Endogenous expression of the renal high-affinity
H+-peptide cotransporter in
LLC-PK1 cells
Uwe
Wenzel,
Daniela
Diehl,
Martina
Herget, and
Hannelore
Daniel
Institute of Nutritional Sciences, University of Giessen, 35392 Giessen, Germany
 |
ABSTRACT |
The reabsorption of filtered di- and
tripeptides as well as certain peptide mimetics from the tubular lumen
into renal epithelial cells is mediated by an
H+-coupled
high-affinity transport process. Here we demonstrate for the first time
H+-coupled uptake of dipeptides
into the renal proximal tubule cell line
LLC-PK1. Transport was assessed
1) by uptake studies using the
radiolabeled dipeptide
D-[3H]Phe-L-Ala,
2) by cellular accumulation of the fluorescent dipeptide D-Ala-Lys-AMCA, and
3) by measurement of intracellular
pH (pHi) changes as a
consequence of H+-coupled
dipeptide transport. Uptake of
D-Phe-L-Ala
increased linearly over 11 days postconfluency and showed all the
characteristics of the kidney cortex high-affinity peptide transporter,
e.g., a pH optimum for transport of
D-Phe-L-Ala
of 6.0, an apparent Km value for
influx of 25.8 ± 3.6 µM, and affinities of differently charged
dipeptides or the
-lactam antibiotic cefadroxil to the binding site
in the range of 20-80 µM.
pHi measurements established the
peptide transporter to induce pronounced intracellular acidification in
LLC-PK1 cells and confirm its
postulated role as a cellular acid loader.
PEPT2; proximal tubule cell line
LLC-PK1; intracellular
acidification; kinetic characterization
 |
INTRODUCTION |
PEPTIDE TRANSPORTERS located in the brush-border
membrane of kidney tubular cells play a pivotal role in preserving
amino acid nitrogen by reabsorption of di- and tripeptides filtered or
generated by enzymatic hydrolysis of larger filtered oligopeptides. Two
peptide transport systems with different substrate affinities have been
described to exist in the brush-border membrane of the tubular cells
(9, 10, 23). Both systems were found to operate in an electrogenic mode
by coupling of substrate influx to an inwardly directed
H+ gradient (9, 23, 26, 27).
Besides short-chain peptides, a number of peptidomimetics carrying a
peptide backbone, such as
-lactam antibiotics of the
aminocephalosporin class or the anti-cancer agent bestatin, serve as
substrates (8, 15, 23). The cDNAs encoding the two distinctly different
H+-peptide cotransporters have
been cloned from intestine (PEPT1) and kidney (PEPT2) of
various species (3, 4, 13, 19, 20, 22, 25). Although the high-affinity
transporter PEPT2 is expressed mainly in the kidney but not in the gut
(3, 20, 25), PEPT1 mRNA is also expressed at low levels in renal tissue (19, 20, 22).
Although the characterization of the renal high-affinity transporter
has been performed after heterologous expression (1, 3), a detailed
analysis of its function in renal epithelial cells with regard to
kinetics and acid-loading mechanisms has not been performed. This lack
of information results mainly from the unavailability of suitable cell
lines. Until now, the only cell line described to express the
kidney-specific high-affinity H+-peptide cotransporter
endogenously is SKPT-0193 Cl.2 obtained by SV40 transformation of rat
proximal tubular cells (6). In the present study we describe for the
first time the endogenous expression of a high-affinity peptide
transporter in the porcine kidney cell line
LLC-PK1. Because
LLC-PK1 cells have been shown to
differentiate into epithelial cells that have been proven to be useful
in the study of selected proximal cell processes (21, 31), they might
provide a valuable model for studies on the characteristics and
regulation of the renal high-affinity peptide transporter.
 |
METHODS |
Materials. Custom-synthesized
D-[3H]Phe-L-Ala (9 Ci/mmol) and unlabeled
D-Phe-L-Ala
were obtained from Zeneca (Cheshire, UK) and Bachem (Heidelberg,
Germany), respectively. All other peptides and
-lactams were
purchased from Sigma Chemical. The pH-sensitive fluorescent dye
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM (BCECF-AM) was obtained from Bioprobes (Leiden, Netherlands). Fluorescent N-hydroxy-succinimidyl
7-amino-4-methylcoumarin-3-acetic acid (AMCA-NHS) was obtained from
Pierce (Rockford, IL). All the materials needed for cell culture were
either from GIBCO (Eggenstein, Germany) or Renner (Dannstadt, Germany).
