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
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ABSTRACT |
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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.
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INTRODUCTION |
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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
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 exchanger AE2
(32) and of the electrogenic Na- HCO
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/HCOImmunocytochemistry
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 atSections 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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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 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
-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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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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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
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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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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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 in the lumen produced a rapid
intracellular alkalinization due to the influx of NH3 into
the cells. On washout of NH3/NH
, a marked
acidification was induced as intracellular NH
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
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
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
, respectively [P = not significant (NS)].
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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-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
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
, respectively
(n = 5; P = NS).
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DISCUSSION |
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Both pH and bicarbonate regulate the motility of spermatozoa
(1, 41, 42, 48). The establishment of an acidic pH and of
a low HCO 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 absorption (7, 8, 28,
40, 52, 56). In view of the transepithelial Na+ and
HCO
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. 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 that is reached in the caput epididymidis (38). In the epididymis, NHE3 expression also correlates
with high levels of the electrogenic Na-HCO
cotransporter NBC and of the Cl/HCO
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
transporters in the
basolateral membrane indicates that the initial segments, intermediate
zone, and proximal caput epididymis are highly specialized for net
HCO
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, 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 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.
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ACKNOWLEDGEMENTS |
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We thank Ndona N. Nsumu for excellent technical help.
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FOOTNOTES |
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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.
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