Characterization of regulatory volume decrease in freshly
isolated mouse cholangiocytes
Won Kyoo
Cho
Department of Medicine, Division of
Gastroenterology/Hepatology, Indiana University School of Medicine
and The Richard L. Roudebush Veterans Affairs Medical Center,
Indianapolis, Indiana 46202 - 5121
 |
ABSTRACT |
Cell volume
regulation plays a vital role in many cell functions. Recent study
indicates that both K+ and Cl
channels are
important for the regulatory volume decrease (RVD) of
cholangiocarcinoma cells, but its physiological significance is unclear
due to the tumorous nature of the cells used. This present study
reports the RVD of normal mouse cholangiocytes by using freshly
isolated bile duct cell clusters (BDCC). A relatively simple and
practical method of measuring the cross-sectional area of BDCCs by
quantitative videomicroscopy was used to indirectly measure their
volumes. Mouse cholangiocytes exhibited RVD, which was inhibited by
5-nitro-2'-(3-phenylpropylamino)-benzoate, DIDS, and glibenclamide,
suggesting its dependence on certain chloride channels, such as
volume-activated chloride channels. It is also inhibited by barium
chloride but not by tetraethylammonium chloride, indicating its
dependence on certain potassium channels. However, cAMP agonists had no
significant effect on the RVD of BDCCs. This indirect method described
can be used to study the RVD of cholangiocytes from normal as well as
genetically altered mouse livers.
ion channel; quantitative videomicroscopy
 |
INTRODUCTION |
OSMOREGULATION PLAYS A
VITAL role in hepatobiliary metabolism, ion transport, and gene
expression and thus is closely regulated (13). Under
physiological conditions, cholangiocytes are subjected to various
osmotic stresses from swelling, due to uptake of solutes and
electrolytes, and bile secretion (12, 15). Recent studies (23) indicate that cholangiocytes, as in other cells, are
able to regulate their cell volumes back to baseline from swelling induced by exposure to hypotonic solution. These adaptive mechanisms of
regulatory volume decrease (RVD) are mediated by concurrent activation
of separate but complementary K+ and Cl
conductances (23). Moreover, this RVD in cholangiocytes is thought to involve activation of PKC (21), ATP release
with purinergic receptor interactions (22), and
phosphoinositol 3-kinase activation (10).
Recently, we have developed a method for preparing novel intact
polarized isolated bile duct units (IBDU) from mouse liver, which
consists of clustered cholangiocytes lining a central lumen (8). This method eliminates the need for difficult and
time-consuming microdissection and produces many functional and
polarized IBDUs from 30- to 100-µm-sized murine bile ducts after
several steps of enzymatic digestion and mechanical separation
(8). In the present study, we used primary murine bile
duct cell clusters (BDCC), which were prepared by the same isolation
method used for IBDUs but which lack enclosed lumens. Because of the
possibility that changes in the osmolarity of the perfusion medium may
alter biliary secretion, and therefore luminal volume, thus possibly invalidating or complicating the interpretation of cell volume measurements, only BDCCs were included selectively in this study for
the purpose of studying changes in cell volume.
In the present study, we present an easy method of measuring changes in
cell volume using quantitative videomicroscopy and the validation of
this method using other cell volume-measurement techniques. We also
present some evidence that the primary BDCCs isolated from normal mouse
liver demonstrate an intact RVD, which involves K+ and
Cl
conductances, thereby confirming the results obtained
from a biliary tumor cell line (23). Furthermore, we
present additional data on further characterization of these
K+- and Cl
-conductance pathways using various
inhibitors and agonists of ion transporters.
 |
MATERIALS AND METHODS |
Materials.
Bovine serum albumin, penicillin/streptomycin, EDTA, HEPES,
D(+)-glucose, insulin, DMSO, hyaluronidase,
deoxyribonuclease (DN-25), tetraethylammonium chloride (TEA), barium
chloride, sucrose, DIDS, and 5-nitro-2'-(3-phenylpropylamino)-benzoate
(NPPB) were purchased from Sigma (St. Louis, MO). Matrigel was from
Collaborative Biomedical (Bedford, MA), collagenase D was from Roche
Applied Sciences (Indianapolis, IN), and pronase was from Calbiochem
(San Diego, CA). Liebowitz-15 (L-15), MEM,
-MEM,
L-glutamine, gentamicin, and fetal calf serum were from
GIBCO (Grand Island, NY). Monoclonal anti-cytokeratin 19 antibody was
from Amersham. All other chemicals were of highest purity commercially available.
Solutions.
