1 Liver Center and Department of Medicine, Yale University School of Medicine, New Haven, Connecticut 06520; and 2 Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, Cincinnati, Ohio 45267-0524
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ABSTRACT |
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Na+/H+ exchanger (NHE) isoforms play important
roles in intracellular pH regulation and in fluid absorption. The
isoform NHE3 has been localized to apical surfaces of epithelia and in
some tissues may facilitate the absorption of NaCl. To determine
whether the apical isoform NHE3 is present in cholangiocytes and to
examine whether it has a functional role in cholangiocyte fluid
secretion and absorption, immunocytochemical studies were performed in
rat liver with NHE3 antibodies and functional studies were obtained in
isolated bile duct units from wild-type and NHE3 (/
) mice after
stimulation with forskolin, using videomicroscopic techniques. Our
results indicate that NHE3 protein is present on the apical membranes
of rat cholangiocytes and on the canalicular membrane of hepatocytes.
Western blots also detect NHE3 protein in rat cholangiocytes and
isolated canalicular membranes. After stimulation with forskolin, duct
units from NHE3 (
/
) mice fail to absorb the secreted fluid from the
cholangiocyte lumen compared with control animals. Similar findings
were observed in isolated bile duct units from wild-type mice and rats
in the presence of the Na+/H+ exchanger
inhibitor 5-(N-ethyl-N-isopropyl)-amiloride. In
contrast, we could not demonstrate absorption of fluid from the
canalicular lumen of mouse or rat hepatocyte couplets after stimulation
of secretion with forskolin. These findings indicate that NHE3 is located on the apical membrane of rat cholangiocytes and that this NHE
isoform can function to absorb fluid from the lumens of isolated rat
and mouse cholangiocyte preparations.
bile secretion; bile duct epithelium; hepatocyte
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INTRODUCTION |
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BILE IS A SECRETORY
PRODUCT of both hepatocytes and cholangiocytes. Cholangiocytes
line the biliary epithelium and are capable of both secretory and
absorptive functions that modify the primary secretion from the liver
parenchymal cells (6, 31). Although the mechanisms that
determine the secretion of bile from hepatocytes have been largely
characterized at the molecular level in recent years, less is known
about the function of cholangiocytes, which represent only 3-5%
of the total liver cell population. Long thought to be little more than
a conduit for the delivery of primary bile to the intestine, this
epithelium is capable of carrying out a variety of both secretory and
absorptive processes, as indicated by more recent studies. Hormones
such as secretin, vasoactive intestinal polypeptide (VIP), and bombesin
have been shown to be direct stimulants of HCO3
secretion after meal-stimulated release of these hormones (1, 11,
13, 19). This process occurs through the activation of a
cystic fibrosis transmembrane conductance regulator (CFTR) Cl
channel coupled with a
Cl
/HCO3
exchanger on the apical domain
(1, 19). Absorptive processes have been less well
characterized, but transporters that mediate the removal of glucose
(Glut-3) and bile salt (Isbt) have been described (16,
17). Purinergic receptors on the apical domain stimulate
calcium-mediated Cl
secretion (20, 28). Less
is known about alternative mechanisms for the transport of fluid and
electrolytes, particularly whether NaCl and water are absorbed by the
biliary epithelium and whether this process has a role in modulation of
basal as well as hormone-stimulated ductular secretion.
In addition to a role in acid-base balance, Na+/H+ exchangers (NHE) play an important role in the absorption of NaCl in a number of epithelia (26). These transporters exist as a family of multiple isoforms with different tissue and regional distributions. At least five isoforms have been described. Recently, NHE3 has been localized to the apical domains of several epithelia, including the kidney (4, 9, 25), intestine (9), and submandibular gland (18), where it facilitates NaCl absorption. In the present study, we have identified NHE3 protein on the apical membranes of cholangiocytes and, for the first time, demonstrated a functional role for NHE3 in the absorption of fluid from the lumen of isolated bile duct units (IBDU) from the mouse. NHE3 was also detected in canalicular membranes of hepatocytes. These findings have important implications for understanding the regulation of salt and water secretion in cholangiocytes, both for the normal physiology of the biliary tree as well as in pathological states such as cholestatic liver disease.
