(Received for publication, June 5, 1995; and in revised form, August 30, 1995)
From the
This study was initiated to determine if there are differences
in the recognition of -lactam antibiotics as substrates between
intestinal and renal peptide transporters, PEPT 1 and PEPT 2. Reverse
transcription-coupled polymerase chain reaction and/or Northern blot
analysis have established that the human intestinal cell line Caco-2
expresses PEPT 1 but not PEPT 2, whereas the rat proximal tubule cell
line SKPT expresses PEPT 2 but not PEPT 1. Detailed kinetic analysis
has provided unequivocal evidence for participation of PEPT 2 in SKPT
cells in the transport of the dipeptide glycylsarcosine and the
aminocephalosporin cephalexin. The substrate recognition pattern of
PEPT 1 and PEPT 2 was studied with cefadroxil (a cephalosporin) and
cyclacillin (a penicillin) as model substrates for the peptide
transporters constitutively expressed in Caco-2 cells (PEPT 1) and SKPT
cells (PEPT 2). Cyclacillin was 9-fold more potent than cefadroxil in
competing with glycylsarcosine for uptake via PEPT 1. In contrast,
cefadroxil was 13-fold more potent than cyclacillin in competing with
the dipeptide for uptake via PEPT 2. The substrate recognition pattern
of PEPT 1 and PEPT 2 was also investigated using cloned human peptide
transporters functionally expressed in HeLa cells. Expression of PEPT 1
or PEPT 2 in HeLa cells was found to induce H
-coupled
cephalexin uptake in these cells. As was the case with Caco-2 cells and
SKPT cells, the uptake of glycylsarcosine induced in HeLa cells by PEPT
1 cDNA and PEPT 2 cDNA was inhibitable by cyclacillin and cefadroxil.
Again, the PEPT 1 cDNA-induced dipeptide uptake was inhibited more
potently by cyclacillin than by cefadroxil, and the PEPT 2 cDNA-induced
dipeptide uptake was inhibited more potently by cefadroxil than by
cyclacillin. It is concluded that there are marked differences between
the intestinal and renal peptide transporters in the recognition of
-lactam antibiotics as substrates.
Peptide transporters are primarily expressed in the small
intestine and kidney. The endogenous substrates for these transporters
are small peptides consisting of two or three amino acids(1) .
These transporters function in the absorption of peptides arising from
digestion of dietary proteins (small intestine) and in the reabsorption
of peptides present in the glomerular
filtrate(2, 3, 4) . It became apparent that
the peptide transporters can serve as carriers for exogenous compounds
which bear structural resemblance to the physiologically occurring
peptide substrates, when the transport of cephalexin, a -lactam
antibiotic, was shown to be mediated in the kidney (5) and
small intestine (6) by the peptide transport system. The
peptide substrates of the peptide transport system and cephalexin share
certain structural features such as a peptide bond with an
-amino
group and a terminal carboxylic acid group. This structural similarity
is apparently the basis for the molecular mimicry, enabling the peptide
transporters to accept cephalexin as a substrate. The pharmacological
relevance of the peptide transporters became immediately evident from
these studies because of the enormous potential of these transporters
to serve as carriers for a variety of peptidomimetic drugs. Subsequent
studies have indeed identified a wide spectrum of pharmacologically
active compounds that are accepted as substrates by the peptide
transporters in the intestine and/or
kidney(7, 8, 9) .
The two organs, the
small intestine and the kidney, in which the peptide transporters are
primarily expressed play an important role in the therapeutic efficacy
of -lactam antibiotics. The intestinal peptide transport system is
responsible for the oral absorption of these drugs. The renal peptide
transport system, which functions in the reabsorption of these drugs
from the glomerular filtrate, enhances the half-life of these drugs in
the circulation. Therefore, detailed studies on the interaction of
-lactam antibiotics with the peptide transporters in these two
organs are vital to the understanding of the pharmacodynamics of these
drugs. Recent molecular cloning studies (10, 11, 12, 13) have shown that the
peptide transporters expressed in the small intestine and kidney are
structurally different. The human intestinal peptide transporter (PEPT
1) and the human kidney peptide transporter (PEPT 2) exhibit only about
50% homology in amino acid sequence. PEPT 1 is expressed primarily in
the small intestine and, to a small extent, in the kidney, whereas PEPT
2 is expressed only in the kidney(12) . Nonetheless, both
transporters accept small peptides as substrates and are driven by a
transmembrane electrochemical H
gradient. The current
investigation was undertaken to study in detail the interaction of
-lactam antibiotics with PEPT 1 and PEPT 2 and to determine
whether there are differences between these two transporters in the
recognition of these drugs as substrates.
