Division of Gastroenterology, Department of Medicine,
University of Washington and Veterans Affairs Puget Sound Health
Care System, Seattle, Washington 98108
Pancreatic duct epithelial cells
(PDEC) mediate the secretion of fluid and electrolytes and are exposed
to refluxed bile. In nontransformed cultured dog PDEC, which express
many ion transport pathways of PDEC, 1 mM taurodeoxycholic acid (TDCA)
stimulated an 125I
efflux inhibited by DIDS
and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and a
86Rb+ efflux inhibited by charybdotoxin.
Inhibition by
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM suggests mediation via increased intracellular Ca2+ concentration, whereas the absence of lactate
dehydrogenase release excludes cellular toxicity. At 1 mM, TDCA
stimulated a larger 125I
efflux than
glycodeoxycholate; two dihydroxy bile acids, taurochenodeoxycholate and
TDCA, were similarly effective, whereas a trihydroxy bile acid,
taurocholate, was ineffective. In Ussing chambers, 1 mM serosal or 2 mM
luminal TDCA stimulated an Isc increase from
confluent PDEC monolayers. TDCA also stimulated 1) a
short-circuit current (Isc) increase from
basolaterally permeabilized PDEC subject to a serosal-to-luminal
Cl
gradient that was inhibited by BAPTA-AM, DIDS, and
NPPB and 2) an Isc increase from
apically permeabilized PDEC subject to a luminal-to-serosal
K+ gradient inhibited by BAPTA-AM and charybdotoxin. Along
with the efflux studies, these findings suggest that TDCA interacts directly with PDEC to stimulate Ca2+-activated apical
Cl
channels and basolateral K+ channels.
Monolayer transepithelial resistance was only minimally affected by 1 mM serosal and 2 mM luminal TDCA but decreased after exposure to higher
TDCA concentrations (2 mM serosal and 4 mM luminal). A secretory role
for bile acids should be considered in pancreatic diseases associated
with bile reflux.
taurocholate; iodide and rubidium efflux; pancreatitis; Ussing
chamber; transepithelial resistance
 |
INTRODUCTION |
PANCREATIC DUCT
EPITHELIAL cells (PDEC) secrete fluid and electrolytes. Together
with acinar cells, which secrete digestive enzymes, they are the main
components of pancreatic exocrine secretion. Because the pancreatic and
the bile ducts share a common outflow in the duodenum, obstruction of
the ampulla of Vater may cause bile to reflux backward into the
pancreatic duct, exposing PDEC to bile acids. Indeed, in 1901, Opie
suggested that refluxed bile is a factor in gallstone pancreatitis.
Bile acids stimulate electrolyte secretion by epithelial cells from
different portions of the gastrointestinal tract, such as the large
bowel and the biliary tree (8, 9, 11, 12). However, to our
knowledge, the secretory effects of bile acids on PDEC have not been
evaluated thoroughly. Using cultured nontransformed dog PDEC that
express many secretory functions characteristic of PDEC (1, 13,
20), such as mucin secretion (21), cAMP- and
Ca2+-activated Cl
channels (14),
and Ca2+-activated K+ channels
(16), we 1) examined whether bile acids
activate Cl
and K+ conductances,
2) identified the signaling pathway mediating this effect,
3) compared the secretory effects of different bile acids, 4) studied the effects of bile acids on monolayer
transepithelial resistance (TER), and 5) compared the
effects of bile acids on the apical or basolateral membrane of the PDEC.
 |
MATERIAL AND METHODS |
Chemicals and reagents.
Taurodeoxycholic acid (TDCA), taurochenodeoxycholic acid,
glycodeoxycholic acid, taurocholic acid, charybdotoxin,
4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), sodium
pyrophosphate, lactate, and tissue culture medium and supplements were
from Sigma (St. Louis, MO).
1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM) was from Calbiochem (San
Diego, CA), and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) was
from Research Biochemicals International (Natick, MA). Na125I (16 mCi/mg iodide) was from Amersham (Arlington
Heights, IL), and 86RbCl (4.66 mCi/mg
86Rb+) was from NEN (Boston, MA).
PDEC culture.
