in the epididymis, thereby providing an optimal microenvironment for the maturation and storage of spermatozoa.
epididymis
INTRODUCTION
Mammalian spermatozoa acquire their capacity for forward motility and fertilizing capacity during their transit through the epididymis. The epithelial cells lining the epididymal ducts create a favorable fluid environment for sperm maturation and storage. This stable epididymal milieu is ensured by the absorptive and secretory functions of the epididymal epithelium [1].
Under basal condition, the epididymis reabsorbs a major part of the testicular fluid. Fluid reabsorption in the epididymis is a passive process secondary to active Na+ transport [2]. This process involves active extrusion of Na+ from the cytosol into the blood by Na+/K+-ATPase on the basolateral membrane [3]. The Na+ efflux creates an inwardly directed Na+ gradient that drives Na+ influx into the cytosol via apical Na+-dependent transporters or Na+ channels. The net absorption of Na+ from lumen into blood then creates an osmotic gradient for isotonic fluid reabsorption. Nevertheless, little is known regarding how Na+ is transported from the apical compartment into the cytosol, but Na+ reabsorption by the epididymal epithelium is largely an electroneutral process [4].
The Na+/nucleoside cotransporters are present in specialized cells such as liver and intestinal and renal epithelia [5–7]. Five subclasses are categorized based on substrate selectivity, and two have been cloned by functional complementation in oocytes. Concentrative nucleoside transporter 1 (CNT1) is a Na+/nucleoside cotransporter that is pyrimidine nucleoside selective but that also accepts uridine, whereas CNT2 is purine nucleoside selective [8, 9]. The present study was carried out to investigate the involvement of Na+/nucleoside cotransporter in the Na+ reabsorption by cultured rat epididymal epithelium.
MATERIALS AND METHODS
Tissue Culture Technique
All experiments were carried out according to the guidelines of the Laboratory Animal Services Centre of the Chinese University of Hong Kong. The procedure of tissue culture has been described previously [10, 11]. In brief, immature male Sprague-Dawley rats weighing 150 g were used as a source of epididymis and were killed by CO2 inhalation. The epididymis was dissected out, finely chopped with scissors, and treated successively with 0.25% (w/v) trypsin and 0.1% (w/v) collagenase type I. The disaggregated epithelial cells were suspended in Eagle's minimum essential medium (EMEM) containing nonessential amino acids (0.1 mM), sodium pyruvate (1 mM), glutamine (4 mM), 5
-dihydrotestosterone (1 nM), 10% fetal bovine serum, penicillin (100 IU/ml), and streptomycin (100 µg/ml) and were seeded into the wells of Millipore filter assemblies (Millipore, Bedford, MA) with a diameter of 0.4 cm2 (cell concentration, 105 cells/ml; plating density, 5 x 104 cells/cm2 filter) floating on 15 ml of culture medium. Cultures were incubated in 5% CO2 for 3 days at 32°C. Thereafter, the monolayers reached confluency and were ready for the measurement of short-circuit current (Isc).
RNA Isolation and Reverse Transcription-Polymerase Chain Reaction
Total RNA was isolated from the epididymal epithelial cells using TRIzol reagent (Gibco BRL, Grand Island, NY). Two micrograms of total RNA were used for first-strand cDNA synthesis using random hexamer primers and Superscript II RNase H- reverse transcriptase (SuperScript Preamplification System; Gibco BRL). The resulting first-strand cDNA was directly used for polymerase chain reaction (PCR) amplification.
Different sets of primers were designed and synthesized for PCR analysis. The two primers for CNT1 were sense 5'-CAACACACAGAGGCAAAGAGAG-3' and antisense 5'-ACACCAGCAGCAAGGGCTAG-3', which yielded a PCR product of 476 base pairs (bp). The two primers for amplifying CNT2 were sense 5'-AGGCCTGGAGCTCATGGAAGTC-3' and antisense 5'-GGCTCCCATGAACACCCTCTTAAG-3', which yielded a PCR product of 399 bp. ß-Actin was used as an internal standard and coamplified in the PCR reaction. The two primers used for amplifying ß-actin were sense 5'-GGTGTGATGGTGGGTATGGGT-3' and antisense 5'-GTGGGTCATCTTTTCACGGT-3', which yield a PCR product of 240 bp. Reactions were carried out with the following parameters: denaturation at 94°C for 30 sec, annealing at 58°C for 1 min, and extension at 72°C for 1 min. Thirty cycles were performed.
