Introduction

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Histamine H₂-receptor antagonists (H₂-antagonists) have enjoyed tremendous success as an important class of therapeutic agents for the treatment of gastric and duodenal ulcers over the last 25 years (Lin, 1991). However, the mechanism of their oral (intestinal) absorption has not been elucidated. Gan et al. (1993) suggested that the absorptive (mucosal to serosal) transport of the H₂-antagonist ranitidine across the intestinal epithelium occurred predominantly via the paracellular pathway based on studies with Caco-2 cell monolayers, a cell line derived from human colorectal adenocarcinoma (Pinto et al., 1983; Hidalgo et al., 1989; Artursson, 1990; Gan and Thakker, 1997 and references therein). Collett et al. (1996) also reported that the absorptive transport of ranitidine and cimetidine occurred predominantly via the paracellular pathway across Caco-2 cell monolayers. We have extended these observations and demonstrated that the paracellular transport of the H₂-antagonists, ranitidine and famotidine, has a saturable component (Lee and Thakker, 1999).

It appears that the intestine also plays a role in the excretion of H₂-antagonists. Ranitidine was secreted into the intestine in humans (Gramatte et al., 1994) and rats (Suttle and Brouwer, 1995). Furthermore, there is some evidence for P-glycoprotein (P-gp)-mediated efflux of the H₂-antagonists, ranitidine and cimetidine, across the apical (AP) membrane of Caco-2 cell monolayers (Cook and Hirst, 1994; Collett et al., 1999). Given that P-gp is located on the AP membrane (Hunter et al., 1993), these results clearly suggest that the secretory transport of the H₂-antagonists occurs via a predominantly transcellular pathway in Caco-2 cells.

A transcellular secretory transport across epithelia involves translocation of compounds across the basolateral (BL) membrane, and then across the AP membrane. Passive diffusion across the BL membrane is not likely to be the predominant mechanism, considering that these drugs are relatively hydrophilic cationic compounds and that their absorptive transport across Caco-2 cell monolayers occurs predominantly via the paracellular route (Gan et al., 1993; Collett et al., 1996, 1999; Lee and Thakker, 1999). It is conceivable that these drugs are transported across the BL membrane by a transporter (e.g., organic cation transporters). Ranitidine, famotidine, and cimetidine are known to be secreted into the renal tubules across the epithelium by an organic cation transporter that is distinct from P-gp and inhibited by tetraethylammonium (TEA) (Grundemann et al., 1994; Somogyi et al., 1994). Possible involvement of a similar transporter in drug secretion into the intestine also has been demonstrated (Saitoh et al., 1996; Koepsell, 1998).

The present study attempts to characterize the mechanism(s) by which the H₂-antagonists, ranitidine and famotidine (Fig. 1), traverse across Caco-2 cell monolayers in the secretory direction. Our results show that the transport of these compounds across the BL membrane of Caco-2 cells is mediated by a saturable transport system that is distinct from known TEA-sensitive organic cation transporters. Furthermore, we confirm the role of P-gp in the secretion of H₂-antagonists across Caco-2 cell monolayers.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Structures of ranitidine and famotidine.

	Materials and Methods

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Materials. Eagle's minimum essential medium (with Earle's salts and L-glutamate), fetal bovine serum, nonessential amino acids (×100), and 0.05% trypsin-EDTA solution were obtained from Invitrogen (Carlsbad, CA). Hanks' balanced salt solution (×1), ranitidine hydrochloride, famotidine, guanethidine monosulfate, mannitol, [¹⁴C]mannitol (43 mCi/mmol), rhodamine 123, (±)-verapamil, TEA bromide, diphenhydramine, chlorquine, l-methyl-4-phenylpyridinium (MPP⁺), antibiotic antimycotic solution (×100), D-(+)-glucose, and Triton X-100 were purchased from Sigma-Aldrich (St. Louis, MO). HEPES (1 M) and phosphate-buffered saline (PBS; ×1) were purchased from Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC. [¹⁴C]TEA (55 mCi/mmol) was obtained from American Radiolabeled Chemicals Inc., St. Louis, MO. [¹⁴C]Ranitidine (10 mCi/mmol) was a gift from GlaxoSmithKline (Research Triangle Park, NC). Cyclosporin A (CsA) was a gift from Dr. Moo J. Cho (School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC).

