Introduction

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-Aminolevulinic acid (-ALA) has gained enormous interest in recent years as an endogenous photosensitizer for fluorescence diagnosis and photodynamic tumor therapy (PDT; Peng et al., 1997; Zöpf and Riemann, 1997). Under physiological conditions, -ALA is required for the synthesis of protoporphyrin IX, a direct precursor of heme. Endogenously generated protoporphyrin IX is an excellent, so-called second-generation photosensitizing agent (Zoepf et al., 2001b). After oral or intravenous application of -ALA, the -ALA synthase is bypassed and porphyrins such as protoporphyrin IX accumulate in cells above normal level. Photoactivation results in cell death. Therefore, in recent years -ALA was established as an important drug in PDT of several types of tumors such as skin basal cell carcinoma, gastrointestinal adenocarcinoma, dysplastic mucosa, and early esophagus cancers (Peng et al., 1997; Zöpf and Riemann, 1997; Gossner et al., 1998; Byrnes and Afdhal, 2002). Recently, Pech et al. (2002) proved the efficiency of PDT with -ALA-protoporphyrin IX in human Barrett's adenocarcinoma. For tumors of the extrahepatic biliary duct, PDT is of particular relevance. Very successfully, photofrin II and photosan-3 were used for treatment of extrahepatic bile duct cancers. Bile duct stenosis could be almost completely eliminated in the treated area by PDT with photosan-3 (Rumalla et al., 2001; Zoepf et al., 2001a). In a pilot study, PDT with -ALA was not effective (Zoepf et al., 2001b). Possible explanations are specific biochemical characteristics in bile duct cancer that are different from other epithelial tumors, thereby requiring optimization of treatment parameters. Gibson et al. (1999) found that -ALA-induced protoporphyrin IX reaches different levels in different tumor cells.

Based on the medical importance of -ALA, several studies of the membrane transport of this substance in human tissues, e.g., at the apical and basolateral membrane of the intestinal epithelium, in brain synaptosomes, and at the choroid plexus have been reported. Surprisingly, quite different transport pathways have been described depending on the specific membrane. Döring et al. (1998) performed a thorough investigation of -ALA transport focused on peptide transporters. They reported that -ALA represents a high-affinity substrate for the intestinal type H⁺/peptide cotransporter PEPT1 and the renal type H⁺/peptide cotransporter PEPT2. When administered orally, intact -ALA is very well absorbed in the gastrointestinal tract (Dalton et al., 1999). In the kidney, -ALA is efficiently reabsorbed after glomerular filtration from the primary filtrate back to the blood. The result was also of high interest for structure-transport considerations of peptide transport because -ALA contains a ketomethylene group instead of a peptide bond. Importantly, neither the structurally related GABA, which has a shorter backbone than -ALA, nor glutamate could inhibit -ALA transport by PEPT1 and PEPT2 (Döring et al., 1998). In rat brain synaptosomes, [³H]-ALA, glutamate, and GABA interacted with the same transporter (McLoughlin and Cantrill, 1984). Similarly, for the human adenocarcinoma cell line WiDr transport of -ALA by -amino acid and GABA carriers has been reported (Rud et al., 2000). In amelanotic melanomas uptake of -ALA is inhibited by glycine (Langer et al., 1999). Glycine, however, did not inhibit -ALA transport in the Döring et al. (1998) study.

Studies regarding the intestinal transport of -ALA were extended by Inui's group. They investigated recognition and transport characteristics of -ALA uptake in intact cells (Caco-2) both at the apical and at the basolateral membrane (Irie et al., 2001). They found that cells grown on filters had greater transport activity from the apical-to-basolateral membrane than in the opposite direction. -ALA was, however, transported by the basolateral system. The authors did not determine whether GABA, glutamate, glycine, or aspartate affected the total [³H]-ALA uptake in Caco-2 cells. At the epithelium of the choroid plexus, it has been described that -ALA is transported by two different uptake mechanisms: PEPT2 and a putative Na⁺/HCO₃-dependent organic anion transporter (Novotny et al., 2000).

As stated above, PDT of tumors of the extrahepatic biliary duct is of particular interest. Recently, we described the expression of the intestinal H⁺/peptide symporter PEPT1 in tumor cells of the extrahepatic biliary duct and in normal rabbit bile duct (Knütter et al., 2002). -ALA inhibited uptake of [¹⁴C]Gly-Sar via PEPT1. In the present study, we investigated directly the transport characteristics of [³H]-ALA in bile duct tumor cells.