The PCR products were analyzed by agarose gel electrophoresis and visualized by staining with ethidium bromide. Amplification products of the expected sizes were purified from the gel, cloned into the vector pCR 2.1 (Invitrogen, Carlsbad, CA), and subjected to fluorescent sequencing according to the manufacturer's protocols (PE/Applied Biosystem 377 Automated DNA sequencer; Foster City, CA).
Isc Measurement
Confluent epididymal monolayers were clamped between two halves of Ussing chambers (World Precision Instrument, New Haven, CT) with a 0.6-cm2 window. The tissue was short-circuited with a voltage-clamp amplifier (DVC 1000; World Precision Instrument). The Isc was displayed on a pen recorder. Transepithelial resistance was obtained from Ohm's law by clamping the tissue intermittently at a voltage at 0.1–0.3 mV displaced from zero. Epithelia with transepithelial resistance of less than 300 
cm2 were discarded. Two channels of the amplifier were mostly used simultaneously on parallel monolayers so that studies could be made under both control and experimental conditions. In most cases, the monolayers were bathed on both sides with Krebs-Henseleit solution, gassed with 95% O2/5% CO2, and warmed to 32°C.
Solutions
Krebs-Henseleit solution had the following composition: NaCl, 117 mM; KCl, 4.7 mM; KH2PO4, 1.2 mM; MgSO4·7H2O, 1.2 mM; CaCl2·2H2O, 2.56 mM; NaHCO3, 24.8 mM; and glucose, 11.1 mM. This solution had a pH of 7.4 when bubbled with 95% O2/5% CO2. In Na+-free solution, NaCl and NaHCO3 were replaced by choline chloride and choline bicarbonate, respectively. In all solutions, the osmolarity was adjusted to 290 mOsm·kg-1 with D-mannitol if necessary.
Materials
The EMEM, fetal bovine serum, glutamine, and nonessential amino acids were purchased from Gibco Laboratories. Penicillin/streptomycin, Hanks balanced salt solution, sodium pyruvate, 5-dihydrotestosterone, trypsin, collagenase type I, bumetanide, phloridzin, forskolin, ATP, guanosine, inosine, thymidine, and cytidine were from Sigma (St. Louis, MO). The diphenylamine-2-carboxylate (DPC) was from Calbiochem (San Diego, CA).
Statistical Analysis
Results are expressed as the mean ± SEM. Comparisons between groups of data were made by Student's t-test. A P value of less than 0.05 was considered to be statistically significant.
RESULTS
Effect of Guanosine on Isc
When bathed in normal Krebs-Henseleit solution containing glucose (11 mM), cultured epididymal epithelia exhibited a transepithelial potential difference of 2.68 ± 0.23 mV (n = 100 monolayers from 10 different cell preparation), a basal Isc of 4.84 ± 0.4 µA/cm2 (n = 100 monolayers from 10 different cell preparation), and a transepithelial resistance approximately 500 cm2. The Na+/glucose cotransport system would have been saturated by the presence of glucose in normal Krebs-Henseleit solution, because addition of glucose (1 and 10 mM) to the bathing solution had no effect on Isc (Fig. 1A). Guanosine (0.1 mM) elicited a rise in Isc when added to the apical side (Fig. 1B). In some cases, this rise appeared to be biphasic (i.e., a rapid increase followed by a slower rise to a maximal plateau level). Occasionally, however, a monophasic increase was seen (Figs. 6B and 7B). No change in Isc was observed when guanosine was added to the basolateral side (Fig. 1B). The response to guanosine was dose dependent (Fig. 2). The maximal Isc response to guanosine was 1.56 ± 0.1 µA/cm2, and the median effective concentration (EC50) was 7 ± 2 µM. In all subsequent experiments, guanosine was added to the apical side of the tissue.
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Involvement of Na+/Nuceloside Cotransporter and Na+/K+-ATPase in the Isc Response to Guanosine
Figure 3 shows that removal of Na+ from the solution bathing the apical side of the epithelium completely abolished the Isc response to guanosine without affecting the response to forskolin, a known stimulant of Cl- secretion. This result indicated that the guanosine-induced Isc was Na+ dependent.
