H+-peptide cotransport in the human bile duct
epithelium cell line SK-ChA-1
Ilka
Knütter1,2,
Isabel
Rubio-Aliaga3,
Michael
Boll3,
Gerd
Hause2,
Hannelore
Daniel3,
Klaus
Neubert1, and
Matthias
Brandsch2
1 Institute of Biochemistry, Department of
Biochemistry/Biotechnology and 2 Biozentrum of the
Martin Luther University Halle-Wittenberg, Halle
D-06120; and 3 Molecular Nutrition Unit,
Institute of Nutritional Sciences, Technical University of
Munich, Freising-Weihenstephan, D-85350 Germany
 |
ABSTRACT |
This study describes for the first time the
presence of H+-peptide cotransport in cells of the bile
duct. Uptake of
[glycine-1-14C]glycylsarcosine
([14C]Gly-Sar) in human extrahepatic cholangiocarcinoma
SK-ChA-1 cells was stimulated sevenfold by an inwardly directed
H+ gradient. Transport was mediated by a low-affinity
system with a transport constant (Kt) value of
1.1 mM. Several dipeptides, cefadroxil, and
-aminolevulinic acid,
but not glycine and glutathione, were strong inhibitors of Gly-Sar
uptake. SK-ChA-1 cells formed tight, polarized monolayers on permeable
membranes. The transepithelial electrical resistance was 856 ± 29
× cm2. The transepithelial flux of
[14C]Gly-Sar in apical-to-basolateral direction exceeded
the basolateral-to-apical flux 11-fold. Uptake was 20-fold higher from
the apical side. RT-PCR analysis using primer pairs specific for the
intestinal-type peptide transporter (PEPT1) or kidney-type (PEPT2)
revealed that the transport system expressed in SK-ChA-1 and also in
cells of the native rabbit bile duct is PEPT1. Immunohistochemistry
localized PEPT1 to the apical membrane of cholangiocytes of mouse
extrahepatic biliary duct. We conclude that the cells of the mammalian
extrahepatic biliary tract epithelium express the intestinal-type
H+-peptide cotransporter in their apical membrane. SK-ChA-1
cells represent a convenient model to study the physiological and
clinical aspects of peptide transport in cholangiocytes.
membrane transport; peptide symporter; peptide transporter-1; cell culture
 |
INTRODUCTION |
IN THE MAMMALIAN
INTESTINE and kidney, transport of di- and tripeptides across the
luminal membrane of epithelial cells occurs via carrier-mediated
mechanisms energized by an inwardly directed H+ gradient
(1, 12, 14, 16). It has been shown that the absorptive
cells of the intestinal epithelium express the low-affinity system
PEPT1, whereas the renal epithelium predominantly expresses the
high-affinity system PEPT2 but also PEPT1. At the intestinal epithelium, PEPT1 is responsible for the absorption of di- and tripeptides originating from external dietary protein digestion. At the
renal proximal tubulus, PEPT1 and PEPT2 are responsible for the
reuptake of filtered peptides. In addition to their natural substrates,
both systems are capable of transporting structurally related
pharmacologically active compounds such as
-lactam antibiotics and
other peptidomimetics (4, 27). Several cell lines such as
Caco-2 (11), MDCK (9), SKPT (8),
and LLC-PK1 (27) have been proven to be very
useful tools for the investigation of function, mechanism, specificity,
and regulatory aspects of peptide transport. In addition to intestine
and kidney, specific mRNAs for H+-dependent peptide
transporters have been found in brain, lung, pancreas, and liver
(14, 16). To our knowledge, nothing has been published so
far about peptide transport in cholangiocytes, the epithelial cells of
the bile duct. This epithelium, however, has gained much attention in
recent years. There have been major advances in our understanding
of physiology and pathophysiology of this barrier (2,
22). Several transport systems have been described,
e.g., Cl
/HCO
exchangers,
Na+/H+ exchangers, the cAMP-dependent
Cl
channel CFTR, aquaporin-1, a Na+-dependent
glucose transporter and the Na+-dependent bile acid
transporter (2). The transporters of the biliary
epithelium are regulated by hormones and neuropeptides (2,
3).
In 1985, three cholangiocarcinoma cell lines were established and
characterized in permanent tissue culture (18). Recently, they have been used for studies of Cl
and K+
transport (7). In the present study, we characterize the
uptake of [glycine-1-14C]glycylsarcosine
([14C]Gly-Sar) in SK-ChA-1 cells. Results reveal that
SK-ChA-1 cells express the H+-dependent low-affinity
transport system for di- and tripeptides PEPT1. The system is also
expressed in normal rabbit and mouse extrahepatic bile duct cells. This
study represents the first description of a H+/peptide
transport in cells of the biliary duct.
 |
MATERIALS AND METHODS |
Cell culture.
