EDITORIAL FOCUS
Riboflavin uptake by human-derived colonic epithelial NCM460
cells
Hamid M.
Said,
Alvaro
Ortiz,
Mary Pat
Moyer, and
Norimoto
Yanagawa
Veterans Affairs Medical Center, Long Beach 90822; Veterans Affairs
Medical Center, Sepulveda 91343; Departments of Medicine and
Physiology/Biophysics, University of California at Irvine, Irvine
92697; Department of Medicine, University of California at Los
Angeles, Los Angeles, California 90024; and INCELL Corporation, San
Antonio, Texas 78249
 |
ABSTRACT |
Normal microflora of
the large intestine synthesize a number of water-soluble vitamins
including riboflavin (RF). Recent studies have shown that colonic
epithelial cells posses an efficient carrier-mediated mechanism for
absorbing some of these micronutrients. The aim of the present study
was to determine whether colonic cells also posses a carrier-mediated
mechanism for RF uptake and, if so, to characterize this mechanism and
study its cellular regulation. Confluent monolayers of the
human-derived nontransformed colonic epithelial cells NCM460 and
[3H]RF were used in the study. Uptake of RF was
found to be 1) appreciable and temperature and energy
dependent; 2) Na+ independent; 3) saturable
as a function of concentration with an apparent Km
of 0.14 µM and Vmax of 3.29 pmol · mg
protein
1 · 3 min
1; 4) inhibited by the structural analogs
lumiflavin and lumichrome (Ki of 1.8 and 14.1 µM,
respectively) but not by the unrelated biotin; 5) inhibited in
a competitive manner by the membrane transport inhibitor amiloride
(Ki = 0.86 mM) but not by furosemide, DIDS, or
probenecid; 6) adaptively regulated by extracellular RF levels with a significant and specific upregulation and downregulation in RF
uptake in RF-deficient and oversupplemented conditions, respectively;
and 7) modulated by an intracellular
Ca2+/calmodulin-mediated pathway. These studies demonstrate
for the first time the existence of a specialized carrier-mediated
mechanism for RF uptake in an in vitro cellular model system of human
colonocytes. This mechanism appears to be regulated by extracellular
substrate level and by an intracellular
Ca2+/calmodulin-mediated pathway. It is suggested that the
identified transport system may be involved in the absorption of
bacterially synthesized RF in the large intestine and that this source
of RF may contribute toward RF homeostasis, especially that of colonocytes.
riboflavin transport; human colonocytes in culture; membrane
transport mechanism; transport regulation; normal colonic epithelial
cells
 |
INTRODUCTION |
RIBOFLAVIN (RF), a water-soluble vitamin, is essential
for normal cellular functions and growth. In its coenzyme forms,
riboflavin-5'-phosphate (FMN) and FAD, the vitamin is involved in
key metabolic reactions including carbohydrate, amino acid, and lipid
metabolism and in the conversion of folic acid and pyridoxine into
their coenzyme forms (2). RF deficiency occurs in humans and leads to a
variety of clinical abnormalities, including degenerative changes in
the nervous system, endocrine dysfunction, anemia, and skin disorders (6, 9, 12, 13, 18).
Humans and other mammals have lost their ability to synthesize RF and
thus must obtain the vitamin from exogenous sources via intestinal
absorption. The intestine is exposed to RF from two sources: 1)
the diet, and 2) the bacterially synthesized RF in the large
intestine. Absorption of dietary RF has been extensively studied over
the past three decades using a variety of intestinal preparations,
including gut everted sacs, perfused intestinal segments, purified
intestinal membrane vesicles, and cultured intestinal epithelial cells
(1, 3, 15, 19-24). Results of these studies have shown that RF
uptake takes place mainly in the proximal part of the small intestine
and involves a specialized, carrier-mediated mechanism.