Rat tail collagen R was purchased from Serva. All reagents for RNA
preparation and RT-PCR were from MBI Fermentas (Heidelberg, Germany),
and the primers were custom synthesized by Eurogentec (Seraing,
Belgium). Tertiary butyl-oxy-carbonyl-D-Ala-L-Lys-tertiary
butylester
(Boc-D-Ala-L-Lys-OtBu) was a generous gift from Prof. H. Brückner (Giessen, Germany).
Cell culture.
LLC-PK1 cells (American Type
Culture Collection, CRL 1392, passage 195) were cultured and passaged
in Dulbecco's modified Eagle's medium (GIBCO 41965) supplemented with
10% fetal calf serum, 2 mM glutamine, 1% MEM nonessential amino acids
(GIBCO 01140), 10 mM HEPES, 1 mM sodium pyruvate, 100 IU/ml penicillin, and 100 µg/ml streptomycin in a humidified incubator at 37°C
under an atmosphere of 5% CO2.
Cells between passages 200 and 215 were seeded at a density of 5 × 105 cells/well on Renner
6-well plastic cell culture plates or 2.2 × 105 cells/well on 12-well plates
subsequent to collagen coating of the wells with rat tail collagen.
Transport studies. Flux studies in
LLC-PK1 cells were performed in a
buffer containing (in mM) 145 NaCl, 5.4 Cl, 1.8 CaCl2, 1.8 MgSO4, 20 glucose, and 25 HEPES/Tris (pH 7.4) or MES/Tris (pH
6.5), respectively. For uptake,
cell monolayers grown in six-well plates were washed free of
serum-containing medium and incubated with substrates or inhibitors for
15 min at 37°C. After the incubation period the cells were washed
three times with ice-cold incubation buffer, scraped off with a rubber
policeman after addition of 600 µl TEN buffer/well (in mM: 150 NaCl,
40 Tris, 1 EDTA), and digested with 20 µl of tissue solubilizer.
Cellular accumulation of
D-[3H]Phe-L-Ala was
measured subsequent to the addition of scintillation cocktail by liquid
scintillation spectroscopy. Binding of tracer to the cells was
determined as the residual radioactivity associated with the cells in
the presence of excess nonlabeled (20 mM) Gly-Gln. Uptake of
D-[3H]Phe-L-Ala over 15 min was linear for all pH values and substrate concentrations tested.
Synthesis of
D-Ala-L-Lys-AMCA
and fluorescence microscopy.
Conjugation of AMCA with the
-amino group of lysine has been carried
out using
Boc-D-Ala-L-Lys-OtBu
and AMCA-NHS as starter molecules (2). After removal of protective
groups,
D-Ala-L-Lys-AMCA was purified by two-dimensional preparative thin-layer chromatography. Determination of the compound's concentration was based on its molar
extinction coefficient (absorption maximum at 340 nm) and fluorescent
properties (emission maximum at 450 nm when excited at 340 nm).
For transport studies with the fluorescent dipeptide analog
D-Ala-L-Lys-AMCA,
LLC-PK1 cells were grown on
collagen-coated coverslips inserted into the six-well plates.
Incubation with 5 µM fluorophore-linked dipeptide was performed as
described above for the radiolabeled peptide. After the cells were
washed with ice-cold buffer, they were fixed with 3%
p-formaldehyde and 1% glutaraldehyde
for 15 min at room temperature. The coverslips were taken from the
wells and washed three times with PBS, pH 7.4, and one drop of
embedding medium was applied to the cells before adhesion to
polylysine-coated coverslips. Specific uptake of
D-Ala-L-Lys-AMCA was assessed by fluorescence
microscopy using a Leitz Aristoplan microscope, and cells were observed
in either fluorescent light using filterblock A (band-pass
filter 340-380 nm for excitation; long-pass filter 425 nm for
emission) or by using Nomarski optics as described previously (12).
Intracellular pH measurements. For
intracelllular pH (pHi)
measurements, LLC-PK1 cells grown
in 12-well plates were loaded with BCECF by preincubation with 5 µM
lipophilic acetoxymethyl-ester (BCECF-AM) at 37°C for 30 min.
Subsequently, the monolayers were washed with buffer at pH 7.4, and the
buffers with or without substrates were changed by superfusion at the
time points indicated in the graphs. Intracellular apparent
H+ activity was determined by
measuring the intensity of emission at 538 nm after excitation of the
fluorophore at 444 nm (isosbestic point) and 490 nm (pH-sensitive
wavelength), respectively, using a microtiter plate reader (Fluoroskan
Ascent-Labsystems, Merlin Diagnostika, Bornheim-Hersel, Germany). The
444/490 fluorescence ratio was converted to
pHi values based on a calibration
curve generated by estimation of the fluorescence ratio in buffers of various pH (5).
