Introduction

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The intestinal absorption of dietary nutrients and oral drugs occurs via the paracellular and transcellular transport pathways. Transcellular transport across the intestinal epithelial cells involves 1) uptake from the lumen across the brush-border membranes, 2) diffusion through the cytoplasm, and 3) exit to the portal blood across the basolateral membranes. Some hydrophilic nutrients and drugs are transported by the specific transport systems localized at the brush-border and basolateral membranes.

The H⁺-coupled peptide transporter (PEPT1) expressed in the brush-border membranes of the intestinal epithelial cells contributes to the supply of dietary amino nitrogen by means of mediating the transport of di- and tripeptides into the cells (Leibach and Ganapathy, 1996; Inui and Terada, 1999). PEPT1 accepts as substrates not only native small peptides, but also some pharmacologically active compounds bearing a resemblance to small peptides such as -lactam antibiotics (Ganapathy et al., 1995; Saito et al., 1995; Terada et al., 1997). Using the human intestinal cell line Caco-2, we found that another peptide transporter was expressed in the basolateral membranes and that this transporter was involved in the transfer of small peptides and peptide-like drugs from the cells to the circulating blood (Inui et al., 1992; Saito and Inui, 1993; Terada et al., 1999).

Recently, it was revealed that PEPT1 was capable of transporting various compounds without peptide bond(s). For example, valacyclovir, the L-valine ester prodrug of the antiherpetic agent acyclovir, was demonstrated to be transported by PEPT1 (Han et al., 1998a,b). We also found that L-valine alkyl ester compounds were recognized by rat PEPT1 (Sawada et al., 1999b). Döring et al. (1998a) demonstrated that -aminolevulinic acid (-ALA), which has a ketomethylene group instead of a peptide bond, was translocated by PEPT1. Döring et al. (1998b) also showed that -fatty amino acids such as 8-aminooctanoic acid were transported by PEPT1. These findings have brought great interest in the molecular design of drugs to achieve a high oral bioavailability using the broad substrate specificity of the intestinal PEPT1. However, in contrast to PEPT1, it is still unknown whether the basolateral peptide transporter can recognize and transport compounds lacking peptide bond(s).

In the present study, we examined the recognition characteristics of several nonpeptidic compounds by the basolateral peptide transporter in Caco-2 cells and compared them with those by the apical PEPT1. In addition, the transport characteristics of -ALA were examined by both transporters, because -ALA is currently used in clinical studies as an oral endogenous photosensitizer of photodynamic therapy.

	Materials and Methods

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Materials. [¹⁴C]Glycylsarcosine (1.78 GBq/mmol) was obtained from Daiichi Pure Chemicals Co., Ltd. (Ibaraki, Japan). [³H]-ALA (25.9 GBq/mmol) was from PerkinElmer Life Science Products, Inc. (Boston, MA). Valacyclovir was supplied by GlaxoSmithKline Research and Development (Hertfordshire, UK). Glycine and -ALA were obtained from Nacalai Tesque Inc. (Kyoto, Japan). Glycylsarcosine, 8-aminooctanoic acid, alanine-4-nitroanilide, and all L-valine alkyl esters were purchased from Sigma Chemical Co. (St. Louis, MO). All other chemicals used were of the highest purity available.

Cell Culture. Caco-2 cells at passage 18 obtained from the American Type Culture Collection (ATCC HTB37) were maintained by serial passage in plastic culture dishes. Complete medium consisted of Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA), supplemented with 10% fetal bovine serum (BioWhittaker, Walkersville, MD) and 1% nonessential amino acids (Invitrogen) 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, Caco-2 cells were seeded on 35-mm plastic dishes (2 × 10⁴ cells/dish, 2 ml of medium) or on 12-well cluster plates (1 × 10⁴ cells/well, 1 ml of 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 6.6 × 10⁴ cells/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 were given fresh medium every 2 to 4 days and were used on the 14th or 15th day for uptake experiments.

Uptake Studies by Cell Monolayers. The uptake of radiolabeled substrates was determined as described previously (Terada et al., 1999). In uptake studies using valacyclovir, the extraction solution (water/methanol, 50:50) was added to the cells after the uptake period. After standing for 1 h at room temperature, the solutions were centrifuged and the supernatants were filtered through a Millipore filter (SGJVL, 0.22 µm). The filtrate was analyzed by high-performance liquid chromatography (HPLC).

