Am J Physiol Renal Physiol
281: F557–F570, 2001.
special communication
Neurotransmitter-stimulated ion transport by cultured
porcine vas deferens epithelium
Received 5 February 2001; accepted in final form 7 May 2001
Sedlacek, Roger L., Ryan W. Carlin, Ashvani K.
Singh, and Bruce D. Schultz. Neurotransmitter-stimulated ion transport by cultured porcine vas deferens epithelium. Am J Physiol Renal Physiol 281: F557–F570, 2001.—A
collagenase-based dissociation technique has been developed
to routinely establish monolayer cultures of freshly isolated
porcine vas deferens epithelium. Cells isolated from each
tissue are transferred to 25-cm2 tissue culture flasks and
grown in a standard cell culture medium. Flasks reach confluency in 3–4 days, and cells are subsequently seeded onto
permeable supports. Cultured cells display a monolayer cobblestone appearance and are immunoreactive to anti-ZO-1
and anti-cytokeratin antibodies. Electron microscopy is employed to demonstrate the presence of junctional complexes
and microvilli. When evaluated in modified Ussing chambers, cultured monolayers exhibit a basal lumen negative
potential difference, high electrical resistance (⬎1,000 ⍀ 䡠
cm2), and respond to norepinephrine, vasopressin, ATP,
adenosine, and histamine, with changes in short-circuit current indicative of anion secretion. Responses are significantly
attenuated in Cl⫺- and/or HCO3⫺-free solutions. Attempts to
further optimize culture conditions have shown that chronic
exposure to insulin increases proliferation rates. Thus the
culture method described will reliably produce viable neurotransmitter-responsive cell monolayers that will allow for the
characterization of vas deferens epithelial function and associated control mechanisms.
male reproductive tract
plays a key role in male fertility. It has been shown
that the epithelium lining the epididymis actively
transports ions to adjust the luminal environment for
sperm development and maturation (2, 8, 11, 27, 28,
34, 35, 53, 54). Based largely on morphology, similar
activity has been attributed to the vas deferens epithelia, although conclusive studies on this portion of the
tract are rare (25, 36, 37, 43). Numerous studies have
evaluated vas deferens epithelial histology (9, 22, 24,
40), morphometry (43), and enzymology (21, 31). However, few studies directly assess the epithelial ion
transport mechanisms of the vas deferens. Furthermore, most studies reported to date have been completed in rodent models (6–8) with only a single large
animal epithelial cell model having been reported (4).
Thus very little is known regarding vas deferens epithelial ion transport in humans and other large animal
species.
The geometry of the vas deferens (small-diameter
lumen, thick muscular wall) has provided obstacles to
its study. Primary epithelial cell cultures circumvent
these difficulties. Three studies (13, 22, 41) employing
fetal human epididymis and vas deferens demonstrated that cells isolated from these tissues were amenable to culturing manipulations, possessed mRNA
transcripts for the cystic fibrosis transmembrane conductance regulator (CFTR), and functionally expressed
both chloride and potassium channels. Epithelial cells
from the genital ducts of perinatal rams have also been
successfully cultured, and exhibited active ion transport when grown on permeable supports. In addition to
CFTR, the cultured cells expressed an epithelial Na⫹
channel (ENaC; 4). Therefore, much can be learned
from additional in vitro epithelial systems regarding
the ion transport mechanisms that contribute to the
luminal environment of the vas deferens.
Changes in vas deferens epithelial ion transport are
associated with the loss of normal ductal development,
ductal maintenance, and/or fertility. This association is
exemplified by cystic fibrosis (CF) where there is an
almost universal correlation of CF with congenital bilateral absence of the vas deferens (CBAVD). The most
perplexing observation regarding CF is that some “patients” present with CBAVD and have no other signs of
Address for reprint requests and other correspondence: B. D.
Schultz, Dept. of Anatomy and Physiology, Kansas State University,
1600 Denison Ave., VMS 228, Manhattan, KS 66506 (E-mail:
[email protected]).
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.
model system; epithelia; anion transport; pH regulation; cystic fibrosis; congenital bilateral absence of the vas deferens
THE EXCURRENT DUCT OF THE
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F557
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ROGER L. SEDLACEK,1 RYAN W. CARLIN,1 ASHVANI K. SINGH,2 AND BRUCE D. SCHULTZ1
1
Department of Anatomy and Physiology, Kansas State University,
Manhattan, Kansas 66506; and 2Aurora Biosciences, San Diego, California 92121
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VAS DEFERENS ION TRANSPORT
MATERIALS AND METHODS
Tissue isolation. Mature porcine reproductive tracts including scrotum, testes, epididymes, and vas deferens (up to
and including a portion of the abdominal ducts) were obtained from a local slaughterhouse ⬃40 min post mortem.
Immediately on removal from the animal, whole reproductive
tracts were immersed in ice-cold Ringer solution of the following composition (in mM): 120 NaCl, 25 NaHCO3, 3.3
KH2PO4, 0.83 K2HPO4, 1.2 CaCl2, and 1.2 MgCl2, supplemented with 100 U/ml penicillin, 100 ␮g/ml streptomycin, 40
␮g/ml gentamicin, and 4 ␮g/ml amphotericin B. Tracts were
maintained in this solution until further processing was
initiated at the laboratory (2–3 h). Because the anatomic
demarcation of the vas deferens from the caudal region of the
epididymis is open to interpretation, we chose to focus on
regions of the excurrent duct that did not exhibit the convoluted geometry common to the caudal region of the epididymis or the transitional portion of the vas deferens. Hence,
regions selected for cell isolation comprised only the
“straight” regions of the vas deferens (Fig. 1). Ducts collected
for epithelial cell isolation ranged in length from 10 to 35 cm.
Once removed, the lumen of each duct was flushed with 3 ml
of fresh ice-cold Hanks’ buffered saline solution (HBSS) of the
following composition (in mM): 137 NaCl, 5.4 KCl, 0.4
KH2PO4, 0.34 NaH2PO4, 5.5 D-(⫹)-glucose, pH 7.0, supplemented with 100 U/ml penicillin, 100 ␮g/ml streptomycin, 40
AJP-Renal Physiol • VOL
Fig. 1. Normal gross morphology of porcine male genital tract including testicle (T), caput epididymis (CaE), cauda epididymis (CE),
and vas deferens (VD). The vertical dashed line indicates the proximal end of tissues used for cell isolation; the convoluted transitional
portion of the epididymis/vas deferens was excluded from experimental procedures. Tissues for cell isolation extended 10–35 cm distal to
the demarcation. A ruler with centimeter graduations is included at
the base of the figure.
␮g/ml gentamicin, and 10 ␮g/ml amphotericin B, and placed
into iced beakers containing the antibiotic-supplemented
HBSS (HBSS⫹A).
