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Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, The State University of New York, Amherst, NY 14260
3To whom correspondence should be addressed. E-mail: memorris{at}buffalo.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • Caco-2 • human intestine • transport • choline
Caco-2 cells, a well differentiated human intestinal cell line, represent a useful in vitro model with which to evaluate the intestinal permeability and transport of potential drug candidates in humans (1,2). Several studies have been conducted to investigate specific small intestinal carriers in Caco-2 cells. These studies have demonstrated the transport of bile salts, glucose, amino acids, cobalamine, peptides, organic anions and cations, and p-glycoprotein (P-gp) substrates (3–7).
Choline is a physiologically important organic cation, which is essential for the biosynthesis of cell membrane components such as phosphatidylcholine and sphingomyelin, as well as the neurotransmitter acetylcholine (8). Additionally, it is important for nerve signaling, cell signaling and lipid transport and metabolism, and represents the predominant source of methyl-groups in the body (8,9). In 1998 the National Academy of Sciences identified choline as a required nutrient for humans and recommended daily intake amounts (9); the main source of choline in the body is from the diet (10). Although choline is a highly hydrophilic organic cation, it is well absorbed from the intestine, suggesting the presence of specialized intestinal transporters (11,12). Extensive investigations of intestinal choline transport in various species including rats, chickens, hamsters and guinea pigs using various in vitro intestinal preparations (everted intestinal sacs, intestinal segments, isolated enterocytes and brush border membrane vesicles) have provided evidence for saturable choline transport (11–16). However, there exist some species differences in the characteristics of choline transport, specifically concerning the dependency on sodium for transport. Sodium-dependent choline uptake was reported in isolated epithelial cells from guinea pig jejunum and ileum (15) and intestinal segments of chick (14). Saitoh and co-workers (11) reported that the choline transporter in rat intestinal brush border membrane vesicles was saturable but was not driven by an inwardly directed sodium- or H+-gradient, nor by an inside negative membrane potential, which represent physiologically relevant conditions in the small intestine. Additionally, an outside-directed H+-gradient did not represent the driving force for choline transport. Cis-inhibition and trans-stimulation of 14C-choline transport occurred with the addition of choline and other small molecular quaternary ammonium compounds including tetramethylammonium, acetylcholine and N1-methylnicotinamide (NMN).
Recently, choline transport across Caco-2 cell monolayers was examined (17). This study suggested an active apical to basal transport mechanism that was pH dependent and inhibited by nifidipine, verapamil, EGTA and cyclosporin. However, no publications examining the characteristics of choline uptake on the apical membrane of Caco-2 cells are available. The overall objective of the present study was to further elucidate the characteristics of the transport of choline in Caco-2 cells. Previous studies with organic cation transport in Caco-2 cells have demonstrated the presence of two distinct sodium-independent transporters for 1-methyl-4-phenylpyridinium (MPP+): these transporters may represent extraneuronal monoamine transporter (hEMT, hOCT3, uptake2) and organic cation transporter 1 (hOCT1) (5). Thiamine is transported by Caco-2 cells by a sodium-independent, pH-sensitive mechanism that is not inhibited by tetraethylammonium (TEA), NMN or choline (6). Our principal goals were to determine the uptake characteristics of the choline transporter in Caco-2 cells and then to investigate the substrate specificity of that transporter. Our results indicated the presence of an Na+-independent carrier-mediated transport system for choline in Caco-2 cells. Choline uptake into Caco-2 cells was dependent on temperature and demonstrated saturability. Other small hydrophilic organic cations such as TEA and NMN did not inhibit choline transport, whereas two typical P-gp substrates, daunomycin (DNM) and verapamil, decreased choline uptake.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Radiolabeled [methyl-3H]-choline (85 Ci/mmol; 3.15 Bq) was obtained from New England Nuclear Research Products (Boston, MA). Biodegradable Counting Scintillant was obtained from Amersham (Arlington Heights, IL). Tissue culture reagents were purchased from GIBCO BRL (Grand Island, NY) and Falcon tissue culture plastic flasks and dishes were used (BectonDickinson, Bedford, MA). Coomassie blue dye reagent was obtained from Bio-Rad (Hercules, CA) and all other chemicals were purchased from Sigma Chemical (St. Louis, MO). Caco-2 cells were obtained from American Type Cell Culture (Rockville, MD).
