Proton secretion in the male reproductive tract: involvement of Clminus -independent HCOminus 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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 (Delta 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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 Delta 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 Delta 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.

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 Delta 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.

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 Delta 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 Delta 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.

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 Delta 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.

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, Delta 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.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Al-Awqati, Q. Plasticity in epithelial polarity of renal intercalated cells: targeting of the H+-ATPase and band 3. Am. J. Physiol. 270 (Cell Physiol. 39): C1571-C1580, 1996[Abstract/Free Full Text].

2.   Au, C. L., and P. Y. Wong. Luminal acidification by the perfused rat cauda epididymidis. J. Physiol. (Lond.) 309: 419-427, 1980[Medline].

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