The compositions of the Krebs-Ringer bicarbonate (KRB) and HEPES buffer
solutions [containing (in mM) 135 NaCl, 4.7 KCl, 1.2 KH2PO4, 1.25 CaCl2, 10 HEPES, 1 MgSO4, and 5 glucose, pH 7.4, 37°C] have been described
previously (1, 26). Isotonic solution was made by
replacing 40% of the NaCl in the HEPES solution (pH 7.4 at 37°C)
with an equimolar amount of sucrose. Hypotonic solution was the same as
the isotonic solution by removing 40% of the NaCl in the HEPES
solution (pH 7.4 at 37°C) but without the sucrose. The actual
osmolarities of the solutions used were determined by a Vapor Pressure
Osmometer 5500 (Wescor, Logan, UT).
Isolation of BDCC.
Normal male C57BL6 (Harlan Laboratory, Indianapolis, IN) mice at age
5-12 wk were housed and allowed free access to water and Purina
rodent chow (St. Louis, MO). Animal care and studies were performed in
compliance with institutional animal care and use committee guidelines.
Mouse BDCCs lacking enclosed lumen were prepared using the same methods
used to isolate mouse IBDUs as previously described by our lab
(8). Briefly, mice were prepared and their livers were
perfused via the portal vein with Hank's buffers with collagenase D,
and were then harvested. After the hepatic capsule was removed, the
intrahepatic bile ducts were mechanically dissociated from hepatic
parenchymal tissue by shaking in cold L-15 medium and then
using pressure from medium forced through a 20-gauge syringe to further
remove the attached hepatocytes. The remaining nonparenchymal tissue
was then finely minced with scissors in solution A, containing various
enzymes including pronase as previously described (8). The
minced tissue was then transferred to a culture flask and incubated in
37°C shaker for ~30 min, then was filtered through 100- and 30-µm
meshes Nitex Swiss nylon monofilament screens (Tetko, Lancaster, NY).
Fragments remaining on the 30-µm mesh were digested for an additional
20-30 min in the enzyme solution, filtered through the sized
meshes as before, then further digested in solution consisting of the
same components as solution A except with hyaluronidase instead of
pronase. After 15-20 min incubation in solution B in a 37°C
shaker, fragments were again filtered through the mesh, and those
remaining on the 30-µm mesh were collected in 3-6 ml modified
-MEM medium as described (8). Fragments were then
plated on small coverslips (2-4 mm), coated with Matrigel (Collaborative Research), in 12-mm-diameter tissue culture wells (Corning) and incubated at 37°C in an air-5%
CO2-equilibrated incubator. Experiments were carried out
36-56 h after plating. Cell viability was assessed by trypan blue
exclusion in plated BDCCs at the end of the functional studies.
Characterization of Mouse BDCC.
Cytokeratin (CK) immunocytochemistry using CK-19 antibody (8,
16, 26) was performed in BDCCs 48-72 hr after plating. Immunofluorescent images were obtained using Olympus IX-70 inverted fluorescent microscope (Olympus America, Melville, NY) with a cooled
charge-coupled device (CCD) video camera (Hamamatsu Photonics Systems,
Bridgewater, NJ) connected to a Power Mac computer with image-analysis
software (Improvision, Boston, MA). In addition, confocal micrograph of
the mouse BDCCs, immunofluorescently stained with CK-19 antibody, were
obtained using a Bio-Rad 1024 MRC laser-scanning confocal microscope
(Hercules, CA) equipped with a Kr/Ar laser.
Quantitation of regulatory volume response with videomicroscopy.
BDCCs cultured overnight on Matrigel-coated glass coverslips were
preincubated in KRB solution for 10-20 min after being placed in a
thermostated specimen chamber on a microscope stage. Coverslips were
scanned for 5-10 min to select relatively spheroid BDCCs with
sharp borders and without connections to other contiguous BDCCs and
without any enclosed lumen. Video images of these BDCCs were obtained
at 1- to 5-min intervals while maintaining the same focal plane at the
maximum cross-sectional area (CSA). Osmoregulatory responses of BDCCs
were determined by assessing the changes in CSA of BDCCs using an
Olympus IX-70 (Olympus America) or Leica DMIR (Leica Microsystems,
Bannockburn, IL) inverted microscope with Nomarski optics equipped with
CCD video camera (Hamamatsu Photonics Systems) connected to a computer
with OpenLab image-analysis software (Improvision). After a 10- to
20-min prestimulation period with isotonic HEPES buffer alone, BDCCs
were exposed to hypotonic HEPES buffer for 40 min with or without
various inhibitors or agonists dissolved in the solution. Each BDCC
served as its own internal control and changes in CSA were expressed as
a percentage of baseline values at time 0. Viability of each
BDCC was assessed by the addition of trypan blue to the specimen
chamber after each experiment. The BDCCs with positive trypan blue
staining were excluded from data analysis. However, there was no
significant change in viability, assessed by trypan blue staining, in
experimental groups exposed to various inhibitors or chemicals compared
with controls.