Analytical Methods. To measure the uptake of valacyclovir by Caco-2 cells, valacyclovir and acyclovir were simultaneously determined using a high-performance liquid chromatograph LC-10A (Shimadzu Co., Kyoto, Japan) equipped with an SPD-6A variable wavelength UV detector (Shimadzu Co.) and an integrator (Chromatopac C-R1A, Shimadzu Co.) under the following conditions: column, TSK-gel ODS 80TM 4.6 mm i.d. × 150 (Tosoh Co., Tokyo, Japan); mobile phase, 30 mM ammonium formate (pH 3.5)/methanol, 95:5; flow rate, 0.8 ml/min; wavelength, 254 nm; injection volume, 50 µl; and temperature, 45°C.

Data Analysis. Each experimental point shown represents the mean ± S.E. of three to nine measurements from one to three separate experiments. When the bars are not shown, they are smaller than the symbols. Data were analyzed statistically by a nonpaired t test or a one-way analysis of variance followed by Scheffé's test when multiple comparisons were needed. IC₅₀ values were determined by nonlinear regression analysis. The inhibition constant (K_i) values were calculated from IC₅₀ values according to the method described by Cheng and Prusoff (1973).

	Results

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Effect of Various Nonpeptidic Compounds on [¹⁴C]Glycylsarcosine Uptake by PEPT1 and the Basolateral Peptide Transporter. We first examined the effect of various nonpeptidic compounds on [¹⁴C]glycylsarcosine uptake by PEPT1 and the basolateral peptide transporter in Caco-2 cells. As shown in Fig. 1A, all compounds examined showed a marked inhibitory effect on [¹⁴C]glycylsarcosine uptake via the apical PEPT1. Similarly, [¹⁴C]glycylsarcosine uptake by the basolateral peptide transporter was substantially inhibited by Ala-4-nitroanilide, -ALA, 8-aminooctanoic acid, valacyclovir, and L-valine benzyl ester (Val-OBz) (Fig. 1B). However, L-valine alkyl esters other than Val-OBz and anti-diabetes agents, glibenclamide, and nateglinide did not show the inhibitory effect (Fig. 1B). L-Valine alkyl esters were suggested to be transported by rat PEPT1 and rat PEPT2 (Sawada et al., 1999b), and glibenclamide and nateglinide were demonstrated to be noncompetitive inhibitors of these transporters (Sawada et al., 1999a; Terada et al., 2000).

View larger version (44K): [in this window] [in a new window]   Fig. 1.   Effects of various nonpeptidic compounds on [14C]glycylsarcosine uptake by PEPT1 (A) and the basolateral peptide transporter (B) in Caco-2 cells. The cell monolayers were incubated at 37°C for 15 min with incubation medium containing [14C]glycylsarcosine (20 µM, 37 kBq/ml) in the absence or presence of each inhibitor added to either the apical (pH 6.0) or basolateral side (pH 7.4). Thereafter, the radioactivity of solubilized cells was determined. Each column represents the mean ± S.E. of three monolayers (A) or of six monolayers from two separate experiments (B). 8-AOA, 8-aminooctanoic acid; Val-ACV, valacyclovir. *P < 0.05, **P < 0.01, significantly different from control.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Dose-dependent inhibition of Ala-4-nitroanilide (A), -ALA (B), and Val-OBz (C) to [14C]glycylsarcosine uptake by PEPT1 () and the basolateral peptide transporter () in Caco-2 cells. The cell monolayers were incubated at 37°C for 15 min with incubation medium containing [14C]glycylsarcosine (20 µM, 37 kBq/ml) in the absence () or presence (, ) of increasing concentrations of various inhibitors added to either the apical (pH 6.0) or basolateral side (pH 7.4). [14C]Glycylsarcosine uptake in the absence of the inhibitor was taken as 100% (apical: A, 356 ± 10; B, 321 ± 6; C, 301 ± 6; basolateral: A and B, 105 ± 4; C, 72 ± 3 pmol/mg of protein · 15 min1). · Each point represents the mean ± S.E. of three monolayers. When error bars are not shown, they are smaller than the symbols.

