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Articles

Transport Mechanisms of the Imino Acid L-Proline in the Human Intestinal Epithelial Caco-2 Cell Line

Valérie Berger^*,12, Nancy De Bremaeker^*, Yvan Larondelle, André Trouet^** and Yves-Jacques Schneider^*

^* Laboratoire de Biochimie Cellulaire, Laboratoire de Biochimie de la Nutrition and ^** Laboratoire de Biologie Cellulaire, Université Catholique de Louvain, B-1348 Louvain-la-Neuve, Belgium

²To whom correspondence should be addressed.

	ABSTRACT

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The intestinal transport of L-proline (L-Pro) has been investigated in various animal species with the use of different tissue preparations. Because major qualitative differences have been observed among the species, it is difficult to extent the results obtained with animal models to humans. In addition, studies on human tissue are lacking because of difficulties in obtaining material for experiments. To characterize the mechanisms involved in the intestinal absorption of L-Pro in humans, the transport of this nonessential imino acid was studied in monolayers of human intestinal Caco-2 cells that were cultivated on microporous membranes. In this model, L-Pro was transported selectively in the apical (AP)-to-basolateral (BL) direction. This transport was significantly reduced by metabolic inhibitors and by an incubation at 4°C; it was Na⁺ dependent and showed competition with (methylamino)--isobutyric acid and L-hydroxyproline. By contrast, transport in the BL-to-AP direction resulted to a large extent from passive movement (paracellular passage and transcellular diffusion). L-Pro accumulation by Caco-2 cells was significantly greater from the AP pole than from the BL pole. About 30–50% of the accumulated molecules were incorporated into newly synthesized proteins in a process inhibited by cycloheximide, whereas the remainder were extensively metabolized into non–amino acid compounds. L-Pro accumulations from the AP and BL poles were both Na⁺ dependent, but they exhibited different characteristics. AP accumulation was inhibited by competition with (methylamino)--isobutyric acid, L-hydroxyproline and, to a lesser extent, D-Pro, whereas BL accumulation was inhibited by competition with L-hydroxyproline, (methylamino)--isobutyric acid, -aminoisobutyric acid, L-histidine and small neutral amino acids. The results indicate that AP-to-BL transport and AP accumulation of L-Pro exhibited very different characteristics than BL-to-AP transport and BL accumulation.

KEY WORDS: • Caco-2 cells • L-proline • intestinal transport • transepithelial transport • intracellular accumulation

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The intestinal epithelium expresses multiple transport systems that are involved in the absorption of various substances present in the gut or the blood. In particular, the absorption from the lumen of the free amino acids generated by protein digestion requires their transport across the brush border membrane of the enterocytes as well as across the basolateral (BL)³ membrane for blood delivery. To perform the vectorial transport of the amino acids from lumen to serosa, the enterocyte is equipped with transport systems at the two poles of its plasma membrane that exhibit different functional characteristics (Ganapathy et al. 1994 , Maillard et al. 1995 ). The amino acid transport systems in the enterocyte membranes are either Na⁺ independent (facilitated transport) or Na⁺ dependent (secondary active transport).

The intestinal transport of imino acids has been investigated in various animal species with the use of different tissue preparations. L-Proline (L-Pro) has been reported to be transported across the brush border membrane of the rat and rabbit small intestine through two Na⁺-dependent carrier systems: the B system, which is selective for neutral amino acids, and the IMINO system (Munck et al. 1994 , Stevens et al. 1982 ). Reports with rabbit jejunal brush border membrane vesicles indicate that the IMINO carrier of the rabbit small intestine is a relatively selective carrier for imino acids: it is strongly inhibited by imino acids, (methylamino)--isobutyric acid (MeAIB) and, to a lesser extent, L-phenylalanine, but it is insensitive to glycine, L-alanine, ß-alanine and most other amino acids (Stevens et al. 1985 and 1987 ). An equivalent Na⁺-dependent transport system with a high affinity for imino acids and a low affinity for neutral amino acids has been described in intact epithelium from the distal ileum of the rabbit (Munck 1985 ). However, in the intact preparation, imino acids were also transported by an Na⁺-dependent system that accepts neutral, cationic and non–-amino acids and that was not detected in brush border membrane vesicles. The imino acid carrier of the guinea pig small intestine resembles the IMINO carrier of the rabbit small intestine (Hayashi et al. 1980 , Satoh et al. 1989 ). In contrast, the imino acid carrier of the rat small intestine differs markedly from those of guinea pig and rabbit small intestine (Munck et al. 1994 ). It transports non–-amino acids such as ß-alanine and -aminobutyric acid as efficiently as imino acids; second, it does not prefer the L over the D configuration, is sensitive to the small neutral amino acid, glycine and to -aminoisobutyric acid (AIB) and is insensitive to inhibition by neutral amino acids with side chains that contain more than one methyl group. Moreover, although in the rabbit and guinea pig small intestine the transport of imino acids is both Cl^- and Na⁺ dependent, in the rat small intestine transport is Cl^- independent (Munck et al. 1994 ). The study of Malo et al. (1991) demonstrated the coexistence of both Na⁺-dependent and Na⁺-independent carrier systems for L-Pro uptake into brush border membrane vesicles from human fetal small intestine. The Na⁺-dependent uptake was attributed to the IMINO system, but this was not confirmed by inhibition studies.

