|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 7 (April 1), 1998:
pp. 2536-2546
Colony-Stimulating Factors Signal for Increased Transport of Vitamin
C in Human Host Defense Cells
By
Juan Carlos Vera,
Coralia I. Rivas,
Rong H. Zhang, and
David W. Golde
From the Program in Molecular Pharmacology and Therapeutics, Memorial
Sloan-Kettering Cancer Center, New York, NY.
 |
ABSTRACT |
Although serum concentrations of ascorbic acid seldom exceed 150 µmol/L, mature neutrophils and mononuclear phagocytes accumulate millimolar concentrations of vitamin C. Relatively little is known about the mechanisms regulating this process. The colony-stimulating factors (CSFs), which are central modulators of the production, maturation, and function of human granulocytes and mononuclear phagocytes, are known to stimulate increased glucose uptake in target
cells. We show here that vitamin C uptake in neutrophils, monocytes,
and a neutrophilic HL-60 cell line is enhanced by the CSFs. Hexose
uptake studies and competition analyses showed that dehydroascorbic
acid is taken up by these cells through facilitative glucose
transporters. Human monocytes were found to have a greater capacity to
take up dehydroascorbic acid than neutrophils, related to more
facilitative glucose transporters on the monocyte cell membrane.
Ascorbic acid was not transported by these myeloid cells, indicating
that they do not express a sodium-ascorbate cotransporter. Granulocyte
(G)- and granulocyte-macrophage colony-stimulating factor (GM-CSF)
stimulated increased uptake of vitamin C in human neutrophils,
monocytes, and HL-60 neutrophils. In HL-60 neutrophils, GM-CSF
increased both the transport of dehydroascorbic acid and the
intracellular accumulation of ascorbic acid. The increase in transport
was related to a decrease in Km for transport of dehydroascorbic acid
without a change in Vmax. Increased ascorbic acid accumulation was a
secondary effect of increased transport. Triggering the neutrophils
with the peptide fMetLeuPhe led to enhanced vitamin C uptake by
increasing the oxidation of ascorbic acid to the transportable moiety
dehydroascorbic acid, and this effect was increased by priming the
cells with GM-CSF. Thus, the CSFs act at least at two distinct
functional loci to increase cellular vitamin C uptake: conversion of
ascorbic acid to dehydroascorbic acid by enhanced oxidation in the
pericellular milieu and increased transport of DHA through the
facilitative glucose transporters at the cell membrane. These results
link the regulated uptake of vitamin C in human host defense cells to
the action of CSFs.
| |
INTRODUCTION |
WIDELY HELD notions connect vitamin C
with host defense against microorganisms.1-4 Because humans
cannot synthesize vitamin C,5 it must be provided
externally and transported intracellularly.6 Vitamin C is
present in human blood at concentrations of about 50 µmol/L almost
exclusively in the reduced form, ascorbic acid. Similarly, ascorbic
acid is present in cells and tissues at concentrations that can exceed
by several orders of magnitude the blood levels of the vitamin. These
observations led to the proposal that the transport of vitamin C by
human cells occurs against a concentration gradient and in a
sodium-dependent manner, with the direct participation of
sodium-ascorbate cotransporters.6 The reduced form of
vitamin C, ascorbic acid, is present in human neutrophils and
mononuclear phagocytes in high (millimolar) concentrations, but the
identity and functional properties of the mechanisms regulating the
uptake and accumulation of vitamin C in these cells are still a matter of controversy.6,7 Studies of the interaction of vitamin C
with host defense cells have been confounded by the lack of standard
procedures for measuring the uptake of ascorbic acid in cells. In
solution, ascorbic acid undergoes reversible oxidation to
dehydroascorbic acid, a process that can be catalyzed and greatly accelerated by traces of metal ions and prevented by metal chelators or
reducing agents.8-10 The uncontrolled oxidation of vitamin C in solution has resulted in contradictory evidence concerning the
mechanism and the chemical form of vitamin C (reduced or oxidized) transported by mammalian cells.6,7,11-20 We recently showed that facilitative hexose transporters are the primary route of cellular
vitamin C transport in myeloid cells and provided evidence that they
transport only the oxidized form of the vitamin, dehydroascorbic acid.10,21,22 In vitro, human neutrophils and HL-60 cells incubated in the presence of dehydroascorbic acid accumulate high intracellular concentrations of reduced ascorbic acid in a matter of
minutes, but no dehydroascorbic acid is detected intracellularly. These
cells transport dehydroascorbic acid down a concentration gradient
through facilitative hexose transporters. Once inside the cell, the
dehydroascorbic acid is reduced to ascorbic acid, a mechanism allowing
for the trapping and accumulation of high intracellular concentrations
of reduced vitamin C.
Granulocyte (G)- and granulocyte-macrophage colony-stimulating factor
(GM-CSF) are important regulators of the growth and maturation of
myeloid precursor cells and they also enhance the function of mature
neutrophils and mononuclear phagocytes.23-29 The myeloid
CSFs stimulate increased glucose uptake in target cells, presumably to
provide increased metabolic fuel for heightened cellular activity. In
the case of GM-CSF, signaling for increased glucose uptake in target
cells is mediated through the GM-CSF receptor  subunit.30,31 Because vitamin C is transported into myeloid
host defense cells as dehydroascorbic acid through the facilitative
glucose transporters, we hypothesized that the myeloid CSFs could be
important regulators of vitamin C uptake in target host defense cells.
