0022-3565/00/2941-0117$03.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics
JPET 294:117–125, 2000 /2399/830131
Vol. 294, No. 1
Printed in U.S.A.
Involvement of a Receptor-Mediated Component in Cellular
Translocation of Riboflavin
SE-NE HUANG and PETER W. SWAAN
Division of Pharmaceutics, College of Pharmacy (S.-N.H., P.W.S.), and The Ohio State Biophysics Program (P.W.S.), The Ohio State University,
Columbus, Ohio
Accepted for publication March 10, 2000
This paper is available online at http://www.jpet.org
The cell membrane imposes a formidable absorption barrier to the translocation of water-soluble vitamins. To accommodate the entry of these essential nutrients, the cell expresses specific membrane proteins on the cell surface. These
proteins are part of a specialized uptake mechanism that can
be broadly categorized as carrier-mediated and receptor-mediated endocytosis (RME). The first mechanism facilitates
the movement of vitamin molecules across the cell membrane
via membrane carrier protein(s) energized by ATP hydrolysis
or the cotransport of ions moving down their electrochemical
gradient. In fact, transport of most B vitamins has been
identified to occur via a Na⫹- or H⫹-dependent carrier-mediated pathway (Dutta et al., 1999). In the second model, the
vitamin molecules may first bind to an endogenous protein
that in turn binds to surface receptors (e.g., vitamin B12) or
directly binds receptors localized in specialized membrane
regions (e.g., folate) before they are internalized into endocytic vesicles (Antony, 1996).
Cellular uptake of riboflavin, also known as vitamin B2,
has been extensively investigated in a variety of cell lines,
and organs and tissues (intestine, liver, and kidney) from
Received for publication December 16, 1999.
mannitol or cholic acid transport, whereas AP-to-BL riboflavin
(252.8%) and FITC-Tf (145.1%) transport was increased.
Brefeldin A significantly enhanced AP-to-BL riboflavin (37.1%)
and bidirectional FITC-Tf transport (AP-to-BL: 13-fold; BL-toAP: 5-fold). without affecting BL-to-AP riboflavin transport.
Combined, these data suggest an essential role of microtubuledependent movement and vesicular sorting component(s) in
the bidirectional transport of riboflavin. Dissociation of riboflavin from the cell surface was pH-dependent with significantly
higher substrate release at acidic pH, indicating the presence of
riboflavin-specific cell surface receptors. In summary, our studies provide biochemical evidence of the involvement of a receptor-mediated mechanism in the cellular translocation of
riboflavin.
several species (human, rat, and rabbit) (Said and Arianas,
1991; Rindi and Gastaldi, 1997). From these studies, it appears that riboflavin is taken up into most cells via an active,
carrier-mediated mechanism that is pH-independent. It has
been suggested that these mechanisms are present on both
surfaces of epithelial cells (Said et al., 1993; Said and Mohammadkhani, 1993). Contradictory results have been reported regarding the influence of Na⫹ on riboflavin uptake.
Although some studies suggest no obvious requirement for
Na⫹ or partial Na⫹ dependence (Middleton, 1990; Said and
Arianas, 1991), most reports indicate riboflavin transport to
be Na⫹-independent (Said and Ma, 1994; Dyer and Said,
1995). Furthermore, riboflavin uptake is not sensitive to the
Na⫹/K⫹-ATPase inhibitor ouabain (Said and Ma, 1994; Dyer
and Said, 1995), confirming the Na⫹-independent behavior of
this transport system. The apparent Na⫹, K⫹, and H⫹ (pH)
independence of the riboflavin transport system would classify this system as a uniporter, challenging the current paradigm that mammalian apical (AP) solute carrier proteins
are predominantly cotransporters (Hediger et al., 1995). The
outlined controversy surrounding riboflavin absorption
prompted us to explore alternative hypotheses to better rationalize the uptake mechanism of this important vitamin.
ABBREVIATIONS: RME, receptor-mediated endocytosis; FITC-Tf, fluorescein isothiocyanate-labeled transferrin; TfR, transferrin receptor; AP,
apical; BL, basolateral; BFA, brefeldin A; Is.c., short-circuit current.