Epithelial cell isolation. Before dissociation of the luminal
epithelial cells, tissues were first carefully cleaned of connective tissue and blood vessels and then flushed with 5 ml of
HBSS⫹A followed by 5 ml of HBSS to remove the antibiotics
from the lumen. To effect dissociation of the epithelial layer,
a small volume (typically ⬍0.5 ml) of phosphate-buffered
saline for cell culture (PBScc) of the following composition (in
mM), i.e., 140 NaCl, 2 KCl, 1.5 KH2PO4, 15 Na2HPO4, containing 300 U/ml collagenase, 0.25% (vol/vol) trypsin, and
2.65 mM disodium EDTA, was perfused through the lumen.
After the initial perfusion, the distal end of the tissue was
occluded with a mosquito hemostat, and the duct was filled
with dissociation solution until distended. The proximal end
was then occluded with a second hemostat. Filled ducts were
placed into 50-ml tubes containing chilled HBSS, immersed
into a 37°C water bath, and incubated for 20–60 min, with
swirling at 10-min intervals. Incubation periods of ⬍30 or
⬎45 min resulted in a lower proportion of isolations yielding
viable epithelial cell cultures. Therefore, incubation periods
of 30–45 min were employed for the majority of this work.
Freshly dissociated cells were collected from the vas deferens by blotting the duct with tissue paper to remove excess
fluid and then draining the luminal contents into a 15-ml
tube. The duct was flushed with 1.0 ml of growth medium
DMEM (GIBCO-BRL, Grand Island, NY), supplemented
with 10% heat-inactivated fetal bovine serum (HyClone, Logan, UT), 100 U/ml penicillin, and 100 ␮g/ml streptomycin,
massaged by rolling it between a gloved thumb and index
finger, and again flushed with 1.0 ml of supplemented growth
medium into the same tube. The resulting cell suspension
was further diluted with 3 ml of growth medium and pipetted
several times to facilitate the disruption of epithelial sheets
that detached from the lumen. After this treatment, numerous small aggregates of cells remained. These multicell aggregates precluded any quantitative determination of cell
viability. The entire volume (nominally 5 ml) of suspension
was then placed into a vented, 25-cm2 tissue culture flask
(Corning, Corning, NY) and incubated at 37°C in a humidified environment containing 5% CO2.
Initial cell culture and routine passage. After 18–24 h of
culture, the growth media and any nonadherent cells were
removed by aspiration, and cells were fed with fresh growth
media. Adherent cells exhibited an epithelioid-like morphol-
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the disease; infertility is their only complaint, and
genotyping is required to determine their CF status (3,
11, 15, 20). Furthermore, men seeking intervention for
infertility have a higher incidence of “mild” mutations
in the CF gene than the general population (26, 51).
These observations suggest that the vas deferens is
more sensitive to the loss of an anion conductance
(CFTR) than any other epithelial tissue.
An aim of the present study was to develop a suitable
and readily available model of vas deferens epithelium
for in vitro ion transport studies. Human male genital
ducts are difficult to obtain for such purposes. Mouse
tissues are readily available in most laboratory settings and, because of genetically modified strains,
readily manipulated at the molecular level. However, it
is particularly important to note that, unlike humans
who lack expression of CFTR and suffer CBAVD, CF
mice have a complete and patent ductal system. Thus
pathological manifestations of epithelial changes are
fundamentally different in humans and mice. The
adult human and porcine vas deferens are similar in
that they are 30–40 cm in length with distinct scrotal
and abdominal components, and it has been suggested
that the porcine reproductive system is an adequate
model for inferences to humans (29, 30).
In this report we detail a method to routinely obtain
and grow cultures of epithelial cells from porcine vas
deferens that can be used as a model for ductal ion
transport. Included are examples of ion transport by
these epithelial cells in response to physiological hormones and neurotransmitters as well as forskolin, a
direct stimulator of adenylyl cyclase activity. This system provides a reliable means by which researchers
may examine the role of ion transport mechanisms and
regulatory cascades that contribute to the luminal environment to which sperm are exposed.
VAS DEFERENS ION TRANSPORT
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fixed overnight in 10% buffered neutral formalin (BNF;
Fisher Scientific, Pittsburgh, PA) at 4°C and rinsed in PBS
for histochemistry [PBSh; (in mM)]: 5 KH2PO4, 15 K2HPO4,
and 150 NaCl, pH 7.2–7.4. Small segments of each specimen
(0.5–1.0 cm) were cut and placed in cryoprotectant [Sörenson’s Phosphate Buffer (in mM: 8 KH2PO4, 42 Na2HPO4)
containing 30% wt/vol sucrose] ⫹ 0.02% wt/vol sodium azide
for 1–2 h, then mounted and frozen onto cryostat mounting
stages using Tris-buffered saline Tissue Freezing Medium
(Triangle Biomedical Sciences, Durham, NC). The tissue was
cut into 6- or 8-␮m thick sections (Leica Kryostat 1720,
Wetzlar, Germany) and picked up on subbed slides (0.5%
wt/vol gelatin ⫹ 0.05% wt/vol chromium potassium sulfate,
single coated). The slides were allowed to air dry on a heated
stage (45°C) and were stained with hematoxylin and eosin Y
(Fisher Scientific) by standard techniques, and coverslips
were applied with Permount (Fisher Scientific). Tissue sections were observed and digital images were acquired with
light microscopy (Leica DM RX, Wetzlar).
Trichrome staining and electron microscopy of cultured
cells. Porcine vas deferens epithelial cells were isolated as
described above and grown to confluency on permeable supports. After verification of epithelial resistance and tissue
responsiveness in the Ussing chamber, inserts were recovered and fixed in 10% BNF overnight at 4°C, followed by
three 15-min rinses in PBSh, and stored in PBSh ⫹ 0.02%
sodium azide. Epithelial cell monolayers were dehydrated in
increasing concentrations of ethanol to absolute, followed by
100% glass-distilled acetone (Electron Microscopy Sciences,
Fort Washington, PA), and embedded in Epon/Araldite (Electron Microscopy Sciences). For light microscopy observations,
embedded tissues were cut into 0.6- to 0.8-␮m semithin
sections (Ultracut E ultramicrotome, Reichert-Jung) using a
diamond knife (Diatome, Fort Washington, PA). Sections
were heat-fixed on glass slides and stained as previously
described (33). For electron microscopy, embedded tissues
were cut into 90- to 95-nm ultrathin sections and picked up
on Formvar-coated copper slot grids (Electron Microscopy
Sciences). The sections were then stained with Reynold’s lead
citrate (44) and uranyl acetate (5% wt/vol in acidic 70%
ethanol) and viewed with an electron microscope at 80 kV
(Philips model EM400, Natick, MA).