Previously, measurements of CSAs of hepatocytes and cholangiocytes were
shown to reflect changes in volume (19). Relative CSA
measurements of BDCCs obtained by quantitative videomicroscopy were
validated by three independent methods: 1) sequential light, 2) fluorescence microscopy of BDCCs loaded with
intracellular fluorescent dye BCECF and computer-assisted measurements
of the corresponding CSA and volume calculations, and 3)
laser-scanning confocal microscopy of BCECF-loaded BDCCs and
computer-assisted three-dimensional reconstruction and volume calculation.
Quantitative fluorescent microscopy.
Fluorescent micrographs were obtained using Olympus IX-70 inverted
fluorescent microscope (Olympus America) with a cooled CCD video camera
(Hamamatsu Photonics Systems) connected to a Power Mac computer with
OpenLab image-analysis software (Improvision). Serial cross-sectional
fluorescent images of BCECF-loaded BDCCs were obtained with fluorescent
excitation at 490 nm, and the emission collected >505 nm as focal
plane was advanced in 1-µm increments through the cell thickness by
using a BioPoint Z-stepper (Ludl Electronic Products, Hawthorne, NY).
The CSAs of the BDCCs from the serial fluorescent images were analyzed
using the image-analysis program and were used to calculate their volumes.
Laser-scanning confocal microscopy.
Cell images were collected with an inverted BioRad laser-scanning
confocal microscope. Serial confocal fluorescence images of
BCECF-loaded BDCCs were collected as the focal plane was advanced in
1-µm increments through the cell thickness. Images from each focal
plane were collected with fluorescence excitation at 488 nm, and the
emission was collected at >520 nm. Confocal images were then analyzed
with computer programs for three-dimensional reconstruction and volume calculation.
Statistical analysis.
All data from videomicroscopic measurements are presented as the
arithmetic means ± SE. Statistical differences were assessed by
the unpaired or paired Student's t-tests using the INSTAT
statistical computer program (GraphPad Software, San Diego, CA). In
addition, the curve fits for linear and power-regression analyses were
done using CA-Cricket Graph III (Computer Associates International, Islandia, NY).
 |
RESULTS |
Characterization of BDCCs.
As is the case with rat tissue, bile duct fragments from mouse liver,
isolated immediately after serial enzymatic digestions, appeared as
tubulelike structures that formed spherical clusters of cells with
24-48 h in culture (Fig. 1).
Viability of the BDCCs studied was >95%, as assessed by trypan blue
exclusion, after 24-72 h in culture. As with the normal mouse
IBDUs characterized previously (8), the cells comprising
these BDCCs were identified as bile duct epithelial cells by positive
immunocytochemistry using an antibody to CK-19, whereas negative
controls using secondary antibody alone were consistently negative for
immunostaining (Fig. 2).

View larger version (101K):
[in this window]
[in a new window]
|
Fig. 1.
Videomicroscopy of normal mouse bile duct cell clusters
(BDCC) with hypotonic maneuver. Normal mouse BDCCs (top and
bottom) were preincubated in modified isotonic HEPES
solution (40% of NaCl replaced with equimolar sucrose)
(top, left), then were exposed to hypotonic solution
(isotonic solution without sucrose) for 40 min. Normal mouse BDCCs
swelled in hypotonic solution by +24.6% (top) and +24.2%
(bottom) of their initial sizes (top, middle),
then slowly recovered toward their initial sizes of +5.6%
(top) and +4.9% (bottom), indicating an intact
RVD.
|
|

View larger version (91K):
[in this window]
[in a new window]
|
Fig. 2.
Cytokeratin immunocytochemistry of 48 h-cultured mouse BDCC.
A: light micrograph of the BDCC in B. B: immunofluorescent micrograph of a BDCC from mouse liver
stained for a-cytokeratin 19 shows brightly positive intracellular
stainings, whereas negative, unstained isolated bile duct units (IBDUs;
not shown) have a minimal background autofluorescence.
|
|
Validation of quantitative videomicroscopic measurements of cell
volume.
As previously used for other cell types (17) and for
hepatocytes and cholangiocytes (19), measurements of CSA
have been used as indirect indexes of cell volumes in BDCCs. As shown
in Fig. 3, the CSAs of BDCCs in isotonic
or hypotonic HEPES solutions, as measured by quantitative
videomicroscopy, were linearly correlated (linear correlation
efficient, R2 = 0.95, n = 20) with the cell volumes of BDCCs, as measured by three-dimensional
volume reconstruction and calculations performed on serial optical
sections of BDCCs. Similar results were obtained by serial fluorescence
microscopy of BCECF-loaded BDCCs followed by three-dimensional
volume-measurement analysis (data not shown). The volumes of
BCECF-loaded BDCCs were also analyzed by laser-scanning confocal
microscopy followed by three-dimensional volume-measurement analysis.