As previously described (21), PDEC isolated from the
accessory pancreatic duct of a dog were cultured on Transwell or
Snapwell inserts (Costar, Cambridge, MA) coated with 0.7 ml (for
Transwell) or 0.15 ml (for Snapwell) of a 1:1 solution of Eagle's
MEM-Vitrogen (Collagen, Palo Alto, CA) over a feeder layer of
myofibroblasts and in Eagle's MEM supplemented with 10% FBS, 2 mM
L-glutamine, 20 mM HEPES, 100 IU/ml penicillin, 100 µg/ml
streptomycin, 5 µg/ml bovine insulin, 5 µg/ml human transferrin,
and 5 ng/ml sodium selenite. These PDEC express a cAMP-activated
Cl
channel, corresponding to the cystic fibrosis
transmembrane conductance regulator, a Ca2+-activated
Cl
channel (14), and
Ca2+-activated K+ channels (16).
Cells used in this report were between passages 9 and
30. Efflux studies, Ussing chamber recordings, TER
measurements, and lactate dehydrogenase (LDH) assays used confluent
PDEC on Transwell or Snapwell filters that were isolated from the
myofibroblast feeder layer.
Efflux studies.
Studies of the activation of Cl
and K+
conductances through cellular 125I
and
86Rb+ effluxes have been validated in T84 cells
(23) and have been used extensively to characterize the
Cl
and K+ conductances on PDEC
(14-19).
PDEC were grown to confluence on Transwell inserts as described. The
membranes and attached cells were excised from the insert and washed
two times with 1 ml of experimental buffer consisting of 140 mM NaCl,
4.7 mM KCl, 1.2 mM CaCl2, 10 mM glucose, and 10 mM HEPES,
pH 7.4. The cells were then loaded with radioactive markers through a
45-min incubation at 37°C with 1.5 ml of experimental buffer
containing either ~2 µCi/ml Na125I or ~1 µCi/ml
86RbCl and washed four times with 2 ml of isotope-free
buffer. The isotope efflux was measured by sequential addition and
removal of 1 ml of isotope-free buffer at 15-s intervals for a 5-min
period. To establish baseline efflux, no secretagogue was added for the first minute; in the remaining 4 min, the secretagogue tested was
included in the buffer. When inhibitors were tested, they were added at
the beginning of the experiment (including the baseline monitoring
period). Treatment with BAPTA-AM also included the 125I
loading period. The radioactivity of
these sequential samples and the radioactivity associated with the
cells at the end of the experiment were measured using a gamma counter
for 125I
and a liquid scintillation counter
for 86Rb+.
The radioactivity contained in the cells at a particular time point was
calculated as the sum of the radioactivities released in subsequent
efflux samples and remaining in the cells at the end of the experiment.
The efflux rate coefficient for a certain time interval was calculated
using the formula
where R1 and R2 are the percentages of
radioactivity initially loaded that remained in the cells at times
t1 and t2.
Ussing chamber studies.
Confluent monolayers of PDEC cultured on Snapwell inserts were mounted
in modified Ussing chambers with an aperture area of 1.0 cm2. Three different configurations were studied. For
studies of net electrogenic transepithelial ion transport, both sides
of the monolayer were bathed in Ringer solution (in mM: 115 NaCl, 1.2 CaCl2, 1 MgCl2, 0.4 KH2PO4, 2.4 K2HPO4, 25 NaHCO3, and 10 glucose), warmed to 37°C with a
circulating water jacket, and gently mixed and aerated with a constant
inflow of 95% O2-5% CO2. For studies of
apical Cl
conductance, the basolateral surface of the
PDEC was permeabilized by adding 0.20 mg/ml nystatin to the serosal
compartment 15 min before the addition of TDCA. A serosal-to-luminal
Cl
gradient (intracellular-to-extracellular gradient
across the apical membrane) was generated by adding, to the serosal
compartment, a buffer consisting of 135 mM NaCl, 1.2 mM
CaCl2, 1.2 mM MgCl2, 2.4 mM
K2HPO4, 0.6 mM KH2PO4,
10 mM HEPES, and 10 mM glucose, titrated to pH 7.4 with NaOH, and, to
the luminal compartment, a buffer of 135 mM sodium gluconate, 1.2 mM
CaCl2, 1.2 mM MgCl2, 2.4 mM
K2HPO4, 0.6 mM KH2PO4,
10 mM HEPES, and 10 mM glucose, titrated to pH 7.4 with NaOH. For
studies of basolateral K+ conductance, the apical membrane
was permeabilized with nystatin (0.20 mg/ml, 15 min) and a
luminal-to-serosal K+ gradient (intra-to-extracellular
gradient across the basolateral membrane) generated using a luminal
buffer of 10 mM NaCl, 1.25 mM CaCl2, 1 mM
MgCl2, 118 mM potassium gluconate, 10 mM HEPES, and 10 mM
glucose, titrated to pH 7.4 with NaOH, and a serosal buffer of 10 mM
NaCl, 1.25 mM CaCl2, 1 mM MgCl2, 4 mM potassium gluconate, 114 mM N-methyl-D-glucamine, 10 mM
HEPES, and 10 mM glucose, titrated to pH 7.4 with acetic acid.