GM-CSF could affect vitamin C uptake in myeloid cells by mechanisms
involving a direct effect on the membrane transporters and also
increase the oxidative generation of the transported substrate,
dehydroascorbic acid, from ascorbic acid. Neutrophils and mononuclear
phagocytes activated by the chemotactic peptide fMetLeuPhe undergo the
oxidative burst.32 GM-CSF primes the cells for the
oxidative response, with the result that myeloid cells exposed to
GM-CSF and then stimulated with fMetLeuPhe generate increased amounts
of oxidant molecules.33
We show here that G- and GM-CSF increase the cellular uptake of vitamin
C by human myeloid host defense cells. Triggering the neutrophils with
the chemotactic peptide fMetLeuPhe also led to increased uptake of
vitamin C, and this effect was increased by priming the cells with
GM-CSF. Our results indicate that the effect of GM-CSF is probably
mediated by changes in the functional activity of the glucose
transporters that permit the cellular uptake of dehydroascorbic acid by
cells. The data also indicate that fMetLeuPhe affects cellular vitamin
C uptake by a mechanism that involves the oxidation of ascorbic acid to
dehydroascorbic acid without affecting the activity of the
transporters. These findings link vitamin C uptake by mature myeloid
cells to the action of CSFs, providing evidence that humoral modulators
of host defense stimulate increased transport of vitamin C in cells central to host defense against microorganisms.
| |
MATERIALS AND METHODS |
Leukopaks were provided by the New York Blood Center. Neutrophils were
purified by Ficoll-Hypaque density centrifugation followed by dextran
sedimentation of the erythrocytes. From these preparations, monocytes
were selected by adherence to culture dishes and cultured overnight in
Iscove's modified Dulbecco's medium (IMDM) supplemented with 10%
fetal calf serum, 1% glutamine and 1% penicillin/streptomycin. A
neutrophilic subline of HL-60 cells34 was cultured in IMDM supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 1% L-glutamine. Cell viability was always greater than 95% as
assessed by trypan blue exclusion.
For uptake assays, the cells were suspended in incubation buffer (15 mmol/L Hepes pH 7.6, 135 mmol/L NaCl, 5 mmol/L KCl, 1.8 mmol/L
CaCl2, 0.8 mmol/L MgCl2), washed by
centrifugation in the same buffer and resuspended at 0.5 to 2 × 107 cells per milliliter. Similarly, adherent cells were
washed with incubation buffer twice. Ascorbic acid uptake assays were
performed in a final volume of 0.2 mL incubation buffer containing 2 to 15 × 106 cells, 0.1 to 0.4 µCi of
L-[14C]-ascorbic acid (specific activity 8.2 mCi/mmol,
NEN-Dupont, Wilmington, DE), and a final concentration of 0.05 to 15 mmol/L ascorbic acid. For dehydroascorbic acid uptake, 1 to 10 U of
ascorbic acid oxidase (50 U/mg protein, Sigma, Milwaukee, WI) were
added to the incubation mixture and incubated for 2 minutes before
adding the cells. The oxidation of ascorbic acid was monitored by the decrease in absorbance at 266 nm and also by high-performance liquid
chromatography (HPLC) (see below).
Uptake was stopped by adding 10 volumes of cold Ca2+ and
Mg2+-free phosphate-buffered saline. The cells were washed
twice with cold phosphate-buffered saline, lysed in 10 mmol/L Tris-HCl
(pH 8.0) containing 0.2% sodium dodecyl sulfate and the incorporated radioactivity was determined by liquid scintillation
counting.10,21 For HPLC analysis, cells were lysed in 60%
methanol, 1 mmol/L EDTA (pH 8.0) at 4°C, and the supernatants
obtained were stored at  70°C until use. For samples
containing vitamin C in solution, the samples were adjusted to final
concentrations of methanol and EDTA of 60% and 1 mmol/L, respectively,
and stored at 70°C. HPLC analysis was performed using a
Whatman strong anion exchange reverse phase column (Partisil 10 SAX,
4.6 mm × 25 cm, 10-µm particle) with a silica preconditioning
column (Whatman guard cartridge anion exchanger). The HPLC system was
equipped with a two-channel ultraviolet (UV) diode
detector and a radioactivity detector arranged in series.35
The conditions of the chromatography were modified to decrease the
total running time to 35 minutes. The elution conditions were:
temperature, 25°C; flow, 1 mL/min; buffer A (7 mmol/L
KH2PO4, 7 mmol/L KCl, pH 4.0) and buffer B
(0.25 mol/L KH2PO4, 0.5 mol/L KCl, pH 5.0);
mobile phase, isocratic buffer A from 0 to 5 minutes, linear gradient
from 100% buffer A and 0% buffer B to 50% buffer B from 5 to 20 minutes, 100% buffer B from 20 to 25 minutes, and 100% buffer A from
25 to 35 minutes to equilibrate the column. Under these conditions,
dehydroascorbic acid eluted at 4.4 minutes and ascorbic acid eluted at
10.4 minutes.
Hexose uptake assays were similarly performed using 1 µCi of
3-O-[methyl-3H]-D-glucose (specific activity 10 Ci/mmol,
NEN-Dupont) and 0.3 to 20 mmol/L 3-O-methyl-D-glucose (methylglucose)
or 1 µCi of 2-[1,2-3H(N)]-deoxy-D-glucose (specific
activity 26.2 Ci/mmol, NEN-Dupont) and 0.3 to 20 mmol/L
2-deoxy-D-glucose (deoxyglucose). When appropriate, competitors and
inhibitors were added to the uptake assays or the cells were
preincubated in their presence. Cells were treated with recombinant
human GM-CSF (a gift from Amgen Inc, Thousand Oaks, CA) as indicated in
the figure legends.
| |
RESULTS |
Facilitated transport of dehydroascorbic acid in neutrophils and
monocytes.
