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Regular Article |
a Department of Physiology, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China
b Department of Medicine, Division of Gastroenterology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
ABSTRACT
Although earlier work has pointed to the presence of Na+/H+ exchangers (NHEs) in the rat epididymis, little is known about the regional distribution of various NHE isoforms and their functions. In the present work, expression of different isoforms of NHE in cultured epithelia of the efferent duct and cauda epdidymidis were studied. Reverse transcription-polymerase chain reaction revealed the presence of NHE1, NHE2, and NHE3, but not NHE4, message in both cultured epithelia. Western blot analysis detected the presence of NHE1 and NHE2 proteins in both cultured epithelia, but NHE3 protein was only detected in the cultured epithelial cells from the efferent duct. Immunohistochemical studies demonstrated that NHE2 was localized in the cytoplasm of the ciliated cells, whereas NHE3 was localized at the apical membrane of the principal cells of the efferent duct. The NHE activities in both cultured epithelia were inhibited by 10 µM HOE-694 (3-methylsulphonyl-4-piperidinobenzoyl guanidine methanesulphonate), a NHE1 inhibitor, by approximately 76%. The HOE-694-resistant NHE activities in the cultured epithelia of efferent duct and cauda epididymidis were completely inhibited by 20 µM S3226 (3-[2-(3-guanidino-2-methyl-3-oxo-propenyl)-5-methyl-phenyl]-N-isopropylidene-2-methyl-acrylamide dihydrochloride), a NHE3 inhibitor, and 300 µM HOE-694 (a dose that can completely block NHE2), respectively. These results indicated that NHE1, NHE2, and NHE3 were expressed in the cultured epithelial cells of the efferent duct, whereas only NHE1 and NHE2 were expressed in the cultured epithelial cells of the cauda epididymidis. It is suggested that NHE1 may provide "housekeeping" functions in both epithelia, whereas NHE2 in the cauda epididymidis and NHE3 in the efferent duct may be involved in Na+ reabsorption and regulation of pH of the luminal fluid.
epididymis
INTRODUCTION
Mammalian spermatozoa gradually acquire their fertilizing capacity and motility as they are transported through the efferent duct and epididymis. The efferent duct reabsorbs most of the fluid leaving the testis and delivers spermatozoa in high concentration to the epididymis, where the composition of the fluid is further modified. These processes are achieved by a number of transport systems that translocate ions, organic solutes, and macromolecules as well as water across the epithelia lining the efferent duct and epididymis [1–3].
The Na+/H+ exchanger (NHE) is a ubiquitous plasma membrane transporter in mammalian cells. At present, six NHE isoforms have been identified. The NHE1 is an ubiquitous NHE isoform that is found in almost all mammalian cells [4]. In epithelial cells, it is present exclusively on the basolateral membrane [5]. It plays a "housekeeping" role in regulating intracellular pH, osmolarity, and cell volume. Both NHE2 and NHE3 are specifically expressed in the apical membrane of epithelial tissues, such as the kidney and intestine [6–8]. They are responsible for luminal Na+ reabsorption [9, 10]. However, in a cell line of the inner medullary collecting duct, NHE2 coexists with NHE1 on the basolateral membrane [11], where they serve similar functions. The NHE4 is localized to the basolateral membrane of the stomach and kidney [12]. A nonepithelial NHE (i.e., NHE5) and a mitochondrial NHE (i.e., NHE6) have also been identified [13, 14], but little is known about their cellular location and physiological functions.
Using the immunohistochemical method, NHE1 and NHE2 have been localized in the basolateral and apical aspect, respectively, of the epithelium lining the rat epididymis [15]. Recently, it was reported that NHE could be a principal transporter for electrolytes and fluid in the efferent duct [16]. Nevertheless, little is known about the isoforms of NHE in this tissue. In the present study, mRNA, protein, and functional expression of different NHE isoforms in cultured epithelial cells from the rat efferent duct were studied. The results were compared with those of the cauda epididymidis, because the physiology of this part of the epididymis has been the most extensively investigated.