As shown in Fig. 4, the cell volumes of
BDCCs, determined using this methodology, showed a significant linear
correlation (R2 = 0.98, n = 11), with the corresponding CSA values obtained using quantitative
videomicroscopy. These results confirmed the validity of using the CSA
measurements of BDCCs as indirect indexes of BDCC cell volume.
Furthermore, as shown in Figs. 3 and 4, the CSAs of BDCCs in the usual
size range of the BDCCs used are linearly correlated with their
corresponding cell volumes.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 3.
Comparison of cross-sectional area (CSA) measurements of
BDCCs by quantitative videomicroscopy with volume measurements by light
microscopy with 3-dimensional volume reconstruction. The CSA of BDCCs,
as measured by quantitative videomicroscopy, showed a very tight
(R2 = 0.95, n = 20) linear
correlation with corresponding volume measurements, obtained by
three-dimensional reconstructions and calculations on serial optical
sections of BDCCs.
|
|

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 4.
Comparison of CSA measurements of BDCCs by quantitative
videomicroscopy with volume measurements by confocal microscopy with
3-dimensional volume reconstruction. The CSA of BDCCs, as measured by
quantitative videomicroscopy, showed a very tight
(R2 = 0.98, n = 11) linear
correlation with their corresponding volume measurements, obtained by
confocal microscopy with 3-dimensional reconstructions and calculations
on serial confocal images of BDCCs.
|
|
Study of RVD in normal mouse cholangiocytes.
As expected from their compositions, the osmolarity of the isotonic
solutions, measured by a vapor pressure osmometer, was 300.9 ± 4.5 mosM (n = 12) and that of the hypotonic solutions was 181.9 ± 3.6 mosM (n = 13). As shown in Fig.
1, exposure of normal BDCCs to hypotonic HEPES solution after isotonic
HEPES solution caused rapid swelling of their cell volumes. These
changes in cell volume were assessed by changes in their corresponding CSAs, as measured by quantitative videomicroscopy. As shown in Fig.
5, the relative CSAs of BDCCs rapidly
increased to 1.24 ± 0.02 (n = 15) in 10 min after
exposure to hypotonic HEPES solution, then gradually returned to a
relative CSA of 1.06 ± 0.02 over the next 30 min. Importantly,
the viability of BDCCs, as assessed by trypan blue exclusion, was
unchanged after exposure to hypotonic solution. These results are
consistent with the previous studies of Mz-ChA-1 cells from a human
cholangiocarcinoma cell line, in which cholangiocytes exhibited an
intact RVD after exposure to hypotonic solution (23).

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 5.
Quantitative videomicroscopic measurement of changes in
CSA of normal mouse BDCC with hypotonic maneuver. The CSAs of BDCCs, as
measured by quantitative videomicroscopy, increased by >20% at 10 min
with hypotonic maneuver then returned toward their initial sizes (6%
in 40 min), indicating the presence of a regulatory volume decrease
(RVD) of normal mouse BDCCs.
|
|
Role of Cl
channels in RVD.
To examine the involvement of Cl
channels in the observed
RVD in cholangiocytes, NPPB, a Cl
-channel inhibitor, was
administered during the perfusion of hypotonic HEPES solution (5,
9, 24). As shown in Fig. 6,
coadministration of 10 µM NPPB with the hypotonic HEPES solution
completely inhibited the RVD in BDCCs, and the relative CSA after
40 min of hypotonic challenge was 1.16 ± 0.02 (n = 29), which was statistically significant (P < 0.01)
compared with the RVD seen with control BDCCs (1.07 ± 0.01, n = 35). These results indicate that the RVD of mouse cholangiocytes is dependent on Cl
conductance, as shown
previously (23). To further characterize the chloride
channels involved in the RVD of BDCCs, the effect of a glibenclamide
(100 µM, n = 12) on RVD was studied, as shown in Fig.
7. With hypotonic challenge, the relative
CSA of the glibenclamide-treated BDCCs increased to about the same
amount as untreated controls but only decreased to 1.14 ± 0.02 of
the initial CSA (n = 26) after 40 min, compared with
1.07 ± 0.01 in untreated controls (n = 32),
indicating significant (P < 0.01) inhibition of the RVD by glibenclamide. In addition, as shown in Fig.
