Na+/H+-exchange activity and immunolocalization of NHE3 in rat epididymis

Corinne Bagnis1, Mireille Marsolais2, Daniel Biemesderfer3, Raynald Laprade2, and Sylvie Breton1,4

1 Program in Membrane Biology, Massachusetts General Hospital, Charlestown, Massachusetts 02129; 2 Groupe de Recherche en Transport Membranaire, University of Montreal, Montreal, Canada H3C3J7; 3 Department of Cellular and Molecular Physiology, Yale University, New Haven, Connecticut 06510; and 4 Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

An acidic luminal pH in the epididymis and vas deferens (VD) helps maintain mature sperm in an immotile state during storage. We have previously shown that the majority of proton secretion in the VD is due to the activity of the vacuolar H+-ATPase. Acidification is dependent on luminal sodium in more proximal regions of the epididymis, and we examined the distribution of the Na+/H+ exchanger, NHE3, by immunofluorescence and measured Na+/H+ exchange (NHE) activity in isolated epididymal tubules. NHE3 was detected in the apical pole of nonciliated cells of the efferent ducts and principal cells (PC) of the epididymis. No staining was seen in the distal cauda epididymidis and the VD. Isolated tubules from the distal initial segment (DIS) and proximal cauda epididymidis were perfused in vitro and loaded with the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6')-carboxyfluorescein. Ethylisopropyl amiloride (EIPA) (50 µM) reduced the initial rate of intracellular pH recovery (dpHi/dt), in response to an acute acid load, by 51% and 45% in the DIS and cauda epididymidis, respectively. In the DIS, removal of luminal sodium reduced dpHi/dt by 52%. HOE694 (50 µM) inhibited all EIPA-sensitive dpHi/dt in the DIS, despite the previously reported absence of NHE2 in this region (Cheng Chew SB, Leung GPH, Leung PY, Tse CM, and Wong PYD, Biol Reprod 62: 755-758, 2000). These data indicate that HOE694- and EIPA-sensitive Na+/H+ exchange may participate, together with the H+-ATPase, in luminal acidification in the male excurrent duct.

male reproductive tract; transepithelial acid-base transport; acidification; immunofluorescence; intracellular pH.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MEMBERS OF THE SODIUM/HYDROGEN exchanger (NHE) family mediate, under physiological conditions, the electroneutral exchange of one extracellular Na+ for one intracellular H+ across the plasma membrane. In eukaryotic organisms, the NHEs are involved in a wide variety of physiological processes including intracellular pH (pHi) regulation, cell volume regulation, as well as transepithelial vectorial transport of Na+ and acid/base equivalents (40, 52, 56). To date, five NHE isoforms (NHE1 to NHE5) have been cloned (3, 43, 44, 49, 50, 53). NHE1 is expressed in virtually all cell types (40), and NHE2 and NHE3 have been detected in intestine and kidney where they are targeted to the apical membrane of epithelial cells (7, 8, 9, 28, 47). NHE3 is involved in sodium absorption in the intestine, and in sodium reabsorption coupled to bicarbonate reabsorption in the kidney. NHE4 mRNA was detected in various organs including stomach, intestine, and kidney (9). In the kidney, it is located on the basolateral membrane of epithelial cells that are in contact with highly concentrated fluids. Because NHE4 is activated under hypertonic conditions, it is likely to be involved in cell volume regulation. Thus NHE1 and NHE4 are proposed to regulate cell pH and volume, respectively, whereas NHE2 and NHE3 participate in net transepithelial transport of Na+ and H+ in several absorptive epithelia.

The epithelium lining parts of the male reproductive tract is involved in active transepithelial transport of water and various solutes. Epithelial cells of the epididymis and vas deferens play a vital role in establishing the luminal environment in which spermatozoa mature and are stored (26, 33, 45). After leaving the testis, spermatozoa enter the efferent ducts, which extend from the rete testis to the epididymis. In the rat, the epididymis is composed of one convoluted tubule and is divided into several regions. The proximal region of the epididymis contains the initial segments, the intermediate zone, and the caput (or head). The corpus (or body) of the epididymis is in the middle part and connects the caput epididymidis to the cauda (or tail) epididymidis, located in the distal region, just before the vas deferens.

The efferent ducts are related embryologically to the kidney (27). They are remnants of the mesonephric kidney ducts that are closely related to renal proximal tubules. Isotonic fluid reabsorption with net sodium and chloride reabsorption occur in the efferent ducts, as in the homologous kidney proximal tubule (21, 29). During embryological development, the efferent ducts link the testis to the Wolffian duct, which differentiates to form the vas deferens. The epididymis derives from the Wolffian duct, as does the kidney collecting duct. The tubular fluid in the lumen of the epididymis undergoes considerable changes in composition as a consequence of net water, Na+, Cl- and HCO<SUB>3</SUB><SUP>−</SUP> absorption, K+ secretion, and acidification (4, 30, 38, 51, 54). As the efferent duct fluid transits through the initial segment and intermediate zone of the epididymis, the luminal bicarbonate concentration becomes significantly lower than that of blood (38), and an acidic pH is established in all regions of the epididymis and vas deferens (18, 37, 38). These factors help to maintain spermatozoa in an immotile state while they mature and are stored in the epididymis (1). Thus the development of the kidney and the excurrent duct system of the male reproductive tract are closely intertwined, and this is reflected by their similar transport properties.

Work from our laboratory has shown that a subpopulation of epithelial cells in the epididymis and vas deferens express high levels of the vacuolar type H+-ATPase on their apical membrane and subapical vesicles (11, 13, 15), and that up to 80% of net proton secretion is inhibited by bafilomycin in isolated vas deferens (11, 13). Cells that express the H+-ATPase are the narrow cells in the initial segments, and the clear cells in the caput, corpus, and cauda epididymidis. These cells also contain high levels of the cytosolic carbonic anhydrase, CAII (11, 13, 22, 34), implicating the involvement of bicarbonate in the acidification process. We have recently shown the presence of the Cl/HCO<SUB>3</SUB><SUP>−</SUP> exchanger AE2 (32) and of the electrogenic Na- HCO<SUB>3</SUB><SUP>−</SUP> cotransporter NBC (31) on the basolateral membrane of epithelial cells lining the lumen of the epididymis and vas deferens, these proteins being expressed at higher levels in the more proximal regions (initial segments, intermediate zone, and caput epididymidis) compared with the cauda epididymidis and vas deferens. Altogether, these results indicate that bicarbonate is being reabsorbed in these tissues and that the relative contribution of various transporters depends on the epididymal regions in which acid/base transport occurs.

