Expression and localization of organic cation/carnitine transporter OCTN2 in Caco-2 cells

Ihsan Elimrani1, Karim Lahjouji1, Ernest Seidman2, Marie-Josée Roy2, Grant A. Mitchell1, and Ijaz Qureshi1

Divisions of 1 Medical Genetics and 2 Gastroenterology, Research Center, Hôpital Sainte-Justine, Department of Pediatrics, Université de Montréal, Montreal, Quebec, Canada H3T 1C5


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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L-Carnitine is derived both from dietary sources and biosynthesis. Dietary carnitine is absorbed in the small intestine and then distributed to other organs. Previous studies using Caco-2 cells demonstrated that the transport of L-carnitine in the intestine involves a carrier-mediated system. The purpose of this study was to determine whether the uptake of L-carnitine in Caco-2 cells is mediated by the recently identified organic cation/carnitine transporter (OCTN2). Kinetics of L-[3H]carnitine uptake were investigated with or without specific inhibitors. L-Carnitine uptake in mature cells was sodium dependent and linear with time. Km and Vmax values for saturable uptake were 14.07 ± 1.70 µM and 26.3 ± 0.80 pmol · mg protein-1 · 6 min-1, respectively. L-carnitine uptake was inhibited (P < 0.05-0.01) by valproate and other organic cations. Anti-OCTN2 antibodies recognized a protein in the brush-border membrane (BBM) of Caco-2 cells with an apparent molecular mass of 60 kDa. The OCTN2 expression was confirmed by double immunostaining. Our results demonstrate that L-carnitine uptake in differentiated Caco-2 cells is primarily mediated by OCTN2, located on the BBM.

intestine; brush-border membrane; organic cation transport; anti-OCTN2 antibodies; valproate.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

beta -HYDROXY-gamma -TRIMETHYLAMINOBUTYRATE (L-carnitine) plays an important role in lipid metabolism by facilitating the transport of long-chain fatty acids across the mitochondrial inner membrane followed by fatty acid beta -oxidation (3, 35). Carnitine, supplied principally in the diet, is absorbed in the intestine and transported to many other tissues by putative transporters. Recently, a new family of organic cation transporters, designated organic cation/carnitine transporter (OCTN) has been described (43). Some novel members of this family, namely OCTN1, -2, and -3, are also capable of transporting carnitine, each with distinct characteristics (18, 41, 43). OCTN2, the most important carnitine transporter, was recently cloned (42, 55, 56). Mutations in this protein cause the autosomal recessive inborn error, systemic carnitine deficiency (SCD) (4, 17, 30, 50). OCTN2 has 12 putative transmembrane domains, is expressed in several tissues, such as kidney, and transports carnitine in an Na+-dependent manner, with high-affinity and pH-dependence (19, 32, 42, 55, 56). OCTN2 also plays an important pharmacological role, because it mediates the transport of many drugs, including tetraethylammonium (TEA), pyrilamine, valproate, and verapamil (55).

Recently, Nakanishi et al. (28) described the identification of a second energy-coupled carnitine transporter. This transporter, called amino acid transporter system B0,+ (ATB0,+), is primarily as an amino acid transporter (29) and plays an important role in their absorption in the intestine. It is expressed in the intestine, lung, and mammary gland and has an Na+- and Cl--coupled transport system for neutral and cationic amino acids (28, 29).

In the intestine, the mechanism of L-carnitine uptake remains unclear. Earlier studies (9) using human intestinal biopsy specimens suggested that intestinal L-carnitine uptake occurred by a carrier-mediated process. However, a subsequent study (21) using rat intestinal brush-border membrane (BBM) vesicles concluded that the uptake process is the result of passive diffusion. Human-derived intestinal epithelial cell lines constitute in vitro models for the study of small intestinal epithelia. Among these, Caco-2 cells readily undergo enterocyte-like differentiation and when grown to confluency, adopt many of the absorptive characteristics of villus absorptive cells (20, 25). In culture, Caco-2 cells progress through distinct states: homogeneously undifferentiated (subconfluence), heterogeneously polarized and differentiated (0-20 days postconfluence), and homogeneously polarized and differentiated (30 days postconfluence) (49). By using these cells in their second state, i.e., with a strong discrepancy in cellular morphology and ultrastructural brush border organization, McCloud et al. (25) have shown that the L-carnitine uptake involves a carrier-mediated system (Km =45 µM) that is temperature, Na+, and energy dependent. However, the exact nature and identity of the carnitine transporter in Caco-2 cells has not been defined.

