Functional polarity of
Na+/H+
and
Cl
/HCO
3
exchangers in a rat cholangiocyte cell line
Carlo
Spirlì1,2,
Anna
Granato1,
Àkos
Zsembery1,
Franca
Anglani1,
Lajos
Okolicsànyi2,
Nicholas
F.
LaRusso3,
Gaetano
Crepaldi1, and
Mario
Strazzabosco1
1 Institute of Internal
Medicine, University of Padova, 35100 Padova;
2 Chair of Gastroenterology,
University of Parma, 43100 Parma, Italy; and
3 Mayo Clinic, Rochester,
Minnesota 55905
 |
ABSTRACT |
Intrahepatic
bile duct cells (cholangiocytes) play an important role in the
secretion and alkalinization of bile. Both
Na+/H+
exchange (NHE) and
Cl
/HCO
3
exchange (AE) contribute to these functions, but their functional
distribution between the apical and basolateral membrane domains
remains speculative. We have addressed this issue in a normal rat
cholangiocyte cell line (NRC-1), which maintains a polarized
distribution of membrane markers. Gene expression of AE and NHE
isoforms was studied by RT-PCR. For functional studies, cells were
placed in a chamber that allowed separate perfusion of the apical and
basolateral aspect of the epithelial sheet; intracellular pH
(pHi) was measured by
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein microfluorometry. In
HCO
3-CO2free
medium and in the presence of apical amiloride,
pHi recovery from an acid load was
Na+ dependent and was inhibited by
basolateral amiloride and by HOE-642 (10 µM), consistent with
basolateral localization of the NHE1 isoform, which had clearly
expressed mRNA. Apical Na+
readmission induced a slow pHi
recovery that was inhibited by apical administration of 1 mM HOE-642 or
amiloride. Among the apical NHE isoforms,
NHE2 but not
NHE3 gene expression was detected. The
AE1 gene was not
expressed, but two different variants of AE2 mRNAs (AE2a and AE2b) were
detected; pHi experiments
disclosed AE activities at both sides of the membrane, but only apical
AE was activated by cAMP. In conclusion, these studies provide the first functional description of acid-base transporters in a polarized cholangiocyte cell line. NHE1, NHE2, AE2a, and AE2b isoforms are expressed and show different membrane polarity, functional properties, and sensitivity to inhibitors. These observations add a considerable level of complexity to current models of electrolyte transport in cholangiocytes.
intracellular pH; reverse transcription-polymerase chain reaction
 |
INTRODUCTION |
AFTER ITS SECRETION BY hepatocytes, bile flows through
a progressively merging network of conduits lined by epithelial cells (cholangiocytes). Cholangiocytes modulate bile flow and alkalinity by
secreting and reabsorbing fluid and electrolytes, mainly
Cl
and
HCO
3 (32), following stimulation with secretin and other hormones modulating intracellular concentrations of cAMP.
A number of ion channels and acid-base carriers have been functionally
identified in the biliary epithelium (32). Among the acid-base
carriers,
Na+/H+
exchange (NHE) and
Na+-HCO
3
symport mediate acid extrusion from the cells, whereas
Na+-independent
Cl
/HCO
3
(anion) exchange (AE) functions as an acid loader. Current models for
cholangiocyte acid-base transport propose that NHE (together with
carbonic anhydrase) and
Na+-HCO
3
symport increase intracellular HCO
3 concentration, whereas AE mediates
HCO
3 efflux. In concert with the
cystic fibrosis transmembrane conductance regulator (CFTR)
Cl
channel, AE is
responsible for cholangiocyte HCO
3 secretion induced by cAMP and secretin (3, 32).
Although epithelial cell function is strictly dependent on the
polarized distribution of ion carriers (13), the above model is derived
from studies in unpolarized cells in which vectorial transport
properties of normal cholangiocytes are lost or from polarized duct
preparations in which the apical membrane is not accessible. Thus the
allocation of acid-base transporters to the apical or basolateral
plasma membrane domains and their putative physiological role remains
speculative. In addition, three isoforms of the
Cl
/HCO
3
exchanger (AE1, AE2, AE3) (27) and four isoforms of the
Na+/H+
exchanger (NHE1, NHE2, NHE3, NHE4) (37) have been cloned in different
tissues. These isoforms differ in terms of functional properties,
sensitivity to inhibitors, regulatory mechanisms, tissue distribution,
and membrane polarity (1, 23, 27, 37, 45). NHE1 and AE1 are involved in
homeostatic functions such as intracellular pH
(pHi) regulation and cell volume
control. AE2 and NHE2 and NHE3 are involved in
HCO
3 secretion and
Na+ reabsorption, respectively.
Using a normal rat cholangiocyte cell line (NRC-1) that, when grown on
semipermeable membrane inserts, maintains differentiated phenotypes and
a polarized distribution of membrane markers (39), we studied the
functional membrane distribution of the different Na+/H+
exchanger and
Cl
/HCO
3
exchanger isoforms expressed in the intrahepatic biliary epithelium.
 |
MATERIALS AND METHODS |
Chemicals.
HEPES, amiloride, DMSO, nigericin, DIDS,
D-gluconolactone, sodium
gluconate, potassium gluconate, calcium gluconate, gluconic acid,
choline chloride, choline bicarbonate, ammonium bicarbonate, sodium
propionate, epidermal growth factor, dexamethasone, triiodothyronine, EDTA, high-activity collagenase, forskolin,
N6,2'-O-dibutyryl-cAMP
(DBcAMP), and IBMX were purchased from Sigma Chemical (Milano, Italy).