To establish unequivocally that Caco-2 cells express PEPT 1 and SKPT
cells express PEPT 2, we performed the following experiments. We
determined the identity of the peptide transporter present in Caco-2
cells by RT-PCR using PEPT 1- and PEPT 2-specific
primers(11, 12) . The specificity of each pair of
primers was established by PCR using respective cDNAs as templates. RNA
samples prepared from human intestine, human kidney, and Caco-2 cells
were subjected to RT-PCR using these primers and the products were
analyzed by agarose gel electrophoresis. The results of these
experiments, given in Fig. 1, show that the PEPT 1-specific PCR
product of expected size (1.2 kb) was generated from all three RNA
samples. In contrast, the PEPT 2-specific PCR product (
0.9 kb in
size) was generated only from kidney RNA. RNA samples from Caco-2 cells
and intestine were negative for this product (Fig. 1). These
data demonstrate that Caco-2 cells express PEPT 1 and not PEPT 2.
Figure 1: RT-PCR with PEPT 1- and PEPT 2-specific primers. RNA samples isolated from human intestine, human kidney, and Caco-2 cells were subjected to RT-PCR using PEPT 1- and PEPT 2-specific primers. The RT-PCR products were analyzed by agarose gel electrophoresis. The expected size of the product was 1.2 kb in the case of PEPT 1 and 0.9 kb in the case of PEPT 2.
The nucleotide sequences of the rat homologs of PEPT 1 and PEPT 2
have not yet been determined. Therefore, we investigated the expression
of PEPT 1/PEPT 2 in the SKPT cell line by Northern blot hybridization
using the human PEPT 1 and PEPT 2 cDNAs as probes (Fig. 2).
Poly(A) RNA prepared from SKPT cells and Caco-2 cells
was size-fractionated and probed with these cDNAs. With the PEPT 1
probe, the presence of a major hybridizing band, 3.1 kb in size, was
evident in Caco-2 cells, but this band was absent in SKPT cells. With
the PEPT 2 probe, there was a primary hybridizing band, 4.2 kb in size,
in SKPT cells. This signal was absent in Caco-2 cells. These results
indicate that the peptide transporter expressed in SKPT cells is PEPT
2. PEPT 1, which is expressed in Caco-2 cells, is not present in SKPT
cells.
Figure 2:
Northern
blot analysis of poly(A) RNA from SKPT cells and
Caco-2 cells using PEPT 1 cDNA and PEPT 2 cDNA as probes.
Poly(A)
RNA, isolated from SKPT cells and Caco-2
cells, was size-fractionated and probed by sequential hybridization
with human PEPT 1 cDNA and human PEPT 2
cDNA.
Figure 3:
Inhibition of
[C]Gly-Sar uptake by
-lactam antibiotics in
SKPT cells. Uptake of 3 µM [
C]Gly-Sar was measured for 10 min in
monolayer cultures of SKPT cells at pH 6.0 in the absence and presence
of increasing concentrations of
-lactam antibiotics
(0.316-10,000 µM). Uptake of Gly-Sar measured in the
absence of the inhibitors was taken as 100% (51.0 ± 1.3 pmol/mg
of protein/10 min). Key:
, cefadroxil;
, cyclacillin;
, cephalexin;
, ampicillin;
, cephalothin;
,
benzylpenicillin.
We then investigated the kinetics of
inhibition of glycylsarcosine uptake by cefadroxil (a cephalosporin)
and cyclacillin (a penicillin). The presence of the antibiotics
decreased the affinity of PEPT 2 for glycylsarcosine, without affecting
the maximal velocity (Fig. 4). The Michaelis-Menten constant (K) for glycylsarcosine in the absence of the
antibiotics was 48 ± 4 µM. This value was increased
3.3-fold to 156 ± 17 µM in the presence of 40
µM cyclacillin. Similarly, the K
value was increased 2.3-fold to 112 ± 3 µM in
the presence of 3 µM cefadroxil. Therefore, the
-lactam antibiotics and the dipeptide substrates apparently
compete for the same binding site on PEPT 2.