RT-PCR from RNA of LLC-PK1 cells.
RNA from LLC-PK1 cells was
isolated by using the Tristar RNA-clean kit from MBI Fermentas
(Heidelberg, Germany). RT-PCR was performed with 5 µg of isolated
RNA. First-strand cDNA synthesis was accomplished with a primer
representing nucleotides 1899-1879 (back primer:
5'-CCTGTGACAGAGAACATGACC-3') of the protein-coding region
of rabbit PEPT2. PCR amplification of a 732-bp product was achieved
with a forward primer, representing nucleotides 1167-1188 (5'-CTAGCATGCCTG GCATTTGCAG-3') of the rabbit
PEPT2 protein-coding region, and the back primer. Amplification was
performed with 35 cycles (95°C denaturation for 1 min, 55°C
hybridization for 2 min, 72°C extensions for 2 min; Personal
Cycler; Biometra, Göttingen, Germany). RT-PCR products were
separated on a 1% agrose gel and visualized by ethidium bromide.
Calculations and statistics. All
calculations (linear as well as nonlinear regression analysis) were
performed by using Prism 2.01 (Graph PAD, Los Angeles, CA). For each
variable, three to nine independent experiments were carried out. Data
are means ± SE. Significance of differences between control and
treated cells was determined by a nonpaired
t-test.
 |
RESULTS |
Uptake of
D-[3H]Phe-L-Ala and
D-Ala-L-Lys-AMCA into
LLC-PK1 cells in the postconfluent state.
Peptide transport into LLC-PK1
cells was measured by uptake of radiolabeled
D-Phe-L-Ala at various time points after the
cells had reached confluency. Although
LLC-PK1 cells have been described not to possess substantial peptide transport activity on the day of
reaching confluency (7), uptake of
D-[3H]Phe-L-Ala at pH
6.0 increased almost linearly for up to 11 days in the postconfluent
state (Fig. 1). Moreover, transport rates were suppressed to rates similar to that of confluent cells (0 days) by
the addition of 1 mM Gly-Gln (Fig. 1). All further uptake experiments
were performed at day 9 of
postconfluency, since cell adherence to the culture wells decreased
subsequent to this time point. The uptake of dipeptides or dipeptide
mimetics into LLC-PK1 cells in the
postconfluent state was also demonstrated by transport studies, using
the fluorescent dipeptide
D-Ala-L-Lys-AMCA
as a substrate. Although cells displayed a bright blue fluorescence when incubated with the fluorophore-conjugated dipeptide, simultaneous application of 5 mM Gly-Gln reduced staining of the cells to background levels (Fig. 2).

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 1.
Uptake of 5 µM
D-[3H]Phe-L-Ala into
LLC-PK1 cells as a function of time after confluence; 5 × 105 cells/well were seeded in collagen-coated
plastic 6-well culture plates. Cells reached confluency on the day
postseeding. Fresh medium was given every second day until
day 4 postconfluently, after which
medium was renewed daily. Uptake of
[3H]D-Phe-L-Ala
is shown at indicated time intervals postconfluently in absence ( )
or presence ( ) of 1 mM Gly-Gln.
|
|

View larger version (167K):
[in this window]
[in a new window]
|
Fig. 2.
Uptake of 5 µM D-Ala-Lys-AMCA
into LLC-PK1 cells. Uptake of the
fluorescent dipeptide was determined 9 days postconfluently at pH 6.0 and is shown in absence (A,
C) or presence
(B,
D) of 5 mM Gly-Gln. Shown are
photomicrographs using Nomarski optics
(A,
B) or conventional fluorescence
microscopy (C,
D).
|
|
Characteristics of
D-[3H]Phe-L-Ala influx
into LLC-PK1 cells.
Transport of D-Phe-L-Ala into
LLC-PK1 cells as a function of apical pH increased fourfold
when buffer pH values were reduced from 7.4 to 6.0 (Fig.
3, inset). At pH values
<5.5, transport rates were moderately reduced when compared with the
transport optimum at pH 6.0-5.5. Uptake of
D-Phe-L-Ala as a function of substrate
concentration followed Michaelis-Menten kinetics with an apparent
Michaelis-Menten constant
(Km) of 25.8 ± 3.6 µM and a maximum velocity
(Vmax) of 33.4 ± 1.7 pmol · cm
2 · 15 min
1 (Fig. 3).