8, another chloride channel blocker, DIDS
(250 µM), also had a significant (P < 0.05)
inhibitory effect on the RVD of BDCCs and the relative CSA of
DIDS-treated BDCCs (n = 7) after hypotonic challenge
was 1.15 ± 0.03, compared with 1.08 ± 0.01 in untreated
controls (n = 11). These results indicate that certain
chloride channels, which are inhibitable by NPPB, glibenclamide, and
DIDS, play some important role in the RVD of BDCCs. There was no
significant difference between the viabilities of BDCCs treated with
these inhibitors and control BDCCs, as assessed by trypan blue
exclusion.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of 5-nitro-2'-(3-phenylpropylamino)-benzoate
(NPPB) on RVD of normal mouse BDCCs. Coadministration of NPPB (10 µM)
during hypotonic (HYPO) maneuver inhibits the RVD of normal mouse
BDCCs, indicating a significant role of chloride channels in the RVD of
mouse cholangiocytes.
|
|

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 7.
Effect of glibenclamide on RVD of normal mouse BDCCs.
Coadministration of glibenclamide (100 µM) during hypotonic maneuver
inhibits the RVD of normal mouse BDCCs, suggesting a significant role
of certain chloride channels in the RVD of mouse cholangiocytes.
|
|

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 8.
Effect of DIDS on RVD of normal mouse BDCCs.
Coadministration of DIDS (250 µM) during hypotonic maneuver inhibits
RVD in normal mouse BDCCs, indicating a significant role of certain
chloride channels, such as volume-activated chloride channels, in RVD
of mouse cholangiocytes.
|
|
Role of K+ channels in RVD.
To study the role of K+ channels in the RVD of
cholangiocytes, the effect of a K+-channel inhibitor, TEA,
on RVD was examined. As shown in Fig. 9,
coadministration of 10-1,000 µM TEA had no significant effect on
the RVD, and the relative CSA at 40 min after hypotonic challenge returned toward basal CSA (n = 13). However, as shown
in Fig. 10, coadministration of barium
chloride (5 mM), which is another K+-channel inhibitor,
during the hypotonic challenge had a significant inhibitory effect on
the RVD. The relative CSA at 40 min after hypotonic challenge only
decreased to 1.16 ± 0.02 in barium chloride-treated BDCCs
(n = 6), compared with that in untreated controls
1.09 ± 0.02 (n = 11), indicating a significant
(P < 0.05) inhibitory effect of barium chloride on the
RVD. These findings suggest important roles for certain TEA-resistant,
barium chloride-sensitive K+ channel(s) mediating RVD.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 9.
Effect of tetraethylammonium chloride (TEA) on RVD of
normal mouse BDCCs. Coadministration of TEA (1 µM) during hypotonic
maneuver had no significant effect on RVD in normal mouse BDCCs.
|
|

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 10.
Effect of barium chloride on RVD of normal mouse BDCCs.
Coadministration of barium chloride (5 mM) during hypotonic maneuver
inhibits RVD in normal mouse BDCCs, indicating a significant role of
potassium channels in RVD of mouse cholangiocytes.
|
|
Effect of cAMP agonists on RVD.
To examine the effect of cAMP agonists on the RVD in cholangiocytes,
IBMX was administered during the hypotonic challenge and the RVD
responses were compared with normal controls. As shown in Fig.
11, there was no significant difference
in the RVD of BDCCs treated with IBMX (1 mM) compared with
untreated controls. In addition, the effect of a more potent cAMP
agonist forskolin on the RVD of BDCCs was studied. As shown Fig.
12, the RVD of BDCCs treated with
forskolin (10 µM; n = 10) appeared to be slower in the initial phase of RVD compared with that of untreated controls (n = 18), but there was no significant difference
between them. These findings indicate that stimulation of
cholangiocytes with cAMP has no significant effect on the RVD.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 11.
Effect of IBMX on RVD of normal mouse BDCCs.
Coadministration of IBMX (1 mM) during hypotonic maneuver had no
significant effect on the RVD of normal mouse BDCCs.
|
|

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 12.
Effect of forskolin on RVD of normal mouse BDCCs.
Coadministration of forskolin (10 µM) during hypotonic maneuver had
no significant effect on the RVD of normal mouse BDCCs.
|
|
 |
DISCUSSION |
In this manuscript, we report the successful isolation and use of
isolated BDCCs from normal mouse liver using the mouse IBDU isolation
method developed and reported recently (8). Although we
have previously made extensive use of IBDUs for the study of biliary
secretion, we have excluded these lumen-containing IBDUs and have used
in this study only BDCCs, which lack enclosed lumens, to facilitate the
measurement of cell cluster volume by eliminating the need to consider
possible changes in luminal volume due to hypotonic challenge. These
BDCCs were determined to be of biliary origin by positive
immunocytochemistry using a cholangiocyte-specific CK-19 antibody.
Similar to mouse IBDUs, these BDCCs have less connective tissue around
them than rat IBDUs and exhibit a more refractory pattern than rat
IBDUs by light microscopy using Nomarski optics. Thus measurement of
CSA by tracing of the borders of individual cell clusters is easier
with mouse BDCCs than with rat IBDUs.