MATERIALS AND METHODS
Culture of Rat Epididymal Efferent Duct Epithelium
The procedures for primary culture of rat cauda epididymidis have been described previously [17, 18]. The procedures for primary cultures of rat efferent duct were modified from the protocol described by Rozewicka et al. [19]. Immature male Sprague-Dawley rats weighing 150–175 g (40 days) were used, because the presence of NHE in sperm [20] may interfere with the interpretation of the results. Rats were killed by CO2 inhalation. The lower abdomens were opened, and the efferent ducts were isolated and microdissected under sterile condition to remove fat and connective tissue. The ductules were cut into several small segments, transferred to Hanks balanced salt solution (HBSS) containing 0.1% (w/v) trypsin and 0.2% (w/v) collagenase I, and incubated in a water bath at 32°C for 1 h with vigorous shaking (150 strokes/min). Thereafter, the tissue was separated by low-speed centrifugation (800 x g, 5 min). The supernatent was discarded, and the pellets were resuspended in HBSS containing 0.2% (w/v) collagenase I for 30 min at 32°C with vigorous shaking. After centrifugation at 800 x g for 5 min, cell plaques were again resuspended in HBSS containing 0.2% (w/v) collagenase I and subjected to repeated pipetting for 15 min. The cells were then centrifuged at 800 x g for 5 min and resuspended 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). The cell suspension was incubated for 5–6 h at 32°C in 5% CO2. During this period, fibroblasts and smooth muscle cells attached to the bottom of the culture flask, whereas epithelial cells remained suspended. The resulting epithelial cell suspension was immediately used for RNA isolation and crude membrane preparation or was seeded on rat-tail collagen, type I (Becton Dickinson Labware, Bedford, MA)-coated glass coverslips for measurement of NHE activity.
RNA Isolation and Reverse Transcription-Polymerase Chain Reaction
Total RNA was isolated from cultured epithelial cells of the efferent duct and cauda epididymidis 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 used for PCR analysis as described by Borensztein et al. [21]. The two primers used for amplifying NHE1 were sense 5'-TCTGCCGTCTCAACTGTCTCTA-3' and antisense 5'-CCCTTCAACTCCTCATTCACCA-3', which generated a 422-base pair (bp) NHE1 PCR product. The two primers for NHE2 were sense 5'-GCAGATGGTAATAGCAGCGA-3' and antisense 5'-CCTTGGTGGGGGCTTGGGTG-3', which generated a 310-bp NHE2 product. The two primers for NHE3 were sense 5'-GGAACAGAGGCGGAGGAGCAT-3' and antisense 5'-GAAGTTGTGTGCCAGATTCTC-3', which yielded a PCR product of 321 bp. The two primers for amplifying NHE4 were sense 5'-GGCTGGGATTGAAGATGTATGT-3' and antisense 5'-GCTGGCTGAGGATTGCTGTAA-3', which yielded a PCR product of 501 bp. Reactions were carried out with denaturation at 94°C for 1 min, annealing at 60°C for 1 min, and extension at 72°C for 1.5 min. A total of 30 cycles were performed.
The PCR products were analyzed by agarose gel electrophoresis and visualized by staining with ethidium bromide. The PCR products of the expected sizes were confirmed by sequencing.
Antibody Production
All polyclonal anti-NHE1, anti-NHE2, and anti-NHE3 antibodies were raised in rabbit as previously described [8, 22]. Anti-NHE1 antibody was raised against a purified fusion protein of maltose-binding protein and the putative cytoplasmic C-terminus of NHE1. Anti-NHE2 antibody was raised against a purified protein of glutathione-S-transferase and the last 87 amino acids of NHE2. Anti-NHE3 antibody was raised against a purified protein of glutathione-S-transferase and last 85 amino acids of NHE3.