	Materials and Methods

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Materials. The human extrahepatic biliary duct tumor cell line SK-ChA-1 (Knuth et al., 1985) was obtained from the Ludwig Institute for Cancer Research (Zurich, Switzerland). -[3,5-³H(N)]aminolevulinic acid ([³H]-ALA, specific activity 0.7 Ci/mmol) was purchased from BioTrend (Köln, Germany), [2-³H]glycine (specific activity, 15 Ci/mmol), and [glycine-1-¹⁴C]glycylsarcosine ([¹⁴C]Gly-Sar, specific radioactivity 53 mCi/mmol) were from Amersham Biosciences UK, Ltd. (Little Chalfont, Buckinghamshire, UK). Phorbol 12-myristate 13-acetate (PMA) was supplied by Calbiochem (Germany). Cell culture reagents were obtained from Invitrogen (Germany). All other chemicals were supplied by Sigma Chemie (Deisenhofen, Germany) or Roth (Karlsruhe, Germany).

Cell Culture. Cells at passage number 31 to 59 were maintained in 75-cm² culture flasks at 37°C in a humidified atmosphere with 5% CO₂. They were cultured in minimum essential medium supplemented with nonessential amino acid solution (1%), fetal bovine serum (10%), and gentamicin (50 µg/ml) as described previously (Knütter et al., 2002). Cells grown to confluence were released by trypsinization (0.05% trypsin/EDTA in modified Pucks solution A) and subcultured in 35-mm disposable Petri dishes (BD Biosciences, Heidelberg, Germany). The medium was replaced every 2 days, the day after trypsinization, and the day before the uptake experiment. With a starting cell density of 0.8 × 10⁶ cells/dish, the cultures reached confluence within 24 h. Uptake was measured 7 days after seeding. Pretreatment of the cells with PMA and/or staurosporine was done in 1.5 ml of medium at 37°C in a humidified atmosphere with 5% CO₂ for 2 h (Brandsch et al., 1994).

Transport Studies. Uptake of [³H]-ALA, [¹⁴C]Gly-Sar, and [³H]glycine was determined at 37°C (Knütter et al., 2002). The uptake buffer was 25 mM 2-(N-morpholino)ethanesulfonic acid/Tris (hydroxymethyl) aminomethane (MES/Tris, pH 6.0) or 25 mM HEPES/Tris (pH 7.5) with 140 mM NaCl or choline chloride, 5.4 mM KCl, 1.8 mM CaCl₂, 0.8 mM MgSO₄, and 5 mM glucose. Uptake experiments were initiated by removing the culture medium from the dishes, washing the cell monolayers twice with 1 ml of buffer, and adding 1 ml of uptake buffer containing [³H]-ALA, [¹⁴C]Gly-Sar, or [³H]glycine and unlabeled compounds at increasing concentrations. After incubation for the desired time (mostly 10 min), the buffer was removed and monolayers were quickly washed with ice-cold uptake buffer four times, dissolved, and transferred into counting vials. The radioactivity associated with the cells was measured by liquid scintillation spectrometry.

Data Analysis. Each experimental point shown represents the mean ± S.E. of three to four measurements. The kinetic constants were calculated by nonlinear regression of the Michaelis-Menten plot. The calculated parameters are shown with their S.E. Inhibition constants (K_i) were calculated from IC₅₀ values (i.e., the concentration of the unlabeled compound necessary to inhibit 50% of specific [³H]-ALA- and [¹⁴C]Gly-Sar uptake) using the K_t value of 2.1 mM (obtained in this study) for -ALA and 1.1 mM for Gly-Sar (Knütter et al., 2002). Statistical analysis was done by the two-tailed nonparametric U test. A p < 0.05 was considered significant.

	Results

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Driving Force of the [³H]-ALA Uptake in SK-ChA-1 Cells. Uptake of [³H]-ALA (1 µM) into cholangiocytes was strongly stimulated by an inwardly directed pH gradient (Fig. 1). At an outside pH of 6.0, the uptake rate was increased 3- to 4-fold compared with transport at an outside pH of 7.5. The total uptake of [³H]-ALA was linear for up to 10 min at pH 6.0. 

View larger version (11K): [in this window] [in a new window]   Fig. 1.   pH and time-dependent uptake of [3H]-ALA in SK-ChA-1 cells. Uptake of [3H]-ALA (1 µM) was measured at pH 7.5 () and pH 6.0 () for up to 60 min. Values represent means ± S.E. (n = 3-4).

