Introduction

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The H⁺/peptide cotransport system expressed in the small intestine mediates the absorption of oligopeptides (Ganapathy and Leibach, 1985; Hoshi, 1985) and peptide-like drugs (Okano et al., 1986). The peptide transport system recognizes structurally diverse drugs, such as -lactam antibiotics (Inui et al., 1992b; Matsumoto et al., 1994; Muranushi et al., 1989; Tamai et al., 1995), antineoplastic agent bestatin (Inui et al., 1992a; Tomita et al., 1989) and the angiotensin-converting enzyme inhibitors (Hu and Amidon, 1988; Swaan et al., 1995). In addition to this broad substrate specificity of the peptide transport system, species differences in substrate recognition and transport activities have been reported (Sugawara et al., 1992).

A cDNA encoding the H⁺/peptide transporter (PEPT1) derived from various mammalian species has been cloned and PEPT1 was identified as an integral membrane-spanning protein that is predominantly expressed in the small intestine and slightly in the kidney (Fei et al., 1994; Liang et al., 1995; Saito et al., 1995). By functional expression of rat PEPT1 in Xenopus oocytes, we demonstrated that the single peptide transporter mediated the uptake of differently charged -lactam antibiotics such as zwitterionic cephradine and anionic ceftibuten (Saito et al., 1995). Rat PEPT1 had much higher affinity for ceftibuten than for cephradine (Saito et al., 1995). Such specificity for -lactams was consistent with results of transport studies with Caco-2 cells (Matsumoto et al., 1994, 1995). Another peptide transporter, PEPT2, also identified by cDNA cloning (Boll et al., 1996; Liu et al., 1995; Saito et al., 1996) was expressed predominantly in the kidney, and it showed the differential recognition of -lactam antibiotics from PEPT1 (Ganapathy et al., 1995). PEPT2 transported bestatin, and its mRNA transcript was expressed in the rat brain and lung in addition to the kidney (Saito et al., 1996).

Immunohistochemically, we found that rat PEPT1 is localized at the brush-border membranes of the small intestine (Ogihara et al., 1996). In contrast to the peptide transporter at this location, little is known about the transporter molecule in the basolateral membranes. We suggested that functionally different peptide transporters exist in the apical and basolateral membranes of the human intestinal epithelial cell line, Caco-2 (Matsumoto et al., 1994; Saito and Inui, 1993). The peptide transporter expressed in renal brush-border membranes has also been characterized (Daniel et al., 1992; Inui et al., 1984; Miyamoto et al., 1988), whereas the basolateral peptide transporter remains to be investigated. Considering the above-mentioned background, an in vitro epithelial model expressing the peptide transporters is required for molecular analysis of their functions and membrane-sorting mechanisms.

In the present study, we established LLC-PK₁ cells stably expressing rat PEPT1 by cDNA transfection and examined the localization and uptake by rat PEPT1 of oral -lactam antibiotics.

	Materials and Methods

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Materials. Ceftibuten (Shionogi and Co., Osaka, Japan), cephradine (Sankyo Co., Tokyo, Japan), bestatin and [³H]bestatin (12.7 GBq/mmol) (Nippon Kayaku Co., Tokyo, Japan) and cefixime (Fujisawa Pharmaceutical Co., Osaka, Japan) were gifts from the respective suppliers. [¹⁴C]Glycylsarcosine (1.78 GBq/mmol) was obtained from Daiichi Pure Chemicals Co., Ltd. (Ibaraki, Japan). Glycylsarcosine and glycylglycylphenylalanine were obtained from Sigma Chemical Co. (St. Louis, MO). Glycyl-L-leucine was purchased from the Peptide Institute Inc. (Osaka, Japan). Glycine, captopril, tetraethylammonium and cimetidine were obtained from Nacalai Tesque Inc. (Kyoto, Japan). All other chemicals were of the highest purity available.

Cell culture. Parental LLC-PK₁ cells obtained from the American Type Culture Collection (ATCC CRL-1392) were cultured in complete medium consisting of Dulbecco's modified Eagle's medium (GIBCO, Life Technologies, Grand Island, NY), containing 10% fetal bovine serum (Whittaker Bioproducts Inc., Walkersville, MD) without antibiotics in an atmosphere of 5% CO₂-95% air at 37°C (Saito et al., 1992). Transfected cell lines were maintained in the same medium containing 1 mg/ml of G418.