We hypothesized that carnitine uptake by Caco-2 cells is mediated by OCTN2 on the basis of its functional properties, i.e., Na+-dependence, pH-dependence, and inhibition by valproate (18, 42, 56). The results of our studies indicate that OCTN2 is indeed expressed on the BBM of Caco-2 cells.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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Cell culture. Caco-2 cells were obtained from the American Type Culture Collection (Manassas, VA) at passage 17. Cells were grown in 75-cm2 plastic flasks (Corning Glass Works, Corning, NY) in MEM (GIBCO-BRL, Grand Island, NY) supplemented with decomplemented FCS 10% (GIBCO-BRL), penicillin/streptomycin 1% (GIBCO-BRL), and DMEM nonessential amino acid solution (GIBCO-BRL). The cells were maintained at 37°C in a 95% air-5% CO2 atmosphere. After confluence (70-90%), cells were split by using 0.05% trypsin (0.5 nM) in EDTA (GIBCO-BRL).

Sucrase activity. To assess cellular differentiation, sucrase activity was measured. The Caco-2 cells (between passages 34 and 37) were plated (5 × 106/well) onto 12-well plates (Corning Glass Works) in MEM supplemented with 5% FCS. Undifferentiated (subconfluent), partially differentiated (7-10 days postconfluence) and mature (10-15 postconfluence) Caco-2 cells, were scraped in maleate buffer and sonicated, and sucrase activity was assayed by the method of Messer and Dahlqvist (26) as modified by Lloyd and Whelan (22).

Carnitine uptake. Carnitine uptake studies were carried out according to McCloud's method (25), with minor modifications. Caco-2 cells (between passages 34 and 37) were plated (5 × 106/well) onto 12-well plates (Corning Glass Works) in MEM supplemented with 5% FCS. The cells were used for uptake studies 10-15 days after confluence, and all the experiments were conducted at 37°C. The mature cells were incubated in 1 ml of prewarmed Krebs-Ringer phosphate buffer (in mM: 123 NaCl, 4.93 KCl, 1.23 MgSO4, 0.85 CaCl2, 5 glucose, 5 glutamine, and 20 NaH2PO4, pH 7.4) containing methyl-L-[3H]carnitine (Amersham Pharmacia Biotech, Buckinghamshire, UK) at a concentration of 50 nM. After different incubation periods, the medium was aspirated and cells were immediately washed with ice-cold phosphate buffer and digested at 70°C with 0.5 ml of 1 N NaOH. The reaction was neutralized after 1 h by adding 0.5 ml of 1 N HCl. The cells were then collected, scintillation liquid was added, and radioactivity was counted. In another series of experiments, the Na+, pH, Cl- dependence, and the effect of some ATB0,+ substrates (leucine, alanine, tryptophan) on carnitine uptake were explored.

Assays with inhibitors. For the inhibitor experiments, mature Caco-2 cells (10-15 postconfluence) were preincubated for 30 min with 500 µM pyrilamine, verapamil, TEA, or valproate. All these drugs were purchased from Sigma (St. Louis, MO). Protein determinations were done on parallel wells by the method of Lowry et al. (23).

