Proton secretion in the male reproductive tract: involvement
of Cl
-independent
HCO
3 transport
Sylvie
Breton1,2,
Katherine
Hammar3,
Peter J. S.
Smith3, and
Dennis
Brown1,2,4
1 Renal Unit,
2 Program in Membrane Biology, and
4 Department of Pathology,
Massachusetts General Hospital and Harvard Medical School, Boston
02129; and 3 BioCurrents Research
Center, Marine Biological Laboratory, Woods Hole, Massachusetts
02543
 |
ABSTRACT |
The lumen of the
epididymis is the site where spermatozoa undergo their final maturation
and acquire the capacity to become motile. An acidic luminal fluid is
required for the maintenance of sperm quiescence and for the prevention
of premature activation of acrosomal enzymes during their storage in
the cauda epididymis and vas deferens. We have previously demonstrated
that a vacuolar H+-ATPase
[proton pump (PP)] is present in the apical pole of apical and narrow cells in the caput epididymis and of clear cells in the
corpus and cauda epididymis and that this PP is responsible for the
majority of proton secretion in the proximal vas deferens. We now show
that PP-rich cells in the vas deferens express a high level of carbonic
anhydrase type II (CAII) and that acetazolamide markedly inhibits the
rate of proton secretion by 46.2 ± 6.1%. The rate of
acidification was independent of
Cl
and was strongly
inhibited by SITS under both normal and
Cl
-free conditions (50.6 ± 5.0 and 57.5 ± 6.0%, respectively). In the presence of
Cl
,
diphenylamine-2-carboxylate (DPC) had no effect, whereas SITS inhibited
proton secretion by 63.7 ± 11.3% when applied together with DPC. In Cl
-free
solution, DPC markedly inhibited proton efflux by 45.1 ± 7.6%,
SITS produced an additional inhibition of 18.2 ± 6.6%, and bafilomycin had no additive effect. In conclusion, we propose that CAII
plays a major role in proton secretion by the proximal vas deferens.
Acidification does not require the presence of
Cl
, but DPC-sensitive
Cl
channels might
contribute to basolateral extrusion of
HCO
3 under
Cl
-free conditions. The
inhibition by SITS observed under both normal and
Cl
-free conditions
indicates that a
Cl
/HCO
3
exchanger is not involved and that an alternative
HCO
3 transporter participates in proton secretion in the proximal vas deferens.
carbonic anhydrase II; self-referencing proton-selective electrode; immunocytochemistry; hydrogen-adenosine
5'-triphosphatase
 |
INTRODUCTION |
MAMMALIAN SPERMATOZOA undergo their final maturation
and acquire the ability to fertilize an ovum during their transit
through the epididymis. As they gain the capacity to become motile
(38), they are maintained in a quiescent state in the cauda epididymis and vas deferens (15, 17). Sperm motility is triggered, during ejaculation, by the neutralization of epididymal fluid by seminal and
prostatic fluid, followed by elevation of intracellular pH (3, 12)
and/or an increase in HCO
3
concentration (26). An acidic fluid in the lumen of the epididymis (11, 23) and a low HCO
3 concentration (27)
are required for the maintenance of sperm quiescence, as well as for the prevention of premature activation of acrosomal enzymes (17, 31).
Impairment of the acidification capacity of the epididymis and vas
deferens might therefore result in deficient sperm maturation and
motility, leading to lower fertility. However, acidification mechanisms
in the epididymis and vas deferens are poorly understood. Previous
studies on perfused epididymis and cultured epididymal principal cells
have implicated an apical
Na+/H+
exchanger in this process (2). In addition, our laboratory has
demonstrated that apical or narrow cells in the caput epididymis and
clear cells in the corpus and cauda epididymis express a high level of
the vacuolar H+-ATPase
[proton pump (PP)] on their apical plasma membrane and on
intracellular vesicles (8). A subpopulation of cells in the vas
deferens also contains apical PP, and we have shown that these cells
are responsible for the majority of proton secretion in this segment of
the male reproductive tract (6). The cytoplasmic enzyme carbonic
anhydrase type II (CAII) is present at high levels in PP-rich cells of
the vas deferens (6) and in a subpopulation of epithelial cells in the
epididymis (13, 18), indicating the potential involvement of
HCO
3 in the proton secretory process.