In the rat efferent ducts microperfused in vivo, up to 70% of net fluid reabsorption is inhibited by amiloride, indicating that Na+/H+ exchange is a major transport pathway for fluid and electrolyte reabsorption in these segments (24). In the cauda epididymidis, net sodium reabsorption is inhibited by amiloride and does not depend on luminal chloride (54). Because luminal sodium was required for the acidification process to take place in this segment (4), an apical Na/H exchanger was then proposed to be involved in this process. Altogether, these results suggest that net transepithelial transport of NaHCO3 occurs in the epididymis, as is the case for other reabsorptive epithelia, such as the kidney proximal tubule. Whereas the contribution of the apical NHE3 has been well established in the proximal tubule (7, 8, 40, 55), a recent study showed that NHE2 (-/-) mice did not have a significant renal acidification defect and that the rate of sodium-dependent proton secretion in isolated proximal tubules in vitro was comparable to control mice (20). It was then concluded that NHE2 does not mediate proximal tubule Na/H antiporter activity. A recent report has shown the presence of NHE2 on the apical membrane of principal cells in some regions of the epididymis, and its absence from the initial segments (19). In the present study, we examined the potential role of NHE3 in Na-related acidification in the epididymis and vas deferens. We used an affinity-purified monoclonal antibody to localize NHE3 in the rat epididymis and vas deferens, and NHE functional activity was assessed on isolated tubules from the initial segment and cauda epididymidis perfused in vitro and loaded with the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6')-carboxyfluorescein (BCECF).


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MATERIALS AND METHODS
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Antibodies

An affinity-purified monoclonal antibody (19F5) against a fusion protein containing amino acids 702-832 of rabbit NHE3 was used. This antibody has been characterized previously (8). It is highly specific for NHE3 and does not recognize other NHE isoforms. To distinguish between principal cells and narrow and clear cells in the epididymis, an affinity-purified chicken antibody raised against the COOH-terminal 14 amino acids of the 31-kDa subunit (E subunit) of the H+-ATPase was used (14). To identify the ciliated cells in the efferent ducts, an affinity-purified rabbit antibody raised against the Cl/HCO<SUB>3</SUB><SUP>−</SUP> AE2 exchanger (provided by Seth Alper, Beth Israel Deaconess Medical Center) was used. A goat anti-mouse IgG conjugated to FITC was used to detect NHE3, and donkey anti-chicken or goat anti-rabbit IgG conjugated to CY3 was used to detect H+-ATPase and AE2, respectively.

Immunocytochemistry

Mature male Sprague-Dawley rats were anesthetized with an intraperitoneal injection of Nembutal (0.2 ml/100 g body wt of a 50 mg/ml solution). The male reproductive organs were fixed via left ventricle perfusion with PBS (0.9% NaCl in 10 mM sodium phosphate buffer, pH 7.4) followed by 150 ml of fixative solution containing 4% paraformaldehyde, 10 mM sodium periodate, 75 mM lysine, and 5% sucrose in 0.1 M sodium phosphate buffer (periodate-lysine-paraformaldehyde; PLP), as described previously (12, 31, 32). The epididymis and proximal vas deferens were dissected and further fixed by immersion at room temperature in PLP for 4-5 h. Tissues were washed 3 times, 10 min each time, in PBS (0.9% NaCl in 10 mM phosphate buffer, pH 7.4), and stored at 4°C in PBS containing 0.02% sodium azide. For cryostat sectioning, the epididymis was separated into two parts, one including the initial segments, the intermediate zone, the caput and proximal corpus epididymidis, and the other including the distal corpus, the cauda epididymidis, and the proximal vas deferens. Tissues were cryoprotected by immersion in 30% sucrose/PBS for at least 4 h, mounted in Tissue-Tek (Miles, Elkhart, IN) and frozen at -29°C in a Reichert Frigocut cryostat (Reichert Jung, Derry, NH). Sections were cut at 4 µm and picked up onto Superfrost/Plus microscope slides (Fisher Scientific, Pittsburgh, PA).

Sections were hydrated 5 min in PBS and treated for 4 min with SDS (1% in PBS), an antigen retrieval technique previously described (16). After three washes in PBS of 5 min each, nonspecific staining was blocked in a solution of 1% BSA/PBS/sodium azide for 15 min. Anti-NHE3 antibody (19F5) was applied at a dilution of 1:10 in PBS/sodium azide for 1.5 h at room temperature. Sections were then washed two times, 5 min each time, in PBS containing 2.7% NaCl to reduce nonspecific binding, followed by one wash in normal PBS.

For immunofluorescence labeling, secondary goat anti-mouse antibody coupled to FITC was applied, diluted 1:30 for 1 h at room temperature, and washes were performed as for the primary antibody. Sections were then incubated with antibody against the E subunit of the H+-ATPase, at a concentration of 10 µg/ml, or anti-AE2 antibody, at a concentration of 3 µg/ml, for 1.5 h at room temperature. After washes, donkey anti-chicken or goat anti-rabbit IgG conjugated with CY3 (Jackson Immunologicals) was applied at a dilution of 1:800 for 1 h and washed. Slides were mounted in Vectashield (Vector Laboratories, Burlingame, CA) diluted 1:2 in Tris buffer (1.5 M, pH 8.9).

For immunoperoxidase labeling, secondary goat anti-mouse antibody coupled to horseradish peroxidase was applied, after the primary antibody incubation, at a dilution of 1:50, for 1 h at room temperature. Washes were performed as above, and sites of peroxidase activity were detected by incubation with a solution of 3,3'-diaminobenzidine (Electron Microscopy Science, Ft. Washington, PA), hydrogen peroxide, and nickel in a Tris buffer, pH 7.6. Sections were then dehydrated in a graded series of ethanol concentrations in water and were cleared by incubating in xylene, two times, 5 min each time. Slides were mounted in Permount.

Sections were photographed on a Nikon Eclipse 800 or a Nikon FXA epifluorescence microscope. Black and white images were taken on Kodak TMAX 400 film exposed at 1600 ASA by using specific CY3 and FITC filters, and color images were taken on Kodak Ektachrome 400 Elite film exposed at 2800 ASA, by using a dual CY3/FITC filter that allows simultaneous visualization of both fluorophores. Color slides were scanned on a Polaroid slide scanner (SprintScan 35 Plus) by using Adobe Photoshop. Some images were collected by using a BioRad Radiance 2000 confocal microscope. A collection of XY images (Z-series collection) was acquired and 3-D projections were performed by using the Radiance 2000 software. Digital images were printed on an Epson Stylus 600 inkjet printer.