We previously showed that uptake of dehydroascorbic acid by human
neutrophils and HL-60 cells is mediated by facilitative glucose
transporters and that these cells do not transport ascorbic acid.10,21,22 Data have been presented, however, implying that human neutrophils and HL-60 cells can transport ascorbic acid
through a sodium-dependent ascorbate cotransporter.7 In the
present studies, we examined the sodium dependence of the transport of
vitamin C by human neutrophils and monocytes. Long uptake assays (30 minutes to 2 hours) showed low level cellular uptake of radioactive
ascorbic acid, but those conditions are unsuited to study transport of
ascorbic acid due to the slow oxidation of ascorbic acid to
dehydroascorbic acid in solution.8-10 Given the high
efficiency of transport of dehydroascorbic acid by the glucose
transporters, the oxidation of even a fraction of a percent of the
ascorbic acid present in the incubation medium will result in the rapid
transport of the generated dehydroascorbic acid.22 Therefore, we used short uptake assays (5 minutes or less) to minimize
the effect of oxidation of ascorbic acid, and used 15 million cells per
assay to increase the sensitivity of the uptake assay. These studies
showed that dehydroascorbic acid was efficiently taken up by human
neutrophils and monocytes and that uptake was unchanged in sodium-free
medium, indicating that it was sodium independent
(Fig 1A and B). The initial rate of
dehydroascorbic acid uptake by monocytes (450 pmol/min/106
cells) was threefold higher than by neutrophils (150 pmol/min/106 cells). Also, for neutrophils, dehydroascorbic
acid uptake increased linearly with time for the length of the uptake
assay (Fig 1A), whereas for monocytes, uptake was biphasic, with a
rapid initial linear phase of uptake that lasted approximately 1 minute
followed by a second slower linear phase with a rate that was constant for the remainder of the uptake assay (Fig 1B).
View larger version (29K):
[in this window]
[in a new window]
| Fig 1.
Transport of dehydroascorbic acid by human neutrophils
and monocytes. (A) Uptake of dehydroascorbic acid (DHA) by human
neutrophils in the presence of NaCl ( ) or choline chloride ( ).
(B) Uptake of dehydroascorbic acid by human monocytes in the presence
of NaCl () or choline chloride (). (C) Uptake of ascorbic acid (AA) by human neutrophils in the presence of NaCl () or choline chloride (). (D) Uptake of ascorbic acid by human monocytes in the
presence of NaCl () or choline chloride (). (E) Ratio of the
uptake of dehydroascorbic acid versus ascorbic acid by human neutrophils in the presence of NaCl () or choline chloride (). (F) Ratio of the uptake of dehydroascorbic acid versus ascorbic acid by
human monocytes in the presence of NaCl () or choline chloride
(). Data represent the mean ± SD of four samples and correspond to
one of three similar experiments.
|
|
In marked contrast to their ability to take up large amounts of
dehydroascorbic acid, human neutrophils and monocytes accumulated only
very small amounts of radioactive material when incubated with
14C-ascorbic acid (Fig 1C and D). Moreover, uptake under
these conditions was independent of the presence of sodium ions in the
incubation medium, which militates strongly against the participation
of a sodium-dependent ascorbate cotransporter. In fact, neutrophils consistently accumulated more radioactive material when incubated with
ascorbic acid in the absence than in the presence of sodium ions (Fig
1C). In both cell types, uptake in cells incubated with ascorbic acid
was only a small fraction of the uptake observed in cells exposed to
dehydroascorbic acid. The initial rate of uptake by neutrophils
incubated with ascorbic acid in the presence of sodium ions was 0.42 pmol/min/106 cells compared with 0.30 pmol/min/106 for monocytes and increased to 0.70 nmol/min/106 cells in the absence of sodium. Thus,
neutrophils take up 150-fold to 250-fold more vitamin C when incubated
with dehydroascorbic acid compared with ascorbic acid, while monocytes
take up approximately 700-fold to 1,200-fold more vitamin C when
presented with dehydroascorbic acid compared with ascorbic acid (Fig 1E
and F).
These data indicate that human neutrophils and monocytes transport only
the oxidized form of vitamin C, dehydroascorbic acid, and do not
express a sodium-dependent cotransporter with the capacity to transport
ascorbic acid. The results also indicate that human monocytes have an
increased capacity to take up dehydroascorbic acid compared with
neutrophils. We hypothesized that the increased cellular uptake of
dehydroascorbic acid by human monocytes could reflect an increased
number of glucose transporters engaged in the transport of
dehydroascorbic acid. Consistent with this hypothesis, monocytes also
showed a greater capacity than neutrophils to take up deoxyglucose
(Fig 2A), a substrate that is transported
only by glucose transporters of the facilitative type.36
Uptake of deoxyglucose by monocytes and neutrophils increased in a
linear fashion for the first 10 minutes and reached a plateau at about 50 minutes. At the end of the 60-minute uptake assay, monocytes accumulated 2.6-fold more deoxyglucose (presumably as deoxyglucose phosphate) than neutrophils. The difference in uptake was even greater
during the initial part of the uptake curve (10 minutes or less), which
more closely reflects transport as opposed to accumulation. The initial
rate of deoxyglucose uptake by monocytes (400 pmol/min/106
cells) was 3.4-fold higher than by neutrophils (117 pmol/min/106 cells) (Fig 2A). A similar increased transport
of the nonmetabolized substrate methylglucose was observed in monocytes
(2.3-fold) compared with human neutrophils (Fig 2B). The initial rate
of methylglucose uptake by monocytes was 140 pmol/min/106
cells compared with 59 pmol/min/106 cells for neutrophils.