The human extrahepatic biliary duct tumor cell line SK-ChA-1
established by Knuth et al. (18) was obtained from the
Ludwig Institute for Cancer Research (Zurich, Switzerland). The human colon carcinoma cell line Caco-2 was obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany).
Cells at passage 27-65 (SK-ChA-1) or
18-23 (Caco-2) were maintained in 75-cm2
culture flasks at 37°C in a humidified atmosphere with 5%
CO2. They were cultured in minimum essential medium
supplemented with nonessential amino acid solution (1%), fetal bovine
serum (10%), and gentamicin (50 µg/ml). All cell culture media were
purchased from Life Technologies (Karlsruhe, Germany). Cells grown to
confluence were released by trypsinization (0.05% trypsin/EDTA in
modified Pucks solution A) and subcultured in 35-mm
disposable petri dishes (Becton, Dickinson). The medium was replaced
every other day. With a starting cell density of 0.8 × 106 cells/dish, the cultures reached confluence within
24 h. Uptake was measured in these cells 7 days after
seeding. SK-ChA-1 cells were also cultured on permeable
polycarbonate Transwell cell culture inserts (24.5-mm diameter, 3 µm-pore size; Costar, Bodenheim, Germany). Subcultures were started
at a cell density of 43,000 cells/cm2 and cultured for
14-18 days. The lower (receiver) compartment contained 2.6 ml
medium and the upper (donor) compartment, 1.5 ml medium.
Transport studies.
Uptake of [14C]Gly-Sar (53 mCi/mmol specific
radioactivity; Amersham International) was determined at 37°C. In
most experiments, the uptake medium was 25 mM
2-(N-morpholino)ethanesulfonic acid/Tris, pH 6.0, or 25 mM
HEPES/Tris, pH 7.5, containing (in mM): 140 NaCl, 5.4 KCl, 1.8 CaCl2, 0.8 MgSO4, and 5 glucose.
Na+-free media were prepared by replacing NaCl in the
uptake medium by choline chloride. The procedure for NH4Cl
prepulse was the same as described previously (8, 9, 11,
21). Uptake was initiated by removing the pretreatment culture
medium from the dish, washing the cell layer with 1-ml buffer and
adding 1-ml uptake medium containing [14C]Gly-Sar
(8-11, 15). After incubation for the desired time in
the presence or absence of unlabeled compounds (Sigma-Aldrich), the
buffer was removed, and monolayers were quickly washed four times with
ice-cold uptake buffer, dissolved, and transferred to counting vials.
Radioactivity associated with the cells was measured by liquid
scintillation spectrometry. Transepithelial flux of
[14C]Gly-Sar across SK-ChA-1 cell monolayers cultured on
permeable filters was measured as follows. After washing the inserts
with buffer (in mM): 25 HEPES/Tris (pH 7.5), 140 NaCl, 5.4 KCl, 1.8 CaCl2, 0.8 MgSO4, and 5 glucose for 10 min,
uptake was started by adding uptake buffer (pH 6.0) containing
[14C]Gly-Sar to the donor side (1.5 ml apical or 2.6 ml
basolateral compartment, respectively). All experiments were performed
at 37°C in a shaking water bath. At time intervals of 10, 30, 60, and
120 min, samples were taken from the receiver compartment and replaced
with fresh buffer. Radioactivity in the samples was measured by liquid
scintillation spectrometry. After 2 h, the filters were quickly
washed four times with ice-cold uptake buffer, cut out of the plastic
insert and transferred to scintillation vials. The integrity of the
SK-ChA-1 grown on permeable filters was confirmed microscopically, by
measuring the transepithelial electrical resistance and by measuring
the apical-to-basolateral transepithelial flux of
[14C]mannitol (10 µM) as a space marker.
Transmission electron microscopy.
SK-ChA-1 cells cultured on polycarbonate cell culture inserts for 14 days were fixed in 3% sodiumcacodylate-buffered glutaraldehyde (pH
7.2), postfixed with 1% OsO4 solution, dehydrated in an
ethanol series, and embedded in epoxy resin (23).
Ultrathin sections (90 nm) were stained with uranyl acetate/lead
citrate and observed with an EM 900 transmission electron microscope (Zeiss).
RNA isolation and RT-PCR.
SK-ChA-1 cells were cultured in 75-cm2 culture flasks for 7 days. Total RNA from the cell line and from rabbit tissues (intestinal mucosa, kidney, gall bladder, and bile duct; Charles River
Laboratories) was isolated with the RNAwiz system according to the
manufacturer's protocol (Ambion, Wiesbaden, Germany). Two micrograms
total RNA were reverse transcribed using the Retrocsript kit (Ambion).
