0022-3565/02/3032-574 –580$7.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics
JPET 303:574–580, 2002
Vol. 303, No. 2
38512/1014530
Printed in U.S.A.
Secretory Transport of Ranitidine and Famotidine across
Caco-2 Cell Monolayers
KIHO LEE, CHEE NG, KIM L. R. BROUWER, and DHIREN R. THAKKER
Division of Drug Delivery and Disposition, School of Pharmacy, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
Received May 9, 2002; accepted July 8, 2002
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 inArticle, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
DOI: 10.1124/jpet.102.038521.
ylammonium (TEA)-sensitive organic cation transporters are
not involved in the uptake of ranitidine and famotidine across
the BL membrane of Caco-2. This conclusion was based on the
evidence that functionally active TEA-sensitive organic cation
transporters did not exist in the BL membranes of the Caco-2
cells, whereas the functionally active TEA-sensitive organic
cation transporter(s) in LLC-PK1 cells did not contribute to the
transport of ranitidine or famotidine across the cell monolayers.
Thus, we conclude that the secretory transport of ranitidine and
famotidine across Caco-2 cell monolayers is mediated by 1) a
carrier in the BL membrane that is distinct from the TEAsensitive organic cation transporter(s) and 2) P-gp in the apical
membrane.
testine 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
ABBREVIATIONS: P-gp, P-glycoprotein; AP, apical; BL, basolateral; CsA, cyclosporin A; 2,4-DNP, 2,4-dinitrophenol; OCT, organic cation
transporter; MPP⫹, 1-methyl-4-phenylpyridinium.
574
Downloaded from jpet.aspetjournals.org at ASPET Journals on July 31, 2017
ABSTRACT
The secretory transport of the H2-antagonists, ranitidine and
famotidine, across Caco-2 cell monolayers was found to be a
saturable process. Both drugs exhibited greater permeability in
the basolateral (BL) to apical (AP) direction than in the AP to BL
direction, indicating apically directed secretion; BL to AP transport was inhibited by P-glycoprotein (P-gp) inhibitors verapamil
and cyclosporin A. The cellular uptake of ranitidine across the
BL membrane was saturable and temperature dependent, indicative of carrier-mediated transport. The Km and Vmax for the
uptake process were estimated to be 66.9 mM and 20.9
nmol/mg of protein/min, respectively. The uptake of [14C]ranitidine across the BL membrane was inhibited by unlabeled
ranitidine and structurally diverse organic cations. The tetraeth-
Secretory Transport of Ranitidine and Famotidine
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.
Materials and Methods
Fig. 1. Structures of ranitidine and famotidine.
port 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.
The radiolabeled compounds were quantified with a liquid scintillation spectrometer (Tri-Carb 4000; PerkinElmer Life Sciences, Boston, MA). Rhodamine 123 was quantified with a spectrofluorometer
(PerkinElmer Limited, Beaconsfield, Buckinghamshire, UK); ␭ex ⫽
500 nm, ␭em ⫽ 524 nm. Ranitidine and famotidine were quantified
with high-performance liquid chromatography (1100 series, Hewlett
Packard, Waldbronn, Germany) on a Prodigy ODS(2) column (150 ⫻
4.6-mm i.d.; Phenomenex, Torrance, CA) of 5 ␮m particle and 150 Å
pore size, eluted with an isocratic mobile phase [80% 50 mM phosphate buffer (pH 6.0) and 20% methanol for ranitidine, and 90% 50
mM phosphate buffer (pH 6.0) and 10% methanol for famotidine]).
The flow rate of the mobile phase was 1.0 ml/min, the injection
volume was 100 ␮l, and the temperature of the column compartment
was maintained at 40°C and 25°C for ranitidine and famotidine,
respectively. Ranitidine and famotidine were detected by UV at 320
nm and 270 nm, respectively. Under these conditions, the retention
times for ranitidine and famotidine were 5.2 and 11.8 min, respectively, and no other peaks were detected after the transport experiments (Lee and Thakker, 1999).