Spontaneous tissue potential differences were short-circuited using an
automatic voltage clamp (model VCC MC2; Physiologic Instruments, San
Diego, CA) and Ag-AgCl2 electrodes, and the corresponding short-circuit current (Isc) was recorded
continuously using the Acquire and Analyze software (Physiologic
Instruments). In this system, the Isc reflects
1) for nonpermeabilized PDEC, a net electrogenic ion
transport across the intact epithelial monolayer; 2) for
basolaterally permeabilized PDEC, Cl
flow down the
serosal-to-luminal Cl
gradient across activated apical
Cl
channels; and 3) for apically permeabilized
PDEC, K+ flow down the luminal-to-serosal K+
gradient across activated basolateral K+ channels.
Instruments were calibrated before each experiment using
Vitrogen-coated membranes devoid of cells.
Measurement of TER.
Confluent PDEC monolayers cultured on Snapwell inserts were replenished
with fresh culture medium and exposed, either apically or serosally, to
different concentrations of TDCA for either 2 min, 5 min, 10 min, or
24 h. TER of the monolayers was measured at the beginning of the
experiment, after TDCA exposure (or after 10 min for the control and
24 h treatments), after 15 min, and after 24 h, using an
epithelial voltohmmeter (EVOM apparatus) from WPI (Sarasota, FL). TER
measurements were calibrated using a Transwell membrane coated with
Vitrogen but devoid of PDEC. For each monolayer, TER measurements were
normalized to the value obtained at the beginning of the experiment.
LDH activity.
To assess for cellular injury, LDH release from PDEC exposed to bile
acids was monitored. The cultured PDEC and supporting filter were
excised from the Transwell inserts and exposed for 5 min to 1 ml of
efflux buffer containing different concentrations of bile acids. LDH
released in the medium was assayed according to method of Amador et al.
(3): 1.4 ml reaction buffer (77.5 mM lactic acid, 5.25 mM
-nicotinamide adenine dinucleotide, and 0.05 M sodium pyrophosphate,
pH 8.8) was mixed with 100 µl supernatant, and absorption at 340 nm
was measured using a 160U ultraviolet spectrophotometer (Shimadzu). The
absorption change after a lag time of 1 min was determined.
Statistics.
All observations were based on at least three experiments. Unless
specified otherwise, results were expressed as means and SE, and
statistical significance was determined with the unpaired Student's
t-tests or ANOVA (concentration-dependence studies) using
the Statview 512+ (Abacus Concepts, Calabasas, CA) or InStat 3 (GraphPad Software) software.
 |
RESULTS |
Iodide efflux studies.
The possible activation of Cl
conductances on PDEC by
TDCA was evaluated by monitoring cellular
125I
efflux. As shown in Fig.
1A, TDCA, at a concentration
of 1 mM, stimulated a robust increase in 125I
efflux to a peak rate coefficient of 0.726 ± 0.072 min
1 30 s after addition of the conjugated bile acid
(vs. 0.155 ± 0. 0.011 min
1 for control treatment,
P < 0.01, n = 3). A much smaller
increase was obtained with 500 µM TDCA with a peak rate coefficient
of 0.224 ± 0.026 min
1 45 s after TDCA addition
(vs. 0.134 ± 0.014 min
1 for control treatment,
P < 0.05, n = 3). At a concentration
of 100 µM, TDCA failed to elicit an increased efflux different from control.

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 1.
Effect of taurodeoxycholic acid (TDCA) on
125I efflux. 125I
efflux from pancreatic duct epithelial cells (PDEC) was determined as
detailed in MATERIALS AND METHODS, and the efflux rate
coefficient was calculated and shown. In A-D, means and SE
from 3 experiments are shown. A: after 1 min of baseline
determination, TDCA was added at the different final concentrations
shown. NPPB, 5-nitro-2-(3-phenylpropylamino)benzoic acid. B:
after 1 min of baseline determination, 1 mM TDCA was added in the
absence ( ) or presence ( ) of 500 µM
NPPB. C: after 1 min of baseline determination, 1 mM TDCA
was added in the absence ( ) or presence
( ) of 500 µM DIDS. D: after 1 min of
baseline determination, 1 mM TDCA was added to cells pretreated with 50 µM BAPTA-AM ( ) or untreated control cells
( ).
|
|
To verify that this effect was mediated through Cl
conductances, the effects of the inhibitors of Cl
channels, NPPB (Fig. 1B) and DIDS (Fig. 1C), were
assessed. Preincubation with 500 µM of either of these two inhibitors
completely abolished the subsequent response stimulated by TDCA. This
inhibitory profile is similar to the one observed with the
Ca2+-activated Cl
conductance previously
characterized on these PDEC (14).