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ABSTRACT
This study addresses the transport mechanism of riboflavin
(vitamin B2) across intestinal epithelium in the presence and
absence of pharmacologically active compounds. A polarized
transport process with a 6-fold higher basolateral (BL)-to-apical
(AP) flux was observed in both a human intestinal cell model
(Caco-2) and rat intestinal tissue. Riboflavin-specific translocation systems on both the AP and BL cell surfaces were saturable with affinity values close to most receptors (Km: 9.72 ⫾
0.85 and 4.06 ⫾ 0.03 nM, respectively). Pharmacological
agents known to alter intracellular endocytic events were used
to examine the potential involvement of receptor-mediated
events. Nocodazole significantly inhibited AP uptake (58.4%),
BL-to-AP riboflavin (56.7%) and fluorescein isothiocyanate-labeled transferrin (FITC-Tf) (31.8%) transport without affecting
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Huang and Swaan
Experimental Procedures
Materials
[3H]Riboflavin (20 Ci/mmol) and [14C]mannitol (60 mCi/mmol)
were purchased from Sigma (St. Louis, MO). [3H]Cholic acid (25
Ci/mmol) was from American Radiolabeled Chemicals Inc. (St. Louis,
MO). Cell culture materials and buffer solutions were obtained from
Life Technologies (Grand Island, NY). Transwell inserts were purchased from Costar (Corning, NY) and rat tail collagen (type I) was
from Becton Dickinson Labware (Bedford, MA). Human transferrin
(Tf), fluorescein isothiocyanate (FITC) conjugation kit, BCA protein
assay kit, nocodazole, and brefeldin A (BFA) were purchased from
Sigma. All other chemicals were from Fisher Scientific (Pittsburgh,
PA).
Cell Culture
Caco-2 cells were obtained from American Type Culture Collection
(Rockville, MD). Cells with passage numbers 23–38 were maintained
at 37°C under 5% CO2 in complete medium consisting of Dulbecco’s
modified Eagle’s medium with 10% heat-inactivated fetal bovine
serum, 1% nonessential amino acids, 100 U/ml penicillin, 100 ␮g/ml
streptomycin, and 10 mM HEPES. Cells were plated at 6 ⫻ 104
cells/cm2 in tissue culture treated flasks and used during the exponential growth phase (day 5). Cells were propagated and cultured as
described previously (Hidalgo and Borchardt, 1990). Cell monolayers
were grown on collagen-coated polycarbonate Transwell membrane
inserts (3.0-␮m pore size) at a density of 63,000 cells/cm2. The culture medium was changed every other day during the 1st week after
seeding and daily thereafter. Protein content was determined by the
BCA method using BSA as standard.
Assessment of Transepithelial Membrane Resistance
An epithelial voltohmmeter with dual electrodes (World Precision
Instruments, New Haven, CT) was used to measure transepithelial
resistances of monolayers grown on filters as described by Rindler
and Traber (1988). Filters were used only if the electrical resistance
of the monolayer exceeded 250 ⍀ 䡠 cm . Coincubation with [ C]mannitol (0.2 ␮Ci/ml) was performed as an independent confirmation
that paracellular transport in monolayers with electrical resistances
higher than 250 ⍀ 䡠 cm2 was indeed minimal.
14
Transepithelial Transport Studies (Caco-2 Cells)
Before transport studies, cell monolayers were washed twice with
warm Dulbecco’s PBS and incubated for 30 min at 37°C with substrate-free bathing medium (Hanks’ balanced salt solution containing 25 mM glucose and 10 mM HEPES, adjusted to pH 7.4). Transport studies were initiated by adding 1.5 to 480 ␮l of 10 ␮Ci/ml
[3H]riboflavin and 30 ␮l of 10 ␮Ci/ml [14C]mannitol stock solutions to
the donor compartment of pre-equilibrated Caco-2 cell monolayers,
thereby achieving final donor concentrations of 0.5 to 160 nM [3H]riboflavin and 3.6 ␮M [14C]mannitol. The donor compartment volume
was adjusted accordingly to maintain a constant 1.5-ml AP bathing
volume. The transepithelial flux of [3H]riboflavin and [14C]mannitol
from either direction was followed over time by withdrawing samples
at t ⫽ 0, 15, 30, 45, 60, 90, 120, 150, and 180 min. To maintain a
constant volume, an identical volume of bathing medium was added
back after every sample. Cell monolayers (14 days postseeding) that
exerted a mannitol flux larger than 0.18%/h/cm2 were excluded from
data analysis (Hidalgo, 1996). At the end of the experiments, the AP
and basolateral (BL) bathing media were removed. Cell monolayers
were washed three times with ice-cold PBS, pH 7.4, and cells were
scraped off the inserts and processed for liquid scintillation counting.
