Functional and molecular evidence for Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter in porcine vas deferens epithelia

Ryan W. Carlin, Rebecca R. Quesnell, Ling Zheng, Kathy E. Mitchell, and Bruce D. Schultz

Department of Anatomy and Physiology, Kansas State University, Manhattan, Kansas 66506


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study focused on the role of sodium-bicarbonate cotransporter (NBC1) in cAMP-stimulated ion transport in porcine vas deferens epithelium. Ion substitution experiments in modified Ussing chambers revealed that cAMP-mediated stimulation was dependent on the presence of Na+, HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, and Cl- for a full response. HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-dependent current was unaffected by acetazolamide, bumetanide, or amiloride but was inhibited by basolateral 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Na+-driven, HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-dependent, stilbene-inhibitable anion flux was observed across the basolateral membrane of selectively permeabilized monolayers. Results of radiotracer flux studies suggest a 4,4'-dinitrostilbene-2,2'-disulfonate-sensitive stoichiometry of 2 base equivalents per Na+. Antibodies raised against rat kidney NBC epitopes (rkNBC; amino acids 338-391 and 928-1035) identified a single band of ~145 kDa. RT-PCR detected NBC1 message in porcine vas deferens epithelia. These results demonstrate that vas deferens epithelial cells possess the proteins necessary for the vectoral transport of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and that these mechanisms are maintained in primary culture. Taken together, the results indicate that vas deferens epithelia play an active role in male fertility and have implications for our understanding of the relationship between cystic fibrosis and congenital bilateral absence of the vas deferens.

NBC1; anion transport; pH regulation; cystic fibrosis; congenital bilateral absence of the vas deferens


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE VAS DEFERENS plays a key role in male fertility by providing an appropriate luminal environment for sperm before ejaculation. However, few studies have directly assessed the epithelial ion transport mechanisms in this portion of the reproductive tract. Furthermore, most studies reported to date have been completed in rodent models (5-7, 33). The expression pattern for the cystic fibrosis transmembrane conductance regulator (CFTR), an apical anion conductance, is distinctly different in rats compared with humans (47), indicating that there may be substantial differences in transport mechanisms between these species. Only two large animal vas deferens epithelial cell models have been reported (4, 44), and direct comparisons to human tissues have appeared only in abstract form (10, 42). Thus very little is known regarding vas deferens epithelial ion transport in humans and other large animal species.

Cystic fibrosis (CF), a genetic disease caused by the loss of an anion channel (CFTR), is almost universally associated with congenital bilateral absence of the vas deferens (CBAVD). The Wollfian duct system appears to form normally during gestational development (21) but undergoes atresia beginning late in gestation or early in postnatal life. The most perplexing observation regarding the relationship between CF and CBAVD is that some "patients" present with CBAVD and have no other signs of the disease; infertility is their only complaint, and genotyping is required to determine their CF status (2, 16, 19, 20). Furthermore, men seeking intervention for infertility, whether due to azoospermia, oligospermia, or asthenozoospermia, have a higher incidence of "mild" mutations in the CF gene than the general population (25, 48, 49). These observations suggest that the epithelium lining the male reproductive tract, and especially the vas deferens, is extremely sensitive to the loss of an anion conductance and that the loss of this anion conductance affects both sperm quality and duct maintenance.

Historically, the male reproductive tract (specifically the epididymis) has been characterized as an acid-secreting organ that promotes the maturation and storage of quiescent sperm (27, 33). Acidification is accomplished in part by secretion of protons into the lumen via a vacuolar H+-ATPase located on the apical membrane of some epithelial cells in the epididymis and, to a lesser extent, the vas deferens (5). Na+/H+ exchanger (NHE) has also been detected in the apical membranes of selected rat epididymal cells but not in the vas deferens (3, 29, 32). Likewise, CFTR is present in rat epididymis but not in rat vas deferens (47). These differences between the epididymis and vas deferens suggest that sperm may require an environment immediately before ejaculation that is unlike that employed for maturation and storage. Increases in pH or an increase in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration (without a change in pH) are reported to activate the soluble form of adenylyl cyclase found in sperm (12) and thus promote sperm motility (34). The timing and/or location of changes in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration likely plays a key role in male fertility. It is generally thought that this activation occurs when sperm are exposed to secretions of the accessory sex organs at the time of ejaculation. However, previous work suggests that HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion can occur in rat epididymis (11, 14), and our work with porcine vas deferens epithelial cells suggests that adrenergic agonists might stimulate HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion (44). Thus it may be time to rethink the timing of sperm activation in the male reproductive tract.

Cultured porcine vas deferens epithelial cells were previously shown to secrete anions in response to cAMP-mediated stimulation (44). Ion substitution studies suggest that both Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> are secreted by the epithelium, and transporter pharmacology provides a preliminary indication that an electrogenic sodium-bicarbonate cotransporter (NBC) participates in the response. Thus we sought to determine the identity and subcellular localization of the ion transporter responsible for HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport across vas deferens epithelial cell monolayers. Results indicate that an NBC1 variant is present in the basolateral membranes of epithelial cells lining the vas deferens and contributes to forskolin-stimulated ion transport.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Tissue acquisition. Reproductive tracts from adult boars were acquired from a local slaughterhouse immediately post mortem and transported to the laboratory as previously described (44). Upon arrival at the laboratory, distal vas deferens (excluding convolutions that might be associated with the transitional vas deferens) were isolated and stripped of connective tissue. Each duct was flushed with Hanks' buffered salt solution (HBSS; Table 1) supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml), gentamicin (40 µg/ml), and amphotericin B (4 µg/ml). Tissues then underwent further processing for epithelial cell isolation to be used in electrophysiological and RT-PCR experiments.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Composition of solutions

Primary cell isolation and culture. Epithelial cells were isolated as previously described in detail (44). Briefly, ducts were filled with a phosphate-buffered saline for cell culture (Table 1) containing 300 U/ml collagenase, 0.25% (vol/vol) trypsin, and 2.65 mM EDTA and allowed to incubate in a small volume (15-25 ml) of HBSS at 37°C for 30-35 min. After incubation, ducts were flushed with 3-5 ml of growth medium (Dulbecco's modified Eagle's medium, DMEM; Invitrogen, Baltimore, MD) supplemented with 10% fetal bovine serum (FBS; Invitrogen), penicillin (100 U/ml), and streptomycin (100 µg/ml), seeded onto 25-cm2 culture flasks (Corning, Corning, NY), and incubated in a humidified chamber containing 5% CO2 at 37°C. After reaching confluency (72-96 h), cells were routinely passed and seeded onto tissue culture inserts (1.13 cm2; Snapwell, Costar, Cambridge, MA) as previously described (44). Growth medium was changed every other day for 2 wk, at which point the inserts were assayed for electrophysiological characteristics. Cells from the first and second passage were used for studies presented herein.