Immunohistochemistry. Isolated vas deferens epithelial
cells were grown on permeable supports for 24–48 h and then
fixed in 10% BNF for 1–2 h at room temperature. For immunofluorescence staining, the cells were treated with 5% normal goat serum (GIBCO BRL) in PBSh ⫹ 0.2% vol/vol Triton
X-100 (PBSh-Tx) to block nonspecific staining. Primary antibody (anti-Pan Cytokeratin Clone C-11, mouse monoclonal;
Sigma, St. Louis, MO; anti-ZO-1, rat monoclonal, Chemicon,
Temecula, CA) was diluted 1:200 in PBSh-Tx and applied for
1 h in a humidified chamber at room temperature. The cells
were then rinsed three times for 15 min in PBSh-Tx, and the
secondary antibody (anti-cytokeratin, Texas red dye-conjugated goat anti-mouse IgG, Jackson ImmunoResearch Laboratories, West Grove, PA; anti-ZO-1, FITC-conjugated goat
anti-rat IgG, Chemicon), diluted 1:100 in PBSh-Tx, was applied for 3 h at room temperature. Cells were rinsed in PBSh
as before and then rinsed in distilled water. After drying,
coverslips were applied in antifading solution (0.6% N-propyl
gallate, 70% glycerin in 30 mM Tris buffer). Cells were
observed and digital images captured with light microscopy
(Leica DM RX microscope using 596-nm polarized light).
Digital images of treated and control cells were acquired with
identical numerical settings and prepared for publication in
parallel using CorelDRAW (version 8.2, Corel, Ottawa, Ontario).
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ogy and were monitored for growth and contamination for an
additional 48 h. Bacterial or fungal contamination was observed in ⬍10% of primary epithelial cell isolations. At the
end of the monitoring period, more than one-half of the flasks
contained confluent or near-confluent monolayers. Routine
cell passage of confluent flasks was performed by aspirating
spent media from the flask, rinsing with PBScc, and then
incubating the cells in 1.5 ml of dissociation media [PBScc
containing 2.5% (vol/vol) trypsin and 2.65 mM EDTA] for 2
min, removing the dissociation solution, and allowing the
cells to incubate at 37°C for an additional 4–5 min. Once the
cells had detached from the flask, 5.0 ml of growth medium
were added and the suspension was pipetted 5–10 times to
facilitate disruption of the cell sheets. One milliliter of the
suspension was added to a 25-cm2 flask containing 4 ml of
growth media (passage 1). Subcultures in flasks grew to
confluency within 5–7 days. At the time of passage, permeable supports (1.13 cm2; Snapwell, Costar, Cambridge, MA)
were seeded with three to four drops of the initial suspension.
The basolateral side of the support was bathed in 2.0 ml of
growth medium. Subsequent passages were carried out as
detailed above. Cells in culture flasks and on permeable
supports were fed every other day by removing the spent
media and replacing it with an equal volume of fresh growth
media. Some isolations have retained the ability to form
high-resistance monolayers and continue to be responsive to
forskolin through ⬎20 passages and after being frozen and
thawed. However, the principal focus of this report is to
describe the morphology and electrophysiology of freshly
isolated and cultured vas deferens epithelial cells. Results
from the first and second passage were not significantly
different from one another. Therefore, data from these initial
cultures have been pooled for presentation.
Electrophysiology. Porcine vas deferens epithelial cells cultured on permeable supports were evaluated for transepithelial potential difference (PDte), transepithelial electrical resistance (Rte), basal short-circuit current (Isc; an indicator of
net ion transport), and changes in Isc stimulated by selected
physiological and pharmacological agents using a modified
Ussing chamber (model DCV9, Navicyte, San Diego, CA). For
electrical measurements, cell monolayers were bathed in
Ringer solution maintained at 39°C and continuously bubbled with 5% CO2-95% O2. After PDte was recorded, the
monolayers were clamped to 0 mV, and Isc was measured
continuously with a voltage-clamp apparatus (model 558C,
University of Iowa, Department of Bioengineering, Iowa
City, IA). Data were digitally acquired at 1 Hz with a Macintosh computer (Apple Computer, Cupertino, CA) using an
MP100A-CE interface and Aqknowledge software (ver. 3.2.6,
BIOPAC Systems, Santa Barbara, CA). For anion substitution studies, symmetrical solutions of the following compositions were employed (in mM): Cl⫺-free, 120 NaC2H5O4S, 25
NaHCO3, 3.3 KH2PO4, 0.83 K2HPO4, 1.2 CaSO4, and 1.2
MgSO4; HCO3⫺-free, 120 NaCl, 20 NaC2H5O4S, 10 HEPES,
3.3 KH2PO4, 0.83 K2HPO4, 1.2 CaCl2, and 1.2 MgCl2; Cl⫺and HCO3⫺-free, 140 NaC2H5O4S, 10 HEPES, 3.3 KH2PO4,
0.83 K2HPO4, 1.2 CaSO4, and 1.2 MgSO4. The pH of HCO3⫺free solutions was adjusted to 7.40 with NaOH, and they
were oxygenated with room air during the experiments. Epithelial monolayers were evaluated for ion transport activity
12–17 days after seeding unless otherwise noted.
Tissue preparation for histochemistry. Vas deferens were
acquired and stripped of connective tissue as described
above. Tissues to be used for predigestion observations were
fixed after stripping (before treatment with collagenase/trypsin/EDTA) whereas postdigestion tissues were fixed immediately after epithelial cell isolation. In both cases, tissues were
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Cell proliferation in supplemented media. Proliferation
rates of passage 1 cells were observed for each of several
growth conditions. Freshly isolated cells (passage 0) were
grown to confluency as described above. Cells were dissociated and dispersed, and 24-well plates were seeded at a
concentration of 1.0 ⫻ 105 cells per well. Growth conditions
were as follows: 1) standard growth media, or growth media
supplemented with 2) 1 mM sodium pyruvate, 3) pyruvate
plus 0.2 U/ml insulin, 4) pyruvate plus 0.1 ␮g/ml hydrocortisone, or 5) pyruvate plus insulin plus hydrocortisone. At 24-h
intervals beginning at 24-h postseeding, cells were dissociated, suspended in 100 ␮l of media, and loaded into a hemocytometer for determination of cell density. Cells from three
wells were counted at each time point. A sigmoidal equation
y ⫽ y0 ⫹
a
⫺ 共x ⫺ x0兲
b
in which y0 was the minimal number of cells (constrained to
100,000), a is the maximal change in cell number, x0 was the
time in hours to reach half maximal response, and ⫺1/b
represents the rate of proliferation at xo, was fitted to the
data (SigmaPlot version 6.00; SPSS, Chicago, IL).
Chemical sources. 4,4⬘-Diaminostilbene-2,2⬘-disulfonic
acid (DNDS) was purchased from Acros Organics (Fairlawn,
NJ). ATP was purchased from Boehringer-Mannheim (Indianapolis, IN). Forskolin (Coleus forskohlii) was purchased
from Calbiochem (La Jolla, CA). Glycerin, 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, bis-Tris propane (BTP), bumetanide, carbachol, charybdotoxin, clotrimazole, histamine, hydrocortisone, insulin, isoproterenol, norepinephrine, ouabain, N-propyl gallate, pyruvate, serotonin, sodium azide, sucrose, vasopressin, and
vasoactive intestinal polypeptide (VIP) were purchased from
Sigma. Uranyl acetate was purchased from Tousimis
Research (Rockville, MD). N-[4-methylphenylsulfonyl]-N⬘-[4trifluoromethy-lphenyl]urea (DASU-02) was synthesized de
novo in the laboratory. All other chemicals were of reagent
grade or better and purchased from reputable sources.
