Abstract
We established stably transfected LLC-PK1 cells expressing the rat H+/peptide cotransporter PEPT1 (designated LLC-rPEPT1) and examined membrane localization and uptake by rat PEPT1 of oral β-lactam antibiotics. The LLC-rPEPT1 cells expressed a novel PEPT1 protein with an apparent molecular mass of 75 kdaltons, which was found in rat intestinal membranes. The cell surface biotinylation of LLC-rPEPT1 cell monolayers grown on membrane filters showed that PEPT1 was localized predominantly on the apical membranes and, to a lesser extent, on the basolateral membranes. The amount of [14C]glycylsarcosine uptake in LLC-rPEPT1 cell monolayers was 3-fold greater from the apical, than from the basolateral side, which suggested that rat PEPT1 expressed on both membranes was functionally active. LLC-rPEPT1 cells grown on plastic dishes transported differently charged oral cephalosporins such as ceftibuten (divalent anion lacking an α-amino group) and cephradine (zwitterion with an α-amino group) in the presence of an inward H+gradient, whereas those transfected with the vector alone did not have transport activity. Kinetic analysis revealed that the LLC-rPEPT1 cells had much higher affinity for ceftibuten than for cephradine. Di- and tripeptides and bestatin, a dipeptide-like antineoplastic drug, potently inhibited the uptake of these cephalosporins. These results suggest that the LLC-rPEPT1 cells serve as a useful model with which to analyze the mechanisms involved in membrane targeting and substrate recognition by rat PEPT1.
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 β-lactam antibiotics (Inui et al., 1992b; Matsumotoet al., 1994; Muranushi et al., 1989; Tamaiet al., 1995), antineoplastic agent bestatin (Inui et al., 1992a; Tomita et al., 1989) and the angiotensin-converting enzyme inhibitors (Hu and Amidon, 1988; Swaanet 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 (Feiet al., 1994; Liang et al., 1995; Saito et al., 1995). By functional expression of rat PEPT1 inXenopus oocytes, we demonstrated that the single peptide transporter mediated the uptake of differently charged β-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 β-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; Saitoet al., 1996) was expressed predominantly in the kidney, and it showed the differential recognition of β-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, anin 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 β-lactam antibiotics.
Materials and Methods
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 CaCl2and 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 γ-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
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 (Ogiharaet 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).
Figure 2 shows the cellular accumulation of [14C]glycylsarcosine by the LLC-pBK and LLC-rPEPT1 cells grown on permeable membranes. Threefold more [14C]glycylsarcosine was accumulated from the apical than from the basolateral side of the monolayers in the presence of an inward H+ gradient (pH 6.0). Transport activity of [14C]glycylsarcosine was not enhanced from each side of the LLC-pBK cell monolayers. These results suggested that rat PEPT1 with functional dipeptide transport activity was localized predominantly on the apical membranes, and to a lesser extent to the basolateral membranes.
Transport characteristics of β-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 α-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).
Figure 4 shows the time courses of uptake of ceftibuten and cephradine by LLC-rPEPT1 cells. The uptake of both drugs was extensively increased with the incubation time in the presence of an inward H+ gradient (pH 6.0), and the uptake rate of ceftibuten was much greater than that of cephradine. In the absence of a H+ gradient (pH 7.4), there was no difference in the uptake rates of either drug.
Figure 5 shows the initial uptake rate of ceftibuten and cephradine at pH 6.0 by LLC-rPEPT1 cells as a function of the substrate concentrations. The uptake of both antibiotics is represented as specific uptake, which was calculated by subtracting the nonspecific uptake by LLC-pBK cells from the total uptake by LLC-rPEPT1 cells. By means of nonlinear least-squares regression analysis (Yamaoka et al., 1981), kinetic parameters were calculated from the Michaelis-Menten equation. The values of apparentK m for the uptake of ceftibuten and cephradine were 1.5 and 8.2 mM, respectively. The V maxvalues for ceftibuten and cephradine were 1.6 and 1.9 nmol/mg protein/min, respectively.
To examine the substrate specificity of rat PEPT1, the effects of various compounds on the uptake of ceftibuten and cephradine in LLC-rPEPT1 cells were examined by cis-inhibition. As shown in figure 6A, the uptake of ceftibuten was inhibited markedly by the presence of oligopeptides such as glycylleucine, glycylsarcosine and glycylglycylphenylalanine, but not by glycine. The uptake of ceftibuten was inhibited slightly, but not significantly by cephradine. The uptake of ceftibuten was inhibited significantly by cefixime, an oral anionic cephalosporin bearing two carboxyl groups like ceftibuten. Bestatin also markedly inhibited ceftibuten uptake. Captopril, an angiotensin-converting enzyme inhibitor, the absorption of which is mediated by the intestinal peptide transport system (Hu and Amidon, 1988), had a weak inhibitory effect on ceftibuten uptake. Organic cations, such as tetraethylammonium and cimetidine, did not inhibit uptake. As shown in figure 6B, cephradine uptake as well as ceftibuten uptake was inhibited by glycylleucine, glycylsarcosine, glycylglycylphenylalanine and bestatin. Cefixime did not inhibit cephradine uptake. Captopril had weakly affected cephradine uptake. Neither tetraethylammonium nor cimetidine inhibited cephradine uptake.
Discussion
Previous studies with isolated intestinal brush-border membranes vesicles revealed that the intestinal peptide transporter recognizes a broad range of drugs including oral β-lactam antibiotics (Inuiet al., 1988; Muranushi et al., 1989; Okanoet al., 1986; Tsuji et al., 1987) and bestatin (Inui et al., 1992a; Tomita et al., 1989). The transport characteristics of β-lactam antibiotics via the H+/peptide cotransporter have been examined in the human intestinal cell line, Caco-2 (Inui et al., 1992b; Matsumotoet 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 β-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 α-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 apparentK m value of cefixime was 0.8 mM in isolated brush-border membrane vesicles from the rat intestine at pH 5.0 (Inuiet 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 β-lactam antibiotics without an α-amino group by PEPT1 might be different from that of antibiotics with an α-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 β-lactams including cefixime (Wenzel et al., 1996). In contrast, anionic β-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 β-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 β-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 β-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.
Footnotes
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Send reprint requests to: Professor Ken-ichi Inui, Ph.D., Department of Pharmacy, Kyoto University Hospital, Sakyo-ku, Kyoto 606–01, Japan.
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↵1 This work was supported by a Grand-in-Aid for Scientific Research on Priority Areas of “Channel-Transporter Correlation” from the Ministry of Education, Science, and Culture of Japan, and by Grants-in-Aid from Japan Health Sciences Foundation.
- Abbreviations:
- TRIS
- 2-amino-(2-hydroxymethyl)-1,3-propanediol
- PAGE
- polyacrylamide gel electrophoresis
- SDS
- sodium dodecyl sulfate
- PBS
- phosphate-buffered saline
- bestatin
- (2R,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-l-leucine
- Received October 22, 1997.
- Accepted February 4, 1997.
- The American Society for Pharmacology and Experimental Therapeutics