Tubule Perfusion In Vitro

Mature Sprague-Dawley rats were anesthetized with Nembutal as described above. Epididymis was harvested and kept either in a cold preservation fluid containing (in mM) 56 Na2HPO4, 13 NaH2PO4, and 140 sucrose, pH 7.4, or they were kept at 37°C in a physiological solution (control solution in Table 1) bubbled with O2. Tubules were dissected from the distal initial segments or the proximal cauda epididymidis by using fine forceps. They were then transferred into the perfusion chamber on the stage of a Zeiss inverted microscope, and peritubular and luminal perfusion were performed, as described previously for kidney proximal tubules (10, 35).

                              
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Table 1.   Solution constituents

pHi Measurements

pHi was measured by using the fluorescent dye BCECF (Molecular Probes), as described previously (5, 10). The acetoxymethyl ester form of the probe was added to the luminal perfusate at a final concentration of 5 µM, and loading was allowed for 5-10 min at 30°C. Dual excitation at 450 and 500 nm was performed by using a PTI spectrofluorometer. Fluorescent light emitted at 530 nm and corrected for background was detected from the epithelial cells lining the tubule lumen. For each experiment, calibration of the dye was performed after equilibration of the tubule in solutions containing 120 mM potassium and 10 µM nigericin at pH 6.75, 7.0, and 7.25. pHi was estimated from the ratio of the emitted light at the two excitation wavelengths.

After the steady-state pHi was monitored under control conditions, an acute acid load was induced by a pulse of 20 mM NH3/NH<SUB>4</SUB><SUP>+</SUP> in the luminal solution (replacing N-methyl-D-glucamine), as described previously (39). NHE function was assayed as the ethylisopropyl amiloride (EIPA)-dependent inhibition of the initial rate of pHi recovery (dpHi/dt). dpHi/dt was estimated from the linear portion of the pHi recovery by using the linear regression function in Excel 5.0. These experiments were performed in the absence of nominal bicarbonate in the peritubular and luminal perfusates to minimize the contribution of HCO<SUB>3</SUB><SUP>−</SUP>-transporting mechanisms in pHi recovery.

Statistics

Data are expressed as means ± SE, where n refers to the number of tubules analyzed. Two tailed paired t-tests were performed by using Excel 5.0.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Immunolocalization of NHE3

Immunocytochemistry on cryostat epididymis sections using horseradish peroxidase revealed that NHE3 is located in the apical membrane of epithelial cells and that its level of expression varies with the epididymal region examined (Fig. 1, A and B). NHE3 is also expressed by connective tissue. As shown in Fig. 1A, proximal and middle initial segments are negative for NHE3 (see Refs. 2 and 25 for nomenclature). NHE3 staining starts to appear in the distal initial segment and intermediate zone, and a strong staining is observed in the proximal caput epididymidis, whereas the distal caput shows a low level of NHE3 expression. The proximal corpus epididymidis also shows a weak staining for NHE3 (data not shown). Figure 1B shows the distal corpus epididymidis, the cauda epididymidis, and the proximal region of the vas deferens stained for NHE3. NHE3 expression also varies in these regions, being moderate in the distal corpus epididymidis and highest in the proximal cauda epididymidis. The distal cauda epididymidis and the vas deferens show no detectable levels of NHE3. Spermatozoa in all regions of the epididymis, also, do not show expression of NHE3.


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Fig. 1.   Immunocytochemical localization of Na+/H+ exchanger (NHE3) on cryostat sections of rat epididymis using horseradish peroxidase. Each figure is a montage of 20 individual images taken with a ×4 objective on a Nikon E800 microscope. Images were assembled by using IPLab Spectrum software. A: proximal portions of the epididymis including the initial segments, intermediate zone, and caput epididymidis. Proximal (PIS) and middle (MIS) initial segments are negative for NHE3, the distal initial segment (DIS) shows a moderate apical staining, the intermediate zone (IZ) and proximal caput epididymidis (PCap) are strongly stained, and the distal caput epididymidis (DCap) shows a very weak apical staining. B: distal portions of the epididymis including the distal corpus (DCor) and the cauda epididymidis. A moderate staining is seen in the DCor, and no staining is seen in the distal cauda epididymidis (DCau). The highest staining is observed in the proximal cauda epididymidis (PCau). Connective tissue is stained for NHE3, and spermatozoa are negative. Bar = 1 mm.

Higher magnification immunofluorescence microscopy revealed the same pattern of NHE3 expression in various regions of the epididymis and vas deferens. The efferent ducts, which connect the testis to the epididymis, express high levels of NHE3 on their apical membrane (Fig. 2). These segments contain two cell types, the ciliated cells, which express high levels of the basolateral Cl-/HCO<SUB>3</SUB><SUP>−</SUP> exchanger AE2 (32), and the nonciliated cells. As shown in Fig. 2, the nonciliated cells show a bright apical staining for NHE3, and cells that are positive for AE2 do not express NHE3. In the epididymis, the proximal and middle initial segments were negative (not shown), but the distal initial segment (Fig. 3A) and the intermediate zone (Fig. 3C) showed a strong staining of the apical membrane of most epithelial cells. Double labeling for H+-ATPase (Fig. 3, B and D) revealed the presence of a few narrow cells (positive for H+-ATPase) in these segments. Due to the presence of long principal cell apical microvilli (or stereocilia), which extend extensively into the tubule lumen, it was difficult to determine whether narrow cells also expressed NHE3 on their apical membrane. However, careful examination of sections at higher magnification by confocal microscopy clearly showed that H+-ATPase-rich cells do not express NHE3 (Fig. 4).


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Fig. 2.   Double-immunofluorescence localization of NHE3 (green) and the Cl/HCO<SUB>3</SUB><SUP>−</SUP> exchanger AE2 (red) in the efferent duct. NHE3 is located in the apical membrane of nonciliated cells (AE2 negative). Two AE2-positive cells (ciliated cells) that were appropriately sectioned through their apical membrane (arrows) show no staining for NHE3. Red blood cells (arrowheads) are strongly stained with the AE2 antibody. Bar = 20 µm.