Major qualitative differences are observed with respect to the transport of L-Pro in the various animal species. This interspecies variation in the intestinal transport of L-Pro prevents the extrapolation of animal data to humans. In addition, studies on human adult tissue are lacking because of difficulties in obtaining material for experiments. The use of a human cell culture system, as an in vitro model of the intestinal barrier, would therefore be a useful approach to characterize the mechanisms and the factors that regulate the transport of imino acids across the human intestinal mucosa. One such system based on the culture of the Caco-2 cells on microporous membranes is increasingly used to investigate the physiology of the enterocytes as well as the mechanisms involved in the transport of nutrients and pharmacological agents. This cell line, although derived from a colon adenocarcinoma, has been shown to express, under routine culture conditions, numerous morphological features of the small intestine absorptive cells, including a brush border at the apical (AP) surface, AP tight junctional complexes, a tall columnar shape and a polarized distribution of membrane components, including enzymes, receptors, transport systems and ion channels (Hidalgo et al. 1989 ). Caco-2 cells have been reported to express several transport systems that are present in the intestinal epithelium. These include transport systems for nutrients such as glucose, amino acids and dipeptides as well as for vitamins such as cobalamin and biotin, nucleosides, bile acids and monocarboxylic acids (for a review, see Hidalgo and Li 1996 ).

The uptake of L-Pro in Caco-2 cells was previously studied by Nicklin et al. (1992). They showed that L-Pro uptake was saturable, energy dependent and Na⁺ dependent. According to the results of competition experiments with amino acids and synthetic analogs, the authors concluded that the carrier system responsible for L-Pro uptake had many features in common with the ubiquitous system A. These results were, however, obtained with monolayers cultured on six-well plates and, most likely, formed of undifferentiated or only partially differentiated Caco-2 cells.

In the present study, the transport of L-Pro was investigated in monolayers of differentiated Caco-2 cells grown on microporous membrane supports. Two amino acid carrier systems that serve L-Pro have been identified and characterized: the Na⁺-dependent IMINO system at the AP membrane and the Na⁺-dependent A system at the BL membrane.

	MATERIALS AND METHODS

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Chemicals.

Iscove’s modified Dulbecco’s medium, Ham’s F12 medium, NCTC 135 medium, Hanks’ balanced salt solution (HBSS, powder form), HEPES, L-glutamine, a mixture of trace elements and minimal essential medium nonessential amino acids were purchased from GIBCO BRL (Life Technologies Ltd., Paisley, UK). L-Phe, L-Leu, L-Pro, D-Pro, L-hydroxyproline, L-His, glycine, L-Ala, ß-Ala, AIB, MeAIB, 2-deoxyglucose (2-DOG), choline chloride, deoxycholic acid sodium salt, cycloheximide, 5-(N,N-hexamethylene)-amiloride, albumin complexed to linoleic acid, ethanolamine, insulin, triiodothyronine, hydrocortisone and o-phthaldialdehyde were purchased from Sigma Chemical Co. (St. Louis, MO). Trichloroacetic acid (TCA), sodium azide, NaHCO₃, NaOH, Tris, 5-sulfosalicylic acid and LiOH were purchased from Merck (Darmstadt, Germany). 2-[N-Morpholino]ethanesulfonic acid was purchased from Sigma-Aldrich (Steinheim, Germany). Fetal bovine serum was purchased from BioWhittaker Europe (Verviers, Belgium). Epidermal growth factor was purchased from Boehringer-Mannheim (Mannheim, Germany). Bovine dermal type I collagen was purchased from Cellon (Strassen, Luxembourg). L-[2,3-³H]Proline (L-[³H]Pro, specific activity 1.63 TBq/mmol) and D-[1-¹⁴C]mannitol (D-[¹⁴C]mannitol, specific activity 2.15 GBq/mmol) were purchased from Amersham Life Sciences (Little Chalfont, U.K.).

Cell culture.

The intestinal Caco-2 cell line was routinely grown, as previously described (Halleux and Schneider 1991 , Sergent-Engelen et al. 1993 ), in plastic tissue culture flasks (175 cm²; Greiner Labortechnik, Frickenhaussen, Germany) using the basal defined medium (Schneider 1989 ). Basal defined medium mainly consists in a 5:5:1 (v/v/v) mixture of Iscove’s modified Dulbecco’s, Ham’s F12 and NCTC 135 media.