Western blot analysis. Rabbit polyclonal antibodies were raised against a synthetic polypeptide from Research Genetics (Huntsville, AL), 5'-QWQIQSQTRMQKDGEESPT-3', corresponding to amino acids 532-550 of mouse OCTN2 (17). Undifferentiated (subconfluence), partially differentiated (7-10 days postconfluence) and mature (10-15 days postconfluence) Caco-2 cells (71-90% confluence) were washed three times with saline and collected with a scraper. Thereafter, cells were centrifuged for 5 min at 24°C (850 g). The pellet was suspended in 200 µl of saline and sonicated for 20 s. BBMs and basolateral membranes (BLMs) of mature (10-15 days postconfluence) Caco-2 cells were isolated as described by Schmitz et al. (37), and the degree of their purity was assessed by measuring the enrichment of marker enzymes, alkaline phosphatase for the BBM (15), and Na+,K+-ATPase for the BLM (27). The enrichment factor of alkaline phosphatase in BBM was 10-fold, similar to that reported recently by Hirohashi et al. (14). For the BLM, the relative enrichment of Na+,K+-ATPase over the homogenate was eight. The membranes were dispersed ultrasonically and sample buffer 2 × [4% SDS, 20% glycerol, 200 mM DTT, 120 mM Tris (pH 6.8), and 0.002% bromphenol blue] was then added to Caco-2 cell homogenates or to the isolated membrane preparations. Proteins were denatured in a boiling water bath for 5 min before use for Western blot analysis. Samples were first separated by electrophoresis on a 7.5% SDS-PAGE. The proteins were transferred to polyvinylidene difluoride (PVDF) membrane Immobilon (Millipore, Bedford, MA). The PVDF membrane was incubated in PBS buffer (in mM: 150 NaCl, 2.5 KCl, 5 Na2HPO4, 1.5 KH2PO4) containing 0.02% sodium azide and 12% skim milk. Subsequently, the membranes were incubated overnight with polyclonal anti-peptide antibody (in PBS buffer containing 0.02% sodium azide and 1% BSA), rinsed with the above buffer with 0.1% Tween 20 without skim milk, then incubated with secondary antibody [goat anti-rabbit IgG, horseradish peroxidase-linked whole antibody (Sigma)]. The membranes were washed as before and the ELISA substrate added (Boehringer Mannheim, Laval, QC, Canada). Molecular weights were estimated by using prestained SDS-PAGE broad-range standards (Bio-Rad, Hercules, CA).

Immunofluorescence. Caco-2 cells (between passages 34 and 37) were grown on glass coverslips and were maintained in 95% air-5% CO2 atmosphere at 37°C. They were grown in a complete medium consisting of MEM containing 1% penicillin/streptomycin, 1% DMEM nonessential amino acids, and 5% decomplemented FCS. The cells were employed for experiments 10-15 days after confluence. They were first fixed in 3.8% paraformaldehyde for 30 min at room temperature. After brief washings, the cells were permeabilized for 10 min at room temperature in 0.2% Triton X-100 and blocked with 3% BSA in PBS. The primary antibodies anti-OCTN2 and anti-alkaline phosphatase (Medicorp, Montreal, QC, Canada), diluted 1:800 and 1:80 in PBS, were added and incubated with cells for 90 min at 4°C. After brief washings, fluorescein-conjugated goat anti-rabbit IgG (Biosource International, Montreal, QC, Canada) (1:500) and rhodamine-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA) (1:400) were used as secondary antibodies, at a dilution of 1:500. The cover glass was washed, mounted in Vectashield medium (Vector Laboratories, Burlingame, CA), and examined by fluorescence microscopy by using a Nikon Eclipse E600 microscope.

Statistical analysis. Results are reported as means ± SE of at least triplicate samples. Statistical analyses were performed by using ANOVA with GraphPad InStat (GraphPad Software, San Diego, CA). For the determination of uptake kinetic parameters, the Graph PRISM version 3.0 program was used. Inhibition studies using valproate were analyzed by using linear regression of Lineweaver-Burk plots.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Assessment of the state of differentiation of Caco-2 cells. Sucrase activity, determined before each experiment, was 0.015 ± 0.005 IU/g protein for undifferentiated (subconfluent cells) and 3.2 ± 1.1 IU/g protein for partially differentiated (7-10 postconfluent cells). Sucrase activity was 7.9 ± 1.5 IU/g protein at 10-15 days postconfluence, confirming cellular maturation and differentiation.

L-[3H]carnitine uptake. We studied carnitine transport in undifferentiated and mature Caco-2 cells using L-[3H]carnitine at a concentration of 50 nM. Uptake by mature Caco-2 cells was linear initially and reached an equilibrium at ~8 min at a rate of 0.426 pmol · mg protein-1 · min-1 (Fig. 1). In undifferentiated Caco-2 cells, uptake values were very low, indicating an absence of carnitine transport (Fig. 1).


