Recognition of
-lactam antibiotics by rat peptide
transporters, PEPT1 and PEPT2, in
LLC-PK1 cells
Tomohiro
Terada,
Hideyuki
Saito,
Mayumi
Mukai, and
Ken-Ichi
Inui
Department of Pharmacy, Kyoto University Hospital, Faculty of
Medicine, Kyoto University, Kyoto 606-01, Japan
 |
ABSTRACT |
PEPT1 and PEPT2 are
H+-coupled peptide transporters
expressed preferentially in the intestine and kidney,
respectively, which mediate uphill transport of oligopeptides and
peptide-like drugs such as
-lactam antibiotics. In the present
study, we have compared the recognition of
-lactam antibiotics by
LLC-PK1 cells stably transfected
with PEPT1 or PEPT2 cDNA. Cyclacillin (aminopenicillin) and ceftibuten
(anionic cephalosporin without an
-amino group) showed potent
inhibitory effects on the glycylsarcosine uptake in the
PEPT1-expressing cells. Other
-lactams, such as cephalexin, cefadroxil, and cephradine (aminocephalosporins), inhibited modestly the PEPT1-mediated glycylsarcosine uptake. Except for ceftibuten, these
-lactams showed much more potent inhibitions
on the glycylsarcosine uptake via PEPT2 than via PEPT1. Comparison of
the inhibition constant
(Ki) values
between cefadroxil and cephalexin suggested that the hydroxyl group at
the NH2-terminal phenyl ring
increased affinity for both PEPT1 and PEPT2. It is concluded that PEPT2 has a much higher affinity for
-lactam antibiotics having an
-amino group than PEPT1 and that substituents at the
NH2-terminal side chain of these
drugs are involved in the recognition by both peptide transporters.
penicillins; cephalosporins; intestinal absorption; renal tubular
reabsorption; complementary deoxyribonucleic acid transfection
 |
INTRODUCTION |
PEPTIDE TRANSPORTERS contribute to the maintenance of
protein nutrition, mediating the efficient absorption of protein
digestive products in the small intestine and kidney (8, 9). Intestinal peptide transporter has also been indicated to recognize a broad range
of peptide-like drugs, such as orally active
-lactam antibiotics (19, 20, 28), anti-cancer agent, Bestatin (10, 22), and angiotensin
converting enzyme inhibitors (26), thereby playing an important
pharmacological role in determining their absorption rates or
half-lives. Using intestinal and renal brush-border membrane vesicles,
others have demonstrated the electrogenic and
H+-gradient-driven dipeptide
cotransport (7, 27). Although intestinal and renal peptide transporters
have similar features, such as substrate recognition and driving force,
it has been suggested that the intestinal and renal peptide transport
systems are not identical (11, 17, 18). In addition, it has been
reported that there are at least two distinct peptide transporters in
the brush-border membranes of the renal proximal tubules (4, 16, 19).
Recent molecular cloning studies revealed the existence of two
homologous peptide transporters, PEPT1 and PEPT2 (12, 13). The tissue
distribution of both transporters were different, i.e., PEPT1 was found
to be expressed mainly in the small intestine and slightly in the
kidney, whereas PEPT2 was expressed predominantly in the kidney but not
in the small intestine. Human PEPT2 possessed a higher affinity for
various dipeptides than human PEPT1 (21). We have also cloned
cDNA encoding rat PEPT1 (23) and PEPT2 (24) and demonstrated that rat
PEPT1 mediates
H+-gradientdependent transport
of differently charged
-lactam antibiotics by measuring uptake in
Xenopus oocytes (23) and in the stably transfected LLC-PK1 cells (30).
When expressed in Xenopus oocytes, both rat PEPT1 and PEPT2 stimulated the uptake of Bestatin, a dipeptide-like antineoplastic drug (24). More recently, we have identified essential residues of rat PEPT1 for its activity (29). By
using chemical modification and site-directed point mutation techniques, we demonstrated that histidine residues at position 57 and
121 of rat PEPT1 were involved in substrate binding and/or were
responsible for intrinsic activity of the transporter (29). To
understand the structure-function relationships and multispecificities of the peptide transporters, we have established the
LLC-PK1 cells stably expressing
rat PEPT2 and compared recognition of
-lactam antibiotics by using
rat PEPT1- and PEPT2-expressing transfectants.