Electrophysiology. Basal short-circuit current (Isc; a measure of net ion flux), changes in Isc, and transepithelial resistance (Rte) were recorded by using a modified Ussing chamber and Acqknowledge software (Biopac Systems, Santa Barbara, CA) as described previously (44). Unless otherwise noted, data were recorded in symmetrical Ringer solution (Table 1) at 39°C. Ion substitution studies were conducted in solutions in which the ion or ions indicated were replaced on an equimolar basis with impermeant ions. The composition of each solution is presented in Table 1. Fluids in each chamber were continuously circulated with a gas-lift system composed of room air for HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free solutions or 5% CO2-95% O2 for HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-containing solutions. The pH of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free solutions was adjusted to 7.40 with either NaOH or KOH.

Nystatin permeabilization was employed to isolate either the apical or the basolateral membrane in the presence of an ionic gradient. Initial experiments were conducted in low symmetrical Cl- with apical K-gluconate and basolateral Na-gluconate (Table 1). Nystatin (180 µg/ml) was applied apically to isolate cation-dependent currents across the basolateral membrane. In subsequent experiments, HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free solutions were employed to assess HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> dependence of the observed fluxes. Similar experiments were conducted with the opposite cation gradient to assess reversibility of the ion transport processes. Additional experiments were conducted in which the basolateral membrane was permeabilized with nystatin (360 µg/ml) to assess gradient-driven flux across the apical membrane. Two ionic configurations were employed for these experiments. In the first, a Na-gluconate/K-gluconate gradient was employed. The second configuration included apical Ringer solution with basolateral Na-gluconate to form a Cl- gradient in symmetrical Na+. This configuration was employed as a positive control to clearly demonstrate gradient-driven flux across the apical membrane.

Radiotracer flux. 22Na+ flux was measured in apically nystatin-permeabilized monolayers in the presence of a basolateral-to-apical Na+ gradient as described in Electrophysiology (Table 1). 22NaCl (3 µCi; Perkin Elmer Life Sciences, Boston, MA) was added to the solution bathing either the apical or basolateral aspect of paired monolayers. Three sequential periods of ~15 min each were assessed [basal, after apical permeabilization with nystatin, and after exposure to 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS)] with sampling occurring at the beginning and end of the period to determine the unidirectional and, by subtraction, net flux, expressed as µeq · h-1 · cm-2. Samples were diluted in 8 ml of Scintiverse (Fisher) and counted in a Tri-Carb 2100TR liquid scintillation analyzer (Packard BioScience). Experiments were conducted in both the absence and presence of clotrimazole (20 µM). No significant differences in DNDS-sensitive flux or Isc were observed. Therefore, data are pooled for presentation.

Immunoblots. Cultured vas deferens epithelial cells and BMC-UV (bovine mammary epithelial cells as a negative control; Ref. 40) were lysed by using RIPA buffer with Protease Inhibitor Cocktail (Sigma-Aldrich, St. Louis, MO), proteins were precipitated, and total protein content was determined via Micro-Bicinchoninic Acid Concentration Assay (Pierce, Rockford, IL). Western analysis was performed according to the Laemmli method. Briefly, after incubation in Laemmli sample buffer, proteins were loaded onto precast 4-15% Tris · HCl polyacrylamide stacking gels (ReadyGel, Bio-Rad, Hercules, CA) and electrophoretically resolved with a current of 190 A for 45 min (Mini-Protean 3, Bio-Rad). Proteins were transferred to Immobilon-P polyvinylidene difluoride membranes (Millipore, Bedford, MA) and preblocked with 5% blocking buffer to reduce background. Membranes were then incubated with NBC1 primary polyclonal antibody raised against two distinct epitopes of the rat kidney Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter (rkNBC1; amino acids 338-391 or 928-1035; Chemicon International, Temecula, CA; Ref. 41). After copious washing with Tween-TBS (0.1% Tween 20, 0.9% NaCl, and 100 mM Tris, pH 7.5; 3-5 times for 5 min per rinse), membranes were exposed to a horseradish peroxidase-conjugated secondary antibody (Pierce). Membranes were then incubated with SuperSignal West Pico Chemiluminescent Substrate (Pierce), exposed to CL-Xposure film (Pierce) for a derived period, and developed for presentation.

RT-PCR. PCR analysis of NBC1 transcripts was assessed in freshly isolated cultured epithelial cells of adult pig. Total RNA was extracted by using the TRIzol method (GIBCO BRL, Rockville, MD) according to the manufacturer's instructions. RNA was quantitated by ultraviolet spectrophotometry at 260/280 nm and resuspended at a concentration of 1 µg/µl. Primers were designed from published sequences (23) and synthesized by Iowa State University (Ames, IA): sense primer, TGG CTC CCA TCT TGA AGT TTA; antisense primer, CAG CTA CAA GTG CCA AGA TCA. For the synthesis of cDNA, 3 µl of extracted total RNA, 1 µl (0.5 µM) of the antisense primer, 2 µl of 10× RT buffer (Promega, Madison, WI), 8.0 µl of 25 mM dNTPs, 4 µl of 25 mM MgCl2, 1 µl of RNase inhibitor, and 1 µl of avian myeloblastosis virus reverse transcriptase (Promega) were mixed. The samples were incubated at 42°C for 1 h followed by 5 min at 95°C to inactivate the enzyme. cDNAs (2 µl) were subjected to PCR to detect transcripts encoding NBC1 by using specific primers. The templates were amplified in a 25-µl reaction containing 1× PCR buffer, 2.5 mM MgCl2, 0.25 mM dNTPs, 0.5 µM oligonucleotide primers, and 2.5 units of Taq DNA polymerase. After an initial 5-min incubation period at 94°C, amplifications were performed by using a PTC-100 programmable thermal cycler (MJ Research) under the following conditions: 94°C for 45 s, 50°C for 45 s, and 72°C for 1 min with 35 cycles. This was followed by a 72°C incubation for 10 min. Products were analyzed by 1.5% agarose gel electrophoresis and visualized by ethidium bromide staining under ultraviolet light. The DNA bands with correct molecular weight were excised from the gel, and the DNA were purified by using the Geneclean system (Bio 101, Carlsbad, CA). The PCR product was then cloned into T-vector (Promega), and recombinant plasmid was used to transform bacteria according to standard procedures (39). The insertion was confirmed by sequencing (Dept. of Plant Pathology, Kansas State University, Manhattan, KS).

Chemical sources. DNDS was purchased from Acros Organics (Fairlawn, NJ). Forskolin (Coleus forskohlii) was purchased from Calbiochem (La Jolla, CA). Penicillin, streptomycin, Tris, and Triton-X 100 were purchased from Fisher Scientific. Gentamicin, collagenase, and trypsin plus EDTA were purchased from GIBCO BRL. Amiloride, amphotericin B, bumetanide, clotrimazole, nystatin, and ouabain were purchased from Sigma. N-[4-methylphenylsulfonyl]-N'-[4-trifluoromethylphenyl]urea (DASU-02) was synthesized de novo in the laboratory. All other chemicals were of the highest grade available and purchased from reputable sources.