Because it has been demonstrated previously that bile acids may
stimulate an increase in intracellular Ca2+ concentration
([Ca2+]i; see Refs. 7,
8, 22), we determined whether the effect of
TDCA on 125I
efflux was mediated by an
increase in [Ca2+]i using the intracellular
Ca2+ chelator BAPTA-AM. As shown in Fig. 1D,
preincubation with 50 µM BAPTA also totally abolished the subsequent
stimulation by 1 mM TDCA.
The 125I
effluxes stimulated by deoxycholic
acid conjugated to either taurine or glycine were compared next. As
shown in Fig. 2A, at 1 mM, the
response obtained with glycodeoxycholic acid was smaller than the
response to TDCA (peak efflux rate coefficient: 0.587 ± 0.082 min
1 with glycodeoxycholic acid vs. 1.233 ± 0.094 min
1 with TDCA, P < 0.01, n = 3).

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 2.
Effect of different bile acids on
125I efflux. 125I
efflux from PDEC was determined as detailed in MATERIALS AND
METHODS, and the efflux rate coefficient was calculated and
shown. In A and B, means and SE from 3 experiments are shown. A: after 1 min of baseline
determination, 1 mM of deoxycholic acid conjugated to either taurine
( ) or glycine ( ) was added.
B: after 1 min of baseline determination, 1 mM of either
deoxycholic acid ( ), chenodeoxycholic acid
( ), or cholic acid ( ), all conjugated
to taurine, was added.
|
|
The effects of the three bile acids that account for ~90% of the
bile acids in the lumen of the bile ducts (chenodeoxycholic acid,
cholic acid, and deoxycholic acid) were also evaluated at the same
concentration of 1 mM and when conjugated to taurine. As shown in Fig.
2B, the 125I
effluxes stimulated
by taurochenodeoxycholic acid and TDCA were equivalent; in contrast, no
efflux was detected with 1 mM taurocholic acid (efflux rate
coefficients 45 s after bile acid addition: 1.09 ± 0.006 min
1 for taurochenodeoxycholic, 1.108 ± 0.011 min
1 for TDCA, and 0.16 ± 0.006 min
1
for taurocholic acid, n = 3).
86Rb+ efflux studies.
As previously reported, these PDEC also express
Ca2+-activated K+ conductances that can be
studied through measurements of 86Rb+ efflux
(16). Because the findings above suggested that TDCA caused a [Ca2+]i increase to stimulate
Ca2+-activated Cl
channels, the effect of
this [Ca2+]i increase on
Ca2+-activated K+ channels was also evaluated.
As shown in Fig. 3A, 1 mM TDCA
also stimulated an increased 86Rb+ efflux in a
concentration-dependent manner. Unlike the
125I
efflux, the
86Rb+ efflux was sustained over time. Indeed, 4 min after the addition of TDCA, the 86Rb+
efflux rate coefficients were at stable values of 0.045 ± 0.007, 0.088 ± 0.008, and 0.145 ± 0.007 min
1 for
control, 0.75 mM, and 1 mM TDCA (P < 0.01 for 1 mM vs.
control and for 1 mM vs. 0.75 mM; P < 0.05 for 0.75 mM
vs. control, n = 3), respectively. This effect was also
inhibited by 100 nM charybdotoxin, an inhibitor of
Ca2+-activated K+ channels, from a efflux rate
coefficient of 0.164 ± 0.009 to 0.049 ± 0.009 min
1 4 min after the addition of 1 mM TDCA
(P < 0.01, n = 3; Fig. 3B).

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 3.
Effect of TDCA on 86Rb+ efflux.
86Rb+ efflux from PDEC was determined as
detailed in MATERIALS AND METHODS, and the efflux rate
coefficient was calculated and shown. In A and B,
means and SE from 3 experiments are shown. A: after 1 min of
baseline determination, TDCA was added at the different final
concentrations shown. B: after 1 min of baseline
determination, 1 mM TDCA was added in the absence ( ) or
presence ( ) of 100 nM charybdotoxin.
|
|
Ussing chamber studies.
As previously reported, the cultured dog PDEC exhibit tight junctions,
generating the high transepithelial monolayer electrical resistance
necessary for studies in Ussing chambers (16, 17, 19).