Figure 4:
Kinetics of inhibition of Gly-Sar uptake
by cefadroxil and cyclacillin in SKPT cells. Uptake of Gly-Sar was
measured in monolayer cultures of SKPT cells in the absence () or
presence (
) of 3 µM cefadroxil or 40 µM cyclacillin (
). The uptake was measured at pH 6.0 with a
10-min incubation period. Concentration of Gly-Sar was varied between 5
and 500 µM, keeping the concentration of
[
C]Gly-Sar constant at 5 µM and
adding unlabeled Gly-Sar to desired concentrations. Non-mediated
component was determined from the uptake of radiolabel measured in the
presence of 10 mM Gly-Pro. This component was subtracted from
total uptake to calculate mediated uptake which was used in kinetic
analysis. Results are given as Eadie-Hofstee plots (uptake rate versus uptake rate/substrate concentration). V,
uptake of Gly-Sar in nmol/mg of protein/10 min; S, Gly-Sar
concentration in µM.
We also employed
radiolabeled cephalexin as a substrate to study the interaction of
-lactam antibiotics with PEPT 2 in SKPT cells. Our initial
characterization studies have established that the uptake of cephalexin
in these cells was stimulated by an inwardly directed H
gradient (Fig. 5). The uptake of the antibiotic (0.1
µM) was stimulated severalfold upon acidification of the
extracellular medium. However, this stimulation was abolished when the
cells were pH-clamped (i.e. intracellular pH =
extracellular pH) with nigericin. Kinetic analysis, done over a
cephalexin concentration of 25-250 µM, showed that
the uptake occurred via a single, saturable process (Fig. 6).
The Michaelis-Menten constant (K
) for the uptake
process was 49 ± 8 µM and the maximal velocity (V
) was 1.5 ± 0.1 nmol/mg of protein/20
min.
Figure 5:
Dependence of cephalexin uptake in SKPT
cells on an inwardly directed transmembrane H gradient. Uptake of cephalexin (100 nM) was measured at
different extracellular pH in control cells (
) and in pH-clamped
cells (
). Incubation time for uptake measurement was 20 min. pH
clamping was done by incubating the cells with 20 µM nigericin for 30 min at respective extracellular pH prior to
initiation of uptake.
Figure 6:
Kinetics of cephalexin uptake in SKPT
cells. Uptake of cephalexin in SKPT cells was measured at pH 6.0 with a
20-min incubation period. Concentration of cephalexin was varied
between 25 and 250 µM, keeping the concentration of
[H]cephalexin constant at 200 nM and
adding unlabeled cephalexin to desired concentrations. Non-mediated
component was determined from the uptake of radiolabel measured in the
presence of 10 mM unlabeled cephalexin. This component was
subtracted from total uptake to calculate mediated uptake which was
used in kinetic analysis. Inset: Eadie-Hofstee plot (V
versus V/S). V, cephalexin uptake in nmol/mg of
protein/20 min; S, cephalexin concentration in
µM.
We then performed detailed kinetic studies to establish that
the uptake of the dipeptide glycylsarcosine and the uptake of the
-lactam antibiotic cephalexin occur via a common transport system (i.e. PEPT 2) in SKPT cells. The experimental approach
employed here for this purpose is the so-called ``A-B-C
test'' which is widely used in the transport
field(20, 21, 22) . We systematically
investigated the interaction between glycylsarcosine and cephalexin
during uptake in SKPT cells with the primary aim to determine whether
the uptake characteristics of these two compounds meet the criteria of
the A-B-C test. The results of these experiments have shown that the
uptake of glycylsarcosine was completely inhibitable by cephalexin.