Methylglucose is a nonmetabolized glucose analog, and therefore changes
in the cellular uptake of this substrate are not associated with
intracellular events that occur after transport.36 In
neutrophils and monocytes, the transport of dehydroascorbic acid was
specifically completed by the transported substrates deoxyglucose and
methylglucose, but not by L-glucose, a hexose incapable of interacting
with the glucose transporters (Fig 2C and D). Additionally, transport
was inhibited by cytochalasin B, a specific inhibitor of facilitated
transport,36-39 but not by the noninhibitory compound
cytochalasin E (Fig 2C and D). The substrate of the sodium-dependent
glucose cotransporter -methyl-D-glucopyranoside40 did
not affect the uptake of dehydroascorbic acid by the cells, arguing
against the participation of this transporter in the transport of
dehydroascorbic acid (data not shown). Overall, the data are consistent
with the direct participation of facilitative glucose transporters in
the transport of dehydroascorbic acid by human neutrophils and
monocytes.
View larger version (39K):
[in this window]
[in a new window]
| Fig 2.
Human neutrophils and monocytes take up dehydroascorbic
acid through facilitative glucose transporters. (A) Uptake of
deoxyglucose (DOG) by human neutrophils () and monocytes (). (B)
Transport of methylglucose (OMG) by human neutrophils () and
monocytes (). (C) Effect of competitors and inhibitors on the uptake
of dehydroascorbic acid by human neutrophils. Uptake was assayed in the
absence (control) or in the presence of 30 mmol/L deoxyglucose, methylglucose, or L-glucose (LG), or the cells were incubated with 20 µmol/L cytochalasin B (CytB) or cytochalasin E (CytE) for 5 minutes
before the uptake assay. (D) Effect of competitors and inhibitors on
the uptake of dehydroascorbic acid by human monocytes. Uptake was
assayed in the absence (control) or in the presence of 30 mmol/L
deoxyglucose, methylglucose, or L-glucose, or the cells were incubated
with 20 µmol/L cytochalasin B or cytochalasin E for 5 minutes before
the uptake assay. For (A and B), the data represent the mean ± SD of
four samples and correspond to one of three similar experiments. For (C
and D), the data represent the average of two experiments performed in
duplicate.
|
|
Effect of G- and GM-CSF on the capacity of human neutrophils and
monocytes to take up dehydroascorbic acid.
Human neutrophils incubated in the presence of 0.5 nmol/L GM-CSF showed
an increased ability to take up dehydroascorbic acid (Fig 3A). The effect of GM-CSF on uptake
developed rapidly, with half-maximal stimulation of uptake observed at
approximately 30 minutes with a maximal effect seen at 60 minutes.
G-CSF at 0.5 nmol/L exerted a similar effect on the ability of human
neutrophils to take up dehydroascorbic acid (Fig 3A). Tumor necrosis
factor (TNF) at 1 nmol/L did not induce a change in the
capability of these cells to take up dehydroascorbic acid (Fig 3A). We
also tested the effect of the neutrophil triggering peptide
fMetLeuPhe.32,33 No effect of 10 µmol/L fMetLeuPhe on the
uptake of dehydroascorbic acid by neutrophils was observed in these
studies, a concentration known to produce a clear biologic response in
these cells. Dose-response experiments showed that GM- and G-CSF
exerted their action at low picomolar concentrations (Fig 3B).
Half-maximal stimulation of uptake was seen at 3 pmol/L GM-CSF, a
result consistent with the expression of high-affinity GM-CSF receptors
in these cells.24-28,41-43 Maximal activation was reached
at 30 pmol/L GM-CSF, with no further increase in uptake at
concentrations of GM-CSF as high as 10 nmol/L. G-CSF also showed a
dose-dependent effect enhancing the ability of human neutrophils to
take up dehydroascorbic acid, with half-maximal and maximal effects
observed at 10 and 100 pmol/L G-CSF (Fig 3B). GM-CSF consistently
induced a larger increase in dehydroascorbic acid uptake compared with
G-CSF, with increases of 60% above control values. TNF did not cause
changes in neutrophil uptake of dehydroascorbic acid at concentrations
from 1 pmol/L to 10 nmol/L (Fig 3B), confirming the specificity of the
effect of G- and GM-CSF.
View larger version (35K):
[in this window]
[in a new window]
| Fig 3.
GM-CSF and G-CSF increase the uptake of dehydroascorbic
acid (DHA) by human neutrophils and monocytes. (A) Time course of the
effect of GM-CSF () and TNF ( ) on the uptake of dehydroascorbic acid by human neutrophils. Cells were treated with 0.5 nmol/L GM-CSF or
1 nmol/L TNF for the time periods indicated in the figure. Uptake of
dehydroascorbic acid was measured afterwards in a 5-minute assay. Data
represent the mean ± SD of four samples. (B) Dose-dependence of the
effect of GM-CSF (), G-CSF () and TNF () on the uptake of
dehydroascorbic acid by human neutrophils. Cells were incubated for 30 minutes in the presence of the indicated concentrations of the
different agonists and uptake of dehydroascorbic acid was measured in a
5-minute assay. (C) Time course of the effect of GM-CSF () and G-CSF
() on the uptake of dehydroascorbic acid by human monocytes. Cells
were treated with 0.5 nmol/L GM- or G-CSF, or left untreated (controls,
), for the time periods indicated in the figure. Uptake of
dehydroascorbic acid was measured afterwards in a 5-minute assay. Data
represent the mean ± SD of four samples. (D) Dose-dependence of the
effect of GM-CSF () and G-CSF () on the uptake of dehydroascorbic
acid by human monocytes. Cells were incubated for 30 minutes in the
presence of the indicated concentrations of the different agonists and
uptake of dehydroascorbic acid was measured in a 5-minute assay. Data
represent the mean ± SD of four samples and correspond to one of
three similar experiments.