They are also polarized with apical and basolateral membranes that may
differ in their ability to mediate the secretory effects of bile acids.
We therefore compared the effects of TDCA added to either the luminal
or serosal compartment of Ussing chambers.
Confluent PDEC monolayers were mounted in Ussing chambers, and the net
electrogenic transepithelial ion transport was stimulated by bile acids
monitored through the Isc. As shown in Fig.
4A, when added to the serosal
compartment contiguous with the basolateral side of the PDEC, TDCA
stimulated a sustained increase in Isc. This
response occurred in a dose-dependent manner, with peak
Isc increases of 1.9 ± 0.1, 3.2 ± 0.5, 5.0 ± 0.5, and 6.6 ± 0.1 µA/cm2,
respectively, for 0.4, 0.6, 0.8, and 1 mM TDCA (n = 3, statistical significances shown in Fig. 4A,
inset). As shown in Fig. 4B, when added to the luminal
compartment, 1 mM TDCA only stimulated a minimal
Isc increase. Higher concentrations, however,
elicited significant responses in a dose-dependent manner, with peak
Isc increases of 0.7 ± 0.2, 1.3 ± 0.2, 1.1 ± 0.2, 2.4 ± 0.4, and 5 ± 0.3 µA/cm2, respectively, for 1, 1.25, 1.5, 1.75, and 2 mM
TDCA (n = 4-5, statistical significances detailed
in Fig. 4B, inset).

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of TDCA on net electrogenic ion transport by PDEC
monolayers. Monolayers of confluent PDEC were mounted in Ussing
chambers, as described in MATERIALS AND METHODS, and the
short-circuit current (Isc) was measured at 10-s
intervals. After ~5 min of baseline determination, TDCA was added at
the different final concentrations shown. The
Isc increases shown (calculated by subtracting
the Isc recorded just before the addition of
TDCA) were obtained from monolayers cultured and studied at the same
time. A: TDCA was added to the serosal compartment in
contact with the basolateral membrane of the PDEC. These tracings are
representative of 3 experiments, and the average peak
Isc increases (highest
Isc within 10 min of TDCA addition Isc just before addition of TDCA) are shown in
the inset [significant differences from 0 ( ), 0.2 (#),
0.4 (@), and 0.6 (&) mM, P < 0.05 by ANOVA].
B: TDCA was added to the luminal compartment in contact with
the apical membrane of the PDEC. These tracings are representative of 5 experiments, and the average peak Isc increases
(highest Isc within 10 min of TDCA addition Isc just before addition of TDCA) are shown
in the inset [significant differences from 0 ( ), 1 (#),
1.25 (@), 1.5 (&), and 1.75 (*) mM, P < 0.05 by
ANOVA].
|
|
The studies above suggest that TDCA stimulated net electrogenic ion
transport from nonpermeabilized PDEC. Because the efflux studies
suggest that TDCA activated Cl
and K+
channels, we characterized the effects of this bile acid on
Cl
and K+ conductances of PDEC mounted in
Ussing chambers. To study apical Cl
conductances, the
basolateral membrane of PDEC was permeabilized with nystatin added to
the serosal compartment, and the monolayer was subjected to a 135 mM
serosal-to-luminal Cl
gradient. As shown in Fig.
5, 1 mM serosal TDCA stimulated an increase in Isc that was inhibited by 50 µM
BAPTA-AM (80% inhibition, Fig. 4A, inset), 500 µM DIDS (98% inhibition, Fig. 4B and inset), and 500 µM NPPB (75% inhibition, data not shown). These findings are
consistent with the 125I
efflux studies,
suggesting that Cl
flow is mediated through apical
Ca2+-activated Cl
channels (14, 17,
18).

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of TDCA on apical Cl channels.
Monolayers of confluent PDEC, cultured and studied at the same time,
were mounted in Ussing chambers, in the presence of a 135 mM
serosal-to-luminal Cl gradient, and basolaterally
permeabilized with 0.2 mg/ml nystatin for 15 min, as described in
MATERIALS AND METHODS. A: monolayer pretreated
for 30 min with 50 µM BAPTA-AM. B: monolayer pretreated
for 30 min with 500 µM DIDS. After ~5 min of baseline
determination, 1 mM TDCA was added to the serosal compartment. The
Isc was measured at 10-s intervals, and the
Isc increases were calculated by subtracting the
Isc recorded just before TDCA addition. The
tracings displayed are representative of 3 experiments, and the average
peak Isc increases (highest
Isc within 10 min of TDCA addition Isc just before addition of TDCA) from these
experiments are shown in the insets. *Significant difference
between Isc increases in the presence or absence
of either BAPTA-AM or DIDS (P < 0.05 by unpaired
1-tailed t-test).
|
|
Activation of basolateral K+ channels was also examined by
permeabilizing the apical membrane of PDEC with luminal nystatin and
subjecting the monolayer to a luminal-to-serosal K+
gradient of 114 mM. As shown in Fig.