Stock solutions of modulators for Ussing chamber experiments. Solutions were prepared as follows: forskolin, 10 mM in ethanol; amiloride and ouabain, 10 mM in H2O; bumetanide, 20 mM in ethanol; DASU-02, 100 mM in dimethyl sulfoxide (DMSO); clotrimazole, 30 mM in DMSO; and DNDS, 30 mM in Ringer solution. Forskolin and bumetanide were stored at -20°C. Amiloride was stored at 4°C. All other modulators were freshly dissolved on the day of the experiment.

Statistical analysis. Numerical data for Ussing chamber experiments are presented as the arithmetical means and standard error of the mean, using a culture well as the experimental unit. Where appropriate, the Student's t-test was employed to assess likelihood of population differences. A probability of a type I error of <5% was considered statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

cAMP-stimulated ion transport is Na+, HCO<UP><SUB><UP>3</UP></SUB><SUP><UP>−</UP></SUP></UP>, and Cl- dependent. Initial experiments were conducted to test for the dependence of forskolin-stimulated changes in Isc on the individual or combined presence of Na+, HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, and Cl- in the bathing media. These experiments directly build on previous observations indicating that a maximal response to forskolin is observed only in the combined presence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and Cl- (44). In complete Ringer solution (Fig. 1A), forskolin caused a transient increase (5.1 ± 0.4 µA/cm2) in Isc that was followed by a sustained plateau (3.2 ± 0.2 µA/cm2; n = 14). A component of the sustained current was sensitive to the basolateral addition of bumetanide (-0.3 ± 0.1 µA/cm2). The present results closely parallel previous observations for the effects of forskolin and bumetanide in the absence of Cl- (Fig. 1C) and in the absence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (Fig. 1E). In the absence of Cl-, the sustained plateau was present (1.7 ± 0.3 µA/cm2) and was not bumetanide sensitive, whereas in the absence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, a transient peak (4.5 ± 0.5 µA/cm2) was observed with a substantially reduced sustained component (0.7 ± 0.1 µA/cm2) that was totally inhibited by bumetanide (-0.6 ± 0.1 µA/cm2). In the absence of Na+, effects of forskolin on Isc were routinely not observed (Fig. 1B), although the increase in pulse size clearly indicates that Rte was reduced, suggesting that a conductive pathway had been activated. The results clearly show that HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-dependent ion transport is Na+ dependent (compare Fig. 1, E and F) and that Cl--dependent ion transport is Na+ dependent (compare Fig. 1, C and D). Summarized in Fig. 1I are results from paired experiments (n = 4-14) to evaluate responses in the absence of selected ions. The peak (transient) effect of forskolin was not significantly reduced by the absence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (P > 0.7; n = 4) but was significantly less in all other conditions (P < 0.05). Compared with control, sustained responses were significantly reduced in all conditions except the absence of Cl-. A significant effect of bumetanide was observed only in control conditions and in the absence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (i.e., only in the presence of both Cl- and Na+). Together, these results strongly suggest that two forskolin-stimulated, Na+-dependent anion secretory pathways are present in vas deferens epithelia; one pathway is Cl- dependent and bumetanide sensitive, whereas the other is HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> dependent and bumetanide insensitive.


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 1.   Forskolin stimulation and bumetanide inhibition of vas deferens epithelial short-circuit current (Isc) depends on the presence of Na+, HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, and Cl- in the bathing media. A-H: typical responses to forskolin (Forsk; 2 µM apical and basolateral) and bumetanide (Bumet; 20 µM basolateral) in Ringer solution with the indicated ions replaced by impermeant ions. Scale bar, 4 µA/cm2, 5 min. I: summarized results from 4 to 14 observations per condition. *Significant difference (P < 0.05) from response in paired control conditions. #Significant (P < 0.05) effect of bumetanide.

Two separate sets of experiments were conducted to test for the contribution of metabolic HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> generation to the forskolin-stimulated response. In the first set of experiments, monolayers were exposed to acetazolamide either before or after forskolin exposure. As shown in Fig. 2, acetazolamide, at a concentration far in excess of that shown to inhibit carbonic anhydrase-dependent HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion in pancreatic cells (13), had no effect on baseline or forskolin-stimulated Isc. The results shown are typical of five to eight observations in each condition. Additional experiments were conducted to test the hypothesis that an effect of acetazolamide might be unmasked in the absence of Cl- and/or HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. The effect of forskolin on Isc was unchanged by acetazolamide regardless of which anions were present (not shown). Although it is highly unlikely, the possibility remained that metabolic production of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> could account for the Isc if a mechanism were also present for the removal of cellular H+. Therefore, a second set of experiments was conducted to test for the presence of an amiloride-inhibitable NHE that might be present at the basolateral membrane. As shown in Fig. 3, monolayers were mounted in the presence and absence of Cl- in the bathing media and stimulated with forskolin. In concordance with Fig. 1, the transient response to forskolin was substantially reduced in the absence of Cl-, and the sustained response was modestly reduced. Most importantly, amiloride (1 mM) had only a modest and transient effect on Isc. Maximal reduction in Isc was 0.9 µA; there was no sustained reduction in Isc in the presence of Cl- and <0.5 µA (i.e., 10%) reduction in Isc in the absence of Cl-. Together, these results indicate that metabolic production of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> does not contribute to forskolin-stimulated Isc across vas deferens epithelial cells.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 2.   Metabolically generated HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> does not contribute to the forskolin-induced increase in Isc. Acetazolamide (Acetazol; 300 µM apical and basolateral) had no effect on basal Isc (left) or forskolin-stimulated (2 µM apical and basolateral) changes in Isc (right). Typical effect of bumetanide (20 µM basolateral) is observed. Results are typical of 8 and 5 observations, respectively. Amiloride (Amil; 10 µM apical) had no effect on the responses.



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 3.   NHE does not contribute to forskolin-stimulated Isc. Amiloride (1 mM, basolateral) had no effect on forskolin-stimulated (2 µM apical and basolateral) changes in Isc in either the presence (control) or absence of Cl-. Results are typical of 2 and 4 observations, respectively.

HCO<UP><SUB>3</SUB><SUP><UP>−</UP></SUP></UP>-dependent ion transport is localized to the basolateral membrane. Selectively permeabilized monolayers were employed to test for the membrane (apical or basolateral) at which a HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-dependent step in ion transport could be identified. In the first set of experiments, monolayers were mounted in the presence of basolateral Na-gluconate and apical K-gluconate in the absence or presence of symmetrical HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (25 meq). Nystatin permeabilization of the apical membrane resulted in a large (107 ± 7 µA/cm2; n = 41) sustained increase in Isc that is consistent with anion secretion or cation absorption, but only in the presence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (Fig. 4). A substantial component of the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-dependent current was inhibited by basolateral addition of DNDS (Fig. 4, A and E), an inhibitor of NBC and anion exchangers (AE). Clotrimazole also inhibited a component of the current (Fig. 4E), consistent with that portion of the current being mediated by gradient-driven K+ absorption across the basolateral membrane. The magnitude of the DNDS-sensitive current was unchanged by clotrimazole pretreatment (Fig. 4E), indicating that these inhibitors affected different transport processes. In the absence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, apical permeabilization failed to unmask any basolateral ion movement and no effects of either DNDS or clotrimazole were observed. Ouabain reduced Isc by 4.0 ± 1.1 and 3.8 ± 3.6 µA/cm2 in the presence and absence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, respectively (n = 3), suggesting that the ongoing activity of the Na+/K+-ATPase does not contribute to the net HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-dependent ion transport across the basolateral membrane. To further test for the basolateral location of the NBC, the cation gradient was reversed (apical Na+, basolateral K+), and the experimental protocol was repeated. In these conditions, currents were consistent with anion absorption or cation secretion and were reduced by both DNDS and clotrimazole (not shown). The magnitude of gradient-driven flux was substantially reduced compared with the reverse cation gradient, and the effects of DNDS and clotrimazole, though consistent and statistically significant, were likewise smaller.