Transport Studies with Rat Intestinal Tissue
Three-month-old male Sprague-Dawley rats fed ad libitum were
used in all experiments. The small intestine was removed after
decapitation, and tissue was prepared for mounting in side-by-side
diffusion chambers (Ussing chamber type) as described previously
(Swaan et al., 1994). Briefly, mucosa was stripped of underlying
muscle, mounted in the Ussing chamber (1-cm2 exposed surface
area), and bathed on both sides with Ringer’s buffer solution. Solutions were circulated by gas lift with carbogen (95%O2, 5%CO2;
Liquid Carbonic, Columbus, OH) and maintained at 38°C (rat body
temperature) by water-jacketed reservoirs. Tissues were equilibrated for 30 min before initiation of experiments. Potential difference (Pd) and short-circuit current (Is.c.) were monitored during the
entire transport experiments to ascertain tissue viability. Tissue
integrity was independently determined by assessing the [14C]mannitol flux (Marks et al., 1991). Tissue viability was further assessed
at the end of the study by spiking AP sides of tissue with a 1 M
glucose solution (100 ␮l); the intestinal tissues with a 2- to 4-fold Is.c.
jump were considered viable. Is.c. values ranged from 10 to ⬃25
␮Eq/h 䡠 cm2 to 40 to ⬃125 ␮Eq/h 䡠 cm2 after addition of D-glucose.
Binding Studies
Caco-2 cells were seeded on 6-well plates and cultured for at least
14 days. Before experiments, cells were washed twice with ice-cold
PBS. After incubation at 4°C for 1 h with 2 nM [3H]riboflavin, cells
were washed twice with ice-cold PBS (6 ml/well for each 2-min wash)
and surface-bound riboflavin was released by incubation with 1 ml of
ice-cold PBS with pH values ranging from 3.0 to 8.0 for 2 min. After
buffer collection, cells were washed again with 6 ml of ice-cold PBS
and finally lysed with 0.5 ml of a 1% Triton X-100 solution. To
ascertain mass balance, cell lysates and samples from each PBS
washing step were analyzed for radiolabeled material. One nanomolar [14C]mannitol was incorporated in the incubation medium as a
control for the specificity of the washing steps. Nonspecific binding
and potential passive diffusion was determined in parallel studies by
measuring radioactivity bound in the presence of a 1000-fold excess
of nonradiolabeled riboflavin. Riboflavin-specific binding was obtained by subtracting nonspecific binding count from the total radioactivity.
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Another possibility for the active cellular uptake of riboflavin
is internalization by surface receptors via a process similar to
the RME of other water-soluble vitamins such as folate and
vitamin B12. In fact, a soluble high-affinity riboflavin-binding
protein has been detected in plasma (Zheng et al., 1988) and
the reproductive organs of either sex (Natraj et al., 1994),
although its function in riboflavin transport remains to be
defined. Interestingly, Low and coworkers recently showed
the facilitated entry of BSA into lung epithelial cells and
other cell cultures after covalently coupling BSA to riboflavin
(Wangensteen et al., 1996; Holladay et al., 1999). Conjugates
were detected in endosomal compartments, suggesting that
BSA-riboflavin conjugates enter the cell via endocytosis.
However, direct evidence of the involvement of endocytosis
and/or transcytosis in the uptake of riboflavin in any cell type
has not been reported previously.
To elucidate the cellular translocation mechanism of riboflavin in the intestine and investigate the potential involvement of an RME component, we have determined its binding
and transport in the absence and presence of pharmacologically active compounds that are known to affect vesicular
trafficking pathways. We used the well characterized Caco-2
cell line as a model for the small intestine. These cells, when
grown to confluence on polymer membrane inserts, mimic
the in vivo intestinal absorption process, enabling us to characterize vectorial substrate movement across both cell membranes and the cytoplasm.
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Receptor-Mediated Endocytosis of Riboflavin
Analytical Methods
Results
Radioactivity. The amount of dual-labeled radioactivity in the
samples was quantitated using a Beckman liquid scintillation
counter (model LS 6000IC; Fullerton, CA) at a counting efficiency of
43 and 75% for 3H and 14C, respectively.