Five microliters of each RT reaction were subjected to PCR reactions (REDTaq; Sigma) with the following primer pairs derived from mouse: 1) PEPT1-F42 (5'-GAGCATCTTCTTCATCGTGGTC-3') and PEPT1-B901
(5'-CCTGCTGGTCAAACAAGGCC-3'), 2) PEPT2-F290
(5'-ACCATGCCTTCAGCAGCCTCT-3') and PEPT2-B1161 (5'-CGCTAGGATCATACCA ACAGC-3'), and 3) GAPDH-F (5'-GACCACAGTCCATGACATCACT-3') and
GAPDH-B (5'-TCCACCACCCTGTTGCTGTAG-3') in 25 µl total volume. PCR
conditions were: 94°C 1 min, 35× (94°C 30 s, 57°C 30 s, 72°C 45 s). Ten microliters of each PCR reaction were
separated on a 1% agarose gel.
Immunohistochemistry.
Murine tissues were fixed in 4% paraformaldehyde overnight at
4°C and processed for embedding in paraffin wax. Deparaffinized sections (5 µm) were used for immunofluorescence analysis. Antigen retrieval was carried out by incubating the slides in citrate buffer
(pH 6.0) in a microwave oven. Slides were blocked for 20 min with 3%
goat serum and incubated overnight with a rabbit polyclonal anti-mouse-PEPT1 serum raised against the amino acids 248-261 (17) diluted 1:100. For detection of the primary antibody,
the slides were incubated 1 h with an anti-rabbit Cy3 coupled
antibody (1:200; Dianova). Control incubations in parallel sections
were carried out to specify the reaction by preabsorption of the
primary antibody with 5 µg of the corresponding antigenic peptide.
Slides were viewed using confocal laser scanning microscopy (model TCS SP2; Leica Microsystems, Heidelberg, Germany).
Data analysis.
Each experimental point shown represents the mean ± SE of three
to four measurements. The kinetic constants were calculated by
nonlinear regression of the Michaelis-Menten plot and confirmed by
linear regression of the Eadie-Hofstee plot. Calculated parameters are
shown with their SE. Inhibition constants (Ki)
were calculated from IC50 values (i.e., concentration of
the unlabeled compound necessary to inhibit 50% of radiolabeled
Gly-Sar uptake).
 |
RESULTS |
Effect of a pH gradient on the [14C]Gly-Sar uptake in
SK-ChA-1 cells.
To determine whether epithelial cells of the mammalian bile duct
express a H+/peptide symport system, we studied the uptake
of [14C]Gly-Sar in the human cholangiocarcinoma cell line
SK-ChA-1, cultured as monolayers on impermeable plastic surfaces. The
uptake activity, expressed as pmol · 10 min
1 · mg protein
1, remained
approximately the same for
12 days (data not shown). Day 7 was chosen for all uptake experiments. [14C]Gly-Sar
uptake was markedly stimulated by changing the extracellular pH (Fig.
1). Uptake measured at pH 6.0 was
sevenfold greater than uptake measured at pH 8.5. An extracellular pH
of 6.0 represents the optimum for the Gly-Sar uptake. The same pH
optimum for H+-dependent dipeptide transport has been found
in the intestinal cell line Caco-2 and in the renal cell lines MDCK and
SKPT (8-11). To determine whether the stimulation
observed at pH 6.0 was due to the inwardly directed H+
gradient or due to the acidic pH per se, we investigated the influence
of intracellular pH on [14C]Gly-Sar uptake. Intracellular
pH was decreased by the NH4Cl prepulse technique
(21). Results of these experiments were performed at both
SK-ChA-1 cells and, for comparison, Caco-2 cells are given in Table
1. [14C]Gly-Sar uptake at
an extracellular pH of 6.0 is inhibited by >70% in both cell lines
when the intracellular pH was made acidic. Stimulation of Gly-Sar
uptake caused by an acidic extracellular pH is thus the effect of an
inwardly directed H+ gradient rather than the acidic
extracellular pH per se. This conclusion is further supported by
the finding that the protonophore carbonyl cyanide
p-(trifluoromethoxy)phenylhydrazone (FCCP, 10 µM, present
during uptake measurement), which dissipates transmembrane electrochemical H+ gradients, inhibited
[14C]Gly-Sar uptake from 199 ± 8 to 95.2 ± 8.2 pmol · 10 min
1 · mg
protein
1 (by 52%) in SK-ChA-1 cells and from 237 ± 9 to 95.5 ± 18.7 pmol · 10 min
1 · mg protein
1 (by 60%) in
Caco-2 cells.

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 1.
pH dependence of
[glycine-1-14C]glycylsarcosine
([14C]Gly-Sar) uptake in SK-ChA-1 cells. Uptake of
[14C]Gly-Sar (10 µM) was measured at varying pH values
(range 5.0-9.0). Values represent means ± SE for 3 determinations. When not indicated, the error lies within the symbol.
|
|
Kinetic parameters.