Stock solutions of modulators for Ussing chamber experiments. Solutions were prepared as follows: forskolin, 10 mM
in ethanol; carbachol and histamine, 100 mM in H2O; charybdotoxin, 100 ␮M in H2O; isoproterenol and norepinephrine,
10 mM in 1 mM ascorbic acid; serotonin, 50 mM in 1 mM
ascorbic acid; VIP and vasopressin, 1 mM in H2O; amiloride
and ouabain, 10 mM in H2O; bumetanide, 20 mM in ethanol;
DASU-02, 100 mM in dimethyl sulfoxide (DMSO); clotrim-
Fig. 2. Photomicrographs of porcine vas deferens tissues before (A)
and after (B) the cell isolation protocol. A pseudostratified columnar
epithelium composed primarily of principal cells is present before
enzyme exposure. After cell isolation, the remaining cells lining the
lumen are presumed to be basal cells (arrows). Six-micrometer-thick
sections, paraffin embedded, hematoxylin and eosin stained, are
shown. Scale bar ⫽ 50 ␮m.
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azole, 300 mM in DMSO; DNDS, 5 mM in Ringer solution;
ATP, 10 mM in 10 mM BTP. Forskolin, bumetanide, and VIP
were stored at ⫺20°C. Amiloride and vasopressin were stored
at 4°C. All other modulators were freshly dissolved on the
day of the experiment.
Statistical analysis. Numerical data from Ussing chamber
experiments are presented as the arithmetical mean and SE
of the mean using the culture well as the experimental unit.
Where appropriate, Student’s t-test was employed to assess
the likelihood of population differences. A probability of a
type I error of ⬍5% was considered statistically significant.
RESULTS
Morphological and histochemical characterization of
cells isolated from deferent ducts. Deferent ducts were
obtained before and after cell isolation and prepared
for histochemical analysis to identify the origin of the
cells that were isolated for in vitro cell culture. Sections
from intact ducts revealed a lumen that was lined by a
pseudostratified columnar epithelium composed predominantly of principal cells (Fig. 2A). Basal cells were
observed near the basement membrane. After cell isolation, tissue sections exhibited no columnar epithelium, although a layer of what appeared to be basal
cells remained in contact with the basement membrane (Fig. 2B). Thus the cell isolation protocol effectively removed the columnar epithelial cells throughout the deferent duct. The vast majority of these cells
are reported to be principal cells, although a small
proportion of the isolated cells likely includes other
types [e.g., clear cells, dark (pencil) cells, narrow cells,
etc.; 2, 43].
Epithelioid cells were routinely observed in the culture flasks after cell isolation. Photomicrographs of a
typical epithelial cell culture at 24 and 48 h postisolation are presented in Fig. 3. Epithelial cell isolations
contained predominantly small clusters of cells, although individual cells were also observed. Cell clusters attached to the culture substrate and spread out to
reach confluency in the 25-cm2 flask within 72–96 h
after isolation. The cultured vas deferens cells exhibited a cobblestone pattern typical of epithelial cells.
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1⫹e
Fig. 3. Demonstration of in vitro attachment and proliferation of
freshly dissociated porcine vas deferens epithelial cells. Phase-contrast photomicrographs of isolated porcine vas deferens cells in
culture at 24-h postisolation (A) and at near-confluence 48-h postisolation (B) are shown. Light-colored masses in A and B are cell
aggregates that did not fully adhere to the substrate. At confluency
(B) the cells display the cobblestone appearance typical of epithelial
cells. Scale bar ⫽ 100 ␮m.
VAS DEFERENS ION TRANSPORT
although the magnitude was significantly greater (P ⬍
0.001) after 11 days in culture (Fig. 5A). After recording PDte, epithelial cell monolayers were clamped to 0
mV, and Isc was continuously recorded. Basal Isc was
near zero and unaffected (P ⬎ 0.5) by the number of
days in culture (Fig. 5B). Transepithelial resistance
was determined by assessing the effects of a 5-mV
bipolar pulse on Isc and calculating the resistance using Ohm’s law. Epithelial cell monolayers displayed a
high Rte (2,400 ⫾ 300 ⍀ 䡠 cm2) after 5–8 days in culture
that increased (P ⬍ 0.001) and reached a plateau
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Additional morphological studies were completed to
determine whether cultured vas deferens cells exhibited characteristics commonly associated with epithelia. Cells grown in culture on permeable supports exhibited a cuboidal epithelial morphology when
assessed by both light and electron microscopy. A
trichrome-stained section of cultured cells is shown in
Fig. 4A. The cells grew as a continuous cuboidal
pseudostratified layer on the permeable support. Portions of at least five cells are displayed with nuclei at
varying heights. With transmission electron microscopy, cellular and subcellular components are observed
(Fig. 4B). Cultured cells exhibited microvilli and tight
junctions apically, and desmosomes dispersed along
the lateral membranes. Additionally, smooth endoplasmic reticulum and many mitochondria are scattered
throughout the cytoplasm. In separate experiments,
cells were cultured on permeable supports and probed
for anti-ZO-1 and anti-cytokeratin immunoreactivity
(Fig. 4, C and E). Cultured cells exhibited epitopes that
were recognized to be each of these antibodies.
In summary, the results show that pseudostratified
columnar epithelial cells (primarily principal cells) are
removed from the vas deferens by the isolation protocol. These cells attach, disperse, and proliferate in
tissue culture flasks to rapidly reach confluency. Subsequently, cells that are passaged onto permeable supports develop into a cuboidal pseudostratified epithelial monolayer in which the cells exhibit morphological
and cytochemical markers commonly associated with
epithelium.