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Fig. 1.   Caco-2 cell uptake of L-[3H]carnitine as a function of time. Cells were grown in 75-cm2 plastic flasks in MEM supplemented with 10% FCS, 1% penicillin/streptomycin, and a 1% DMEM nonessential amino acid solution. They were maintained at 37°C in a 95% air-5% CO2 atmosphere. After confluence (70-90%), the Caco-2 cells (between passages 34 and 37) were plated (5 × 106/well) onto 12-well plates in MEM supplemented with 5% FCS. Mature Caco-2 cells (10-15 days postconfluence) were incubated at 37°C with Krebs-Ringer phosphate buffer at pH 7.4 in the presence of 50 nM L-carnitine. Open squares represent carnitine uptake by mature Caco-2 cells (10-15 days postconfluence) (r = 0.97), whereas filled squares display carnitine uptake by undifferentiated cells (n = 3 in each case).

L-[3H]carnitine uptake by Caco-2 cells was affected significantly (P < 0.001) when the luminal Na+ concentration was reduced. Uptake was inhibited significantly (P < 0.001) in an Na+-depleted buffer, prepared by substituting KCl for NaCl (Fig. 2A). Although L-[3H]carnitine uptake by Caco-2 cells was not significantly inhibited when the buffer pH was acidic (5.5), it was significantly reduced (P < 0.05) at a pH of 8.5 (Fig. 2B). These results strongly suggest that L-[3H]carnitine uptake was Na+ dependent and slightly proton-gradient dependent. To verify whether the carnitine transporter is a Cl--coupled amino acid transporter (ATB0,+), we investigated the influence of Cl- on the carnitine uptake. Removal of Cl- from the uptake buffer by replacement with NaH2PO4 did not abolish the transport of carnitine. Thus the Cl- had no effect on this system (Fig. 2C). We also tested known amino acid substrates of ATB0,+ and noted that neither leucine, alanine, nor tryptophan inhibited carnitine transport in Caco-2 cells (Fig. 2C).


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Fig. 2.   Na+ and pH dependence of Caco-2 cell L-carnitine uptake. Caco-2 cells (between passages 34 and 37) were maintained in 75 cm2 flasks in 95% air-5% CO2 atmosphere at 37°C. They were grown in a complete medium consisting of MEM containing 1% penicillin/streptomycin, 1% DMEM nonessential amino acids, and 10% decomplemented FCS. After confluence, cells were split by using 0.05% trypsin and plated (5 × 106 cells/well) on plastic 12-well culture plates in complete medium supplemented with 5% FCS. Cells were used for experiments 10-15 days after confluence. A: L-[3H]carnitine uptake was measured in Krebs-Ringer phosphate buffer containing varying concentrations of Na+ (n = 6). B: uptake of L-[3H]carnitine by mature Caco-2 cells was measured in Krebs-Ringer phosphate buffer at pH varying from 5.5 to 8.5 at 37°C for 6 min (n = 3). C: transport of L-[3H]carnitine by mature Caco-2 cells in the presence of Na+ and absence of Cl- (NaH2PO4), and in the presence of leucine, alanine, and tryptophan. Each point represents the mean ± SE of separate determinations. *P < 0.05, ***P > 0.001.

Fig. 3A shows carnitine uptake as a function of concentration in the presence and absence of 500 µM valproate. The data indicate that Caco-2-cell carnitine transport is a saturable process with a Km of 14.07 ± 1.70 µM and Vmax of 26.3 ± 0.80 pmol · mg protein-1 · 6 min-1. A Lineweaver-Burk plot (Fig. 3B) revealed that valproate competitively inhibited Caco-2 cell uptake of L-[3H]carnitine, Km of 62.0 ± 11.1 µM, and Vmax of 24.91 ± 2.19 pmol · mg protein-1 · 6 min-1.


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Fig. 3.   Concentration-dependent uptake of L-carnitine by Caco-2 cells. Cells were grown in 75 cm2 plastic flasks in MEM supplemented with 10% FCS, 1% penicillin/streptomycin, and 1% DMEM nonessential amino acid solution. They were maintained at 37°C in a 95% air-5% CO2 atmosphere. After confluence (70-90%), the Caco-2 cells (between passages 34 and 37) were plated (5 × 106/well) onto 12-well plates in MEM supplemented with 5% FCS. A: uptake of L-carnitine by mature Caco-2 cells was measured at 37°C for 6 min in Krebs-Ringer phosphate buffer at pH 7.4 in the presence of different concentrations of L-carnitine in the absence or the presence of 500 µM of valproate. B: Lineweaver-Burk plot of L-carnitine uptake by mature Caco-2 cells in the absence and the presence of 500 µM of valproate. Each point represents the mean ± SE of 3 separate transport determinations.