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 4.   HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-dependent ion transport is observed in the basolateral membrane of selectively permeabilized vas deferens epithelial monolayers. Effect of apical nystatin (Nyst; 18 µg/ml) to isolate the basolateral membrane in the presence of a basolateral-to-apical Na+ concentration gradient and in the presence (A) or absence (B) of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. 4,4'-Dinitrostilbene-2,2'-disulfonate (DNDS; 3 mM basolateral) reduces HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-dependent ion transport. C and D: effect of nystatin (36 µg/ml) on basolateral membrane to isolate gradient-driven flux across the apical membrane. In the presence of a cation gradient (C; apical Na+, basolateral K+), no changes in Isc were observed, whereas in the presence of an apical-to-basolateral Cl- gradient (D), Isc was stimulated by forskolin (2 µM apical and basolateral) and inhibited by DASU-02 (DASU; 100 µM apical and basolateral). E and F: results summarized from experiments similar to those presented in A and B, respectively. A total of 9 or more observations are summarized for each condition. BL, basolateral; Clotrim, clotrimazole.

Similar experiments were conducted to test for the presence of Na+- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-dependent ion transport across the apical membrane. In the presence of a cation gradient (apical Na+, basolateral K+), no Cl-, and symmetrical HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, basolateral permeabilization decreased transepithelial resistance without a change in Isc (Fig. 4C). Neither DNDS nor DASU-02 had any effect on net ion flux in these conditions, although Rte was reduced by both treatments (Fig. 4C). Concurrent experiments were conducted in symmetrical Na+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, but with an apical-to-basolateral-directed Cl- gradient (basolateral gluconate). Basolateral nystatin unmasked an anion absorptive current (-2.1 ± 0.2 µA/cm2) that was significantly increased by forskolin (-21.2 ± 1.9 µA/cm2; Fig. 4D). The current across the apical membrane was unaffected by DNDS or clotrimazole (not shown) but substantially reduced by DASU-02 (8.5 ± 1.6 µA/cm2), an inhibitor of the CFTR anion channel (43). Together, these results indicate that Na+-dependent HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport is not evident in the apical membrane, although control experiments clearly demonstrate that the experimental protocol isolates functional activity across the apical membrane. Results presented in Figs. 1 and 4 indicate that an electrogenic basolateral NBC is likely present in vas deferens epithelial cells. Subsequent experiments were conducted to learn the stoichiometry and molecular identity of this ion transporter.

Radiotracer flux suggests a stoichiometry of 2HCO<UP><SUB>3</SUB><SUP><UP>−</UP></SUP></UP> to 1Na+ for DNDS-sensitive transport. 22Na+ fluxes were measured in three conditions (basal, apically permeabilized in the absence of DNDS, or apically permeabilized in the presence of DNDS), to determine the ratio between Na+ movement and Isc. As shown in Fig. 5, unidirectional basal fluxes were <0.2 µeq · h-1 · cm-2 with flux in the serosal-to-mucosal (secretory) direction being greater such that a net secretory flux was observed. Isc was positive, indicative of net anion secretion. Thus the residual flux (i.e., difference between Na+ flux and Isc) was greater than the Isc and consistent with anion secretion. Nystatin permeabilization of the apical membrane was associated with an increase in both the mucosal-to-serosal (statistically significant) and the serosal-to-mucosal flux. Net Na+ flux was not significantly changed and was near zero. Isc, however, was significantly increased to ~6 µeq · h-1 · cm-2, resulting in a nearly equal residual flux that is consistent with anion secretion. Overall, these results (i.e., increased Isc without increased Na+ secretion) were surprising but might be explained by the ongoing activity of other membrane transporters (see DISCUSSION). Application of DNDS resulted in a further significant increase in unidirectional mucosal-to-serosal and net Na+ absorption while decreasing the Isc and residual flux. The ratio of change in mucosal-to-serosal and net absorptive Na+ flux to change in Isc was -1.02 ± 0.32 and -1.34 ± 0.81, respectively. An increase in net Na+ absorption associated with a reduction in Isc can occur only if the Na+ is accompanied by a net negative charge. The reported ratios of charge movement are consistent with the DNDS-sensitive component comprising two negative charges moving in parallel with each Na+; a stoichiometry of 2HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> to 1Na+.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 5.   Unidirectional and net 22Na+ flux along with Isc and residual flux in 3 conditions. A positive value indicates a net absorptive cationic flux (or a net secretory anionic flux). *Significant difference from previous period. Results from 9 experiments are summarized.

Vas deferens epithelial cells express NBC immunoreactivity. Antibodies raised against two different epitopes of rkNBC1 (amino acids 338-391 and 928-1035) were employed with immunoblots to test for NBC1 immunoreactivity in vas deferens epithelial cell lysates. These antibodies have previously been described in detail (41) and are commercially available. Shown in Fig. 6A is an immunoblot of cultured cell lysates from three separate isolations probed with anti-NBC1 amino acids 338-391. A single band at ~145 kDa was recognized by the antibody in vas deferens cell lysates, whereas no bands were present in bovine mammary cell lysates. The gel was stripped and subsequently probed with anti-NBC1 amino acids 928-1035, and an identical pattern was observed (Fig. 6B). In additional control experiments, NIH-3T3 cell lysates displayed no immunoreactivity to either of these antibodies, and labeling of the 145-kDa band was not observed in vas deferens cell lysates when primary antibody was replaced with anti-bovine serum albumin or when primary antibodies were absent from the assay protocol (not shown).


View larger version (64K):
[in this window]
[in a new window]
 
Fig. 6.   Sodium-bicarbonate cotransporter (NBC1) immunoreactivity is present in cultured vas deferens epithelial cell lysates. Cell lysates were resolved by SDS-PAGE and probed with antibodies raised against epitopes of rat kidney NBC1 fused to maltose binding protein. A: a prominent band of ~145 kDa was recognized by anti-kNBC1 amino acids 338-391 in cultured porcine vas deferens epithelia cells, but no band was observed in 2 separate BME-UV (bovine mammary) cell lysates. Arrows indicate the mobility of commercial molecular mass markers. Cell isolations are indicated from 2 animals (1 and 2) and from 2 ducts within 1 animal (A and B). B: identical results were observed when the gel was stripped and probed with anti-kNBC1 amino acids 928-1035. Results are typical of 3 separate experiments.

