Department of Pharmacy, Kyoto University Hospital, Faculty of Medicine, Kyoto University, Kyoto 606-8507, Japan
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ABSTRACT |
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The apical
H+-coupled peptide transporter (PEPT1) and basolateral
peptide transporter in human intestinal Caco-2 cells were functionally
compared by the characterization of [14C]glycylsarcosine
transport. The glycylsarcosine uptake via the basolateral peptide
transporter was less sensitive to medium pH than uptake via PEPT1 and
was not transported against the concentration gradient.
Kinetic analysis indicated that glycylsarcosine uptake across the
basolateral membranes was apparently mediated by a single peptide
transporter. Small peptides and -lactam antibiotics inhibited
glycylsarcosine uptake by the basolateral peptide transporter, and
these inhibitions were revealed to be competitive. Comparison of the
inhibition constant values of various
-lactam antibiotics between
PEPT1 and the basolateral peptide transporter suggested that the former
had a higher affinity than the latter. A histidine residue modifier,
diethyl pyrocarbonate, inhibited glycylsarcosine uptake by both
transporters, although the inhibitory effect was greater on PEPT1.
These findings suggest that a single facilitative peptide transporter
is expressed at the basolateral membranes of Caco-2 cells and that
PEPT1 and the basolateral peptide transporter cooperate in the
efficient transepithelial transport of small peptides and peptidelike drugs.
intestinal absorption; -lactam antibiotics; human intestinal
Caco-2 cells
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INTRODUCTION |
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SMALL INTESTINAL EPITHELIAL CELLS are the primary site of absorption of nutrients such as glucose and amino acids. Absorption through the intestine requires that such molecules cross two distinct membranes, i.e., be taken up by the epithelial cells from the lumen across the brush-border membranes followed by transfer to the blood across the basolateral membranes. Numerous studies have indicated that asymmetric distribution of amino acid and glucose transporters between these two plasma membranes contributes to the transepithelial transport of glucose and amino acids in the small intestine (1, 4, 8).
It has been demonstrated that di- and tripeptides as well as amino
acids are also actively transported into the cells by the peptide
transporter after ingestion of protein (13). The peptide transporter in
the brush-border membranes was shown to be driven by H+
gradient (7) and to mediate the transport of peptidelike drugs such as
-lactam antibiotics (15). Recently, the H+-coupled
peptide transporters (PEPT1 and PEPT2) have been cloned and well
characterized (5, 10). In the small intestine, only PEPT1 is expressed
and localized at the brush-border membranes (14, 17, 18). PEPT1 is also
expressed in the human intestinal cell line Caco-2 (11). Although a
large amount of functional and molecular information is available about
the peptide transporter localized at the brush-border membranes, little
is known regarding the basolateral peptide transporter.
Previously, it was commonly believed that only free amino acids entered
the portal blood from intestinal epithelial cells (13). However, recent
studies demonstrated that ~50% of circulating plasma amino acids
were peptide bound, and the majority were in the form of di- and
tripeptides (19), suggesting the existence of a basolateral peptide
transporter in the small intestine. In addition, orally active
-lactam antibiotics, which are impermeable to membranes because of
their low lipophilicity and are not broken down like small peptides,
are efficiently absorbed through the intestine. This fact also leads to
the idea that a peptide transporter exists in the basolateral membranes
to efflux drugs into the blood.
On the basis of this background, we characterized the basolateral transport of peptidelike drugs in Caco-2 cells (9, 12, 16). Our findings suggested that a facilitative, not an H+-coupled, peptide transport system was localized in the basolateral membranes of Caco-2 cells. In contrast to our results with dipeptides as substrates, there have been a few reports that the peptide transporter in the basolateral membranes was H+ dependent (6, 23, 24). To clarify the reason for this discrepancy, we used glycylsarcosine to examine the transport system of the basolateral peptide transporter in Caco-2 cells. Furthermore, we functionally characterized the apical and basolateral peptide transporters by comparing the substrate affinity and the effect of chemical modification of both transporters.