Effect of cationic drugs on L-carnitine uptake. Subsequent uptake experiments were performed by using mature Caco-2 cells, in the presence of Na+. Preincubation for 30 min with 500 µM of verapamil, pyrilamine, valproate, or TEA revealed in each case, a significant decrease in L-[3H]carnitine uptake (Table 1). As shown in Table 1, unlabeled carnitine but not proline (at a concentration of 500 µM, 30 min preincubation) inhibited L-[3H]carnitine uptake (P < 0.01) (Table 1).

                              
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Table 1.   Effect of competitive inhibition by cationic drugs and unlabeled L-carnitine on L-[3H]carnitine uptake by mature Caco-2 cells

OCTN2 identification and localization. Western blot analysis revealed a protein with an apparent molecular weight of 60 kDa labeled by the mouse OCTN2-specific antibody in partially and mature Caco-2 cell homogenates (Fig. 4A). In contrast, undifferentiated Caco-2 cells showed no immunoreactivity with this antibody (Fig. 4A). To localize the OCTN2, BBM and BLM were isolated from mature Caco-2 cells and probed with the specific antibodies. The OCTN2 antibody recognized a 60-kDa protein in immunoblots of BBM from mature Caco-2 cells (Fig. 4B). No bands were detected in BLM (Fig. 4B).


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Fig. 4.   Immunolocalization of OCTN2 by Western blotting. Antibodies specific to OCTN2 were used against undifferentiated, partially differentiated, and mature Caco-2 cells (A) against brush-border membranes (BBM) and basolateral membranes (BLM) of mature Caco-2 cells (B). Caco-2 cells, cultures (passages 34-37) were grown until 70-90% confluence in 175-cm2 flasks. They were maintained at 37°C in a 95% air-5% CO2 atmosphere and left to differentiate for 10 to 15 days. Homogenates of differentiated, partially differentiated, and mature Caco-2 cells were prepared as described in MATERIALS AND METHODS. Isolation of mature Caco-2 cell BBM and BLM was carried out as described under Methods. The antigen-antibody complexes were revealed by using an enhanced chemiluminescence detection method.

Immunofluorescence. Immunostaining with anti-OCTN2 polyclonal antibodies revealed strongly positive fluorescence (Fig. 5A). The distribution of fluorescence with anti-OCTN2 was approximately the same when the cells were stained by anti-alkaline phosphatase (Fig. 5B), which is specific to the apical membrane. Colocalization of OCTN2 and alkaline phosphatase (Fig. 5C) revealed that OCTN2 was restricted to the apical membrane of mature Caco-2 cells.


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Fig. 5.   Detection of OCTN2 by immunofluorescence in Caco-2 cells. Caco-2 cells (between passages 34 and 37) were grown on glass coverslips and were maintained in 95% air-5% CO2 atmosphere at 37°C. They were grown in a complete medium consisting of MEM containing 1% penicillin/streptomycin, 1% DMEM nonessential amino acids, and 5% decomplemented FCS. Cells were used for experiments 10-15 days after confluence. They were fixed in 3.8% paraformaldehyde, permeabilized with 0.2% Triton, immunostained for OCTN2 (A) and for alkaline phosphatase (B) and examined by fluorescence microscopy at a magnification of ×100. C: superposition of A and B.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study demonstrates the presence of a specific transporter for the uptake of L-carnitine in mature Caco-2 cells. To pursue our hypothesis that OCTN2 may be responsible for this observation, we have determined its characteristics in the presence and absence of inhibitors of OCTN2-mediated transport and confirmed the presence of OCTN2 by Western blot analysis and immunofluorescence. We observed that mature Caco-2 cells are able to transport L-carnitine, whereas undifferentiated cells are not. Transport occurs only when the cells attain their ultrastructural brush-border organization, up to 10-15 days postconfluence (Fig. 1). Previous studies on Caco-2 cells done with a variety of soluble molecules have shown that transport occurs only after cell polarization (13). Caco-2 cells have been shown to express the transport systems of nutrients, such as carbohydrates (1, 24), amino acids (11, 31), dipeptides (45), and bile acids (12), only when they acquire other properties of differentiated enterocytes. In our studies (Fig. 1), the carnitine transport in Caco-2 cells appears to be mediated by a membrane transporter, because its uptake was only observed in mature cells (polarized epithelia). Hamilton et al. (9) reported that human small intestine tissue is capable of carnitine uptake, whereas colonic tissue is not. Of note, although undifferentiated Caco-2 cells resemble the colon cancer from which they are derived, mature Caco-2 cells are morphologically and functionally like human enterocytes (13, 20, 34). Our results are therefore consistent with those of Hamilton et al. (9).