In many proton secretory epithelia, including the kidney, two
HCO
3 transporters have been described,
the electrogenic
Na+-HCO
3
cotransporter (4, 5, 32) and the electroneutral
Cl
/HCO
3
exchanger (1, 7). In kidney type A intercalated cells, a basolateral
Cl
/HCO
3
exchanger (AE1) coupled to basolateral Cl
channels, intracellular
CAII, and apical PP all contribute to proton secretion and
HCO
3 reabsorption. The aim of this
study was to further characterize proton secretory mechanisms in the
vas deferens by determining the role of CAII and of
Cl
and
HCO
3 transporters in this process.
 |
MATERIALS AND METHODS |
Immunofluorescence.
Sprague-Dawley rats were perfused via the left ventricle with a
physiological Hanks' buffer followed by a fixative solution containing
4% paraformaldehyde, 10 mM sodium periodate, 75 mM lysine, and 5%
sucrose for 10 min. The proximal vas deferens were dissected and
further fixed overnight at 4°C with the same fixative solution.
They were then washed in PBS (0.9% NaCl in 10 mM
PO3
4 buffer; pH 7.4) and stored in PBS
containing 0.02% sodium azide; 4-µm cryostat sections were cut after
the tissues had been cryoprotected in 30% sucrose, using a Reichert
Frigocut cryostat. Sections were picked up on Fisher Superfrost Plus
charged glass slides and stored at 4°C.
Vas deferens sections were double labeled to localize
H+-ATPase and CAII. Sections were
rehydrated in PBS for 5 min and treated with 1% SDS for 5 min to
enhance fluorescence staining, as previously described (9). Nonspecific
staining was blocked with PBS containing 1% BSA for 15 min. A
monoclonal antibody against the 31-kDa subunit of the
H+-ATPase (provided by Steven
Gluck, Washington University, St. Louis, MO), diluted 1:1, was applied
overnight at 4°C. Sections were washed twice in PBS containing
2.7% NaCl, to reduce background staining, and once in normal PBS. Goat
anti-mouse IgG conjugated to FITC (20 µg/ml; Kirkegaard & Perry,
Gaithersburg, MD) was applied for 1 h at room temperature and washed as
for the primary antibody. The sections were then incubated with rabbit
polyclonal anti-CAII (provided by William S. Sly, St. Louis University
Medical Center and School of Medicine, St. Louis, MO), diluted 1:200,
for 1.5 h at room temperature, followed by goat anti-rabbit IgG
conjugated with indocarbocyanine (CY3; 2 µg/ml; Jackson
ImmunoResearch, West Grove, PA). Sections were mounted in Vectashield
diluted 2:1 in 0.1 M Tris · HCl (pH 8.0). Control
experiments were conducted using preimmune serum or secondary
antibodies alone to show specificity of the primary antibodies.
An Optronics 3-bit charge-coupled device color camera attached to a
Nikon FXA photomicroscope was used to capture images directly; they
were stored on an Apple Macintosh Power PC 8500 using IP Lab Spectrum
software (Scanalytics, Vienna, VA). The digitized images were printed
on a Tektronix Phaser 440 dye sublimation color printer.
Detection of proton secretion.
Proton fluxes across the epithelium of the vas deferens were detected
using an extracellular proton-selective, self-referencing electrode, as
described previously (6). Briefly, the initial region of rat vas
deferens was microdissected, and most of the surrounding connective and
muscular tissue was removed. A longitudinal incision was made, and the
opened vas deferens was bent over a block of dental wax to expose the
apical surface of the epithelium. The vas deferens was bathed in the
same low-PO3
4 buffered saline (2 mM
PO3
4) as we used in our previous
report (6), and no bath perfusion was performed in order to allow the
establishment of a proton gradient near the apical surface of the
PP-rich cells. The tissue preparation was mounted on the stage of an
inverted microscope, and a proton-selective electrode was moved to a
distance <5 µm from the epithelium. Glass microelectrodes were
constructed from 1.5-mm borosilicate tubes (TW150-4, World Precision
Instruments, New Haven, CT) pulled on a Sutter Model PC90 pipette
puller to reach a final tip diameter of 2-4 µm (34, 35).