With regard to the bacterially synthesized RF, it has been known that
the normal microflora of the large intestine synthesize considerable
amounts of RF and that a significant portion of this RF exists in the
free form, i.e., available for absorption (7, 17). The amount of RF
provided through this source varies depending on the diet, being
significantly higher following consumption of a vegetable-based diet
compared with a meat-based diet (7). Studies in humans and rats have
shown that the large intestine is capable of absorbing a large amount
of luminally introduced RF (8, 28). Nothing, however, is known about
the mechanism and regulation of the RF uptake process in the large
intestine. The existence of a specialized, carrier-mediated mechanism
for RF uptake in the large intestine is possible in light of the recent findings of specialized carrier-mediated systems for uptake of other
water-soluble vitamins that are synthesized by the normal microflora of
the large intestine, such as folate, biotin, and pantothenic acid (10,
26). Our aim in this study was, therefore, to test this possibility
using the human-derived colonic epithelial cell line NCM460 (16) as an
in vitro experimental model system. These cells were chosen because
they possess characteristics similar to that of the native colonocytes,
including similar uptake mechanisms (4, 10). Our study demonstrates the
existence of a specialized, carrier-mediated mechanism for RF uptake in
NCM460 cells and provides evidence to suggest that the process is
regulated by extracellular substrate levels and by an intracellular
Ca2+/calmodulin-mediated pathway.
 |
MATERIALS AND METHODS |
[3H-(G)]RF (sp act 27.5 Ci/mmol; radiochemical
purity determined by the manufacturer and by us was >98%) was
purchased from Moravek Biochemicals (Brea, CA). Trypsin, fetal bovine
serum (FBS), and other cell culture materials were obtained from Irvine
Scientific (Santa Ana, CA). Ham's F-12 growth medium was obtained from
Sigma Chemical. Unlabeled RF and all other chemicals and reagents were obtained from commercial sources and were of analytical quality.
The human-derived nontransformed colonic epithelial cell line NCM460
was routinely maintained in M3:10 medium (INCELL, San Antonio, TX) but
was shifted to Ham's F-12 culture medium supplemented with 20%
(vol/vol) FBS and antibiotics for the experiments described in this
study. NCM460 cells were used between passages 29 and 39 in this study. The cells were grown in 75-cm2
plastic flasks (Costar) at 37°C in a 5% CO2-95% air
atmosphere with media changes every 4 days. NCM460 cells were
subcultured by trypsinization with 0.05% trypsin and 0.9 mM EDTA in
Ca2+- and Mg2+-free PBS and plated onto 12-well
plates at a concentration of 5 × 105 cells/well.
Uptake of RF was studied 2-4 days following confluence. Cell
growth was observed by periodic monitoring with an inverted microscope.
Cell viability was tested by the trypan blue dye exclusion method and
found to be >95%.
Uptake experiments were performed at 37°C, unless otherwise stated.
Incubation was performed in Krebs-Ringer buffer containing (in mM) 123 NaCl, 4.93 KCl, 1.23 MgSO4, 0.85 CaCl2, 5 glucose, 5 glutamine, 10 HEPES, and 10 MES (pH 7.0), unless otherwise
stated. [3H]RF was added to the incubation
buffer at the onset of experiments and uptake was terminated after 3 min of incubation (unless otherwise specified) by the addition of 1 ml
of ice-cold buffer followed by immediate aspiration. The monolayers
were rinsed twice with ice-cold buffer and digested with 1 ml of 1 N
NaOH, neutralized by HCl, and then counted for radioactivity in a
liquid scintillation counter. Protein contents of cell digests were
estimated on parallel wells by a Bio-Rad protein assay kit. Uptake data
are means ± SE of multiple separate monolayers performed on at least
two different occasions and are expressed in femtomoles or picomoles
per milligram protein per unit time. P values of experimental
vs. simultaneously performed control groups were calculated using the
Student's t-test. Kinetic parameters of RF uptake, i.e.,
maximal velocity (Vmax) and the apparent Michaelis
constant (Km), were calculated using a computerized
nonlinear regression analysis program of the Michaelis-Menten equation
as described previously (29).