|
|
We next examined the effect of G- and GM-CSF on the transport of
dehydroascorbic acid by human monocytes. Human monocytes responded to
G- and GM-CSF with a rapid increase in their ability to take up
dehydroascorbic acid (Fig 3C). Half-maximal stimulation was observed at
30 minutes, with maximal effect observed after 60 minutes of exposure
to the cytokines. The effect of G- and GM-CSF was dose dependent, with
half-maximal stimulation observed at 3 pmol/L and maximal activation of
uptake observed at 10 pmol/L (Fig 3D).
Effect of GM-CSF on dehydroascorbic acid uptake by HL-60 neutrophils.
We recently characterized the transport of dehydroascorbic acid by
HL-60 cells and showed the direct participation of the facilitative
glucose transporter GLUT1 in this process.10,22 We
performed a detailed analysis of vitamin C uptake in these cells and
established that transport of dehydroascorbic acid is the only
mechanism by which the HL-60 cells acquire vitamin C, and that they do
not express sodium-dependent ascorbate cotransporters. Thus, the HL-60
cells represent an ideal in vitro model to address the issue of the
regulation of vitamin C uptake in myeloid cells. Similar to human
neutrophils, a neutrophilic clone of HL-60 cells responded to GM-CSF
with an increased uptake of dehydroascorbic acid that was time and dose
dependent (Fig 4A and B). Half-maximal activation was observed at 60 minutes, with maximal effect evident after 90 minutes of exposure to 0.5 nmol/L GM-CSF (Fig 4A). GM-CSF induced half-maximal activation of uptake at approximately 10 pmol/L,
and maximal activation was observed at 100 pmol/L, with no further
activation of uptake seen at concentrations of GM-CSF as high as 10 nmol/L (Fig 4B). The effect of GM-CSF on the uptake of dehydroascorbic
acid was most evident during the first 5 minutes of uptake, suggesting
that it was related to the transport step rather than to the secondary
step leading to the intracellular accumulation of elevated
concentrations of ascorbic acid. Consistent with this interpretation,
short uptake experiments indicated that the transport of
dehydroascorbic acid was increased by treating the HL-60 neutrophils
with GM-CSF (Fig 4C). Kinetic analysis using 30-second uptake assays
showed that the transport of dehydroascorbic acid was characterized by
a single functional component with an apparent Km of 0.72 ± 0.32 mmol/L and a Vmax of 0.5 ± 0.1 nmol/min/106 cells for
the transport of dehydroascorbic acid by control HL-60 neutrophils. On
the other hand, cells treated with 0.5 nmol/L GM-CSF had an apparent Km
of 0.49 mmol/L and a Vmax of 0.48 nmol/min/106 cells (Fig
4D). These results support the concept that GM-CSF treatment induces a
change in the capacity of the glucose-vitamin C transporters to
transport dehydroascorbic acid.
View larger version (38K):
[in this window]
[in a new window]
| Fig 4.
GM-CSF increases the transport of dehydroascorbic acid by
HL-60 neutrophils. (A) Time-course of the effect of GM-CSF. Cells were
incubated for varied times in the absence () or in the presence () of 0.5 nmol/L GM-CSF before measuring the uptake of
dehydroascorbic acid (DHA). Data represent the average of two
experiments with three replicates each. (B) Dose-response of the effect
of GM-CSF. Cells were incubated for 30 minutes in the presence of
different concentrations of GM-CSF and uptake of dehydroascorbic acid
was measured afterwards. Data represent the mean ± SD of four samples and correspond to one of three similar experiments. (C) Effect of
GM-CSF on the transport of dehydroascorbic acid. Cells were incubated
for 30 minutes in the absence () or in the presence () of 0.5 nmol/L GM-CSF and transport of dehydroascorbic acid was measured
afterwards. Data represent the average of two experiments with three
replicates each. (D) Double reciprocal plot of the effect of GM-CSF on
the substrate dependence for dehydroascorbic acid transport. Cells were
incubated for 30 minutes in the absence () or in the presence ()
of 0.5 nmol/L GM-CSF and transport of dehydroascorbic acid was measured
for 30 seconds. Data represent the mean of four samples and correspond
to one of three similar experiments.
|
|
We showed previously that HL-60 promyelocytic cells transport
dehydroascorbic acid and accumulate high intracellular concentrations of ascorbic acid.22 In experiments in which the HL-60
neutrophils were incubated with dehydroascorbic acid, we identified the
chemical form of vitamin C present in the incubation medium and in cell extracts by HPLC. Analysis of the incubation medium showed that greater
than 99% of the radioactivity eluted at 4.4 minutes, the position of
dehydroascorbic acid (Fig 5A). When the
samples were treated with 1 mmol/L dithiothreitol (DTT) before the HPLC
separation, the radioactive peak at 4.4 minutes was no longer visible
and the radioactivity eluted at 10.4 minutes, the position of ascorbic acid (Fig 5B). Greater than 95% of the radioactive material taken up
by HL-60 neutrophils incubated with dehydroascorbic acid eluted in the
position corresponding to ascorbic acid (Fig 5C). A small peak
containing approximately 3% of the radioactive material eluted at the
expected position of dehydroascorbic acid (Fig 5C). This peak
disappeared when the extracts were treated with 1 mmol/L DTT before the
HPLC separation, and all the radioactivity eluted in the position of
ascorbic acid at 10.4 minutes (data not shown). The ascorbic acid peak
disappeared when the cell extracts were treated with ascorbic acid
oxidase before the HPLC separation, and a new peak eluting in the
position of dehydroascorbic acid was observed (data not shown). The
above experiments were performed using 50 µmol/L dehydroascorbic
acid, but essentially identical results were obtained, namely greater
than 95% of the intracellularly accumulated vitamin C was in the form
of ascorbic acid, when the HL-60 cells were incubated with
dehydroascorbic acid concentrations as high as 10 mmol/L (Fig 5D).