To assess changes in cell cluster volume during hypotonic challenge, we
have used CSA as an indirect measure of volume, as done previously in
other cell types by other investigators (17, 19). The
validity of CSA measurements of BDCCs by quantitative videomicroscopy
as indirect measurements of cell cluster volume was confirmed by three
independent methods: sequential light microscopy, fluorescence
microscopy, and laser-scanning confocal microscopy of BCECF-loaded
BDCCs, each of which was followed by computer-assisted measurements
of the corresponding CSAs and volumes. As shown in Figs. 2 and 3, CSA
measurements of BDCCs are very well correlated with their corresponding
volume measurements, confirming the validity of CSA measurements of
BDCCs as indirect measurements of their corresponding volumes. It is
notable that the CSA measurements of BDCCs are linearly correlated with
the corresponding volume measurements in the usual ranges of volumes of
BDCCs. Thus this relatively simple method of quantitative
videomicroscopy will allow accurate measurements of volume changes by
measuring CSA to further study underlying mechanisms of cell volume
regulation in primary bile duct cell preparations. Furthermore, given
the inherent limitations of mouse cholangiocyte preparations due to their low cell number and yield in mouse liver, this method provides the only practical method to study cell volume regulation in primary mouse cholangiocytes from normal as well as from various knockout mouse
livers. In fact, we tried to use a coulter counter with cell sizer to
measure cell volume changes of primary cholangiocyte preparations but
were not successful due to low amount and suboptimal purity of the
cholangiocytes obtained from mouse livers and some methodological
problems with cellular debris resulting from isolation procedure.
As previously reported in other cell types (3, 14, 17, 24)
as well as in Mz-ChA-1 cells from human cholangiocarcinoma cell lines
(23), normal mouse cholangiocytes exhibit an intact RVD,
dependent on both Cl
and K+ conductances. As
shown in Fig. 1, normal mouse BDCCs swell rapidly after exposure to
hypotonic buffer, then gradually return toward their original cell
volumes, as reflected by the changes in CSA shown in Fig. 5, indicating
an intact RVD in these cell clusters. The RVD observed in normal BDCCs
is significantly inhibited by general Cl
-channel blockers
such as NPPB and DIDS, indicating the significant role of
Cl
channels in the RVD of cholangiocytes (Figs. 5 and 7).
In various cell types, DIDS has been shown to inhibit volume-activated
chloride channels (24), calcium-activated chloride
channels (2, 4), outwardly rectifying chloride channels
(6), and ATP release (6) but does not inhibit
CFTR (2, 11). Thus the inhibition of RVD by DIDS implies
that chloride conductance via CFTR may not play a major role in RVD in
cholangiocytes, and this conclusion is consistent with the finding that
cAMP agonists had no significant effect on RVD. The RVD of BDCCs is
also inhibited by glibenclamide (Fig. 7), which is a known inhibitor of
CFTR, volume-activated chloride channels, as well as ATP-sensitive
K+ (KATP) channels (25). However,
RVD was not affected by coadministration of TEA at 10-1,000 µM
(Fig. 9), suggesting that the KATP channel had no
significant role in RVD because the KATP channel is
inhibited by TEA (18). Therefore, these findings as a
whole indicate an important role of the volume-sensitive chloride
channel in RVD in cholangiocytes but cannot exclude a potential
contributory role of other chloride channels such as calcium-activated
chloride channels.
Next, the role of K+ channels in the RVD of BDCCs is
examined. As shown in Fig. 10, the RVD of BDCCs is inhibited by
BaCl2, a K+-channel blocker, whereas it is
resistant to TEA, another K+-channel blocker, indicating
the significant role of certain K+ channels in the RVD of
cholangiocytes. A recent study of the RVD of Mz-ChA-1 cells from a
human cholangiocarcinoma cell line (20) showed that a
volume-sensitive SKCa channel is involved in the RVD of human
cholangiocarcinoma cells. It is notable that these SKCa channels are
known to be sensitive to BaCl2 but resistant to TEA,
consistent with our results from normal mouse cholangiocytes (27). However, further characterization of these ion
channels in normal cholangiocytes, using detailed patch-clamping
studies, is needed to better define these pathways.
Our present study also examined the effects of cAMP agonists on the RVD
of cholangiocytes. The cAMP agonists IBMX (Fig. 11) and forskolin (Fig.