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MATERIALS AND METHODS |
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Cell culture. Caco-2 cells at passage 18 obtained from the American Type Culture Collection (ATCC HTB-37) were maintained by serial passage in plastic culture dishes. Complete medium consisted of DMEM (GIBCO Life Technologies, Grand Island, NY), supplemented with 10% fetal bovine serum (Whittaker Bioproducts, Walkersville, MD) and 1% nonessential amino acids (GIBCO) without antibiotics. Monolayer cultures were grown in an atmosphere of 5% CO2-95% air at 37°C. To measure the uptake of [14C]glycylsarcosine from the apical side of Caco-2 cells, 35-mm plastic dishes were inoculated with 2 × 105 cells in 2 ml of complete culture medium. To measure the uptake of [14C]glycylsarcosine from the basolateral side, Caco-2 cells were seeded on microporous membrane filters (3-µm pores, 1 cm2) inside Transwell cell culture chambers (Costar, Cambridge, MA) at a cell density of 3.3 × 104 cells per filter. Each Transwell chamber was filled with 0.33 ml and 1 ml of medium in the apical and basolateral compartments, respectively. The cell monolayers grown in 35-mm plastic dishes or in the Transwell chamber were given complete medium every 2-4 days and were used on the 15th day for uptake studies.
Uptake studies by monolayers.
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 (MES; pH 6.0) or HEPES (pH 7.4). Uptake by monolayers grown in
35-mm plastic dishes was determined as described previously (22).
Uptake by monolayers grown in the Transwell chambers was measured as
follows. Caco-2 cell monolayers were preincubated apically and
basolaterally with 1 ml of incubation medium (pH 7.4) for 10 min at
37°C. The medium was then removed, and 1 ml of incubation medium
containing [14C]glycylsarcosine (20 µM, 37 kBq/ml, pH
7.4) was added to the basolateral side, with 0.5 ml of unlabeled
incubation medium (pH 6.0) added to the apical side. Incubation
proceeded for the indicated periods at 37°C. The incubation medium
was aspirated at the end of the incubation period, and the monolayers
were rapidly washed twice on both sides with 1 ml of ice-cold
incubation medium (pH 7.4). The filters with monolayers were detached
from the chambers, and cells were solubilized in 0.5 ml of 1 N NaOH.
The radioactivity of the solubilized cells was determined by liquid
scintillation counting. The protein content of the solubilized cell
monolayers was determined by the method of Bradford (3), using a
Bio-Rad protein assay kit with bovine -globulin as the standard. The protein content of the intact monolayers was 0.8-1.1 mg/filter.
Statistical analysis. Data were analyzed for statistical significance by one-way ANOVA followed by Scheffé's test.
Materials. Amoxicillin and cefixime (Fujisawa Pharmaceutical, Osaka, Japan), cefadroxil (Bristol Meyers, Tokyo, Japan), cephalexin and ceftibuten (Shionogi, Osaka, Japan), cephradine (Sankyo, Tokyo, Japan), cyclacillin (Takeda Chemical Industries, Osaka, Japan), and [(2R,3S)-3-amino-2-hydroxy-4-phenylbutanoyl]-L-leucine (Bestatin; Nippon Kayaku, Tokyo, Japan) were gifts from the respective suppliers. [14C]glycylsarcosine (1.78 GBq/mmol) was obtained from Daiichi Pure Chemicals (Ibaraki, Japan). Ampicillin, glycylsarcosine, and glycylglyclyphenylalanine were obtained from Sigma Chemical (St. Louis, MO). Glycyl-L-leucine was purchased from Peptide Institute (Osaka, Japan). Diethyl pyrocarbonate (DEPC) was obtained from Nacalai Tesque (Kyoto, Japan). All other chemicals used were of the highest purity available.
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RESULTS |
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pH dependence of glycylsarcosine uptake.
We first examined the pH dependence of
[14C]glycylsarcosine uptake by the apical peptide
transporter PEPT1 and the basolateral peptide transporter in Caco-2
cells (Fig. 1). In both transporters, [14C]glycylsarcosine uptake was inhibited by 20 mM
glycyl-L-leucine at all pHs examined, indicating
transporter-mediated uptake. Figure 1, A and B insets,
shows the transporter-mediated specific uptakes, which were calculated
by subtracting the nonspecific uptake estimated in the presence of 20 mM glycyl-L-leucine from the total uptake. PEPT1-mediated
[14C]glycylsarcosine uptake was markedly influenced by
the medium pH with the maximal uptake at pH 6.0. In contrast, specific
[14C]glycylsarcosine uptake by basolateral peptide
transporter was less sensitive to the medium pH than that by PEPT1.