Cell Membrane Preparation and Western Blot Analysis
Isolated efferent ductal or cauda epididymal epithelial cells were sonicated at 4°C in 5 mM (Na2PO4) sodium phosphate buffer and centrifuged at 2500 x g for 15 min. The protein content of the supernatant was determined spectrophotometrically using a commercial bicinchoninic acid assay (Pierce Biochemicals, Rockford, IL). Fifty micrograms of protein were resolved on 9% (w/v) SDS-polyacrylamide gel and electrotransferred to nitrocellulose membranes. The membranes were subsequently blocked with 5% (w/v) nonfat dry milk in PBS overnight at 4°C, followed by incubation with polyclonal rabbit anti-NHE1, anti-NHE2, or anti-NHE3 antibody (1:1000 [v/v] dilution in blocking solution) for 2 h at room temperature. Nitrocellulose membranes were then washed extensively with 0.02% (v/v) Triton X-100 in PBS. After washing, the membranes were incubated with horseradish-conjugated goat anti-rabbit secondary antibody (1:5000 [v/v] dilution in blocking solution) for 2 h at room temperature. Excess secondary antibody was again washed, and the bound secondary antibody was detected by enhanced chemiluminescence (Western Blot Chemiluminescence Reagent Plus; NEN Life Science Products, Boston, MA).
Immunohistochemistry
Adult, male Sprague-Dawley rats weighing 350–400 g (80 days of age) were used in this study. Paraffin sections were dewaxed and hydrated. Antigens were retrieved by treatment in 0.01 M citrate buffer (pH 6.0) for 5 min in a microwave oven. They were then rinsed twice with pure water and incubated in methanol containing 3% (v/v) H2O2 for 15 min. After rinsing with pure water and PBS, sections were incubated in normal blocking serum (vectastain Elite ABC kit, Vector PK-6101; Vector Laboratories, Burlingame, CA) for 30 min and then with the polyclonal rabbit anti-NHE2 or anti-NHE3 antibody diluted 1:100 (v/v) with diluting buffer (PBS with 0.01% [v/v] Triton X-100, 0.01% [v/v] Tween 20, and 0.1% [w/v] BSA) at 4°C overnight. Sections were washed three times with PBS and incubated with biotinylated secondary antibody (ABC kit) for 30 min. After washing three times with PBS, the sections were incubated with Vectastain Elite ABC reagent (ABC kit) for 30 min and finally washed three times with PBS again. Visualization was achieved by immersing sections in a peroxidase-substrate solution (Vector VIP substrate kit) until the desired stain intensity developed. Slides were rinsed with pure water for 5 min, counterstained with Lillie-Mayer's hematoxylin (Merck, Darmstadt, Germany), dehydrated, and mounted for observation. Negative controls were obtained by omission of primary antibodies.
Measurement of NHE Activity
Cultured cells were seeded on rat-tail collagen-coated glass coverslips and incubated at 32°C in 5% CO2 for 3 days, until they reached 50%–70% confluency. The cells were loaded with 5 µM pH-sensitive dye, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-acetoxymethyl ester (BCECF-AM; Molecular Probes, Eugene, OR), in the presence of 40 mM NH4Cl (to promote subsequent intracellular acidification) for 30 min at room temperature. The coverslips were then mounted in a cuvette and inserted into the fluorometer (SPF 500C; SLM, Urbana, IL). The cells were alternately excited with light at 440 and 500 nm, and light emission at 530 nm was measured. The ratio of the emitted light at an excitation of 500 nm to that at 440 nm was determined.
The cultured cells were superfused with TMA buffer (130 mM tetramethylammonium chloride, 5 mM KCl, 2 mM CaCl2, 1 mM MgSO4, 1 mM tetramethylammonium phosphate, 25 mM glucose, 20 mM Hepes, pH 7.4), which resulted in acidification of the cells. The TMA solution was subsequently replaced with Na+-buffer (identical to TMA buffer, except that tetramethylammoniun salts are replaced by Na+ salts) to induce alkalinization of the cells. At the end of each experiment, the fluorescence ratio (500:440) was calibrated to intracellular pH (pHi) by equilibrating the cells in pH-clamping solutions containing 20 mM Hepes, 20 mM 2-(4-morpholino)-ethane-sulfonic acid, 75 mM KCl, 35 mM potassium gluconate, 14 mM sodium gluconate, 1 mM CaCl2, 1 mM MgSO4, 2 mM tetramethylammonium chloride, 10 mM sodium phosphate, and 10 µM nigericin at pH 6.1 or 7.1. The NHE activity, expressed as 
pHi/min, was calculated from the slope of the initial 10–15 sec of Na+-dependent pHi recovery by linear least square analysis.