Electrodes were then silanized, front filled with a proton-selective
liquid ionophore (30-µm column; Fluka hydrogen ionophore cocktail B),
and back filled with 100 mM KCl. Electrode potentials were measured
with a unity gain, high-input-impedance preamplifier (model AD515;
Analog Devices), followed by a 1,000-fold gain amplifier, and low- and
high-pass filters to remove the large static voltages associated with
the Nernstian behavior of the electrode and signal of frequencies higher than 30 Hz. The circuit was completed with a 3 M KCl agar bridge. Signals were digitized using an analog-to-digital board (DT
2800 series, Data Translations) and were stored and analyzed in a
Pentium computer. Square wave oscillations of the electrode were
performed, perpendicular to the apical membrane, with an amplitude of
50 µm and a frequency of 0.3 Hz, as previously described (6, 35).
Proton equilibrium potentials were measured by the selective electrode
at the extreme points of oscillation. The difference between these two
values (
V) reflects the proton
concentration gradient that is established over a distance of 50 µm
and is therefore proportional to the proton flux generated by the
epithelium. The Nernstian slope of the electrode was determined before
and after each experiment using calibration solutions at pH 6.0, 7.0, and 8.0. The amplifiers, motion controllers, and IonView software used
in these experiments are products of the BioCurrents Research Center
(Marine Biological Laboratory, Woods Hole, MA;
http://www.mbl.edu/BioCurrents).
Chemicals.
Bafilomycin (Alexis, San Diego, CA) was dissolved in ethanol at a
concentration of 100 µM and was added to the bath to make a final
concentration of 1 µM. A 2 mM stock solution of acetazolamide (Sigma)
was made in the bath buffer and was used at a final concentration of
100 µM. A 10 mM stock solution of SITS (Sigma) in bath buffer was
used to make a final concentration of 1 mM. Diphenylamine-2-carboxylate (DPC) was first dissolved in ethanol at a stock concentration of 50 mM
and was added to the bath to make a final concentration of 0.5 mM.
Statistics.
Data are means ± SE. Two-tailed paired
t-tests or unpaired
t-tests were used as appropriate using
Statistical Package for Social Sciences software (Chicago, IL).
 |
RESULTS |
Role of carbonic anhydrase II in proton secretion.
Double labeling of proximal vas deferens using anti-PP and anti-CAII
antibodies showed, as we previously described (6), that all PP-rich
cells express a high level of CAII (Fig.
1). To determine the involvement of CAII
in proton secretion, we examined the effect of acetazolamide, a
carbonic anhydrase inhibitor, on the apical proton efflux detected by
the proton-selective electrode. For each experiment, the
vas deferens was scanned for the presence of PP-rich cells by measuring
V at different locations along the
surface of the tissue. As we have previously reported (6), variable
rates of proton secretion were detected. The highest rates corresponded
to PP-rich cells located beneath the tip of the electrode because they
were abolished by bafilomycin, a specific inhibitor of the vacuolar PP.
All experiments were conducted after location of one region of high
acidification (hot spot); the electrode then remaining at this
location. In this series of experiments, the highest acidification
rates detected, under control conditions, in six different preparations
had a mean
V value of 913 ± 129 µV. Figure 2 shows a
representative trace of the effect of acetazolamide on proton
secretion. Addition of 0.1 mM acetazolamide markedly reduced the rate
of proton secretion by 46.2 ± 6.1%
(P = 0.008; Fig.
3), and, when 1 µM bafilomycin was
applied at the end of the experimental period, a nonsignificant,
additional inhibition of 20.7 ± 10.6% was observed
(P = 0.121), indicating that hydration of CO2 by CAII represents a major
source of protons for the PP. Control experiments were conducted to
determine the stability of the vas deferens over time. These vas
deferens were bathed in control solutions for periods up to 1 h, after
which bafilomycin was added. As shown in Fig. 2, proton secretion
remained constant over a period of 40 min and was significantly
inhibited by bafilomycin. A residual proton secretion was always
observed after bafilomycin inhibition. The nature of this flux is under
investigation in our laboratory. We have tested the possibility that an
H+-K+-ATPase
might be involved. However, omeprazole (10-50 µM) failed to
inhibit proton secretion, and immunocytochemical studies using antibodies raised against both gastric and colonic
H+-K+-ATPase
failed to show any detectable antigenicity in epithelial cells (not
shown).

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Fig. 1.
Double immunostaining to detect 31-kDa subunit of vacuolar
H+-ATPase
(A; green) and carbonic anhydrase type
II (CAII; B; red) in proximal vas
deferens epithelial cells.