Similarly, the uptake of cephalexin was completely inhibitable by
glycylsarcosine. The interaction between the two compounds during
uptake was strictly competitive. The results of the kinetic experiments
are summarized in Table 1. The K
value for
glycylsarcosine determined from its uptake was 48 ± 4 µM which is approximately the same as the K
value (64 ± 4 µM) for the inhibition of
cephalexin uptake by glycylsarcosine. Similarly, the K
value for cephalexin determined from its uptake was 49 ± 8
µM which is very close to the K
value
(68 ± 5 µM) for the inhibition of glycylsarcosine
uptake by cephalexin. Cyclacillin inhibited the uptake of
glycylsarcosine and the uptake of cephalexin with similar potency, the K
values being 39 ± 1 µM and
37 ± 5 µM, respectively. Cefadroxil also inhibited
the uptake of these two compounds with similar potency, the K
values being 2.8 ± 0.2 µM and 2.5 ± 0.1 µM, respectively. The conclusion
from these experiments is that the uptake characteristics of
glycylsarcosine and cephalexin strictly meet every requirement of the
classical A-B-C test, thus strongly indicating that these two compounds
are transported by the same transporter (i.e. PEPT 2) in the
SKPT renal cell line.
Since it has now
become clear from molecular biological studies that the intestinal and
renal peptide transporters are distinct proteins with significant
differences in their primary
structure(10, 11, 12, 13) , we
initiated studies to see if there are differences in the substrate
recognition pattern between these two transporters. In our studies to
compare the substrate recognition pattern of PEPT 1 and PEPT 2, we
selected cefadroxil (a cephalosporin) and cyclacillin (a penicillin) as
model substrates. Initially, we carried out the experiments with Caco-2
cells (PEPT 1) and SKPT cells (PEPT 2) by determining the relative
potency of these two peptidomimetic drugs for the inhibition of the
uptake of the dipeptide glycylsarcosine. In Caco-2 cells, the dipeptide
uptake was inhibited by cefadroxil and by cyclacillin in a
dose-dependent manner (Fig. 7A). The respective
IC values for the inhibition were 5.4 ± 0.6 and 0.6
± 0.1 mM. Thus, cyclacillin is severalfold more potent
than cefadroxil in competing with glycylsarcosine for uptake via PEPT
1. Interestingly, even though the uptake of glycylsarcosine in SKPT
cells was inhibited by both cyclacillin and cefadroxil as in Caco-2
cells, there were important differences (Fig. 7B). The
potency with which these drugs inhibited the uptake in SKPT cells was
much greater than in Caco-2 cells. The IC
values were in
the micromolar range rather than in millimolar range. In addition,
there was a significant difference in the relative inhibitory potency
between the two drugs. The IC
values for cefadroxil and
cyclacillin in SKPT cells were 3.0 ± 0.2 and 41.6 ± 1.5
µM, respectively. In other words, cefadroxil is manyfold
more potent than cyclacillin in competing with the dipeptide for uptake
via PEPT 2. Thus, the relative affinities of PEPT 1 and PEPT 2 for
cyclacillin and cefadroxil are reversed. These results show that the
substrate recognition pattern is significantly different between PEPT 1
and PEPT 2.
Figure 7:
Differential recognition of cefadroxil and
cyclacillin by PEPT 1 in Caco-2 cells and PEPT 2 in SKPT cells. Uptake
of [C]Gly-Sar was measured at pH 6.0 in Caco-2
cells (A) and in SKPT cells (B) in the absence and
presence of increasing concentrations of cefadroxil (
) and
cyclacillin (
). Concentration of Gly-Sar was 5 µM for
Caco-2 cells and 3 µM for SKPT cells. Incubation time for
uptake measurement was 10 min for both cell types. Uptake of Gly-Sar
measured in the absence of inhibitors was taken as 100%. This value was
81.6 ± 8.8 pmol/mg of protein/10 min for Caco-2 cells and 53.7
± 3.1 pmol/mg of protein/10 min for SKPT
cells.