For transport experiments, efflux experiments and biotransformation studies, the cells were seeded onto a poly(ethylene terephthalate) microporous membrane (1-µm pore diameter, 11-µm thickness, 1.6 x 10⁶ pores/cm²; Whatman SA, Louvain-la-Neuve, Belgium) as previously described (Halleux and Schneider 1991 , Sergent-Engelen et al. 1993 ). All cells used in this study were between passages 194 and 239. Experiments were always performed 24 h after the last feeding because the time after feeding is known to affect the transport of amino acids. The integrity of the monolayers was checked by measurement of the transepithelial flux of D-[¹⁴C]mannitol and the transepithelial electrical resistance (TEER) (Millicell-ERS Resistance System; Millipore, Bedford, MA and Endohm 24; World Precision Instruments, Sarasota, FL). All of the monolayers used in this study exhibited TEER values of 400–600 · cm² and D-[¹⁴C]mannitol clearances of <30 µL · insert^-1 · h^-1.

Transport studies.

The culture medium was replaced by HBSS containing 5 mmol/L glucose and supplemented with 10 mmol/L HEPES and 5 mmol/L NaHCO₃ (transport medium). The pH value of the transport medium was adjusted to pH 7.4 with NaOH or Tris for sodium-free medium. Before transport experiments, the cells were depleted of amino acids by incubation for 30 min at 37°C in the transport medium, after which the donor and acceptor solutions were replaced with the appropriate solutions prewarmed at 37°C.

Transepithelial passage was quantified by the addition of D-[¹⁴C]mannitol and L-[³H]Pro in either the upper or lower compartment of the bicameral culture insert and analysis of the solution of the other compartment. The upper and lower compartments contained 1.8 and 2.8 mL transport medium, respectively. The donor solution contained the radioactive compounds (8.4–16.7 MBq/L L-[³H]Pro and 16.7 MBq/L D-[¹⁴C]mannitol) dissolved in transport medium in the presence of a varying amount of nonlabeled L-Pro to give the required final concentration. At each sampling time (60, 120 and 180 min), 100 µL transport medium was withdrawn from the acceptor compartment and replaced with fresh transport medium. The rates of transport were calculated from the slope of L-Pro or D-mannitol appearance curves.

To determine sodium dependency of transport, sodium chloride in the transport medium was replaced with an equimolar amount of choline chloride and sodium salts were replaced with their potassium equivalents. Alternatively, amiloride (100 µmol/L) was added to the sodium-containing transport medium in both compartments to block the Na⁺/H⁺ exchanger. To ensure that choline did not induce muscarinic effects, atropine (10 µmol/L) was added to the Na⁺-free medium. For energy depletion, transport medium was depleted in glucose and supplemented with 1 mmol/L NaN₃ and 50 mmol/L 2-DOG in both compartments. For protein synthesis inhibition, 50 mg/L cycloheximide was added to the transport medium in both compartments. For sodium replacement, energy depletion and protein synthesis inhibition, the cells were preincubated in the appropriate transport medium for 30 min. For the experiments at 4°C, cells were preincubated for 30 min at 4°C before the addition of the labeled probes. The effect of acidification of the donor solution on L-Pro transport was studied by adding HBSS at pH 6.0 (buffered with 10 mmol/L 2-[N-morpholino]ethanesulfonic acid) in the donor compartment and HBSS at pH 7.4 in the acceptor compartment. The absence of toxicity of these experimental conditions was checked by optical phase contrast microscopy examination and assayed by measurement of the TEER at the end of the transport experiment. For the competition experiments, an excess of unlabeled amino acid or synthetic analog (1 mmol/L) was added to the donor compartment together with the radioactive L-Pro.

Intracellular accumulation.

At the end of the transport experiment (180 min), the cell monolayers were washed six times with phosphate-buffered saline (PBS) and then resuspended in 1 mL of 10 g/L deoxycholic acid sodium salt adjusted to pH 11.3 with NaOH. After 10 min at room temperature, the cells were removed by scraping and disrupted by ultrasonication. The radioactivity of 100 µL of the cell homogenate was measured to determine the amount of L-Pro taken up. The amount of cell protein was measured by the method of Lowry (Lowry et al. 1951 ) using bovine serum albumin as a standard. Then, 100 µL of the cell homogenate was also precipitated with 100 µL of a 250 g/L TCA solution in the presence of 10 µL fetal bovine serum as a protein carrier for 30 min at 4°C. At the end of the incubation, the TCA-insoluble pellet, representing amino acid incorporation into newly synthesized proteins, was separated from the soluble fraction through centrifugation at 5200 x g for 10 min, and the soluble fraction was assessed for radioactivity.

Efflux experiments.