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 ␮M 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).
To examine the effect of the metabolic inhibitor 2,4-dinitrophenol
(2,4-DNP) on BL uptake of ranitidine in Caco-2 cells, the cell monolayers were preincubated with 2,4-DNP (1 mM), and the uptake
studies were carried out as described above in the presence of 2,4DNP (1 mM). The uptake of ranitidine (0.1 mM) in the presence and
absence of 2,4-DNP was compared.
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.
Downloaded from jpet.aspetjournals.org at ASPET Journals on July 31, 2017
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 ␮g/ml streptomycin, and 0.25 ␮g/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 ␮m 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 ⍀ 䡠
cm2 after 20⬃25 days postseeding and LLC-PK1 cell monolayers
with transepithelial electrical resistance values above 110 ⍀ 䡠 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 trans-
575
576
Lee et al.
Results
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 Thak-
Downloaded from jpet.aspetjournals.org at ASPET Journals on July 31, 2017
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.
ker, 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).
The effect of P-gp inhibitors on the BL to AP transport of
ranitidine and famotidine was investigated in Caco-2 cell
monolayers to confirm the involvement of P-gp in the secretory transport of these H2-antagonists. Consistent with the
previous findings (Collett et al., 1999), the P-gp inhibitors
such as CsA (20 ␮M) and (⫾)-verapamil (100 ␮M) caused a
significant decrease in the BL to AP transport of ranitidine
(51% and 68%, respectively) (data not shown). These inhibitors also decreased BL to AP transport of famotidine by 27
and 62%, respectively. These results imply that the transport
of ranitidine and famotidine in the BL to AP direction must
have a significant transcellular component, distinct from the
previously reported paracellular transport of those compounds in the AP to BL direction (Gan et al., 1993; Lee and
Thakker, 1999).
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
␮M) (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
(Vmax) and the apparent Michaelis-Menten constant (Km) 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
Secretory Transport of Ranitidine and Famotidine
577
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).
As shown in Fig. 4A, the BL to AP transport of [14C]TEA
across Caco-2 cell monolayers was significantly lower than
that of ranitidine or famotidine, and similar to that of
[14C]mannitol at equimolar concentrations. Thus, it appears
Downloaded from jpet.aspetjournals.org at ASPET Journals on July 31, 2017
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
(F) or presence (E) of CsA (20 ␮M) 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 (E, F)
or [14C]mannitol (f) was measured at 37°C (F, f) or 4°C (E) 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 ␮M) 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.
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.
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 H2antagonists, an organic cation transporter(s), such as OCTs
or OCTNs (Koepsell, 1998), may be involved in the transport
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 ␮M [14C]TEA (f), ranitidine (F), 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 (F) or 4°C (䡺) for the initial 2 min after addition of TEA to the BL
side. Data represent mean ⫾ S.D.; n ⫽ 3.
578
Lee et al.
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 ␮M [14C]TEA (dotted diamond), ranitidine (F), famotidine
(dotted triangle), or [14C]mannitol (f) to the BL side. The transported
amount was expressed as percentage of the initial amount in the BL
compartment.
Discussion
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 (Km 95– 463 ␮M) 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 paracel-
Downloaded from jpet.aspetjournals.org at ASPET Journals on July 31, 2017
that BL to AP transport of TEA across Caco-2 cell monolayers, unlike that of ranitidine and famotidine, is predominantly via paracellular passive diffusion. These results suggested that either a TEA-sensitive transporter is not present
in the BL membrane of Caco-2 cells, or TEA could not exit the
AP membrane after being taken up by a transporter in the
BL membrane. To determine whether a TEA-sensitive transporter is functionally present in the BL membrane of Caco-2
cells, BL [14C]TEA uptake was measured. The uptake was
linear over a wide range of concentrations (5–10,000 ␮M)
(Fig. 4B) and did not show a saturable uptake component,
typical of a transporter-mediated process. These results suggested that TEA-sensitive organic cation transporters are not
functionally present, and thus cannot be responsible for the
BL uptake of ranitidine or famotidine into Caco-2 cells.