 |
MATERIALS AND METHODS |
Cell culture and
transfection. The parental
LLC-PK1 cells obtained from the
American Type Culture Collection (CRL-1392) were cultured in complete
medium consisting of Dulbecco's modified Eagle's medium (GIBCO Life
Technologies, Grand Island, NY), supplemented with 10% fetal bovine
serum (Whittaker Bioproducts, Walkersville, MD) without antibiotics in
an atmosphere of 5% CO2-95% air
at 37°C (25). The clonal LLC-rPEPT1 cells were used as described previously (29, 30). In the case of LLC-rPEPT2 cells, a cDNA encoding
rat PEPT2 was subcloned into the Sal
I- and Not I-cut mammalian expression
vector pBK-CMV (Stratagene, La Jolla, CA) and transfected into
LLC-PK1 cells by the
CaPO4 coprecipitation method (29,
30). G-418 (1 mg/ml)-resistant cells were picked up, and
PEPT2-expressing cells (LLC-rPEPT2) were selected by measuring [14C]glycylsarcosine
transport activity.
Uptake studies by cell monolayers.
Uptake of
[14C]glycylsarcosine
was measured in cells grown on 60-mm plastic dishes as described previously (29). The composition of the incubation medium was as
follows (in mM): 145 NaCl, 3 KCl, 1 CaCl2, 0.5 MgCl2, 5 D-glucose, and 5 2-(N-morpholino)ethanesulfonic acid (pH 6.0) or
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH 7.4). The pH of the medium was adjusted with a solution of
NaOH or HCl. The cells were preincubated for 10 min at 37°C with 2 ml of the incubation medium. After the medium was removed, the cells
were incubated with 2 ml of incubation medium (pH 6.0) containing
[14C]glycylsarcosine
(20 µM, 37 kBq/ml) for 15 min at 37°C. After incubation, the
medium was aspirated, and the cells were rapidly rinsed twice with 5 ml
of ice-cold incubation medium. The cells were scraped with a rubber
policeman into 1 ml of 1 N NaOH, and the cell-associated radioactivity
was determined in ACS II (Amersham International, Buckinghamshire, UK)
by liquid scintillation counting. The protein content of cell
monolayers solubilized in 1 N NaOH was determined by the method of
Bradford (2), using a Bio-Rad protein assay kit (Bio-Rad, Richmond, CA)
with bovine
-globulin as the standard.
Materials. Amoxicillin, cefdinir, and
cefixime (Fujisawa Pharmaceutical, Osaka, Japan), ceftibuten and
cephalexin (Shionogi, Osaka), cefadroxil (Bristol Meyers, Tokyo,
Japan), cephradine (Sankyo, Tokyo), cyclacillin (Takeda Chemical
Industries, Osaka), and Bestatin
[(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-L-leucine; Nippon Kayaku, Tokyo] were gifts from the respective suppliers. [14C]glycylsarcosine
(1.78 GBq/mmol) was obtained from Daiichi Pure Chemicals (Ibaraki,
Japan). Glycylsarcosine and ampicillin were obtained from Sigma
Chemical (St. Louis, MO). All other chemicals used were of the highest
purity available. Figure 1 shows the general molecular structures of penicillin and cephalosporin
antibiotics.
 |
RESULTS |
Characteristics of glycylsarcosine uptake by
LLC-rPEPT1 and LLC-rPEPT2 cells. The pBK-CMV expression
vector with the rat PEPT2 cDNA inserted was transfected into
LLC-PK1 cells, and approximately 50 G-418-resistant cells were picked up and examined for
[14C]glycylsarcosine
transport activity. A clone with the highest transport activity was
selected (LLC-rPEPT2 cells) and used for further characterization of
rat PEPT2. First, we examined the time course of
[14C]glycylsarcosine
uptake at pH 6.0 by LLC-rPEPT1 (rat PEPT1-expressing LLC-PK1 cells) and LLC-rPEPT2
cells. As shown in Fig. 2, the rate of
glycylsarcosine uptake in LLC-rPEPT1 cells was much greater than that
in LLC-rPEPT2 cells. On the other hand, the uptake of glycylsarcosine
by the parental LLC-PK1 cells
transfected with control vector (LLC-pBK cells) was negligible compared
with that by LLC-rPEPT1 and LLC-rPEPT2 cells.

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Fig. 2.
Time course of
[14C]glycylsarcosine
uptake by LLC-rPEPT1 ( ), LLC-rPEPT2 ( ), and LLC-pBK cells ( ).