The cells monolayers were loaded with 10 µmol/L L-[³H]Pro for 15 min at 37°C and then washed three times with ice-cold PBS. The intracellular accumulation of L-[³H]Pro was determined on three samples as described earlier. For the other inserts, the loaded L-[³H]Pro was then allowed to efflux into the media bathing the AP and BL sides of the monolayers. After various durations, ranging from 10 to 90 min, the amounts of L-[³H]Pro released were determined by measuring the radioactivity of 100 µL samples withdrawn from the AP and BL media.

Radioactive measurements.

For all of the experiments, the amount of radioactive material was analyzed by liquid scintillation spectrometry with a Packard Tri-Carb 1600 TR instrument (Packard Instrument Company, Meriden, CT) after dispersion in 2 mL Aqualuma cocktail (Lumac; LSC, Groningen, the Netherlands).

Biotransformation.

The extent of L-Pro biotransformation by the Caco-2 cells was checked at the end of the transport experiment (180 min). For this purpose, the identity of the ³H label (either in the donor compartment or collected from the acceptor compartment, as well as that of the non-TCA precipitable cell fraction) was determined at the end of the incubation period by thin layer chromatography (TLC) on silica plates (0.25 mm thick on glass supports, 20 x 20 cm; Merck, Darmstadt, Germany) using n-butanol/acetic acid/water (4:1:1) as the mobile phase. A solution of L-Pro (2 g/L) dissolved in HBSS was used as a reference to determine the position of the imino acid on the silica plate. At the end of the migration, the plates were stained with ninhydrin reagent (0.2% in ethanol; Sigma-Aldrich NV/SA, Bornem, Belgium), and each lane was divided vertically into 1-cm blocks. Each block of silica was scraped and poured into a scintillation vial together with 2 mL Aqualuma cocktail and underwent liquid scintillation counting as described.

The level of L-Pro biotransformation into other amino acids by the Caco-2 cells was checked through HPLC analysis of cellular extracts and culture media. Increases in various amino acids were examined by comparison with a reference sample. After a preincubation of 30 min at 37°C in HBSS, the cells were incubated for an additional 3 h at 37°C with 100 µmol/L L-Pro in HBSS or with HBSS alone (reference sample) in the donor compartment and with HBSS in the acceptor compartment. Samples of cellular extracts and culture media were deproteinized and underwent HPLC analysis as described by Fekkes et al. (1995) with minor modifications. The HPLC system consisted of a P4000 pump and a Thermo Separation Products degassing device (Thermo Separation Products, San Jose, CA), a RF-551 fluorescence detector (Shimadzu, Kyoto, Japan) and a Carlo Erba Instruments integrator (Milano, Italy). A 5-µm Spherisorb ODS 2 column (125 x 3 mm I.D.) (Waters, Milford, MA) was used.

Data analysis.

Results are expressed as means ± SD. Statistical analysis was performed using one-way ANOVA with a Dunnett post hoc test for comparisons between test assays and controls or a Tukey post hoc test for multiple comparisons. The computer program was Systat 5.2.1 (Systat, Evanston, IL).

	RESULTS

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Passage of D-mannitol and L-Pro.

The passages of D-[¹⁴C]mannitol and L-[³H]Pro were studied on Caco-2 cells cultivated for 17–19 d after seeding over the time range of 0–180 min with a 60-min sampling interval (Fig. 1 ). To facilitate direct comparison, the results are presented as clearance, which is, by definition, the volume of plasma containing the amount of substance that is removed by the kidney per unit of time. By analogy, in this study, we defined clearance as the equivalent volume of solution (µL) containing the amount of labeled material that passed across the cell monolayer. The results indicate that the passages of D-[¹⁴C]mannitol and L-[³H]Pro were proportional to the duration of the experiment. The clearance rate of D-[¹⁴C]mannitol was similar in the AP-to-BL and BL-to-AP directions, reaching values of 16 and 11 µL · insert^-1 · h^-1, respectively. The passage of L-Pro was systematically higher than that of D-[¹⁴C]mannitol. In addition, the transport of L-Pro in the AP-to-BL direction was significantly higher than that in the BL-to-AP direction, suggesting that the distribution of the transport carriers is polarized.

View larger version (25K): [in this window] [in a new window]   Figure 1. Kinetics of the apical (AP)-to-basolateral (BL) and BL-to-AP passages of 7 µmol/L D-mannitol and 10 µmol/L L-Pro across Caco-2 cell monolayers. After the application of D-[14C]mannitol and L-[3H]Pro to one side, monolayers were incubated at 37°C and samples were withdrawn from the other side, at different durations. Values are means ± SD (n = 20 or 21). +P < 0.001 and *P < 0.05, significantly different.