In the study examining the effect of growing cells in the presence of
different RF levels in the growth medium on subsequent uptake of
[3H]RF, cells were incubated for 24 h in a
control growth medium [a medium that contains a total of 38.15 ng/ml RF, of which 38 ng/ml RF was added to the DMEM cultured medium
and the rest (~0.15 ng/ml) was contributed by the added 5%
FBS], a RF-deficient medium (no RF added to the growth medium
except that contributed by 5% FBS), and a RF oversupplemented medium
(which contains 50-fold the normal amount of RF added to the control
growth medium, i.e., 1.91 µg/ml). When examining the effect of
growing the cells in a medium containing different levels of the RF
structural analog lumiflavin (LF), the 38 ng/ml of RF added to the
normal control growth medium was replaced with an equal amount of LF
("control LF state"). The "LF-deficient state" contained no
added LF, whereas the "LF-oversupplemented state" contained
50-fold the normal amount of LF added to the control LF state growth
medium (i.e., 1.9 µg/ml). Therefore, in all the studies with LF
levels, the RF level was ~0.15 ng/ml, which is provided by the added
5% FBS. No change in cell viability or appearance was observed
compared with normal control NCM460 monolayers in all the studies with
the different RF and LF levels.
On examining the metabolic form of the 3H radioactivity
taken up by NCM460 cells following incubation with
[3H]RF, silica-gel-precoated TLC plates and a
solvent system of ethanol and water (9:1 vol/vol) were used as
described previously (5).
 |
RESULTS |
Uptake of RF with time, and effect of temperature, incubation buffer
pH, and Na+.
Uptake of low (5.5 nM) and high (1 µM) concentrations of RF by NCM460
cells was examined as a function of time. At both concentrations, RF
uptake was linear over the entire 5 min of incubation and
occurred at a rate of 0.032 and 0.870 pmol · mg
protein
1 · min
1,
respectively (Fig. 1). A 3-min incubation
time was used as the standard incubation time in all subsequent
studies.

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Fig. 1.
Uptake of riboflavin (RF) as a function of incubation time. NCM460
cells were incubated in Krebs-Ringer buffer at 37°C in the presence
of low (5.5 nM; A) and high (1 µM; B) concentrations
of RF. Results are means ± SE of 4-8 separate uptake
determinations. When not shown, SE bars are within the symbol size.
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The metabolic form of the 3H radioactivity taken up by
NCM460 cells following a 3-min incubation with 17 nM
[3H]RF was examined using a TLC system
described in MATERIALS AND METHODS. The result showed that
the majority (97%) of the transported 3H radioactivity was
in the form of intact RF. We also examined the effect of incubation
temperature on the uptake of RF (5.5 nM) and found significant
(P < 0.01) decreases in RF uptake on decreasing buffer
temperature from 37 to 22 to 4°C (129 ± 3.14, 78.16 ± 3.05, and
36.48 ± 4.24 fmol · mg
protein
1 · 3 min
1, respectively).