Thus, the HL-60 neutrophils transported dehydroascorbic acid, but
accumulated ascorbic acid intracellularly.
View larger version (38K):
[in this window]
[in a new window]
| Fig 5.
GM-CSF increases the accumulation of ascorbic acid by
HL-60 neutrophils. (A) HPLC separation of dehydroascorbic acid
generated by treating a sample of ascorbic acid with ascorbic acid
oxidase. The arrows indicate the elution positions of dehydroascorbic
acid (DHA) and ascorbic acid (AA). (B) HPLC separation of a commercial preparation of ascorbic acid after treatment with 1 mmol/L DTT. (C)
HPLC separation of a cellular extract from cells incubated with 100 µmol/L dehydroascorbic acid. (D) HPLC separation of a cellular
extract from cells incubated with 10 mmol/L dehydroascorbic acid. Data
(A through D) represent the result of one of three similar experiments.
(E) Accumulation of ascorbic acid. Cells were incubated for 30 minutes
in the absence () or in the presence () of 0.5 nmol/L GM-CSF and
afterwards the accumulation of ascorbic acid as a function of different
concentrations of dehydroascorbic acid was measured for 10 minutes.
Data are expressed as the ratio of the intracellular concentration of
ascorbic acid to the extracellular concentration of dehydroascorbic
acid. Data represent the mean ± SD of four samples and correspond to
one of three similar experiments. (F) Double reciprocal plot of the
substrate dependence for ascorbic acid accumulation. Cells were
incubated for 30 minutes in the absence () or in the presence ()
of 0.5 nmol/L GM-CSF and accumulation of ascorbic acid as a function of
different concentrations of dehydroascorbic acid was measured at 10 minutes. Data represent the mean of four samples and correspond to one
of three similar experiments.
|
|
Studies using long uptake assays (30 minutes) showed that GM-CSF
treatment induced increased accumulation of ascorbic acid in HL-60
neutrophils incubated with dehydroascorbic acid (Fig 5E). Unstimulated
cells accumulated intracellular concentrations of ascorbic acid that
greatly exceeded the extracellular concentrations of dehydroascorbic
acid to which they were exposed. The ratio of intracellular ascorbic
acid to extracellular dehydroascorbic acid reached a maximal value of
29 at 30 µmol/L extracellular dehydroascorbic acid (Fig 5E). Although
the ratio decreased as extracellular dehydroascorbic acid increased to
0.1 mmol/L or more, it still maintained a value of 2.2 at 10 mmol/L.
GM-CSF-treated cells showed an increased ratio of intracellular
ascorbic acid to extracellular dehydroascorbic acid at all
dehydroascorbic acid concentrations, reaching a value of 39 at 30 µmol/L dehydroascorbic acid, and 2.6 at 10 mmol/L dehydroascorbic
acid. In other words, at 10 mmol/L dehydroascorbic acid, the HL-60
cells accumulated intracellular concentrations of ascorbic acid from 22 to 26 mmol/L. The accumulation data are consistent with the concept
that the rate-limiting step of the overall uptake process in long
uptake studies is the intracellular reduction of the transported
substrate. We performed a kinetic analysis of this step using 30-minute
assays. These studies showed the presence of two functional components involved in the intracellular accumulation of ascorbic acid in HL-60
neutrophils incubated with dehydroascorbic acid, as indicated by a
break in the slope of the Michaelis and Menten double reciprocal plot
(Fig 5F). The low-affinity component, evident at dehydroascorbic acid
concentrations of 1 mmol/L or greater, showed a small increase in Vmax,
from approximately 0.25 nmol/min/106 cells in untreated
cells to 0.31 nmol/min/106 cells in GM-CSF-treated cells,
but no change in the apparent Km (3.3 mmol/L) was observed (Fig 5F). On
the other hand, the Km of the second kinetic component that was
observed at concentrations of dehydroascorbic acid of 1 mmol/L or
lower, decreased from 0.68 mmol/L in untreated cells to 0.45 mmol/L in
GM-CSF-treated cells.
The above studies established that the HL-60 neutrophils possessed a
remarkable capacity to reduce the transported dehydroascorbic acid to
ascorbic acid, which is then accumulated intracellularly at high
concentrations. The value of 0.68 mmol/L for the high-affinity component was similar to the Km for the transport of dehydroascorbic acid determined in untreated cells using short uptake assays, and the
decrease in the high-affinity Km in the GM-CSF-treated cells mirrored
the respective decrease observed in the Km for transport in
GM-CSF-treated cells. The data are compatible with the concept that
GM-CSF regulates accumulation through its direct effect on transport,
which is the rate-limiting step for the overall accumulation process at
extracellular dehydroascorbic acid concentrations of 1 mmol/L or less.