2',7'-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-AM was purchased from Molecular Probes (Eugene, OR). Culture medium DMEM, Ham's F-12, fetal bovine serum, MEM nonessential amino
acids solution, glyceryl monostearate, chemically defined lipid
concentrate, MEM vitamin solutions, trypsin inhibitor soybean, penicillin-streptomycin, gentamicin, trypsin-EDTA, and glutamine were
purchased from GIBCO (Grand Island, NY). NuSerum and bovine pituitary
extract were from Beckton Dickinson (Milano, Italy). Membrane inserts
were purchased from Nunc (Mascia Brunelli, Milano, Italy); RNAzol B
solution from Biotex (Milano, Italy); Maloney murine leukemia virus
(MMLV) RT was from Perkin-Elmer (Milano, Italy). Qiagen QIAquick PCR
purification kit and Qiagen QIAquick gel extraction kit were purchased
from Qiagen (Ilden, Germany). 4-Isopropyl-3-methylsulfonylbenzoyl
guanidine methanesulfonate (HOE-642) was a kind gift from Drs. A. Weichert and H. J. Lang (Hoechst Marion Russel, Frankfurt/Main, Germany).
Cell culture.
NRC-1 cells were routinely grown on the top of rat tail collagen in
culture flasks (Corning, NY) in DMEM-Ham's F-12 medium as described by
Vroman and LaRusso (39). Experiments were performed in cells cultured
for at least 1 wk after confluence was reached over collagen-coated
semipermeable membrane inserts (Nunc Anophore, 0.2 µm pore-size). As
described previously (39), these culture conditions allow the
establishment of confluent monolayers with apical microvilli and
polarized distribution of phenotypic and functional markers.
Establishment of a confluent monolayer with competent tight junction
was routinely checked by measuring transepithelial resistance and
membrane potential difference (Millicell ERS system). In cells used for
functional studies, transepithelial resistance was above 800
· cm2 and
membrane potential difference was
6.77 ± 1.5 mV
(n = 87). (Values obtained with
collagen-coated inserts alone were subtracted from values recorded with
monolayers.) In preliminary studies, we showed that the percentage of
experiments in which apical recovery could be recorded increased from
33% at day 3 to 100% at
day 8. Thus, to reduce variability due
to culture conditions, cells were studied 8 days postconfluency.
pHi measurement.
pHi was measured with the
fluorescent pHi indicator BCECF
(33). Cells were incubated with the cell permeant tetraacetoxymethyl ester BCECF-AM (12 µmol/l) for 30 min at 37°C followed by a
10-min wash in BCECF-free medium. NRC-1 cell membrane inserts
containing monolayers of proper transepithelial resistance (see above)
were transferred into a thermostated (37°C) perfusion chamber
placed on the stage of a Nikon (Galileo, Siscam, Florence, Italy)
inverted microscope. The chamber was modified to allow separate
perfusion of the apical vs. basolateral aspects of the inserts. HEPES
solutions were in the nominal absence of
HCO
3; in HEPES experiments, the cells
were equilibrated for 50 min in nominally HCO
3-free HEPES before starting the
experiments. In experiments with
HCO
3-CO2-buffered
Ringer medium, solutions were continuously gassed with 5%
CO2-95%
O2; perfusion tubes were made with
CO2-impermeant materials. The
equipment and procedures were essentially as described previously (33). Briefly, the microscope was connected to an SPEX-AR-CM microsystem (Spex Industries, Edison, NJ, or ISA Instruments, Milan, Italy) equipped with a 150 W xenon lamp, and the sample was excited at 495 and
440 nm. Emitted light was then captured by a Nikon ×40 Achromat
LWD with a 1.3 numerical aperture objective and read by a photon
counting photometer. Cell autofluorescence was not higher than
background values of collagen-coated inserts, which, at the end of each
experiment, was subtracted from fluorescence readings.
Signal-to-background ratio at 440 nm was ~40:1. The 495 nm-to-440 nm
fluorescence ratio data were converted to
pHi values using a calibration
curve generated at the end of each experiment by exposing cells
to the
K+-H+
ionophore nigericin (12 µmol/l) in a
Na+-free medium containing a high
K+ concentration (135 mM) and
buffered at three different pHi
values (6.8, 7.2, and 7.6) (34).
Cellular intrinsic buffering power
(
i) was determined at
different pHi values by exposing
cells to various stepwise decreasing concentrations of a permeant weak
base (NH4Cl) as described (7, 34,
44). To exclude all transport systems able to counterregulate pHi, experiments were performed in
the absence of Na+ and
HCO
3. The
i values measured from
seven experiments were pooled and correlated to the
respective pHi values using a
best-fit program (Graph-Pad, Biosoft, Cambridge, UK). Buffering power
data are shown in Fig. 1. Similar
relationships between
i and
pHi have also been reported in
previous studies in isolated rat cholangiocytes (3, 34). The total
intracellular buffering power
(
tot) in the presence of the
open buffering system (H2CO3-CO2)
was then calculated from
i as
tot =
i + 2.302 × [HCO
3]i,
where intracellular HCO
3 concentration
([HCO
3]i)
is derived from the measured pHi
with the Henderson-Hasselbach equation. Rates of pHi changes in the alkaline or
acidic directions
(
pHi/
t)
were measured by hand drawing a tangent from the experimental plot. Transmembrane H+ fluxes
(JH+)
were calculated from
i and
pHi/
t
as
JH+ =
i ×
pHi/
t
(6, 33).