Different NHE isoforms show different sensitivities to inhibitors. Amiloride, a conventional NHE inhibitor, lacks the ability to distinguish between NHE1 and NHE2 (IC50 for both isoforms is 3 µM) [23]. In contrast, HOE-694 (3-methylsulphonyl-4-piperidinobenzoyl guanidine methanesulphonate), a benzoylguanidine derivative of amiloride, is a useful tool to differentiate between NHE1 and NHE2 (IC50 values for NHE1, NHE2, and NHE3 are 0.25, 20.5, and 650 µM, respectively) [23, 24]. Recently, S3226 (3-[2-(3-guanidino-2-methyl-3-oxo-propenyl)-5-methyl-phenyl]-N-isopropylidene-2-methyl-acrylamide dihydrochloride) was found to be a specific inhibitor of NHE3, with an IC50 of 0.23 µM (IC50 values for NHE1 and NHE2 are 3.55 and 80 µM, respectively) [25]. Sequential addition of these inhibitors was used to differentiate between NHE1, NHE2, and NHE3 activities in cultured efferent duct and epididymal epithelial cells. In our protocol, 1 mM amiloride, which would inhibit all isoforms [23], was added to the superfusate to confirm that the Na+-dependent recovery of pHi after acid challenge was due to NHEs. Then, 10 µM HOE-694 was added to inhibit NHE1 activity, which was calculated from the difference between the total NHE activity (measured without adding any drugs) and the activity that remained after addition of HOE-694. Any HOE-694-resistant activity was further characterized by addition of 300 µM HOE-694 and 20 µM S3226, which are specific inhibitors of NHE2 and NHE3, respectively [10, 24].
Materials
Fetal bovine serum, EMEM, and nonessential amino acids were purchased from Gibco BRL. Penicillin/streptomycin, HBSS, sodium pyruvate, 5-dihydrotestostestone, trypsin, collagenase I, amiloride, and nigericin were from Sigma (St. Louis, MO). the HOE-694 and S3226 were kindly provided by Dr. H.J. Lang (Hoechst, Frankfurt, Germany).
Statistical Analysis
Data are expressed as mean ± SEM. Comparisons between groups of data were made by Student's paired t-test, with P < 0.05 considered to be statistically significant.
RESULTS
Identification of NHE Isoforms in Epithelial Cells of the Efferent Duct and Cauda Epididymidis by Reverse Transcription-PCR
Reverse transcription (RT)-PCR was used to study the expression of NHE1, NHE2, NHE3, and NHE4 in the cultured epithelial cells of efferent duct and cauda epididymidis (Fig. 1). The rat jejunum cDNA was used as a positive control, because all four NHE isoforms were expressed in this tissue [4]. The PCR products of NHE1 (422 bp), NHE2 (310 bp), and NHE3 (321 bp) were amplified by RT-PCR of RNA isolated from epithelial cells in efferent duct and cauda epididymal epithelia. In contrast, amplification failed to produce NHE4 a PCR product from the cultured epithelia of the efferent duct and cauda epididymidis.
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Western Blot Analysis of NHE Proteins
Western blot analysis was performed to study the protein expression of different NHE isoforms in cultured efferent ductal and cauda epididymal epithelial cells. As shown in Figure 2A, two protein bands of approximately 100 and 80 kDa were expressed in both the cultured efferent ductal and cauda epididymal epithelial cells when using the NHE1 antibody. The two detected bands were consistent with the predicted values of the immature, intracellular form of NHE1 and the mature, glycosylated form of NHE1 [26]. The NHE2 antibody detected a 85-kDa protein in both cultured efferent ductal and cauda epididymal epithelial cells. The size of NHE2 protein is in line with the predicted molecular mass and is similar to that previously described in rat intestine [8]. In contrast, a protein band of approximately 85-kDa, which corresponds to NHE3 protein [8], was only expressed in the cultured epithelial cells of the efferent duct, not the cauda epididymidis.