Cell Culture. Caco-2 and LLC-PK₁ cells were obtained from GlaxoSmithKline and Lineberger Comprehensive Cancer Center, respectively. Both cell lines were cultured at 37°C in minimum essential medium, supplemented with 10% fetal bovine serum, 1% nonessential amino acids, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B in an atmosphere of 5% CO₂ and 90% relative humidity, and passaged at about 90% confluence, using trypsin-EDTA. Caco-2 (passage 50~60) and LLC-PK₁ (passage 215~223) cells were seeded at a density of 60,000 cells/cm² and 500,000 cells/cm², respectively, on polycarbonate membranes of Transwells (12 mm i.d., 3.0 µm pore size; Costar, Cambridge, MA). Medium was changed the day after seeding and every other day thereafter (AP volume 0.5 ml, BL volume 1.5 ml). Caco-2 cell monolayers with transepithelial electrical resistance values above 350  · cm² after 20~25 days postseeding and LLC-PK₁ cell monolayers with transepithelial electrical resistance values above 110  · cm² after 10~12 days postseeding were used in this study.

Transport Studies. Transport experiments using cell monolayers were performed as described previously (Lee and Thakker, 1999). The cell monolayers were incubated for 30 min at 37°C in the transport buffer (Hanks' balanced salt solution supplemented with 25 mM D-glucose and 10 mM HEPES, pH 7.2) (AP volume 0.5 ml, BL volume 1.5 ml). AP to BL transport was initiated by replacing the AP buffer with 0.4 ml of the transport buffer containing the compound being investigated. The inserts were then transferred at selected times to a 12-well cell culture cluster (Costar) containing 1.5 ml of prewarmed transport buffer in each well. BL to AP transport was initiated by replacing the BL buffer with 1.5 ml of the drug solution after the AP buffer had been replaced with 0.4 ml of the transport buffer. AP solution (0.2 ml) was withdrawn and the same volume of prewarmed transport buffer was added to the AP sides at selected times. The temperature was maintained at 37°C during the transport experiments. All transport experiments were carried out under sink conditions because the concentrations of drugs in the receiver compartment remained at least 10-fold lower than those in the donor compartment. For the P-gp inhibition studies, the cell monolayers were incubated for 30 min at 37°C in the presence of a P-gp inhibitor added to both sides of the cell monolayers after the 30-min preincubation in the transport medium. The transport experiments were then initiated by replacing the BL solution with 1.5 ml of the transport buffer containing the test compound and the P-gp inhibitor.

Cellular Uptake Studies. The cell monolayers were preincubated for 30 min at 37°C as described under Transport Studies. For P-gp inhibition, the cell monolayers were further incubated for 10 min in the presence of 20 µM CsA. The BL solution was then replaced with the drug solution to initiate the studies. Both sides of the cell monolayers were washed five times with ice-cold PBS (0.5 ml and 1.5 ml for AP and BL sides, respectively) at selected times. After the washing step, 0.3 ml of 1% Triton X-100 was added to the AP side, and the cell monolayers were incubated for 1 h at room temperature under shaking. The cell lysate was centrifuged (10,000g, 5 min), and the supernatant was analyzed by liquid scintillation spectrometry or high-performance liquid chromatography in the same manner as described under Transport Studies. The amount of protein in the cell lysate was determined with a bicinchoninic acid protein assay kit (Pierce, Rockford, IL) using bovine serum albumin as a standard (Smith et al., 1985).

Data Analysis. Data were expressed as mean ± S.D. from three measurements. The statistical significance of differences between control and treatment was evaluated using unpaired Student's t tests.

	Results

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Secretory Transport of Ranitidine and Famotidine across Caco-2 Cell Monolayers. The flux of ranitidine and famotidine across Caco-2 cell monolayers was determined at several concentrations over the range of 0.1~2.0 mM in both AP to BL and BL to AP directions. The AP to BL transport of the H₂-antagonists involved a saturable component (Fig. 2, A and B), consistent with the previous report (Lee and Thakker, 1999). The flux in the BL to AP direction was greater than the corresponding AP to BL flux for both ranitidine and famotidine (Fig. 2, A and B). Furthermore, the BL to AP transport of ranitidine and famotidine also exhibited a saturable component (Fig. 2, A and B). In contrast, the mannitol (paracellular marker) flux was the same in both directions at equimolar concentrations, and was linear over the entire concentration range examined (Fig. 2C). Significantly greater BL to AP flux of ranitidine and famotidine, compared with their AP to BL flux, is consistent with the hypothesis that the secretory (BL to AP) transport of both ranitidine and famotidine may be mediated by an apically directed efflux pump(s) such as P-gp (Cook and Hirst, 1994; Collett et al., 1999).