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Fig. 1.
pH dependence of intrinsic buffering power
( i) in NRC-1 cells ( ). An
inverse relationship between i
and intracellular pH (pHi) is
clearly shown [best fit gave the following polynomial curve:
4,178 + ( 1,108)x + 73.69x2].
Curve with filled triangles describes the calculated total
buffering power [polynomial curve: 6,787 + ( 1,108)x + 134x2]. See
text for methodological details.
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|
The composition of perfusion buffers (all in mmol/l) used for
pHi studies was essentially as
described (6, 33). HEPES-buffered Ringer solution contained 135 NaCl,
4.7 KCl, 1.2 KH2PO4,
1 MgSO4, 1.5 CaCl2, 10 HEPES, 5 glucose, and 1 sodium pyruvate and was titrated to pH 7.4 with NaOH. In
NaHCO3-CO2-buffered
Ringer solution (KRB), NaCl was 115 mmol/l and 25 mmol/l
NaHCO3 substituted for HEPES. In
Cl
-free KRB, equimolar
gluconate substituted for
Cl
. In
Na+-free KRB and HEPES, equimolar
choline substituted for Na+. In
KRB and HEPES used to acid load cells, 30 mmol/l
NH4Cl substituted for equal
amounts of NaCl. In propionate KRB, 50 mmol/l sodium propionate
substituted for 50 mmol/l NaCl. BCECF was prepared as 1 mmol/l stock
solution dissolved in DMSO. Amiloride was dissolved in DMSO and then
added to the different solutions at the desired concentration, whereas
nigericin was solubilized in ethanol. HOE-642 was solubilized directly
into the solutions at the desired concentrations.
NHE and AE gene expression.
NHE and AE gene expression was assessed by RT-PCR. Total RNA from NRC-1
cells and rat kidney (used as positive control) (17, 27, 37) was
isolated using the RNAzol B solution (Biotex) according to
manufacturer's instructions on the basis of the guanidinium thiocyanate-phenol chloroform method (8). The quantity of total RNA was
measured by spectrophotometry, and 1 µg was electrophoresed on 2%
NuSieve 3:1 agarose gel with ethidium bromide to check the RNA integrity.
Total RNA (1 µg) was reverse transcribed with MMLV RT (2.5 U/µl) in
a final volume of 20 µl containing buffer (500 mM KCl and 100 mM
Tris · HCl, pH 8.3),
MgCl2 (5 mM), dNTPs (1 mM), random examers (2.5 µM), and RNase inhibitor (1 U/µl). cDNA synthesis was performed in a thermalcycler (M. J. Research, M-Medical, Firenze, Italy); tubes were incubated for
10 min at room temperature and then for 30 min at 42°C. At the end
of the incubation period, RT was inactivated by heating at 99°C for
5 min.
To examine the expression of AE and NHE isoforms, PCR was
performed with the specific primers reported in Table
1. To increase the specificity and the
efficiency of the PCR reaction, the hot start procedure
was applied by using a Taq-specific
antibody (Clontech). Two units of Taq
DNA polymerase from a freshly prepared 28:1 mixture of
Taq antibody and
Taq polymerase were added to a final
volume of 50 µl. For AE isoforms, the thermalcycler profile for AE1
consisted of an initial denaturation at 95°C per 5 min, followed by
35 cycles, with denaturation at 94°C for 1 min, primer annealing at
55°C, and primer extension at 72°C for 1 min. For the
AE2 isoform and its variants AE2a and AE2b, PCR conditions were as
follows: denaturation at 94°C for 1 min for AE2 and 45 s
for AE2a and AE2b, annealing at 60°C for 1 min for AE2 and 2 min
for AE2a and AE2b, primer extension at 72°C for 1 min for AE2 and 2 min for AE2a and AE2b. A final extension of 7 min was terminated by
rapidly cooling to 4°C after 32 cycles for AE2 and AE2a and 35 cycles for AE2b. For NHE isoforms, after the initial denaturation
(95°C for 5 min), the thermalcycler profile consisted of a
denaturation at 94°C for 30 s, annealing at 65°C for 30 s, and
primer extension 72°C for 45 s. PCR conditions for each isoform are
summarized in Table 2. In all
amplifications, contamination by genomic DNA was ruled out by running
samples without a previous reverse transcription phase.
Amplification products were electrophoresed on 7% acrylamide gel and
visualized by ethidium bromide and then silver stained to enhance the
sensitivity of the detection system. To confirm their identity, PCR
products were then sequenced. To this aim, PCR samples were purified
using either the Qiagen QIAquick PCR purification kit or gel purified
using the Qiagen QIAquick gel extraction kit. Purified PCR samples were
sequenced on an ABI 373A Stretch automated sequencer using the PRISM
dye terminator cycle sequencing kit according to the manufacturer's
instructions. Approximately 10 ng per 100 bp of cDNA were used in each
sequencing reaction. Sequences were analyzed using the Perkin-Elmer
sequencing analysis program 2.1.2.
Statistical analysis.
Results of continuous variables are shown as means ± SD. Paired and
unpaired t-tests were carried out
using STATGRAPH software (STSC).
 |
RESULTS |
Gene expression of AE and NHE isoforms.
Gene expression of
Na+/H+
exchanger isoforms (NHE1, NHE2, NHE3) and of
Cl
/HCO
3
exchanger isoforms (AE1 and AE2) was tested by RT-PCR using total RNA
extracted from NRC-1 cells and rat kidneys, which were used as positive
controls (17, 27, 37). As expected, RT-PCR of rat kidney RNA produced
amplicons of the proper size for all isoforms tested on agarose gel. On
the contrary, as shown in Fig. 2, only
NHE1, NHE2 (Wang sequence) (40), and AE2 were expressed in NRC-1 cells.