HPLC analysis. A Beckman HPLC system, consisting of a model
166 UV detector and an LPSX gradient pump, was coupled in series
with a Packard radiometric flow scintillation analyzer (Packard Instruments, Meriden, CT). A Merck reversed phase RP-C18 column
(10 cm, 5 ␮m) was eluted at 3 ml/min with a scintillation cocktail and
1 ml/min with 87% 10 mM ammonium phosphate (pH 5.5)/acetonitrile (Pietta et al., 1982).
Data Analysis and Statistics
冉冘 冊
n
Q ⫽ Vs
C n ⫺ 1 ⫹ V tC n
(1)
n⫽1
where Q is the total amount of radioactive ligand in the donor
compartment, Vs is the sample volume, Vt is the volume of Transwell, and C1,2,. . . ,n represents the concentration of sample 1, 2,. . . , n.
Transport parameters [Michaelis-Menten constant (Km), maximum
flux (Jmax), and passive permeability coefficient (Pm)] were calculated using the NONLIN module in SYSTAT (version 8.0, SPSS Inc.,
Chicago, IL) by nonlinear regression analysis of the obtained data to
the general expression for transepithelial flux (J):
J max ⫻ C
⫹ Pm ⫻ C
Km ⫹ C
(2)
Statistical analyses were performed by one-way ANOVA and significant differences were reported with a confidence interval of 95%.
Effect of Culture Time on Riboflavin Transport.
Within 10 days after achieving confluency, Caco-2 cells undergo a differentiation program that involves formation of
tight junctions (as assessed by changes in electrical resistance across monolayers grown on filters) and marked elevations in the levels of several brush border hydrolases and
transport proteins (Delie and Rubas, 1997). When grown on
filters, this proliferation and differentiation program yields a
functional monolayer of cells strongly resembling the small
intestinal epithelium. It has been shown previously that the
transepithelial transport level of some nutrients such as
biotin and vitamin B12 across these monolayers can vary
significantly with days in culture, depending on their specific
membrane transport protein expression during differentiation (Dix et al., 1990; Ng and Borchardt, 1993). Thus, we first
investigated the optimal culture time for maximal expression
of riboflavin transport protein(s). In this experiment, we
chose a [3H]riboflavin concentration in accordance with the
baseline riboflavin concentration in human plasma (⬃12 nM)
(Zempleni et al., 1996). [14C]Mannitol was used as a control
to ascertain monolayer integrity. This compound uniquely
diffuses across the cell membrane via the paracellular pathway, providing a quantitative measure of tight junctional
development in maturing Caco-2 cell monolayers (Marks et
al., 1991, Swaan et al., 1994).
A relatively high mannitol flux 7 days after seeding indicates that a cohesive monolayer has not yet been established
(Fig. 1). This supports the premise that membrane leakiness
accounts for the apparent maximal riboflavin flux in the 1st
week postseeding. After 7 days in culture, however, no significant difference can be observed in the transepithelial flux
of either riboflavin or mannitol in both transport directions
with regard to culture time (AP-to-BL or BL-to-AP; P ⬎ .05;
one-way ANOVA). Accordingly, all subsequent transport
studies were performed at or after 14 days in culture. The
BL-to-AP riboflavin flux is consistently higher, approxi-
Fig. 1. Effect of culture time on riboflavin transport in Caco-2 cell monolayers. On days 7, 14, 21, and 28 postseeding, transport studies were
initiated by spiking AP or BL sides of
Caco-2 cell monolayers with [3H]riboflavin (Rf) and [14C]mannitol (Ma) (final concentrations 5 nM and 3.6 ␮M,
respectively). Flux values were calculated according to methods described
under Experimental Procedures. Each
bar/point represents the mean ⫾ S.D.
of triplicate cell monolayers.