Dependence of the Gly-Sar uptake rate on the substrate concentration
was investigated to determine the kinetic parameters of the transport
system. Uptake rates were measured over a substrate concentration range
of 20 µM-10 mM. Carrier-mediated uptake calculated by
subtracting the nonmediated component from the total uptake was used in
the kinetic analysis. The nonmediated component, which represents
diffusion plus binding, was determined from the uptake of
[14C]Gly-Sar in the presence of excess amount (50 mM) of
unlabeled Gly-Sar. This component was 10.9% of total uptake at 20 µM
of [14C]Gly-Sar. The relationship between
carrier-mediated uptake rate and substrate concentration was found to
be hyperbolic over the Gly-Sar concentration range (Fig.
2), indicating saturability of the
transport system. When the results were expressed in the form of an
Eadie-Hofstee plot (uptake rate/substrate concentration vs. uptake
rate), a straight line (r2 = 0.98) was
obtained (Fig. 2, inset). The apparent Michaelis-Menten constant of transport processes (Kt) was
1.1 ± 0.1 mM and the maximal velocity
(Vmax) was 21.6 ± 2.2 nmol · 10 min
1 · mg protein
1. We found no
evidence for the presence of a high affinity/low capacity transport
system as described in SKPT cells (8) and in renal
brush-border membrane vesicles (12).

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 2.
Kinetics of Gly-Sar uptake in SK-ChA-1 cells. Uptake of
[14C]Gly-Sar was measured with a 10-min incubation over a
Gly-Sar concentration range of 20 µM-10 mM. The
diffusional/binding component was determined by measuring uptake in the
presence of an excess amount (50 mM) of unlabeled Gly-Sar. This
component (10.9%) was subtracted from the total uptake to calculate
the carrier-mediated uptake used in the kinetic analysis. Values
represent means ± SE for 4 determinations. Inset:
Eadie-Hofstee transformation of the data. v, Uptake rate in
nmol · 10 min 1 · mg
protein 1; S, Gly-Sar concentration in mM.
|
|
Substrate specificity.
To determine the substrate specificity of the H+-dependent
transport system responsible for the uptake of Gly-Sar in SK-ChA-1 cells, the effect of unlabeled peptides, peptidomimetics, and glycine
on the uptake of Gly-Sar was measured at pH 6.0 (Fig. 3). Table
2 shows the resulting
Ki values. Ki values of
potential substrates range from 0.25 ± 0.01 mM for Ala-Ala to
3.3 ± 0.3 mM for
-aminolevulinic acid. Glycine and glutathione
were not recognized. Results indicate that dipeptides and several
peptidomimetics are recognized by the peptide transport system as
potential transport substrates.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 3.
Specificity of H+-peptide cotransport in SK-ChA-1
cells. Uptake of 10 µM [14C]Gly-Sar was measured in
monolayer cultures of SK-ChA-1 cells at pH 6.0 in the absence and
presence of increasing concentrations of unlabeled peptides and
peptidomimetics (0-31.6 mM). Uptake of [14C]Gly-Sar
measured in the absence of the inhibitors (197 ± 18 pmol · 10 min 1 · mg
protein 1) was taken as 100%.
|
|
View this table:
[in this window]
[in a new window]
|
Table 2.
Inhibition constants (Ki) for different peptides
and peptidomimetics for the inhibition of
[14C]Gly-Sar uptake in SK-ChA-1 cells
|
|
Transepithelial flux.
The inner layer of the extrahepatic bile duct is formed by
cholangiocytes in polarized, epithelial formation. Microscopic studies
by Knuth et al. (18) have shown that SK-ChA-1 cells also
polarize when grown on cover glasses. Here, we cultured SK-ChA-1 cells
on permeable filters for transepithelial flux studies. Transmission electron micrographs (Fig. 4)
show that within 14 days the cells form an epithelial monolayer
with polarized cells. They establish a brush border at their apical
membrane and form junctional complexes between cells. Transport studies
were performed after 18 days. At this stage, the transepithelial
electrical resistance of the SK-ChA-1 monolayers was 856 ± 29
× cm2. The transepithelial flux of
[14C]mannitol through the SK-ChA-1 cell monolayers was
0.37 ± 0.01% · h
1 · receiver
well
1. These results demonstrate that SK-ChA-1 cell
monolayers on permeable filters are well suited as a model for biliary
tract flux studies as are Caco-2 cells for studies of intestinal
transport. Figure 5 summarizes the
results of Gly-Sar flux studies. When added to the basolateral
compartment in an uptake buffer (pH 6.0), the [14C]Gly-Sar flux to the apical compartment is only
insignificantly higher than the flux of the space marker
[14C]mannitol. However, from the apical side,
transepithelial [14C]Gly-Sar flux to the basolateral side
(5.6 ± 0.4% · h
1 · receiver
well
1) exceeds the [14C]mannitol flux
15-fold and the basolateral-to-apical [14C]Gly-Sar flux
11-fold. Hence, the [14C]Gly-Sar transport is dominantly
directed absorptively, and almost neglectable in the excretory
direction. As expected, the transepithelial flux of
[14]Gly-Sar is mainly carrier-mediated. Addition of an
excess amount of unlabeled Gly-Sar (10 mM) to the apical compartment
inhibits the apical-to-basolateral [14C]Gly-Sar flux by
79% (from 5.6 ± 0.4 to 1.2 ± 0.07% · h
1 · receiver
well
1). The flux results correspond very well with the
uptake into the cells on the filter. These filters were cut out after
2 h and analyzed. Figure 5 (inset) shows that the
[14C]Gly-Sar uptake from the apical side exceeds the
uptake from the basolateral side by a factor of 20. Unlabeled Gly-Sar
at a concentration of 10 mM at the apical side inhibits the apical [14C]Gly-Sar uptake into the cells by 78%.