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MATERIALS AND METHODS |
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Materials.
DNase, hyaluronidase, BSA, forskolin, penicillin-streptomycin,
dexamethasone, insulin, and nigericin were purchased from Sigma Chemicals (St. Louis, MO). MEM, -MEM, Williams' E medium, Liebowitz 15 (L-15), and gentamicin were from GIBCO (Grand Island, NY). FCS was
from Gemini Bioproducts. Collagenase B was from Boehringer Mannheim,
and 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) was
from RBI (Natick, MA). Pronase was from Calbiochem (La Jolla, CA), and
Matrigel was purchased from Collaborative Biomedical.
Primary antibodies. Isoform-specific monoclonal antibodies (MAbs) and polyclonal (raised in guinea pig or goat) anti-B1 antibodies were raised to a maltose-binding protein fusion protein representing amino acids 702-831 of rabbit NHE3 (5, 27). The epitope for MAb 2B9 has been mapped to a region between amino acids 702 and 756, whereas the regions for MAbs 4F5 and 19F5 lie between amino acids 756 and 792. Anti-NHE3 MAbs 2B9, 4F5, and 19F5 were obtained from Chemicon International (Temecula, CA).
MAbs were used either as undiluted hybridoma supernatant or as purified IgG (stock solutions at ~1 mg/ml) at 1:50 for immunocytochemistry or at 1:1,000 for immunoblotting. Rabbit antisera was used at 1:50 for immunocytochemistry or at 1:1,000 for immunoblotting.Secondary antibodies. For indirect immunofluorescence microscopy, Alexa 488 goat anti-mouse IgG and Alexa 594 goat anti-rabbit IgG (Molecular Probes) were used. For immunoblotting, horseradish peroxidase (HRP)-conjugated rabbit anti-goat IgG (heavy and light chain specific) was purchased from Zymed Laboratories (San Francisco, CA) and goat anti-rabbit and sheep anti-mouse peroxidase-conjugated F(ab')2 IgG was from Sigma. For immunoelectron microscopy an HRP-conjugated goat anti-mouse (heavy and light chain specific) F(ab')2 (Zymed Laboratories) was also used.
Cell isolation and culture.
Male Sprague-Dawley rats (150-250 g) were purchased from Camm
Laboratory Animals (Wayne, NJ). Female (+/+) and (/
) NHE3 mice were
established as previously described (29). Isolated rat
hepatocyte couplets (IRHC), cholangiocytes, and IBDU from rats and mice
were isolated as previously described (8, 12, 14, 23).
Briefly, the livers were perfused via the portal vein with Hanks' A
buffer for 10 min at 40 ml/min for rats and 5 ml/min for mice. This was
followed by Hanks' B buffer supplemented with 0.03% collagenase B for
10 min or until livers appeared digested. The livers were excised, and
the hepatocytes were removed by gently shaking in 4°C L-15; the
remaining nonparenchymal tissue was used for IBDU isolation. The
hepatocytes were passed through 80- and 40-µm mesh to remove large
clusters of cells and then washed three times in L-15 by
centrifugation. Hepatocytes were plated on Matrigel-coated coverslip
fragments in Williams' E medium supplemented with 26 mM
HCO3
, penicillin-streptomycin (100,000 units, 100 mg/l), 50 µg/ml gentamicin, 0.1 µM insulin, 0.3 µM dexamethasone,
and 10% FCS and maintained in culture in a 5% CO2
air-balanced incubator for 24 h at 37°C.
Immunocytochemistry.