H+-ATPase is present in a
subpopulation of cells and is concentrated in apical region. These
cells express a high level of cytoplasmic CAII. Colocalization of
H+-ATPase and CAII in same cells
is illustrated in merged image (C).
Scale bar, 10 µm.
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Fig. 2.
Representative traces showing rate of proton secretion detected with an
extracellular proton-selective self-referencing electrode, under
control conditions (dashed line) and after addition of 0.1 mM
acetazolamide (solid line). Acetazolamide markedly reduced rate of
proton secretion to a level that was not further inhibited by 1 µM
bafilomycin. In contrast, control proton secretion remained stable
during periods of up to 1 h and was markedly inhibited by bafilomycin
when added at end of experimental period. Signal is expressed relative
to control value.
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Fig. 3.
Mean effects of acetazolamide (Actz) and bafilomycin (Bafilo) on proton
secretion from a series of 6 experiments. Addition of 0.1 mM
acetazolamide significantly reduced rate of proton secretion, and
bafilomycin induced an additional but nonsignificant inhibition. Data
are expressed relative to control value. NS, not significant vs.
acetazolamide; * P = 0.008 vs.
control.
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Role of HCO
3 transporter(s)
in proton secretion.
The marked reduction of proton secretion by acetazolamide strongly
suggests that HCO
3 participates in
proton secretion. We therefore examined the effect of SITS, an
inhibitor of HCO
3 transporters, in
this process. As shown in Fig. 4, addition
of 1 mM SITS rapidly inhibited proton secretion. In this series of six
experiments, the control
V values
were 817 ± 105 µV, SITS inhibited proton secretion by 50.6 ± 5.0% (P = 0.003), and bafilomycin
produced a small additional inhibition of 8.5 ± 2.3%
(P = 0.014; Fig.
5).

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Fig. 4.
Representative trace showing effect of 1 mM SITS on rate of proton
secretion. SITS markedly inhibited proton secretion to a level that was
no longer sensitive to bafilomycin. Transient increase observed
initially is attributed to a disturbance of proton gradient upon SITS
addition. Signal is expressed relative to control value.
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Fig. 5.
Mean effects of SITS and bafilomycin on proton secretion from a series
of 6 experiments. Addition of 1 mM SITS significantly inhibited rate of
proton secretion, and 1 µM bafilomycin produced a small additional
inhibition. Data are expressed relative to control value.
* P = 0.003 vs. control;
** P = 0.014 vs. SITS.
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|
To determine whether a
Cl
/HCO
3
exchanger was involved in proton secretion by the vas deferens, SITS
inhibition experiments were repeated in the absence of
Cl
(replaced by cyclamate).
To ensure maximal depletion of intracellular Cl
, some vas deferens were
dissected and mounted in
Cl
-free buffer, allowing a
preincubation of at least 45 min in the absence of
Cl
. Under these conditions,
the
V value was 705 ± 131 µV
(n = 5), remained unchanged after
washes of the bath with fresh
Cl
-free solution, and was
not statistically different from the value of 822 ± 54 µV
(n = 22) measured in the presence of
Cl
(P = 0.37). In a separate series of
experiments, the effect of Cl
removal was determined
for each vas deferens. Vas deferens were dissected and mounted in
normal Cl
-containing
buffer, and proton fluxes were measured before and after
Cl
removal. The
V value was 813 ± 92 µV under
control conditions and 822 ± 103 µV after 10 min in
Cl
-free solutions
(n = 7). These values were not
significantly different (P = 0.83),
indicating that proton secretion by the proximal vas deferens is not
dependent on Cl
.
As shown in Figs. 6 and
7, under
Cl
-free conditions,
addition of SITS strongly inhibited proton secretion by 57.5 ± 6.0% (n = 6;
P = 0.006), and bafilomycin had no
further effect. On average, SITS inhibition and bafilomycin-sensitive
signals were identical to the values measured in the presence of
Cl
(Figs. 5 and 7). These
results indicate that a
Cl
/HCO
3
exchanger does not seem to participate in bafilomycin-sensitive proton
secretion by the vas deferens.

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Fig. 6.
Representative trace showing effect of 1 mM SITS on rate of proton
secretion under Cl -free
conditions. SITS strongly inhibited proton secretion to a level that
was not further reduced by 1 µM bafilomycin. Signal is expressed
relative to control value.
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Fig. 7.