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To test whether the Isc response to guanosine was mediated by a Na+/nucleoside cotransporter, the tissue was pretreated with a Na+/nucleoside cotransporter inhibitor, phloridzin (1 mM) [12], before stimulation with guanosine. As shown in Figure 4, pretreatment with phloridzin completely inhibited the Isc response to guanosine, but the Isc response to forskolin was not affected. Pretreatment with ouabain (1 mM), a Na+/K+-ATPase inhibitor, completely abolished the Isc response to guanosine (Fig. 5).
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Effects of ATP, DPC, and Bumetanide
Adenosine, a nucleoside, can stimulate Cl- secretion in epididymal epithelium via purinoceptors on the apical side of the epithelium [13], and purinoceptors are subjected to rapid desensitization by repeated stimulation [13]. To test whether the Isc response to guanosine resulted from activation of purinoceptors, purinoceptors were desensitized by previous addition of ATP. As shown in Figure 6, ATP (10 µM), a potent purinoceptor agonist, was first added to the apical side to elicit an Isc response. When the response subsided, ATP (10 µM) was again added to the apical side, yielding a significantly smaller response than the first. This indicated that repeated stimulation with ATP caused tachyphylaxis of the apical purinoceptors; however, pretreatment with ATP had no effect on the Isc response to guanosine. Before ATP, 
Isc = 1.20 ± 0.18 µA/cm2 (n = 4); after ATP, Isc = 1.13 ± 0.10 µA/cm2 (n = 4; not significantly different) (Fig. 6, B and C).
To further confirm that the Isc response did not result from Cl- secretion, the effects of bumetanide, a Na+/K+/2Cl- symport inhibitor, and of DPC, a Cl- channel blocker, were examined. Pretreatment with DPC (2 mM) (Fig. 7) and bumetanide (0.2 mM) (Fig. 8) had no effect on the Isc response to guanosine. Before DPC, Isc = 1.30 ± 0.10 µA/cm2 (n = 4); after DPC, Isc = 1.20 ± 0.16 µA/cm2 (n = 4; not significantly different). Before bumetanide, Isc = 1.20 ± 0.23 µA/cm2 (n = 4); after bumetanide, Isc = 1.05 ± 0.19 µA/cm2 (n = 4; not significantly different).
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Substrate Specificity of Na+/Nucleoside Cotransporter
Because guanosine is a substrate of CNT2 (i.e., purine nucleoside-selective system), the experiment was extended to elucidate whether CNT1 (i.e., pyrimidine nucleoside-selective system) was also present in the cultured epididymal epithelium. As shown in Figure 9, Isc responses to guanosine and inosine, which are substrates of CNT2, were much greater than those to thymidine and cytidine, which are substrates of CNT1, suggesting that CNT2 is the predominant Na+/nucleoside transporter in epididymal epithelium.
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Identification of mRNA in the Rat Epididymis by Reverse Transcription-PCR
Reverse transcription (RT)-PCR was used to study the gene expression of CNT1 and CNT2 in the epididymis. Rat kidney cDNA was used as positive control, because both Na+/nucleoside cotransporters are expressed in this tissue [14]. As shown in Figure 10, CNT2 (399 bp) was amplified by RT-PCR from RNA isolated from epithelial cells of the rat epididymis. No amplified CNT1 PCR product was expressed from the same tissue, whereas the internal standard, ß-actin, was expressed. The PCR products were confirmed by sequencing (data not shown).
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DISCUSSION
Our study shows that the gene encoding CNT2, a purine nucleoside-selective Na+/nucleoside cotransporter, was expressed in the epididymis (Fig. 10). In a parallel functional study, guanosine, a substrate for CNT2, stimulated a dose-dependent rise in Isc in cultured rat epididymal epithelium, whereas the pyrimidine nucleoside (e.g., thymidine and cytidine) had a negligible effect (Fig. 9). The Na+/glucose cotransporter was present in the epididymis [15], and this Na+/glucose cotransporter shows a certain degree of homology with the Na+/nucleoside cotransporter [16]. However, the response to guanosine in our study was not mediated by the electrogenic Na+/glucose cotransporter, because this transport system would have been saturated by the presence of glucose in the Krebs-Henseleit solution (Fig. 1A). Moreover, stimulation of Isc by guanosine was restricted to the apical side (Fig. 1B). These results are consistent with the presence of Na+/nucleoside cotransporters in the luminal membranes, as has been reported in other epithelia [17, 18].