Identity of the amplification products obtained from kidney and NRC-1
RNA was confirmed by sequence analysis. These findings are in agreement
with those of Marti et al. (21) who detected NHE1 and NHE2 in freshly
isolated, partially purified cholangiocytes and with those of
Martinez-Ansò et al. (22) who reported expression of AE2, but not
AE1, in RNA extracted from human liver biopsies. Given the presence of
functional Cl
/HCO
3
exchange both at the apical and basolateral membranes (see
Cl
/HCO
3
exchange activity), we also looked for
expression of two variants of AE2 (AE2a and AE2b), which are generated
by different promoters. These two variants differ at their
NH2-terminal sequence and may be
differently targeted and regulated (1). As shown in Fig.
3, kidney and NRC-1 RNA amplification
products of the proper size were detected for AE2a and AE2b using
RT-PCR.

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Fig. 2.
Anion exchanger (AE) and
Na+/H+
exchanger (NHE) isoform gene expression in NRC-1 cells and rat kidneys.
Total RNAs (1 µg) from NRC-1 cells and rat kidneys were reverse
transcribed with random primers and then PCR amplified with NHE1, NHE2,
and NHE3 primers and with AE1 and AE2 primers (see Table 1 for primer
sequences and Table 2 for amplification conditions); 5 µl of the
amplification products were directly stained with ethidium bromide and
then silver stained on 7% acrylamide gel. Lane
1: DNA marker phiX
174/Hae III. Lane
2: rat kidney NHE1. Lane
3: NRC-1 NHE1. Lane 4:
rat kidney NHE2 (Wang et al.). Lane 5:
NRC-1 NHE2 (Wang et al.). Lane 6: rat
kidney NHE2 (Collins et al., Ref. 10). Lane
7: NRC-1 NHE2 (Collins et al.). Lane
8: rat kidney NHE3. Lane
9: NRC-1 NHE3. Lane
10: rat kidney AE1. Lane
11: NRC-1 AE1. Lane
12: DNA marker phiX
174/Hae III. Lane
13: rat kidney AE2. Lane
14: NRC-1 AE2. Contrary to kidney, NRC-1 cells express
only AE2, NHE1, and the 12-transmembrane domain NHE2 transcripts cloned
by Wang et al. (40).
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Fig. 3.
Expression of AE2 gene variants in NRC-1 cells. Total RNAs (1 µg)
from NRC-1 cells and rat kidneys were reverse transcribed with random
primers and then PCR amplified with AE2a and AE2b primers (see Table 1
for primer sequences and Table 2 for amplification conditions); 5 µl
of the amplification products were directly stained with ethidium
bromide and then silver stained on 7% acrylamide gel. Lanes from
left to
right are as follows.
Lane 1: NRC-1 genomic AE2a.
Lane 2: rat kidney genomic AE2a.
Lane 3: NRC-1 AE2a.
Lane 4: rat kidney AE2a.
Lane 5: NRC-1 AE2b.
Lane 6: rat kidney AE2b.
Lane 7: DNA marker phiX
174/Hae III. Lane
8: NRC-1 genomic AE2b. Lane
9: rat kidney genomic AE2b. RT-PCR detected
amplification products of the proper size for both AE2a and AE2b.
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|
Na+/H+
exchange activity.
Mechanisms responsible for H+
extrusion were studied by measuring
pHi recovery after intracellular
acidification in nominally HCO
3-CO2-free
medium (HEPES) (Fig. 4). Removal of
basolateral Na+ (substitution with
the impermeant monovalent cation choline), in the presence of the
Na+/H+
exchange inhibitor amiloride (1 mM) on the apical side, induced a rapid
acidification of pHi (from
pHi 7.13 ± 0.12 to
pHi 6.58 ± 0.19). After
readmission of basolateral Na+,
still in the presence of apical amiloride, cells recovered to basal
pHi, extruding protons
[JH+ = 27.5 ± 9.6 mmol · l
1 · min
1
and
pH/
t = 0.392 ± 0.123/min at pHi 6.7 (n = 8) and
JH+ = 9.79 ± 5.6 mmol · l
1 · min
1
and
pH/
t = 0.297 ± 0.172/min
(n = 11) at
pHi 7.0]. In another set of
experiments, basolateral amiloride was added at different concentrations simultaneously with
Na+ readmission;
pHi recovery (at
pHi 6.7) was inhibited in a
dose-dependent way (with 0.1 mM amiloride: 85% inhibition; 0.5 mM
amiloride: 94% inhibition; 1 mM amiloride: 95% inhibition). To
further characterize the NHE isoform expressed at the basolateral
membrane, we tested the effects of HOE-642, a new benzoyl-guanidinium
inhibitor similar to HOE-694 (30). Because of its specific and potent
inhibitory activity toward NHE1, HOE-642 can be used to discriminate
between different NHE isoforms (30). As shown in Fig.
5A,
cells were acidified by pulsing with 30 mM
NH4Cl and thereafter perfused from
both sides with Na+-free HEPES
medium. The basolateral side was then exposed to various concentrations
of HOE-642 in the presence of
Na+-containing HEPES medium. As
shown in Table 3 and Fig.