The effect of varying incubation buffer H+ concentration
([H+]) and [Na+] on the
uptake of RF was also tested. [H+] was varied
by changing buffer pH over the range 5.0-8.0. The results showed
that lowering the incubation buffer pH from 8.0 to 7.0 leads to an
increase in RF (5.5 nM) uptake; no further increase, however, was
observed at lower pH values (65.79 ± 6.84, 105.62 ± 5.26, 133.50 ± 4, 131.55 ± 2.59, 133 ± 4.68, and 141.98 ± 3.31 fmol · mg
protein
1 · 3 min
1 at buffer pH 8.0, 7.5, 7.0, 6.5, 6.0, and 5.0, respectively). With regard to an effect of Na+, this was
tested by replacing the [Na+] in the incubation
medium isosmotically with Li+ or
NH+4 on RF (5.5 nM) uptake. The results
showed no inhibition in RF uptake on such replacement [121 ± 3.2, 127.19 ± 8.98, and 135.17 ± 4.11 fmol · mg
protein
1 · 3 min
1 in the presence of Na+ (control),
Li+, and NH+4,
respectively]. This is in contrast to uptake of the unrelated
biotin by these cells, which showed its known Na+
dependence [14.77 ± 0.84, 6.60 ± 0.16, and 5.36 ± 0.43 fmol · mg
protein
1 · 3 min
1 in the presence of Na+ (control),
Li+, and NH+4,
respectively]. We also examined the effect of pretreating (for 30 min) the cells with the Na+-K+-ATPase inhibitor
ouabain (1 and 10 mM) on the uptake of RF (5.5 nM). The results showed
no effect of such treatment on the vitamin uptake (128.3 ± 3.4, 134.7 ± 4.9, and 124.5 ± 8.6 fmol · mg
protein
1 · 3 min
1 in the absence and presence of 1 and 10 mM
ouabain, respectively).
Uptake of RF as a function of concentration. The initial rate
of RF uptake (3 min) by NCM460 cells was examined as a function of
increasing the substrate concentration in the incubation medium (5.5-1,000 nM). Uptake was found to include a saturable component. Uptake by this component was determined by subtracting diffusion uptake
(calculated from the slope of the line between uptake at high
pharmacological concentration of 100 µM and the point of origin) from
total uptake. Kinetic parameters of the saturable component, i.e., the
apparent Km and Vmax, were then
calculated as described in MATERIALS AND METHODS and found
to be 0.14 ± 0.004 µM and 3.29 ± 0.58 pmol · mg
protein
1 · 3 min
1, respectively (Fig.
2).

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Fig. 2.
Initial rate of RF uptake as a function of substrate concentration.
NCM460 cells were incubated for 3 min in Krebs-Ringer buffer at
37°C in the presence of different concentrations of RF. Uptake by
the saturable component was determined as described in text. Each data
point is mean ± SE of at least 7 separate uptake determinations.
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Specificity of the RF uptake system: effect of RF structural analogs
on [3H]RF uptake.
The effect of different concentrations of the structural analogs LF and
lumichrome (LC) and that of the unrelated biotin (1 mM) on the initial
rate of [3H]RF (5.5 nM) uptake was examined.
The results showed that both LF and LC cause a concentration-dependent
inhibition in [3H]RF uptake with an inhibition
constant (Ki) of 1.8 and 14.1 µM, respectively
(Fig. 3). The unrelated biotin, on the
other hand, showed no effect on RF uptake (144.2 ± 2.8 and 142 ± 2.2 fmol · mg
protein
1 · 3 min
1 for control and in the presence of biotin,
respectively).

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Fig. 3.
Dixon plot of the inhibitory effect of lumiflavin (LF) and lumichrome
(LC) on [3H]RF uptake. NCM460 cells were
incubated for 3 min at 37°C in Krebs-Ringer buffer in the presence
of 5.5 nM [3H]RF and different concentrations
of lumiflavin (A) and lumichrome (B). Results are from
a mean of at least 6 separate uptake determinations.
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Energy requirement of the RF uptake process and effect of membrane
transport inhibitors. In this study, we examined the effect of
pretreating cells for 30 min with the metabolic inhibitors p-cholomercuriphenylsulfonate (p-CMPS; 1 mM),
dinitrophenol (DNP, 10 mM), and azide (10 mM) on the uptake of RF (5.5 nM). The results showed significant (P < 0.01 for all)
inhibition in RF uptake by all tested compounds (128.3 ± 3.4, 47.8 ± 2, 74 ± 5.3, and 91.8 ± 8.5 fmol · mg
protein
1 · 3 min
1 for control and on pretreatment with p-CMPS,
DNP, and azide, respectively).