Effect of DEPC on [¹⁴C]Glycylsarcosine Uptake in Caco-2 Cells. Previously, we demonstrated that the excess peptidic substrates prevented the diethylpyrocarbonate (DEPC)-induced inactivation of PEPT1 (Terada et al., 1998) and the basolateral peptide transporter (Terada et al., 1999). Because DEPC is a histidine residue-modifier reagent, the histidine residue was proposed to be the substrate binding site of both transporters. We then examined whether the nonpeptidic compounds also showed the protective effect on DEPC-induced inactivation. As shown in Fig. 3, the DEPC-induced inhibition of [¹⁴C]glycylsarcosine uptake by both transporters was substantially protected in the presence of nonpeptidic compounds such as valacyclovir and -ALA.

View larger version (34K): [in this window] [in a new window]   Fig. 3.   Effects of DEPC pretreatment on [14C]glycylsarcosine uptake by PEPT1 (A) and the basolateral peptide transporter (B) in Caco-2 cells. The cell monolayers were preincubated at 25°C for 10 min with 1 mM DEPC (pH 6.0) added to either the apical or basolateral side in the absence (opened column) or presence (closed columns) of various compounds (10 mM). After preincubation, Caco-2 monolayers were washed once with the incubation medium and then incubated at 37°C for 15 min with [14C]glycylsarcosine (20 µM, 37 kBq/ml) added to either the apical (pH 6.0) or basolateral side (pH 7.4). Thereafter, radioactivity of solubilized cells was determined. A, each column represents the mean ± S.E. of three monolayers. B, each column represents the mean ± S.E. of 6 to 12 monolayers from three separate experiments. Val-ACV, valacyclovir.

Direct Measurements of Transport of Valacyclovir and [³H]-ALA in Caco-2 Cells. Inhibition and DEPC protection studies showed that some nonpeptidic compounds were recognized by the basolateral peptide transporter, but these findings did not necessarily indicate that this transporter had the transport ability of nonpeptidic compounds. Thus, we carried out the direct measurements of valacyclovir and [³H]-ALA transport in Caco-2 cells. As shown in Fig. 4, A and B, valacyclovir uptake from both sides was decreased in the presence of excess glycyl-leucine, but not of glycine. Figure 4, C and D, show the time course of [³H]-ALA uptake through both membranes, and its uptake was inhibited by the excess glycylsarcosine. These findings suggested that both valacyclovir and -ALA were transported by the basolateral peptide transporter in addition to PEPT1. Although valacyclovir was found to be transported by the basolateral peptide transporter, valacyclovir taken by the cells from lumen was mainly metabolized to valine and acyclovir (Han et al., 1998a). Thus, it is unlikely that the investigation of the transport characteristics of valacyclovir by the basolateral peptide transporter is pharmacologically important. For this reason, the following studies were carried out using [³H]-ALA alone.

View larger version (41K): [in this window] [in a new window]   Fig. 4.   Valacyclovir (A, B) and -ALA (C, D) uptake from the apical (A, C) or basolateral side (B, D) in Caco-2 cells. A and B, Caco-2 monolayers were incubated at 37°C for 1 h with valacyclovir (A, 1 mM; B, 3 mM) in the absence or presence of glycine or glycyl-leucine added to the apical (pH 6.0) or basolateral (pH 7.4) side. Thereafter, valacyclovir and acyclovir extracted from the cell monolayers were simultaneously measured by HPLC as described under Materials and Methods. C and D, Caco-2 monolayers were incubated at 37°C with incubation medium containing [3H] -ALA (5 µM, 18.5 kBq/ml) in the absence () or presence () of 5 mM glycylsarcosine added to either the apical (pH 6.0) or basolateral side (pH 7.4). Thereafter, the radioactivity of solubilized cells was determined. Each column and point represents the mean ± S.E. of three monolayers. **P < 0.01, significantly different from control.

Transport Characteristics of [³H]-ALA in Caco-2 Cells. -ALA is a precursor of porphyrins and heme and plays an important role in the production of heme-containing proteins. Recently, there has been growing interest in the transport and metabolism of -ALA, because this compound has been successfully used in treating various tumors by photodynamic therapy (Loh et al., 1993; Fromm et al., 1996; Peng et al., 1997). When -ALA was administered orally, it showed high oral bioavailability and rapid increases in the circulating plasma level (Fromm et al., 1996; Dalton et al., 1999), suggesting the efficient intestinal transport systems involved in its delivery. At first, to evaluate the transport systems of -ALA, inhibition studies were carried out. Figure 5 shows the effect of various compounds on the [³H]-ALA uptake from both sides. [³H]-ALA uptake by the apical side was markedly inhibited by -ALA and glycylsarcosine and slightly inhibited by -aminobutyric acid (GABA) and glycine. In contrast, [³H]-ALA uptake by the basolateral side was inhibited by dipeptides, peptide-like drugs, and valacyclovir, but not by amino acids and GABA.