Cell culture.
Caco-2 cells (passages 24–40) were grown on tissue culture flasks in DMEM (4500 mg/L glucose) supplemented with 10% fetal bovine serum, 1% nonessential amino acids, 2 mmol/L L-glutamine, 5 mmol/L HEPES, penicillin (10 mg/L) and streptomycin (10 mg/L). The cells were maintained at 37°C in a humidified atmosphere with 5% CO2/95% air. For uptake studies, cells were seeded onto 35-mm2 plastic culture dishes and used 15 d after seeding.
Uptake studies.
Cell layers in the 35-mm2 plastic dishes were washed with 3 mL uptake buffer (137 mmol/L NaCl, 5.4 mmol/L KCl, 2.8 mmol/L CaCl2, 1.2 mmol/L MgCl2, 10 mmol/L HEPES, pH 7.4). Incubation buffer (1 mL) containing ([3H]-choline dissolved in uptake buffer) was added to each dish and incubated at 37°C for the designated time periods. The uptake was stopped by aspirating the incubation buffer and washing the cells three times with ice-cold stop solution (137 mmol/L NaCl, 14 mmol/L Tris, pH 7.4). Zero-time values were determined immediately after the buffer addition to the dish by aspirating the buffer and rinsing with cold stop solution. One milliliter of 0.5% Triton-X-100 was added to each dish to lyse the cells. Radioactivity was determined by adding 3 mL of scintillant liquid to 150 µL of the solubilized cell solution and counting it in a liquid scintillation counter (Packard Instruments, Downers Grove, IL). To ensure that changes in uptake values were not due to changes in nonspecific binding of [3H]-choline to the cell membrane, zero-time values were subtracted from the uptake values.
The dependency of choline uptake on Na+ was examined by performing the studies in the presence and absence of NaCl. The NaCl in the uptake buffer was replaced with N-methylglucamine (NMG, 137 mmol/L). The maximum uptake rate (Vmax) and Michaelis-Menton constant (Km) values for choline uptake were determined by examining the 10-min uptake at various concentrations of choline. Temperature dependence of choline transport was determined by comparing transport at 4 and 37°C. Studies to determine whether various compounds could inhibit choline transport were performed by examining the 10-min uptake of 0.5 µmol/L choline (0.005 µmol/L [3H]-choline + 0.495 µmol/L unlabeled choline) in the presence of 100 µmol/L concentration of various compounds.
Protein measurement.
The protein concentration was determined by the Bradford method (18) using the Bio-Rad protein assay kit. Bovine serum albumin was used as a standard.
Data analysis.
The transport parameters were obtained by fitting the data to the following Michaelis-Menten equation plus a diffusion component using the computer program PCNONLIN (Pharsight, Mountain View, CA).
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where Vmax is the maximum rate, Km is the Michaelis-Menten constant, KD is the diffusion clearance and C is the choline concentration.
Ki (inhibitory rate constant) values were calculated using the following equation:
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where V0 is the initial uptake rate and I represents the inhibitor concentration.
Use of this equation assumes that the choline concentration used in the studies <<Km and that there is competitive or noncompetitive inhibition (19).
One-way ANOVA followed by Dunnett's post-test was used to compare the data. Differences were considered significant when P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The uptake of 0.5 µmol/L [3H]-choline was examined up to 5 h. Choline uptake at 37°C was significantly higher than the uptake at 4°C (Fig. 1). This suggests the involvement of an energy-requiring process for choline uptake into Caco-2 cells. The 10-min time point (which is in the linear range of uptake) was used for all subsequent experiments to study the concentration dependence as well as the effect of various compounds on uptake. The time course of [3H]-choline (0.5 µmol/L) was studied in the presence and absence of 1000-times excess unlabeled choline. At all of the time points studied, the presence of excess choline inhibited [3H]-choline uptake into the cells, indicating the presence of a transporter for choline in Caco-2 cells (Fig. 2).