On the other hand, the lack of effect of GM-CSF at high extracellular
dehydroascorbic acid levels, at which the reduction of the transported
dehydroascorbic acid is the rate-limiting step, suggest that GM-CSF
does not directly regulate the reduction-trapping mechanism, and that
the increased accumulation of ascorbic acid seen in GM-CSF-treated
cells is an effect secondary to increased transport. We further
explored this issue by examining the effect of GM-CSF on the cellular
uptake of deoxyglucose and methylglucose. A detailed time-course of
transport showed that the effect of GM-CSF on the uptake of
deoxyglucose was evident during the first 5 minutes of uptake, but no
effect was evident at time intervals longer than 20 minutes. Kinetic
analyses of the transport of deoxyglucose in cells treated with GM-CSF
showed no changes in the maximal velocity for transport (2.3 nmol/min/106 cells) compared with untreated cells (2.2 nmol/min/106 cells) (Fig 6A).
GM-CSF treatment, however, induced a change in the transport Km, from
4.5 mmol/L in untreated cells to 2.9 mmol/L in GM-CSF-treated cells
(Fig 6A). A similar analysis indicated that the GM-CSF-induced
increase in the initial rate of transport of methylglucose was
accompanied by a decrease in the Km, from 9 mmol/L in untreated cells
to 5.1 mmol/L in GM-CSF-treated cells (Fig 6B). The data are
compatible with the concept that, in HL-60 neutrophils, GM-CSF
increases the intrinsic functional activity of the facilitative
transporters involved in the transport of dehydroascorbic acid.
View larger version (23K):
[in this window]
[in a new window]
| Fig 6.
Effect of GM-CSF on the uptake of deoxyglucose (DOG) and
methylglucose (OMG) by HL-60 neutrophils. (A) Double reciprocal plot of
the effect of GM-CSF on the substrate dependence for deoxyglucose transport. Cells were incubated for 30 minutes in the absence () or
in the presence () of 0.5 nmol/L GM-CSF and transport of
deoxyglucose was measured at 30 seconds. Data represent the mean of
four samples. (B) Double reciprocal plot of the effect of GM-CSF on the
substrate dependence for methylglucose transport. Cells were incubated
for 30 minutes in the absence () or in the presence () of 0.5 nmol/L GM-CSF and transport of methylglucose was measured at 30 seconds. Data represent the mean of four samples and correspond to one
of three similar experiments.
|
|
Effect of GM-CSF and fMetLeuPhe on dehydroascorbic acid uptake in
HL-60 neutrophils.
The uptake experiments described were performed under conditions
designed to provide the cells with nonlimiting concentrations of the
transported substrate, dehydroascorbic acid. Thus, the CSF-mediated
increased ability of the cells to take up dehydroascorbic acid was not
related to increased availability of the transported substrate in the
extracellular milieu, but rather to a specific effect of the CSF on the
uptake of the vitamin. The distinction is important, as a process able
to generate dehydroascorbic from ascorbic acid will result in increased
uptake of the vitamin by the cells20 because the glucose
transporters are specific for dehydroascorbic acid and do not transport
reduced ascorbic acid.10,21,22 Neutrophils and mononuclear
phagocytes activated by various physiologic signals such as the
chemotactic peptide fMetLeuPhe produce molecules that act as strong
oxidants, which can generate the transported species, dehydroascorbic
acid, from ascorbate.23,32 On the other hand, changes in
the number or the intrinsic activity of the glucose transporters
involved in the cellular uptake of dehydroascorbic acid at the level of
the plasma membrane, will directly affect the transport of
dehydroascorbic acid.
We explored these issues using HL-60 neutrophils.34
Unstimulated HL-60 neutrophils produced basal levels of
H2O2, and stimulation with fMetLeuPhe activated
the oxidative burst that could be measured by an increased production
of H2O2 and the highly reactive superoxide anion (data not shown). GM-CSF primed the cells for the oxidative response with the result that cells exposed to GM-CSF and then stimulated with fMetLeuPhe generated even larger amounts of both compounds.33 Because the oxidative burst leads to the
oxidation of ascorbic acid to dehydroascorbic acid,20 these
results predicted that the generated dehydroascorbic acid would be
transported intracellularly and reduced back to ascorbic acid,
resulting in the intracellular accumulation of ascorbic acid. There was
a substantial accumulation of ascorbic acid in cells activated with
fMetLeuPhe under these conditions, and the amount of ascorbic acid
accumulated was even greater in cells preincubated with GM-CSF before
stimulation with fMetLeuPhe (Fig 7A).
Measurable uptake was also observed in untreated cells, an observation
consistent with the oxidation of ascorbic acid to dehydroascorbic acid
under basal conditions.8-10 In all cases, uptake was only a
fraction of that observed in cells directly incubated in the presence
of dehydroascorbic acid (Fig 7A and G).
View larger version (51K):
[in this window]
[in a new window]
| Fig 7.
Effect of fMetLeuPhe on the uptake of dehydroascorbic
acid (DHA) and ascorbic acid (AA) by HL-60 neutrophils. (A) Effect of GM-CSF and fMetLeuPhe on the uptake of ascorbic acid (AA). Cells were
incubated in the absence (,  ) or in the presence ( ) of GM-CSF
for 30 minutes. Afterwards, uptake of ascorbic acid was assayed in the
absence (control, ) or in the presence (, ) of 5 µmol/L
fMetLeuPhe. (B) Effect of fMetLeuPhe on the initial uptake of ascorbic
acid. Uptake of ascorbic acid was assayed in the absence () or in
the presence () of 5 µmol/L fMetLeuPhe. (C) Effect of superoxide
dismutase and catalase on the uptake of ascorbic acid uptake. Uptake
was measured for 10 minutes in the presence of fMetLeuPhe and
superoxide dismutase (SOD) or catalase. (D) Effect of deoxyglucose
(DOG) on the uptake of ascorbic acid in cells treated with fMetLeuPhe.