6A, 1 mM serosal TDCA also stimulated an increased Isc in this
configuration. This effect was inhibited by 50 µM BAPTA-AM (98%
inhibition, Fig. 6A and inset) and 100 nM
charybdotoxin (92% inhibition, Fig. 6B and
inset), suggesting mediation through the
Ca2+-activated K+ channels previously
identified in these cells (16).

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of TDCA on basolateral K+ channels.
Monolayers of confluent PDEC, cultured and studied at the same time,
were mounted in Ussing chambers in the presence of a 114 mM
luminal-to-serosal K+ gradient and apically permeabilized
with 0.2 mg/ml nystatin for 15 min as described in MATERIALS AND
METHODS. A: monolayer pretreated for 30 min with 50 µM BAPTA-AM. B: monolayer pretreated for 5 min with 100 nM
charybdotoxin. After ~5 min of baseline determination, 1 mM TDCA was
added to the serosal compartment. The Isc was
measured at 10-s intervals, and the Isc
increases were calculated by subtracting the Isc
recorded just before TDCA addition. The tracings displayed are
representative of 3 experiments, and the average peak
Isc increases (highest
Isc within 10 min of TDCA addition Isc just before addition of TDCA) from these
experiments are shown in the inset. *Significant difference
between Isc increases in the presence and
absence of either BAPTA-AM or charybdotoxin (P < 0.05 by unpaired 2-tailed t-test).
|
|
Measurements of TER.
TER is another electrophysiological property of PDEC monolayers that
may be affected by bile acids. Figure 7
shows that the effects of TDCA on monolayer TER also differ according
to the side of exposure. From the serosal compartment (Fig.
7A), transient treatments with 1 mM TDCA (2, 5, and 10 min)
only produced temporary TER decreases of up to 12 ± 2%
(n = 6), resolving when TDCA was removed (15-min time
point). However, 24 h after treatment, TER was only 40-45%
of the initial value. Continuous exposure to 1 mM TDCA resulted in a
marked TER decrease of 95% after 24 h. When PDEC were exposed
serosally to 2 mM TDCA, the resulting decrease in TER increased with
time. Indeed, after only 10 min of TDCA exposure, the TER was 22 ± 3% of its original value; after 24 h, this value decreased to
5%.

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 7.
Effects of TDCA on transepithelial resistance (TER) of
PDEC monolayers. PDEC monolayers were either untreated (control) or
treated with 1, 2, 3, or 4 mM TDCA added to either the serosal
(A) or luminal (B) compartment for 2 min, 5 min,
10 min, or 24 h. Monolayer TERs were measured, as described in
MATERIALS AND METHODS, before treatment, at the end of
treatment (or at 10 min for the control and 24-h exposures), at 15 min,
and at 24 h. The mean ± SE of the TERs, normalized to the
initial value before treatment, are shown (n = 6).
Statistical difference from 100% (P < 0.05, t-test, 5 degrees of freedom). For the serosal
exposure, the initial TERs were, respectively, 706 ± 13, 604 ± 9, 592 ± 19, 653 ± 24, 796 ± 29, 621 ± 55, 711 ± 67, 693 ± 94, and 757 ± 109 · cm2 for the control, 1 mM/2 min, 1 mM/5 min,
1 mM/10 min, 1 mM/24 h, 2 mM/2 min, 2 mM/5 min, 2 mM/10 min, and 2 mM/24 h treatments. For the luminal exposure, the initial TERs were,
respectively, 839 ± 35, 861 ± 58, 849 ± 67, 807 ± 43, 860 ± 44, 929 ± 90, 904 ± 112, 929 ±
142, 917 ± 130, 901 ± 119, 875 ± 145, 943 ±
149, and 907 ± 160 · cm2 for the control,
2 mM/2 min, 2 mM/5 min, 2 mM/10 min, 2 mM/24 h, 3 mM/2 min, 3 mM/5 min,
3 mM/10 min, 3 mM/24 h, 4 mM/2 min, 4 mM/5 min, 4 mM/10 min, and 4 mM/24 h treatments.