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Uptake of riboflavin or cholic acid by cell monolayers was expressed as femtomoles per milligram of protein. Unidirectional flux
was estimated over a period of 0 to 180 min with an observed lag time
of approximately 10 to 15 min. Linearity was observed up to at least
180 min. Values in figures and tables are means ⫾ S.D. of at least
three different experiments with cells from different passages. The
effect of sample withdrawal was taken into account for the calculation of fluxes using the following equation:
J⫽
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Huang and Swaan
Effect of Endocytosis Inhibitors on Riboflavin
Transport. The affinity values (Km) for carrier-mediated
transport pathways are generally in the micromolar range,
whereas most receptor-mediated processes exert substrate
affinity values in the low nanomolar range (Feener and King,
1998). Concentration-dependence studies reveal two highaffinity riboflavin transport systems with Km values in the
low nanomolar range (Table 1). Therefore, our results suggest the involvement of a receptor-mediated mechanism in
the transepithelial transport of riboflavin. Moreover, most
receptor-mediated transcytosis systems transport their substrates preferentially in the BL-to-AP direction (Okamoto,
1998). The interesting consistency between these observations and our current results led us to hypothesize that
cellular uptake of riboflavin may use an RME/transcytosis
mechanism similar to that of other vitamins in the B group,
e.g., folate and vitamin B12. Without the availability of a
cDNA clone of the postulated transporter/receptor, it is relatively difficult to distinguish between RME and carriermediated pathways using contemporary biochemical or electrophysiological techniques because both mechanisms reveal
characteristics of active transport processes. However, several inhibitors are known to affect specific processes and/or
organelles involved in vesicle trafficking, protein sorting,
RME, and transcytosis. To directly test our hypothesis, two
inhibitors with different mechanisms of action were chosen:
nocodazole and BFA. At the cellular level, nocodazole inhibits
endocytosis by depolymerizing microtubules (Hamm-Alvarez
and Sheetz, 1998), which are necessary for endocytic vesicles
to move within the cell, and BFA induces missorting of vesicles in the trans-Golgi network (Wan et al., 1992).
To confirm the specificity of endocytosis inhibitors in distinguishing carrier-mediated processes from RME, we also
examined the transepithelial transport of cholic acid (negative control) and Tf (positive control). Cholic acid is a known
substrate of the intestinal bile acid transporter, a well characterized carrier-mediated transport system (reviewed by
Swaan, 1996). In Caco-2 cells, it has been demonstrated that
cholic acid is absorbed via a bile acid transporter with a Km
of 49.7 ␮M (Hidalgo and Borchardt, 1990). Furthermore, the
transepithelial flux of cholic acid can serve as an indicator for
functional integrity and viability of a Caco-2 cell culture
system (Hidalgo, 1996). Tf, an iron transport protein internalized by the Tf receptor (TfR), is used as a positive control
for endocytosis because the RME mechanism of TfR has been
well established (reviewed by Huebers and Finch, 1987).
Endocytosis of FITC-labeled Tf (FITC-Tf) has been characterized in different cell lines, and FITC-Tf is widely used as
an endosome marker in microscopic analysis of an RME
event (Teter et al., 1998). Thus, to visualize and detect the
transport of Tf across the Caco-2 cell monolayer, a FITC-Tf
conjugate was synthesized.
Figure 5 shows the effect of endocytosis inhibitors on bidirectional transepithelial transport of riboflavin, cholic acid,
and FITC-Tf. Compared with the control, nocodazole inhibits
BL-to-AP transport of riboflavin and FITC-Tf (56.7 and
31.8%, respectively) but does not affect cholic acid transport
from both directions (Fig. 5A). AP-to-BL transport of riboflavin and FITC-Tf is significantly increased after treatment
with nocodazole (Fig. 5B). Corresponding mannitol fluxes
indicate that nocodazole does not compromise the intactness
of Caco-2 cell monolayers during these transport studies
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mately 6-fold, compared with the AP-to-BL flux, regardless of
culture time.
Metabolic Stability of [3H]Riboflavin. In addition to
formation of tight junctions, functional differentiation in
Caco-2 cells is accompanied by elevated expression of several
metabolic enzymes. Multiple studies have shown that enzymatic activities gradually increase during differentiation and
reach maximal levels 15 to 21 days after confluency (reviewed by Delie and Rubas, 1997). It has also been suggested
that phosphorylation of riboflavin inside enterocytes is an
essential step of its AP absorption (Kasai et al., 1988). To
investigate whether putative enzymatic conversion and metabolic instability of [3H]riboflavin (which contains a general
tritium label) could contribute to the observed transport polarity, we analyzed samples from transport experiments on
an HPLC system coupled to a radiometric liquid scintillation
detector. Chromatograms of samples after a 3-h transport
study (Fig. 2) indicated that most radiolabeled riboflavin on
the donor side remains unchanged (Fig. 2, B and C), although
we cannot exclude the possibility that any metabolites are
below the limit of detection on this system. On the AP acceptor side, 85% of transported ligand remains in the form of
unchanged riboflavin (Fig. 2A), whereas the remaining activity cannot be ascribed to the major coenzyme forms of riboflavin, FMN and FAD, which would elute at 2.5 and 3.0 min,
respectively.