Mean effects of SITS and bafilomycin on proton secretion under
Cl -free conditions from a
series of 6 experiments. Addition of 1 mM SITS significantly inhibited
rate of proton secretion, and bafilomycin did not further reduce
signal. Data are expressed relative to control value. NS, not
significant vs. SITS; * P = 0.006 vs. control.
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Role of Cl
channels in proton
secretion.
Cl
channels, including the
cystic fibrosis transmembrane conductance regulator, have been
described in the epididymis (37), and this epithelium secretes
Cl
via a DPC-sensitive
conductance (21). In many proton secretory epithelial cells such as
kidney intercalated cells,
Cl
transport is essential
for proton secretion to maintain electroneutrality. We therefore
examined the effect of the
Cl
channel inhibitor DPC on
proton secretion by the vas deferens. In this series of three
experiments, control
V was 668 ± 88 µV, and addition of DPC had no effect on proton efflux (753 ± 69 µV; P = 0.24; Figs.
8 and 9). In
contrast, SITS induced an inhibition of 63.7 ± 11.3%
(P = 0.002) to reach a level that was
not further reduced by bafilomycin.

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Fig. 8.
Representative trace showing effects of diphenylamine-2-carboxylate
(DPC), SITS, and bafilomycin on rate of proton secretion. Addition of
0.5 mM DPC had no effect, and 1 mM SITS strongly inhibited proton
secretion to a level that was not further reduced by 1 µM
bafilomycin. As also seen in Figs. 4 and 10, an initial disturbance of
proton gradient resulted from addition of SITS. Signal is expressed
relative to control value.
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Fig. 9.
Mean effects of DPC, SITS, and bafilomycin on proton secretion from a
series of 3 experiments. Addition of 0.5 mM DPC produced a small,
nonsignificant increase, 1 mM SITS significantly inhibited rate of
proton secretion, and bafilomycin did not further reduce signal. Data
are expressed relative to control value. NS1, not significant vs.
control; NS2, not significant vs. DPC-SITS;
* P = 0.002 vs. DPC.
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Many Cl
channels are
permeable to HCO
3 (36), and we
determined whether such channels might be involved in proton secretion
by contributing to basolateral HCO
3 efflux. Under Cl
-free
conditions,
V was 676 ± 93 µV
(n = 7). As shown in Figs. 10 and
11, DPC markedly inhibited proton
secretion by 45.1 ± 7.6% (P < 0.001), SITS produced an additional inhibition of 18.2 ± 6.6% (P = 0.05), and bafilomycin had
no additive effect.

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Fig. 10.
Representative trace showing effects of DPC, SITS, and bafilomycin on
rate of proton secretion under
Cl -free conditions.
Addition of 0.5 mM DPC markedly inhibited proton secretion, 1 mM SITS
induced a small additional inhibition, and 1 µM bafilomycin had no
further effect. Signal is expressed relative to control value.
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Fig. 11.
Mean effects of DPC, SITS, and bafilomycin on proton secretion under
Cl -free conditions from a
series of 7 experiments. Addition of 0.5 mM DPC markedly inhibited
proton secretion, 1 mM SITS produced an additional inhibition, and 1 µM bafilomycin did not further reduce signal. Data are expressed
relative to control value. NS, not significant vs. DPC-SITS;
* P < 0.001 vs. control;
** P = 0.05 vs. DPC.
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 |
DISCUSSION |
Although the significance of luminal acidification in the
epididymis/vas deferens is well established, the mechanisms responsible for net proton secretion are still poorly understood. We have previously shown that a subpopulation of cells in the epididymis and
proximal vas deferens express a high level of the vacuolar H+-ATPase on their apical plasma
membrane and intracellular vesicles (6, 8). In the vas deferens, this
bafilomycin-sensitive H+-ATPase is
responsible for the majority of proton secretion, indicating a crucial
role in luminal acidification in this segment (6). In the present
study, detection of apical proton fluxes using a noninvasive
proton-selective electrode was coupled to immunofluorescent techniques
to further characterize the intracellular and basolateral pathways
involved in net proton secretion in the proximal vas deferens.
Role of carbonic anhydrase II.
Previous studies have shown a high level of CAII expression in a
distinct population of epithelial cells in the epididymis, an
indication that these cells are involved in luminal proton secretion
(13, 18, 25). We have recently demonstrated that PP-rich cells in the
vas deferens contain a high amount of CAII (6). These cells therefore
represent an interesting model for characterization of proton secretory
mechanisms in the male reproductive tract.