Rat livers were cut into 5-mm cubes and quick frozen in liquid
N2-cooled Freon before sectioning. Cryosections were fixed in 20°C acetone for 10 min, washed in 0.1 M PBS, and blocked in 1%
BSA for 30 min followed by 1-h incubation in anti-NHE3 (2B9) primary
antibody diluted 1:50. After washing in PBS, an anti-mouse NHE3
secondary antibody (1:500) was added for 1 h and the sections were
washed and mounted in Vectashield (Vector Laboratories, Burlingame, CA). Fluorescence was visualized on a Zeiss LSM 510 confocal microscope or a Nikon Microphot FX epifluorescence microscope.
Tissue preparation for electron microscopy. Sprague-Dawley rats were starved overnight but were allowed free access to drinking water. The animals were anesthetized with pentobarbital sodium and perfusion fixed via the left ventricle. Perfusion was performed first with PBS, pH 7.4, at 37°C to remove blood, followed by PLP fixative containing 2% paraformaldehyde, 75 mM lysine, and 10 mM sodium periodate in phosphate buffer, pH 7.4 (21). Blocks of tissue (2- to 4-mm cubes) from fixed livers were cut and postfixed in PLP for an additional 4-6 h at room temperature. Tissue was then washed in PBS and stored at 4°C in PBS containing 0.5% paraformaldehyde. Blocks of fixed tissue were incubated overnight in 30% sucrose in PBS, then frozen in liquid N2-cooled isopentane.
Immunoelectron microscopy. Immunoelectron microscopy was performed using the HRP method as described previously (3). Briefly, 300-µm cryosections of fixed liver were incubated in primary antibody overnight at 4°C in buffer containing 1% BSA in PBS (pH 7.4). The next morning, sections were washed in PBS and incubated in HRP-conjugated secondary antibody in the same buffer for 2 h at room temperature. After washing, the sections were fixed in 3% glutaraldehyde, reacted with diaminobenzidine, postfixed in OsO4, and embedded in Epon. Sections were cut, stained with lead citrate, and examined with a Zeiss 910 electron microscope.
Preparation of membrane fractions. Sprague-Dawley rats (Charles River) were killed by injection of pentobarbital sodium (Butler, Columbus OH). Brush border membrane vesicles (BBMV) were prepared from renal cortex using the Mg2+ precipitation method described previously (2).
Canalicular membranes from hepatocytes were isolated, as previously described by this laboratory (22). BBMV and canalicular membranes were used for SDS-PAGE and Western blotting.SDS-PAGE and Western blotting. Protein for SDS-PAGE from hepatocytes and IBDU was isolated by washing away culture media with ice-cold Tris-sucrose (0.1 M Tris, 0.25 M sucrose) and then homogenizing by repeatedly passing through a 30-g needle in Tris-sucrose containing the protease inhibitors 0.2 mM phenylmethylsulfonyl fluoride, 0.5 mM benzamidine, 5 µM leupeptin, and 1 µM aprotinin. The suspension was centrifuged at 1,000 g for 8 min, and the supernatant was assayed to quantitate protein. Samples were run on a 7.5% polyacrylamide gel and transferred to nitrocellulose paper or polyvinylidene difluoride (PVDF) membranes (Millipore Immobilon-P). Immunoblotting was performed as follows. Sheets of nitrocellulose or PVDF containing transferred protein from gels were incubated first in Blotto (5% nonfat dry milk in PBS or Tris-buffered saline, pH 7.4) for 1-3 h to block nonspecific binding of antibody and incubated with primary antibody (anti-B1 or MAb 2B9) diluted 1:500 overnight at 4°C. They were next washed in Blotto for 3 h, incubated in anti-goat (B1) or anti-rabbit (2B9) peroxidase-conjugated IgG secondary antibody 1:2,000 for 1 h, washed again three times in Blotto and once in PBS or Tris-buffered saline, and detected using the ECL chemiluminescence system (Amersham, Arlington Heights, IL) according to manufacturer's protocols.
Videomicroscopy.