|
|
Monolayer TER was more resistant to luminal TDCA exposure. Only
transient decreases by 15-20% from the original TER were detected when PDEC were exposed to 2 mM luminal TDCA for up to 10 min. Different
from transient serosal exposure to 1 or 2 mM TDCA, PDEC monolayers
recovered their original TER value after 24 h when exposed
transiently to 2 mM luminal TDCA. However, after continuous exposure,
the corresponding TER decreased to 74 ± 6.3% of its original
value after 24 h. With 3 mM luminal TDCA, the transient TER
decreases were more pronounced; 5 min after a 10-min exposure to TDCA,
the TER was 16.5 ± 4.1% of its original value. However, after
transient exposure, the PDEC monolayers still regained their original
TER values after 24 h. Only at a higher concentration of 4 mM
luminal TDCA were there marked sustained TER decreases to <10 and 5%
of the initial values 15 min and 24 h, respectively, after exposure.
Cellular LDH release.
Because bile acids are detergents, the toxicity of these bile acids
against PDEC was also evaluated by monitoring LDH release from cells
exposed to bile acids. As shown in Fig.
8, after treatment with 0.5 and 1 mM TDCA
for 5 min, minimal LDH was released in the supernatant, similar to the
control treatment (LDH activity in arbitrary units; control: 120 ± 20.8, 1 mM TDCA: 96.7 ± 28.5, n = 3). A
significant increase in LDH was released in the supernatant with 2 mM
TDCA; with 5 mM TDCA, the amount of LDH released was equivalent to the
amount released with 1 mM Triton X-100, used as positive control (LDH
activity: 2 mM TDCA, 893 ± 91; 5 mM TDCA, 8,803 ± 2,112;
and Triton X-100, 7,316 ± 1,839).

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 8.
Lactate dehydrogenase (LDH) leakage from PDEC treated
with TDCA. PDEC were treated with different concentrations of TDCA or
with 1% Triton X-100, and the LDH released into the supernatant was
assayed as described in MATERIALS AND METHODS. The means
and SE from 3 determinations are shown.
|
|
 |
DISCUSSION |
PDEC mediate the secretion of fluid and electrolytes (mainly
bicarbonate), one of the two main functions of the exocrine pancreas. Because these cells are exposed to refluxed bile acids after ampullary obstruction and because bile acids stimulate secretion from colonic and
biliary epithelia, we examined the secretory effects of bile acids on
PDEC. Nontransformed, well-differentiated, and polarized cultured dog
PDEC served as the model since they were used successfully to
characterize the secretory effects of histamine (acting via H1 receptors), UTP and ATP (via P2Y2 and
P2Y11 receptors), and trypsin (via proteinase-activated
receptor-2; see Refs. 15, 17,
18, and 19). In addition, these PDEC are derived
from the main pancreatic duct of the dog, the first pancreatic
structure exposed to refluxed bile.
In this model, TDCA activated both Cl
and K+
conductances. Indeed, it stimulated an increased
125I
efflux, inhibited by the
Cl
channel blockers NPPB or DIDS, and a
86Rb+ efflux inhibited by the K+
channel blocker charybdotoxin. When PDEC were examined in Ussing chambers, TDCA also stimulated an Isc increase
from basolaterally permeabilized cells subject to a serosal-to-luminal
Cl
gradient and from apically permeabilized cells subject
to a luminal-to-serosal K+ gradient. These
Isc increases were also inhibited, respectively, by the Cl
channel inhibitors NPPB and DIDS and the
K+ channel inhibitor charybdotoxin. As previously reported,
these efflux and Isc responses are
characteristic of those mediated by the apical
Ca2+-activated Cl
channels and basolateral
Ca2+-activated K+ channels (14,
16). Inhibition by BAPTA-AM, a cell-permeant Ca2+
chelator, of TDCA-stimulated 125I
efflux and
Isc increases from basolaterally permeabilized
PDEC subject to a serosal-to-luminal Cl
gradient and
apically permeabilized PDEC subject to a luminal-to-serosal K+ gradient further verifies that activation of the
Cl
and K+ channels is mediated through an
increase in [Ca2+]i. A toxic effect of TDCA
at the concentrations tested was excluded by the absence of increased
LDH leakage after exposure to
1 mM TDCA. These findings are
consistent with the activation, through increased
[Ca2+]i, by similar concentrations of bile
acids of K+ and/or Cl
conductances on colonic
T84 cells and biliary cells and the stimulation by tauroursodeoxycholic
acid of exocytosis in hepatocytes (6, 8, 9, 22).