Electrophysiology. Electrophysiological techniques
provide a stringent test of cellular differentiation and
epithelial function. Experiments were conducted 5–22
days postseeding in modified Ussing chambers to test
for the development of PDte, Rte, and ion transport
activity of the epithelial cells. Cultured cell monolayers
exhibited a lumen negative PDte at all time points
Fig. 4. Porcine vas deferens cells exhibit morphological and biochemical characteristics typical of epithelia. A: brightfield micrograph of
cells exhibiting a pseudostratified cuboidal morphology. Portions of
numerous cells are present, as indicated by their nuclei with (arrow)
or without (arrowhead) nucleoli. Full thickness of the substrate is
shown. Scale bar ⫽ 10 ␮m. A section 0.8-␮m thick embedded in
Epon-Araldite and trichrome stained is shown. B: electron micrograph showing portions of at least 3 cells. Visible are microvilli (mv)
on the apical surface, nucleus (N), numerous mitochondria (arrow),
and desmosomes (arrowhead). Electron-dense junctional complexes
are present near the apical membrane, but are not fully resolved at
this magnification. Scale bar ⫽ 2 ␮m. Inset: higher resolution image
of portions of 2 cells including an apical juctional complex and a
subapical desmosome. Scale bar ⫽ 0.1 ␮M. C: indirect immunofluorescence employing anti-ZO-1 antibody revealed an immunoreactive
pattern outlining the periphery of each epithelial cell. D: no fluorescence was observed when the primary antibody to ZO-1 was omitted
from the protocol. E: indirect immunofluorescence employing antipan cytokeratin antibody (human cytokeratins 4–6, 8, 10, 13, and 18
as well as cross-species reactivity for the same peptides) revealed an
immunoreactive pattern consistent with intermediate filaments
within each epithelial cell. F: no fluorescence was observed when the
primary antibody to cytokeratins was omitted from the protocol. C-F:
porcine vas deferens cells grown on permeable supports viewed on
fos from the apical perspective. Scale bar ⫽ 50 ␮m.
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VAS DEFERENS ION TRANSPORT
of 3,900 ⫾ 300 ⍀ 䡠 cm2 by 11–14 days postseeding
(Fig. 5C).
Forskolin was employed as a stimulant of the cAMP
second messenger cascade to assess the responsiveness
of the cell monolayers. The addition of forskolin (2 ␮M)
to the bathing solution caused a prototypical response
that was characterized by a rapid transient peak in Isc,
followed by a sustained plateau (e.g., see Fig. 8). The
increase in Isc was consistently accompanied by a reduction in Rte suggesting that a conductive pathway
had been activated. Forskolin-stimulated increases in
Isc were routinely observed for periods in excess of 1 h
(not shown). As shown in Fig. 5, B and C, the responsiveness of epithelial monolayers to forskolin was fully
developed at the earliest time point tested. Monolayers
universally responded to forskolin-dependent stimulation of adenylyl cyclase with an increase in Isc that was
paralleled by a reduction in Rte. Results of subsequent
experiments strongly suggest that the increase in Isc is
indicative of Cl⫺ and/or HCO3⫺ secretion.
To determine whether vas deferens epithelial cell
monolayers could respond to physiological stimuli, various neurotransmitters, paracrine agents, and pharmaceuticals were employed as stimulants. As shown in
Fig. 6, vasopressin, norepinephrine, and isoproterenol
caused increases in Isc (as well as decreases in transepithelial resistance) that were qualitatively indistinguishable from a forskolin-stimulated response. Responses to adenosine were also indistinguishable from
that of forskolin (not shown). Responses to the subsequent addition of forskolin were reduced in every case.
VIP, ATP, and histamine were associated with smaller,
yet significant (P ⬍ 0.04) increases in Isc whereas
serotonin and carbachol were without effect (P ⬎ 0.13)
AJP-Renal Physiol • VOL
on basal current. Results are summarized in Fig. 7A
and show that cultured vas deferens epithelial cell
monolayers consistently respond to only selected neurotransmitters. The receptor subtypes and second messenger cascades that modulate these responses remain
to be determined.
Experiments were completed to pharmacologically
test for the presence of selected ion transport proteins.
As shown in Fig. 8A, basolateral addition of ouabain
reversed forskolin-stimulated ion transport, indicating
that Na⫹-K⫹ ATPase plays a crucial role in the response of the monolayer. Bumetanide reduced, but did
not abolish, both forskolin- and norepinephrine-stimulated ion transport (Fig. 8, B and E), suggesting that a
component of the response requires the activity of the
Na⫹-K⫹-2Cl⫺ cotransporter. Importantly, the inhibitory effect of bumetanide was manifested only from the
basolateral side of the monolayer (Fig. 8B), strengthening the conclusion that at least a portion of the Isc
reflects Cl⫺ secretion. Further support of this conclusion is presented in Fig. 8C. DASU-02, an inhibitor of
the CFTR anion channel (38, 46–48), reduced forskolin-stimulated ion transport whereas DNDS (0.5 mM),
an inhibitor of Ca2⫹-activated Cl⫺ channels, was without effect. DNDS has also been reported to inhibit
Na⫹/HCO3⫺ cotransporter (NBC) at greater concentrations (18). Basolateral exposure to DNDS resulted in a
biphasic response that included both inhibition of forskolin-stimulated Isc and in a modest stimulation of Isc
(Fig. 8D). This result suggests that NBC participates
in the response to forskolin. The stimulatory component could reflect stimulation of other conductances
(see DISCUSSION). Amiloride, an inhibitor of epithelial
Na⫹ channels, was without effect on basal Isc (Fig. 8C)
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Fig. 5. Porcine vas deferens epithelial cells cultured on permeable supports differentiate to become an iontransporting epithelium. A: a lumen negative transepithelial potential difference (PDte) was always observed and
was significantly greater 11 or more days postseeding compared with the earliest time point tested (P ⬍ 0.001). B:
basal short-circuit current (Isc) was ⬍0.6 ␮A/cm2 throughout and no significant developmental changes were
observed. C: a relatively high basal transepithelial resistance (Rte) was seen at all time points and was significantly
greater 11 or more days postseeding (P ⬍ 0.001). Forskolin-stimulated changes in Isc [peak and sustained (B); e.g.,
Fig. 8] and Rte (C) were invariably observed and exhibited no significant developmental changes in magnitude.
Results are summarized from 24–26 wells per condition, representing the first and second passage of isolations
from 12 ducts from 8 animals.
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VAS DEFERENS ION TRANSPORT
and had no effect on either ATP-stimulated or forskolin-stimulated Isc (data not shown). Amiloride’s lack of
effect appears to directly contradict an earlier report
employing ovine vas deferens (4). The basis of this
difference will be addressed in the DISCUSSION.
Basolateral K⫹ conductance(s) are responsible for
maintaining membrane potential during ongoing ion
transport. Ba2⫹, a nonselective K⫹ channel blocker,
rapidly and significantly inhibited forskolin-stimulated Isc; inhibition was concentration dependent, and
Fig. 7. A: peak and sustained changes
in Isc (measured as a change from baseline) to various agonists. Adenosine,
isoproterenol, norepinephrine, vasopressin, ATP, and histamine were associated with significant transient (P ⬍
0.003) and sustained (P ⬍ 0.04) increases in Isc. VIP, carbachol, and serotonin produced no consistent effect
on Isc. Results from 4–21 experiments for
each condition are summarized. B: inhibitors of various ion transport proteins were
evaluated. Amiloride (ENaC inhibitor)
had no effect on basal Isc (P ⬎ 0.8). When
introduced after cAMP-dependent stimulation with forskolin, N-[4-methylphenylsulfonyl]-N⬘-[4-trifluoromethy-lphenyl]rea
(DASU-02; CFTR inhibitor) and bumetanide (Bum; Na⫹-K⫹-2Cl⫺ cotransporter
inhibitor) significantly reduced Isc (P ⬍
0.001; ‚Isc ⫽ 1 to 3 ␮A/cm2). Results from
20–26 experiments for each condition are
summarized.