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Immunolocalization of NHE Proteins in Efferent Duct and Cauda Epididymidis
Previous work has shown that NHE2 is localized in the apical membrane of the principal cells of the cauda epididymal epithelium [15]. Immunohistochemistry showed that the cytoplasm of ciliated cells of efferent duct of the adult rat was highly stained by NHE2 antibody (Fig. 3B). In contrast, immunoreactive NHE3 was localized on the apical membrane of principal cells of the efferent ductal epithelium (Fig. 3C). Inconsistent with the result of Western blotting, cauda epididymal epithelium was negatively stained by the NHE3 antibody (Fig. 3D).
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NHE Activity in Efferent Ductal and Cauda Epididymal Epithelial Cells
Figure 4A showed a representative pattern of intracellular pH recovery from acid-loaded, cultured efferent ductal epithelial cells. Exposure of the epithelial cells to Na+ resulted in a rapid intracellular alkalization. The pHi increased from 6.1 ± 0.1 to a steady value of 8 ± 0.11 (n = 18). Alkalization was completely inhibited by 1 mM amiloride (Fig. 4B), indicating that the pH recovery is attributed to the activity of NHE. The acid-loaded, cultured cauda epididymal epithelial cells showed a similar pHi recovery, from 6.1 ± 0.1 to 8.03 ± 0.1 (n = 16), after addition of Na+ (Fig. 5A). The intracellular alkalization was completely blocked by 1 mM amiloride (Fig. 5B).
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Effect of Different NHE Inhibitors on NHE Activity
Two NHE inhibitors, HOE-694 and S3226, were used to differentiate NHE1, NHE2, and NHE3 activities. The 10 and 300 µM HOE-694 have been shown to completely inhibit NHE1 and NHE2 activity, respectively [23, 24]. In contrast, NHE3 activity was found to be inhibited by 20 µM S3226 [10, 25]. As shown in Figure 6A, the NHE activity in the efferent ductal epithelial cells was suppressed with 10 µM HOE-694 by 76.07% ± 2.42% (n = 14). Similarly, as shown in Figure 7A, the NHE activity of cauda epididymal epithelial cells was also inhibited with HOE-694 by 74.79% ± 3.78% (n = 14). Therefore, NHE1 was the predominant NHE isoform in these cultured epithelia. In both cases, the residual activity was completely inhibited by 1 mM amiloride (Figs. 6A and 7A), indicating the expression of NHE subtypes other than NHE1. Because the pHi recovery rate gradually decreased as the intracellular pH rose, we repeated the experiment by initiating intracellular alkalization with Na+ buffer in the presence of 10 µM HOE-694 (Figs. 6, B and C, and 7, B and C). Similar HOE-694-resistant NHE activities in both cultured epithelia were obtained (Table 1). The HOE-694-resistant NHE activity in cultured efferent ductal epithelial cells was completely inhibited by 20 µM S3226 (Fig. 6B), whereas 300 µM HOE-694 had no effect (Fig. 6C), indicating that NHE3 was functionally expressed in the cultured efferent ductal epithelial cells. On the other hand, the HOE-694-resistant NHE activity in the cultured cauda epididymal epithelial cells was completely inhibited by 300 µM HOE-694 (Fig. 7B), but not by 20 µM S3226 (Fig. 7C). These results indicate that NHE2 was functionally expressed in the cultured cauda epididymal epithelial cells.