After a 3-h incubation with 10 µmol/L L-[³H]Pro, the Caco-2 cells accumulated ³H-labeled material corresponding to 1 100 ± 114 pmol/mg cell protein from the AP pole and to 467 ± 169 pmol/mg cell protein from the BL pole. The proportion of ³H-labeled material precipitable by TCA was 50 ± 14 and 36 ± 15% from the AP and BL poles, respectively. The intracellular concentration of ³H label recovered as radioactive TCA-soluble material, calculated on the basis of a cellular volume for Caco-2 cells of 3.66 µL/mg cell protein (Brunham et al. 1989 ), was 148 ± 48 µmol/L from the AP side and 85 ± 47 µmol/L from the BL side. These results suggest that Caco-2 cells concentrated the ³H-labeled material from both the AP and BL sides of the monolayers and accumulated it preferentially from the AP pole. In both cases, 30–50% of the accumulated ³H-labeled material was incorporated into newly synthesized proteins.

Biotransformation of L-Pro.

After 3 h of incubation of Caco-2 cell monolayers with 10 µmol/L L-[³H]Pro, the identity of the ³H label in the AP and BL bathing solutions as well as in the TCA-soluble cell-associated fraction was determined with TLC. Regardless of the compartment in which L-[³H]Pro was added, the ³H signal in the solution bathing the BL side was typical of L-[³H]Pro and no other major radioactive products were detected. In contrast, several other radioactive products were detected in the solutions bathing the AP side: they amounted to 73 and 57% of the ³H-labeled material recovered when L-[³H]Pro was added at the AP and BL sides, respectively. These various radioactive products were also detected in the TCA-soluble cell-associated fraction: they represented 66 and 61% of the TCA-soluble ³H-labeled material accumulated from the AP and BL sides, respectively. The results suggest that L-Pro was extensively metabolized by the Caco-2 cells and that the products of metabolism were selectively excreted at the AP side of the cells.

HPLC analysis gave no indication that the final products of the metabolism of L-Pro by the Caco-2 cells would consist of some of the amino acids commonly found in proteins.

Because of this extensive metabolism, the radioactive material detected in the solution bathing the AP side as well as accumulated by the cells does not correspond exclusively to L-[³H]Pro and is expressed as L-Pro equivalents in the next sections.

Concentration dependence of transport and accumulation.

The transport of L-Pro was investigated at different concentrations ranging from 100 µmol/L to 50 mmol/L in the AP-to-BL and BL-to-AP directions. The transport rate of L-Pro in the AP-to-BL direction increased at first and then tapered off at concentrations of >5 mmol/L (Fig. 2 ). This curve likely represents the contribution of a saturable process as well as a nonsaturable one to total L-Pro transport. In contrast, transport in the BL-to-AP direction was strictly proportional to the concentration over the entire range.

View larger version (16K): [in this window] [in a new window]   Figure 2. Effect of concentration on the apical (AP)-to-basolateral (BL) and BL-to-AP transport rates of L-Pro across Caco-2 cell monolayers. The monolayers were incubated at 37°C for 3 h, and the rates were calculated from the slope of the appearance curves with time points every hour. Values are means ± SD (n = 3).

View larger version (22K): [in this window] [in a new window]   Figure 3. Effect of concentration on the intracellular accumulation of L-Pro equivalents from apical (A) and basolateral (B) sides of Caco-2 cell monolayers in the range of 0.1–50 mmol/L. Monolayers were incubated for 3 h at 37°C, washed with phosphate-buffered saline and collected for determination of the intracellular accumulation levels. L-[3H]Pro in the trichloroacetic acid (TCA)-insoluble portion was determined. Values are means ± SD (n = 3).

Supplementation of the transport medium with 1 mmol/L NaN₃ and 50 mmol/L 2-DOG significantly decreased the AP-to-BL transport of L-Pro, whereas it increased the BL-to-AP transport (Table 1 ). The results also show that treatment of the monolayers with these metabolic inhibitors increased the passage of D-mannitol through tight junctions.

The transport of amino acids has been described to be coupled with Na⁺ (Christensen 1990 , Maillard et al. 1995 ). Therefore, we studied the transport of L-Pro in the absence of Na⁺. The substitution of sodium chloride in the transport medium with choline chloride decreased the AP-to-BL transport of L-Pro: the transport in the absence of Na⁺ was decreased to 25% of control (Table 1) . In contrast, the substitution of Na⁺ with choline slightly increased the BL-to-AP transport. The addition of atropine (10 µmol/L) in the Na⁺-free medium, to abolish the possible muscarinic effects of choline, did not significantly affect the transport of L-Pro in either direction (data not shown). A similar inhibition of the AP-to-BL transport (to 29% of control) was observed on the addition of 100 µmol/L amiloride to the Na⁺-containing transport medium.

Treatment of the monolayers with cycloheximide did not significantly affect the transepithelial transport of L-Pro (Table 1) .

Acidification of the donor compartment to pH 6.0 (with pH of the acceptor compartment being maintained at pH 7.4) did not affect the transport of L-[³H]Pro in the AP-to-BL direction or of L-[³H]Pro equivalents in the BL-to-AP direction (data not shown).