View larger version (9K): [in this window] [in a new window]   Fig. 2.   Comparison of AP to BL and BL to AP transport of ranitidine (A), famotidine (B), and [14C]mannitol (C) across Caco-2 cell monolayers. The flux (J) was determined in a linear region of the time course (30-60 min) of AP to BL (filled symbols) or BL to AP (open symbols) transport at various concentrations.

Uptake Characteristics of Ranitidine across the BL Membrane of Caco-2 Cell Monolayers. Given that both of the H₂-antagonists are hydrophilic cations (see Fig. 1 for structures), it is unlikely that these compounds can cross the BL cell membrane by passive diffusion during their transcellular transport. It is conceivable, however, that their translocation across the BL membrane of Caco-2 cells is mediated by a transporter. The initial rapid uptake of ranitidine across the BL membrane of Caco-2 cells reached a plateau after 20 min (Fig. 3A). The uptake (initial and maximal) was significantly greater in the presence of the P-gp inhibitor CsA (20 µM) (Fig. 3A). This was expected because inhibition of P-gp would reduce the efflux of ranitidine, a substrate for P-gp (Cook and Hirst, 1994; Collett et al., 1999), and thus would allow greater cellular accumulation during the uptake studies. Initial BL uptake of ranitidine, determined over a wide range of concentrations (1-200 mM), was saturable as evidenced by a good fit of the Michaelis-Menten equation to the uptake data (Fig. 3B). The estimates of the maximal velocity (V_max) and the apparent Michaelis-Menten constant (K_m) for the BL uptake of ranitidine were 20.9 nmol/mg of protein/min and 66.9 mM, respectively. Over the same concentration range, uptake of mannitol was linear with respect to concentration and significantly lower than that of ranitidine (Fig. 3B). Furthermore, the BL uptake of ranitidine at 4°C was much lower than that at 37°C over the entire concentration range and exhibited a linear relationship with concentration, rather than the hyperbolic relationship observed at 37°C (Fig. 3B). These results are consistent with a carrier-mediated translocation of ranitidine across the BL membrane of Caco-2 cells. Interestingly, the BL uptake of ranitidine (0.1 mM) in the absence or presence of the metabolic inhibitor 2,4-DNP (1 mM) was very similar: 0.14 ± 0.014 and 0.13 ± 0.008 nmol/mg protein/2 min (p > 0.05), respectively.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   A, time course of cellular uptake of ranitidine across the BL membrane of Caco-2 cells. Cellular uptake of 0.1 mM ranitidine was measured at selected times after addition to the BL side in the absence () or presence () of CsA (20 µM) added to both AP and BL sides. B, cellular uptake characteristics of ranitidine and [14C]mannitol across the BL membrane of Caco-2 cells. Cellular uptake of 0.1 mM ranitidine (, ) or [14C]mannitol () was measured at 37°C (, ) or 4°C () for the initial 2 min, after addition to the BL side in the presence of CsA added to the AP side. For this study, CsA (20 µM) was added only to the AP side, since it was similarly effective as compared with its addition to both the AP and BL sides. The Michaelis-Menten equation was fit to the cellular uptake data obtained at 37°C using nonlinear least-squares regression analysis (WinNonlin 1.1; Scientific Consulting Inc., Apex, NC). Data represent mean ± S.D.; n = 3.

The Lack of Involvement of TEA-Sensitive Organic Cation Transporters in the Secretory Transport of Ranitidine and Famotidine across Caco-2 Cell Monolayers. Because of the hydrophilic cationic nature of the H₂-antagonists, an organic cation transporter(s), such as OCTs or OCTNs (Koepsell, 1998), may be involved in the transport process. However, to date, the presence of such a transporter in the BL membrane of Caco-2 cells has not been reported. Hence, BL to AP transport of ranitidine and famotidine across Caco-2 cell monolayers was compared with that of [¹⁴C]TEA, a prototypical substrate for organic cation transporters (Gorboulev et al., 1997; Tamai et al., 1997; Zhang et al., 1997; Koepsell, 1998; Wagner et al., 2000; Wu et al., 2000).