PDEC polarization and the presence of monolayer resistance allowed us
to examine the polarity of TDCA effects. At 1 mM, only serosal, but not
luminal, TDCA stimulated an Isc increase from intact monolayers; a higher concentration (2 mM) was required to
produce a luminal effect. In T84 cells, <1 mM TDCA also produced only
a serosal effect, and
1 mM TDCA was required for luminal action. It
was postulated that the higher luminal concentrations of bile acids
disrupted T84 cell monolayer integrity, allowing bile acids to reach
the basolateral membrane and stimulate secretion (9). In
this report, it is possible that TDCA interacts directly with the
apical membrane of PDEC to stimulate secretion. Indeed, 2 mM luminal
TDCA decreased monolayer TER by only 15-20%, which may be
inadequate for rapid TDCA leakage in the serosal compartment. Furthermore, the onsets of action after serosal and luminal TDCA addition were similar, without the delay expected for transepithelial leakage.
The secretory effects of the different bile acid types were examined.
Because biliary bile acids are also conjugated to glycine, we
demonstrated that 1 mM glycodeoxycholic acid also stimulated an
increase in 125I
efflux, albeit of smaller
amplitude. The effects of cholic acid and chenodeoxycholic acid, which,
together with deoxycholic acid, account for 90% of biliary bile acids,
were also evaluated. Conjugated to taurine and tested at 1 mM,
chenodeoxycholic acid and deoxycholic acid were equivalent in their
secretory response; in contrast, cholic acid did not produce a
significant response. Of note, taurocholic acid also did not activate
conductances in T84 cells (8). Because taurocholic acid,
like TDCA, is a strong detergent, its lack of activity further argues
against the secretory effect of TDCA resulting from a nonspecific
detergent action. Structural differences may account for these
different effects as follows: both chenodeoxycholic acid and
deoxycholic acid are dihydroxylated (at positions 3 and 7 for chenodeoxycholic acid and at positions 3 and 12 for deoxycholic acid), whereas cholic acid is
trihydroxylated (at positions 3, 7, and
12).
The effects of TDCA on PDEC TER are consistent with the previous
findings that bile acids at concentrations >1 mM increased the
permeability of bovine pancreatic duct explants (2) and of
the main pancreatic duct in different animal models of bile-induced pancreatitis (4, 5, 10). Again, higher luminal TDCA
concentrations were also necessary to affect monolayer TER. Marked
immediate and delayed decreases in TER followed transient treatment
with 2 mM serosal TDCA and with 4 mM luminal TDCA. Of note, transient exposure to 1 mM serosal TDCA caused delayed TER decreases after 24 h, suggesting that serosal bile acids produce long-lasting effects. On the other hand, transient exposure to 2-3 mM luminal TDCA only caused a transient TER decreases, with full recovery after
24 h. The PDEC used in this study are well differentiated and
polarized, with apical and basolateral membranes differentially exhibiting receptors for ATP, UTP, histamine, and trypsin (15, 17, 18, 19). The differential effects of luminal and serosal TDCA most likely reflect different interaction with apical and basolateral membranes.
Because serum concentrations of bile acids are in the micromolar range,
it is unlikely that systemic bile acids will cause any serosal effect
on PDEC under physiological conditions. On the other hand, our findings
may be relevant to gallstone pancreatitis in which refluxed bile may
yield conjugated bile acid concentrations in pancreatic juice in excess
of 2 mM. The stimulation of secretion by subtoxic concentrations of
TDCA may be a defense mechanism for diluting and washing off refluxed
bile acids before critically toxic concentrations are reached. The
relative resistance of the apical membrane to the toxic effects of TDCA
further illustrates the importance of the barrier function of the
pancreatic ductal mucosa.
In summary, we have shown that dihydroxy bile acids activate
Cl
and K+ conductances on PDEC and at higher
concentrations impair epithelial barrier function. These effects will
be relevant to conditions associated with bile reflux and disruption of
the pancreatic ductal epithelial barrier.
We thank Dr. Sum Lee (University of Washington) for advice on bile
acids and on the culture and characterization of pancreatic duct
epithelial cells.
This research was partially funded by a Merit Review from the
Department of Veterans Affairs, the Cystic Fibrosis Foundation RDP
program Grant R565, and National Institute of Diabetes and Digestive
and Kidney Diseases Grants RO1-DK-55885 (to T. D. Nguyen) and
DK-07742 (C. Okolo).
Address for reprint requests and other correspondence:
T. D. Nguyen, GI Section (S-111-Gastro), VA Medical Center,
1660 S. Columbian Way, Seattle, WA 98108 (E-mail:
t1nguyen{at}u.washington.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.