The TEA-sensitive organic cation transporters are known
to be present in LLC-PK1 cells (Fauth et al., 1988; Fouda et
al., 1990; McKinney et al., 1992; Saito et al., 1992; Tomita et
al., 1997). Hence, as a positive control, BL to AP flux of
[14C]TEA across LLC-PK1 cell monolayers was determined
and, as expected, was found to be much greater than that of
[14C]mannitol (Fig. 5). In contrast, the BL to AP flux of
ranitidine and famotidine across LLC-PK1 cell monolayers
was similar to that of [14C]mannitol, and much lower than
that of [14C]TEA (Fig. 5).
Inhibition of BL Uptake of [14C]Ranitidine across
Caco-2 Cell Monolayers by Organic Cations. The uptake
of [14C]ranitidine (0.1 ␮M) 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.
Secretory Transport of Ranitidine and Famotidine
TABLE 1
Effect of organic cations on the BL uptake of [14C]ranitidine across
Caco-2 cell monolayers
Organic Cations
None
Ranitidine
Diphenhydramine
Chloroquine
Guanethidine
Famotidine
TEA
MPP⫹
Concentration
Uptakea
mM
% of control
0
10
1
1
10
10
10
5
100 ⫾ 6.0
61 ⫾ 2.0**
55 ⫾ 3.7**
55 ⫾ 10.3**
74 ⫾ 8.4*
57 ⫾ 3.7**
78 ⫾ 25b
84 ⫾ 7b
a
The uptake of 0.1 mM [14C]ranitidine was measured for the initial 2 min after
addition to the BL side in the absence or presence of organic cations added to the BL
side. The control uptake was 0.093 nmol/mg of protein (mean ⫾ S.D.; n ⫽ 3).
b
The control uptake was 0.1 nmol/mg of protein.
*p ⬍ 0.05, **p ⬍ 0.01 compared to control.
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.
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 concentrationtime 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,
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.
Downloaded from jpet.aspetjournals.org at ASPET Journals on July 31, 2017
lular 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 Km value of this substrate
for known organic cation transporters is 463 ␮M (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
579
580
Lee et al.
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.
Acknowledgments
We thank Dr. Pieter Annaert for valuable discussion on cellular
uptake studies.
References
Address correspondence to: Dr. Dhiren R. Thakker, Division of Drug Delivery and Disposition, School of Pharmacy, CB no. 7360, Beard Hall, The
University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7360.
E-mail: [email protected]
Downloaded from jpet.aspetjournals.org at ASPET Journals on July 31, 2017
Arimori K and Nakano M (1998) Transport of furosemide into the intestinal lumen
and the lack of effect of gastrointestinal dialysis by charcoal in rats with acute
renal failure. J Pharmacobio-Dyn 11:1– 8.
Artursson P (1990) Epithelial transport of drugs in cell culture. I: A model for
studying the passive diffusion of drugs over intestinal absorptive (Caco-2) cells.
J Pharm Sci 79:476 – 482.
Bleasby K, Chauhan S, and Brown CDA (2000) Characterization of MPP⫹ secretion
across human intestinal Caco-2 cell monolayers: role of P-glycoprotein and a novel
Na⫹-dependent organic cation transport mechanism. Br J Pharmacol 129:619 –
625.
Collett A, Higgs NB, Sims E, Rowland M, and Warhurst G (1999) Modulation of the
permeability of H 2 receptor antagonists cimetidine and ranitidine by Pglycoprotein in rat intestine and the human colonic cell line Caco-2. J Pharmacol
Exp Ther 288:171–178.
Collett A, Sims E, Walker D, He Y-L, Ayrton J, Rowland M, and Warhurst G (1996)
Comparison of HT29 –18-C1 and Caco-2 cell lines as models for studying intestinal
paracellular drug absorption. Pharm Res (NY) 13:216 –221.
Cook MJ and Hirst BH (1994) Transepithelial secretion of the histamine H-2 receptor antagonists ranitidine in human intestinal epithelial Caco-2 monolayers is
mediated by P-glycoprotein. J Physiol (Lond) 479P:103.