NBC mRNA is present in vas deferens epithelial cells. RT-PCR was employed to test for the expression of NBC1 message in cultured vas deferens epithelial cells. Primers expected to recognize both human kidney NBC1 (kNBC1, bp 2695-2973; Ref. 8) and human pancreatic NBC1 (pNBC1, bp 2795-3073; Ref. 1) as previously described (23) were employed. A PCR product of expected length (279 bp; Fig. 7A) was identified and sequenced. BLAST analysis indicated >90% identity with a 279-bp segment of human, bovine, rabbit, mouse, and rat electrogenic sodium-bicarbonate cotransporter (SLC4A4; Fig. 7B). The deduced amino acid sequence of porcine vas deferens NBC1 was also subjected to BLAST analysis. Identity was observed for 91 or 92 of 93 amino acids with NBC from the indicated species (Fig. 7C). This segment corresponds to the ninth and tenth putative membrane-spanning domains along with the intervening extracellular loop (38). Although additional experiments are required to document which NBC1 variant is expressed in vas deferens, pNBC1 or kNBC1, these results clearly indicate that the porcine homologue of NBC1 exhibits substantial homology to NBC1 from other mammalian species and is expressed in vas deferens epithelia.


View larger version (86K):
[in this window]
[in a new window]
 
Fig. 7.   Partial sequence of porcine vas deferens NBC1. A: amplification of cDNA from RT-PCR product using total RNA isolated from cultured porcine vas deferens epithelial cells. A band of expected size (279 bp) is observed. B: base sequence of PCR product with comparisons to published sequences derived from the indicated mammalian species. C: deduced amino acid sequence of porcine vas deferens NBC1 fragment with comparisons to published sequences derived from the indicated mammalian species. PCR product was generated and sequenced on 2 occasions with identical results.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The current results provide molecular and functional evidence for the presence of NBC1 in the basolateral membrane of porcine vas deferens epithelial cells. Results demonstrate that, in the context of an intact, confluent monolayer, cAMP-mediated stimulation results in activation of Na+-dependent HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion that is inhibited by DNDS but unaffected by acetazolamide or high concentrations of amiloride. Radiotracer flux experiments suggest a transporter stoichiometry of 2HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> to 1Na+. NBC1 protein is shown to be present in freshly isolated cultured cell lysates via Western analysis. RT-PCR provides compelling evidence that NBC1 mRNA is expressed in vas deferens epithelia and shows that porcine NBC1 exhibits substantial homology to NBC1 cloned from other mammalian species.

We and others have previously reported HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-dependent ion transport across male reproductive epithelial cell monolayers. Like cultured porcine vas deferens epithelia (44), cultured perinatal ovine (4) and rat (11) epididymal epithelia were shown to have a robust response to forskolin in the absence of Cl- but not in the absence of both Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-dependent ion transport has also been observed across cultured human vas deferens epithelia (R. W. Carlin and B. D. Schultz, unpublished observation). The current report extends these observations by demonstrating the presence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-dependent ion transport in distal portions of the porcine reproductive tract. Additionally, the results enhance previous observations by showing a codependence on the presence of Na+, inhibition by DNDS, basolateral localization of the ion transport activity, and an apparent stoichiometry of 2:1. Apical permeabilization in the presence of a cation gradient unmasked ion transport across the basolateral membrane that we first attributed to K+ absorption. However, substantial current remained in the presence of K+ channel inhibitors (e.g., Ba2+ or clotrimazole). Ouabain had little effect on the remaining current, indicating that the remaining ion flux could not be attributed to the electrogenic activity of Na+/K+- ATPase, although the results clearly indicate ongoing activity of this transporter. Subsequently, results indicated that current remaining in the presence of K+ channel blockers required the presence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> in the bathing media but that the flux was not driven by an anion gradient. The Na+ gradient provided electromotive force for the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-dependent net movement of anions in the secretory direction. As expected, the Na+-dependent HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion was sensitive to DNDS. Together, these new observations provide strong evidence to support the conclusion that NBC participates in cAMP-mediated anion transport across the epithelial basolateral membrane in the distal male reproductive tract.

It was, at first, surprising to observe a large Isc with little net Na+ flux. What is the basis of the current if Na+ is not apparently involved? Experiments were conducted in the absence of Cl-, ruling out this ion as a charge carrier. Alternatively, a K+ gradient might drive the absorption of this ion to account for the Isc. However, a portion of the experiments was conducted in the presence of clotrimazole (20 µM) with the experimental outcomes not being significantly changed. The other permeant ions present are Na+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. The gradient for Na+ is in the opposite direction of the residual flux, and flux measurements do not support its movement accounting for any residual current (by definition). This leaves HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> as the only readily available permeant monovalent ion that might account for the measured current. However, the lack of a matched Na+ secretion in the same conditions is perplexing. One possible explanation is that the cytoplasm of the nystatin-permeabilized cells is a barrier to the ready diffusion of Na+ entering from the basolateral side to freely mix with Na+ that is present on the apical face of the epithelium. Ongoing activity of the Na+/K+- ATPase could preferentially extrude Na+ of extracellular origin back to the extracellular solution. Thus, for every three cycles of NBC1, the Na+/K+-ATPase could cycle once, resulting in the secretion of three HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, no net Na+ movement across the basolateral membrane, and the uptake of two K+. The activity of the Na+/K+-ATPase would be expected to increase the Isc only if K+ were not allowed to freely recycle across the basolateral membrane via a K+ conductance. Importantly, DNDS, an inhibitor of NBC, unmasked ongoing Na+ absorption and reduced overall ion flux. The ratio of DNDS-induced change in Na+ flux to DNDS-induced change in Isc is ~1:1. However, increased Na+ absorption would tend to increase Isc, not decrease it as was observed. Thus the DNDS-induced change in Na+ flux is equal in magnitude but opposite in direction to the measured change in Isc. These observations can be resolved if the DNDS-sensitive component couples two negative charges to every Na+ ion that moves in a serosal-to-mucosal direction. Thus we can conclude that DNDS inhibits a transporter that functions in the absence of Cl- with a stoichiometry of two base equivalents per Na+, a stoichiometry that has been reported for pancreas, duodenum, and other epithelia that secrete HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> into the lumen of a hollow organ.

The identification of NBC in the male reproductive tract is not without precedent. Rat epididymis has previously been shown to exhibit NBC1 immunoreactivity by employing one of the same antibodies that was employed in the present studies (26). In situ hybridization (26) and stilbene-sensitive luminal acidification (5) provided additional evidence implicating the presence of NBC1 in this epithelium. The authors (26) concluded that NBC1 was present in the basolateral membranes of principal and narrow cells of the epididymis, where this transporter was hypothesized to provide a base exit route while bafilomycin-sensitive H+-ATPase extruded acid across the apical membrane. In sharp contrast to the present study, which unequivocally indicates NBC1 mRNA as well as protein in porcine vas deferens epithelial cell lysates, the authors (26) reported that both in situ hybridization and immunocytochemistry provided evidence for only modest NBC1 expression in the rat vas deferens. More recently, Pushkin et al. (37) employed RT-PCR and immunocytochemistry to implicate an electroneutral NBC (NBC3) in the apical membranes of rat epididymis. The authors speculated that NBC3 participated in Na+-dependent base uptake from the lumen, the net result of which would be luminal acidification. Because this transporter is electroneutral, the present studies cannot address the possibility of its functionality in porcine vas deferens epithelia. Certainly, an electrogenic NBC was not observed in the isolated apical membrane of porcine vas deferens.