5C,
JH+ was exquisitely sensitive to HOE-642, being almost completely inhibited
by a concentration of 10 µM [apparent inhibition constant (Ki) = 1 µM]. This high sensitivity to HOE-642 clearly identifies NHE1
(23, 30) as the isoform functionally expressed at the basolateral
membrane of cholangiocytes.

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Fig. 4.
Representative experiments showing functional evidence for basolateral
NHE1. Apical medium changes are shown above the tracing; basolateral
changes are shown below. In HCO 3-free
medium (HEPES), removal of basolateral
Na+ (substitution with the
impermeant monovalent cation choline) in the presence of apical
amiloride (1 mM) acidified pHi
(1), which rapidly recovered
following basolateral Na+
readmission; pHi recovery induced
by Na+ readmission was inhibited
by basolateral amiloride (1 mM)
(2).
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Fig. 5.
Effect of HOE-642 on basolateral and apical NHE activities.
A: representative experiments showing
basolateral Na+-dependent
H+ extrusion after intracellular
acid load. In HCO 3-free medium
(HEPES), administration and withdrawal of 30 mM
NH4Cl induced an acute
intracellular acidification (1).
Cells were perfused at both sides with
Na+-free HEPES buffer
(2); when basolateral
Na+ was then readmitted, a
complete recovery at baseline pHi
was achieved (3), indicating
basolateral NHE exchanger. HOE-642 inhibition curve is shown in
C. B:
representative experiments showing apical
Na+-dependent
H+ extrusion after intracellular
acid load. In HCO 3-free medium
(HEPES), administration and withdrawal of 30 mM of
NH4Cl induced an acute
intracellular acidification (1).
Cells were perfused at both sides with
Na+-free HEPES buffer
(2), while the basolateral side was
also exposed to 0.5 mM HOE-642.
Na+ was then readmitted at the
apical side. At this point, a partial recovery was evident
(3).
C: dose-dependent effect of HOE-642 on
apical (open bars) and basolateral (solid bars) NHE activities. HOE-642
was administered 1 min before Na+
readmission at the apical or basolateral aspect of the cells following
the protocol described in A and
B. Data are expressed in % with
respect to NHE activity in control experiments. Clearly, basolateral
NHE is much more sensitive to HOE-642 than apical NHE. See also Table
3.
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In Na+-reabsorbing
gastrointestinal epithelia, including the gallbladder (11, 43), an
Na+/H+
exchanger belonging to the NHE2 or NHE3 isoform is located at the
apical pole of the cell. Given the presence of the NHE2 message in
NRC-1 cells, we looked for functional
Na+/H+
exchanger activity also on the apical membrane. In a preliminary report
by Singh et al. (31), an apical
Na+-dependent, amiloride-sensitive
H+ mechanism, active from pH 6.2 and 6.5, was described in microperfused bile ducts. To achieve a
comparable acidification, cells were pulsed with 30 mM
NH4Cl and then perfused with
Na+-free HEPES buffer (Fig.
5B), with average nadir
pHi of 6.38 ± 0.13 (n = 12), while the basolateral side
was perfused with an Na+-free
HEPES containing 0.5 mM HOE-642. When
Na+ was readmitted at the apical
side, a slow but measurable pHi recovery
(JH+ = 3.57 ± 1.34 mmol · l
1 · min
1,
pHi/
t = 0.027 ± 0.016/min at pHi
6.5) was present. Using a similar experimental protocol, but in the
presence of 1 mM basolateral amiloride, we also evaluated the effects
of apical amiloride (1 mM). A concentration of 1 mM amiloride fully
inhibited pHi recovery [JH+ = 3.71 ± 1.36 mmol · l
1 · min
1,
pHi/
t = 0.045 ± 0.015/min at pHi 6.5 (n = 5)] in controls (not
shown), indicating that H+ efflux
was mediated by an
Na+/H+
exchange isoform. In another set of experiments (see Fig.
5B), 1 min before apical
Na+ readmission, the apical side
of the monolayer was also exposed to HOE-642 at different
concentrations. As shown in Table 3 and Fig.
5C, apical
pHi recovery was inhibited by
HOE-642 concentrations 100 times higher than basolateral NHE (apparent
Ki = 100 µM). Although NHE3 is ten times less sensitive to HOE-642, with a reported apparent Ki of 1 mM (30), these data are consistent with the presence of NHE2 at the
apical membrane of cholangiocytes.
Cl
/HCO
3
exchange activity.