IRHC and IBDU were cultured on coverslip fragments for 24 and
48 h, respectively, before being transferred to the stage of an
inverted microscope (IM35 Zeiss, Thornwood, NY) equipped with Nomarski
optics, where they were perfused at 37°C with
Krebs-Ringer-bicarbonate buffer gassed with 95% O2-5%
CO2. IRHC and IBDU were imaged with a
charge-coupled device camera (Dage-MTI, Michigan City, IN) connected to
a computer, where they were measured using image analysis software. The
maximal cross-sectional area was measured and converted to volume using
the formula 4/3 r3, where r
(radius) was derived from the maximal areas (MA): MA =
r2 or r =
MA/
. This
calculation assumes that the luminal area is spherical as judged from
previous cross-sectional analyses of the IBDU lumens (10).
Data was standardized by expressing the changes in volume as percentage
of baseline (time 0) values. Changes in luminal volume were
assessed as a measure of net secretion (+) or absorption (
) by the
cholangiocyte epithelium. Both IBDU and IRHC were stimulated to secrete
with 10 µM forskolin, which was infused as specified in each
experiment. In experiments in which the NHE inhibitor EIPA (1 µM) was
used, the coverslips were preincubated for 30 min before the start of
the experiment and infused throughout the experiment.
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RESULTS |
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Immunolocalization of
Na+/H+
exchanger isoform NHE3 in rat liver.
To localize Na+/H+ exchanger isoform NHE3 in
the rat liver we used isoform-specific monoclonal antibody 2B9 combined
with indirect immunofluorescence microscopy. Liver sections showed
strong immunoreactivity to NHE3 (Fig. 1).
In hepatocytes, strong positive staining was limited to the canalicular
membrane and no basolateral staining was observed (Fig.
1A). Staining was similar throughout the lobule and
was not restricted to any particular zone. Bile ducts, regardless of
their size, also stained intensely at their apical membrane (Fig.
1B). Sinusoidal endothelial cells demonstrated weak
immunoreactivity to the NHE3 antibody (Fig. 1A).
Furthermore, expression of NHE3 was maintained in cultured hepatocyte
couplets (Fig. 1C), where it was also observed at the
canaliculus. The specificity of staining for NHE3 in rat liver was
verified by the observation (not shown) that identical staining
resulted from use of MAbs 4F5 and 19F5, which react with a different
epitope on NHE3 than does MAb 2B9 (5).
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Immunoblotting.
Western blot analysis of protein extracted from cultured IBDU revealed
a positive band that migrated to the same position as protein from the
kidney brush border positive control (Fig. 3A). The molecular weight was
~83-85, consistent with previous reports for NHE3 (4,
5). Although protein extracted from hepatocytes failed to
demonstrate a definitive positive band regardless of whether the cells
were freshly isolated or cultured for 24 h, detection of a
positive band in purified canalicular membranes (Fig. 3B)
confirmed that NHE3 protein is present in hepatocytes.
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Functional assessment of NHE3.
To determine a functional role for NHE3 we analyzed hepatocyte couplets
and IBDU from normal and NHE3 (/
) mice by videomicroscopy using
techniques previously described in detail by this laboratory (7,
11, 23, 32). As illustrated in Fig.
4, when IBDU isolated from the wild-type
NHE3 mouse (+/+) were perfused with forskolin (10 µM) for 10 min, the
duct lumen rapidly expanded in response to this secretagogue, reaching
a maximum expansion of +87 ± 20 (relative to baseline,
n = 13) 5 min after forskolin was withdrawn.