AJP-Renal Physiol • VOL
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Fig. 6. Cultured vas deferens epithelial cell monolayers selectively responded to neurotransmitters, paracrine
agents, and pharmaceuticals with
changes in ion transport, as indicated by
changes in Isc. All epithelial monolayers
exhibited basal currents of ⬍1 ␮A/cm2
(the dotted line represents the 0-current
level). Periodic deflections in the Isc trace
result from a 5-mV bipolar pulse (Rte ⱖ
2,000 ⍀䡠cm2). Norepinephrine (10 ␮M),
vasopressin (1 ␮M), isoproterenol (10
␮M), and forskolin (For; 2 ␮M) caused an
increase in Isc (and concomitant decrease
in monolayer resistance) that was characterized by a transient peak of 5–10
␮A/cm2 followed by a plateau phase of 3
to 6 ␮A/cm2. Other compounds were
used at the following concentrations: vasoactive intestinal polypeptide (VIP), 1
␮M; ATP, 10 ␮M; serotonin, carbachol,
and histamine, 100 ␮M. In these experiments, all compounds were simultaneously added to both the apical and
basolateral compartments. Results are
typical of 4–21 observations for each agonist.
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VAS DEFERENS ION TRANSPORT
⬎60% inhibition was observed at the highest concentration tested (3 mM, Fig. 9, A and E). Clotrimazole
and charybdotoxin were employed as selective inhibitors of an intermediate-conductance K⫹ channel, IK.
Clotrimazole significantly reduced forskolin-stimulated Isc at the highest concentration tested (30 ␮M,
Fig. 9, B and E) but was without effect at 3 ␮M (data
not shown), a concentration reported to inhibit IK in
other secretory epithelia (17, 18). A more potent and
selective inhibitor of IK, charybdotoxin, was employed
to further test for the presence of this conductance and
was found to be without effect (Fig. 9, C and E). Subsequently, the chromanol 293B, a blocker of the cAMPdependent, small-K⫹ conductance (IsK), was employed to
test for a contribution to forskolin-stimulated ion transport from this conductance. As shown in Fig. 9, D and E,
293B was without effect. Taken together, these results
indicate that a basolateral K⫹ conductance is involved in
ongoing ion transport, but that it is neither IK nor IsK.
Further experiments are required to determine the idenAJP-Renal Physiol • VOL
tity of this Ba2⫹- and clotrimazole-sensitive K⫹ conductance(s).
Anion substitution experiments were conducted to
further characterize the ionic basis of the Isc. As shown
in Fig. 10, a maximal response to ATP and bumetanide
requires the presence of Cl⫺ in the bathing media.
Similarly, the forskolin-stimulated transient increase
in ion transport required Cl⫺ to be present. The absence of HCO3⫺ did not adversely affect these responses. Alternatively, the prolonged effect of forskolin
was dependent on HCO3⫺ in the bathing media, and, in
the absence of Cl⫺, was bumetanide insensitive. In the
absence of Cl⫺ and HCO3⫺, no sustained effects of ATP
or forskolin are observed. The transient peak effects of
ATP and forskolin on Isc, however, are significantly
different from zero and might reflect the loss of anions
(i.e., trapped Cl⫺ and HCO3⫺) from epithelial cell cytoplasm into the apical media. The sustained effect of
forskolin (0.51 ⫾ 0.23 ␮A) is not significantly different
from zero in these conditions. These results suggest
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Fig. 8. Ion transport by cultured vas deferens epithelial cell monolayers is selectively reduced by ion transport
inhibitors. All epithelial monolayers exhibited basal currents of ⬍1 ␮A/cm2 (the dotted line represents the 0current level). Periodic deflections in the Isc trace result from a 5-mV bipolar pulse (Rte ⱖ 1,500 ⍀ 䡠 cm2).
Forskolin-stimulated (2 ␮M, symmetrical) ion transport was reduced by basolateral ouabain (10 ␮M; A), basolateral bumetanide (Bum, 20 ␮M; Ap, apical; BL, basolateral; B), and DASU-02 (300 ␮M; C), but not by apical
amiloride (10 ␮M), apical Bum, or by symmetrical 4,4⬘-diaminostilbene-2,2⬘-disulfonic acid (DNDS; 0.5 mM; C).
Concentration-dependent inhibition by basolateral DNDS was observed (D). Norepinephrine-stimulated ion
transport was also reduced by Bum (E). Results are typical of 4–26 observations for each inhibitor.
VAS DEFERENS ION TRANSPORT
F565
that at least two sustained ion transport pathways are
stimulated by forskolin. The first requires the presence
of Cl⫺ and is bumetanide sensitive, and the second
requires HCO3⫺ and is bumetanide insensitive.
Taken together, these results demonstrate that cultured epithelial cell monolayers develop an electrically
tight barrier, actively transport ions to develop a lumen-negative potential in basal conditions, and behave
as functional syncichum that acutely and selectively
responds to neurotransmitters and peptide hormones,
with changes in ion transport that would be expected to
change the composition and/or volume of luminal fluid.
Optimization of culture conditions. A relatively simple medium has proven itself extremely effective for
the initial culture of vas deferens epithelial cells. However, we sought to determine whether commonly employed supplements would affect cell proliferation.
Thus cells isolated from individual vas deferens were
seeded onto 24-well plates and cultured in selected
supplements, and, at specified intervals, cells were
dissociated and counted (Fig. 11). The fit of a sigmoidal
function to the data suggests that insulin supplementation significantly decreases the doubling time during
log growth (control, 43.9 ⫾ 9.5 h/generation vs. pyruAJP-Renal Physiol • VOL
vate ⫹ insulin, 20.0 ⫾ 3.3 h/generation) and increases
the number of cells present at growth climax (control,
1.5 ⫾ 0.3 ⫻ 106 cells/well vs. pyruvate ⫹ insulin, 5.1 ⫾
0.3 ⫻ 106 cells/well). Pyruvate and pyruvate ⫹ hydrocortisone differed little from control. Doubling time and
maximal number of cells in the presence of pyruvate ⫹
insulin ⫹ hydrocortisone was similar to that observed
in the presence of pyruvate and insulin. Lag time was
not affected by media supplementation. Additional
studies are required to determine whether the proliferative effects of insulin would be duplicated with cells
cultured on permeable supports and to determine
whether ion transport by epithelial monolayers would
be affected.