Each cell was incubated for the specified periods at 37°C with
incubation medium of pH 6.0 containing
[14C]glycylsarcosine
(20 µM, 37 kBq/ml). Thereafter, the radioactivity of solubilized
cells was determined. Each point is the mean ± SE of 3 experiments.
When the error bar is not shown, it is smaller than the symbol.
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Figure 3 shows effects of the medium pH on
glycylsarcosine uptake by LLC-rPEPT1 and LLC-rPEPT2 cells. In
LLC-rPEPT1 cells, glycylsarcosine uptake was maximal at pH
5.5-6.0, whereas it was maximal at pH 6.5-7.0 in LLC-rPEPT2
cells, suggesting that LLC-rPEPT1 cells prefer a more acidic pH region
to transport glycylsarcosine than LLC-rPEPT2 cells.

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Fig. 3.
pH dependence of
[14C]glycylsarcosine
uptake by LLC-rPEPT1 (A) and
LLC-rPEPT2 cells (B). Each cell was
incubated for 15 min at 37°C with incubation medium of various pH
containing
[14C]glycylsarcosine
(20 µM, 37 kBq/ml). Thereafter, radioactivity of solubilized cells
was determined. Each point is the mean ± SE of 3 experiments. When
the error bar is not shown, it is smaller than the symbol.
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Figure 4 illustrates the concentration
dependence of glycylsarcosine uptake at pH 6.0 by both transfectants.
The specific uptake was calculated by subtracting the nonspecific
uptake, which was estimated in the presence of excess unlabeled
dipeptide, from the total uptake, and kinetic parameters were
calculated according to the Michaelis-Menten equation. The values of
the apparent Michaelis constant
(Km) for the
glycylsarcosine uptake were 1.1 mM for LLC-rPEPT1 cells and 0.11 mM for
LLC-rPEPT2 cells, suggesting that rat PEPT2 has 10-fold higher affinity
for glycylsarcosine uptake than rat PEPT1.

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Fig. 4.
Concentration dependence of
[14C]glycylsarcosine
uptake by LLC-rPEPT1 (A) and
LLC-rPEPT2 cells (B).
[14C]glycylsarcosine
uptake by LLC-rPEPT1 or LLC-rPEPT2 cells was measured at various
concentrations (20-5,000 µM for LLC-rPEPT1 and 5-1,000 µM
for LLC-rPEPT2 cells) for 15 min at 37°C in absence ( ) or
presence ( ) of 10 mM unlabeled glycylsarcosine. Thereafter,
radioactivity of solubilized cells was determined. Each point is the
mean ± SE of 3 experiments. When the error bar is not shown, it is
smaller than the symbol. Insets:
Eadie-Hofstee plots of the uptake after correction for the nonsaturable
component; V, uptake rate (nmol · mg
protein 1 · 15 min 1); S, glycylsarcosine
concentration (mM).
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|
Inhibition of glycylsarcosine uptake by
various
-lactam antibiotics. To
compare substrate specificity of PEPT1 and PEPT2, the ability of
several
-lactam antibiotics to inhibit the uptake of glycylsarcosine
was examined. Figure 5 shows dose-dependent inhibition of glycylsarcosine uptake by several
-lactam antibiotics in the LLC-rPEPT1 and LLC-rPEPT2 cells. The PEPT1-mediated
glycylsarcosine uptake was inhibited by cyclacillin (aminopenicillin) > ceftibuten (anionic cephalosporin without an
-amino group) > cefadroxil (aminocephalosporin), in the order of inhibitory potency.
Ampicillin (aminopenicillin) had much less effect on the uptake. In
contrast, the PEPT2-mediated glycylsarcosine uptake was suppressed by
cefadroxil > cyclacillin > ampicillin = ceftibuten, in the order of
inhibition. The inhibition patterns of these antibiotics
were apparently different between LLC-rPEPT1 and LLC-rPEPT2 cells,
suggesting that PEPT2 had a much higher affinity for
-lactams except
for ceftibuten.

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Fig. 5.