The kinetic characteristics of
D-Phe-Ala influx into LLC-PK1 cells therefore closely
resemble those found for the same substrate when assessed in
Xenopus oocytes expressing the cloned renal PEPT2 transporter (11).

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 3.
Uptake of
D-[3H]Phe-L-Ala into
LLC-PK1 cells as a function of substrate concentration and
incubation pH. Transport was measured at pH 6.0 at day
9 postconfluently in the presence of increasing
concentrations of
D-Phe-L-Ala.
Radioactivity associated with the cells in the presence of 20 mM
Gly-Gln was assumed to represent substrate diffused or bound, and the
amount was subtracted from total counts. Curves were fitted to a
Michaelis-Menten equation by nonlinear regression analysis, and the
apparent Km value
was derived by the least-squares method.
Inset: transport of 5 µM
D-[3H]Phe-L-Ala when
measured in a range between pH 5.0 and 8.0.
|
|
Substrate specificity. The substrate
specificity of the expressed activity in LLC-PK1 was
assessed by determining the ability of a variety of dipeptides and
peptidomimetics to inhibit influx of
D-[3H]Phe-L-Ala (Table
1). Gly-Gln, Gly-Asp, and Gly-Lys were all strong inhibitors of
D-Phe-L-Ala
uptake, irrespective of the different net charges they bear at pH 6.0. Although a dipeptide and a tripeptide consisiting of alanine also
inhibited dipeptide transport, the free amino acid failed to reduce
D-Phe-Ala uptake significantly. The ability of the transporter to interact also with peptide mimetics is shown by the inhibition of transport by the amino
-lactam antibiotic cefadroxil. In contrast, benzylpenicillin, another
-lactam, failed to reduce transport. Moreover, captopril, an angiotensin-converting enzyme inhibitor that has been demonstrated to
interact with PEPT1 (4) but not with PEPT2 (3), was also unable to
inhibit
D-Phe-L-Ala
influx into LLC-PK1 cells. The
high-affinity phenotype interaction of cefadroxil with the transporter
was confirmed by its dose-dependent inhibition with an apparent
Ki value of 15.2 ± 1.1 µM (Fig. 4). Cefadroxil uptake
in oocytes mediated by the cloned PEPT2 (3) occurs with a
Km of 25.8 ± 6.3 µM but showed an
~30-fold lower affinity when studied with the cloned intestinal
isoform PEPT1 (4). In addition, the apparent
Ki values of
three differently charged dipeptides (Gly-Gln, Gly-Asp, Gly-Lys) were
determined. This was of interest since we recently demonstrated for
PEPT2, when expressed in oocytes, that affinities for the peptides
decreased in the order Gly-Asp, Gly-Gln, Gly-Lys (1). From Fig. 4 it
becomes evident that all substrates displayed a high affinity to the
binding site of the peptide transporter in
LLC-PK1 cells. Moreover,
affinities of the differently charged dipeptides clearly revealed
PEPT2-like characteristics with
Ki values of
8.0 ± 1.3 µM for Gly-Asp, 30.3 ± 1.2 µM for
Gly-Gln, and 151.8 ± 1.4 µM for Gly-Lys, respectively.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 4.
Ki values for
differently charged dipeptides and the -lactam, cefadroxil, with
regard to
D-[3H]Phe-L-Ala uptake
into LLC-PK1 cells. Uptake of 5 µM
D-Phe-L-Ala at pH 6.0 was measured in the
presence of increasing concentrations of cefadroxil ( ), Gly-Asp
( ), Gly-Gln ( ), or Gly-Lys ( ), respectively. Data represent
means ± SE from 3-6 wells per concentration tested.
|
|
Intracellular acidification of LLC-PK1
cells as a consequence of
H+-peptide
cotransport.
H+-coupled peptide transport with
a concomitant decline in pHi has
so far been demonstrated for the renal transporter only for the acidic
substrate Ala-Asp (17).
By using the pHi indicator BCECF,
here we show that superfusion of
LLC-PK1 cells with acidic
(Gly-Asp), neutral (Gly-Gln), or basic (Gly-Lys) dipeptides at
extracellular pH 6.0 leads to strong intracellular acidification that
reached steady-state levels at a
pHi of 6.25 and did not differ
significantly between those peptides chosen (Fig.
5). In contrast, the acidification induced by cefadroxil was markedly smaller (Fig. 5). After the substrates were
washed out by perfusion with buffer pH 7.4, cells totally recovered
from the peptide or peptide mimetic-induced acid load, and
pHi returned to its initial
values.

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 5.
Intracellular acidification of
LLC-PK1 cells as a consequence of
apical addition of cefadroxil, Gly-Asp, Gly-Gln, or Gly-Lys (all 1 mM).
