Department of Anatomy and Physiology, Kansas State University, Manhattan, Kansas 66506
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ABSTRACT |
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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 for a full response.
HCO
NBC1; anion transport; pH regulation; cystic fibrosis; congenital bilateral absence of the vas deferens
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INTRODUCTION |
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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
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
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MATERIALS AND METHODS |
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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.
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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
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 · h1 · 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
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.
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RESULTS |
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cAMP-stimulated ion transport is Na+,
HCO 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
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
(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
, the sustained
plateau was present (1.7 ± 0.3 µA/cm2) and was not
bumetanide sensitive, whereas in the absence of HCO
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
-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
. A significant effect
of bumetanide was observed only in control conditions and in the
absence of HCO
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
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HCO
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Radiotracer flux suggests a stoichiometry of
2HCO1 · 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
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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).
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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.
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DISCUSSION |
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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
We and others have previously reported HCO but not in the absence of both
Cl
and HCO
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
with a stoichiometry of two base
equivalents per Na+, a stoichiometry that has been reported
for pancreas, duodenum, and other epithelia that secrete
HCO
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
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 conductance working in concert with an anion
exchanger to recycle Cl
and extrude
HCO
NBC1 has been reported to participate in both the absorption and the
secretion of HCO60 mV) is required to drive the
net movement of two negative charges from the cell against the
concentration gradient of both Na+ and
HCO
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
and HCO
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
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
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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