Considering the physiological conditions,
[14C]glycylsarcosine was added to the apical side at pH
6.0 and to the basolateral side at pH 7.4 in the following experiments.
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Time course of glycylsarcosine uptake.
As shown in Fig. 2, the rate of
[14C]glycylsarcosine uptake by PEPT1 was much greater
than that by the basolateral peptide transporter. In both cases, the
uptake of [14C]glycylsarcosine was inhibited by 10 mM
unlabeled glycylsarcosine.
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Concentration dependence of glycylsarcosine uptake.
Figure 3 illustrates the concentration
dependence of glycylsarcosine uptake by PEPT1 and the basolateral
peptide transporter in Caco-2 cells. The specific uptake was calculated
by subtracting the nonspecific uptake, which was estimated in the
presence of excess unlabeled dipeptide, from the total uptake. With the
use of nonlinear least squares regression analysis, kinetic parameters were calculated according to the Michaelis-Menten equation. The apparent Michaelis-Menten constant (Km) values
for PEPT1 and the basolateral peptide transporter were 0.65 and 2.1 mM,
respectively. Maximal uptake rate (Vmax) values
for PEPT1 and the basolateral peptide transporter were 13 and 9.5 nmol · mg
protein1 · min
1,
respectively.
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Effect of various compounds on glycylsarcosine uptake.
Next, we examined the effects of various compounds on the
[14C]glycylsarcosine uptake from the basolateral side
(Fig. 4). The [14C]glycylsarcosine uptake was slightly inhibited by
glycylsarcosine, glycylleucine, and glycylglycylphenylalanine at the
concentration of 1 mM but markedly inhibited at 20 mM (Fig.
4A). Glycine at 20 mM did not significantly inhibit
[14C]glycylsarcosine uptake (Fig. 4A). Figure
4B shows the inhibitory effects of peptidelike drugs (20 mM).
Cyclacillin and Bestatin markedly inhibited
[14C]glycylsarcosine uptake similarly to native small
peptides. Cefadroxil, cephradine, ceftibuten, and cefixime showed
significant inhibitory effects on [14C]glycylsarcosine
uptake. The [14C]glycylsarcosine uptake was inhibited
slightly but not significantly by cephalexin and amoxicillin.
Ampicillin did not have an inhibitory effect.
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Kinetics of inhibition of glycylsarcosine uptake.
Figure 5 shows the kinetics of inhibition
of glycylsarcosine uptake by basolateral peptide transporter in the
presence of cephradine and ceftibuten. Kinetic analysis revealed that
the presence of cephradine or ceftibuten increased the
Km values of glycylsarcosine [in mM: 5.2 ± 0.5
(cephradine) or 6.3 ± 1.4 (ceftibuten) vs. 3.0 ± 0.4
(control)] for the basolateral peptide transporter without
significantly affecting the Vmax values [in
nmol · mg protein1 · min
1:
8.5 ± 0.9 (cephradine) or 7.9 ± 1.5 (ceftibuten) vs.
8.2 ± 0.5 (control)]. These results indicated that cephradine and
ceftibuten inhibited glycylsarcosine uptake by the basolateral peptide
transporter competitively. Cyclacillin and cefadroxil also inhibited
the [14C]glycylsarcosine uptake competitively (data not
shown).
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Substrate affinities of PEPT1 and the basolateral peptide
transporter.
To compare the substrate affinities of PEPT1 and the basolateral
peptide transporter, we estimated inhibition constant
(Ki) values of several -lactam antibiotics and
Bestatin from the competition curves by nonlinear least square
regression analysis as described (22). The estimated
Ki values of these drugs are summarized in Table
1. All of these drugs showed much more
potent inhibition of [14C]glycylsarcosine uptake via
PEPT1 than via the basolateral peptide transporter. Among the drugs
examined, ceftibuten showed the highest affinity for PEPT1 but lower
affinity for the basolateral peptide transporter.
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Effect of DEPC on glycylsarcosine uptake.