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Kinetic Parameters. The uptake of [³H]-ALA was found to be saturable. In the presence of an excess amount of unlabeled -ALA (32 mM), uptake was decreased by 85% of total uptake at a 2 µM tracer concentration. This value represents the linear, nonsaturable, nonmediated transport, most likely simple diffusion plus tracer binding. To determine the kinetic parameters of specific -ALA uptake, SK-ChA-1 cells were incubated for 10 min with [³H]-ALA (2 µM) and increasing concentrations of -ALA ranging from 0.3 to 32 mM. The relationship between total uptake rate and substrate concentration is shown in Fig. 2. Kinetic analysis performed by nonlinear regression of total uptake data revealed for the saturable component an apparent affinity constant (Michaelis-Menten constant, K_t) of 2.1 ± 0.3 mM and a maximal velocity of transport (V_max) of 60.1 ± 3.7 nmol · 10 min¹ · mg of protein¹. The linear, nonsaturable transport constant (K_d) was 1.9 ± 0.1 µl · 10 min¹ · mg of protein¹. Kinetically, there was no evidence for the participation of a second saturable transport component (p < 0.05).

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Substrate concentration dependence of total -ALA uptake in SK-ChA-1 cells. Uptake of [3H]-ALA (2 µM) was measured for a 10-min incubation time over a -ALA concentration range of 0.316 to 31.6 mM. Inset, kinetic parameters of the saturable transport component. Values represent means ± S.E. (n = 4).

Substrate Specificity of -ALA Uptake. The uptake of [³H]-ALA (1 µM) into SK-ChA-1 cells (pH 6.0) could be inhibited by 10 mM unlabeled -ALA, Ala-Ala, Gly-Sar, and cefadroxil (Fig. 3). All these compounds are well known substrates of H⁺/peptide symporters. In contrast, no significant inhibition was found for glycine, GABA, aspartic acid, and glutamic acid at 10 mM concentrations (p < 0.05). These compounds, although structurally related to -ALA, were not recognized by the system responsible for -ALA uptake. Figure 4 shows the results of competition assays performed to determine the apparent affinity constants of the effective inhibitors. From the displacement curves, IC₅₀ values were determined by nonlinear regression. From these, the K_i values shown in Table 1 were calculated. The dipeptides and peptidomimetics used displayed apparent affinity constants between 0.2 and 3.6 mM.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Substrate specificity of [3H]-ALA uptake in SK-ChA-1 cells. Total uptake of 1 µM [3H]-ALA was measured in monolayer cultures of SK-ChA-1 cells for 5 min at pH 6.0 in the presence of 10 mM of unlabeled compounds (n = 4).

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Substrate specificity of [3H]-ALA uptake in SK-ChA-1 cells. Uptake of 1 µM [3H]-ALA was measured in monolayer cultures of SK-ChA-1 cells at pH 6.0 in the absence and presence of increasing concentrations of unlabeled peptides and peptidomimetics (0-31.6 mM, n = 4). Uptake of [3H]-ALA measured in the absence of the inhibitors (23.8 ± 0.6 pmol · 10 min1 · mg of protein1) was taken as 100%.

View this table: [in this window] [in a new window]   TABLE 1 Inhibition constants of different compounds to compete with the uptake of radiolabeled -aminolevulinic acid and glycylsarcosine Uptake of [3H]-ALA (1 µM) and [14C]Gly-Sar (10 µM, pH 6.0) in confluent monolayer cultures of SK-ChA-1 cells was measured in the absence or presence of increasing concentrations of unlabeled -ALA, Gly-Sar, Ala-Ala, and cefadroxil. The incubation time for the uptake measurements was 10 min. IC50 values (i.e., the concentration of unlabeled compounds to inhibit 50% of the carrier-mediated uptake of radiolabeled substrates) were determined from dose-response inhibition curves shown in Fig. 4 for [3H]-ALA. From these values Ki were calculated.