Thereafter, the lumen volume progressively declined, returning to
baseline (
5 ± 10%) by 30 min, consistent with either absorption or leakage of fluids from the closed luminal space. However,
in the NHE3 (
/
) IBDU, the luminal volume did not decline after
reaching maximum expansion during the 10-min forskolin infusion compared with controls (+69 ± 14%, n = 22) but
remained expanded compared with controls (68 ± 21% above
baseline) for the duration of the experiment. This strongly suggests
that NHE3 and not leakage is responsible for the observed fluid
absorption from the cholangiocyte lumen. The role of cholangiocyte NHE3
in facilitating fluid absorption was examined further in rat and
wild-type mouse IBDU by assessing the effect of the
Na+/H+ exchange inhibitor EIPA (1 µM). In
control experiments in NHE3 (+/+) mouse IBDU, forskolin stimulated
expansion of the luminal space by 126 ± 38% (n = 10) (Fig. 5A). Luminal
expansion progressively diminished after withdrawal of forskolin to
1 ± 23% above baseline values. EIPA slowed forskolin's
maximum secretory response (+80 ± 21%, n = 12).
However, in contrast to the control experiment, EIPA reduced the
decline in luminal expansion after forskolin to 61 ± 16% by 45 min, consistent with involvement of an NHE in fluid reabsorption from
the apical surface of NHE3 (+/+) mouse cholangiocytes. A similar
pattern was observed in rat IBDU (Fig. 5B). On stimulation
with 10 µM forskolin, rat IBDU expanded by 137 ± 29%
(n = 15), followed by a progressive decrease to 48 ± 21% above baseline values by the end of the experiment. Normal rat
IBDU incubated in 1 µM EIPA maximally expanded by 103 ± 29%, and their luminal expansion only decreased to 88 ± 22% above
baseline values by 50 min (n = 12).
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DISCUSSION |
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In the present study, we identified NHE3 on the luminal membranes
of cholangiocytes and hepatocytes and demonstrated a role for this NHE
isoform in fluid secretion and absorption from the luminal space of
isolated mice and rat cholangiocyte preparations. NHE3 was localized by
immunofluorescence staining to the apical membrane in both rat
cholangiocytes and hepatocytes using isoform-specific MAbs. These sites
of membrane expression of NHE3 were also demonstrated by immunoelectron
microscopy. Western blot analysis confirmed the presence of NHE3
protein in cholangiocytes and in purified canalicular membranes,
consistent with the findings of immunocytochemistry. We next used mice
in which the NHE3 gene had been disrupted by gene targeting
(29) to determine whether the NHE3 isoform was involved in
absorption of fluid from the lumen of isolated cholangiocyte preparations. Net fluid secretion/absorption was assessed using IBDU
from NHE3 (+/+) and (/
) mice and videomicroscopic techniques. IBDU
from rat and mice have been characterized extensively and are useful in
vitro models to assess secretory responses in this epithelium. IBDU
form closed spaces in short term culture and accumulate fluid within
the spaces after exposure to various secretagogues including secretin,
dibutyryl cAMP, forskolin, VIP, and bombesin (1, 13, 23,
32). Measurements of cross-sectional diameters of this space by
videomicroscopy have been validated as quantitative determinations of
changes in volume within the duct lumens (32).
Using this approach, we have demonstrated that the NHE3 isoform is responsible for fluid absorption in the IBDU, because fluid was not absorbed if NHE3 was inactivated by gene knockout or by the NHE inhibitor EIPA. This finding contrasts markedly with observations in the wild-type mouse and rat, in which the expanded luminal spaces of these IBDU progressively diminish immediately after cessation of the forskolin infusion. The studies with EIPA further support a role for NHE isoforms in fluid absorption because a qualitatively similar pattern of inhibition of fluid absorption was observed after its administration. However, because EIPA would be expected to inhibit all NHE isoforms at this dose, we cannot exclude an indirect effect of other NHE isoforms on the luminal expansion in those inhibitor experiments. Also, because stimulation of luminal expansion during forskolin administration was slightly less in both the knockout and EIPA experiments, rather than enhanced above controls as might have been expected if absorption was inhibited, other nonspecific effects on the degree of luminal expansion cannot be excluded in these experiments.