To explore the substrate specificity of the L-Pro carrier systems, we compared the transport of 10 µmol/L L-[³H]Pro in the absence or presence of a 1 mmol/L concentration of various unlabeled amino acids or synthetic analogs in the donor compartment (Fig. 4 ). The most potent inhibitors of the AP-to-BL transport were MeAIB and L-hydroxyproline. The stereoisomer D-Pro showed lower inhibition. The small neutral amino acids (L-Ala and glycine), the non–-amino acid ß-Ala, the cationic amino acid L-His, the large neutral amino acids (L-Phe and L-Leu) and AIB did not interfere with the AP-to-BL transport of L-Pro. BL-to-AP transport was unaffected by any of the tested compounds, indicating that AP-to-BL and BL-to-AP transports were mediated by different mechanisms.

View larger version (31K): [in this window] [in a new window]   Figure 4. Effect of various amino acids and synthetic analogues on apical (AP)-to-basolateral (BL) and BL-to-AP transport of L-Pro across Caco-2 cell monolayers. L-[3H]Pro (10 µmol/L) was applied to the AP or BL side of the monolayers alone (control) or in the presence of a 1 mmol/L concentration of another amino acid or synthetic analog. Transport was measured for 3 h, and the rates were calculated from the slope of the appearance curves with time points every hour. The control values for AP-to-BL and BL-to-AP transport were 26.2 ± 3.0 and 3.4 ± 0.8 pmol · mg cell protein-1 · min-1, respectively. A control value was determined for each set of cells, and treatment values were normalized against this control value and expressed in percent. Values are means ± SD (n = 3–6). +P < 0.001 and *P < 0.01, compared with the control condition.

The accumulation of L-Pro equivalents from both the AP and BL poles of the Caco-2 cells was significantly reduced by metabolic inhibitors, by an incubation at 4°C and, to a lesser extent, by cycloheximide (Table 2 ). Under these conditions, the incorporation of L-[³H]Pro into TCA-precipitable proteins became very low.

The amount of intracellular ³H-labeled material in the TCA-soluble fraction was drastically reduced by the metabolic inhibitors, by an incubation at 4°C and in the absence of Na⁺ (not shown).

Acidification of the transport medium in the donor compartment to pH 6.0 significantly decreased the accumulation from the BL side of the Caco-2 cells but not from the AP side (Table 2) .

Substrate selectivity of the systems responsible for L-Pro accumulation was determined by coincubation of 10 µmol/L L-[³H]Pro with a 100-fold excess of various unlabeled amino acids or synthetic analogs. The results (Fig. 5 ) indicate that MeAIB and L-hydroxyproline inhibited accumulation from both AP and BL sides of the cells; D-Pro inhibited accumulation from the AP side but not from the BL side; glycine, L-Ala, L-His and AIB inhibited accumulation from the BL side but not from the AP side and ß-Ala, L-Leu and L-Phe had no effect on accumulation from either side of the cells.

View larger version (31K): [in this window] [in a new window]   Figure 5. Effect of various amino acids and synthetic analogs on intracellular accumulation of L-Pro equivalents from apical (AP) and basolateral (BL) sides of Caco-2 cell monolayers. After 3 h of incubation with transport medium containing 10 µmol/L L-[3H]Pro alone (control) or in the presence of a 1 mmol/L concentration of another amino acid or synthetic analog, the intracellular accumulation levels were recorded. The control values for AP and BL accumulation were 1036 ± 147 and 465 ± 151 pmol of L-Pro equivalents/mg cell protein, respectively. A control value was determined for each set of cells, and treatment values were normalized against this control value and expressed in percent. Values are means ± SD (n = 3–6). +P < 0.001, *P < 0.01 and *P < 0.05, compared with the control condition.

Uptake and efflux of L-Pro.

Monolayers were preloaded from either the AP side (for BL efflux studies) or the BL side (for AP efflux studies) of the cells with 10 µmol/L L-[³H]Pro for 15 min. The accumulation level of L-Pro equivalents achieved under these conditions is given in Figure 6 (inset). After washing the cell monolayers with ice-cold PBS, the efflux of the preloaded L-Pro equivalents from the BL or AP side of the monolayers was monitored as a function of time at 37°C (Fig. 6) . The amount effluxed at the AP side did not significantly increase after 20 min, whereas the amount effluxed at the BL side continued to increase until the end of the experiment (90 min). Most of the preloaded ³H-labeled material from the AP side of the cells (60%) was released at their BL side. This radioactivity corresponded exclusively to L-[³H]Pro, because no metabolites were detected by TLC in the medium bathing the BL pole of the Caco-2 cells (see earlier). In addition, 62 ± 4% of the cell-associated radioactivity at the end of the efflux experiment was precipitable by TCA, indicating that the ³H label not effluxed principally corresponded to L-[³H]Pro incorporated into proteins. Some of the preloaded ³H-labeled material from the BL side (35%) was released at the AP side, and 33 ± 10% of the cell-associated radioactivity at the end of the efflux experiment was precipitable by TCA. These results indicate that the accumulated ³H-labeled material was rather selectively effluxed at the BL side of the Caco-2 cells, in the form of intact L-[³H]Pro.