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Fig. 3.   Double-immunofluorescence localization of NHE3 (A-C) and the vacuolar H+-ATPase (B-D) in the rat epididymis. A-B: DIS; C-D: IZ. Principal cells in both DIS and IZ show a strong apical staining (A-C). A few narrow cells, stained for H+-ATPase, are present in these segments (B-D). Due to the presence of the very long principal cell stereocilia, it is very difficult to determine whether narrow cells express NHE3 (see Fig. 4). Bar = 20 µM.



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Fig. 4.   Confocal microscopy showing double-immunofluorescence labeling for NHE3 (green) and H+-ATPase (red) in the DIS. A collection of 15 XY images (Z-series collection) was acquired. A, B, and C show 3-D projections at 3 different angles. NHE3 is present in the apical stereocilia (microvilli) of principal cells, whereas narrow cells (arrow), positive for H+-ATPase, do not express NHE3. Bar = 10 µM.

A strong apical staining was detected in principal cells of the proximal caput epididymidis (Fig. 5A), and a weaker NHE3 staining was observed in the distal caput epididymidis (Fig. 5C). In these regions, H+-ATPase-rich cells are wider and principal cell microvilli are shorter than in the more proximal regions. Double labeling for the H+-ATPase revealed that NHE3 is restricted to principal cells (Fig. 5, A-D). Confocal microscopy confirmed that NHE3 staining was absent from H+-ATPase-rich cells in these regions (not shown). NHE3 labeling became progressively weaker in the corpus epididymidis (data not shown) and reintensified in the proximal cauda epididymidis, where principal cells showed a strong apical staining (Fig. 6 A-C). In the transition region between the proximal and distal cauda epididymidis, some principal cells became negative for NHE3 (Fig. 6A, arrows), and no staining was detected in the principal cells of the distal cauda epididymidis and the vas deferens (Fig. 6B). Figure 6C shows that H+-ATPase-rich cells do not express NHE3 in the proximal cauda epididymidis.


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Fig. 5.   Double-immunofluorescence labeling for NHE3 (A-C), and the vacuolar H+-ATPase (B-D) in the PCap (A and B) and DCap (C and D). A strong staining for NHE3 is seen in the apical membrane of principal cells of the PCap (A), and a weaker apical staining is seen in principal cells of the DCap (C). H+-ATPase-rich cells do not express NHE3. In the PCap, the apical membrane of several H+-ATPase-rich cells is masked by the long apical microvilli of principal cells. Bars = 20 µm.



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Fig. 6.   Double-immunofluorescence labeling for NHE3 (green) and the vacuolar H+-ATPase (red) in the PCau (A) and the proximal vas deferens (VD) (B). In the PCau, a strong apical staining is seen in principal cells, exclusively. In the transition region between the PCau and DCau, some principal cells are negative for NHE3 (A, arrows), and principal cells of the VD are negative (B). A larger magnification of the PCau (C), shows that clear cells, positive for H+-ATPase, do not contain NHE3. Bars = 20 µm.

Na+/H+ Exchange Functional Activity in Isolated Tubules

Distal initial segment and intermediate zone. In a first series of experiments, the steady-state pHi measured in 10 tubules isolated from the distal initial segment or the intermediate zone was 7.06 ± 0.04 under control conditions. As shown in Fig. 7A, addition of NH3/NH<SUB>4</SUB><SUP>+</SUP> in the lumen produced a rapid intracellular alkalinization due to the influx of NH3 into the cells. On washout of NH3/NH<SUB>4</SUB><SUP>+</SUP>, a marked acidification was induced as intracellular NH<SUB>4</SUB><SUP>+</SUP> dissociated into NH3 and H+, followed by a spontaneous pHi recovery. After pHi has returned to its control value, the Na+/H+ exchanger inhibitor, EIPA, was added to the luminal perfusate at a final concentration of 50 µM for 5 min. A second pulse of NH3/NH<SUB>4</SUB><SUP>+</SUP> was then applied in the continuous presence of EIPA. On average, dpHi/dt was 0.14 ± 0.01 under control conditions and was reduced to 0.07 ± 0.01 in the presence of EIPA (51% inhibition; n = 10; P < 0.005; Fig. 7B). Control experiments were also performed in which two consecutive pulses of NH3/NH<SUB>4</SUB><SUP>+</SUP> were induced without addition of EIPA (not shown). In this series of five tubules, dpHi/dt was 0.12 ± 0.02 and 0.15 ± 0.02 pH unit/min in response to the first and second pulse of NH3/NH<SUB>4</SUB><SUP>+</SUP>, respectively [P = not significant (NS)].


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Fig. 7.   A: representative trace showing the effect of ethylisopropyl amiloride (EIPA) on intracellular pH (pHi) recovery after an acid load induced by a prepulse of NH3/NH<SUB>4</SUB><SUP>+</SUP> in the lumen of a tubule isolated from the DIS. The rate of pHi recovery is significantly reduced by EIPA. B: histogram showing the mean effect of 50 µM EIPA on pHi recovery in response to an acute acid load in 10 tubules isolated from the DIS and intermediate zone. Ctl, control. EIPA significantly reduced the rate of pHi recovery (* P < 0.005).

To determine whether the Na+/H+ exchange activity measured in isolated tubules from the distal initial segments was generated by an apical or basolateral Na+/H+ exchanger, the luminal sodium was removed and pHi and dpHi/dt were measured. As shown in Fig. 8, removal of sodium (replaced by N-methyl-D-glucamine; Na = 0 in Table 1) in the luminal perfusate induced a progressive intracellular acidification from an initial value of 7.27 ± 0.04 to reach 6.97 ± 0.07 after 5 min (n = 5, P < 0.005). In this series of experiments, three pulses of NH3/NH<SUB>4</SUB><SUP>+</SUP> were performed: the first and third were applied before and after the sodium removal period (controls), and the second was applied in the absence of sodium. The dpHi/dt values from the two control pulses were averaged and compared with the value measured under sodium-free conditions. dpHi/dt was 0.14 ± 0.02 pH unit/min under control conditions, and was reduced to 0.06 ± 0.01 pH unit/min in the absence of luminal sodium (52% inhibition; n = 5; P < 0.0005). Therefore, both EIPA and luminal sodium removal induced an inhibition of dpHi/dt of the same magnitude, indicating that an apical Na+/H+ exchange activity was being detected.


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Fig. 8.   Representative trace showing the effect of luminal sodium removal on pHi recovery in response to an acid load in a tubule isolated from the DIS. Three NH3/NH<SUB>4</SUB><SUP>+</SUP> pulses were performed. The first and third were control pulses applied before and after the sodium removal period, and the second pulse was applied in the absence of sodium. The removal of luminal sodium induced an intracellular acidification and significantly reduced pHi recovery.