Functional Demonstration of -ALA Uptake via PEPT1 in SK-ChA-1 Cells. To obtain further evidence that -ALA is transported exclusively by the intestinal type H⁺/peptide symporter PEPT1 in SK-ChA-1 cells, several types of experiments were performed. In the first series, we determined the type of inhibition of the uptake of the PEPT1 substrate Gly-Sar by -ALA and its inhibitory constant in a Dixon type of transport study. Uptake of [¹⁴C]Gly-Sar was measured at two different Gly-Sar concentrations (50 and 500 µM) in the presence of increasing amounts of -ALA (0-10 mM). The results are presented as Dixon plot (Fig. 5). They reveal linearity at both Gly-Sar concentrations with lines intersecting above the abscissa in the fourth quadrant, as expected for a competitive inhibitor. A K_i value of 1.7 mM was calculated from the point of intersection.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Determination of the inhibition constant with a Dixon plot. The uptake rate of [14C]Gly-Sar (10 µM) was measured at pH 6.0 for a 10-min incubation time at two different concentrations of unlabeled Gly-Sar. The diffusional component of 11.6%, measured in the presence of an excess amount of Gly-Sar (50 mM), was subtracted from the total rate of uptake to calculate the mediated rate of uptake (n = 3).

Involvement of Other Transport Systems for -ALA Uptake in SK-ChA-1 cells. As stated above, transport of -ALA in SK-ChA-1 cells is sodium-independent. Moreover, there was no significant interaction of glycine, GABA, glutamate, or aspartate with [³H]-ALA uptake at pH 6.0 (Fig. 3). To show unequivocally that PEPT1 is the major or only transport system available for -ALA transport in these cells, we also studied the effect of glycine, GABA, glutamate, and aspartate on [³H]-ALA uptake at pH 7.5, i.e., in the absence of a proton gradient. At this pH, uptake of -ALA is lower than at pH 6.0. However, just as at pH 6.0, glycine, glutamate, GABA, or aspartate (all 10 mM) did not affect [³H]-ALA uptake to any significant extent (Table 2). As expected, unlabeled -ALA (10 mM) inhibits [³H]-ALA uptake even in the absence of a pH gradient because under these conditions PEPT1 still transports its substrates to equilibrium. Next, uptake of [³H]glycine (70 nM) into SK-ChA-1 cells was measured for 10 min. Unlabeled glycine at a concentration of 10 mM inhibited [³H]glycine uptake by 81% (from 3.5 ± 0.1 to 0.68 ± 0.01 pmol · 10 min¹ · mg of protein¹). For -ALA, we found a weak inhibition of [³H]glycine uptake by 15% (to 2.9 ± 0.1 pmol · 10 min¹ · mg of protein¹) when used at a concentration of 10 mM.

View this table: [in this window] [in a new window]   TABLE 2 Substrate specificity of [3H]-ALA uptake in SK-ChA-1 cells in the absence of a pH gradient Uptake of [3H]-ALA (3 µM) in confluent monolayer cultures of SK-ChA-1 cells was measured for 10 min at 37°C at pH 7.5 in the presence of a sodium gradient (n = 4).

Effect of Phorbol Ester on -ALA Uptake in SK-ChA-1 Cells. For apical Gly-Sar uptake via PEPT1 in the intestinal cell line Caco-2 it has been described that pretreatment of the cells with phorbol esters inhibits transport of the dipeptide. The inhibition could be blocked by staurosporine (Brandsch et al., 1994). In the present study, we investigated whether uptake of [³H]-ALA is affected by modulators of protein kinase C (Table 3). SK-ChA-1 cells were preincubated with 1 µM PMA or 0.5 µM staurosporine or 1 µM PMA and 0.5 µM staurosporine together, respectively, for 2 h in medium. After washing the cells, [³H]-ALA transport was measured at pH 6.0 using an incubation time of 10 min. Treatment with PMA resulted in a significant inhibition of the [³H]-ALA transport to 73% (Table 3). Cotreatment with staurosporine completely blocked the PMA effect. Staurosporine alone stimulated the uptake rate significantly by 16%.

View this table: [in this window] [in a new window]   TABLE 3 Effect of the protein kinase C modulators PMA and staurosporine on the [3H]-ALA uptake in SK-ChA-1 cells SK-ChA-1 cells were preincubated with or without PMA (1 µM) and/or staurosporine (0.5 µM) at 37°C in a humidified atmosphere with 5% CO2 for 2 h in medium. Uptake of [3H]-ALA (1 µM) was measured in cells for 10 min at pH 6.0. Values represent means ± S.E. (n = 3).