Cells were loaded with intracellular pH
(pHi) indicator BCECF to allow
determination of pHi. Fluorescence
of cells was measured at 1-min intervals. At the beginning of the
experiment, cells were incubated with buffer pH 7.4, which was changed
for buffer pH 6.0 after 5 min (arrows). When cells reached an
equilibrium of pHi, buffer was
changed for cefadroxil (A), Gly-Asp
(B), Gly-Gln
(C), or Gly-Lys
(D) containing buffer pH 6.0 (1). Substrates were washed out by
superfusion with buffer pH 7.4 (2).
Data represent means ± SE from 4 independent wells. Of each well 5 fluorescence spots were measured over the time period indicated and
given as the mean per well.
|
|
Expression of a PEPT2 isoform in LLC-PK1
cells.
Although the functional data obtained suggested that the transporter
expressed in LLC-PK1 cells is
PEPT2-like, the porcine transporter has not been cloned, and therefore
it is not known whether the transporter is expressed in these cells. We
therefore performed RT-PCR analysis with specific primers derived from
highly conserved regions of cloned PEPT2. It becomes evident from Fig. 6 that a product of 732 bp (specific for
PEPT2 but not PEPT1) was amplified from
LLC-PK1 RNA samples. In addition,
the amplified product revealed the same
EcoR V restriction site as rabbit
PEPT2, suggesting that at least a very similar gene product is
expressed in LLC-PK1 cells.

View larger version (98K):
[in this window]
[in a new window]
|
Fig. 6.
RT-PCR with PEPT2-specific primers using RNA from
LLC-PK1 cells. RT-PCR products
were amplified from total RNA using specific forward and backward
primers from the PEPT2 coding region to yield a 732-bp fragment. The
product was digested with EcoR V to
449- and 283-bp fragments, indicating the same restriction site as the
cloned PEPT2 from rabbit renal proximal tubule.
|
|
 |
DISCUSSION |
Reabsorption of short-chain peptides in the mammalian renal tubule has
been described as mediated by two different
H+-coupled transport systems that
differ considerably in substrate affinities (9, 10, 23). Although the
high-affinity-type transport system PEPT2 is prominent on the mRNA
level and the functional level, Northern blot analysis suggested that
the mRNA of the low-affinity-type transporter PEPT1 is also expressed
in kidney but at low levels (19, 20, 22). Brandsch et al. (6) suggested
that PEPT1 and PEPT2 may be located in different sections of the
nephron. According to their hypothesis, the concentration of small
peptides increases from proximal to distal parts of the nephron, due to
progressive hydrolysis of filtered oligopeptides by brush-border
membrane peptidases. Therefore, the presence of the high-affinity-type
PEPT2 in more proximal and of the low-affinity-type PEPT1 in more
distal parts of the tubule would be advantageous with regard to the
most efficient conservation of amino acid nitrogen. However, so far the
proposed different localizations of both transporter isoforms along the
tubule, e.g., by in situ hybridization techniques or
immunolocalization, have not been reported.
Madin-Darby canine kidney cells, which display characteristics of cells
of the distal tubule (18) or collecting duct (24), have been
demonstrated to express a low-affinity-type peptide (PEPT1-like)
transport activity, suggesting that distal parts of the tubule express
solely functional PEPT1 carriers. Moreover, it was shown that this
transporter activity is regulated by calmodulin-dependent processes
(7).
Although, recently, expression of the high-affinity PEPT2 transporter
in rat proximal tubular cells immortalized by SV40 transformation (6)
has been demonstrated, these cells are not readily available. The
well-established renal proximal cell line
LLC-PK1 (16, 21, 28), on the other
hand, was reported not to possess endogenous peptide transport activity
(7, 29, 30), a feature that was exploited to use LLC-PK1 as
a host for heterologous expression of PEPT1 and PEPT2 (29-31).
Confirming the results of these studies, it was shown here that, before
reaching the confluent state, LLC-PK1 cells possess indeed
only very low peptide transport activity. However, we also clearly
demonstrate that endogenous peptide transport activity resembling the
PEPT2 type is expressed in LLC-PK1
after cells have reached confluency. Phenotypical characteristics of PEPT2, such as the pH optimum for
D-[3H]Phe-L-Ala influx,
the kinetics of dipeptide influx, and the existence of a PEPT2-specific
mRNA present in LLC-PK1 cells,
justify this conclusion. In addition, the apparent affinities for
dipeptides and cefadroxil determined in
LLC-PK1 are almost identical to
those reported in Xenopus laevis
oocytes (3, 11) or Pichia pastoris (12) expressing the cloned rabbit renal PEPT2 or in Hela cells expressing the human PEPT2 (14). Affinities of the same substrates for
interaction with the intestinal transporter are >20-fold lower when
determined in oocytes expressing PEPT1 (4, 11). In the postconfluent
state LLC-PK1 cells not only transport
D-[3H]Phe-L-Ala but
also the dipeptide mimetic
D-Ala-L-Lys-AMCA.