In another study, we examined the effect of the membrane transport
inhibitors furosemide, DIDS, probenecid, and amiloride (all at 1 mM) on
the uptake of RF (5.5 nM). The results showed that, with the exception
of amiloride, which caused a significant (P < 0.01)
inhibition in RF uptake, none of the other compounds inhibited RF
uptake (135. 7 ± 2.4, 137.6 ± 2.3, 139.2 ± 10.4, 145.28 ± 3.9, and 73.1 ± 5.4 fmol · mg
protein
1 · 3 min
1 for control, and in the presence of furosemide,
DIDS, probenecid, and amiloride, respectively). The inhibitory effect
of amiloride (1 mM) was observed both in the presence and absence of
Na+ in the incubation medium (134.55 ± 10.9, 45.76 ± 3.59, and 33.59 ± 2.22 fmol · mg
protein
1 · 3 min
1 for control, in the presence of Na+
and amiloride, and in the absence of Na+ and presence of
amiloride, respectively). The effect of amiloride (1 mM) was
investigated further by the Dixon method to determine the type of the
inhibition and inhibition constant. The result showed the inhibition to
be competitive in nature with a Ki of 0.86 mM (Fig.
4).

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Fig. 4.
Dixon plot of the inhibitory effect of amiloride on
[3H]RF uptake. NCM460 cells were incubated for
3 min in Krebs-Ringer buffer in the presence of different
concentrations of amiloride and [3H]RF ( ,
0.1 µM; , 1.0 µM). Results are from a mean of at least 4 separate uptake determinations.
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Regulation of RF uptake by colonic NCM460 cells: effect of
extracellular substrate levels. In this study, we examined the effect of growing NCM460 cells for 24 h under RF-deficient and RF-oversupplemented conditions on the subsequent uptake of the vitamin.
The results were compared with that of control (i.e., cells grown in
RF-sufficient medium). Growing the cells in RF-deficient medium was
found to lead to significant (P < 0.01)
upregulation of [3H]RF (5.5 nM) uptake compared
with control cells, whereas growing them under RF oversupplemented
conditions leads to a significant downregulation in uptake (182.8 ± 3.7, 124.7 ± 2.2, and 72.9 ± 1.1 fmol · mg
protein
1 · 3 min
1, respectively). Uptake of the unrelated biotin
(6.4 nM), however, was not affected by growing the cells under such
conditions (34.4 ± 0.8, 35.8 ± 0.1, and 34.6 ± 1.2 fmol · mg
protein
1 · 3 min
1 for cells grown under control, RF-deficient,
and RF oversupplemented conditions, respectively). To better define the
mechanism involved in this adaptive regulation of RF uptake, we
determined whether the effect of substrate level is mediated via
changes in the Vmax and/or the apparent
Km of the RF uptake process. This was performed by
examining the initial rate of [3H]RF uptake as
a function of concentration in cells grown under control, RF-deficient,
and RF oversupplemented conditions. Kinetic parameters were then
determined as described in MATERIALS AND METHODS. The
results (Fig. 5) showed a significant
(P < 0.01) increase in the Vmax of the RF
uptake process in cells grown under RF-deficient conditions compared
with control, while a significant (P < 0.01)
decrease in Vmax was observed in cells grown under RF oversupplemented conditions (Vmax of 3.64 ± 0.08, 5.17 ± 0.19, and 2.29 ± 0.14 pmol · mg
protein
1 · 3 min
1 for control and in cells grown under
RF-deficient and oversupplemented conditions, respectively). On the
other hand, no significant change in the apparent
Km of the RF uptake system was observed under the
different conditions (apparent Km of 0.14 ± 0.01, 0.15 ± 0.02, and 0.18 ± 0.03 µM for control and in cells grown
under RF-deficient and oversupplemented conditions, respectively). In
another study, we examined the effect of adding (at the time of media
change, i.e., 24 h before uptake studies) the transcription inhibitor actinomycin D (50 µM) on [3H]RF uptake by
cells grown in RF-deficient and RF-oversupplemented growth media.