In NRC-1 cells, AE2 but not AE1 RNA amplicons were detected. In the
human liver, monoclonal antibodies directed toward a synthetic peptide
specific for the AE2 isoform of
Cl
/HCO
3
exchange decorates the apical membrane of intrahepatic cholangiocytes
(22); thus we looked for the functional presence of AE2 at the apical
pole of NRC-1 cells. Removal of apical
Cl
in NRC-1 cells
pretreated with basolateral DIDS (1 mM per 40 min) did not cause a
significant pHi alkalinization,
even in the presence of agents raising intracellular cAMP levels (not
shown). Although Cl
removal
may be incomplete following this maneuver, or the activity of the acid
loader AE2 may be too low at the baseline
pHi, mechanisms of
HCO
3 extrusion were also investigated
by measuring recovery after an intracellular alkalinization induced by
administration and withdrawal of the permeant weak acid propionate (6)
(Fig. 6) in cells pretreated with
basolateral DIDS (1 mM). As expected, cell pH alkalinized following
propionate withdrawal (from pHi
7.11 ± 0.175 to pHi 7.37 ± 0.181); pHi recovered to baseline
pHi
[JOH
= 7.5 ± 2.43 mmol · l
1 · min
1,
pH/
t = 0.116 ± 0.05/min
(n = 17) at
pHi 7.3] by a mechanism that
was inhibited by apical Cl
removal
[JOH
= 1.08 ± 1.63 mmol · l
1 · min
1,
pH/
t = 0.018 ± 0.02/min
(n = 9) at
pHi 7.3] and by pretreatment with apical DIDS (1 mM)
[JOH
= 3.12 ± 1.43 mmol · l
1 · min
1,
pH/
t = 0.05 ± 0.02/min
(n = 5)] (Table
4), consistent with the operation of
Cl
/HCO
3
exchange. Administration of agents increasing intracellular cAMP levels
(100 µM DBcAMP, 3 µM forskolin, and 100 µM IBMX) (3, 5, 14)
significantly increased the rate of
pHi recovery
[JOH
= 12.65 ± 4.27 mmol · l
1 · min
1,
pH/
t = 0.216 ± 0.07/ min
at pHi 7.3 (n = 12);
P < 0.001 vs. controls]. Taken
together, these data indicate that a cAMP-activated Cl
/HCO
3
exchanger is located at the apical pole of cholangiocytes expressing
AE2 transcripts.

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[in a new window]
|
Fig. 6.
Representative tracings of pHi
recovery after acute intracellular alkalinization in cells pretreated
with basolateral (BL) DIDS. In the presence of
HCO 3-CO2
(KRB), administration and withdrawal of propionate (prop; 50 mM), in
cells pretreated basolaterally with DIDS, induced an intracellular
alkalinization. pHi recovery
(1) was inhibited by apical (AP)
Cl removal
(2) and partly inhibited by apical
DIDS pretreatment (3). After agents
that raise intracellular cAMP concentration [100 µM
dibutyryl-cAMP (DBcAMP), 3 µM forskolin, 100 µM IBMX 100]
were administered, HCO 3 efflux
increased significantly (4).
|
|
To investigate the functional presence of a basolateral
Cl
/HCO
3
exchange in NRC-1 cells, pHi
transients were monitored during acute removal of extracellular
Cl
. This maneuver, in the
presence of an active
Cl
/HCO
3
exchange, causes intracellular alkalinization because
HCO
3 enters the cells in exchange with intracellular Cl
, which is
forced to exit (34). In the presence of
HCO
3-CO2 (KRB buffer), in cells pretreated with 1 mM DIDS on the apical side,
removal of basolateral Cl
induced a rapid pHi alkalinization
(Fig. 7) (from
pHi 7.21 ± 0.12 to
pHi 7.54 ± 0.12)
[JOH
= 21.6 ± 13.46 mmol · l
1 · min
1,
pH/
t = 0.368 ± 0.22/min
(n = 19) at 7.3],
followed by a quick recovery when
Cl
was readmitted
[JOH
= 38.01 ± 14.52 mmol · l
1 · min
1,
pH/
t = 0.707 ± 0.42/min
(n = 19) at
pHi 7.3]. This
alkalinization was inhibited by basolateral DIDS (1 mM for 40 min)
pretreatment [JOH
= 4.61 ± 3.1 mmol · l
1 · min
1,
pH/
t = 0.062 ± 0.03/min
(n = 6)], indicating that
Cl
/HCO
3
exchange is also located at the basolateral membrane. Contrary to the
apical anion exchanger, an increase in intracellular cAMP concentration
did not stimulate base fluxes during the alkalinization phase
[JOH
= 22.6 ± 10.31 mmol · l
1 · min
1,
pH/
t = 0.397 ± 0.17/min
(n = 19) at
pHi 7.3] or the
pHi recovery phase
[JOH
= 38.36 ± 16.6 mmol · l
1 · min
1,
pH/
t = 0.767 ± 0.42/min
(n = 19) at
pHi 7.3] (Table 4).

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|
Fig. 7.
Representative experiments demonstrating the functional
Cl /HCO 3
exchanger on the basolateral aspect of the monolayer. In the presence
of
HCO 3-CO2,
in cells pretreated apically with DIDS, removal of basolateral
Cl induced a rapid
pHi alkalinization
(1), which was not increased by cAMP
(that is, 100 µM DBcAMP, 3 µM forskolin, 100 µM IBMX)
(2);
pHi recovery was inhibited by
basolateral DIDS (3).
|
|
 |
DISCUSSION |
Epithelial cell function depends on the structural and functional
diversity of its apical and basolateral plasma membrane domains.
Establishment of a polarized distribution of membrane carriers and
other proteins involved in specialized epithelial function is a
fundamental characteristic of epithelia that enables the vectorial
transport of ions, fluid, and other constituents from one compartment
to another (13). In addition, loss of membrane polarity is an important
pathogenetic mechanism in a number of diseases, including cholestasis
(32).
A number of studies have addressed the mechanisms of cholangiocyte ion
transport. However, acid-base transport studies in cholangiocytes have,
thus far, been restricted largely to nonpolarized cell preparations or
to cell preparations in which the apical side is not easily accessible.