View larger version (120K):
[in this window]
[in a new window]
|
Fig. 4.
Transmission electron micrographs of SK-ChA-1 cells
cultured on permeable supports. Cells were seeded on polycarbonate
filters at a density of 0.2 × 106 cells/filter. After
14 days, filters were fixed, stained, embedded, and cut. Note the
monolayer character, the polarized differentiation of cells with
brush-border membranes (A) and the junctional complexes
between the cells (B).
|
|

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 5.
Transepithelial flux and intracellular uptake of
[14C]Gly-Sar and [14C]mannitol at SK-ChA-1
cell monolayers. Cells were seeded on polycarbonate filters at a
density of 0.2 × 106 cells/well in 1.5-ml medium and
cultured for 18 days. Tracers were added to the donor compartment
(apical or basolateral) of the Transwell systems in uptake buffer (pH
6.0). After time intervals indicated, samples were taken from the
receiver compartment (pH 7.5) and replaced with buffer.
Inset: uptake of tracer after 2 h into the cells on the
filter membrane. A, [14C]Gly-Sar (20 µM) flux in
apical-to-basolateral direction (Ja-b) and
uptake into the cells from the apical side
(Ja-c, inset). B,
[14C]Gly-Sar (20 µM) flux in apical-to-basolateral
direction (Ja-b) and uptake into the cells from
the apical side (Ja-c, inset) in the
presence of an excess amount of unlabeled Gly-Sar (10 mM). C,
[14C]Gly-Sar (20 µM) flux in basolateral-to-apical
direction (Jb-a) and uptake into the cells from
the basolateral side (Jb-c, inset).
D, [14C]mannitol (10 µM) flux in apical-to-basolateral
direction (Ja-b) and uptake into the cells from
the apical side (Ja-c, inset). Data
shown are means ± SE, n = 3.
|
|
Expression of PEPT1 in SK-ChA-1 cells and native extrahepatic
biliary duct.
Distinct H+-peptide cotransporters have been
cloned from human tissues: PEPT1 from intestine and PEPT2 from the
kidney epithelial cells (1, 4, 12, 16). To identify
conclusively the peptide transporter found in SK-ChA-1 cells, RT-PCR
analysis of mRNA isolated from these cells using primers specific for
PEPT1 and PEPT2, respectively, was carried out. Results are given in
Fig. 6. As expected, PEPT1 is
expressed both in the small intestine and the kidney. PEPT2 is
expressed only in the kidney but not in the intestine. SK-ChA-1 cells,
cells of the gall bladder epithelium, and cells of native rabbit
extrahepatic biliary duct express PEPT1.

View larger version (88K):
[in this window]
[in a new window]
|
Fig. 6.
RT-PCR with peptide transporter (PEPT)1- and PEPT2-specific primers
and GAPDH primer. RNA samples isolated from SK-ChA-1 cells and rabbit
tissues were subjected to RT-PCR. Products were analyzed by agarose gel
electrophoresis. Expected size of the product was 860 bp, PEPT1
(top); 870 bp, PEPT2 (middle); and 430 bp, GAPDH
(bottom). Lane 1, Gene Ruler DNA ladder marker;
lane 2, small intestinal mucosa; lane 3, kidney
cortex; lane 4, extrahepatic biliary duct; lane
5, gall bladder epithelium; lane 6, SK-ChA-1 cell
line.
|
|
Transwell experiments gave strong functional evidence for the
expression of PEPT1 at the apical side of cholangiocytes. This is
supported by immunohistochemistry using a polyclonal antibody specific for PEPT1 (17) (Fig.