This fluorescent dipeptide has recently been demonstrated to display influx characteristics in PEPT2-expressing P. pastoris that were almost identical to those obtained by use
of radiolabeled D-Phe-L-Ala (12). The
coumarin-conjugated dipeptide therefore might allow investigators to
study peptide transport in LLC-PK1 cells independently of
expensive custom synthesis of radiolabeled substrates.
Although the pH dependency of dipeptide transport and the rheogenic
character of PEPT2-mediated influx of neutral dipeptides already
suggested that H+ is the cotransported ion species, this
had been verified experimentally in renal cells only for Ala-Asp (17).
Here we show that translocation of peptide substrates is associated
with H+ influx that reduces
pHi markedly, irrespective of the
net charge of the substrates. By using the
pHi indicator BCECF, we
demonstrate that intracellular acidification rates following perfusion
with Gly-Asp, Gly-Gln, and Gly-Lys are very similar, whereas those generated by the
-lactam cefadroxil are significantly lower. When
currents associated with transport of Gly-Asp, Gly-Gln, and Gly-Lys
were determined in voltage-clamped oocytes expressing the rabbit PEPT2,
we observed that the same three substrates generated the same maximal
current responses, independently of the net charge of the substrates at
extracellular pH 6.5 (1). Although
pHi could not be measured in
oocytes expressing PEPT2, we suggested that the different dipeptides
were transported by the same
peptide-H+ flux-coupling ratio and
that this is the consequence of transport of only the zwitterionic form
of the substrates. Our present findings in
LLC-PK1 cells confirm this
hypothesis by almost identical intracellular acidification rates in the
presence of the three differently charged peptides, which may also
result from similar if not identical flux-coupling ratios and maximal
transport rates.
That pHi is more reduced by
dipeptides than by cefadroxil suggests a higher maximal transport
capacity for dipeptides. This may be a consequence of the
configuration, e.g., when peptides consisting of
L-amino acids are compared with
substrates with a
D-configuration in the
amino-terminal position, such as in cefadroxil or
D-Phe-L-Ala.
This hypothesis is supported by the fact that pHi changes induced by
D-Phe-L-Ala
are comparable with those of cefadroxil but smaller than those induced
by dipeptides consisting of
L-amino acids only (data not
shown). However, it needs to be emphasized that a rapid intracellular
hydrolysis of the dipeptides consisting of
L-
-amino acids could also
contribute to the more pronounced decrease in
pHi observed for the natural dipeptides.
The demonstration that pHi is
markedly reduced when dipeptides are taken up by the renal peptide
transporter addresses the physiological importance of these
transport-mediated pHi changes. Because di- and tripeptides are present in plasma and are continuously filtered in the glomerulus, the renal peptide transporter operates as a
constant acid loader in tubular cells. This is important for both the
pHi recovery systems and their
regulation, as well as for other metabolic events such as increased
renal ammoniagenesis in response to a low
pHi. Because a number of protein
kinase recognition sites have been identified in the coding sequence of
PEPT2, regulation of transport activity, in particular in relation to
changes in pHi, needs to be
investigated. For this purpose
LLC-PK1 might provide a very
useful cellular model.
 |
ACKNOWLEDGEMENTS |
We gratefully acknowledge Prof. Dr. H. Brückner (University
of Giessen, Germany) for supplying
Boc-D-Ala-L-Lys-OtBu.
 |
FOOTNOTES |
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. §1734 solely to indicate this fact.
Address for reprint requests: H. Daniel, Institute of Nutritional
Sciences, Wilhelmstr. 20, 35392 Giessen, Germany.
Received 15 June 1998; accepted in final form 21 August 1998.
 |
REFERENCES |
1.
Amasheh, S.,
U. Wenzel,
W.-M. Weber,
W. Clauss,
and
H. Daniel.
Electrophysiological analysis of the function of the mammalian renal peptide transporter expressed in Xenopus laevis oocytes.
J. Physiol. (Lond.)
504:
169-174,
1997[Abstract].
2.
Anderson, G. W.,
J. E. Zimmermann,
and
F. M. Callahan.
N-hydroxy-succinimide esters in peptide synthesis.