DISCUSSION
In this report, we define techniques to routinely
isolate and culture vas deferens epithelial cells in sufficient quantities to conduct numerous tightly paired
experiments to better understand the role of vas deferens epithelium in fertility. Results show that a population of columnar epithelial cells, predominantly principal cells, are isolated from the duct. These isolated
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Fig. 9. Ion transport by cultured vas deferens epithelial cell monolayers is selectively reduced by K⫹ channel
blockers. All epithelial monolayers exhibited basal currents of ⬍1 ␮A/cm2 (the dotted line represents the 0-current
level). Periodic deflections in the Isc trace result from a 5-mV bipolar pulse (Rte ⱖ 1,000 ⍀ 䡠 cm2). Forskolinstimulated (2 ␮M, symmetrical) ion transport was reduced by basolateral BaCl2 (Ba2⫹, 3 mM; A), symmetrical
clotrimazole (Clot, 30 ␮M; B) but was unaffected by charybdotoxin (CTX, 0.1 ␮M; C) or 293B (30 ␮M; D). Results
from 4–7 observations for each compound are summarized in E.
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VAS DEFERENS ION TRANSPORT
cells attach to standard tissue culture flasks and
rapidly form a monolayer with epithelioid morphology. When passed to permeable supports, the cells
express both ZO-1 and cytokeratin, biochemical
markers of epithelial cells, and exhibit epithelial
morphology when assessed by either light or electron
microscopy. Results from electrophysiological experiments demonstrate that cultured cells exhibit functional characteristics of epithelial tissues, including a
basal transepithelial potential difference, a relatively
high transepithelial resistance, and the ability to actively transport ions. Most importantly, the results
demonstrate that cultured porcine vas deferens epithelial cell monolayers provide differential responses to
physiological and pharmacological stimuli. Thus a system has been developed to identify and study physiological mechanisms that regulate the luminal environment of the distal duct through which sperm must pass.
The results provide a framework from which to propose a basic cellular model of ion transport (Fig. 12).
Included in this model are components for which
highly selective inhibitors are available: the ouabaininhibitable Na⫹-K⫹-ATPase and the bumetanide-inhibitable Na⫹-K⫹-2Cl⫺ cotransporter. DASU-02 has
AJP-Renal Physiol • VOL
been shown to inhibit CFTR-mediated anion transport
in other systems (38, 46–48), and there is good reason
to expect CFTR to be functional in this epithelium
because the vas deferens is severely affected in virtually all CF patients. Thus the presence and functional
activity of these three components in vas deferens
epithelia are reasonably assured.
A basolateral K⫹ conductance is included in Fig. 12.
Such a pathway is required to maintain the membrane
potential in the presence of other ion transport mechanisms. Thus K⫹ entering the cell via the bumetanidesensitive cotransporter can be recycled to the basolateral compartment and can allow for electrogenic anion
secretion at the apical membrane. The identity of the
conductance remains to be determined. Blocker pharmacology is consistent with neither IK nor IsK because
of the lack of inhibition by charybdotoxin and 293B,
respectively. However, inhibition by high concentrations of clotrimazole suggests some similarities with
IK, and stimulation of Isc by DNDS (Fig. 8D) suggests
that some similarities to IsK may also be present (1,
49). Additional experiments are required to determine
whether IK, IsK, and/or other K⫹ channels contribute
to vas deferens ion transport.
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Fig. 10. Agonist-induced changes in Isc are dependent on the anion composition of the bathing solution. Typical
responses to ATP (10 ␮M, apical only), forskolin (2 ␮M, symmetrical), and Bum (20 ␮M, basolateral only) in Ringer
solutions of the indicated composition are shown in A-D. Data from this and 6 similar experiments are summarized
in E. Responses to ATP, Bum, and the peak response to forskolin are significantly reduced (P ⬍ 0.007) by the
absence of Cl⫺. The sustained (Sust) effect of forskolin is significantly reduced in the absence of HCO3⫺ (P ⬍ 0.001).
All epithelial monolayers exhibited basal currents of ⬍1 ␮A/cm2 (the dotted line represents the 0-current level).
Periodic deflections in the Isc trace result from a 5-mV bipolar pulse (Basal Rte ⱖ 3,500 ⍀cm2).
VAS DEFERENS ION TRANSPORT
F567
Anion substitution studies indicate that both Cl⫺and HCO3⫺-dependent ion transport processes are
stimulated by forskolin. Activation of CFTR could account for this response, although other mechanisms
must also be invoked. Two components of the response,
transient and sustained, exhibited differential sensitivity to the absence of Cl⫺. Transient effects require
the presence of Cl⫺ whereas sustained effects require
that both anions are present. To partially account for
these results, we propose that Cl⫺ enters the cell at the
basolateral membrane through a bumetanide-sensitive
cotransporter and exits cells across the apical membrane through CFTR. An additional ion transport
mechanism is required to account for the HCO3⫺-dependent Isc observed in the absence of Cl⫺. HCO3⫺ may
enter the cell at the basolateral membrane via NBC
(inhibited by DNDS) and leave the cell through CFTR
or perhaps another pathway as indicated. Alternatively, the forskolin-stimulated current in the absence
of Cl⫺ could be cation absorption. However, the lack of
an amiloride effect on these cells suggests that Na⫹ is
not being absorbed. There are other possible interpretations of these data, including the presence of an
anion exchanger or the placement of an alternative
NBC (i.e., NBC3) in the apical membrane (42). However, definitive experiments to separate these possibilities and elucidate the identity and location of the
transport proteins are beyond the scope of the present
publication. Regardless, the sustained current in normal Ringer solution appears to be a combination of Cl⫺
and HCO3⫺ secretion. This conclusion would also account for the rather modest effect of bumetanide in
Fig. 12. Proposed model of vas deferens epithelial cells. Ion pumps,
cotransporters, and channels with their selective inhibitors are included. Neurotransmitters that significantly affect ion transport are
included with the proposed location (apical or basolateral) of their
receptors (R).
AJP-Renal Physiol • VOL
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Fig. 11. Quantification of vas deferens epithelial cell proliferation in
supplemented growth media. Cells were seeded onto 24-well plates
and bathed in growth media containing various supplements (C,
control media; P, 1 mM sodium pyruvate; I, 0.2 U/ml bovine insulin;
H, 0.1 ␮g/ml hydrocortisone) alone or in combinations as indicated.
At regular intervals, cells were trypsin dissociated from selected
wells in triplicate and counted on a hemocytometer. Symbols represent the average number of cells present in each well for each growth
condition and time point. Lines represent the best fit of a 4-parameter sigmoidal equation to the data points. Results are typical of 2
repetitions of the protocol.
control conditions, but its virtually complete inhibition
in the absence of HCO3⫺.
Forskolin, a compound known to activate adenylyl
cyclase, consistently increases Isc, indicating that
transport mechanisms (likely including CFTR) are
modulated by this second-messenger cascade. Ion substitution studies indicated that forskolin stimulated
more than one ion transport pathway. Numerous neurotransmitters were also shown to stimulate epithelial
ion transport, with their receptors being discretely
localized to either the apical or basolateral membrane.