Dudley AJ and Brown CD (1996) Mediation of cimetidine secretion by P-glycoprotein
and a novel H(⫹)-coupled mechanism in cultured renal epithelial monolayers of
LLC-PK1. Br J Pharmacol 117:1139 –1144.
Fauth C, Rossier B, and Roch-Ramel F (1988) Transport of tetraethylammonium by
a kidney epithelial cell line (LLC-PK1). Am J Physiol 23:F351–F357.
Fouda A-K, Fauth C, and Roch-Ramel F (1990) Transport of organic cations by
kidney epithelial cell line LLC-PK1. J Pharmacol Exp Ther 252:286 –292.
Gan L-S, Hsyu P-H, Pritchard JF, and Thakker DR (1993) Mechanism of intestinal
absorption of ranitidine and ondansetron transport across Caco-2 cell monolayers.
Pharm Res (NY) 10:1722–1725.
Gan L-S and Thakker DR (1997) Applications of the Caco-2 model in the design and
development of orally active drugs: elucidation of biochemical and physical barriers posed by the intestinal epithelium. Adv Drug Deliv Rev 23:77–98.
Gorboulev V, Ulzheimer JC, Akhoundova A, Ulzheimer-Teuber I, Karbach U,
Quester S, Baumann C, Lang F, Busch AE, et al. (1997) Cloning and characterization of two polyspecific organic cation transporters from man. DNA Cell Biol
16:871– 881.
Gramatte T, el Desoky E, and Klotz U (1994) Site-dependent small intestinal absorption of ranitidine. Eur J Clin Pharmacol 46:253–259.
Gramatte T, Oertel R, Terhaag B, and Kirch W (1996) Direct demonstration of small
intestine secretion and site-dependent absorption of the beta-blocker talinolol in
humans. Clin Pharmacol Ther 59:541–549.
Grundemann D, Gorboulev V, Gambaryan S, Veyhl M, and Koepsell H (1994) Drug
excretion mediated by a new prototype of polyspecific transporter. Nature (Lond)
372:549 –552.
Hidalgo J, Raub TJ, and Borchardt RT (1989) Characterization of the human colon
carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability. Gastroenterology 96:736 –749.
Hunter J, Jepson MA, Tsuruo T, Simmons NL, and Hirst BH (1993) Functional
expression of P-glycoprotein in apical membranes of human intestinal Caco-2 cells.
Kinetics of vinblastine secretion and interaction with modulators. J Biol Chem
268:14991–14997.
Inui K, Saito H, and Hori R (1985) H⫹-gradient-dependent active transport of
tetraethylammonium cation in apical-membrane vesicles isolated from kidney
epithelial cell line LLC-PK1. Biochem J 227:199 –203.
Koepsell H (1998) Organic cation transporters in intestine, kidney, liver and brain.
Annu Rev Physiol 60:243–266.
Koepsell H and Arndt VG (1999) Molecular pharmacology of organic cation transporters in kidney. J Membr Biol 167:103–117.
Lee K and Thakker DR (1999) Saturable transport of H2-antagonists ranitidine and
famotidine across Caco-2 cell monolayers. J Pharm Sci 88:680 – 687.
Lennernas H and Regardh CG (1993) Dose-dependent intestinal absorption and
significant intestinal excretion (exsorption) of the beta-blocker pafenolol in the rat.
Pharm Res (NY) 10:727–731.
Lin JH (1991) Pharmacokinetic and pharmacodynamic properties of histamine H2receptor antagonists. Clin Pharmacokinet 20:218 –236.
McKinney TD, Scheller MB, Hosford M, Lesniak ME, and Haseley TS (1992) Basolateral transport of tetraethylammonium by a clone of LLC-PK1 cells. J Am Soc
Nephrol 2:1507–1515.