7). PEPT1 is localized to cholangiocytes of mouse extrahepatic biliary duct with the highest intensity at the
apical membrane.

View larger version (105K):
[in this window]
[in a new window]
|
Fig. 7.
Localization of PEPT1 immunoreactivity in murine extrahepatic
biliary duct. Murine bile duct was prepared for immunofluorescence
analysis as described in Immunohistochemistry. A polyclonal
anti-mouse-PEPT1 serum raised against amino acids 248-261 was
used. Confocal laser scanning microscopy revealed the presence of PEPT1
(A) at the brush-border membrane of cholangiocytes.
B: control incubations in parallel sections by preabsorption
of the primary antibody with the corresponding antigenic peptide.
C: phase contrast micrograph of A. D:
hemalaun/eosin staining.
|
|
 |
DISCUSSION |
So far, in mammalian tissues, H+/peptide cotransport
activity has been found by functional assays in kidney and intestine
and mostly on the mRNA level in brain, liver, lung, and pancreas. In
our present study, we describe for the first time, a
H+/dependent peptide cotransport system in the extrahepatic
biliary duct. SK-ChA-1 cells express a system in their apical membrane, which transports Gly-Sar in a pH-dependent manner into the cell. The
existence of H+-Gly-Sar cotransport is evident from the
results: 1) Gly-Sar transport is stimulated by an
extracellular acidic pH; 2) inner acidification reduces
Gly-Sar transport; and 3) protonophore FCCP, which
dissipates transmembrane electrochemical H+ gradients
inhibits [14C]Gly-Sar uptake. The
Kt value of Gly-Sar transport of 1.1 mM qualifies the system responsible for Gly-Sar uptake in these cells as
the low-affinity intestinal-type system PEPT1.
Ki values of competing peptides and
peptidomimetics also support the conclusion that the system expressed
is PEPT1. RT-PCR analysis using RNA from several tissues confirmed the
expression of PEPT1 in SK-ChA-1 cells and demonstrated that the
expression of the H+/peptide symporter PEPT1 is a
physiologically occurring fact in rabbit bile duct tissue not
restricted to the tumorous biliary epithelial cell line. From analysis
of the kinetic studies and the PCR results, we conclude that at
extrahepatic bile duct cells, predominantly PEPT1 is expressed. We
cannot rule out, however, that in addition to PEPT1, PEPT2 is
coexpressed to a very minor extent. From transepithelial flux studies
showing that flux and uptake of [14C]Gly-Sar is
carrier-mediated and that apical-to-basolateral flux and apical uptake
exceeded the flux in the opposite direction and the basolateral uptake
11- to 20-fold, we postulate that the carrier is located in the apical
membrane of SK-ChA-1 cells. This was confirmed by
immunohistochemistry. By using a PEPT1 antibody, we localized PEPT1 to
the apical membrane of mouse bile duct epithelial cells.
What could be the physiological function of peptide transport at the
biliary epithelium? Very little is known about the presence of small
peptides in bile fluid. Glutathione is secreted into bile and is almost
completely broken down (6). The authors of this study
suggested that the resulting products are reabsorbed either as
peptides, conjugates, or free amino acids. Furthermore, efficient
hepatobiliary excretion has been described for amino-acylated di- and
tripeptides (5). Glutathione and N-protected
di- and tripeptides, however, do not represent substrates for
H+-peptide cotransporters. Lacking knowledge about the
presence of di- and tripeptides in bile fluid does not necessarily mean that the concentration of potential substrates for peptide transporters in the biliary epithelium is neglectable. For example, by the use of
reverse-phase chromatography, mass spectrometry and Edman degradation,
several hydrophobic polypeptides have been unexpectedly identified in
human bile (25). Furthermore, biologically active peptides, such as atrial natriuretic factor, have been found in bile
fluid (19). A similar situation prevailed for many years regarding the physiological function of the H+/peptide
cotransport (peptide reabsorption) process in the kidney. Concentration
of small peptides in the circulation was considered to be very low
until Seal and Parker (23) could show that the plasma
levels of peptide-bound amino acids are manyfold higher than once
thought. Therefore, it became obvious that the renal reabsorptive
process for small peptides does play a significant role in the
conservation of peptide-bound amino nitrogen under physiological
conditions. It remains to be elucidated whether PEPT1 functions as a
recovery system of di- and tripeptides excreted from hepatocytes into
the bile. Further studies will be needed to clarify the existence of
significant amounts of small peptides in bile. The potential
pharmacological relevance of peptide transport is apparent from the
observation that several pharmacologically active peptidomimetic drugs,
such as certain
-lactam antibiotics, are substrates for this process
(1, 4, 12, 16, 26). Not surprisingly, PEPT1 in SK-ChA-1
cells recognizes cefadroxil (Ki = 3 mM) as
a potential substrate in our study. Of special interest is the
observation that
-aminolevulinic acid is able to inhibit Gly-Sar
uptake (Ki = 3.3 mM). This compound, a
precursor of porphyrin synthesis used as an endogenous photosensitizer
for photodynamic therapy of various tumors (20) has been
shown to be a good substrate of intestinal and renal peptide
transporters (13). This explains its high oral
bioavailability. Accumulation of
-aminolevulinic acid via PEPT1 in
bile duct epithelial cells would allow the use of this compound for
treatment of extrahepatic biliary tract carcinoma (28).