Our results showing strong apical staining for NHE3, together with the previously reported absence of NHE2 in the initial segments (19), pointed toward a role for NHE3, and not NHE2, in isolated tubules from the distal initial segment. In an attempt to identify the NHE isoform responsible for the apical Na/H exchange activity, we used the inhibitor HOE694 (Hoechst, Frankfurt, Germany). Previous reports have shown that, at a concentration of 50 µM, HOE694 should reduce most of the NHE2 activity and should not affect NHE3 (23). To our surprise, HOE694 inhibited dpHi/dt by 51 ± 12% (n = 4; P < 0.005; Fig. 9), and EIPA, when added after HOE694, did not induce any additional reduction of dpHi/dt (48 ± 2.4% inhibition compared with control value: n = 4; P < 0.005 vs. control; P = ns vs. HOE694).


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Fig. 9.   Histogram showing the mean effect of 50 µM HOE694 and 50 µM EIPA on pHi recovery in response to an acute acid load in 4 tubules isolated from the DIS. HOE694 significantly inhibited pHi recovery, and EIPA, when added after HOE694, did not induce any additional reduction. (* P < 0.005 vs. control; P = NS vs. HOE694).

Proximal cauda epididymidis. Na+/H+ exchange activity was also measured on tubules isolated from the proximal cauda epididymidis. In this series of 11 experiments, the control steady-state pHi was 7.19 ± 0.05. dpHi/dt in response to an NH3/NH<SUB>4</SUB><SUP>+</SUP>-induced acid load was 0.23 ± 0.02 pH unit/min under control conditions and was reduced to 0.12 ± 0.01 pH unit/min (45% inhibition; P < 0.005; Fig. 10) by 50 µM EIPA. Control experiments including two consecutive NH3/NH<SUB>4</SUB><SUP>+</SUP> pulses without EIPA were also performed. dpHi/dt were 0.25 ± 0.05 and 0.20 ± 0.03 pH unit/min in response to the first and second pulse of NH3/NH<SUB>4</SUB><SUP>+</SUP>, respectively (n = 5; P = NS).


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Fig. 10.   Histogram showing the mean effect of 50 µM EIPA on pHi recovery in response to an acute acid load in 7 tubules isolated from the cauda epididymidis. EIPA significantly reduced the rate of pHi recovery (* P < 0.005).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Both pH and bicarbonate regulate the motility of spermatozoa (1, 41, 42, 48). The establishment of an acidic pH and of a low HCO<SUB>3</SUB><SUP>−</SUP> concentration in the luminal fluid of the epididymis and vas deferens (18, 37, 38) contribute to the maintenance of sperm in a quiescent state while they mature and are stored in these organs (26, 33, 45). Our laboratory has shown that narrow and clear cells express high levels of the vacuolar H+-ATPase in their apical pole and are responsible for the majority of proton secretion in isolated vas deferens (11, 13, 15). In addition, an earlier study has shown that acidification was dependent on the presence of sodium in the lumen of cauda epididymidis perfused in vivo (4), and an Na+/H+ exchanger was proposed to participate in this process. The purpose of the present study was to determine whether the Na+/H+ exchanger NHE3 was involved, in addition to the H+-ATPase, in the acidification of the epididymal lumen.

NHE3 plays a major role in acid-base balance and Na-fluid homeostasis (46). It is highly expressed on the apical membrane of kidney proximal tubules and intestinal epithelial cells, where it is involved in NaCl and HCO<SUB>3</SUB><SUP>−</SUP> absorption (7, 8, 28, 40, 52, 56). In view of the transepithelial Na+ and HCO<SUB>3</SUB><SUP>−</SUP> reabsorption and H+ secretion mechanisms that take place in the epididymis and vas deferens, we investigated whether this isoform was present in these tissues. By using a highly purified monoclonal antibody, we found that NHE3 is expressed in nonciliated cells of the efferent ducts and in principal cells of the epididymis, where it is located on the apical membrane. Narrow and clear cells, which were identified by their high expression of the vacuolar H+-ATPase, do not contain NHE3. Interestingly, the level of expression of NHE3 varies in different regions of the epididymis. In the proximal parts of the epididymis, NHE3 is most abundant in the distal initial segment, the intermediate zone, and the proximal caput epididymidis. In the distal caput and the corpus epididymidis, NHE3 is barely detectable. In the distal regions of the epididymis, NHE3 is expressed in the proximal cauda epididymidis only, being absent from the distal cauda epididymidis and vas deferens.

Functional assays using BCECF on isolated tubules from the initial segment/intermediate zone region, and from the cauda epididymidis showed that Na+/H+ exchange accounts for ~50% of acid extrusion, in the nominal absence of extracellular HCO<SUB>3</SUB><SUP>−</SUP>. In the initial segments, pHi recovery in response to an acid load was greatly reduced in the absence of luminal sodium. The inhibition induced by the removal of Na+ was similar to that induced by EIPA (in the presence of Na+) indicating that the EIPA-sensitive pHi recovery was due to the activity of an apical Na+/H+ exchanger. We then tried to identify pharmacologically the NHE isoform responsible for this activity. Because the initial segments were reported to be negative for NHE2 by immunocytochemistry (19), NHE3 was the most likely isoform to be involved in this region. However, HOE694, used at a concentration that was expected to inhibit most NHE2 activity but not NHE3 (23), inhibited all the EIPA-sensitive pHi recovery. This result can be interpreted in several ways: 1) NHE2 is present in the initial segments, but has so far remained undetected; 2) an NHE3 splice variant, sensitive to HOE694, is expressed in the epididymis; and 3) epididymal posttranslational modification confers to NHE3 sensitivity to HOE694. NHE2 might have remained undetected by immunocytochemistry in the initial segments for different reasons, including reduced immunoreactivity by masking of the antigenic site, due to protein-protein interaction. This has been shown for some membrane proteins, such as NHE3, the detection of which is reduced by its interaction with megalin (6). It is, therefore, possible that a lower NHE2 immunoreactivity might have precluded its detection in the initial segments compared with the rest of the epididymis. In that case, the HOE694 effect observed in the present study would be due to inhibition of NHE2. Alternatively, sensitivity of NHE3 to HOE694 has been recently shown in the main pancreatic duct (36). In this study, most of the NHE3 activity was inhibited by 50 µM HOE694 in pancreatic ducts isolated from normal and NHE2 (-/-) mice, whereas no inhibition of the kidney proximal tubule NHE3 was observed. The authors concluded that sensitivity to HOE694 (and possibly other amiloride analogs) "cannot be used in all cases to discern the NHE isoform expressed in a given tissue or cell type." It is, therefore, conceivable that, similar to the pancreatic duct, an HOE694-sensitive NHE3 is expressed in the initial segments of the epididymis. Additional experiments using NHE2 (-/-), NHE3 (-/-), and double knockout mice will be required to answer these questions.