View larger version (46K): [in this window] [in a new window]   Fig. 5.   Effects of various compounds on [3H] -ALA uptake from the apical (A) and the basolateral side (B) in Caco-2 cells. The cell monolayers were incubated at 37°C for 10 min with incubation medium containing [3H] -ALA (5 µM, 18.5 kBq/ml) in the absence or presence of each inhibitor added to either the apical (pH 6.0) or basolateral side (pH 7.4). Thereafter, the radioactivity of solubilized cells was determined. Each column represents the mean ± S.E. of three monolayers. *P < 0.05, **P < 0.01, significantly different from control.

View larger version (23K): [in this window] [in a new window]   Fig. 6.   pH Dependence of [3H] -ALA uptake from the apical (A) and the basolateral side (B) in Caco-2 cells. The cell monolayers were incubated at 37°C for 10 min with [3H] -ALA (5 µM, 18.5 kBq/ml) in the absence () or presence () of 5 mM glycylsarcosine added to either the apical or basolateral side at various pH values. Thereafter, the radioactivity of solubilized cells was determined. Each point represents the mean ± S.E. of six monolayers from two separate experiments. When error bars are not shown, they are smaller than the symbols. Insets: pH dependence of PEPT1-mediated (A) and the basolateral peptide transporter-mediated specific -ALA uptake (B). Values () were calculated by subtracting nonspecific uptake estimated in the presence of 5 mM glycylsarcosine () from total uptake ().

View larger version (21K): [in this window] [in a new window]   Fig. 7.   Concentration dependence of [3H] -ALA uptake from the apical (A) and basolateral side (B) in Caco-2 cells. The cell monolayers were incubated at 37°C for 10 min with various concentrations of [3H] -ALA (18.5 kBq/ml) added to either the apical (pH 6.0) or basolateral side (pH 7.4) in the absence () or presence () of 50 mM glycylsarcosine. Thereafter, the radioactivity of solubilized cells was determined. Each point represents the mean ± S.E. of six monolayers from two separate experiments. When error bars are not shown, they are smaller than the symbols. Insets: Eadie-Hofstee plots of -ALA uptake after correction for nonsaturable component (). V, uptake rate (nmol/mg of protein · 10 min1); S, -ALA concentration (mM).

View larger version (21K): [in this window] [in a new window]   Fig. 8.   Transcellular transport (A) and accumulation (B) of [3H] -ALA by Caco-2 cells. A, the cell monolayers were incubated at 37°C with [3H] -ALA (5 µM, 18.5 kBq/ml) added to either the apical (pH 6.0; , ) or basolateral side (pH 7.4; , ) in the absence (, ) or presence (, ) of 10 mM glycylsarcosine. After the incubation, the appearance of radioactivity on the opposite side was measured. Transcellular transport of -ALA was calculated by subtracting the flux of mannitol from the net flux of -ALA. B, after 60 min of incubation, the cell monolayers were washed twice with ice-cold incubation medium, and the radioactivity of solubilized cells was determined. Each point and column represent the mean ± S.E. of six monolayers from two separate experiments. When error bars are not shown, they are smaller than the symbols.

	Discussion

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In this study, we examined the interaction of nonpeptidic compounds with the intestinal basolateral peptide transporter using Caco-2 cells. Inhibition studies showed that the basolateral peptide transporter, as well as PEPT1, interacted with various nonpeptidic compounds, although there is a slight difference in their recognition characteristics, as discussed below. The basolateral peptide transporter had lower affinity for nonpeptidic compounds than did PEPT1, and this finding was corresponded well with our previous findings using peptidic substrates (Terada et al., 1999). DEPC protection studies revealed that not only peptidic substrates but also nonpeptidic compounds bound to a histidine residue of PEPT1 and the basolateral peptide transporter. The direct uptake measurements of valacyclovir and [³H]-ALA clearly demonstrated that nonpeptidic compounds were able to be transported by the basolateral peptide transporter. Overall results indicate that the intestinal basolateral peptide transporter can accept nonpeptidic compounds as substrates, in a way similar to that of peptidic substrates. Preliminarily, we found that the basolateral peptide transporter in Madin-Darby canine kidney cells, which is distinct from the basolateral peptide transporter in Caco-2 cells, could also recognize various nonpeptidic compounds. Therefore, it is reasonable to assume that transport abilities of nonpeptidic compounds were general characteristics of various peptide transporters.