To rule out the possibility that the observed
differences in substrate recognition of the peptide transporters
between these two cell lines may be due to species differences rather
than real differences between PEPT 1 and PEPT 2, we performed similar
experiments with the cloned human PEPT 1 and human PEPT 2. These two
transporters were functionally expressed in HeLa cells using the
vaccinia virus expression system and the uptake of cephalexin was
determined using 0.5 µM [H]cephalexin. As shown in Fig. 8A, cephalexin uptake measured at pH 6.0 in HeLa
cells expressing PEPT 1 was 1.89 ± 0.05 pmol/10
cells/10 min which was 11-fold greater than the uptake in control
cells (i.e. HeLa cells transfected with empty vector) under
similar conditions. The H
gradient-dependent nature of
the uptake process was evident from the findings that the uptake
decreased drastically in PEPT 1-expressing cells when measured at pH
9.0 instead of pH 6.0. Similar results were obtained with PEPT 2 (Fig. 8B). These data demonstrate that PEPT 1 and PEPT
2 catalyze the transport of the
-lactam antibiotic cephalexin. We
then used this experimental system to compare the substrate recognition
pattern of PEPT 1 and PEPT 2. These transporters were individually
expressed in HeLa cells and the uptake of glycylsarcosine was
determined in the presence of increasing concentrations of cyclacillin
and cefadroxil. As was the case in Caco-2 cells and SKPT cells, the
dipeptide uptake via PEPT 1 and PEPT 2 was inhibited by both
cyclacillin and cefadroxil. In the case of PEPT 1, the IC
values for cefadroxil and cyclacillin were 0.87 ± 0.12 and
0.35 ± 0.09 mM, respectively (Fig. 9A).
In contrast, the corresponding IC
values were 66 ±
4 and 610 ± 100 µM in the case of PEPT 2 (Fig. 9B). These data with the cloned human PEPT 1 and
PEPT 2 show that there are significant differences between the two
peptide transporters in substrate selectivity.
Figure 8: Uptake of cephalexin in HeLa cells transfected with human PEPT 1 cDNA (A) or with human PEPT 2 cDNA (B). Cells were transfected with either empty pBluescript alone (pBS) or with PEPT 1 cDNA or PEPT 2 cDNA (pBS-cDNA). The cDNAs were functionally expressed in these cells by the vaccinia virus expression technique. Uptake of cephalexin (0.5 µM) was measured in these cells at pH 6.0 or 9.0 with a 10-min incubation.
Figure 9:
Differential recognition of cefadroxil and
cyclacillin by human PEPT 1 and human PEPT 2 functionally expressed in
HeLa cells. Cells were transfected either with human PEPT 1 cDNA (A) or with human PEPT 2 cDNA (B). The cDNAs were
functionally expressed in these cells by the vaccinia virus expression
technique. Uptake of [C]Gly-Sar was measured at
pH 6.0 with a 3-min incubation in the absence and presence of
increasing concentrations of cefadroxil (
) and cyclacillin
(
). Concentration of Gly-Sar was 25 µM for cells
expressing PEPT 1 and 50 µM for cells expressing PEPT
2.
Significant differences were noted in relative potency of cefadroxil and cyclacillin as inhibitors of glycylsarcosine uptake in Caco-2 and SKPT cells which express PEPT 1 and PEPT 2 natively and in HeLa cells which express the cloned human PEPT 1 and PEPT 2. It is possible that post-translational modifications (e.g. N-glycosylation) of PEPT 1 and PEPT 2 expressed in HeLa cells are not identical to those of the native transporters in Caco-2 and SKPT cells. This may contribute to the observed differences. With respect to PEPT 2, species differences may also be a factor because SKPT cells were derived from rat kidney whereas the PEPT 2 cDNA was cloned from human kidney.
In
addition to PEPT 1 and PEPT 2, mammalian tissues may express other
peptide transporters. A cDNA clone (HPT-1) has been recently isolated
from a Caco-2 cell cDNA library and expression of this cDNA in
mammalian cells leads to increased uptake of the peptidomimetic drugs
cephalexin and bestatin(24) . Interestingly, there is no
sequence homology between the HPT-1 protein and the peptide
transporters PEPT 1 and PEPT 2. It has also been shown that Caco-2
cells express two functionally distinct peptide transporters, one in
the apical membrane and the other in the basolateral
membrane(25) . Among these multiple peptide transporters, PEPT
1 and PEPT 2 have been characterized in detail, both at the functional
level and at the molecular level. The present study, which focuses on
the handling of peptidomimetic drugs by PEPT 1 and PEPT 2, documents a
major functional difference between these two transporters in terms of
recognition of -lactam antibiotics as substrates.