Actinomycin D did not affect RF uptake by cells grown in
RF-oversupplemented medium (48.51 ± 1.3 and 48.34 ± 1.6 fmol · mg
protein
1 · 3 min
1 in the absence and presence of actinomycin,
respectively); however, actinomycin D caused a significant
(P < 0.01) inhibition in RF uptake by cells grown in
RF-deficient medium (147.28 ± 4.1 and 117.33 ± 4.0 fmol · mg
protein
1 · 3 min
1 in the absence and presence of actinomycin D,
respectively).

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Fig. 5.
Effect of growing cells in RF-deficient and RF-oversupplemented media
on initial rate of [3H]RF uptake as a function
of concentration. NCM460 cells were grown for 24 h in control, i.e.,
RF-sufficient ( ), RF-deficient ( ), and RF-oversupplemented ( )
media before use in uptake study. Uptake was assayed by incubating
cells for 3 min in Krebs-Ringer buffer at 37°C in the presence of
different concentrations of RF. Uptake by the saturable component was
determined as described in the text. Each data point is mean ± SE of
3-4 separate uptake determinations. When not shown, SE bars are
within the symbol size.
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In a related study, we examined the effect on
[3H]RF uptake of replacing the added RF in the
study described above with identical amounts of its structural analog
LF as described in MATERIALS AND METHODS. The results
showed that growing the cells for 24 h under an LF-deficient state
leads to a significant (P < 0.01) increase in
[3H]RF uptake compared with cells grown under
an LF control state, whereas growing them under an LF-oversupplemented
state leads to a significant (P < 0.01) decrease in
[3H]RF uptake (131.8 ± 2.73, 112 ± 3.58, and 76.2 ± 3.58 fmol · mg
protein
1 · 3 min
1 for LF-deficient, LF control, and
LF-oversupplemented states, respectively).
Regulation of RF uptake by colonic NCM460 cells: possible role of
intracellular regulatory pathways. In this study, we examined the
possible involvement of Ca2+/calmodulin- and protein kinase
C (PKC)-mediated pathways in cellular regulation of the RF uptake
process of NCM460 cells. The study was performed using specific
modulators of these pathways. The role of the
Ca2+/calmodulin-mediated pathway in the regulation of RF
uptake by NCM460 cells was investigated by examining the effect of
pretreating (for 1 h) the cells with the Ca2+/calmodulin
inhibitor calmidazolium on the uptake of RF (5.5 nM). The results
showed significant (P < 0.01) and concentration-dependent inhibition in RF uptake by this compound [108 ± 2.5, 71.5 ± 12.6, and 46.2 ± 8.2 fmol · mg
protein
1 · 3 min
1 for control (containing DMSO) and in the
presence of 10 and 50 µM of calmidazolium, respectively].
Similarly, the Ca2+/calmodulin inhibitor trifluoperazine
(TFP) also caused a significant (P < 0.01) and
concentration-dependent inhibition in RF uptake (128.7 ± 2.4, 99.4 ± 5.7, and 64.9 ± 4.9 fmol · mg
protein
1 · 3 min
1 for control and in cells pretreated with 50 and
100 µM TFP, respectively). In another experiment, we determined
whether the inhibitory effect of calmidazolium on RF uptake is mediated
via an effect on the apparent Km and/or the
Vmax of the uptake process. This was done by
examining the effect of 10 µM calmidazolium on the uptake of RF as a
function of concentration. The results (Fig.
6) showed a significant (P < 0.01) decrease in the Vmax of RF uptake and a
significant (P < 0.01) increase in apparent
Km (Vmax of 2.61 ± 0.042 and
1.93 ± 0.092 pmol · mg
protein
1 · 3 min
1; apparent Km of 0.135 ± 0.007 and 0.22 ± 0.03 µM for control and calmidazolium-pretreated
cells, respectively).

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Fig. 6.