Polarization of Riboflavin Transport in Rat Tissue.
Although the Caco-2 cell culture system is known to closely
mimic small intestinal enterocytes, it has been reported that
these cells exert slightly different biochemical indices and
constitute altered protein composition and expression when
compared with human small intestinal enterocytes. To further validate that the observed transport polarity of riboflavin did not reflect a cellular aberration inherent to the biochemical differences between Caco-2 cells and human
intestinal epithelial cells in vivo, we conducted transport
studies using rat intestinal tissues mounted in side-by-side
diffusion chambers (Ussing chamber type). Figure 3 shows
that riboflavin transport in rat intestine from both directions
is linear up to 180 min with a significantly greater BL-to-AP
flux (approximately 5-fold, 577.5 fmol/h/cm2; R2 ⫽ 0.99) over
AP-to-BL flux (124.9 fmol/h/cm2; R2 ⫽ 0.98).
Concentration Dependence of Riboflavin Transport.
The observed polarization in transepithelial transport of riboflavin could be attributed to the presence of two different
transport systems located on the AP and BL sides of the
membrane. To test this possibility, concentration-dependence experiments were carried out in both transport directions. Figure 4 demonstrates that riboflavin is transported by
a binary mechanism consisting of both a saturable and a
linear (passive) component. At concentrations near basal
plasma riboflavin concentrations, riboflavin is transported
predominantly by the saturable component. Table 1 lists the
kinetic parameters of the transport systems on both sides of
the epithelium. Consistent with our findings (previous sections) that riboflavin transport is greater in the BL-to-AP
direction, we determined that a BL transport system had a
2-fold higher affinity (Km) compared with the AP translocation system. These results suggest that the higher BL-to-AP
riboflavin transport is partly due to the higher affinity of
riboflavin for the BL system.
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Receptor-Mediated Endocytosis of Riboflavin
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Fig. 2. Stability of [3H]riboflavin in 14day postseeding Caco-2 cells. AP and BL
samples after a 3-h transport study were
analyzed by HPLC with radiometric detection. [3H]Riboflavin elutes at 6.0 to
6.2 min, and [14C]mannitol has a retention time of 1.6 to 1.7 min. A, AP sample
chromatogram from the 3-h BL-to-AP
transport study. B, BL sample chromatogram from the 3-h BL-to-AP transport study. A yet-to-be-defined peak was
detected in AP samples at 5.1 min. C, AP
sample chromatogram from the 3-h APto-BL study.
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Huang and Swaan
Vol. 294
(control: 5.31 ⫾ 1.33 pmol/h/cm2; nocodazole treated: 4.42 ⫾
1.55 pmol/h/cm2). Figure 5B demonstrates that after exposure of cell monolayers to BFA, AP-to-BL riboflavin flux
increases 37.1%, but the transport of cholic acid is not affected. Polarized transport of FITC-Tf is observed in the
control Caco-2 cells with a 6-fold greater BL-to-AP transport.
BFA significantly enhances FITC-Tf transport in both directions (AP-to-BL: 13-fold; BL-to-AP: 5-fold). Elevated riboflavin fluxes are not the result of monolayer leakiness as evidenced by constant mannitol fluxes (control: 5.31 ⫾ 1.33
pmol/h/cm2; BFA treated: 6.19 ⫾ 1.33 pmol/h/cm2). Compared with control, the BL-to-AP transport of riboflavin is
slightly decreased although not statistically different (P ⬎
.05), whereas cholic acid transport is not affected by BFA
treatment (Fig. 5A). To investigate the effect of nocodazole on
AP uptake of riboflavin without the interference of BL-to-AP
backflux, we performed uptake studies using tissue culture
plates. As shown in Fig. 5C, 58.4% of AP uptake of riboflavin
is blocked by nocodazole within 20 min. The combined effects
of nocodazole and BFA on both the transport and uptake of
riboflavin demonstrated the involvement of an RME mechanism. This observation is further substantiated by the effects
of these compounds on FITC-Tf and cholic acid.