Transfection. The cDNA-encoding rat PEPT1 was subcloned into the SalI- and NotI-cut mammalian expression vector pBK-CMV (Stratagene, La Jolla, CA). LLC-PK₁ cells were transfected by calcium phosphate precipitation (Terada et al., 1996). LLC-PK₁ cells were plated at a density of 3 × 10⁶ cells per 100-mm plastic dish and incubated overnight. The DNA-calcium phosphate precipitate formed by 10 µg of pBK-CMV with or without the rat PEPT1 cDNA insert was added to the cells and incubated at 37°C. Fifteen hours later, cells were rinsed twice with Ca⁺⁺- and Mg⁺⁺-free Dulbecco's phosphate-buffered saline (pH 7.4) [PBS() buffer (in mM): 137, NaCl; 3, KCl; 8, Na₂HPO₄; and 1.5, KH₂PO₄]. Thereafter, 3 ml of PBS() containing 15% glycerol was added to the cells and incubated for 5 min at room temperature. After washing once with PBS(), the cells were cultured under normal conditions. Forty-eight hours later, the cells were split at dilutions of 1:75, 1:30 and 1:15. Twelve hours later, G418 (1 mg/ml) was added to the culture medium, which was replaced with fresh medium containing G418 (1 mg/ml) every 3 days. Between 14 and 21 days, single colonies were selected for subsequent screening.

Immunoblotting. Crude plasma membrane fractions of small intestine (Ogihara et al., 1996) and transfected cells (Terada et al., 1996) were prepared as described. Brush-border and basolateral membranes were purified simultaneously from rat renal cortex as described (Takano et al., 1984). The membrane fractions were separated by SDS-PAGE and analyzed by immunoblotting with specific rabbit antibodies against C-terminal synthetic peptide of rat PEPT1 as described (Ogihara et al., 1996; Saito et al., 1995).

Cell surface biotinylation. LLC-rPEPT1 cells (LLC-PK₁ transfected with rat PEPT1 cDNA) were inoculated on collagen-coated membrane filters (0.4-µm pores) inside Transwell cell culture chambers (Costar, Cambridge, MA) at a density of 2.5 × 10⁶ cells per well. Medium was replaced every 2 days, and the cell surface was biotinylated 7 days after seeding as described by Gottardi et al. (1995). LLC-rPEPT1 cells were placed on ice and washed with ice-cold Dulbecco's modified Eagle's medium followed by Dulbecco's phosphate-buffered saline (PBS(+), pH 7.4). Cells were then incubated with N-hydroxysuccinimide-ss-biotin (Pierce, Rockford, IL) at a concentration of 1.5 mg/ml, twice consecutively for 25 min at 4°C. Biotin (0.5 and 1.5 ml) was added to the apical and basolateral sides of the Transwell chambers, respectively. Biotinylation reactions proceeded at pH 9.0 in 10 mM triethanolamine, 2 mM CaCl₂ and 150 mM NaCl. Cells were then rinsed twice with PBS(+) with 100 mM glycine, then washed in this buffer for 20 min at 4°C. After rinsing twice with PBS(+), the filters were excised from the cup, and monolayers were solubilized in 1 ml of lysis buffer (1.0% Triton X-100, 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid, 50 mM TRIS, pH 7.5) for 60 min on ice. Cells were scraped from the filter, and the lysates were centrifuged at 14,000 × g for 10 min at 4°C. To 900 µl of upper supernatant, 100 µl of packed streptavidin-agarose beads (Pierce, Rockfold, IL) were added and incubated for 16 hr with gentle agitation at 4°C. The beads were then washed three times with lysis buffer, twice with high-salt wash buffer (500 mM NaCl, 5 mM ethylenediaminetetraacetic acid, 50 mM TRIS, pH 7.5) and once with no-salt wash buffer (10 mM TRIS, pH 7.5). Proteins were eluted from the beads in 80 µl of SDS-containing sample buffer, then separated by SDS-PAGE and immunoblotted.

Uptake measurements by cell monolayers. We examined the uptake of [¹⁴C]glycylsarcosine from the apical and basolateral side in the transfected cells inoculated on polycarbonate membrane filters (3-µm pores) inside Transwell chambers (Costar, Cambridge, MA) (Saito and Inui, 1993), and the uptake of other drugs in the cells cultured in 60-mm plastic dishes (Matsumoto et al., 1995). The protein content of cell monolayers solubilized in 1 N NaOH was determined by the method of Bradford (1976) with a Bio-Rad Protein Assay Kit (Bio-Rad, Richmond, CA) with bovine -globulin as the standard.