Effect of calmidazolium on initial rate of RF uptake as a function of
substrate concentration. NCM460 cells were pretreated for 1 h in the
absence ( ) and presence ( ) of 10 µM calmidazolium. Incubation
was then continued for 3 min at 37°C in the presence of different
concentrations of RF. Uptake by the saturable component was determined
as described in the text. Each data point is mean ± SE of at least 7 separate uptake determinations. When not shown, SE bars are within the
symbol size.
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The possible role of a PKC-mediated pathway in the regulation of RF
uptake by NCM460 cells was also examined by investigating the effect of
pretreating the cells with modulators of this pathway on RF uptake. The
results showed that neither activation [with the use of phorbol
12-myristate 13-acetate (PMA; 10 uM) and
sn-1,2-dioctanoylglycerol (DAG; 10 µM)] nor inhibition
(with the use of chelerythrine) of this pathway causes significant
changes in RF uptake (141.6 ± 2.6, 149.3 ± 3.1, 152 ± 6, and
150.7 ± 7.4 fmol · mg
protein
1 · 3 min
1 for control and in the presence of PMA, DAG,
and chelerythrine, respectively).
 |
DISCUSSION |
The aim of the present study was to test for the existence of a
transport mechanism for the water-soluble vitamin RF in an in vitro
model system of human colonocytes, the NCM460 cells, and to
determine the characteristics and regulation of any existing system.
The results showed that uptake of RF by these cells is temperature
dependent, with minimal metabolic alteration in the transported
substrate. Uptake was Na+ independent, as indicated by lack
of inhibition in the vitamin uptake on replacing Na+ with
other monovalent cations and by the lack of effect on RF uptake of
pretreatment of the cells with the
Na+-K+-ATPase inhibitor ouabain. Incubation
buffer pH had a limited effect on RF uptake over the pH range of 5 and
7, but uptake decreased at higher pHs. The cause of this decrease is
not clear and requires further investigation.
The uptake process of RF by NCM460 cells was found to involve a
specialized, high-affinity carrier-mediated system. This conclusion was
supported by observations of saturation in RF uptake as a function of
concentration (apparent Km = 0.14 µM) and by the
inhibition in RF uptake by the structural analogs LF and LC
(Ki values of 1.8 and 14.1 µM, respectively). The
closer agreement of the Ki value of LF to the
apparent Km of the RF uptake process compared with
the Ki value of LC suggests that replacing the
ribityl side chain at nitrogen-10 of the isoalloxazin ring by a smaller
group (i.e., -CH3) does not markedly affect the ability of
the new compound (i.e., LF) to interact with the vitamin membrane
transporter. On the other hand, complete removal of the side chain from
position 10 of the isoalloxazin ring may lead to a decrease in the
ability of the new compound (i.e., LC) to interact with the RF membrane transporter.
The RF uptake process of NCM460 cells was energy dependent, as
indicated by the significant inhibition in the substrate uptake by
metabolic inhibitors. The study on the effect of membrane transport inhibitors on RF uptake produced interesting findings. Although furosemide, DIDS, and probenecid had no effect on RF uptake,
competitive inhibition of RF uptake was observed by amiloride. This
inhibition was observed whether the experiment was performed in the
presence or absence of Na+ in the incubation medium,
suggesting that the effect of amiloride is not mediated through an
inhibition of its main target, i.e., the Na+/H+ exchanger.
The above-described findings on the existence of a specialized,
carrier-mediated mechanism for RF uptake in cultured human colonocytes
are similar to those seen previously with human and animal small
intestinal preparations (1, 3, 15, 19-24). This suggests that the
same uptake mechanism may be operating in the large intestine to absorb
the bacterially synthesized RF. After the identification of a
carrier-mediated mechanism for RF uptake in cultured human colonocytes,
we investigated possible regulation of the identified uptake process by
extracellular RF levels. The nutritional importance of addressing this
issue stems from the finding that intraluminal RF levels in the large
intestine vary depending on the type of the diet consumed, being higher following a vegetable-based diet compared with a meat-based diet (7).