Inhibition of
[14C]glycylsarcosine
uptake by -lactam antibiotics in LLC-rPEPT1
(A) and LLC-rPEPT2 cells
(B). Each transfectant was incubated
for 15 min at 37°C with incubation medium containing
[14C]glycylsarcosine
(20 µM, 37 kBq/ml) in absence ( ) or presence of increasing
concentrations of ampicillin ( ), cyclacillin ( ), cefadroxil
( ), and ceftibuten ( ). Thereafter, radioactivity of solubilized
cells was determined. Each point is the mean of 2 experiments.
|
|
Therefore, we further examined the inhibitory effects of other oral
-lactam antibiotics on the glycylsarcosine uptake via these
transporters, and the inhibition constant
(Ki) values for the competitors were estimated by nonlinear regression analysis of the
competition curves with one component. The chemical structures and
Ki values for the
antibiotics to compete with the glycylsarcosine uptake are summarized
in Table 1. The
Ki values
suggested that the affinities of PEPT1 for the antibiotics were in the
following order: cyclacillin > ceftibuten > cefadroxil > cephalexin = cefixime = cephradine > cefdinir = amoxicillin > ampicillin. On the other hand, the affinities of PEPT2 were in the
following order: cefadroxil > cyclacillin = cephradine = cephalexin > amoxicillin > ampicillin > ceftibuten > cefixime = cefdinir.
The Ki values of
cefdinir (monocarboxylic cephalosporin) for PEPT1 and PEPT2 suggested
that cefdinir had low affinities for both transporters compared with other cephalosporins examined. Except for ceftibuten and cefixime, these aminopenicillins and aminocephalosporins showed much more potent
inhibitions on the glycylsarcosine uptake via PEPT2 than via PEPT1
(P < 0.05 by the Mann-Whitney
U test). Notably, the Ki value of
cefadroxil, an aminocephalosporin with a hydroxyl group at the phenyl
ring of cephalexin, on the glycylsarcosine uptake via both PEPT1 and
PEPT2 was much smaller than that of cephalexin. Another peptide-like
drug, Bestatin, an antineoplastic drug possessing a
-amino group, had potent inhibitory effects on the dipeptide uptake
via both PEPT1 and PEPT2. The
Ki values of
Bestatin were 505 µM for PEPT1 and 20 µM for PEPT2.
 |
DISCUSSION |
Studies using isolated membrane vesicles have shown that the uptake of
oligopeptides and
-lactam antibiotics in renal brush-border membranes is mediated by at least two distinct transport systems, i.e.,
the high-affinity/low-capacity and the low-affinity/high-capacity peptide transporters (4, 19). In contrast, the peptide transport studies with intestinal brush-border membrane vesicles have suggested that a single peptide transporter contributes to translocate the oligopeptides (15). Most recently, two distinct but homologous oligopeptide transporters, PEPT1 of human (12), rabbit (5), and rat
(23) and PEPT2 of human (13), rabbit (1), and rat (24), have been
identified by a cDNA cloning technique. The rat PEPT1 mRNA was
expressed in both the small intestine and kidney (23), whereas rat
PEPT2 mRNA was expressed abundantly in the kidney but not in the small
intestine (24). Therefore, both PEPT1 and PEPT2 are coexpressed in the
kidney, suggesting that the two transporters contribute to tubular
reabsorption of oligopeptides. However, substrate specificities of the
two peptide transporters have not yet been fully characterized.
In the present study, we compared the apparent
Km values of
glycylsarcosine uptake by the rat PEPT1- and PEPT2-expressing transfectants and affirmed that PEPT2 is the high-affinity type transporter, whereas PEPT1 is the low-affinity type transporter in the
kidney. Daniel et al. (4) reported the existence of two types of
oligopeptide transport systems in the brush-border membranes isolated
from rat kidney. Therefore, it seems reasonable to consider that the
two distinct peptide transport systems in the renal brush-border
membranes are mediated via the PEPT1 and PEPT2. The differences in the
substrate affinity and in the pH dependence (Fig. 3) of PEPT1 and PEPT2
for glycylsarcosine transport may suggest that these transporters have
the distinct roles in tubular reabsorption of oligopeptides. The
physiological and pharmacological roles of both transporters should be
clarified by determining their localizations along the nephron.
Importantly, the present findings revealed that rat PEPT1 and PEPT2 had
different characteristics not only in substrate affinity for native
dipeptide but also in recognition of a variety of peptide-like drugs.