Statistical analysis. Data were analyzed for statistical significance by the one-way analysis of variance followed by Scheffé's test.

	Results

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Isolation of LLC-PK₁ cells stably expressing rat PEPT1. After transfection with pBK-CMV vector without (LLC-pBK) or with rat PEPT1 cDNA (LLC-rPEPT1), we isolated 10 G418-resistant clones. The crude plasma membrane fractions prepared from them were immunoblotted against anti-rat PEPT1 antibodies (Ogihara et al., 1996; Saito et al., 1995). Among these G418-resistant cells, two clones (LLC-rPEPT1) expressed immunoreactive protein, whereas LLC-pBK cells did not. Figure 1A shows a typical immunoblot of crude membranes isolated from the rat duodenum, LLC-rPEPT1 and LLC-pBK cells, with antiserum directed against the carboxy-terminal synthetic peptide of rat PEPT1 (Saito et al., 1995). A 75-kdalton protein was detected in membranes of the duodenum and LLC-rPEPT1 cells, but not in the LLC-pBK cells. Furthermore, the localization of rat PEPT1 transporter in the LLC-rPEPT1 cells was determined by cell surface biotinylation. As shown in figure 1B, the PEPT1 protein appeared to be expressed in both the apical and basolateral membranes of LLC-rPEPT1 cells, although the expression level of PEPT1 in the apical membranes was greater than in the basolateral membranes. In addition, the immunoreactive protein with the anti-rat PEPT1 antibodies, which showed the same molecular size of 75 kdaltons as that in LLC-rPEPT1 cells, was detected in both the brush-border and basolateral membranes from rat renal cortex (fig. 1C).

View larger version (39K): [in this window] [in a new window]   Fig. 1.   Immunoblots of crude plasma membranes isolated from the rat duodenum, LLC-rPEPT1 and LLC-pBK cells (A), of biotinylated proteins from LLC-rPEPT1 cells (B) and isolated rat renal brush-border and basolateral membranes (C). (A) Membrane proteins were separated by SDS-PAGE (10%) and blotted to polyvinylidene difluoride membranes. Antiserum for rat PEPT1 (Saito et al., 1995) (1:1000 dilution) was the primary antibody. A peroxidase-conjugated anti-rabbit IgG antibody detected bound antibodies, and blots were visualized by chemiluminescence on X-ray film. The arrow indicates the position of PEPT1. (B) LLC-rPEPT1 cells were grown to confluence for 7 days on 0.4-µm polycarbonate membrane filters and biotinylated from either the apical (AP) or the basolateral (BL) side at pH 9.0. After solubilization and streptavidin-agarose precipitation, biotinylated proteins were separated by SDS-PAGE and immunoblotted. (C) Brush-border (BBM) and basolateral (BLM) membranes were purified from rat renal cortex, separated by SDS-PAGE (100 µg protein per lane) and immunoblotted.

View larger version (44K): [in this window] [in a new window]   Fig. 2.   Accumulation of [14C]glycylsarcosine in LLC-pBK cells and LLC-rPEPT1 cells. The cell monolayers were incubated for 60 min at 37°C with the incubation medium at pH 6.0 containing of [14C]glycylsarcosine (20 µM, 37 kBq/ml) from either the apical (shaded columns) or the basolateral side (open columns). Thereafter, the radioactivity levels in the solubilized cells were determined. Each column represents the mean ± S.E. of three independent monolayers. N.D., not detectable.

Transport characteristics of -lactams in LLC-rPEPT1 cells. We measured the uptake of cephalosporins and bestatin in LLC-pBK (control) and LLC-rPEPT1 cells. As shown in figure 3A, the LLC-pBK cells did not show enhanced uptake of oral cephalosporin antibiotics such as ceftibuten, an anionic cephalosporin lacking an -amino group, and cephradine, a zwitterionic aminocephalosporin. Uptake of cefotiam, a parenteral cephalosporin, and bestatin, a dipeptide-like oral antineoplastic agent, was also low and not stimulated by an inward H⁺ gradient. In contrast, LLC-rPEPT1 cells transported ceftibuten, cephradine and bestatin in a H⁺-gradient-dependent manner, but not cefotiam (fig. 3B).