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DISCUSSION
Different NHE isoforms have been found in different tissues, where they have varying cellular and subcellular distributions. This is consistent with a broad range of physiological functions for the NHEs. In this study, we have used pharmacological, molecular, and immunohistochemical approaches to demonstrate the presence of NHE1, NHE2, and NHE3 in cultured epithelial cells of the efferent duct. The cellular distribution of various NHE isoforms in these cells was different from that in cultured cauda epididymal epithelium.
One major consideration in the present experimental design is that epithelia from immature rats were used in the RT-PCR, Western blot, and functional analysis, whereas immunohistochemistry was performed on adult rats. Epididymal spermatozoa have been known to contain NHE [20], which would have rendered interpretation of the results difficult if adult rats had been used in the cell culture. On the other hand, it is technically difficult to obtain intact histological sections of the efferent duct from immature rats for immunohistochemical study because of the fragility and small size of these ducts. Therefore, precaution should be taken when results (e.g., RT-PCR, Western blotting, and intracellular pH measurement) obtained from in vitro studies are compared with those of immunohistochemistry carried out in adult rats. However, such concern may be allayed by the previous findings that localization of NHE proteins in other tissues does not appear to vary in animals of different ages [27–30].
The results of RT-PCR confirmed the presence of NHE1 message in both the cultured epithelial cells of the efferent duct and the cauda epididymidis (Fig. 1A). The existence of NHE1 at the protein level was also confirmed by Western blot analysis (Fig. 2B). Moreover, the intracellular pH measurement confirmed that NHE1 protein was functionally expressed in both cultured epithelia, because approximately 76% of the NHE activity was inhibited by 10 µM HOE-694, a specific NHE1 inhibitor [23, 24] (Figs. 6A and 7A, Table 1). The presence of functional NHE1 protein in the cultured epithelia of efferent duct and cauda epididymidis is consistent with the ubiquitous nature of this NHE isoform. Our previous work has shown that NHE1 is localized on the basolateral membrane of the epithelium lining the epididymis [15], and a similar distribution may occur in the efferent duct. It is generally held that the widespread NHE1 performs a number of "housekeeping" functions, such as maintenance of intracellular pH, regulation of cell volume, and cell proliferation [4]. In addition, it may also be involved in transepithelial electrolyte transport. The epithelia of efferent duct and cauda epididymidis have been shown to secrete anions (i.e., chloride and bicarbonate) when stimulated by secretory agonists [18, 31]. During this process, a basolaterally placed NHE works in concert with the Na+/K+ pump, Na+/K+/2Cl- symport, and basolateral K+ channel to accumulate Cl- and HCO3- intracellularly above their respective electrochemical equilibrium [18]. On stimulation, the apical anion channels open, and these anions then diffuse across the apical membrane along their electrochemical gradients.
Efferent duct is homologous with the proximal tubule of the kidney. During development of the metanephric kidney, the wolffian duct undergoes transformation into the efferent duct and proximal kidney tubule, but not the loop of Henle and distal tubule [32–35]. More than 95% of the testicular fluid is reabsorbed by the efferent duct [36]. Fluid reabsorption is Na+ dependent, and 5 mM amiloride added intraluminally reduced reabsorption by 70% [16], indicating that Na+ reabsorption by the efferent duct is mainly mediated by NHE located in the apical membrane. Functional and immunocytochemical studies [6, 10] indicate that NHE3 is the principal NHE isoform expressed in the brush border of the proximal tubule, where it plays a major role in Na+ and fluid reabsorption. As with the proximal kidney tubule, cultured epithelial cells of the efferent duct also express NHE3 message and protein (Figs. 1C and 2C). Functionally, NHE3 accounted for approximately 24% of the total NHE activity in the cultured efferent ductal epithelial cells (Fig. 6B and Table 1). Immunohistochemical study showed that NHE3 was localized in the apical membrane of the principal cells (Fig. 3C). Therefore, NHE3 is a likely candidate to be involved in Na+ and fluid reabsorption by the efferent duct. The Na+ entering the cells via NHE3 are pumped across the basolateral membrane by the basolaterally placed Na+/K+-ATPase [37, 38].