View larger version (12K): [in this window] [in a new window]   Fig. 4.   A, comparison of the BL to AP transport of ranitidine and famotidine with that of [14C]TEA and [14C]mannitol across Caco-2 cell monolayers. The amount in the AP side was measured at timed intervals after addition of 10 µM [14C]TEA (), ranitidine (), famotidine (dotted triangle), or [14C]mannitol (dotted diamond) to the BL side. The transported amount was expressed as a percentage of the initial amount in the BL compartment. B, cellular uptake characteristics of [14C]TEA across the BL membrane of Caco-2 cells. Cellular uptake of TEA was measured at 37°C () or 4°C () for the initial 2 min after addition of TEA to the BL side. Data represent mean ± S.D.; n = 3.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Comparison of the BL to AP transport of ranitidine and famotidine with that of [14C]TEA and [14C]mannitol across LLC-PK1 cell monolayers. The amount in the AP side was measured at timed intervals after addition of 10 µM [14C]TEA (dotted diamond), ranitidine (), famotidine (dotted triangle), or [14C]mannitol () to the BL side. The transported amount was expressed as percentage of the initial amount in the BL compartment.

Inhibition of BL Uptake of [¹⁴C]Ranitidine across Caco-2 Cell Monolayers by Organic Cations. The uptake of [¹⁴C]ranitidine (0.1 µM) across the BL membrane of Caco-2 cell monolayers was determined in the presence of several structurally diverse cations to determine the selectivity of the putative BL transporter for organic cations (Table 1). The concentrations of the organic cations were 10- to 100-fold (consistent with their solubility) in excess of the ranitidine concentration. Structurally diverse organic cations inhibited BL uptake of [¹⁴C]ranitidine, suggesting a broad ligand selectivity of the putative transporter. Interestingly, TEA and MPP⁺, substrates for organic cation transporters, OCTs and OCTNs, did not inhibit ranitidine uptake as effectively as did other organic cations.

	Discussion

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In the present study, we observed that the flux of ranitidine and famotidine across Caco-2 cell monolayers in the BL to AP direction was significantly greater than that in the AP to BL direction (Fig. 2). Considering the previous results, indicative of paracellular AP to BL transport of ranitidine (Gan et al., 1993; Collett et al., 1996; Lee and Thakker, 1999), these results were surprising because they implied transcellular transport of both ranitidine and famotidine in the BL to AP direction. Inhibition of ranitidine and famotidine transport in the BL to AP direction by the P-pg inhibitors, CsA and verapamil, confirmed the role of the efflux pump in the secretory transport of ranitidine (Cook and Hirst, 1994; Collett et al., 1999) as well as famotidine. The obvious question one must ask is: how did these compounds permeate the BL membrane? It is unlikely that H₂-antagonists traverse the BL membrane via a passive diffusion process, given their cationic hydrophilic nature. Hence, we have investigated the possibility that ranitidine and famotidine enter Caco-2 cells across the BL membrane via a carrier-mediated transport mechanism.

Ranitidine accumulation in Caco-2 cell monolayers was examined, after addition of ranitidine to the BL compartment, to determine the role of a transporter-dependent process in its uptake across the BL membrane. Our data clearly demonstrate that a saturable transport system mediates the transport of ranitidine across the BL membrane of Caco-2 cell monolayers (Fig. 3). However, this is not an active transport process as evidenced by the observation that 2,4-DNP did not inhibit BL uptake of ranitidine, unless the apparent lack of uptake inhibition was an experimental artifact due to inhibition of both the BL uptake transporter and the AP efflux transporter (P-gp).

Organic cation transporters are a family of polyspecific transporters, involved in the absorption and secretion of organic cations in various tissues such as intestine, liver, and kidney (Koepsell, 1998; Koepsell and Arndt, 1999). Five human organic cation transporters have been successfully cloned, namely, hOCT1, hOCT2, hOCT3, hOCTN1, and hOCTN2 (Gorboulev et al., 1997; Tamai et al.,1997, 1998; Yabuuchi et al., 1999; Wu et al., 2000). Among the cloned transporters of this family, mRNA of hOCT1, hOCT2, and hOCTN2 was detected in both small intestine and Caco-2 cells (Tamai et al., 1998; Zhang et al., 1999; Bleasby et al., 2000), raising the possibility that hOCT1, hOCT2, and/or hOCTN2 may serve as the BL membrane transporter for organic cations across Caco-2 cell monolayers. All of these cloned organic cation transporters mediate transmembrane transport of TEA (K_m 95-463 µM) and are referred to as TEA-sensitive organic cation transporters (Gorboulev et al., 1997; Tamai et al., 1997; Zhang et al., 1997; Wagner et al., 2000; Wu et al., 2000). Consequently, we explored the possibility that the TEA-sensitive organic cation transporters were involved in the transport of the H₂-antagonsits, ranitidine and famotidine, across the BL membrane of Caco-2 cell monolayers.