The current results suggest a shift in our understanding of the vas deferens and its role in fertility. The vas deferens has been viewed both historically and more recently as an acid secreting organ. Micropuncture studies provided initial evidence that the luminal environment of rat epididymis and proximal vas deferens was acidic (33). Likewise, more recent reports indicate that proton-secreting mechanisms are present in rat epididymis and vas deferens, although lesser expression is observed in the vas deferens (5, 6). These studies were conducted with unstimulated tissues. HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-dependent secretion has been observed in the presence of hormone, neurotransmitter, or second-messenger stimulation (4, 11, 44). Likewise, the present results address only stimulated conditions and suggest that stimulation of the vas deferens contributes to an alkalization of the lumen that could occur during the period of arousal prior to ejaculation. Vas deferens epithelia have adrenergic neurons associated with them (18, 28, 30), and we previously showed that norepinephrine stimulates Isc across vas deferens epithelia (44). Such an effect would be expected to activate sperm motility and initiate the acrosome reaction so that at the time of ejaculation, sperm are fully equipped for fertilization. The return of an acid environment for the accumulation and storage of quiescent sperm could be accomplished by base recovery and/or by ongoing acid secretion, as has been described for unstimulated tissues.

The porcine reproductive tract offers the opportunity for insights that complement observations made on rodent systems. Rodent models have provided a tremendous amount of information regarding epididymal ion transport. However, rodents lack the extensive vas deferens (up to 40 cm that can be divided into transitional, scrotal, inguinal, and abdominal portions) that is present in humans or other large species. In this regard, the pig offers an excellent model because the relative proportions of these vas deferens regions are similar to those observed in humans. In the present studies, we worked diligently to exclude transitional vas deferens that may include functional aspects of the epididymis (see Ref. 44) and instead have focused on distal portions of the duct. As stated above, experiments employing human vas deferens epithelial cells show similar responses to those observed with porcine cells (9, 42). The present results suggest that porcine and likely human vas deferens can rapidly alkalinize the lumen and thus activate sperm just prior to ejaculation.

Two models have evolved for HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion by organs such as the pancreas (13, 45), cornea (46), or duodenum (24, 36; for review see Ref. 45). A fundamental difference between these models is the mechanism by which intracellular HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> is generated. In one case, CO2 diffuses across the basolateral membrane and, under the influence of carbonic anhydrase, is combined with water and spontaneously dissociates to HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and H+. H+ can then leave the cell across the basolateral membrane either by active transport (H+-ATPase) or via Na+ exchange (e.g., NHE1), leaving intracellular HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> above its electrochemical equilibrium. HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> then leaves the cell across the apical membrane by one of two mechanisms, a conductive channel or an anion exchange process. Controversy remains regarding the ability of CFTR to conduct HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (45). Thus the exit pathway is speculated to be CFTR, an undefined HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> conductance, or CFTR Cl- conductance working in concert with an anion exchanger to recycle Cl- and extrude HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. The second cell model depends on a stilbene-sensitive NBC at the basolateral membrane to harness the electrochemical gradient for Na+ to bring HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> into the cell. Na+ can be recycled across the basolateral membrane by Na+/K+-ATPase while HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> leaves the cell across the apical membrane by a mechanism similar to that described above. The present data are consistent with the latter model of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion. Acetazolamide and high concentrations of amiloride were without effect, and basolateral DNDS significantly reduced anion secretion. These results stand in contrast to a report regarding ion transport by rat epididymis where acetazolamide inhibited acid secretion (5) or where 500 µM amiloride almost completely inhibited cAMP-stimulated ion transport (11). The latter observation is particularly intriguing in light of observed differences between the mechanisms of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion in the pancreas of rats and humans. The rat pancreas employs the first model with CO2 diffusion, carbonic anhydrase, and NHE to provide for HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion. Humans employ NBC to increase cytosolic HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> for secretion. The present results suggest that the pig vas deferens employs a cellular model that is like that of the human pancreas and different from that of the rat pancreas or epididymis. At this point, one should note that murine models of cystic fibrosis do not exhibit pancreatic or male reproductive pathology. The present results suggest that the porcine (and likely human; Refs. 9 and 42) reproductive tract differs from the rat reproductive tract in the mechanism that is employed for HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport in much the same way that the human pancreas differs from the rat pancreas.

NBC1 has been reported to participate in both the absorption and the secretion of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> across epithelial monolayers. In the kidney, NBC1 exhibits a stoichiometry of 3HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> to 1Na+ and moves ions from the intracellular to the extracellular basolateral compartment. A strong electronegative potential (e.g., more negative than -60 mV) is required to drive the net movement of two negative charges from the cell against the concentration gradient of both Na+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. Alternatively, in the pancreas and throughout much of the rest of the body, a stoichiometry of 2HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> to 1Na+ is observed, and the electrochemical driving force for Na+ drives the uptake of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> against its electrochemical gradient. Cellular uptake of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> occurs in cells that are somewhat depolarized (e.g., more positive than -50 mV). Initially, it was postulated that the renal and pancreatic variants of NBC1, kNBC1 and pNBC1, which differ by <100 amino acids in their NH2 terminus, might selectively absorb or secrete HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, respectively. Alternatively, regulation of these variants might differ on the basis of phosphorylation sites that are differentially present in their NH2 terminus. However, it has been shown recently that the stoichiometry of transport across a cell membrane is cell-type specific rather than variant specific (22). There remains the possibility that, in the reproductive tract, epididymal cells may provide an environment for HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption (i.e., luminal acidification), whereas cells of the vas deferens provide an environment for HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion (i.e., luminal alkalinization) when exposed to neurotransmitters. Alternatively, membrane potential may be used to "switch" between Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion depending on the regulators affecting the epithelial cells. Such a mechanism has been demonstrated in vitro employing Calu-3 airway cells where forskolin stimulation resulted in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion in the absence of human intermediate-conductance Ca2+-activated K+ (hIK) channel activators, but resulted in Cl- secretion when K+ channel activators were present (17). The presence of a large, clotrimazole-sensitive basolateral K+ conductance (see Fig. 4E) suggests that a similar system may be present in vas deferens epithelia. However, other hIK channel modulators (e.g., charybdotoxin, 1-ethyl-2-benzimidazalinone; Ref. 44) were without effect, suggesting that an alternative K+ conductance is present in vas deferens. Clearly, additional experiments are required to delineate the cellular mechanisms that modulate HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport across vas deferens epithelial cells.