Na+ and pH dependence of carnitine uptake. Carnitine transport by mature Caco-2 cells was Na+ dependent (Fig. 2A). At low concentrations or in the absence of Na+, L-carnitine transport was significantly lower. These results support the involvement of an Na+-dependent carrier-mediated system for L-carnitine uptake in Caco-2 cells (25). Studies using everted rings of rat intestine, gut sacs, and perfused intestinal segments in vivo (39), as well as those performed in isolated guinea pig enterocytes (7) and using intestinal biopsies (9) as well as human skin fibroblasts (44), have demonstrated the presence of an Na+-dependent carnitine transporter. This distinguishes it from the majority of organic cation uptake mechanisms so far reported at apical membranes of the intestine, most of which are Na+ independent (16). In guinea pig jejunal cells (7) and in HLF cells (58), L-carnitine uptake was also strongly diminished when Na+ was depleted from the buffer or replaced isosmotically by other monovalent cations, such as Li+, K+, or choline.

We also examined the effect of pH variation on L-carnitine uptake by mature Caco-2 cells. Results revealed a modest pH dependence (Fig. 2B). Raising the pH of the extracellular buffer from 7.4 (control) to 8.5 reduced L-carnitine uptake. In contrast, acidification of the extracellular buffer from 7.4 to 5.5 had no significant effect. Variation in pH also had a significant effect on carnitine uptake in cultured human hepatoma HLF cells (58). Previous reports (33) exhibited a variable and slight pH-dependency for the transport of L-carnitine in human embryonic kidney (HEK)-293 cells transfected by human OCTN2 (hOCTN2).

Kinetics and inhibition of carnitine transport. L-Carnitine uptake in Caco-2 cells is a saturable process (Fig. 3A) with high affinity (Km, 14.07 ± 1.70 µM) similar to the known properties of OCTN2. In humans, OCTN2 is expressed in many tissues including intestine (42). OCTN2 is unique in that it transports carnitine with a high affinity (Km approx  4.3 µM) in an Na+-dependent manner, and organic cations in an Na+-independent manner. Recently, Bleasby et al. (2) reported that 1-methyl-4-phenylpyridinium absorption by Caco-2 cells is mediated by an Na+-dependent transport mechanism. This raises the possibility of OCTN2 in these cells, because 1-methyl-4-phenylpyridinium can inhibit carnitine uptake via OCTN2 (55, 56).

Similar high-affinity transport of L-carnitine was observed in HEK-293 cells transfected by hOCTN2 (42) and in HLF cells (58). Clinically important cationic drugs, such as pyrilamine, quinidine, verapamil, and valproate, are transported by hOCTN2 and significantly inhibit OCTN2-mediated L-[3H]carnitine uptake in other culture cells (33, 55). Verapamil was also observed to inhibit carnitine uptake in human retinal pigment epithelial (HRPE) cells transfected by hOCTN2 cDNA (55). In mature Caco-2 cells, we observed that TEA, valproate, verapamil, and pyrilamine all significantly inhibit L-carnitine uptake (Table 1), also consistent with OCTN2-mediated carnitine transport in Caco-2 cells. Other investigators (46, 47) have also tested the effect of valproate on carnitine transport by human fibroblasts and found inhibition. We also examined the effect of unlabeled L-carnitine and proline on the L-[3H]carnitine uptake in Caco-2 cells (Table 1). We found that, whereas unlabeled L-carnitine significantly inhibited (P < 0.01) uptake, proline did not (Table 1). The lack of inhibition by proline was also observed in rat intestine (8) and in human intestinal biopsies (9). This absence of proline inhibition in the three models ruled out the implication of an Na+-dependent cationic amino acid transport in carnitine uptake.