View larger version (28K): [in this window] [in a new window]   Figure 6. Time course of L-Pro equivalent efflux from apical (AP) and basolateral (BL) sides of Caco-2 cell monolayers. Caco-2 cells were preloaded from the AP or BL side with 10 µmol/L L-[3H]Pro for 15 min at 37°C. The intracellular amount of L-Pro equivalents achieved in these conditions is given (inset). After the monolayers were washed with ice-cold PBS, efflux experiments were conducted at 37°C. The results are expressed in percent of the amount present in the cells immediately after loading. Values are means ± SD (n = 3).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

TLC analysis showed that L-Pro was extensively metabolized after uptake by the Caco-2 cells with selective excretion of the metabolites at the AP side of the cells. HPLC analysis indicated that the metabolites did not consist of some of the other amino acids commonly found in proteins. In contrast, ³H-labeled material recovered in the medium bathing the BL pole of the cells was associated with molecules of intact L-[³H]Pro. In pig enterocytes, L-Pro has been shown to be degraded mainly into ornithine, citrulline and arginine: 80–90% of utilized L-Pro carbons was recovered in ornithine plus citrulline plus arginine (Wu 1997 ). In addition, Wu (1997) showed that the mitochondrial enzyme proline oxidase was present in the pig small intestine. It was therefore proposed that proline oxidase catalyzes the oxidation of L-Pro into pyrroline-5-carboxylate (Dillon et al. 1999 , Wu 1997 ). Ornithine aminotransferase then catalyzes the transamination of pyrroline-5-carboxylate with glutamate to form ornithine, which is subsequently converted to citrulline by ornithine carbamoyltransferase. Citrulline is then converted to arginine through the successive action of arginosuccinate synthase and arginosuccinate lyase. On the basis of these findings from pigs, a widely used animal model to study human intestinal physiology, it can be expected that L-Pro would also be converted to ornithine, citrulline and arginine in the human intestine. However, this could not be confirmed in our model because the HPLC method used in this study did not detect ornithine and citrulline.

The transport of L-Pro in the AP-to-BL direction was much faster than that in the opposite direction, indicating the presence in the Caco-2 cells of one or more specific polarized systems for the transepithelial transport of L-Pro. In addition, the passage of L-Pro in the AP-to-BL direction was much faster than that of D-mannitol, suggesting that the transcellular pathway of L-Pro in the AP-to-BL direction outweighed the paracellular pathway.

The AP-to-BL transport of L-Pro was demonstrated to be a carrier-mediated process by the following observations: 1) it exhibited strong temperature dependence, 2) it was significantly reduced by energy depletion, 3) it was saturable and 4) it was Na⁺ dependent. The presence of metabolic inhibitors reduced the AP-to-BL transport to 57% of control, although the real energy dependence may be higher. Indeed, the increase in the D-[¹⁴C]mannitol passage through treatment of the monolayers with metabolic inhibitors could result from tight junction alteration, because the gate function of tight junctions is itself ATP dependent and probably was affected by this strong energy depletion (Mandel et al. 1993 ). An alternative explanation could be that the 2-DOG at the high concentration used (50 mmol/L) could have an osmotic effect on the cells, resulting in a stimulation of the paracellular passage of L-Pro. This hypothesis was tested by measuring the transepithelial passage of D-[¹⁴C]mannitol in the presence of 50 mmol/L D-mannitol. The results (not shown) did not indicate any differences from the control condition. Kinetics of the AP-to-BL transport suggest the contribution of a saturable component and a nonsaturable one to total L-Pro transport. The nonsaturable component likely corresponds to the paracellular pathway and the transcellular diffusion of L-Pro across Caco-2 cell monolayers.

In contrast, BL-to-AP transport of L-Pro was not inhibited by energy depletion or by removal of sodium from the transport medium and was unsaturable. This suggests that transport in the BL-to-AP direction mainly results from passive movement by transcellular passive diffusion and/or through the paracellular pathway. The reduction in the BL-to-AP transport (to 51%) observed when the temperature of incubation was lowered from 37°C to 4°C may in part result from a decrease in the cellular membrane fluidity at 4°C.