Finally, we compared the effects of the histidine residue modifier,
diethylpyrocarbonate (DEPC), on the function of PEPT1 and the
basolateral peptide transporter. When the Caco-2 cells were treated
with various concentrations of DEPC added to either the apical or
basolateral side, half-maximal inhibition was observed at ~0.8 mM for
PEPT1 and at 2.3 mM for the basolateral peptide transporter (Table
2). These DEPC-induced inhibitions of
[14C]glycylsarcosine uptake by PEPT1 and the basolateral
peptide transporter were abolished in the presence of unlabeled 10 mM glycylsarcosine (Fig. 6).
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DISCUSSION |
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Thwaites et al. (23, 24) characterized the transepithelial intestinal
transport of glycylsarcosine using human intestinal Caco-2 cell
monolayers. In their reports, it was demonstrated that the transport
and accumulation of glycylsarcosine from the basolateral side were
enhanced in the presence of a pH gradient and that basolateral
application of glycylsarcosine (pH 6.0) caused a cytosolic
acidification. Therefore, they concluded that the H+-coupled peptide transporter was expressed in the
basolateral membrane surface of Caco-2 cells (24). In addition, they
suggested that the multiple transport systems were involved in the
cephalexin (an oral -lactam antibiotic) transport at the basolateral
surface of Caco-2 cells (23). On the other hand, we have clearly
indicated that a facilitative peptide transport system, not a
H+-coupled peptide transporter, mediates the transport of
peptidelike drugs in the basolateral membranes of Caco-2 cells (9, 12, 16). To confirm whether these, our previous findings, apply to not only
peptidelike drugs but also small peptides, we used the dipeptide
glycylsarcosine as a probe to characterize the basolateral peptide transporter.
Glycylsarcosine uptake mediated by the basolateral peptide transporter was less sensitive to the pH of the medium than that mediated by PEPT1, suggesting that an inward H+ gradient may not be involved in glycylsarcosine transport across the basolateral membranes. These findings are consistent with our previous demonstration using peptidelike drugs (9, 12, 16). Although a slight pH-dependent uptake was observed in the basolateral peptide transporter, it may reflect the change of transport activity due to the different protonation of this transporter protein at various pHs. When a Caco-2 cellular volume of 3.66 µl/mg protein (2) was assumed, the ratios of the intracellular to apical and to basolateral extracellular concentrations of glycylsarcosine were 12.9 and 1.25, respectively. Intracellular accumulations of [14C]glycylsarcosine were calculated by subtracting the uptake in the presence of 10 mM unlabeled glycylsarcosine from the uptake in the absence of inhibitor for a 60-min incubation. These findings suggest that glycylsarcosine fluxes across apical and basolateral membranes are mediated by active and facilitative transport systems, respectively. However, there are discrepancies about transport mechanisms of the basolateral peptide transporter between our results and those of Dyer et al. (6) and Thwaites et al. (23, 24). Dyer et al. (6) demonstrated that the uptake of glycyl-L-proline by intestinal basolateral membrane vesicles was stimulated by an inward H+ gradient. Thwaites et al. (23, 24) demonstrated that the basolateral glycylsarcosine uptake was mediated by H+-coupled peptide transporter using Caco-2 cells. These differences may be due to the experimental systems, culture conditions, and experimental techniques. All of this information indicates that further studies are needed to elucidate the transport mechanisms of glycylsarcosine uptake via basolateral membranes.
Kinetic analysis demonstrated that a single peptide transporter was
involved in basolateral glycylsarcosine transport, and glycylsarcosine
uptake via the basolateral peptide transporter was competitively
inhibited by -lactam antibiotics. Furthermore, the estimated
Ki values of cephradine (13 mM), ceftibuten (39 mM), and Bestatin (0.8 mM) for the basolateral peptide transporter were
consistent with Km values of those obtained in
Caco-2 cells (cephradine, 5.9 mM; ceftibuten, >20 mM; Bestatin, 0.34 mM) (12, 16). These findings indicated that the transport of both
glycylsarcosine and peptidelike drugs was mediated via a single
transport system in the basolateral membranes of Caco-2 cells.