To better define L-carnitine transport via OCTN2, we examined the effect of valproate, known to inhibit L-carnitine uptake in HEK-293 cells transfected by the hOCTN2 cDNA (33). Valproate significantly inhibited L-carnitine uptake, suggesting that it competes for the same transporter (Table 1). In the presence of valproate, the affinity of L-carnitine transport was modified (Km = 62.0 ± 11.1 µM) although the maximum velocity remained unchanged (Vmax = 24.9 ± 2.19 pmol · mg protein-1 · 6 min-1) (Fig. 3B). These results indicate competitive inhibition of L-carnitine uptake by valproate in mature Caco-2 cells. Previous studies (33, 42) using HEK-293 cells transfected by hOCTN2 demonstrated that the inhibition of L-carnitine transport by valproate was of a competitive type, because introduction of this drug induced carnitine deficiency (36). When valproate was administered as an antiepileptic drug, serum and tissue carnitine depletion was induced (2, 5, 51). Furthermore, L-carnitine uptake studies performed either in cultured human skin fibroblasts (45) and in primary cultured rat hepatocytes (40) implied the inhibition of L-carnitine uptake by valproate and revealed the positive effect of L-carnitine on valproate hepatotoxicity.

Immunochemical identification and localization. To further explore the concept that the uptake of L-carnitine in Caco-2 cells is mediated by OCTN2, a mouse OCTN2-specific antibody was produced and used against undifferentiated, partially differentiated, and mature Caco-2 cells. Western blot experiments in homogenates of partially differentiated and mature Caco-2 cells showed immunoreactivity to the antibodies against OCTN2 (Fig. 4A). The molecular size (60 kDa) of the detected bands is close to the estimated size (63 kDa) of OCTN2. Comparing mature Caco-2 cells (10 to 15 days postconfluence) with the partially differentiated Caco-2 cells (7 to 10 days postconfluence), the amount of the protein increased, suggesting that the OCTN2 expression level depends on the differentiation stage. Previous studies (25), reported the involvement of a carrier-mediated system, that is Na+ and energy-dependent in partially differentiated Caco-2 cells. Our results, as well as those of McCloud et al. (25) done on Caco-2 cells, demonstrate the primary role of OCTN2 in the transport of carnitine.

Absence of reactivity with the anti-OCTN2 antibody in immature Caco-2 cells supports the absence of transport of L-carnitine in undifferentiated cells and also confirms that OCTN2 is expressed only when the cells attain their BBM organization. Indeed, the anti-OCTN2 antibodies recognized a protein of 60 kDa only in immunoblots of BBM preparation from mature Caco-2 cells. Staining was absent in BLM preparations (Fig. 4B). Western blot analysis provided further evidence for the presence of OCTN2 in mature Caco-2 cells BBM.

To confirm OCTN2 and its localization, double immunohistochemical staining was undertaken. Staining with anti-OCTN2 polyclonal antibodies (Fig. 5A) revealed the presence of positive punctates on the apical surface. The same fluorescence distribution was observed with the staining with antialkaline phosphatase, which is a specific marker of BBM. When the images were superposed, OCTN2 and alkaline phosphatase, were colocalized (Fig. 5C). These results indicate that OCTN2 is localized on the BBM of mature Caco-2 cells.

Besides OCTN2, the OCTN transporter subfamily includes two other members, OCTN1 and OCTN3, each with the ability to transport carnitine with differing characteristics (18, 41). Human OCTN1 is expressed in several tissues including intestine and transports carnitine weakly in an Na+-dependent manner (41, 43, 57). Rat intestinal OCTN1 interacts with carnitine with a very low affinity and in an Na+-independent manner (54). In mice, OCTN1 mediates carnitine uptake in an Na+-dependent mode with low affinity, although its expression in intestine has not been reported (41). OCTN3 has only been cloned in mice (41) and mediates carnitine uptake in an Na+-independent mode. It is expressed predominantly in testis and weakly in kidney and was not detectable in gut.