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Uptake of -lactam antibiotics and [3H]bestatin by LLC-pBK cells (A) and LLC-rPEPT1 cells (B). The cell monolayers were incubated for 60 min at 37°C in incubation medium at pH 6.0 (shaded columns) or pH 7.4 (open columns) containing each drug (1 mM, 37 kBq/ml for [3H]bestatin). Cellular uptake of drugs was then measured. Each column represents the mean ± S.E. of three independent monolayers.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Time courses of ceftibuten (A) and cephradine (B) uptake by LLC-rPEPT1 cells. LLC-rPEPT1 cells were incubated for the specified periods at 37°C with incubation medium containing 1 mM ceftibuten or cephradine at pH 6.0 () or pH 7.4 (). After incubation, cellular uptake of drugs was measured. Each point represents the mean ± S.E. of three independent monolayers. When the error bar is not shown, it is smaller than the symbol.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Concentration dependence of ceftibuten (A) and cephradine (B) uptake by LLC-rPEPT1 cells. LLC-rPEPT1 cells were incubated for 15 min at 37°C with incubation medium (pH 6.0) containing the indicated concentrations of ceftibuten or cephradine. Nonspecific uptake was evaluated by measuring the uptake of ceftibuten and cephradine by LLC-pBK cells and was subtracted from the total uptake of each drug by LLC-rPEPT1 cells at the indicated concentrations to calculate specific uptake. Each point represents the mean of three independent monolayers.

View larger version (48K): [in this window] [in a new window]   Fig. 6.   Effects of various compounds on ceftibuten (A) and cephradine (B) uptake by LLC-rPEPT1 cells. LLC-rPEPT1 cells were incubated for 15 min at 37°C with 1 mM ceftibuten (pH 6.0) or cephradine (pH 6.0) in the absence or presence of each inhibitor (10 mM), except for cimetidine and ceftibuten (5 mM). Cellular uptake of drugs was then measured. Each column represents the mean ± S.E. of three independent monolayers. GLY, glycine; GLY-LEU, glycylleucine; GLY-SAR, glycylsarcosine; GLY-GLY-PHE, glycylglycylphenylalanine; TEA, tetraethylammonium. *P < .05, significant difference from control.

	Discussion

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Previous studies with isolated intestinal brush-border membranes vesicles revealed that the intestinal peptide transporter recognizes a broad range of drugs including oral -lactam antibiotics (Inui et al., 1988; Muranushi et al., 1989; Okano et al., 1986; Tsuji et al., 1987) and bestatin (Inui et al., 1992a; Tomita et al., 1989). The transport characteristics of -lactam antibiotics via the H⁺/peptide cotransporter have been examined in the human intestinal cell line, Caco-2 (Inui et al., 1992b; Matsumoto et al., 1994). Complementary DNAs encoding the intestinal oligopeptide transporter (PEPT1) from rabbit (Boll et al., 1994; Fei et al., 1994), human (Ganapathy et al., 1995; Liang et al., 1995) and rat (Saito et al., 1995) have been isolated and functionally characterized in terms of their products. By directly measuring ceftibuten and cephradine uptake in Xenopus oocytes, we demonstrated that the rat PEPT1 mediates translocation of differently charged cephalosporin antibiotics (Saito et al., 1995).

To study mechanisms of membrane localization and function in the transcellular transport of PEPT1, a stable epithelial cell line expressing the transporter protein is required. In the present study, we stably transfected rat PEPT1 cDNA into the renal epithelial cell line LLC-PK₁, which lacks intrinsic peptide transport activity. We then examined membrane localization of PEPT1 and transport characteristics of -lactam antibiotics in the transfectants, LLC-rPEPT1 cells. LLC-rPEPT1 cells expressed rat PEPT1 with an apparent molecular mass of 75 kdaltons similar to the protein found in the rat intestinal membranes. To determine localization of PEPT1 protein in LLC-rPEPT1 cells, we biotinylated the cell surface. This procedure was applied to assess the polarized localization of exogenously transfected transporters in cultured epithelial cells (Gu et al., 1996; Pietrini et al., 1994). In this study, the PEPT1 was expressed predominantly in apical membranes and to a lesser, but appreciable extent in basolateral membranes (fig. 1B). The finding that [¹⁴C]glycylsarcosine accumulated from both the apical and basolateral sides of LLC-rPEPT1 cell monolayers provided evidence of a functionally active transporter in both membranes (fig. 2). We showed by immunoblotting and staining that rat PEPT1 was localized on the brush-border membranes of the rat small intestine but not to the basolateral membranes (Ogihara et al., 1996). In contrast, we found that rat PEPT1 was expressed in both the brush-border and basolateral membranes from rat renal cortex (fig. 1C).