Various
-lactam antibiotics showed the different inhibitory potencies against the glycylsarcosine uptake between rat PEPT1 and
PEPT2. Ganapathy et al. (6) evaluated the
Ki values of cefadroxil (aminocephalosporin) and cyclacillin (aminopenicillin) for
glycylsarcosine uptake by Caco-2 cells (human adenocarcinoma cell line
expressing human PEPT1), SKPT cells (rat kidney-derived cell line
expressing PEPT2), and HeLa cells transfected transiently with human
PEPT1 or PEPT2 cDNA. They suggested the differential recognition of
these antibiotics by the two peptide transporters; PEPT1 had a much
higher affinity for cyclacillin than for cefadroxil, whereas PEPT2
preferred cefadroxil to cyclacillin. The inhibition patterns for these
antibiotics observed by Ganapathy et al. (6) agreed with our findings
for rat PEPT1 and PEPT2. In addition, it should be emphasized that
affinities of aminopenicillins and aminocephalosporins for rat PEPT1 in
this study closely correlated with the absorption of these antibiotics
from in situ rat small intestinal loops (31).
Among oral
-lactam antibiotics, there are some cephalosporins
without an
-amino group, such as ceftibuten, cefixime, and cefdinir.
Boll et al. (1) reported that
-lactam antibiotics without an
-amino group appeared not to be transported by rabbit PEPT2; in
other words, the
-amino group of substrates was required to be
recognized by rabbit PEPT2. In contrast, it was demonstrated that
ceftibuten uptake by the rat renal brush-border membrane vesicles was
mediated via two peptide transport systems (19), probably by PEPT1 and
PEPT2. We also reported previously that ceftibuten and cefixime were
transported by human PEPT1 in Caco-2 cells (14) and by rat PEPT1
expressed in oocytes (23) and in transfected cells (30) with relatively
high affinities. Therefore, it has been controversial as to how PEPT1
and PEPT2 are involved in the renal transport of the cephalosporins
without an
-amino group. Interestingly, we found in the
present study that ceftibuten and cefixime inhibited the
glycylsarcosine uptake both via rat PEPT1 and via rat PEPT2, suggesting
that both antibiotics were recognized not only by PEPT1 but also by
PEPT2. In addition, those drugs were twofold more potent in inhibiting
the glycylsarcosine uptake via rat PEPT1 than via rat PEPT2, suggesting
that
-lactam antibiotics without an
-amino group had low affinity
for the rat PEPT2.
Daniel et al. (3) previously reported that increasing the
hydrophobicity of the NH2-terminal
side chain increased the affinities of aminocephalosporins and
aminopenicillins to the renal
H+/oligopeptide cotransporter.
Because cyclacillin with a very hydrophobic NH2-terminal side chain showed
relatively higher affinities to the PEPT1 and PEPT2 than other
-lactams used, the hydrophobicity of the
NH2-terminal side chain was
suggested to contribute to the interaction of
-lactams to the
peptide transporters (Fig. 5; Table 1). In comparing the
Ki values for
cefadroxil and cephalexin or for amoxicillin and ampicillin, we found
that cefadroxil and amoxicillin had much higher affinities for both
PEPT1 and PEPT2 than did cephalexin and ampicillin,
respectively. Cefadroxil and cephalexin (aminocephalosporins) or
amoxicillin and ampicillin (aminopenicillins) have very similar
structures; the former two have a
p-hydroxyphenyl group, and the latter
two have a phenyl group at the side chain in the
position 7 or
6 of cephalosporin and penicillin
structure, respectively. Because the
p-hydroxyphenyl group is much less
hydrophobic than the phenyl group, the hydroxyphenyl group at the
NH2-terminal side chain of some
-lactams would make a stronger interaction of the peptide
transporter than hydrophobic interaction.
In conclusion, the present findings suggest that rat PEPT2 has a much
higher affinity for
-lactam antibiotics with an
-amino group than
rat PEPT1 and that not only hydrophobicity but also the hydroxyphenyl
group at the side chain of these antibiotics is involved in substrate
recognition by both rat PEPT1 and PEPT2. Further studies on mechanisms
of drug recognition by stably transfected cells expressing rat PEPT1
and PEPT2 will provide useful information for drug design and delivery
system to improve the efficiency of chemotherapy.
 |
ACKNOWLEDGEMENTS |
This work was supported in part by a Grant-in-Aid for Scientific
Research (B) and a Grant-in-Aid for Scientific Research on Priority Areas of "Channel-Transporter Correlation" from the
Ministry of Education, Science, and Culture of Japan, and by the
Mochida Memorial Foundation for Medical and Pharmaceutical Research.
 |
FOOTNOTES |
Address for reprint requests: K. Inui, Dept. of Pharmacy, Kyoto Univ.
Hospital, Sakyo-ku, Kyoto 606-01, Japan.
Received 24 February 1997; accepted in final form 19 June 1997.
 |
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