12) had no significant effects on the RVD of BDCCs. These findings
indicate that an activation of cAMP-dependent Cl
or
K+ conductive pathways does not significantly stimulate the
RVD of cholangiocytes and/or that cAMP-dependent pathways are not rate-limiting steps in the RVD of cholangiocytes. Thus CFTR may not
play a major role in providing chloride conductive pathways for the RVD
of cholangiocytes, but volume-activated chloride channels may have a
more important role in the RVD of cholangiocytes. However, it does not
exclude the possibility that CFTR may be important for regulation of
various Cl
or K+ channels and conductive
pathways mediating RVD of cholangiocytes. In fact, our preliminary data
in Cftr
/
mouse BDCCs indicate that CFTR is important for
regulating K+ conductive pathways involved in the RVD of
cholangiocytes (unpublished observation).
In conclusion, our present study provides a simple method to quantify
cell volume changes by measuring CSAs of the primary BDCCs isolated
from mouse livers. It is the only practical method to study cell volume
regulation in mouse cholangiocytes. In the present study, we have shown
that cholangiocytes from normal mouse livers can regulate their cell
volumes toward their basal cell volumes during hypotonic challenges, as
shown previously in cholangiocarcinoma cells (23). As in
cholangiocarcinoma cell lines, the RVDs of normal cholangiocytes are
mediated by chloride channels, which are inhibited by NPPB, DIDS, and
glibenclamide but are not stimulated by cAMP agonists and by potassium
channels, which are inhibited by barium chloride but not by TEA. With
the use of this method and primary BDCC preparations, one can further
study the underlying ion transports and mechanisms involved in the RVD
of cholangiocytes from normal as well as genetically altered mouse livers.
 |
ACKNOWLEDGEMENTS |
We appreciate T. Beyer, B. Poteat, and B. C. Mgboh for
technical support performing videomicroscopy and cell preparation.
 |
FOOTNOTES |
W. K. Cho was supported by an Indiana Univ. biomedical research grant,
National Institute of Diabetes and Digestive and Kidney Diseases Grants
KO8-DK-02613 and R03-DK-61409, and a cystic fibrosis research grant.
Address for reprint requests and other correspondence: W. K. Cho, Indiana Univ. School of Medicine, Roudebush VA Medical Center, Div. of GI/Hepatology (111G), 1481 W. 10th St., Indianapolis, IN 46202 (E-mail: wkcho{at}iupui.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.
10.1152/ajpgi.00256.2002
Received 1 July 2002; accepted in final form 2 September 2002.
 |
REFERENCES |
1.
Alvaro, D,
Cho WK,
Mennone A,
and
Boyer JL.
Effect of secretin on intracellular pH regulation in isolated rat bile duct epithelial cells.
J Clin Invest
92:
1314-1325,
1993[ISI][Medline].
2.
Anderson, MP,
and
Welsh MJ.
Calcium and cAMP activate different chloride channels in the apical membrane of normal and cystic fibrosis epithelia.
Proc Natl Acad Sci USA
88:
6003-6007,
1991[Abstract].
3.
Banderali, U,
and
Roy G.
Activation of K+ and Cl
channels in MDCK cells during volume regulation in hypotonic media.
J Membr Biol
126:
219-234,
1992[ISI][Medline].
4.
Basavappa, S,
Middleton J,
Mangel AW,
McGill JM,
Cohn JA,
and
Fitz JG.
Cl
1 and K+ transport in human biliary cell lines.
Gastroenterology
104:
1796-1805,
1993[ISI][Medline].
5.
Best, L,
Sheader EA,
and
Brown PD.
A volume-activated anion conductance in insulin-secreting cells.
Pflügers Arch
431:
363-370,
1996[ISI][Medline].
6.
Braunstein, GM,
Roman RM,
Clancy JP,
Kudlow BA,
Taylor AL,
Shylonsky VG,
Jovov B,
Peter K,
Jilling T,
Ismailov II,
Benos DJ,
Schwiebert LM,
Fitz JG,
and
Schwiebert EM.
Cystic fibrosis transmembrane conductance regulator facilitates ATP release by stimulating a separate ATP release channel for autocrine control of cell volume regulation.
J Biol Chem
276:
6621-6630,
2001[Abstract/Free Full Text].
7.
Cho, WK,
Beyer T,
Backleund DC,
Cressman VM,
and
Koller BH.
Impaired regulatory volume decrease (RVD) in cholangiocytes from cystic fibrosis (CF) mice (Abstract).
Hepatology
30:
461A,
1999.
8.
Cho, WK,
Mennone A,
and
Boyer JL.
Isolation of functional polarized bile duct units from mouse liver.
Am J Physiol Gastrointest Liver Physiol
280:
G241-G246,
2001[Abstract/Free Full Text].
9.
Ehring, GR,
Osipchuk YV,
and
Cahalan MD.
Swelling-activated chloride channels in multidrug-sensitive and -resistant cells.
J Gen Physiol
104:
1129-1161,
1994[Abstract].
10.
Feranchak, AP,
Roman RM,
Doctor RB,
Salter KD,
Toker A,
and
Fitz JG.