Pinto M, Robine-Leon S, Appay MD, Kedinger M, Triadou N, Dussaulx EB, Croix B,
Simmon-Assmann P, Haffen K, Fogh J, et al. (1983) Enterocyte-like differentiation
and polarization of the human colon carcinoma cell line Caco-2 in culture. Biol Cell
47:323–330.
Piyapolrungroj N, Li C, Pisoni RL, and Fleisher D (1999) Cimetidine transport in
brush-border membrane vesicles from rat small intestine. J Pharmacol Exp Ther
289:346 –353.
Plusquellec Y, Campistron G, Staveris S, Barre J, Jung L, Tillement JP, and Houin
G (1987) A double-peak phenomenon in the pharmacokinetics of veralipride after
oral administration: a double-site model for drug absorption. J Pharmacokinet
Biopharm 15:225–239.
Saitoh H, Gerard C, and Aungst BJ (1996) The secretory intestinal transport of some
beta-lactam antibiotics and anionic compounds: a mechanism contributing to poor
oral absorption. J Pharmacol Exp Ther 278:205–211.
Saito H, Yamamoto M, Inui K, and Hori R (1992) Transcellular transport of organic
cation across monolayers of kidney epithelial cell line LLC-PK1. Am J Physiol
262:C59 –C66.
Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD,
Fujimoto EK, Goeke NM, Olson BJ, and Klenk DC (1985) Measurement of protein
using bicinchoninic acid. Anal Biochem 150:76 – 85.
Somogyi AA, Simmons N, and Gross AS (1994) In vitro potencies of histamine
H2-receptor antagonists on tetraethylammonium uptake in rat renal brush-border
membrane vesicles. J Pharm Pharmacol 46:375–377.
Suttle AB and Brouwer KLR (1994) Bile flow but not enterohepatic recirculation
influences the pharmacokinetics of ranitidine in the rat. Drug Metab Dispos
22:224 –232.
Suttle AB and Brouwer KLR (1995) Gastrointestinal transit and distribution of
ranitidine in the rat. Pharm Res (NY) 12:1316 –1321.
Tamai I, Ohashi R, Nezu J, Yabuuchi H, Oku A, Shimane M, Sai Y, and Tsuji A
(1998) Molecular and functional identification of sodium ion-dependent, high affinity human carnitine transporter OCTN2. J Biol Chem 273:20378 –20382.
Tamai I, Yabuuchi H, Nezu J, Sai Y, Oku A, Shimane M, and Tsuji A (1997) Cloning
and characterization of a novel human pH-dependent organic cation transporter,
OCTN1. FEBS Lett 419:107–111.
Tomita Y, Otsuki Y, Hashimoto Y, and Inui K (1997) Kinetic analysis of tetraethylammonium transport in the kidney epithelial cell line, LLC-PK1. Pharm Res (NY)
14:1236 –1240.
Wagner C, Lukewille U, Kaltenbach S, Moschen I, Broer A, Risler T, Broer S, and
Lang F (2000) Functional and pharmacological characterization of human Na⫹carnitine cotransporter hOCTN2. Am J Physiol 279:F584 –F591.
Wu X, Huang W, Ganapathy M, Wang H, Kekuda R, Conway S, Leibach F, and
Ganapathy V (2000) Structure, function and regional distribution of the organic
cation transporter OCT3 in the kidney. Am J Physiol 279:F449 –F458.
Yabuuchi H, Tamai I, Nezu J, Sakamoto K, Oku A, Shimane M, Sai Y, and Tsuji A
(1999) Novel membrane transporter OCTN1 mediates multispecific, bidirectional
and pH-dependent transport of organic cations. J Pharmacol Exp Ther 289:768 –
773.
Zhang L, Dresser MJ, Gray AT, Yost SC, Terashita S, and Giacomini KM (1997)
Cloning and functional expression of a human liver organic cation transporter. Mol
Pharmacol 51:913–921.
Zhang L, Gorset W, Dresser MJ, and Giacomini KM (1999) The interaction of
n-tetraalkylammonium compounds with a human organic cation transporter,
hOCT1. J Pharmacol Exp Ther 288:1192–1198.