Previous studies using isolated, perfused segments of rat bile ducts have also provided evidence for a functional NHE in the apical membrane that is distinct from a basolateral NHE because it is activated only at pH values <7.0 (30). NHE3 has also been detected in the apical membranes of several other polarized epithelial structures including the kidney, intestine, gallbladder, and salivary glands (4, 9, 18, 25). Limited functional studies have suggested that NHE3 is primarily involved in NaCl and NaHCO3 absorption from these epithelia rather than pHi regulation. The functional analyses in the present study are consistent with this property and provide the first direct demonstration suggesting that 1) fluid secretion is counterbalanced by fluid absorption in cholangiocytes in the normal resting bile duct epithelia and 2) inhibition of the process of absorption may contribute to net secretion after the stimulation of adenyl cyclase by forskolin. These findings add a new level of complexity to our understanding of the mechanisms of regulation of fluid secretion by the cholangiocyte epithelium.
Heretofore, fluid secretion from cholangiocytes has been thought to be
regulated by cAMP-mediated activation of Cl secretion via
a CFTR homologue in the apical membrane of cholangiocytes. This
Cl
channel is coupled to an apical
Cl
/HCO3
exchanger on the apical domain,
resulting in net secretion of HCO3
in the bile duct
lumen (1, 19, 31). On the basis of the current studies and
observations from other epithelia, a cAMP- and protein kinase
A-mediated inhibition of NHE3 now also seems to be involved. How this
effect of cAMP is mediated is not clear. However, studies in fibroblast
cell lines suggest that for this phosphorylation to occur, additional
regulatory proteins are required, known as NHE3 regulatory factor or
NHE3 kinase A regulatory protein, that bind to NHE3 via PDZ domains
(33). Recycling of NHE3 between subapical
compartments and the apical domain of cholangiocytes might also be
involved in the regulation of NHE3 activity, as described in other
tissues (15). Further studies will be necessary to
evaluate whether these mechanisms have a similar regulatory role for
NHE3 in fluid secretion by cholangiocytes.
The finding of a NHE3 protein by immunofluorescence, immunoelectron microscopy, and Western blotting in the hepatocyte apical canalicular membrane of rat liver was unexpected. Previous functional studies in isolated basolateral and canalicular membrane vesicles from rat liver had found NHE activity only in basolateral membranes and not at the apical canalicular domain (24). Therefore, in the present study, we assessed this possibility further using isolated hepatocyte couplets from mouse and rat liver. This well-characterized model, like IBDU, is a polarized secretory unit that can be maintained in short-term culture and permits assessment of fluid secretion from the bile canaliculus of the hepatocyte by videomicroscopy (7). As shown in this study, NHE3 was also detected by immunofluorescence microscopy on the apical canalicular domain of hepatocyte couplets (Fig. 1C). However, unlike IBDU from normal rats and mice, normal mouse and rat hepatocyte couplets did not demonstrate a decline in luminal expansion immediately after cessation of the forskolin-induced secretory response, which would have been expected if absorption of fluid in the canalicular space were significant. Although we cannot exclude the possibility of fluid absorption over longer time periods, more prolonged observation of couplets is technically difficult because of cytoskeleton-mediated contractions that result in leakage from the closed canalicular space in this model. Thus both our previous studies in membrane vesicles (24) and the present findings suggest that NHE3 is not functional at the apical canalicular domain. Future studies will need to determine what factors may be responsible. In summary, the present study provides the first evidence for the presence of NHE3 in cholangiocytes and hepatocytes and demonstrates a functional role for NHE3 in fluid absorption from cholangiocyte epithelia. Further studies of the regulation of biliary fluid production must take into account the role of apical NHE3 in the net secretion of bile.
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FOOTNOTES |
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Address for reprint requests and other correspondence: James L. Boyer, PO Box 208019, Yale Univ. School of Medicine, 333 Cedar St., Rm. 1080 LMP, New Haven, CT 06520-8019 (E-mail: james.boyer{at}yale.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 30 June 2000; accepted in final form 8 September 2000.
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