The high levels of NHE3 in the proximal regions of the excurrent duct (efferent ducts, initial segment, intermediate zone, and proximal caput epididymidis) correlate with the very low concentration of HCO<SUB>3</SUB><SUP>−</SUP> that is reached in the caput epididymidis (38). In the epididymis, NHE3 expression also correlates with high levels of the electrogenic Na-HCO<SUB>3</SUB><SUP>−</SUP> cotransporter NBC and of the Cl/HCO<SUB>3</SUB><SUP>−</SUP> exchanger AE2 in the basolateral membrane of epithelial cells in these regions (31, 32). Although no direct evaluation of the role of NHE3 in transepithelial transport was performed in the present study, the polarized expression of this proton-extruding protein in the apical membrane and of various HCO<SUB>3</SUB><SUP>−</SUP> transporters in the basolateral membrane indicates that the initial segments, intermediate zone, and proximal caput epididymis are highly specialized for net HCO<SUB>3</SUB><SUP>−</SUP> reabsorption. Efferent ducts reabsorb the majority of the fluid that originates from the testis, and a previous study has shown that amiloride inhibits up to 70% of fluid reabsorption in these segments (24). These results, together with our data, suggest that NHE3 might participate in fluid and electrolyte reabsorption in the efferent duct.

NHE3 can also participate in net sodium reabsorption in various regions of the epididymis. NHE3 is expressed in the proximal cauda epididymidis (present study) and Na+ reabsorption is inhibited by amiloride in the perfused cauda epididymidis (54). However, NHE2 has also been localized on the apical membrane of epithelial cells of the cauda epididymidis (19), and the Na+/H+ exchange activity that we measured in this segment might reflect NHE2 activity, in addition to NHE3. The apparent sensitivity of the epididymal NHE3 to HOE694 precludes the use of this inhibitor (or possibly other amiloride analogs) to determine the relative contribution of these two isoforms in this segment. Both NHE2 and NHE3 might, therefore, contribute to net HCO<SUB>3</SUB><SUP>−</SUP>, Na+, and water absorption in this region of the epididymis. Na+-dependent proton secretion is not altered in kidney proximal tubules (20) and main pancreatic ducts (36) from NHE2 (-/-) mice compared with normal animals, and the role of NHE2 in these organs remains to be elucidated. Further experiments using NHE2 (-/-) and NHE3 (-/-) mice will be required to determine the relative contribution of these transporters in the epididymis.

Overall, with the exception of the corpus epididymidis, NHE3 is abundant in segments of the epididymis that contain fewer H+-ATPase-rich cells, and is not detectable in the distal cauda epididymidis, where H+-ATPase-rich cells are numerous. In the distal cauda epididymidis, H+-ATPase-rich cells are poised to play a central role in the final acidification mechanisms in the regions where spermatozoa are stored. Our laboratory has shown that in the vas deferens, H+-ATPase-rich cells are responsible for up to 80% of proton secretion (13). In addition, the absence of NHE3 in the vas deferens correlates with our previous results showing no effect of amiloride on net proton secretion in this segment (17).

In summary, our study shows a high expression of the Na+/H+ exchanger, NHE3, in efferent ducts, initial segments, intermediate zone, proximal caput, and proximal cauda epididymidis, whereas NHE3 was not detectable in the distal cauda epididymidis and vas deferens. These results suggest that transepithelial acid/base transport involves different sets of proteins in various regions of the excurrent duct system, NHE3 being involved in HCO<SUB>3</SUB><SUP>−</SUP> reabsorption in the proximal regions of the epididymis, and the vacuolar H+-ATPase participating in net acid secretion in the distal portions of the epididymis.


    ACKNOWLEDGEMENTS

We thank Ndona N. Nsumu for excellent technical help.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-38452 (S. Breton) and DK-33793 (D. Biemesderfer).

Address for reprint requests and other correspondence: Sylvie Breton, Renal Unit and Program in Membrane Biology, Massachusetts General Hospital East, 149 13th St., Charlestown, MA 02129 (E-mail: sbreton{at}receptor.mgh.harvard.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 18 June 2000; accepted in final form 10 November 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Acott, TS, and Carr DW. Inhibition of bovine spermatozoa by caudal epididymal fluid: II. Interaction of pH and a quiescence factor. Biol Reprod 30: 926-935, 1984[Abstract].

2.   Adamali, HI, and Hermo L. Apical and narrow cells are distinct cell types differing in their structure, distribution, and functions in the adult rat epididymis. J Androl 17: 208-222, 1996[Abstract/Free Full Text].

3.   Attaphitaya, S, Park K, and Melvin JE. Molecular cloning and functional expression of a rat Na+/H+ exchanger (NHE5) highly expressed in brain. J Biol Chem 274: 4383-4388, 1999[Abstract/Free Full Text].

4.   Au, CL, and Wong PY. Luminal acidification by the perfused rat cauda epididymidis. J Physiol (Lond) 309: 419-427, 1980[ISI][Medline].

5.   Beck, JS, Breton S, Giebisch G, and Laprade R. Potassium conductance regulation by pH during volume regulation in rabbit proximal convoluted tubules. Am J Physiol Renal Fluid Electrolyte Physiol 263: F453-F458, 1992[Abstract/Free Full Text].

6.   Biemesderfer, D, Nagy T, DeGray B, and Aronson PS. Specific association of megalin and the Na+/H+ exchanger isoform NHE3 in the proximal tubule. J Biol Chem 274: 17518-17524, 1999[Abstract/Free Full Text].

7.   Biemesderfer, D, Pizzonia J, Abu-Alfa A, Exner M, Reilly R, Igarashi P, and Aronson PS. NHE3: a Na+/H+ exchanger isoform of renal brush border. Am J Physiol Renal Fluid Electrolyte Physiol 265: F736-F742, 1993[Abstract/Free Full Text].