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The effect of sodium ions on choline uptake into Caco-2 cells was examined at the 10-min time point by replacing the sodium in the incubation buffer with NMG. The uptake of choline did not increase in the presence of Na+, indicating that choline uptake was independent of Na+ (data not shown). Furthermore, ouabain, which is an inhibitor of Na+-K+-ATPase, also did not inhibit choline uptake at a concentration of 100 µmol/L, providing further evidence that choline uptake into Caco-2 cells is not driven by a sodium gradient.
Determination of kinetic parameters of choline uptake.
The concentration dependence of choline uptake was examined at 10 min (initial uptake, Fig. 3). Choline concentrations up to 1000 µmol/L were examined. A simple Michaelis-Menten equation did not adequately fit the data, thus requiring the addition of a linear component to the equation. The kinetic parameters for choline uptake in Caco-2 cells were: Vmax = 2800 ± 250 pmol/(mg protein · 10 min), Km = 110 ± 3 µmol/L and KD = 2.0 ± 0.2 µL/(mg protein · 10 min) (mean ± SEM, n = 3 separate studies).
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To determine whether the choline transporter was specific for choline or could transport other organic cations, typical substrates of the OCT (organic cation transporters) family (e.g., TEA and NMN) were studied. The effect of a 200-fold higher concentration of the compounds on the 10-min uptake of choline was examined. Neither TEA or NMN affected choline uptake, whereas a 200-fold higher concentration of choline significantly inhibited its own uptake (Ki = 124.7 µmol/L, Fig. 4). Two structural analogs of choline, succinylcholine and hemicholinium-3, were also studied at a 200-fold higher concentration and had differing results. Succinylcholine had no effect on choline uptake, whereas hemicholinium-3, a known competitive inhibitor of both sodium-dependent and sodium-independent choline transport (20,21), inhibited choline uptake significantly (72% decrease in choline uptake; Ki = 53.1 µmol/L).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The accumulation of choline was saturable (Fig. 3) and could be characterized by a Michaelis-Menten equation plus a diffusion component. The Km value obtained (110 µmol/L) was in the same range as that reported in the rat intestinal brush border membrane vesicles (Km = 159 µmol/L) (7), rabbit intestinal brush border membrane vesicles (Km = 83 µmol/L) (14) and guinea pig enterocytes (Km = 110 µmol/L) (15). Additionally, the studies that have characterized choline transport in animal intestinal preparations indicated the presence of passive diffusion at high choline concentrations (13,25), as found in Caco-2 cells.
Two structural analogs of choline, succinylcholine and hemicholinium-3, had differing actions on choline transport. Succinylcholine had no effect on choline transport, whereas hemicholinium-3 significantly inhibited choline uptake by 72%. In the liver, both succinylcholine and hemicholinium-3 significantly inhibited choline uptake in both sinusoidal and canalicular membrane vesicles isolated from rats (26). This suggests that the choline transporter in Caco-2 cells may have a narrower substrate specificity than that in rat liver. Hemicholinium-3 is a competitive inhibitor of both the sodium-dependent and sodium-independent transport of choline in many tissues (20,21).
TEA and NMN, both typical substrates of the OCT family, did not inhibit choline uptake. Moreover, uptake of 25 µmol/L [14C]-TEA in Caco-2 cells was not inhibited by an excess of choline (500 µmol/L) (data not shown). On the basis of these results, it is unlikely that TEA and NMN utilize the same transporter as choline in Caco-2 cells. This is similar to the condition seen in the rat intestine in which TEA had no effect on choline transport (11,25). However, in considering NMN inhibition, choline transport differed from that in the rat intestine in which NMN inhibited choline transport (11). Choline was reported to be a substrate for OCT in brain and kidney preparations. The transport of choline across the blood brain barrier in mice is dependent on membrane potential and is inhibited by substrates and inhibitors of the OCT family, including TEA, NMN, procainamide, quinine and dopamine (27). OCT2 has been reported to mediate choline transport across the ventricular membrane of the choroid plexus and in the kidney of rats (28–30).