Both human and mouse OCTN1 and OCTN2 transport carnitine in an Na+-dependent manner (41, 43, 57). The carnitine transport activity of OCTN1 is significantly lower than OCTN2 (57). On the basis of this affirmation, OCTN1 is not likely to be responsible for the 50% retained carnitine uptake activity in Na+-deprived conditions seen in Fig. 2A. Tamai et al. (41) have also cloned a new carnitine transporter, OCTN3, from the mouse. When expressed in HEK-293 cells, it exhibits Na+-independent carnitine uptake and shows a higher specificity and affinity for carnitine. Among mouse tissues examined, OCTN3 was strongly expressed in testis and weakly in kidney and not in the intestine. This transporter has been investigated only in mouse and not yet in humans. Before implicating OCTN3 in the retained carnitine uptake activity in Caco-2 cells in Na+-depleted condition, one would have to demonstrate that this transporter exists in humans with specific tissue distribution and functional characteristics. In our studies, the 50% retained carnitine uptake activity in Na+-deprived conditions could be explained by the implication of some other unidentified carnitine transporter, which may be Na+-independent and expressed only when the Caco-2 cells attain differentiation.

Nakanishi et al. (28) reported the cloning of a new carnitine transporter, called ATB0,+, from mouse colon and expressed functionally in mammalian cells (HRPE). The process of carnitine transport by ATBo,+ was Na+ and Cl- dependent and inhibited by several amino acids (e.g., alanine, leucine, isoleucine, phenylalanine and tryptophan). The Km for carnitine was 0.83 ± 0.4 mM. On the basis of the functional characteristics of this transporter, we now demonstrate (Fig. 2C) that in the absence of Cl-, the transport of carnitine was not affected in mature Caco-2 cells. In contrast to ATB0,+, which transports carnitine in an Na+- and Cl--coupled manner, Cl- did not have any role in the function of OCTN2 in our cell model. Furthermore, we found that the carnitine transport in Caco-2 cells was not inhibitable either by leucine, alanine, or tryptophan (Fig. 2C) amino acid substrates of ATB0,+. On the basis of the low affinity of ATB0,+ toward carnitine (0.83 ± 0.04 mM) (28) and the Km of 14.07 ± 1.70 µM found in our studies, we can exclude the involvement of this new transporter.

The original report on cloning hOCTN2 investigated the expression of OCTN2 in several human cell lines (56). With the use of Northern blot analysis, Wu et al. (56) showed that hOCTN2 was expressed in many cell lines including Caco-2 cells. Moreover, they showed that OCTN2 is expressed in another human intestinal cell line (HT-29), suggesting strongly that OCTN2 is expressed in human intestine. Recently, Watanabe et al. (52, 53) demonstrated that in the presence of some OCTN2 substrates, the apical-to-basolateral transport of sulpiride in Caco-2 cell monolayers was significantly inhibited. These results showed that OCTN2 on the apical membrane in Caco-2 cells takes part in the apical-to-basolateral transport of sulpiride. These findings support our result that OCTN2 is expressed in BBM of Caco-2 cells and is responsible for carnitine transport. Our results are also in accord with the recently reported data by Duran et al. (6), which showed a localization of the OCTN2 in isolated chicken enterocyte BBM. In addition, it was confirmed that OCTN2 is expressed in human and rat intestine (38, 42).

In conclusion, on the basis of the transport characteristics of OCTN2 determined by this, as well as previous studies, we affirm that in mature Caco-2 cells, L-carnitine transport primarily occurs via OCTN2. We demonstrate the presence of OCTN2 immunoreactivity on the BBM that mediated transport in mature Caco-2 cells. By extension, this may be seen as occurring physiologically in the human small intestine. The results of this study have clinical implications, because identifying this transporter will facilitate studies on intestinal carnitine absorption and will also open new avenues for treating patients with SCD. Any interaction of OCTN2 with the pharmacology and absorption of other cationic drugs would also be better understood.


    ACKNOWLEDGEMENTS

We acknowledge the technical support given by Dr. Idriss Djilali-Saiah and the secretarial assistance of Sylvie Julien.


    FOOTNOTES

This work was supported by grants from the Dairy Producers of Canada (to E. Seidman and I. Qureshi) and Canadian Institute of Health Research Grant MT-15448 (to I. Qureshi and G. A. Mitchell).

Address for reprint requests and other correspondence: I. A. Qureshi, Division of Medical Genetics, Hôpital Sainte-Justine, 3175, Cote Sainte-Catherine, Montreal, QC, Canada H3T 1C5 (E-mail: aqureshi{at}justine.umontreal.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published January 10, 2003;10.1152/ajpgi.00220.2002

Received 7 June 2002; accepted in final form 4 January 2003.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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