Recently, a differentiated normal rat cholangiocyte cell line (NRC-1)
that maintains a polarized distribution of a number of membrane markers
has become available (39). The NRC-1 cell line is positive for
cholangiocyte phenotypic markers such as
-glutamyltranspeptidase,
CK-7, and CK-19 and is negative for the mesenchymal markers vimentin
and desmin. Ultrastructural examination of cells grown over
semipermeable membrane inserts reveals competent tight junction, apical
microvilli and cilia, basolaterally restricted nuclei, and membrane
interdigitations; somatostatin receptors are expressed basolaterally,
staining for
-glutamyltranspeptidase decorates the apical membrane
(39). NRC-1 cells absorb bile acids and glucose at their apical domain via the ileal Na+-dependent bile
acid transporters (19) and the
Na+-glucose cotransporter SGLT-1
(20), respectively. Activation of purinergic
P2Y2 receptors located at the
apical membrane stimulated short-circuit currents and produced a
basolateral-to-apical Cl
efflux that is inhibited by apical administration of
Cl
channel blockers (28).
Expression of CFTR protein, in NRC-1 cells, has been demonstrated in
apical plasma membrane vesicles by immunoblotting (36).
We have thus exploited this cell model to investigate the functional
topographic distribution and gene expression of two cholangiocyte acid-base carriers, the
Na+/H+
exchanger and the
Cl
/HCO
3
exchanger. Our results show that, in NRC-1 cells, functional NHE1
activity is restricted at the basolateral membrane, whereas an NHE2
isoform is likely to be active on the side facing the lumen.
Furthermore, two transcripts (AE2a and AE2b) (1) of the AE2 gene are
expressed in NRC-1 cells, consistent with the functional presence of
Cl
/HCO
3
exchanger activities with different sensitivity to stimulation by cAMP
and inhibition by DIDS at the apical and basolateral side of the monolayer.
The presence of the basolateral
Na+/H+
exchanger is suggested by the decrease in
pHi after basolateral
Na+ removal in the presence of
apical amiloride (1 mM) and by the basolateral amiloride-inhibitable
pHi recovery recorded when
basolateral Na+ was readmitted.
JH+
was also inhibited by HOE-642 (30), a recently developed compound that
is not derived from amiloride but possesses a highly specific and
potent inhibitory activity toward the NHE1 isoform (Fig.
5C). The high sensitivity of
basolateral pHi recovery to
amiloride and HOE-642 is consistent with basolateral expression of the
mitogen-activated isoform of the
Na+/H+
exchanger NHE1, whose mRNA was clearly expressed (14, 23, 33). In fact,
NHE2 is known to be much less sensitive to HOE-642 (see below). NHE1 is
an electroneutral carrier that extrudes
H+ from the cell, energized by the
transmembrane Na+ gradient. In
epithelial cells, NHE1 is usually located at the basolateral membrane
where it is involved in homeostatic functions such as
pHi, control of cell volume, and
regulation of cellular ionic milieu following stimulation with growth
factors (15). In addition, NHE1 participates in transepithelial
HCO
3 fluxes by loading
HCO
3 into the cell.
HCO
3 uptake in rat cholangiocytes is,
in fact, performed by two mechanisms, which has a still not clear
relative importance: intracellular CO2 diffusion followed by carbonic
anhydrase-catalized CO2 hydration and backward transport of H+ via
NHE1 or direct uptake of HCO
3 by the
electrogenic Na+-HCO3(n)
cotransport (4, 32).
Recently, Marti et al. (21) have reported the presence of NHE2
transcripts in partially purified rat cholangiocytes. Among the two
different published cDNAs coding for rat NHE2, the one reported by
Collins et al. (10) encodes for a protein possessing 10 hydrophobic
transmembrane domains; primers specific for this truncated NHE2 isoform
that misses the 356 NH2-terminal
amino acids (16, 26) gave negative results in NRC-1 cells. On the other
hand, the set of primers used by Marti et al. (21), recognizing also a
12-transmembrane domain NHE2 isoform (40), produced amplimers of the
expected size and sequence. In addition, in a preliminary report, Singh
et al. (31) showed that, in microperfused bile ducts, an apical
Na+/H+
exchanger functions as a pHi
regulator at very acidic pHi
values. In this study, we have shown that apical
Na+ readmission induces a slow but
significant pHi recovery in cells acidified by exposition to NH4Cl
and perfused in Na+-free medium.
We were also able to consistently show inhibition by high
concentrations of apical amiloride and HOE-642 (see Table 3 and Fig.
5C). As expected for the NHE2
isoform (30), apical pHi recovery
was ~100 times less sensitive to HOE-642 with respect to basolateral
recovery. NHE3 is known to be inhibited only by HOE-642 concentrations
10 times higher than NHE2 (30); thus, given the clear expression of
NHE2 mRNA, it seems reasonable to conclude that NHE2 is the isoform
expressed at the apical membrane of NRC-1 cells. The discrepancy
between our data and the preliminary report of Singh et al. (31) may be
explained by the known heterogeneity of cholangiocyte transport systems
along the biliary tree (2). In addition, the slow and incomplete
pHi recovery performed by NHE2 is
consistent with the idea that the primary function of the NHE2 and NHE3
isoforms, expressed on the apical membrane of gastrointestinal
epithelia including gallbladder (11), is
Na+ reabsorption rather than
pHi regulation. The incomplete
recovery mediated by NHE may indicate that cellular acid production
matches NHE activity at pH 6.7 so that, if basolateral
NHE1 is inhibited, further recovery can be mediated by NHE2. NHE2 is
likely involved in Na+
reabsorption and biliary acidification also in cholangiocytes, and it
is interesting to note that luminal pH recorded in isolated ductules
microinjected with BCECF-dextran (pH 7.8) (28) is higher than the pH of
bile recorded from bile fistula rats (pH 7.3) (35). In agreement with
current views on morphofunctional heterogeneity of cholangiocytes (2),
we speculate that a segment of the biliary tree may indeed be devoted
to biliary acidification and fluid reabsorption.