The lipid products of phosphoinositide 3-kinase contribute to regulation of cholangiocyte ATP and chloride transport.
J Biol Chem
274:
30979-30986,
1999[Abstract/Free Full Text].
11.
Fitz, JG,
Basavappa S,
McGill J,
Melhus O,
and
Cohn JA.
Regulation of membrane chloride currents in rat bile duct epithelial cells.
J Clin Invest
91:
319-328,
1993[ISI][Medline].
12.
Graf, J,
and
Haussinger D.
Ion transport in hepatocytes: mechanisms and correlations to cell volume, hormone actions and metabolism.
J Hepatol
24:
53-77,
1996[ISI][Medline].
13.
Haussinger, D.
Regulation and functional significance of liver cell volume.
Prog Liver Dis
14:
29-53,
1996[Medline].
14.
Hazama, A,
and
Okada Y.
Ca2+ sensitivity of volume-regulatory K+ and Cl
1 channels in cultured human epithelial cells.
J Physiol
402:
687-702,
1988[Abstract].
15.
Lira, M,
Schteingart CD,
Steinbach JH,
Lambert K,
McRoberts JA,
and
Hofmann AF.
Sugar absorption by the biliary ductular epithelium of the rat: evidence for two transport systems.
Gastroenterology
102:
563-571,
1992[ISI][Medline].
16.
Mennone, A,
Alvaro D,
Cho W,
and
Boyer JL.
Isolation of small polarized bile duct units.
Proc Natl Acad Sci USA
92:
6527-6531,
1995[Abstract].
17.
Mignen, O,
Le Gall C,
Harvey BJ,
and
Thomas S.
Volume regulation following hypotonic shock in isolated crypts of mouse distal colon.
J Physiol
515:
501-510,
1999[Abstract/Free Full Text].
18.
Quayle, JM,
Bonev AD,
Brayden JE,
and
Nelson MT.
Pharmacology of ATP-sensitive K+ currents in smooth muscle cells from rabbit mesenteric artery.
Am J Physiol Cell Physiol
269:
C1112-C1118,
1995[Abstract/Free Full Text].
19.
Roberts, SK,
Yano M,
Ueno Y,
Pham L,
Alpini G,
Agre P,
and
LaRusso NF.
Cholangiocytes express the aquaporin CHIP and transport water via a channel-mediated mechanism.
Proc Natl Acad Sci USA
91:
13009-13013,
1994[Abstract/Free Full Text].
20.
Roman, R,
Feranchak AP,
Troetsch M,
Dunkelberg JC,
Kilic G,
Schlenker T,
Schaack J,
and
Fitz JG.
Molecular characterization of volume-sensitive SKCa channels in human liver cell lines.
Am J Physiol Gastrointest Liver Physiol
282:
G116-G122,
2002[Abstract/Free Full Text].
21.
Roman, RM,
Bodily KO,
Wang Y,
Raymond JR,
and
Fitz JG.
Activation of protein kinase C
couples cell volume to membrane Cl
1 permeability in HTC hepatoma and Mz-ChA-1 cholangiocarcinoma cells.
Hepatology
28:
1073-1080,
1998[ISI][Medline].
22.
Roman, RM,
Feranchak AP,
Salter KD,
Wang Y,
and
Fitz JG.
Endogenous ATP release regulates Cl
1 secretion in cultured human and rat biliary epithelial cells.
Am J Physiol Gastrointest Liver Physiol
276:
G1391-G1400,
1999[Abstract/Free Full Text].
23.
Roman, RM,
Wang Y,
and
Fitz JG.
Regulation of cell volume in a human biliary cell line: activation of K+ and Cl- currents.
Am J Physiol Gastrointest Liver Physiol
271:
G239-G248,
1996[Abstract/Free Full Text].
24.
Schmid, A,
Blum R,
and
Krause E.
Characterization of cell volume-sensitive chloride currents in freshly prepared and cultured pancreatic acinar cells from early postnatal rats.
J Physiol
513:
453-465,
1998[Abstract/Free Full Text].
25.
Sheppard, DN,
and
Welsh MJ.
Effect of ATP-sensitive K+ channel regulators on cystic fibrosis transmembrane conductance regulator chloride currents.
J Gen Physiol
100:
573-591,
1992[Abstract].
26.
Strazzabosco, M,
Mennone A,
and
Boyer J.
Intracellular pH regulation in isolated rat bile duct epithelial cells.
J Clin Invest
87:
1503-1512,
1991[ISI][Medline].
27.
Vogalis, F,
and
Goyal RK.
Activation of small conductance Ca2+-dependent K+ channels by purinergic agonists in smooth muscle cells of the mouse ileum.
J Physiol
502:
497-508,
1997[Abstract].
Am J Physiol Gastrointest Liver Physiol 283(6):G1320-G1327