Some nonpeptidic compounds did not exhibit inhibitory effects on [¹⁴C]glycylsarcosine uptake by the basolateral peptide transporter, although these compounds showed the inhibitory effect on PEPT1. For example, L-valine alkyl esters such as Val-OMe were unable to diminish the [¹⁴C]glycylsarcosine uptake. Considering that Val-OBz and valacyclovir showed the inhibitory effect on [¹⁴C]glycylsarcosine uptake by the basolateral peptide transporter, the recognition abilities of molecular size might be different between PEPT1 and the basolateral peptide transporter. Other examples were oral anti-diabetes agents, glibenclamide, and nateglinide. In our previous studies, these agents were demonstrated to be noncompetitive inhibitors of PEPT1 and PEPT2 (Sawada et al., 1999a; Terada et al., 2000), and it was suggested that the binding site for glibenclamide and nateglinide was different from that for glycylsarcosine. In the present study, these agents did not show the inhibitory effect on [¹⁴C]glycylsarcosine uptake by the basolateral peptide transporter. It is therefore assumed that the basolateral peptide transporter may not have the binding site for anti-diabetes agents.

The histidine residue of PEPT1 (Terada et al., 1998, 1999; Chen et al., 2000) and the basolateral peptide transporter (Terada et al., 1999) was suggested to be involved in the recognition of peptidic substrates. In the present study, nonpeptidic substrates also protected the inactivation of DEPC. This finding indicates that the histidine residue takes the same functional role in the recognition of peptidic and nonpeptidic compounds. Previously, we suggested that the histidine residue of PEPT1 and PEPT2 was the binding site of an -amino group of peptidic substrates (Terada et al., 1998). Because all nonpeptidic substrates used in the DEPC protection study have an -amino group of their chemical structures, it is likely that the histidine residue of PEPT1 and the basolateral peptide transporter is involved in the binding site for the -amino group of nonpeptidic substrates.

Photodynamic therapy for cancer patients has developed into an important new clinical treatment modality in which photosensitized cells can be selectively eradicated by exposure to light (Fromm et al., 1996; Peng et al., 1997). -ALA is an endogenous precursor of protoporphyrin IX (PpIX), the potent photosensitizer, and -ALA-derived PpIX is highly accumulated in rapidly proliferating tumor cells (Wyss-Desserich et al., 1996). In a number of studies, -ALA uptake by tumor cells was shown to be mediated by various transport systems such as GABA (Rud et al., 2000), glycine (Langer et al., 1999), and -amino acid transport systems (Rud et al., 2000). In contrast, there are few reports about the transport mechanisms of -ALA in healthy tissues. Although providing -ALA by the oral route causes significant increases in the concentration of -ALA and porphyrins in peripheral tissues and plasma (Loh et al., 1993; Peng et al., 1997), the intestinal absorption mechanisms of -ALA are not clear. Some kinds of amino acid transporters were suggested to be involved in these mechanisms (Dalton et al., 1999). Recently, using oocyte expression systems, Döring et al. (1998a) provided the first evidence that -ALA is transported by PEPT1 and PEPT2 and suggested that these transporters may serve as the tissue-uptake systems for -ALA. The [³H]-ALA uptake studies here have demonstrated that PEPT1 plays a major role in the -ALA transport across the brush-border membranes of the intestinal epithelial cells and that the transport systems for GABA and glycine may contribute in a minor part. In addition, we found that the basolateral peptide transporter took a major role in the [³H]-ALA transport across the basolateral membranes. Moreover, transcellular transport of -ALA from the apical to basolateral side, namely the absorptive direction, was much greater than that of the opposite direction, indicating that -ALA could be absorbed efficiently at the small intestine. Therefore, it is suggested that the transport abilities of -ALA by PEPT1 and the basolateral peptide transporter can explain the good bioavailability of -ALA.

In conclusion, this study provided the definite evidence that not only PEPT1 but also the intestinal basolateral peptide transporter can recognize and transport nonpeptidic compounds. Thus, the basolateral peptide transporter plays an important role not only for the transfer of peptide-like drugs, but also for that of nonpeptidic drugs such as -ALA to achieve a high oral bioavailability. These findings may provide useful information for improving the intestinal absorption of poorly absorbed drugs.