The following arguments indicate that uptake of L-Pro from both the AP and BL sides of the cells occurred via a carrier-mediated pathway. 1) Accumulation from both sides was significantly reduced in the presence of metabolic inhibitors or after a decrease in the incubation temperature to 4°C. These reductions seem to be the result of a simultaneous inhibitory action on both protein synthesis and carrier-mediated transport because the incorporation of L-[³H]Pro into newly synthesized proteins and the amount of ³H-labeled material recovered in the TCA-soluble fraction were significantly decreased under these experimental conditions. 2) AP and BL accumulations were both shown to be markedly Na⁺ dependent. Kinetics of AP accumulation suggest the contribution of a saturable and a nonsaturable, probably diffusional, component to total transport, whereas BL accumulation was not saturable.

L-Pro is largely cationic at pH values below its isoelectric point, pI = 6.3 (Boyer 1993 ). Therefore, the decrease in the amount of L-Pro equivalents accumulated from the BL side of the cells at pH 6.0 could result from a decrease in the affinity of L-Pro for the carriers.

To determine the type of amino acid carriers involved in the transepithelial transport of L-Pro, the cross-inhibition profiles of L-Pro transport and accumulation were compared with those of the Na⁺-dependent neutral amino acids carriers described by Christensen (1990) . The strong inhibition of the AP-to-BL transport and of the AP and BL accumulations by MeAIB indicates that system ASC is not responsible for L-Pro transport and accumulation in the Caco-2 cells. Similarly, AP-to-BL transport and AP accumulation are not mediated by system A, because they were not inhibited by AIB and small aliphatic neutral amino acids. In contrast, the cross-inhibition profile of BL accumulation resembles system A: BL accumulation was strongly inhibited by AIB, MeAIB and small aliphatic neutral amino acids (L-Ala, glycine) but not by branched (L-Leu) or aromatic neutral amino acids (L-Phe). Moreover, as observed here, the activity of system A has been shown to be highly sensitive to the extracellular pH and to decrease markedly with a decrease in pH (McGivan et al. 1994 ). AP-to-BL transport and AP accumulation seem to be mediated by a system similar to the rabbit IMINO system: they were Na⁺-dependent and strongly inhibited by MeAIB and L-hydroxyproline but were insensitive to ß-Ala, L-Ala and glycine.

In a previous report on L-Pro transport in Caco-2 cells (Nicklin et al. 1992 ), the uptake of L-Pro has been shown to be saturable, energy dependent and Na⁺ dependent. In competition experiments, strong inhibition of L-Pro uptake was found with MeAIB, AIB, L-Ala and L-Ser, whereas moderate inhibition was observed with glycine, D-Pro and -aminoisobutyric acid. Aromatic and branched amino acids showed no interaction with the carrier system. The authors concluded that the carrier system responsible for L-Pro uptake had many features in common with the ubiquitous system A. These results were, however, obtained with monolayers cultured on six-well plates and most likely formed at least in part of undifferentiated Caco-2 cells. Mordrelle et al. (1996) examined the transport of L-Pro in the rat epithelial cell line IEC-17, which presents the characteristics of the undifferentiated crypt cells. Their results suggested that the transport of L-Pro was also mediated through system A. In contrast, systems B and IMINO, which mediate the transport of L-Pro in the rat small intestine, were absent or expressed at a very low level in the IEC-17 cell line.

Characterization of the transport of the nonessential imino acid L-Pro in the Caco-2 cell line was the aim of this study. L-Pro was transported selectively in the AP-to-BL direction by a system that has many features in common with the IMINO system of the rabbit small intestine. By contrast, transport in the BL-to-AP direction results mainly from passage through nonspecific pathways (paracellular route and/or transcellular passive diffusion). However, the transport of L-Pro across the BL membrane was shown to be, at least in part, mediated by system A, which has also been shown to transport L-Pro in nonepithelial cells (Christensen 1990 ) as well as in undifferentiated intestinal cells (Mordrelle et al. 1996 ). This suggests that L-Pro that enters the intestinal cells from the BL side via a carrier-mediated process is not delivered at their AP side but is for the large part used for their own maintenance and metabolism.

Finally, the results of this report confirm the usefulness of the Caco-2 cell culture system to study intestinal absorption and transport regulation of the amino acids.

	ACKNOWLEDGMENTS

 
The authors would like to thank Geneviève Schmitz-Drévillon for her technical support and Eric Mignolet for the amino acid HPLC analysis.

	FOOTNOTES

 
¹ Supported by the National Foundation for Scientific Research of Belgium (FNRS).

³ Abbreviations used: AIB, -aminoisobutyric acid; AP, apical; BL, basolateral; 2-DOG, 2-deoxyglucose; HBSS, Hanks’ balanced salt solution; MeAIB, (methylamino)--isobutyric acid; PBS, phosphate-buffered saline; TCA, trichloroacetic acid; TEER, transepithelial electrical resistance; TLC, thin layer chromatography.

Manuscript received February 8, 2000. Initial review completed March 22, 2000. Revision accepted July 25, 2000.

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TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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