Adenosine, another substrate for CNT2, stimulated Cl- secretion by interacting with the purinoceptors on the apical side of the epithelium. Thus, the Isc response to guanosine could be attributed to electrogenic Na+ reabsorption via Na+/nucleoside cotransporter, to Cl- secretion via purinoceptors, or both. To elucidate the nature of the Isc response to guanosine, a series of experiments were performed. First, Na+ removal from the apical bathing solution resulted in complete loss of the Isc response to guanosine (Fig. 3), indicating that the guanosine response was Na+ dependent. Such treatment did not affect the response to forskolin, however, which causes Cl- secretion (Fig. 3). Involvement of the Na+/nucleoside cotransporter is further corroborated by the effectiveness of the Na+/nucleoside cotransporter inhibitor phloridzin in abolishing the response to guanosine (Fig. 4). It would seem to be highly probable that Na+ reabsorbed via the Na+/nucleoside cotransporter on the apical side was extruded through the Na+/K+-ATPase on the basolateral side [3]. This conclusion is based on the observation that the Isc response to guanosine was greatly attenuated by pretreatment with ouabain, a Na+/K+-ATPase inhibitor (Fig. 5). Moreover, purinoceptors undergo tachyphylaxis on repeated stimulation (Fig. 6). If the Isc response to guanosine resulted from the stimulation of purinoceptors, then tachyphylaxis of purinoceptors from previous ATP stimulation would be expected to reduce the response to subsequent addition of guanosine. However, this was not the case (Fig. 6). These experiments ruled out the possibility that guanosine stimulated Cl- secretion via purinoceptors. The chloride channels located on the apical membrane and the Na+/K+/2Cl- symport located on the basolateral membrane play a key role in chloride secretion in the epididymis. The lack of effect of DPC, a Cl- channel blocker (Fig. 7), and of bumetanide, a Na+/K+/2Cl- symport inhibitor (Fig. 8), on Isc response to guanosine lends further support to the notion that guanosine-induced Isc response was not caused by Cl- secretion.
Fluid reabsorption in the epididymis is a passive process secondary to active Na+ transport [2]. Reabsorption of Na+ in the epididymis is linked to secretion of H+ [19–22] and is inhibited by apical addition of the diuretic drug amiloride [2, 20]. In addition, immunohistochemical studies revealed the presence of sodium hydrogen exchanger-2 on the apical membrane of the epididymal epithelium [21]. Thus, the Na+/H+ exchanger should be a major candidate involved in Na+ and fluid reabsorption by the epididymis. In addition to the Na+/H+ exchanger, the Na+/glucose cotransporter also participates in Na+ reabsorption in the epididymis [15]. This study shows that the Na+/nucleoside cotransporter may also contribute to Na+ and fluid reabsorption across the epididymal tubules. It is conceivable that a high concentration of nucleosides exists in the epididymal fluid, and they could be derived from the hydrolysis of nucleotides by 5'-ectonucleotidase, which has been found on the plasma membranes of spermatozoa [23–25] and epididymal epithelial cells [26]. Large amounts of nucleosides are, indeed, present in the male reproductive system [27, 28]. For instance, the concentration of guanosine in seminal plasma is 22.4 ± 26.7 µM [28], which is close to the EC50 value for the Isc response to guanosine (7 ± 2 µM) found in the present study (Fig. 2). Therefore, the effect of guanosine on the epididymis could be of physiological significance.
In addition to Na+ reabsorption, other functions of the Na+/nucleoside cotransporter cannot be excluded. Adenosine regulates Cl- secretion via apical adenosine receptors in epididymal epithelial cells [13]. Adenosine also modulates sperm motility [29, 30], possibly by acting on A2 receptors on the sperm membrane [31, 32]. Therefore, the Na+/nucleoside cotransporter may play a role in fine-tuning these adenosine effects by regulating luminal nucleoside content through salvaging them from the epididymal fluid. Such a salvage pathway may also be involved in nucleoside biosynthesis [33].
In conclusion, our investigation indicates the presence of Na+/nucleoside cotransporter (i.e., CNT2) in the epididymis. This membrane protein may not be the major contributor to sodium and fluid reabsorption, but it may work along with the Na+/H+ exchangers [19–22] and other Na+-dependent transport systems [15] to create a favorable environment for sperm maturation.
FOOTNOTES
First decision: 21 August 2000.
1 Supported by the Research Grant Council of Hong Kong. 
2 Correspondence. FAX: 852 2603 5022; patrickwong{at}cuhk.edu.hk
Accepted: October 10, 2000.
Received: July 17, 2000.
REFERENCES
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