Depending on the neurotransmitter, both transient
and sustained responses are observed, suggesting that
unique pathways could be independently modulated by
the various ligands. These results are particularly intriguing in light of the fact that vas deferens muscle
contraction is particularly sensitive to sympathetic
stimulation (i.e., norepinephrine) and that numerous
transmitters (especially ATP) are coreleased with norepinephrine (52). We have demonstrated that these
and other neurotransmitters acutely modulate epithelial function in this portion of the deferent duct, as
well. More recent studies indicate that adenosine stimulates DASU-02-sensitive anion secretion across
freshly excised vas deferens (45), providing evidence
that the cultured cell system mimics the native system
in this regard.
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VAS DEFERENS ION TRANSPORT
1
Inhibition by higher concentrations of DNDS at the basolateral
membrane might indicate that a Cl⫺ conductance is present at the
basolateral membrane. However, such a Cl⫺ conductance would not
be expected to contribute to the apical secretion of anions.
AJP-Renal Physiol • VOL
lin RW and Schultz BD, personal observations). Thus
different culture conditions clearly account for some of
the differences in results between this study and previously published reports. Differences in species and
the stage of sexual maturation likely contribute to
distinct outcomes as well.
The present results support and extend earlier observations in which membrane patch-voltage clamp
techniques were used to identify ion conductances in
vas deferens epithelial cells. Two Cl⫺ channels were
described in human vas deferens epithelial cells that
might contribute to electrolyte and fluid secretion (41).
The smaller of the two channels was activated by
forskolin and exhibited a conductance that might be
expected for CFTR. Additionally, an apical Ca2⫹-activated K⫹ channel was observed (50). These observations show that mechanisms are present that might
support epithelial anion or cation transport. The
present results suggest that forskolin stimulates anion
secretion across this epithelia via apical Cl⫺ channels
(CFTR) that are electrically coupled to K⫹ conductances in the basolateral membrane. A HCO3⫺-dependent component was also observed. This could indicate
that HCO3⫺ secretion accounts for a portion of the Isc.
The excurrent duct of the rat is probably the most
widely studied of any species and thus the best characterized. Breton, Brown, and co-workers (6, 7, 10)
have proposed that the epididymis and, to a much
lesser extent, the vas deferens is an acid-secreting
and/or bicarbonate-absorbing epithelium. In stark contrast, Wong and co-workers (34, 53) propose a mechanism for cAMP-stimulated Cl⫺ and HCO3⫺ secretion in
rat epididymis. Our results from the porcine vas deferens support and extend observations reported by Wong
et al. (54), in that we observe inhibition of cAMPstimulated ion transport by DASU-02, ouabain, and
bumetanide. Similarly, we observed that ATP (apical)
and norepinephrine (basolateral) stimulated anion secretion. The apparently conflicting results can be reconciled by noting that in one case [Breton et. al. (6, 7,
10)] basal ion transport in freshly excised tissues was
assessed, whereas in the other cases [Wong et. al. (34,
54) and the present study] stimulated ion transport
across cultured epithelial cell monolayers was evaluated. Previous reports suggest that, in the absence of
stimulation, the epididymis and the proximal vas deferens establishes an acid environment for the storage
of quiescent sperm. Our results suggest that sympathetic stimulation of the vas deferens contributes to an
alkalization of the lumen that could occur during the
period of arousal before ejaculation. If one assumes a
duct diameter of 2 mm, a length of 30 cm, and a
stimulated current of 5 ␮A with bicarbonate secretion
accounting for 80% of the current, one then calculates
that during 15 min of preejaculatory stimulation, the
duct might secrete as much as 0.7 ⫻ 10⫺6 base equivalents into a volume of ⬍1 ml. This magnitude of base
secretion could raise the luminal pH substantially and
contribute to the timely activation of sperm (12).
Congenital bilateral absence of the vas deferens
(CBAVD) accounts for a significant proportion of male
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Ion channels present in vas deferens epithelial membranes remain to be fully determined. Inhibition by
DASU-02 and the association of CBAVD with CF
strongly suggest that CFTR participates in the response. However, we previously reported that
DASU-02 inhibits CFTR with an IC50 of ⬃10 ␮M (47).
Thus the basis of the Isc remaining in the presence of
100 ␮M DASU-02 is not likely mediated by CFTR.
Neither Cd2⫹ (300 ␮M; an inhibitor of ClC2; data not
shown) nor DNDS (500 ␮M; an inhibitor of Ca2⫹activated Cl⫺ channels and outwardly rectifying Cl⫺
channels) had any effect on forskolin-stimulated Isc,
suggesting that these conductances cannot account for
the forskolin-stimulated ion transport.1 Given that a
major component of the Isc is Cl⫺ independent, these
results suggest that a novel HCO3⫺-conductive pathway
might be present. Certainly, additional experiments
are required to test this hypothesis.
Stimulation of ion transport was consistently accompanied by a reduction in Rte. The simplest interpretation of these data is that a conductive pathway(s) is
activated by stimulation. All measurements of ion flux
were made in short-circuited conditions such that
changes in Isc reflect only active transcellular transport
that includes the activation of conductive pathways in
the cell membranes (e.g., CFTR). Inhibition by
DASU-02 was accompanied by an increase in Rte that
further supports the presence of a conductive pathway
in the activated cell monolayers. However, stimulants
of ion transport have also been reported to modulate
the resistance or ion selectivity of the paracellular
pathway in leaky epithelia (5, 32). Similar effects of
forskolin on the paracellular pathway of the relatively
high-resistance porcine vas deferens remain a possibility but are not directly addressed by the present data.
The procedure that we describe for the culture of
adult porcine vas deferens epithelial cells is less demanding than methods reported for the primary culture of either fetal human (22) or neonatal ovine (4) vas
deferens epithelial cells. The collagenase technique for
cell isolation provides a starting population of cells
that is highly enriched for epithelial cells and includes
extremely few fibroblasts. The growth media employed
for porcine cells was simpler than media employed for
either the ovine or human cells. Harris et al. (22),
reported that insulin, cholera toxin, and hydrocortisone were “essential” for maintenance of cell morphology in these systems. These supplements were not
essential for the culture of porcine cells in the present
study, although insulin was shown to dramatically
enhance proliferation rate. As previously stated, additional studies are required to identify the effects of
these supplements on ion transport. Additionally, we
have observed that chronic treatment with hydrocortisone induces the expression of an amiloride-sensitive
current in basal conditions (i.e., ENaC expression; Car-
VAS DEFERENS ION TRANSPORT
The authors thank Steve Becker for tissue procurement, Dr. Mark
Weiss, Maureen Phillips, Christopher Schmidt, James Broughman,
and Rebecca Quesnell for technical assistance, Ginger Biesenthal,
Pam Say and Bonnie Thompson for clerical support, and Mary Jane
Hayes, Don Hayes and Amy Carpenter for ongoing encouragement.
This research was supported by the Cystic Fibrosis Foundation
(SCHULT99P0) and the Kansas State University College of Veterinary Medicine Dean’s Research Fund (contribution no. 01–276-J
from the Kansas Agricultural Experiment Station).
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infertility (19, 20, 39). Men affected by CF, a disease of
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39.
VAS DEFERENS ION TRANSPORT