Our results indicated that TEA-sensitive organic cation transporters are not functional in the BL membrane of Caco-2 cells because 1) the substrate for these transporters, TEA, was transported in the BL to AP direction of Caco-2 cell monolayers at approximately the same rate as the paracellular marker, mannitol (Fig. 4A), and 2) the BL [¹⁴C]TEA uptake was linear over a wide range of concentrations (Fig. 4B), perhaps indicative of nonspecific binding to cell surface. BL uptake of [¹⁴C]TEA was determined up to a concentration of 10 mM because the maximum K_m value of this substrate for known organic cation transporters is 463 µM (Tamai et al., 1997). As a positive control, BL transport of [¹⁴C]TEA was examined in LLC-PK₁ cell monolayers. These cells express TEA-sensitive transporter(s) in both AP and BL membranes (Inui et al., 1985; McKinney et al., 1992). As expected, [¹⁴C]TEA exhibited significantly greater flux in the BL to AP direction than did mannitol (Fig. 5). In contrast, the flux of ranitidine and famotidine in this system was similar to that of mannitol (Fig. 5). The low transport rate of ranitidine and famotidine was not due to the lack of P-gp-mediated efflux, since the presence of this efflux system in the AP membranes of LLC-PK1 cells has been reported (Dudley and Brown, 1996). These results with LLC-PK1 clearly show what type of a secretory transport profile we should expect if a compound is a substrate for the TEA-sensitive organic cation transporters. Comparison of the secretory transport kinetics of ranitidine and TEA across Caco-2 cells and across LLC-PK1 cells (with established presence of organic cation transporters) confirms that the saturable BL to AP transport of ranitidine and famotidine across Caco-2 cell monolayers is not mediated via the TEA-sensitive organic cation transporter family. Interestingly, Piyapolrungroj et al. (1999) have previously reported that cimetidine, a structurally related H₂-antagonist, is taken up by a TEA-insensitive active transport mechanism across the brush-border (AP) membrane in vesicles from rat small intestine.

In summary, our results suggest that the H₂-antagonists, ranitidine and famotidine, are transported across the BL membrane of Caco-2 cells by a transport system that is distinct from the known organic cation transporters in the intestinal and kidney epithelia. This transporter appears to have fairly broad substrate selectivity, as evidenced by inhibition of ranitidine uptake across the BL membrane of Caco-2 cells by diverse organic cations (Table 1). As expected from our results, TEA and MPP⁺, two known substrates for OCTs and/or OCTNs, do not inhibit BL uptake of ranitidine. The secretory transport mechanism for ranitidine and famotidine appears to be distinct from the proposed absorptive transport mechanism, mediated by the anionic cellular constituents in the paracellular space (Lee and Thakker, 1999) (Fig. 6). As proposed in Fig. 6, a BL transporter and P-gp both play a role in the secretory transport of ranitidine and famotidine across Caco-2 cell monolayers, and presumably across the intestinal epithelium.

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Proposed mechanism of the predominant component of the absorptive and secretory transport of ranitidine and famotidine across Caco-2 cell monolayers. The absorptive transport (dotted arrow) occurs via a paracellular facilitated diffusion process, mediated by anionic cellular constituents in the paracellular space (Lee and Thakker, 1999). The secretory transport (solid arrow) occurs via the transcellular pathway, where the transport across the BL membrane is mediated by the TEA-insensitive transporter and the efflux across the AP membrane is mediated by P-gp.

Direct excretion of drugs into the intestinal lumen across the epithelium may be an important elimination route for certain drugs (Lennernas and Regardh, 1993; Gramatte et al., 1994, 1996; Suttle and Brouwer, 1995; Arimori and Nakano, 1998). In fact, intestinal secretion of ranitidine in rats (Suttle and Brouwer, 1995) and humans (Gramatte et al., 1994) has been demonstrated. The results in the present study provide a mechanistic basis for the observed secretion of ranitidine into the intestine of rats and humans. Such a secretory mechanism may contribute to the secondary peaks and prolonged plateaus observed in plasma concentration-time profiles after oral administration of ranitidine (Plusquellec et al., 1987; Suttle and Brouwer, 1994). The TEA-insensitive organic cation transporter(s), implicated by our results in the BL uptake of ranitidine and famotidine, may play a significant role in the intestinal secretion of these H₂-antagonists as well as other hydrophilic organic cations that are secreted into the intestine (Koepsell, 1998). Clearly, additional characterization of this putative transport mechanism takes up added significance in light of its possible role in the intestinal excretion (secretion) of therapeutic agents.