However, there are substantial species differences in OCT distribution and specificity of substrates (31). Human OCT1 (hOCT1) is restricted predominantly to liver, whereas hOCT2 is found predominantly in kidney. Martel and co-workers (5) reported the presence of mRNA of hOCT1 and hEMT in Caco-2 cells; TEA and NMN are substrates for both hOCT1 and extraneuronal monoamine transporter (EMT). Choline is a substrate for hOCT1, but not hOCT2, hOCTN2 or hEMT (32). However, the importance of hOCT1as a transporter in Caco-2 cells is not known. We found that choline does not inhibit the transport of TEA (which is a substrate for hOCT1 and hEMT), and TEA and NMN do not inhibit the transport of choline. This suggests that choline may not be transported by hOCT1 or hEMT in Caco-2 cells. A recent publication evaluating MPP+ transport in Caco-2 cells suggested that MPP+ may be a substrate for a transporter distinct from the characterized OCT family of transporters; choline could inhibit the transport of MPP+, but whether MPP+ and choline share the same transport mechanism is unknown (33). In a rat brain microvessel endothelial cell line (RBE4) that expresses low levels of rOCT1, but not rOCT2 or rOCT3, choline uptake was inhibited by hemicholinium-3, but not TEA (20). On the basis of the functional characteristics of choline uptake in these cells, the choline transporter also appears to be distinct from the known OCT family of transporters (20). These reports are consistent with our findings in Caco-2 cells and suggest that the choline transporter in Caco-2 cells may also be distinct from the known OCT transporters.
Another interesting observation in this study was the inhibition of choline uptake by the typical P-gp substrates, DNM and verapamil. However, colchicine, another typical P-gp substrate, showed no inhibition of choline transport. Also, a 2000-fold excess of choline had no effect on [3H]-DNM transport in Caco-2 cells. These results suggest that DNM may inhibit the choline transporter either through a noncompetitive binding interaction or through some indirect mechanism. Inhibition could be the result of a nonspecific effect, such as DNM- or verapamil-induced changes in membrane fluidity due to alterations on the membrane lipid bilayer. Canaves and co-workers (34) reported that when DNM and verapamil were present simultaneously in a dipalmitoyl phosphatidylcholine vesicle bilayer, verapamil prevented alterations in the phospholipid phase transition normally seen after DNM treatment. A recent publication reported that verapamil, DNM and rhodamine 123, but not other P-gp substrates including vinblastine, digitoxin and cyclosporine A, can inhibit the transport of an OCT substrate, MPP+, in a cell line transfected with the hEMT (33,35); the authors suggested that these P-gp substrates may bind to hEMT but are not transported (35). However, choline is not a substrate for hEMT; thus, it is unlikely that the inhibition of choline transport by DNM and verapamil is mediated by hEMT. Additionally, Crowe and co-workers found that choline transport across Caco-2 cell monolayers was inhibited by nifedipine, verapamil and cyclosporin (17), all P-gp substrates and/or inhibitors. These authors suggested that the mechanism of inhibition may involve extracellular Ca++ depletion because these compounds can alter extracellular calcium concentrations, and EGTA also inhibited choline flux in Caco-2 cells (17). The mechanism underlying this interaction between specific P-gp substrates and choline in Caco-2 cells is presently unknown.
In summary, the results of the present study demonstrated the presence of a carrier-mediated transport system for choline in Caco-2 cells. This transporter had substrate specificity that differs from that in rat intestinal brush border membrane vesicles (11), renal and brain tissues (27–30), and liver membrane vesicles (26). In addition, it differs from the sodium-dependent, high-affinity choline transporter present in cholinergic neurons (21). Further studies are required to investigate the characteristics of the choline transporter in human intestinal preparations and to evaluate the potential for drug-choline interactions in the intestine.
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FOOTNOTES |
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2 Present address: Bristol Myers Squibb, P.O. Box 4000, Princeton, NJ 08543.
4 DNM, daunomycin; EMT, extraneuronal monoamine transporter; KD, diffusion clearance; Ki, inhibitory rate constant; Km, Michaelis-Menten constant; MPP+, 1-methyl-4-phenylpyridinium; NMG, N-methylglucamine; NMN, N1-methylnicotinamide; OCT, organic cation transporters; P-gp, p-glycoprotein; TEA, tetraethylammonium; Vmax, maximum uptake rate.
Manuscript received 11 April 2003. Initial review completed 4 May 2003. Revision accepted 3 June 2003.
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