The current results suggest that the relationship of CFTR to CBAVD may not simply be related to a loss of Cl- secretion as has previously been suggested (31). Rather, modulation of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion and luminal pH that occur in typical vas deferens may be compromised by the loss of CFTR. Certainly, semen from CF patients (although clearly not of vas deferens origin) is known to exhibit a much lower pH than semen of healthy men (15, 16). Furthermore, we have recently shown that vas deferens epithelia express amiloride-sensitive current when exposed to glucocorticoids (35). Because amiloride-sensitive current is elevated in CF tissues, one might also expect heightened amiloride-sensitive current to contribute to a loss of the vas deferens in CF patients. Thus one might envision attempting to manipulate HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion as a chronic treatment to prevent CBAVD in CF patients. Additionally, vas deferens ion transport may provide a target for interventions to modulate male fertility.


    ACKNOWLEDGEMENTS

We thank Dr. Chris Ross for assistance in design and generation of PCR primers, Steve Becker for tissue procurement, Dr. James Broughman and Roger Sedlacek for technical assistance, and Ginger Biesenthal, Pam Say, and Bonnie Thompson for clerical support.


    FOOTNOTES

This research was supported by the Cystic Fibrosis Foundation (SCHULT99P0) and the Kansas State University College of Veterinary Medicine Dean's Research Fund. This manuscript is contribution no. 02-151-J from the Kansas Agricultural Experiment Station.

Address for reprint requests and other correspondence: B. D. Schultz, Dept. of Anatomy and Physiology, Kansas State Univ., 1600 Denison Ave., VMS 228, Manhattan, KS 66506 (E-mail: bschultz{at}vet.ksu.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.

May 22, 2002;10.1152/ajpcell.00493.2001

Received 13 October 2001; accepted in final form 29 April 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abuladze, N, Lee I, Newman D, Hwang J, Boorer K, Pushkin A, and Kurtz I. Molecular cloning, chromosomal localization, tissue distribution, and functional expression of the human pancreatic sodium bicarbonate cotransporter. J Biol Chem 273: 17689-17695, 1998[Abstract/Free Full Text].

2.   Anguiano, A, Oates RD, Amos JA, Dean M, Gerrard B, Stewart C, Maher TA, White MB, and Milunsky A. Congenital bilateral absence of the vas deferens. A primarily genital form of cystic fibrosis. JAMA 267: 1794-1797, 1992[Abstract].

3.   Bagnis, C, Marsolais M, Biemesderfer D, Laprade R, and Breton S. Na+/H+-exchange activity and immunolocalization of NHE3 in rat epididymis. Am J Physiol Renal Physiol 280: F426-F436, 2001[Abstract/Free Full Text].

4.   Bertog, M, Smith DJ, Bielfeld-Ackermann A, Bassett J, Ferguson DJ, Korbmacher C, and Harris A. Ovine male genital duct epithelial cells differentiate in vitro and express functional CFTR and ENaC. Am J Physiol Cell Physiol 278: C885-C894, 2000[Abstract/Free Full Text].

5.   Breton, S, Hammar K, Smith PJS, and Brown D. Proton secretion in the male reproductive tract: involvement of Cl--independent HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport. Am J Physiol Cell Physiol 275: C1134-C1142, 1998[Abstract/Free Full Text].

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

7.   Brown, D, and Breton S. H+ V-ATPase-dependent luminal acidification in the kidney collecting duct and the epididymis/vas deferens: vesicle recycling and transcytotic pathways. J Exp Biol 203: 137-145, 2000[Abstract].

8.   Burnham, CE, Amlal H, Wang Z, Shull GE, and Soleimani M. Cloning and functional expression of a human kidney Na+:HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter. J Biol Chem 272: 19111-19114, 1997[Abstract/Free Full Text].

9.   Carlin, RW, Mitchell ME, Sedlacek RL, and Schultz BD. Bicarbonate-dependent ion transport by vas deferens epithelial cell monolayers (Abstract). J Gen Physiol 116: 26a, 2000.

10.   Carlin, RW, Mitchell ME, Sedlacek RL, and Schultz BD. Distinct bicarbonate- and chloride-dependent secretion mechanisms are stimulated by ATP and forskolin in vas deferens epithelial cell monolayers (Abstract). Pediatr Pulmonol Suppl 17: 208-209, 2000.

11.   Chan, HC, Ko WH, Zhao W, Fu WO, and Wong PY. Evidence for independent Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion and involvement of an apical Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter in cultured rat epididymal epithelia. Exp Physiol 81: 515-24, 1996[Abstract].

12.   Chen, Y, Cann MJ, Litvin TN, Iourgenko V, Sinclair ML, Levin LR, and Buck J. Soluble adenylyl cyclase as an evolutionarily conserved bicarbonate sensor. Science 289: 625-628, 2000[Abstract/Free Full Text].

13.   Cheng, HS, Leung PY, Cheng Chew SB, Leung PS, Lam SY, Wong WS, Wang ZD, and Chan HC. Concurrent and independent HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and Cl- secretion in a human pancreatic duct cell line (CAPAN-1). J Membr Biol 164: 155-167, 1998[ISI][Medline].

14.   Cuthbert, AW, and Wong PY. Electrogenic anion secretion in cultured rat epididymal epithelium. J Physiol 378: 335-345, 1986[Abstract].

15.   Daudin, M, Bieth E, Bujan L, Massat G, Pontonnier F, and Mieusset R. Congenital bilateral absence of the vas deferens: clinical characteristics, biological parameters, cystic fibrosis transmembrane conductance regulator gene mutations, and implications for genetic counseling. Fertil Steril 74: 1164-1174, 2000[ISI][Medline].

16.   De la Taille, A, Rigot JM, Mahe P, Vankemmel O, Gervais R, Dumur V, Lemaitre L, and Mazeman E. Correlation between genito-urinary anomalies, semen analysis and CFTR genotype in patients with congenital bilateral absence of the vas deferens. Br J Urol 81: 614-619, 1998[ISI][Medline].

17.   Devor, DC, Singh AK, Lambert LC, DeLuca A, Frizzell RA, and Bridges RJ. Bicarbonate and chloride secretion in Calu-3 human airway epithelial cells. J Gen Physiol 113: 743-760, 1999[Abstract/Free Full Text].

18.   Dixon, JS, Jen PY, and Gosling JA. Structure and autonomic innervation of the human vas deferens: a review. Microsc Res Tech 42: 423-432, 1998[ISI][Medline].

19.   Durieu, I, Bey-Omar F, Rollet J, Calemard L, Boggio D, Lejeune H, Gilly R, Morel Y, and Durand DV. Diagnostic criteria for cystic fibrosis in men with congenital absence of the vas deferens. Medicine (Baltimore) 74: 42-47, 1995[ISI][Medline].

20.   Ferrari, M, and Cremonesi L. Genotype-phenotype correlation in cystic fibrosis patients. Ann Biol Clin (Paris) 54: 235-241, 1996[ISI][Medline].

21.   Gaillard, DA, Carre-Pigeon F, and Lallemand A. Normal vas deferens in fetuses with cystic fibrosis. J Urol 158: 1549-1552, 1997[ISI][Medline].