J. Am. Soc.
86:
1839-1849,
1963.
3.
Boll, M.,
M. Herget,
M. Wagener,
W.-M. Weber,
D. Markovich,
J. Biber,
W. Clauss,
H. Murer,
and
H. Daniel.
Expression cloning and functional characterization of the kidney cortex high-affinity proton-coupled peptide transporter.
Proc. Natl. Acad. Sci. USA
93:
284-289,
1996[Abstract/Free Full Text].
4.
Boll, M.,
D. Markovich,
W.-M. Weber,
H. Korte,
H. Daniel,
and
H. Murer.
Expression cloning of a cDNA from rabbit small intestine related to proton-coupled transport of peptides,
-lactam antibiotics and ACE-inhibitors.
Pflügers Arch.
429:
146-149,
1994[Medline].
5.
Boyarski, G.,
C. Hanssen,
and
L. Clyne.
Superiority of in vitro over in vivo calibrations of BCECF in vascular smooth muscle cells.
FASEB J.
10:
1205-1212,
1996[Abstract/Free Full Text].
6.
Brandsch, M.,
C. Brandsch,
P. D. Prasad,
V. Ganapathy,
U. Hopfer,
and
F. H. Leibach.
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].
7.
Brandsch, M.,
V. Ganapathy,
and
F. H. Leibach.
H+-peptide cotransport in Madin-Darby canine kidney cells: expression and calmodulin-dependent regulation.
Am. J. Physiol.
268 (Renal Fluid Electrolyte Physiol. 37):
F391-F397,
1995[Abstract/Free Full Text].
8.
Daniel, H.,
and
S. A. Adibi.
Transport of
-lactam antibiotics in kidney brush border membrane.
J. Clin. Invest.
92:
2215-2223,
1993[Medline].
9.
Daniel, H.,
E. L. Morse,
and
S. A. Adibi.
The high and low affinity transport systems for dipeptides in kidney brush border membrane respond differently to alterations in pH gradient and membrane potential.
J. Biol. Chem.
266:
19917-19924,
1991[Abstract/Free Full Text].
10.
Daniel, H.,
E. L. Morse,
and
S. A. Adibi.
Determinants of substrate affinity for the oligopeptide/H+ symporter in the renal brush border membrane.
J. Biol. Chem.
267:
9565-9573,
1992[Abstract/Free Full Text].
11.
Döring, F.,
D. Dorn,
U. Bachfischer,
S. Amasheh,
M. Herget,
and
H. Daniel.
Functional analysis of a chimeric mammalian peptide transporter derived from the intestinal and renal isoforms.
J. Physiol. (Lond.)
497:
773-779,
1996[Abstract].
12.
Döring, F.,
T. Michel,
A. Rösel,
M. Nickolaus,
and
H. Daniel.
Expression of the mammalian renal peptide transporter PEPT2 in the yeast Pichia pastoris and applications of the yeast system for functional analysis.
Mol. Membr. Biol.
15:
79-88,
1998[Medline].
13.
Fei, Y.-J.,
Y. Kanai,
S. Nussberger,
V. Ganapathy,
F. H. Leibach,
M. F. Romero,
S. K. Singh,
W. F. Boron,
and
M. A. Hediger.
Expression cloning of a mammalian proton-coupled oligopeptide transporter.
Nature
368:
563-566,
1994[Medline].
14.
Ganapathy, M. E.,
M. Brandsch,
P. D. Prasad,
V. Ganapathy,
and
F. H. Leibach.
Differential recognition of beta-lactam antibiotics by intestinal and renal peptide transporters, PEPT1 and PEPT2.
J. Biol. Chem.
270:
25672-25677,
1995[Abstract/Free Full Text].
15.
Hori, R.,
Y. Tomita,
T. Katsura,
M. Yasuhara,
K.-I. Inui,
and
M. Takano.
Transport of bestatin in rat renal brush-border membrane vesicles.
Biochem. Pharmacol.
45:
1763-1768,
1993[Medline].
16.
Hull, R. N.,
W. R. Cherry,
and
G. W. Weaver.
The origin and characteristics of a pig kidney cell strain, LLC-PK1.
In Vitro
12:
670-677,
1976[Medline].
17.
Jin, W.,
and
U. Hopfer.
Dipeptide-induced Cl
secretion in proximal tubule cells.
Am. J. Physiol.
273 (Cell Physiol. 42):
C1623-C1631,
1997[Abstract/Free Full Text].
18.
Kersting, U.,
A. Schwab,
M. Treidtel,
W. Pfaller,
G. Gstraunthaler,
W. Steigner,
and
H. Oberleithner.
Differentiation of Madin-Darby canine kidney cells depends on cell culture conditions.