Unlike the proximal tubule of the kidney [7, 10, 39], efferent duct cells expressed NHE2 message and protein in addition to NHE3 (Figs. 1B and 2B). However, it was unclear why NHE2 protein was expressed in the cytoplasm of the ciliated cells of the efferent duct (Fig. 3B). Intracellular pH measurement did not detect a functional NHE2 in cultured efferent duct epithelia (Fig. 6C). The majority (>80%) of cells in cultured rat efferent duct epithelia are principal cells, with the remainder being ciliated cells [19, 40]. Failure to detect NHE2 function could, therefore, be due to a low number of ciliated cells in the cultured epithelium. Functionally, ciliated cells are thought to move fluid and spermatozoa through the efferent duct [35]. They possess an endocytic apparatus that appears to be involved in modifying the composition of luminal fluid by endocytosis [41]. Acidic pH in the endosomes plays a critical role in mediating the orderly trafficking of receptors and ligands during endocytosis [42]. The NHE2 in the ciliated cells may be involved in the control of the intracellular pH in the endosomes, as has been described for the liver and renal cortex [43, 44]. Further work (e.g., electron microscopy) is required to confirm the localization and role of NHE2 in the efferent duct.
It is interesting to note that NHE2 and NHE3 were differentially distributed in the efferent duct and cauda epididymidis. The latter has been shown to resemble the distal nephron in many respects [45, 46]. In the cauda epididymidis, a net absorption of Na+ and water and secretion of K+ are observed under the basal condition [46, 47]. However, the transport of Na+ was linked to H+ secretion and was inhibited by amiloride, suggesting the presence of NHE at the luminal side of the epididymal tubule [48]. It has been reported that NHE2, but not NHE3, is present in the distal tubule [7, 10]. Our results are consistent with these findings, in that functional NHE2 protein was expressed in cultured epithelium of the cauda epididymidis (Figs. 1B, 2B, and 7B). Although NHE3 mRNA was revealed by RT-PCR (Fig. 1C), its protein was not detectable (Figs. 2C, 3D, and 7C). This discrepancy may be due to a very low NHE3 expression level that was below the detection thresholds of Western analysis, immunohistochemistry, and functional study. Immunohistochemical study revealed that NHE2 was localized apically in cauda epididymal epithelium [15]. Therefore, it is believed that NHE2 may participate in Na+ reabsorption in the cauda epididymidis.
As the testicular fluid flows down the male excurrent duct, the pH of the fluid lowers [49–51]. It has been proposed that the low pH is important for maintaining sperm in a quiescent state during epididymal transit [52, 53]. It is plausible that NHE3 and NHE2 in the efferent duct and epididymis, respectively, are involved in regulation of the pH of luminal fluid [48]. Recently, H+-ATPase has been found in the apical cells and clear cells of the epididymal epithelium [54, 55]. This indicates that more than one H+ transporter may be involved in the regulation of luminal pH in the male excurrent duct, as has been found in the kidney tubules.
In conclusion, NHE1, NHE2, and NHE3 were expressed by cultured epithelial cells of the efferent duct, whereas only NHE1 and NHE2 were expressed by cultured epithelial cells of the cauda epididymidis. It is thought that NHE1 participates in the homeostatic control of intracellular pH, cell volume, and anion secretion in both tissues. The NHE2 was present in the cytoplasm of ciliated cells of the efferent duct, where its function is unknown. The NHE3 was present at the apical membrane of the principal cells of the efferent duct, and it may have an important role in regulating the fluidity and pH of epididymal fluid.
FOOTNOTES
1 Supported by Research Grant Council of Hong Kong (P.Y.D.W.) and by National Cancer Institute grant R01CA85428 and American Heart Association, Maryland Affiliate, grant-in-aid S98645M (C.M.T.). 
2 Correspondence. FAX: 852 2603 5022;patrickwong{at}cuhk.edu.hk
Accepted: September 11, 2000.
Received: June 7, 2000.