In conclusion, mammalian cholangiocytes express the
H+/peptide symporter PEPT1 in their apical membranes.
SK-ChA-1 cells represent a convenient model to study both the
physiological role and the possible clinical applications of the
peptide transport system in the extrahepatic biliary tract epithelium.
 |
ACKNOWLEDGEMENTS |
We thank Dr. A. Knuth (Krankenhaus Nordwest, Frankfurt, Germany)
and the Ludwig Institute for Cancer Research (Zurich, Switzerland) for
providing the cell line and Dr. Frank Hirche for preliminary studies.
The technical assistance of Daniela Kolmeder, Ilka Runkel, and Regina
Franke is greatly appreciated.
 |
FOOTNOTES |
This work was supported by Land Sachsen-Anhalt Grant 2880A/0028G and a
Fellowship (to I. Knütter), by Grant Da-190/6-1 of the Deutsche
Forschungsgemeinschaft, and by the Fonds der Chemischen Industrie.
Address for reprint requests and other correspondence:
M. Brandsch, Membrane Transport Group, Biozentrum of the
Martin Luther University, Halle-Wittenberg, Weinbergweg 22, D-06120
Halle, Germany (E-mail:
brandsch{at}biozentrum.uni-halle.de).
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.
First published March 13, 2002;10.1152/ajpgi.00534.2001
Received 20 December 2001; accepted in final form 4 February 2002.
 |
REFERENCES |
1.
Adibi, SA.
The oligopeptide transporter (Pept-1) in human intestine: biology and function.
Gastroenterology
113:
332-340,
1997[ISI][Medline].
2.
Alvaro, D.
Biliary epithelium: a new chapter in cell biology.
Ital J Gastroenterol Hepatol
31:
78-83,
1999[ISI][Medline].
3.
Alvaro, D,
Gigliozzi A,
Fraioli F,
Romeo R,
Papa E,
Delle Monache M,
and
Capocaccia L.
Hormonal regulation of bicarbonate secretion in the biliary epithelium.
Yale J Biol Med
70:
417-426,
1997[ISI][Medline].
4.
Amidon, GL,
and
Lee HJ.
Absorption of peptide and peptidomimetic drugs.
Annu Rev Pharmacol Toxicol
34:
321-341,
1994[ISI][Medline].
5.
Anderson, RP,
Butt TJ,
and
Chadwick VS.
Hepatobiliary excretion of bacterial formyl-methionyl peptides in rat. Structure activity studies.
Dig Dis Sci
37:
248-256,
1992[ISI][Medline].
6.
Ballatori, N,
Jacob R,
Barrett C,
and
Boyer JL.
Biliary catabolism of glutathione and differential reabsorption of its amino acid constituents.
Am J Physiol Gastrointest Liver Physiol
254:
G1-G7,
1988[Abstract/Free Full Text].
7.
Basavappa, S,
Middleton J,
Mangel AW,
McGill JM,
Cohn JA,
and
Fitz JG.
Cl
and K+ transport in human biliary cell lines.
Gastroenterology
104:
1796-1805,
1993[ISI][Medline].
8.
Brandsch, M,
Brandsch C,
Prasad PD,
Ganapathy V,
Hopfer U,
and
Leibach FH.
Identification of a renal cell line that constitutively expresses the kidney-specific high-affinity H+/peptide cotransporter.
FASEB J
9:
1489-1496,
1995[Abstract/Free Full Text].
9.
Brandsch, M,
Ganapathy V,
and
Leibach FH.
H+-peptide cotransport in Madin-Darby canine kidney cells: expression and calmodulin-dependent regulation.
Am J Physiol Renal Fluid Electrolyte Physiol
268:
F391-F397,
1995[Abstract/Free Full Text].
10.
Brandsch, M,
Knütter I,
Thunecke F,
Hartrodt B,
Born I,
Börner V,
Hirche F,
Fischer G,
and
Neubert K.
Decisive structural determinants for the interaction of proline derivatives with the intestinal H+/peptide symporter.
Eur J Biochem
266:
502-508,
1999[Abstract/Free Full Text].
11.
Brandsch, M,
Miyamoto Y,
Ganapathy V,
and
Leibach FH.
Expression and protein kinase C-dependent regulation of peptide/H+ co-transport system in the Caco-2 human colon carcinoma cell line.