The marked inhibition of proton secretion by acetazolamide and the
colocalization of CAII in PP-rich cells strongly indicate that this
cytoplasmic enzyme plays a major role in supplying protons to the PP.
It is interesting to note that this strong inhibition of proton
secretion by acetazolamide was observed in the nominal absence of
CO2/HCO
3
from the bathing solution. It thus appears that the activity of CAII is
substantial under these conditions and that significant formation of
HCO
3 from endogenous
CO2 still occurs. This
contribution of CAII to proton secretion is consistent with the very
high expression of CAII in PP-rich cells. In this respect, PP-rich
cells in the vas deferens share common characteristics with
proton-secreting cells in other epithelia, including kidney
intercalated cells (1, 7) and proximal tubule cells (33). A small
bafilomycin-sensitive proton secretion was still observed after CAII
inhibition, suggesting a parallel but less important source of protons
in PP-rich cells of the vas deferens. Identification of the source of
this alternative supply of protons requires further investigation, but
one explanation is that CAII was not completely inhibited under our
experimental conditions. Previous studies on the effect of
acetazolamide on luminal acidification have resulted in conflicting
results. In vivo microperfusion of the cauda epididymal duct showed a
marked inhibition of the rate of luminal acidification by acetazolamide (2). Subsequent work examining more proximal portions of the epididymis
(caput, corpus, and proximal cauda epididymis) showed no effect of
acetazolamide on luminal pH (11). These results, together with our
observation that acetazolamide strongly reduces proton secretion in the
vas deferens, suggest a role of CAII in the luminal acidification of
the distal segments of the male reproductive tract (cauda epididymis
and vas deferens), whereas the proximal segments (caput and corpus
epididymis) may rely on different acidification mechanisms.
Role of Cl
and
HCO
3 transporter(s).
The role of CAII in proton secretion described above indicates the
involvement of HCO
3 reabsorptive
mechanisms in this process. The concentration of
HCO
3 reaches low values in the lumen
of the epididymis (it is <14% that of the blood in the cauda
epididymis) (10, 17), which further suggests that
HCO
3 reabsorption occurs in this tissue. In the present study, an almost complete inhibition of the
bafilomycin-sensitive proton flux by SITS was observed, which supports
the notion that HCO
3 transport is
involved. In the kidney, two SITS-sensitive, basolateral transporters
responsible for HCO
3 reabsorption have
been described: an electrogenic
Na+-HCO
3
cotransporter in proximal tubule cells (4, 5, 29) and an electroneutral
Cl
/HCO
3
exchanger that works in parallel with a basolateral
Cl
channel in intercalated
cells (7). In addition, SITS not only inhibits
HCO
3 transporters but can also
decrease the activity of some
Cl
channels (16, 20). In
the present study, removal of
Cl
from the bathing
solution had no effect on proton secretion, and SITS inhibition
measured in Cl
-free
solution was of the same magnitude as that observed in the presence of
Cl
. It remains possible
that, under Cl
-free
conditions, a small source of extracellular
Cl
might still be provided
to a
Cl
/HCO
3
exchanger by the surrounding connective and muscular tissue, despite
the long preincubation in
Cl
-free buffer. However,
this local Cl
concentration
must be high enough to allow the
Cl
/HCO
3
exchanger to function as effectively as it does under normal
Cl
concentration
conditions, because SITS inhibition of acidification was not affected
under Cl
free conditions.
Given the relatively high Michaelis-Menten constant values for
Cl
of 10 mM for the
basolateral and 19 mM for the apical
Cl
/HCO
3
exchanger, in kidney type A and B intercalated cells, respectively
(14), we believe that this possibility is unlikely. In addition, we
observed no modification of the net proton secretion after repeated
washes in Cl
-free buffer,
indicating that Cl
concentration had already reached a level that could not support significant exchanger activity. On the other hand, in the present study, HCO
3 production by
proton-secreting cells might have been reduced in the nominal absence
of
CO2/HCO
3, due to a possibly lower intracellular
CO2 concentration. The resulting decrease in intracellular HCO
3
concentration might have been sufficient to reduce the activity of a
Cl
/HCO
3
exchanger to such an extent that it would no longer be detectable.
Nevertheless, our results indicate that, under the experimental
conditions described in this study, the Cl
/HCO
3
exchanger does not play a significant role in proton secretion by
PP-rich cells.