22.   Gross, E, Hawkins K, Abuladze N, Pushkin A, Cotton CU, Hopfer U, and Kurtz I. The stoichiometry of the electrogenic sodium bicarbonate cotransporter NBC1 is cell-type dependent. J Physiol 531: 597-603, 2001[Abstract/Free Full Text].

23.   Hara, C, Satoh H, Usui T, Kunimi M, Noiri E, Tsukamoto K, Taniguchi S, Uwatoko S, Goto A, Racusen LC, Inatomi J, Endou H, Fujita T, and Seki G. Intracellular pH regulatory mechanism in a human renal proximal cell line (HKC-8): evidence for Na+/H+ exchanger, Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger and Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter. Pflügers Arch 440: 713-720, 2000[ISI][Medline].

24.   Jacob, P, Christiani S, Rossmann H, Lamprecht G, Vieillard-Baron D, Muller R, Gregor M, and Seidler U. Role of Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter NBC1, Na+/H+ exchanger NHE1, and carbonic anhydrase in rabbit duodenal bicarbonate secretion. Gastroenterology 119: 406-419, 2000[ISI][Medline].

25.   Jakubiczka, S, Bettecken T, Stumm M, Nickel I, Musebeck J, Krebs P, Fischer C, Kleinstein J, and Wieacker P. Frequency of CFTR gene mutations in males participating in an ICSI programme. Hum Reprod 14: 1833-1834, 1999[Abstract/Free Full Text].

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

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

28.   Kaleczyc, J. Origin and neurochemical characteristics of nerve fibres supplying the mammalian vas deferens. Microsc Res Tech 42: 409-422, 1998[ISI][Medline].

29.   Kaunisto, K, Moe OW, Pelto-Huikko M, Traebert M, and Rajaniemi H. An apical membrane Na+/H+ exchanger isoform, NHE-3, is present in the rat epididymal epithelium. Pflügers Arch 442: 230-236, 2001[ISI][Medline].

30.   Kihara, K, Sato K, and Oshima H. Sympathetic efferent pathways projecting to the vas deferens. Microsc Res Tech 42: 398-408, 1998[ISI][Medline].

31.   Leung, AYH, and Wong PYD The epididymis as a chloride-secreting organ. News Physiol Sci 9: 31-35, 1994[Abstract/Free Full Text].

32.   Leung, GP, Tse CM, Chew SB, and Wong PY. Expression of multiple Na+/H+ exchanger isoforms in cultured epithelial cells from rat efferent duct and cauda epididymis. Biol Reprod 64: 482-490, 2001[Abstract/Free Full Text].

33.   Levine, N, and Marsh DJ. Micropuncture studies of the electrochemical aspects of fluid and electrolyte transport in individual seminiferous tubules, the epididymis and the vas deferens in rats. J Physiol 213: 557-570, 1971[ISI][Medline].

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

35.   Phillips, ML, and Schultz BD. Steroids modulate transepithelial resistance and Na+ absorption across cultured porcine vas deferens epithelia. Biol Reprod 66: 1016-1023, 2002[Abstract/Free Full Text].

36.   Praetorius, J, Hager H, Nielsen S, Aalkjaer C, Friis UG, Ainsworth MA, and Johansen T. Molecular and functional evidence for electrogenic and electroneutral Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporters in murine duodenum. Am J Physiol Gastrointest Liver Physiol 280: G332-G343, 2001[Abstract/Free Full Text].

37.   Pushkin, A, Clark I, Kwon TH, Nielsen S, and Kurtz I. Immunolocalization of NBC3 and NHE3 in the rat epididymis: colocalization of NBC3 and the vacuolar H+-ATPase. J Androl 21: 708-720, 2000[Abstract/Free Full Text].

38.   Romero, MF, and Boron WF. Electrogenic Na+/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporters: cloning and physiology. Annu Rev Physiol 61: 699-723, 1999[ISI][Medline].

39.   Sambrook, J, Fritsch EF, and Maniatis T. Molecular Cloning: A Laboratory Manual. Plainview, NY: Cold Spring Harbor, 1989.

40.   Schmidt, CR, Carlin RW, Sargeant JM, and Schultz BD. Neurotransmitter-stimulated ion transport across cultured bovine mammary epithelial cell monolayers. J Dairy Sci 84: 2622-2631, 2001[Abstract/Free Full Text].

41.   Schmitt, BM, Biemesderfer D, Romero MF, Boulpaep EL, and Boron WF. Immunolocalization of the electrogenic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter in mammalian and amphibian kidney. Am J Physiol Renal Physiol 276: F27-F38, 1999[Abstract/Free Full Text].

42.   Schultz, BD, Carlin RW, Sedlacek RL, Mitchell ME, Lee JH, and Marcus DC. Adenosine stimulates anion secretion across both human and porcine vas deferens epithelia (Abstract). FASEB J 15: A439, 2001[ISI].

43.   Schultz, BD, Singh AK, Devor DC, and Bridges RJ. Pharmacology of CFTR chloride channel activity. Physiol Rev 79: S109-S144, 1999[Medline].

44.   Sedlacek, RL, Carlin RW, Singh AK, and Schultz BD. Neurotransmitter-stimulated ion transport by cultured porcine vas deferens epithelium. Am J Physiol Renal Physiol 281: F557-F570, 2001[Abstract/Free Full Text].

45.   Soleimani, M, and Ulrich CD, 2nd. How cystic fibrosis affects pancreatic ductal bicarbonate secretion. Med Clin North Am 84: 641-655, 2000[ISI][Medline].

46.   Sun, XC, Bonanno JA, Jelamskii S, and Xie Q. Expression and localization of Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter in bovine corneal endothelium. Am J Physiol Cell Physiol 279: C1648-C1655, 2000[Abstract/Free Full Text].

47.   Tizzano, EF, Silver MM, Chitayat D, Benichou JC, and Buchwald M. Differential cellular expression of cystic fibrosis transmembrane regulator in human reproductive tissues. Clues for the infertility in patients with cystic fibrosis. Am J Pathol 144: 906-914, 1994[Abstract].

48.   Traystman, MD, Schulte NA, MacDonald M, Anderson JR, and Sanger WG. Mutation analysis for cystic fibrosis to determine carrier status in 167 sperm donors from the Nebraska Genetic Semen Bank. Hum Mutat 4: 271-275, 1994[ISI][Medline].

49.   Van der Ven, K, Messer L, van der Ven H, Jeyendran RS, and Ober C. Cystic fibrosis mutation screening in healthy men with reduced sperm quality. Hum Reprod 11: 513-517, 1996[Abstract].


Am J Physiol Cell Physiol 283(4):C1033-C1044
0363-6143/02 $5.00 Copyright © 2002 the American Physiological Society




This Article
Abstract
Full Text (PDF)
All Versions of this Article:
283/4/C1033    most recent
00493.2001v1
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Search for citing articles in:
ISI Web of Science (1)
Google Scholar
Articles by Carlin, R. W.
Articles by Schultz, B. D.
Articles citing this Article
PubMed
PubMed Citation
Articles by Carlin, R. W.
Articles by Schultz, B. D.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online