8.   Biemesderfer, D, Rutherford PA, Nagy T, Pizzonia JH, Abu-Alfa AK, and Aronson PS. Monoclonal antibodies for high-resolution localization of NHE3 in adult and neonatal rat kidney. Am J Physiol Renal Physiol 273: F289-F299, 1997[Abstract/Free Full Text].

9.   Bookstein, C, Xie Y, Rabenau K, Musch MW, McSwine RL, Rao MC, and Chang EB. Tissue distribution of Na+/H+ exchanger isoforms NHE2 and NHE4 in rat intestine and kidney. Am J Physiol Cell Physiol 273: C1496-C1505, 1997[ISI][Medline].

10.   Breton, S, Beck JS, and Laprade R. cAMP stimulates proximal convoluted tubule Na+-K+-ATPase activity. Am J Physiol Renal Fluid Electrolyte Physiol 266: F400-F410, 1994[Abstract/Free Full Text].

11.   Breton, S, Hammer K, Smith PJS, and Brown D. Proton secretion in the male reproductive tract: involvement of Cl-independent HCO3 transport. Am J Physiol Cell Physiol 275: C1134-C1142, 1998[Abstract/Free Full Text].

12.   Breton, S, Nsumu NN, Galli T, Sabolic I, Smith PJ, and Brown D. Tetanus toxin-mediated cleavage of cellubrevin inhibits proton secretion in the male reproductive tract. Am J Physiol Renal Physiol 278: F717-F725, 2000[Abstract/Free Full Text].

13.   Breton, S, Smith PJS, Lui B, and Brown D. Acidification of the male reproductive tract by a proton pumping H+-ATPase. Nat Med 2: 470-472, 1996[ISI][Medline].

14.   Breton, S, Wiederhold T, Marshansky V, Nsumu NN, Ramesh V, and Brown D. The B1 subunit of the H+-ATPase is a PDZ domain-binding protein. Colocalization with NHE-RF in renal B-intercalated cells. J Biol Chem 275: 18219-18224, 2000[Abstract/Free Full Text].

15.   Brown, D, Lui B, Gluck S, and Sabolic I. A plasma membrane proton ATPase in specialized cells of rat epididymis. Am J Physiol Cell Physiol 263: C913-C916, 1992[Abstract/Free Full Text].

16.   Brown, D, Lydon J, McLaughlin M, Stuart-Tilley A, Tyzskowski R, and Alper SL. Antigen retrieval in cryostat sections and cultured cells by treatment with sodium dodecyl sulfate (SDS). Histochem Cell Biol 105: 261-267, 1996[ISI][Medline].

17.   Brown, D, Smith PJ, and Breton S. Role of V-ATPase-rich cells in acidification of the male reproductive tract. J Exp Biol 200: 257-262, 1997[Abstract/Free Full Text].

18.   Caflisch, CR, and DuBose TJ. Direct evaluation of acidification by rat testis and epididymis: role of carbonic anhydrase. Am J Physiol Endocrinol Metab 258: E143-E150, 1990[Abstract/Free Full Text].

19.   Cheng Chew, SB, Leung GPH, Leung PY, Tse CM, and Wong PYD Polarized distribution of NHE1 and NHE2 in the rat epididymis. Biol Reprod 62: 755-758, 2000[Abstract/Free Full Text].

20.   Choi, JY, Shah M, Lee MG, Schultheis PJ, Shull GE, Muallem S, and Baum M. Novel amiloride-sensitive sodium-dependent proton secretion in the mouse proximal convoluted tubule. J Clin Invest 105: 1141-1146, 2000[Abstract/Free Full Text].

21.   Clulow, J, Hansen LA, and Jones RC. In vivo microperfusion of the ductuli efferentes testis of the rat: flow dependence of fluid reabsorption. Exp Physiol 81: 633-644, 1996[Abstract].

22.   Cohen, JP, Hoffer AP, and Rosen S. Carbonic anhydrase localization in the epidimymis and testis of the rat: histochemical and biochemical analysis. Biol Reprod 14: 339-346, 1976[ISI][Medline].

23.   Counillon, L, Scholz W, Lang HJ, and Pouyssegur J. Pharmacological characterization of stably transfected Na+/H+ antiporter isoforms using amiloride analogs and a new inhibitor exhibiting anti-ischemic properties. Mol Pharmacol 44: 1041-1045, 1993[Abstract].

24.   Hansen, LA, Clulow J, and Jones RC. The role of Na+-H+ exchange in fluid and solute transport in the rat efferent ducts. Exp Physiol 84: 521-527, 1999[Abstract].

25.   Hermo, L. Structural features and functions of principal cells of the intermediate zone of the epididymis of adult rats. Anat Rec 242: 515-530, 1995[ISI][Medline].

26.   Hinton, BT, and Palladino MA. Epididymal epithelium: its contribution to the formation of a luminal fluid microenvironment. Microsc Res Tech 30: 67-81, 1995[ISI][Medline].

27.   Hinton, BT, and Turner TT. Is the epididymis a kidney analogue? News Physiol Sci 3: 28-31, 1988[Abstract/Free Full Text].

28.   Hoogerwerf, WA, Tsao SC, Devuyst O, Levine SA, Yun CH, Yip JW, Cohen ME, Wilson PD, Lazenby AJ, Tse CM, and Donowitz M. NHE2 and NHE3 are human and rabbit intestinal brush-border proteins. Am J Physiol Gastrointest Liver Physiol 270: G29-G41, 1996[Abstract/Free Full Text].

29.   Ilio, KY, and Hess RA. Structure and function of the ductuli efferentes: a review. Microsc Res Tech 29: 432-467, 1994[ISI][Medline].

30.   Jenkins, AD, Lechene CP, and Howards SS. Concentrations of seven elements in the intraluminal fluids of the rat seminiferous tubules, rete testis, and epididymis. Biol Reprod 23: 981-987, 1980[ISI][Medline].

31.   Jensen, LJ, Schmitt BM, Berger UV, Nsumu NN, Boron WF, Hediger MA, Brown D, and Breton S. Localization of sodium bicarbonate co-transporter (NBC) protein and mRNA in rat epididymis. Biol Reprod 60 (3): 573-579, 1999[Abstract/Free Full Text].

32.   Jensen, LJ, Stuart-Tilley AK, Peters LL, Lux SE, Alper SL, and Breton S. Immunolocalization of AE2 anion exchanger in rat and mouse epididymis. Biol Reprod 61: 973-980, 1999[Abstract/Free Full Text].