pH-Dependent Dissociation of Surface-Bound Riboflavin. An additional distinct mechanistic feature of ligands
for RME pathways is pH-dependent dissociation of these
molecules from their receptors (reviewed by Mukherjee et al.,
1997). Consequently, we performed surface binding experiments at 4°C using wash buffers with pH values of 3 to 8. At
4°C, the cellular internalization of riboflavin is significantly
blocked (data not shown). Nonspecific binding is determined
from parallel studies in the presence of a 1000-fold excess of
unlabeled riboflavin, and [14C]mannitol binding serves as a
negative control. A decrease in pH from 8 to 3 resulted in
elevated dissociation of riboflavin from its specific binding
site(s) with significantly greater amounts of riboflavin released at pH 3 and 4, whereas the release of mannitol is
pH-independent (Fig. 6). At physiological pH of the small
intestinal lumen (pH 6 –7), riboflavin shows higher binding
interaction to its cell surface receptors.
Fig. 4. Concentration dependence of transport of riboflavin (Rf) in Caco-2
cell monolayers. Radiolabeled riboflavin (0.5–160 nM) and mannitol (3.6
␮M) were added to the BL (A) or AP (B) side of Caco-2 cell monolayers on
the 14th day postseeding. Each point represents the mean ⫾ S.D. of
triplicate cell monolayers. The solid line represents the calculated fit of
eq. 2 as described in the experimental procedure. The estimated active
transport component (Jactive) to the total riboflavin flux (Jtotal) is indicated
with a dotted line; the estimated passive component (Jpassive) is indicated
with a dashed line.
TABLE 1
Transport parameters of bidirectional transport of riboflavin in Caco-2
cell monolayers
Transport parameters were calculated by nonlinear regression analysis of data
obtained from the concentration-dependence study using eq. 2. Parameters are
presented as estimates ⫾ asymptotic S.E.
Km
BL-to-AP
AP-to-BL
Jmax
nM
fmol/hr/cm2
4.06 ⫾ 0.03
9.72 ⫾ 0.85
168.2 ⫾ 0.38
192.98 ⫾ 0.85
Discussion
Although multiple studies have characterized the uptake
mechanism of riboflavin in a variety of tissues and species,
there is surprisingly little information on the transepithelial
transport of riboflavin across different cell types. In this
study, we find that at concentrations around its Km, riboflavin transport from the BL to the AP surface is 6-fold greater,
compared with the flux in the AP-to-BL direction. This ele-
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Fig. 3. Time course of riboflavin transport in rat ileum. Rat ileal mucosa
was mounted in diffusion chambers, circulated with 95% O2, 5% CO2 and
with Ringer’s solution containing 0.1 ␮M nonradioactive riboflavin on
both sides. Transport studies were initiated by spiking the mucosal (F) or
serosal (E) bathing solution with [3H]riboflavin and [14C]mannitol (final
concentrations 5 nM and 3.6 ␮M, respectively).
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Receptor-Mediated Endocytosis of Riboflavin
123
Fig. 5. Effect of nocodazole and BFA on transport and uptake of riboflavin (Rf), cholic acid (CA), and FITC-Tf (Tf) in Caco-2 cell monolayers.
BL-to-AP transport (A), AP-to-BL transport (B) of riboflavin, cholic acid,
and FITC-Tf in nocodazole (3.3 ␮M)- or BFA (1.65 ␮M)-treated Caco-2
cells. Five nanomolar [3H]Rf or 60 ␮g/ml FITC-Tf was added to the BL or
AP side of cell monolayers. Eight nanomolar [3H]cholic acid was applied
to the BL or AP side of cell monolayers with 50 ␮M nonradioactive cholic
acid on both sides. C, uptake of ribiflavin, cholic acid, and mannitol (Ma)
in nocodazole-treated Caco-2 cells. One nanomolar [3H]riboflavin or
[3H]cholic acid and [14C]mannitol were added to nocodazole-pretreated
(3.3 ␮M, 15 min) Caco-2 cells seeded in 12-well plates. Cells were washed
three times with ice-cold PBS, lysed with 0.1% Triton X-100, and measured for radioactivity after a 20-min uptake study. Each bar represents
mean ⫾ S.D. of triplicate cell monolayers (**P ⬍ .01; *P ⬍ .05).
vated transport from the systemic circulation into gut lumen
has not been observed previously either in cell culture systems (Fig. 1) or in intestinal tissue preparations (Fig. 3). The
fact that transport polarity is conserved between two different species (rat versus human) further confirms the physiological and biological relevance of this observation. It also
validates that the striking polarity of riboflavin transport
observed in Caco-2 cells is not due to a transport anomaly
specific to this particular cell line.