The present results suggest that rat PEPT1 expressed bidirectionally in renal epithelial cells is involved in transcellular transport of oligopeptides and peptide-like drugs. Caco-2 cells undergo intracellular acidification in response to the apical uptake of both cefadroxil, a zwitterionic cephalosporin, and cefixime, depending on the apical pH (Wenzel et al., 1996). Therefore, LLC-rPEPT1 cells may be acidified in response to the cumulative uptake of ceftibuten or cephradine in the presence of an inward H⁺ gradient. If so, an outward H⁺ gradient from the cytoplasm to the basolateral side would be produced, thereby stimulating efflux of these antibiotics via basolaterally localized PEPT1. In the kidney, reabsorption of oligopeptides and peptide mimetics filtered through glomerulus in the renal proximal tubules may be mediated by the same H⁺-coupled peptide transporter PEPT1 expressed in both the brush-border and basolateral membranes. Immunohistochemical analysis and/or transport studies with isolated renal basolateral membranes are required to further clarify peptide transport in these membranes.

The uptake of ceftibuten and cephradine was inhibited by di- and tripeptides. Among these, glycylsarcosine potently inhibited the uptake of both drugs, which suggests that rat PEPT1 has much higher affinity for glycylsarcosine. It was notable that cefixime, an anionic cephalosporin without an -amino group, inhibited significantly the uptake of ceftibuten but not of cephradine. These results can not be explained only by the affinity of PEPT1 for cefixime, because PEPT1 has high affinity for cefixime as well as for ceftibuten; the apparent K_m value of cefixime was 0.8 mM in isolated brush-border membrane vesicles from the rat intestine at pH 5.0 (Inui et al., 1988) and 1.4 mM in Caco-2 cells at pH 6.0 (Matsumoto et al., 1995). One possible explanation is that the mechanism for recognition of -lactam antibiotics without an -amino group by PEPT1 might be different from that of antibiotics with an -amino group. Alternatively, cefixime possesses two carboxylic acids, thereby bearing divalent anionic charge with the pH range between 5.0 and 7.5. Therefore, the inhibitory effect of cefixime on ceftibuten uptake might be caused by charge-based interaction of these anionic drugs with the substrate recognition site of rat PEPT1. Similar results were observed in the mutual inhibition of cephradine and ceftibuten; cephradine (10 mM) very weakly inhibited ceftibuten uptake, whereas ceftibuten (5 mM) potently inhibited that of cephradine.

In Xenopus oocytes expressing rabbit PEPT1, cefadroxil transport is inhibited by zwitterionic compounds such as cephalexin and amoxicillin at pH 6.5, but not by anionic -lactams including cefixime (Wenzel et al., 1996). In contrast, anionic -lactams potently inhibited cefadroxil transport at pH 5.5. Considering the ionic forms at various pH ranges, the PEPT1-mediated uptake of cefixime and mutual inhibition, Wenzel et al. (1996) concluded that only the zwitterionic species of -lactams are transported efficiently by the intestinal peptide transporter. We assumed that the recognition and/or binding site of PEPT1 as well as the degree of ionization of -lactams is related to the pH dependence of their transport, because at least two histidine residues in positions 57 and 121 located at the deduced second and fourth transmembrane domains of rat PEPT1 may be involved in substrate recognition by the transporter (Terada et al., 1996).

In conclusion, we established the stably transfected LLC-rPEPT1 cells expressing the rat PEPT1. Functionally active PEPT1 was detected not only at the apical membranes but at the basolateral membranes. The transport characteristics of -lactam antibiotics were mostly similar to those found in isolated brush-border membranes of the rat intestine, which suggests that the LLC-rPEPT1 cells will serve as a useful model with which to study the molecular mechanisms involved in membrane localization and structural requirement for substrate recognition by PEPT1.