Functional characteristics of basolateral peptide transporter in the human intestinal cell line Caco-2

Tomohiro Terada, Kyoko Sawada, Hideyuki Saito, Yukiya Hashimoto, and Ken-Ichi Inui

Department of Pharmacy, Kyoto University Hospital, Faculty of Medicine, Kyoto University, Kyoto 606-8507, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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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 beta -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 beta -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; beta -lactam antibiotics; human intestinal Caco-2 cells


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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 beta -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 gamma -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.


    RESULTS
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MATERIALS AND METHODS
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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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Fig. 1.   pH dependence of [14C]glycylsarcosine uptake by PEPT1 (A) and the basolateral peptide transporter (B) in Caco-2 cells. Caco-2 monolayers were incubated for 15 min at 37°C with [14C]glycylsarcosine (20 µM, 37 kBq/ml), in the absence (open circle ) or presence (bullet ) of 20 mM glycyl-L-leucine, added to either the apical or basolateral side at various pH values. Thereafter, radioactivity of solubilized cells was determined. Each point represents mean ± SE of 3 independent monolayers. When error bars are not shown, they are smaller than symbol. Insets: pH dependence of PEPT1-mediated (A) and basolateral peptide transporter-mediated specific glycylsarcosine uptake (B). Data (black-triangle) were calculated by subtracting nonspecific uptake estimated in presence of 20 mM glycyl-L-leucine (bullet ) from total uptake (open circle ).

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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Fig. 2.   Time course of [14C]glycylsarcosine uptake via PEPT1 (A) and the basolateral peptide transporter (B) in Caco-2 cells. Caco-2 monolayers were incubated with [14C]glycylsarcosine (20 µM, 37 kBq/ml) in absence (open circle ) or presence (bullet ) of 10 mM unlabeled glycylsarcosine added to either the apical (pH 6.0) or basolateral side (pH 7.4). Incubation proceeded for specified period at 37°C. Thereafter, radioactivity of solubilized cells was determined. Each point represents mean ±SE of 3 independent monolayers. When error bars are not shown, they are smaller than symbol.

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 protein-1 · min-1, respectively.


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Fig. 3.   Concentration dependence of [14C]glycylsarcosine uptake by PEPT1 (A) and the basolateral peptide transporter (B) in Caco-2 cells. Caco-2 monolayers were incubated at 37°C for 15 min with varying concentrations of [14C]glycylsarcosine (37 kBq/ml) added to either the apical (pH 6.0) or basolateral side (pH 7.4) in the absence () or presence (open circle ) of 50 mM unlabeled glycylleucine. Thereafter, radioactivity of solubilized cells was determined. Each point represents mean ±SE of 3 independent monolayers. When error bars are not shown, they are smaller than symbol. Insets: Eadie-Hofstee plots of glycylsarcosine uptake after correction for nonsaturable component. V, uptake rate (nmol · mg protein-1 · 15 m in-1); S, glycylsarcosine concentration (mM).

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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Fig. 4.   Effects of glycine and small peptides (A) and various beta -lactam antibiotics and Bestatin (B) on [14C]glycylsarcosine uptake by the basolateral peptide transporter in Caco-2 cells. Caco-2 monolayers were incubated at 37°C for 15 min with [14C]glycylsarcosine (20 µM, 37 kBq/ml) in absence (open bar) or presence of 1 mM (hatched bars) or 20 mM (filled bars) inhibitors added to the basolateral side (pH 7.4). Thereafter, radioactivity of solubilized cells was determined. Each bar represents mean ±SE of 3 independent monolayers. Gly, glycine; Gly-Sar, glycylsarcosine; Gly-Leu, glycylleucine; Gly-Gly-Phe, glycylglycylphenylalanine. *P < 0.05, significantly different from control.

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 protein-1 · 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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Fig. 5.   Concentration dependence of [14C]glycylsarcosine uptake via the basolateral peptide transporter in presence of cephradine or ceftibuten by Caco-2 cells. A: Caco-2 monolayers were incubated at 37°C for 15 min with varying concentrations of [14C]glycylsarcosine (37 kBq/ml) added to the basolateral side (pH 7.4) in the absence (open circle ) or presence of 15 mM cephradine (bullet ) or 30 mM ceftibuten (black-triangle). Thereafter, radioactivity of solubilized cells was determined. B: Eadie-Hofstee plots of glycylsarcosine uptake after correction for nonsaturable component. V, uptake rate (nmol · mg protein-1 · 15 min-1); S, glycylsarcosine concentration (mM). Each point represents mean ±SE of 7 monolayers of 3 separate experiments. When error bars are not shown, they are smaller than symbol.

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 beta -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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Table 1.   Inhibition constants of beta -lactam antibiotics and Bestatin for PEPT1 and the basolateral peptide transporter

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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Table 2.   Effects of DEPC concentration on [14C]glycylsarcosine uptake by PEPT1 and the basolateral peptide transporter in Caco-2 cells



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Fig. 6.   Effects of diethyl pyrocarbonate (DEPC) pretreatment on [14C]glycylsarcosine uptake by PEPT1 (A) and the basolateral peptide transporter (B) in Caco-2 cells. The cells were preincubated at 25°C for 10 min with 1 mM (A) or 5 mM (B) DEPC (pH 6.0) in the absence or presence of unlabeled 10 mM glycylsarcosine. After preincubation, Caco-2 monolayers were rinsed once with the incubation medium and then incubated with [14C]glycylsarcosine (20 µM, 37 kBq/ml) added to either the apical (pH 6.0) or basolateral side (pH 7.4). Radioactivity of solubilized cells was determined. Each bar represents mean ±SE of 3 independent monolayers.


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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 beta -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 beta -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 beta -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 alpha -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.


    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 Biomolecular Design for Biotargeting (no. 296) from the Ministry of Education, Science, Sports, and Culture of Japan.


    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 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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Gastroint Liver Physiol 276(6):G1435-G1441
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