In the present study, we compared the apparent Km
values of glycylsarcosine with Ki values of various
-lactam antibiotics and Bestatin between the apical
H+-coupled peptide transporter PEPT1 and the basolateral
peptide transporter. All of the substrates examined showed higher
affinity for PEPT1 than for the basolateral peptide transporter. As
PEPT1 mediates the active transport of these substrates against a
concentration gradient, the intracellular concentrations of these
substrates are higher than luminal concentrations. If the basolateral
peptide transporter had a higher affinity for substrates, the
basolateral peptide transporter would always be saturated by
intracellular substrates. Although we only measured the kinetics of
uptake across the basolateral membranes, not the kinetics of efflux
from the cells, it is reasonable physiologically for the basolateral
peptide transporter to have a lower affinity to substrates than PEPT1. Similarly, Na+-glucose cotransporter (SGLT1) and
facilitated glucose transporter (GLUT2) were shown to be localized at
brush-border and basolateral membranes of small intestinal epithelial
cells, respectively, and the apparent Km values of
D-glucose were reported to be 0.8 mM for SGLT1 and
15-20 mM for GLUT2 (8).
The inhibitory effect of DEPC on the basolateral peptide transporter
was smaller than that on PEPT1, although these inhibitions of both
transporters were abolished in the presence of unlabeled 10 mM
glycylsarcosine. In our previous studies, two essential histidine
residues of rat PEPT1 were identified (21) and suggested to be involved
in the binding of H+ and an -amino group of the
substrates (20). As the transport activity of basolateral peptide
transporter seemed to be independent of inward H+ gradient,
the histidine residues of the basolateral peptide transporter might not
function as the H+-binding site. However, histidine
residues of the basolateral peptide transporter might be involved in
the substrate recognition because unlabeled 10 mM glycylsarcosine
prevented DEPC-induced inhibition of [14C]glycylsarcosine
transport. As a result of the differences in a number of essential
histidine residues, the effects of DEPC might be different between
PEPT1 and the basolateral peptide transporter. Sulfhydryl groups were
also reported to be essential components of PEPT1 and the basolateral
peptide transporter, although the latter was more sensitive to
sulfhydryl groups modifier (16). The distinct transport mechanisms of
PEPT1 and the basolateral peptide transporter may be regulated by these
functional components.
In conclusion, the present findings suggested that one facilitative peptide transporter was involved in the transport of small peptides and peptidelike drugs across the basolateral membrane of Caco-2 cells. PEPT1 and basolateral peptide transporter can be functionally distinguished by their transport mechanisms (active and facilitative) and substrate affinities, and these differences may be responsible for the efficient transcellular flux, i.e., intestinal absorption, of small peptides and peptidelike drugs.
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ACKNOWLEDGEMENTS |
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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 Biomolecular Design for Biotargeting (no. 296) from the Ministry of Education, Science, Sports, and Culture of Japan.
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FOOTNOTES |
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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 and other correspondence: K. Inui, Department of Pharmacy, Kyoto University Hospital, Sakyo-ku, Kyoto 606-8507, Japan (E-mail: inui{at}kuhp.kyoto-u.ac.jp).
Received 7 October 1998; accepted in final form 24 February 1999.
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REFERENCES |
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---|
1.
Baldwin, S. A.
Mammalian passive glucose transporters: members of an ubiquitous family of active and passive transport proteins.
Biochim. Biophys. Acta
1154:
17-49,
1993[Medline].
2.
Blais, A.,
P. Bissonnette,
and
A. Berteloot.
Common characteristics for Na+-dependent sugar transport in Caco-2 cells and human fetal colon.
J. Membr. Biol.
99:
113-125,
1987[Medline].
3.
Bradford, M. M.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem.
72:
248-254,
1976[Medline].
4.
Christensen, H. N.
Role of amino acid transport and countertransport in nutrition and metabolism.
Physiol. Rev.
70:
43-77,
1990
5.
Daniel, H.,
and
M. Herget.
Cellular and molecular mechanisms of renal peptide transport.
Am. J. Physiol.
273 (Renal Physiol. 42):
F1-F8,
1997
6.
Dyer, J.,
R. B. Beechey,
J.-P. Gorvel,
R. T. Smith,
R. Wootton,
and
S. P. Shirazi-Beechey.
Glycyl-L-proline transport in rabbit enterocyte basolateral-membrane vesicles.
Biochem. J.
269:
565-571,
1990[Medline].
7.
Ganapathy, V.,
and
F. H. Leibach.
Role of pH gradient and membrane potential in dipeptide transport in intestinal and renal brush-border membrane vesicles from the rabbit. Studies with L-carnosine and glycyl-L-proline.