Biochem J
299:
253-260,
1994[ISI][Medline].
12.
Daniel, H,
and
Herget M.
Cellular and molecular mechanisms of renal peptide transport.
Am J Physiol Renal Physiol
273:
F1-F8,
1997[Abstract/Free Full Text].
13.
Döring, F,
Walter J,
Will J,
Focking M,
Boll M,
Amasheh S,
Clauss W,
and
Daniel H.
Delta-aminolevulinic acid transport by intestinal and renal peptide transporters and its physiological and clinical implications.
J Clin Invest
101:
2761-2767,
1998[Abstract/Free Full Text].
14.
Fei, YJ,
Ganapathy V,
and
Leibach FH.
Molecular and structural features of the proton-coupled oligopeptide transporter superfamily.
Prog Nucleic Acid Res Mol Biol
58:
239-261,
1998[ISI][Medline].
15.
Ganapathy, ME,
Brandsch M,
Prasad PD,
Ganapathy V,
and
Leibach FH.
Differential recognition of
-lactam antibiotics by intestinal and renal peptide transporters, PEPT 1 and PEPT 2.
J Biol Chem
270:
25672-25677,
1995[Abstract/Free Full Text].
16.
Ganapathy, V,
Ganapathy ME,
and
Leibach FH.
Intestinal transport of peptides and amino acids.
Curr Top Membr
50:
379-412,
2001[ISI].
17.
Groneberg, DA,
Döring F,
Eynott PR,
Fischer A,
and
Daniel H.
Intestinal peptide transport: ex vivo uptake studies and localization of peptide carrier PEPT1.
Am J Physiol Gastrointest Liver Physiol
281:
G697-G704,
2001[Abstract/Free Full Text].
18.
Knuth, A,
Gabbert H,
Dippold W,
Klein O,
Sachsse W,
Bitter-Suermann D,
Prellwitz W,
and
Meyer zum Büschenfelde KH.
Biliary adenocarcinoma characterisation of three new human tumor cell lines.
J Hepatol
1:
579-596,
1985[ISI][Medline].
19.
Oh, SH,
Cho KW,
Kim SH,
Jeong GB,
Kang CW,
Hwang YH,
Seul KH,
and
Cho BH.
Identification of immunoreactive atrial natriuretic peptide in the gallbladder and bile juice of rabbit, pig and human.
Regul Pept
49:
217-223,
1994[ISI][Medline].
20.
Peng, Q,
Warloe T,
Berg K,
Moan J,
Kongshaug M,
Giercksky KE,
and
Nesland JM.
5-Aminolevulinic acid-based photodynamic therapy. Clinical research and future challenges.
Cancer
79:
2282-2308,
1979.
21.
Ramamoorthy, S,
Tiruppathi C,
Nair CN,
Mahesh VB,
Leibach FH,
and
Ganapathy V.
Relative sensitivity to inhibition by cimetidine and clonidine differentiates between the two types of Na+-H+ exchangers in cultured cells.
Biochem J
280:
317-322,
1991[ISI][Medline].
22.
Roberts, SK,
Ludwig J,
and
Larusso NF.
The pathobiology of biliary epithelia.
Gastroenterology
112:
269-279,
1997[ISI][Medline].
23.
Seal, CJ,
and
Parker DS.
Isolation and characterization of circulating low molecular weight peptides in steer, sheep and rat portal and peripheral blood.
Comp Biochem Physiol B
99:
679-685,
1991[ISI][Medline].
24.
Spurr, AR.
A low-viscosity epoxy resin embedding medium for electron microscopy.
J Ultrastruct Res
26:
31-43,
1969[ISI][Medline].
25.
Stark, M,
Jornvall H,
and
Johansson J.
Isolation and characterization of hydrophobic polypeptides in human bile.
Eur J Biochem
266:
209-214,
1999[Abstract/Free Full Text].
26.
Tsuji, A.
Intestinal absorption of
-lactam antibiotics.
In: Peptide-Based Drug Design, , edited by Taylor MD,
and Amidon GL.. Washington, DC: American Chemical Society, 1995, p. 101-134.
27.
Wenzel, U,
Diehl D,
Herget M,
and
Daniel H.
Endogenous expression of the renal high-affinity H+-peptide cotransporter in LLC-PK1 cells.
Am J Physiol Cell Physiol
275:
C1573-C1579,
1998[Abstract/Free Full Text].
28.
Zopf, T,
and
Riemann JF.
The change in laser usage in gastroenterology-the status in 1997.
Z Gastroenterol
35:
987-997,
1997[ISI][Medline].
Am J Physiol Gastrointest Liver Physiol 283(1):G222-G229
0193-1857/02 $5.00
Copyright © 2002 the American Physiological Society