33.   Jones, RC, and Murdoch RN. Regulation of the motility and metabolism of spermatozoa for storage in the epididymis of eutheran and marsupial mammals. Reprod Fertil Dev 8: 553-568, 1996[ISI][Medline].

34.   Kaunisto, K, Parkkila S, Parkkila AK, Waheed A, Sly WS, and Rajaniemi H. Expression of carbonic anhydrase isoenzymes IV and II in rat epididymal duct. Biol Reprod 52: 1350-1357, 1995[Abstract].

35.   Laprade, R, Lapointe JY, Breton S, Duplain M, and Cardinal J. Intracellular potassium activity in mammalian proximal tubule: effect of perturbations in transepithelial sodium transport. J Membr Biol 121: 249-259, 1991[ISI][Medline].

36.   Lee, MG, Ahn W, Choi JY, Luo X, Seo JT, Schultheis PJ, Shull GE, Kim KH, and Muallem S. Na(+)-dependent transporters mediate HCO3- salvage across the luminal membrane of the main pancreatic duct. J Clin Invest 105: 1651-1658, 2000[Abstract/Free Full Text].

37.   Levine, N, and Kelly H. Measurement of pH in the rat epididymis in vivo. J Reprod Fertil 52: 333-335, 1978[Abstract].

38.   Levine, N, and Marsh DJ. Micropuncture studies of the electrochemical aspects of fluid and electrolytes transport in individual seminiferous tubules, the epididymis and the vas deferens in rats. J Physiol (Lond) 213: 557-575, 1971[ISI][Medline].

39.   Nakhoul, NL, Lopes AG, Chaillet JR, and Boron WF. Intracellular pH regulation in the S3 segment of the rabbit proximal tubule in HCO3- -free solutions. J Gen Physiol 92: 369-393, 1988[Abstract].

40.   Noel, J, and Pouyssegur J. Hormonal regulation, pharmacology, and membrane sorting of vertebrate Na+/H+ exchanger isoforms. Am J Physiol Cell Physiol 268: C283-C296, 1995[Abstract/Free Full Text].

41.   Okamura, N, Tajima Y, Ishikawa H, Yoshii S, Koiso K, and Sugita Y. Lowered levels of bicarbonate in seminal plasma cause the poor sperm motility in human infertile patients. Fertil Steril 45: 265-272, 1986[ISI][Medline].

42.   Okamura, N, Tajima Y, Soejima A, Masuda H, and Sugita Y. Sodium bicarbonate in seminal plasma stimulates the motility of mammalian spermatozoa through direct activation of adenylate cyclase. J Biol Chem 260: 9699-9705, 1985[Abstract/Free Full Text].

43.   Orlowski, J, Kandasamy RA, and Shull GE. Molecular cloning of putative members of the Na/H exchanger gene family. cDNA cloning, deduced amino acid sequence, and mRNA tissue expression of the rat Na/H exchanger NHE-1 and two structurally related proteins. J Biol Chem 267: 9331-9339, 1992[Abstract/Free Full Text].

44.   Reilly, RF, Hildebrandt F, Biemesderfer D, Sardet C, Pouyssegur J, Aronson PS, Slayman CW, and Igarashi P. cDNA cloning and immunolocalization of a Na+-H+ exchanger in LLC-PK1 renal epithelial cells. Am J Physiol Renal Fluid Electrolyte Physiol 261: F1088-F1094, 1991[Abstract/Free Full Text].

45.   Robaire, B, and Viger RS. Regulation of epididymal epithelial cell functions. Biol Reprod 52: 226-236, 1995[Abstract].

46.   Schultheis, PJ, Clarke LL, Meneton P, Miller ML, Soleimani M, Gawenis LR, Riddle TM, Duffy JJ, Doetschman T, Wang T, Giebisch G, Aronson PS, Lorenz JN, and Shull GE. Renal and intestinal absorptive defects in mice lacking the NHE3 Na+/H+ exchanger. Nat Genet 19: 282-285, 1998[ISI][Medline].

47.   Sun, AM, Liu Y, Dworkin LD, Tse CM, Donowitz M, and Yip KP. Na+/H+ exchanger isoform 2 (NHE2) is expressed in the apical membrane of the medullary thick ascending limb. J Membr Biol 160: 85-90, 1997[ISI][Medline].

48.   Tajima, Y, Okamura N, and Sugita Y. The activating effects of bicarbonate on sperm motility and respiration at ejaculation. Biochim Biophys Acta 924: 519-529, 1987[ISI][Medline].

49.   Tse, CM, Brant SR, Walker MS, Pouyssegur J, and Donowitz M. Cloning and sequencing of a rabbit cDNA encoding an intestinal and kidney-specific Na+/H+ exchanger isoform (NHE-3). J Biol Chem 267: 9340-9346, 1992[Abstract/Free Full Text].

50.   Tse, CM, Levine SA, Yun CH, Montrose MH, Little PJ, Pouyssegur J, and Donowitz M. Cloning and expression of a rabbit cDNA encoding a serum-activated ethylisopropylamiloride-resistant epithelial Na+/H+ exchanger isoform (NHE-2). J Biol Chem 268: 11917-11924, 1993[Abstract/Free Full Text].

51.   Turner, TT. Resorption versus secretion in the rat epididymis. J Reprod Fertil 72: 509-514, 1984[Abstract].

52.   Wakabayashi, S, Shigekawa M, and Pouyssegur J. Molecular physiology of vertebrate Na+/H+ exchangers. Physiol Rev 77: 51-74, 1997[Abstract/Free Full Text].

53.   Wang, Z, Orlowski J, and Shull GE. Primary structure and functional expression of a novel gastrointestinal isoform of the rat Na/H exchanger. J Biol Chem 268: 11925-11928, 1993[Abstract/Free Full Text].

54.   Wong, PY, and Yeung CH. Absorptive and secretory functions of the perfused rat cauda epididymidis. J Physiol (Lond) 275: 13-26, 1978[Abstract].

55.   Wu, MS, Biemesderfer D, Giebisch G, and Aronson PS. Role of NHE3 in mediating renal brush border Na+-H+ exchange. Adaptation to metabolic acidosis. J Biol Chem 271: 32749-32752, 1996[Abstract/Free Full Text].

56.   Yun, CH, Tse CM, Nath SK, Levine SA, Brant SR, and Donowitz M. Mammalian Na+/H+ exchanger gene family: structure and function studies. Am J Physiol Gastrointest Liver Physiol 269: G1-G11, 1995[Abstract/Free Full Text].


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