The results showed that uptake of [3H]RF by
cells grown in a RF-deficient medium is significantly higher than
uptake by cells grown in control (i.e., RF-sufficient) medium. On the
other hand, uptake by cells grown in a RF-oversupplemented medium was
significantly lower than that of control. This effect of extracellular
RF substrate levels on RF uptake appears to be specific in nature,
since uptake of the unrelated biotin was found to be similar under the
three differing RF growth conditions. The adaptive regulatory effect of
extracellular RF level on RF uptake was mediated through a significant
increase in the Vmax of the RF uptake process, with
no significant changes in the apparent Km. These
findings suggest that these adaptive regulatory effects are mediated
via changes in the number (and/or activity) and not the affinity of the
RF uptake system. The observation that the transcription inhibitor
actinomycin D caused significant inhibition in the induced RF uptake in
cells grown under RF-deficient conditions suggests possible involvement
of a transcriptional regulatory mechanism in mediating the observed
adaptive regulatory increase in RF uptake by RF-deficient conditions in
these cells. Further studies, however, are needed to confirm this
suggestion. An interesting observation was the ability of extracellular
levels of the RF structural analog LF to induce similar adaptive
regulatory changes in the RF uptake process. Because LF cannot be
phosphorylated or used as a vitamin by mammalian cells (14) and because
it appears to share the same transport system as RF [as indicated by results of the present study and previous findings (25, 23)], this finding suggests that it is the availability of a recognizable substrate by the uptake system that is important for the induction of
the adaptive regulatory effect in RF uptake by substrate levels. Further studies are needed to delineate the exact molecular
mechanism(s) involved in this regulation.
We also investigated possible regulation of the RF uptake process of
NCM460 by intracellular regulatory pathways focusing on a role for
Ca2+/calmodulin and PKC-mediated pathways. Our results
suggested a role for a Ca2+/calmodulin but not a
PKC-mediated pathway in the regulation of colonic uptake of RF. This
conclusion is based on the observations that modulators of the
Ca2+/calmodulin-mediated pathway significantly affect RF
uptake. An inhibition in the vitamin uptake was observed in cells
pretreated with calmidazolium and TFP. The effect of calmidazolium
appears to be mediated via a significant inhibition in the
Vmax of the RF uptake process and an increase in
its apparent Km. These findings suggest that the
effect is mediated via a decrease in the activity of the RF uptake
system and a decrease in its affinity, respectively. The physiological
mechanism(s) through which the Ca2+/calmodulin-mediated
pathway exerts its regulatory effect on RF uptake is not clear.
However, different mechanisms of action for this pathway have been
advanced, including activation of specific protein kinases and the
direct effect on the uptake system involved. Further studies at the
molecular level are needed to address this issue. The possible
regulation of RF uptake in NCM460 cells by a
Ca2+/calmodulin-mediated pathway is similar to what has
been observed in human-derived renal epithelial HK-2 cells and liver
Hep G2 cells (11, 25).
In summary, this is the first report showing the existence of a
specialized, carrier-mediated mechanism for RF uptake in the human-derived colonic epithelial cell line NCM460. This system appears
to be regulated by extracellular RF levels and by an intracellular Ca2+/calmodulin-mediated pathway. It is suggested that the
identified RF transport mechanism is involved in absorption of the
bacterially synthesized RF in the large intestine and that this source
of RF may contribute toward RF homeostasis, especially the localized homeostasis of colonocytes.
 |
ACKNOWLEDGEMENTS |
This study was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grants DK-47203 and DK-56061 and by the
Department of Veterans Affairs.
 |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: H. M. Said,
UCI-Long Beach VA Medical Program, VA Medical Center-151, Long Beach,
CA 90822 (E-mail: hmsaid{at}uci.edu).
Received 1 September 1999; accepted in final form 12 October 1999.
 |
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