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X.F. Wang, M.K. Yu, S.Y. Lam, K.M. Leung, J.L. Jiang, P.S. Leung, W.H. Ko, P.Y. Leung, S.B.C. Chew, C.Q. Liu, et al. Expression, Immunolocalization, and Functional Activity of Na+/H+ Exchanger Isoforms in Mouse Endometrial Epithelium Biol Reprod, January 1, 2003; 68(1): 302 - 308. [Abstract] [Full Text] [PDF]
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Y. Araki, K. Suzuki, R. J. Matusik, M. Obinata, and M.-C. Orgebin-Crist Immortalized Epididymal Cell Lines From Transgenic Mice Overexpressing Temperature-Sensitive Simian Virus 40 Large T-Antigen Gene J Androl, November 1, 2002; 23(6): 854 - 869. [Abstract] [Full Text] [PDF]
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R. W. Carlin, R. R. Quesnell, L. Zheng, K. E. Mitchell, and B. D. Schultz Functional and molecular evidence for Na+-HCO3- cotransporter in porcine vas deferens epithelia Am J Physiol Cell Physiol, October 1, 2002; 283(4): C1033 - C1044. [Abstract] [Full Text] [PDF]
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 C. A. Oliveira, Q. Zhou, K. Carnes, R. Nie, D. E. Kuehl, G. L. Jackson, L. R. Franca, M. Nakai, and R. A. Hess ER Function in the Adult Male Rat: Short- and Long-Term Effects of the Antiestrogen ICI 182,780 on the Testis and Efferent Ductules, without Changes in Testosterone Endocrinology, June 1, 2002; 143(6): 2399 - 2409. [Abstract] [Full Text] [PDF]
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N. Pastor-Soler, C. Isnard-Bagnis, C. Herak-Kramberger, I. Sabolic, A. Van Hoek, D. Brown, and S. Breton Expression of Aquaporin 9 in the Adult Rat Epididymal Epithelium Is Modulated by Androgens Biol Reprod, June 1, 2002; 66(6): 1716 - 1722. [Abstract] [Full Text] [PDF]
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H. H. Badran and L. S. Hermo Expression and Regulation of Aquaporins 1, 8, and 9 in the Testis, Efferent Ducts, and Epididymis of Adult Rats and During Postnatal Development J Androl, May 1, 2002; 23(3): 358 - 373. [Abstract] [Full Text] [PDF]
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K.-H. Lee, C. Finnigan-Bunick, J. Bahr, and D. Bunick Estrogen Regulation of Ion Transporter Messenger RNA Levels in Mouse Efferent Ductules Are Mediated Differentially Through Estrogen Receptor (ER) {alpha} and ER{beta} Biol Reprod, November 1, 2001; 65(5): 1534 - 1541. [Abstract] [Full Text] [PDF]
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 Q. Zhou, L. Clarke, R. Nie, K. Carnes, L.-W. Lai, Y.-H. H. Lien, A. Verkman, D. Lubahn, J. S. Fisher, B. S. Katzenellenbogen, et al. Estrogen action and male fertility: Roles of the sodium/hydrogen exchanger-3 and fluid reabsorption in reproductive tract function PNAS, October 31, 2001; (2001) 241245898. [Abstract] [Full Text] [PDF]
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G.P.H. Leung, X.D. Gong, K.H. Cheung, S.B. Cheng-Chew, and P.Y.D. Wong Expression of Cystic Fibrosis Transmembrane Conductance Regulator in Rat Efferent Duct Epithelium Biol Reprod, May 1, 2001; 64(5): 1509 - 1515. [Abstract] [Full Text]
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Q. Zhou, L. Clarke, R. Nie, K. Carnes, L.-W. Lai, Y.-H. H. Lien, A. Verkman, D. Lubahn, J. S. Fisher, B. S. Katzenellenbogen, et al. From the Cover: Estrogen action and male fertility: Roles of the sodium/hydrogen exchanger-3 and fluid reabsorption in reproductive tract function PNAS, November 20, 2001; 98(24): 14132 - 14137. [Abstract] [Full Text] [PDF]
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