Cell. Physiol. Biochem.
3:
42-55,
1993.
19.
Liang, R.,
Y.-J. Fei,
P. D. Prasad,
S. Ramamoorthy,
H. Han,
T. L. Yang-Feng,
M. A. Hediger,
V. Ganapathy,
and
F. H. Leibach.
Human intestinal H+/peptide cotransporter.
J. Biol. Chem.
270:
6456-6463,
1995[Abstract/Free Full Text].
20.
Liu, W.,
R. Liang,
S. Ramamoorthy,
Y.-J. Fei,
M. E. Ganapathy,
M. A. Hediger,
V. Ganapathy,
and
F. H. Leibach.
Molecular cloning of PepT2, a new member of the H+/peptide cotransporter family, from human kidney.
Biochim. Biophys. Acta
1235:
461-466,
1995[Medline].
21.
Malmström, K.,
and
H. Murer.
Parathyroid hormone inhibits phosphate transport in OK cells but not in LLC-PK1 and JTC-12.P3 cells.
Am. J. Physiol.
251 (Cell Physiol. 20):
C23-C31,
1986[Abstract/Free Full Text].
22.
Miyamoto, K.-I.,
T. Shiraga,
K. Morita,
H. Yamamoto,
H. Haga,
Y. Taketani,
I. Tamai,
Y. Sai,
A. Tsuji,
and
E. Takeda.
Sequence, tissue distribution and developmental changes in rat intestinal oligopeptide transporter.
Biochim. Biophys. Acta
1305:
34-38,
1996[Medline].
23.
Ries, M.,
U. Wenzel,
and
H. Daniel.
Transport of cefadroxil in rat kidney brush-border membranes is mediated by two electrogenic H+-coupled systems.
J. Pharmacol. Exp. Ther.
271:
1327-1333,
1994[Abstract].
24.
Rindler, M. J.,
L. M. Chuman,
L. Shaffer,
and
M. H. Saier.
Retention of differentiated properties in an established dog kidney epithelial cell line (MDCK).
J. Cell Biol.
81:
635-648,
1981[Abstract].
25.
Saito, H.,
T. Terada,
M. Okuda,
S. Sasaki,
and
K.-I. Inui.
Molecular cloning and tissue distribution of rat peptide transporter PEPT2.
Biochim. Biophys. Acta
1280:
173-177,
1996[Medline].
26.
Silbernagl, S.,
V. Ganapathy,
and
F. H. Leibach.
H+ gradient-driven dipeptide reabsorption in proximal tubule of rat kidney. Studies in vivo and in vitro.
Am. J. Physiol.
253 (Renal Fluid Electrolyte Physiol. 22):
F448-F457,
1987[Abstract/Free Full Text].
27.
Skopicki, H. A.,
K. Fisher,
D. Zikos,
R. Bloch,
G. Flouret,
and
D. R. Peters.
Multiple carriers for dipeptide transport: carrier mediated transport of glycyl-L-proline in renal BBMV.
Am. J. Physiol.
261 (Renal Fluid Electrolyte Physiol. 30):
F670-F678,
1991[Abstract/Free Full Text].
28.
States, B.,
D. Harris,
and
S. Segal.
Differences between OK and LLC-PK1 cells: cystine handling.
Am. J. Physiol.
261 (Cell Physiol. 30):
C8-C16,
1991[Abstract/Free Full Text].
29.
Terada, T.,
H. Saito,
M. Mukai,
and
K.-I. Inui.
Identification of the histidine residues involved in substrate recognition by a rat H+/peptide cotransporter, PEPT1.
FEBS Lett.
394:
196-200,
1996[Medline].
30.
Terada, T.,
H. Saito,
M. Mukai,
and
K.-I. Inui.
Characterization of stably transfected kidney epithelial cell line expressing rat H+/peptide cotransporter PepT1: localization of PepT1 and transport of
-lactam antibiotics.
J. Pharmacol. Exp. Ther.
281:
1415-1421,
1997[Abstract].
31.
Terada, T.,
H. Saito,
M. Mukai,
and
K.-I. Inui.
Recognition of
-lactam antibiotics by rat peptide transporters, PEPT1 and PEPT2, in LLC-PK1 cells.
Am. J. Physiol.
273 (Renal Physiol. 42):
F706-F711,
1997[Abstract/Free Full Text].
Am J Physiol Cell Physiol 275(6):C1573-C1579
0002-9513/98 $5.00
Copyright © 1998 the American Physiological Society