Interestingly, a similar transport polarity has been reported for intestinal Tf absorption (Shah and Shen, 1994)
with a 40-fold difference in BL-to-AP versus AP-to-BL translocation activity and receptor expression. Enterocytes are
known to obtain iron from dietary iron uptake and, during
periods of low dietary iron intake, also from body iron stores.
Analogous to intestinal iron homeostasis, we anticipate that
intracellular riboflavin levels in the gut are tightly regulated
as well. Therefore, the putative physiological function of the
riboflavin receptor on the BL surface of enterocytes is to
participate in the supply of riboflavin to maturing epithelial
cells. Because the intestine has the highest cell turnover rate
of any tissue, a continuous supply of riboflavin would be
required to support cell growth and differentiation as well as
the synthesis of essential flavoproteins such as digestive
enzymes.
The Km values we reported from transport experiments
(Table 1) are 100-fold lower than those reported by Said and
coworkers, who found values in the low micromolar range
(0.3 ␮M) via uptake studies (Said and Ma, 1994). It is essential, however, to point out two consequential differences between the previous studies and our data. First, transepithelial transport in polarized cells comprises a series of
sequential events, namely ligand-binding, internalization, ligand movement through the cytoplasm, and translocation
across two cell membranes with different lipid compositions.
Uptake experiments can only discern binding and movement
through the AP membrane. Second, we selected riboflavin
concentrations according to the reported basal human serum
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 16, 2017
Fig. 6. pH dependence of riboflavin-specific binding to cell surface. Cells
were incubated with 2 nM [3H]riboflavin (Rf; F) and 1 nM [14C]mannitol
(‚) at 4°C for 1 h. After removal of free [3H]riboflavin with two ice-cold
PBS washes, cells were incubated for 2 min at 4°C with 1 ml of PBS at
various pH values. Total ligand-binding activity was determined by measuring radioactivity in PBS release buffers. The nonspecific binding component was determined in parallel studies in which cells were incubated
with a 1000-fold excess of nonradioactive riboflavin for 1 h. A specific
binding component was calculated by subtracting the nonspecific component from total binding values. Each point represents mean ⫾ S.D. of
three experiments.
124
Huang and Swaan
transport directions (Shah and Shen, 1994). These observations are also in agreement with the findings that BFA treatment leads to enhanced transcytosis of ricin and Clostridium
botulinum neurotoxin (Prydz et al., 1992; Maksymowych and
Simpson, 1998). Shen and colleagues demonstrated that enhancement of Tf transcytosis by BFA is accompanied by a
significant increase in the number of TfRs on the AP cell
membrane and a decrease in the number of TfRs on the BL
cell membrane without affecting total TfR expression (Wan
et al., 1992; Shah and Shen, 1994). Whether a similar mechanism is responsible for increased riboflavin transport remains to be investigated.
Our finding that dissociation of riboflavin from its specific
surface binding site(s) is pH-dependent (Fig. 6) provides additional biochemical evidence corroborating our hypothesis.
This observation is consistent with a hallmark feature of
RME in which acidification of endosomes during the endocytic sorting process causes dissociation of ligands from receptors. pH-sensitive binding has been reported previously
for folate and vitamin B12, both well-characterized ligands
using RME mechanisms (Kamen and Capdevila, 1986).
In summary, our experiments demonstrate that a specialized, high-affinity riboflavin translocating system(s) exists in
the intestinal cells with affinity values and biochemical characteristics similar to most receptor-mediated systems reported in the literature. This system is primarily expressed
on the BL surface. Although the physiological role behind
this type of polarity needs to be further investigated, our
findings suggest that the BL uptake system could play an
important role in supplying nutrients to the highly proliferative intestinal epithelium. These data aid in our understanding of the transepithelial transport mechanism of riboflavin and the proteins involved in its uptake and contribute
to our knowledge of human riboflavin homeostasis and the
physiology of B vitamins in general.
Acknowledgment
We thank Dr. Michael Darby at the Ohio State University Comprehensive Cancer Center for kind assistance with the use of the
radiometric HPLC system.
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Receptor-Mediated Endocytosis of Riboflavin