The presence of the
Cl
/HCO
3
exchanger at the apical side is demonstrated by the
pHi recovery from an acute alkali
load, which is inhibited by apical
Cl
removal and by
application of DIDS on the apical side (Fig. 6). In addition, the
DIDS-inhibitable alkalinization during basolateral Cl
removal in cells treated
with apical DIDS suggests that
Cl
/HCO
3
exchange activity is also expressed on the basolateral side (Fig. 7).
Expression of the anion exchange in both plasma membrane domains has
been described in other epithelial cells, such as interlobular
pancreatic ducts (45) and kidney
-intercalated cells (12, 42), where
the AE1 is present either apically or basolaterally, depending on the
plating density of the cells (38). In our experiments, basolateral
Cl
/HCO
3
exchange activity was present in cells plated at both low and high
density (not shown). The reported negative immunohistochemistry for AE1
or AE2 on the basolateral membrane in human liver biopsies is not
surprising; for example, whereas all cells in the kidney outer cortical
collecting duct possess basolateral
Cl
/HCO
3
exchange activity, antibodies to band-3 proteins label only a subset of
cells, indicating that there can be immunological heterogeneity for
Cl
/HCO
3
exchange within a single region of the collecting duct (12). In spite
of the presence of AE activity at both sides of the monolayer, only
transcripts for AE2 were detected in NRC-1 cells. It is known that AE
genes can generate a variety of transcripts able to perform different
physiological functions as a result of their tissue-specific expression
and subcellular location. Recently, the
AE2 gene has been shown to contain
three different promoters that lead to the production of mRNAs encoding
for three variants of the exchanger encoded with different sorting
properties (41). By RT-PCR, we have shown that both AE2a and AE2b are
expressed in NRC-1 cells; these two variants are known to differ in
their NH2-terminal sequences, where the AE2a but not AE2b sequence contains a potential
phosphorylation site for protein kinase A (41). Interestingly, on the
basis of their tissue distributions, it has been proposed that AE2a may
be apically targeted (41). Clarke and Harline (9) have reported data
suggesting that cAMP-stimulated HCO
3 transport in the duodenum involves two mechanisms: electrogenic secretion via CFTR-mediated HCO
3
conductance and electroneutral secretion involving carbonic anhydrase
and CFTR-dependent
Cl
/HCO
3
exchange. The functional location of AE2 on the apical membrane and its
sensitivity to stimulation by cAMP suggest it may play a role in
biliary HCO
3 secretion (5, 22, 32)
similar to that proposed by Novak and Greger (24) for pancreatic duct
cells in which, following cAMP stimulation, CFTR recycles
Cl
to a luminal
Cl
/HCO
3
exchange resulting in cAMP-stimulated HCO
3 secretion. On the other hand, the
physiological function of basolateral
Cl
/HCO
3
exchanger in cholangiocytes is unclear and it may be limited to
pHi and volume regulation.
Availability of the NRC-1 cell line will facilitate further studies of
this important aspect of epithelial cell physiology.
In conclusion, we have provided functional evidence in a polarized rat
cholangiocyte cell line that different NHE and AE isoforms are
expressed at different plasma membrane domains. NHE1 is expressed on
the basolateral membrane where it is likely involved in
HCO
3 cell loading as well as in
pHi and volume housekeeping
functions. On the other hand, NHE2 appears to be located at the apical
membrane where it may serve to reabsorb
Na+ and acidify the biliary fluid.
Two different AE2 transcripts were detected in NRC-1 cells, consistent
with the observation that
Cl
/HCO
3
exchange activity was present at both the apical and basolateral sides.
Apical AE was activated by cAMP, consistent with its proposed role in
HCO
3 extrusion, whereas basolateral AE
is likely involved in pHi
homeostatic functions. To the extent that data on cell lines can be
relevant to the in vivo situation, these observations add a
considerable level of complexity to current models of electrolyte
transport in cholangiocytes. This study also indicates that the NRC-1
cell line can be a useful tool to investigate the mechanisms of
transport protein sorting in the biliary epithelium.
 |
ACKNOWLEDGEMENTS |
We are indebted to James L. Boyer, M.D., Elena Ossi, M.D., and J.F.
Medina, M.D., for helpful discussion. À. Zsembery thanks Prof. Làszlò Rosivall for helpful discussion and his
constant support.
 |
FOOTNOTES |
This work was sponsored by Grant 96.0344.04 from Consiglio Nazionale
delle Ricerche. The financial support of Telethon (Grant E-430) is
gratefully acknowledged. This work was also supported in part by
National Institute of Diabetes and Digestive and Kidney Diseases Grant
DK-24031 to N. F. LaRusso.
À. Zsembery was the recipient of an International Fellowship
from the University of Padova.
Part of this work was presented at the 47th Annual Meeting of the
American Association for the Study of Liver Diseases in Chicago, IL,
November 8-12, 1996, and at the 32nd Annual Meeting of the
European Association for the Study of the Liver in London, UK, April
9-12, 1997, and published in abstract form
(Hepatology 24: 146, 1996;
J. Hepatol. 26: 63, 1997).
Present address of À. Zsembery: Institute of Pathophysiology,
University of Medicine, Budapest, Hungary.
Address for reprint requests: M. Strazzabosco, Institute of Internal
Medicine, Univ. of Padova, Via Giustiniani, 2, I-35100 Padova, Italy.
Received 6 November 1997; accepted in final form 30 July 1998.
 |
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