	Discussion

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It has been shown unequivocally by H. Daniels' group that -ALA, although containing a ketomethylene group instead of a peptide bond, represents a substrate for the cloned H⁺/peptide symporters PEPT1 and PEPT2. Among the various substrates for the intestinal type peptide transporter PEPT1 -ALA displays an appreciable affinity of 0.5 mM when the carrier is expressed in yeast cells and 0.4 mM when it is expressed in Xenopus oocytes (Döring et al., 1998). Irie et al. (2001) later confirmed this result by showing that -ALA is transported apically via PEPT1 in intact intestinal cells (Caco-2) with an apparent affinity constant of 1.6 mM. A major consequence of these findings is that one can now explain the high serum level of -ALA after oral administration.

In mammalian tissues H⁺/peptide cotransport activity has been found in several tissues and organs such as kidney, intestine, lung, and choroid plexus. In a recent study, we extended the list by the H⁺-dependent peptide cotransport system PEPT1 in the extrahepatic biliary duct cells SK-ChA-1 (Knütter et al., 2002). We do not know the exact physiological role of PEPT1 in the biliary duct; however, reverse transcription-polymerase chain reaction analysis using RNA from several tissues demonstrated that PEPT1 is a physiologically occurring transport system in normal extrahepatic bile duct epithelium. In the context of the present study, SK-ChA-1 cells, however, are not just one more cell culture model for our studies of H⁺/peptide symport in intact epithelial cells such as Caco-2 for the intestinal PEPT1 (Brandsch et al., 1994), Madin-Darby canine kidney for the renal PEPT1 (Brandsch et al., 1995b), or SKPT for the renal PEPT2 (Brandsch et al., 1995a). We demonstrate that one could perhaps use this transport system in a highly relevant therapeutic connection, the PDT of bile duct tumors. Whether PDT of such cancers can be recommended is still under discussion (Zoepf et al., 2001b), however, demonstration of active transport of -ALA into bile duct tumor cells by a specific system should allow the optimization of treatment parameters. Hypothetically, after oral administration of high doses, -ALA might undergo biliary excretion followed by accumulation uphill from bile into cholangiocarcinoma cells. According to our data, an uphill accumulation requires a local pH gradient across the apical membrane of cholangiocytes comparable with the microclimate at the intestinal and renal epithelia. Indeed, in SK-ChA-1 cells and in intrahepatic cholangiocytes the expression and function of the Na⁺/H⁺ exchanger NHE2 has been demonstrated apically where it may serve to reabsorb Na⁺ and acidify the biliary fluid (Strazzabosco et al., 1994; Spirli et al., 1998). However, the therapeutic concentration needed has to be tested (Gibson et al., 1999).

There are several other systems involved in the transport of -ALA such as glycine, GABA, aspartic acid, and -amino acid transporters (McLoughlin and Cantrill, 1984; Langer et al., 1999; Novotny et al., 2000; Rud et al., 2000). In SK-ChA-1 cells, we found no evidence for the participation of transport systems for glycine, GABA, glutamic acid, or aspartic acid at the total -ALA transport neither at pH 6.0 nor 7.5. None of these compounds affected the transport of the labeled [³H]-ALA. We found a small inhibition of the [³H]glycine transport by -ALA, but compared with the affinity constants of glycine transporting systems, this interaction, in our opinion, is of no physiological significance. Moreover, the kinetic analysis did not suggest presence of more than one transport system. Instead, our results show that the PEPT1 substrate Gly-Sar and -ALA are transported by the same system. The transport of -ALA was proton-dependent. The inhibitory constants of Ala-Ala, Gly-Sar, cefadroxil, and -ALA versus [³H]-ALA and [¹⁴C]Gly-Sar transport correspond very well to values published for transport of the dipeptides and peptidomimetics via PEPT1 at other cell types (Ganapathy et al., 1995; Irie et al., 2001). The inhibition of Gly-Sar transport by -ALA was strictly competitive. Furthermore, for the intestinal peptide transporter it has been shown that it is under direct or indirect regulatory control of protein kinase C (Brandsch et al., 1994; Chen et al., 2002). Results presented in this study show that mediators of protein kinase C also affect the -ALA uptake in bile duct cells. The mechanism could be a direct phosphorylation/dephosphorylation of the protein or an indirect effect on the H⁺ gradient as the driving force of transport (Kennedy et al., 2002).

In conclusion, we obtained evidence that in human cholangiocarcinoma cells SK-ChA-1 -ALA is transported via the H⁺/peptide symporter PEPT1 into the cells. Because PEPT1 is an active transport system, -aminolevulinic acid might be accumulated in biliary duct epithelial cells against a concentration gradient under physiological or pharmacological conditions before PDT of bile duct cancer.