J. Biol. Chem.
258:
14189-14192,
1983
8.
Hediger, M. A.,
and
D. B. Rhoads.
Molecular physiology of sodium-glucose cotransporters.
Physiol. Rev.
74:
993-1026,
1994
9.
Inui, K.,
M. Yamamoto,
and
H. Saito.
Transepithelial transport of oral cephalosporins by monolayers of intestinal epithelial cell line Caco-2: specific transport systems in apical and basolateral membranes.
J. Pharmacol. Exp. Ther.
261:
195-201,
1992[Abstract].
10.
Leibach, F. H.,
and
V. Ganapathy.
Peptide transporters in the intestine and the kidney.
Annu. Rev. Nutr.
16:
99-119,
1996[Medline].
11.
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. Cloning, functional expression, and chromosomal localization.
J. Biol. Chem.
270:
6456-6463,
1995
12.
Matsumoto, S.,
H. Saito,
and
K. Inui.
Transcellular transport of oral cephalosporins in human intestinal epithelial cells, Caco-2: interaction with dipeptide transport systems in apical and basolateral membranes.
J. Pharmacol. Exp. Ther.
270:
498-504,
1994[Abstract].
13.
Matthews, D. M.
Intestinal absorption of peptides.
Physiol. Rev.
55:
537-608,
1975
14.
Ogihara, H.,
H. Saito,
B.-C. Shin,
T. Terada,
S. Takenoshita,
Y. Nagamachi,
K. Inui,
and
K. Takata.
Immuno-localization of H+/peptide cotransporter in rat digestive tract.
Biochem. Biophys. Res. Commun.
220:
848-852,
1996[Medline].
15.
Okano, T.,
K. Inui,
H. Maegawa,
M. Takano,
and
R. Hori.
H+ coupled uphill transport of aminocephalosporins via the dipeptide transport system in rabbit intestinal brush-border membranes.
J. Biol. Chem.
261:
14130-14134,
1986
16.
Saito, H.,
and
K. Inui.
Dipeptide transporters in apical and basolateral membranes of the human intestinal cell line Caco-2.
Am. J. Physiol.
265 (Gastrointest. Liver Physiol. 28):
G289-G294,
1993
17.
Saito, H.,
M. Okuda,
T. Terada,
S. Sasaki,
and
K. Inui.
Cloning and characterization of a rat H+/peptide cotransporter mediating absorption of -lactam antibiotics in the intestine and kidney.
J. Pharmacol. Exp. Ther.
275:
1631-1637,
1995[Abstract].
18.
Saito, H.,
T. Terada,
M. Okuda,
S. Sasaki,
and
K. Inui.
Molecular cloning and tissue distribution of rat peptide transporter PEPT2.
Biochim. Biophys. Acta
1280:
173-177,
1996[Medline].
19.
Seal, C. J.,
and
D. S. Parker.
Isolation and characterization of circulating low molecular weight peptides in steer, sheep and rat portal and peripheral blood.
Comp. Biochem. Physiol.
99B:
679-685,
1991.
20.
Terada, T.,
H. Saito,
and
K. Inui.
Interaction of -lactam antibiotics with histidine residue of rat H+/peptide cotransporters, PEPT1 and PEPT2.
J. Biol. Chem.
273:
5582-5585,
1998
21.
Terada, T.,
H. Saito,
M. Mukai,
and
K. Inui.
Identification of the histidine residues involved in substrate recognition by a rat H+/peptide cotransporter, PEPT1.
FEBS Lett.
394:
196-200,
1996[Medline].
22.
Terada, T.,
H. Saito,
M. Mukai,
and
K. 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
23.
Thwaites, D. T.,
C. D. A. Brown,
B. H. Hirst,
and
N. L. Simmons.
H+-coupled dipeptide (glycylsarcosine) transport across apical and basal borders of human intestinal Caco-2 cell monolayers display distinctive characteristics.
Biochim. Biophys. Acta
1151:
237-245,
1993[Medline].
24.
Thwaites, D. T.,
C. D. A. Brown,
B. H. Hirst,
and
N. L. Simmons.
Transepithelial glycylsarcosine transport in intestinal Caco-2 cells mediated by expression of H+-coupled carriers at both apical and basal membranes.
J. Biol. Chem.
268:
7640-7642,
1993