Cl
channels, including the
cystic fibrosis transmembrane conductance regulator, have been
described in the epididymis (37). Electrogenic
Cl
secretion mediated by a
predominant, cAMP-activated, DPC-sensitive Cl
conductance and a
smaller, Ca2+-activated
Cl
conductance has been
reported in primary cultures of mouse and rat epididymal cells (21,
22). In many proton secretory epithelial cells, such as kidney
intercalated cells, proton secretion is coupled to
Cl
transport to maintain
electroneutrality. In the present study, the absence of effect of
Cl
removal on proton
secretion and the lack of inhibition by DPC in the presence of
Cl
argue against a role of
Cl
in proton secretion by
the vas deferens.
However, some Cl
channels
are significantly permeable to HCO
3
(19, 24, 28, 36), and we examined the possibility that an efflux of
HCO
3 through these channels might be
involved in proton secretion. Our results showing a marked inhibition
of proton secretion by DPC in
Cl
-free solutions indicate
that a significant basolateral efflux of
HCO
3 occurs via
Cl
channels under these
conditions, such an efflux being facilitated in the absence of
competition by Cl
. Under
conditions of normal Cl
concentration, the lack of an inhibitory effect of DPC indicates that
HCO
3 transport via
Cl
channels is minor,
possibly due to a higher and more selective permeability to
Cl
. Alternatively,
DPC-insensitive Cl
channels
might be involved in this process. It remains possible that, upon
Cl
removal, a significant
basolateral efflux of HCO
3 through
Cl
channels compensated for
the inactivation of a
Cl
/HCO
3
exchanger and therefore masked the role of this exchanger. In that
case, the SITS inhibition observed under
Cl
-free conditions might be
interpreted in terms of reduction of basolateral
HCO
3 efflux through SITS-sensitive Cl
channels. However, the
lack of inhibition by DPC observed under normal
Cl
conditions argues
against the participation of a
Cl
/HCO
3
exchanger in this process because this exchanger requires the recycling
of Cl
through
Cl
channels. In addition,
DPC has also been shown to inhibit a
Cl
/HCO
3
exchanger (30). The absence of effect of DPC observed in the presence
of Cl
therefore confirms
the absence of effect of Cl
removal per se and supports the notion that a
Cl
/HCO
3
exchanger is not involved in proton secretion by the vas deferens.
The additional inhibition of proton secretion by SITS observed in the
presence of DPC under both normal and
Cl
-free conditions
indicates the involvement of another
HCO
3 transporter. This additional
inhibition by SITS was lower in the absence of
Cl
than in normal
Cl
solutions, indicating
that this DPC-insensitive, SITS-sensitive alternative pathway for
HCO
3, although it is predominant under
normal Cl
conditions,
represents a less important pathway under
Cl
-free conditions. By
analogy with kidney proximal tubule cells, this transporter might be a
Na+-HCO
3
cotransporter (4, 5, 29). This transporter is electrogenic (32) and
would contribute to the basolateral efflux of
HCO
3 while maintaining electroneutrality, so that no transport of anions such as
Cl
is required. Further
studies are now required to clearly identify the nature of the
HCO
3 transporter that participates in
net proton secretion in the proximal vas deferens.
In summary, the present study indicates that proton secretion by the
proximal rat vas deferens requires the enzymatic activity of CAII and
is not dependent on Cl
and
that the concomitant basolateral efflux of
HCO
3 does not seem to involve a
Cl
/HCO
3
exchanger. These results, together with our previous observation that
the
Cl
/HCO
3
exchanger AE1 is not detectable in the vas deferens (6), indicate that
PP-rich cells in the rat epididymis and vas deferens are not completely
analogous to renal type A intercalated cells.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-38452 (to S. Breton and D. Brown). S. Breton was partially supported by a grant from the National
Kidney Foundation and by a Claflin Distinguished Scholar Award from the
Massachusetts General Hospital. P. J. S. Smith was supported by
National Center for Research Resources Grant P41-RR-O1395.
 |
FOOTNOTES |
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. §1734 solely to indicate this fact.
Address for reprint requests: S. Breton, Renal Unit, Massachusetts
General Hospital, 149 13th St., 8th Floor, Charlestown, MA 02129.
Received 9 April 1998; accepted in final form 15 June 1998.
 |
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