Uptake of ascorbic acid was assayed in the absence () or in the
presence (, ) of 5 µmol/L fMetLeuPhe and 50 mmol/L deoxyglucose
(). (E) Effect of deoxyglucose on the initial phase of transport of
ascorbic acid (AA) by cells treated with fMetLeuPhe. Cells were
incubated in medium containing radiolabeled ascorbic acid and 5 µmol/L fMetLeuPhe with () or without () 50 mmol/L deoxyglucose.
(F) Effect of fMetLeuPhe on the transport of dehydroascorbic acid.
Transport was assayed in the absence () or in the presence () of
5 µmol/L fMetLeuPhe. (G) Effect of fMetLeuPhe on the long-term uptake
of dehydroascorbic acid. Uptake was assayed in the absence () or in
the presence () of 5 µmol/L fMetLeuPhe. (H) Effect of fMetLeuPhe
on transport of deoxyglucose. Transport was assayed in the absence
() or in the presence () of 5 µmol/L fMetLeuPhe. (I) Effect of
fMetLeuPhe on the long-term uptake of deoxyglucose. Uptake was assayed
in the absence () or in the presence () of 5 µmol/L fMetLeuPhe. (I) Effect of fMetLeuPhe on the transport of methylglucose (OMG). Transport was assayed in the absence () or in the presence () of
5 µmol/L fMetLeuPhe. Data represent the mean ± SD of four samples and correspond to one of two to four similar experiments.
|
|
Short uptake studies in cells incubated with reduced ascorbic acid
showed the existence of a short lag period before transport was
detected, and transport increased with time in an exponential fashion
(Fig 7B). Transport under these conditions, however, was less than 3%
of transport observed in cells incubated with dehydroascorbic acid (Fig
7B and F). We interpreted these results as indicating the
time-dependent generation of small quantities of the transported substrate, dehydroascorbic acid, in the samples containing ascorbic acid. The presence of fMetLeuPhe increased the initial rate of transport by twofold to threefold, although the rate of transport was
still a small fraction of that observed in cells incubated with
dehydroascorbic acid (Fig 7B and F). The data suggest that the
increased rate of transport observed after treating the HL-60 neutrophils with fMetLeuPhe is due to the generation of the transported substrate dehydroascorbic acid from ascorbic acid and not to an effect
on the glucose transporters. Consistent with this notion, the presence
of the enzyme superoxide dismutase during the activation-uptake assay
markedly decreased the effect of fMetLeuPhe on the cellular uptake of
ascorbic acid, suggesting the direct involvement of the reactive
superoxide anion in this process (Fig 7C). Uptake decreased from 4.7 pmol/min/106 cells in fMetLeuPhe-treated cells to 3.2 pmol/min/106 cells in cells treated with fMetLeuPhe in the
presence of superoxide dismutase. No effect of the enzyme catalase was
observed in these studies (uptake was 5.5 pmol/min/106
cells), a finding that suggests that the generated
H2O2 did not induce the oxidation of ascorbic
acid (Fig 7C).
An alternative explanation could be that fMetLeuPhe induces the rapid
activation of a still unidentified transport system not related to the
glucose transporters and capable of transporting ascorbic acid. If that
were the case, however, the putative transporter would not be expected
to be sensitive to competition by substrates that enter the cell
through the glucose transporters. We observed that deoxyglucose
completely blocked the increased transport observed in cells incubated
with ascorbic acid in the presence of fMetLeuPhe (Fig 7D). The
inhibitory effect of deoxyglucose was evident during the first seconds
of uptake, confirming the direct participation of the glucose
transporters in the uptake of the dehydroascorbic acid generated by the
fMetLeuPhe treatment (Fig 7E). We further analyzed this issue by
studying the effect of fMetLeuPhe on the ability of the HL-60
neutrophils to transport dehydroascorbic acid. Short uptake studies
indicated that fMetLeuPhe exerted no effect on the ability of the HL-60
neutrophils to transport dehydroascorbic acid (Fig 7F), a result
consistent with the concept that fMetLeuPhe does not affect the ability
of the glucose transporters to transport dehydroascorbic acid. Kinetic
analysis indicated that fMetLeuPhe had no affect on the apparent Km and
Vmax for the transport of dehydroascorbic acid by the HL-60
neutrophils. The Km and the Vmax for dehydroascorbic acid transport
were 0.72 mmol/L and 0.50 nmol/min/106 cells for untreated
HL-60 neutrophils, and 0.69 mmol/L and 0.52 nmol/min/106
cells for activated HL-60 neutrophils. Longer uptake assays, up to 60 minutes, showed that fMetLeuPhe did not affect the accumulation of
vitamin C in HL-60 neutrophils incubated with dehydroascorbic acid
(data not shown). Moreover, HL-60 neutrophils incubated for up to 60 minutes in the presence of 1 µmol/L fMetLeuPhe showed no changes in
their ability to accumulate ascorbate compared with untreated control
cells (Fig 7G).
We next examined the effect of fMetLeuPhe on the cellular uptake of
deoxyglucose and methylglucose by the HL-60 neutrophils. These studies
showed that fMetLeuPhe did not affect the initial rate or the Km of
transport of deoxyglucose by HL-60 neutrophils (Fig 7H). The Km and the
Vmax for deoxyglucose transport were 4.8 mmol/L and 1.9 nmol/min/106 cells for untreated HL-60 neutrophils, and 4.7 mmol/L and 2.1 nmol/min/106 cells for activated HL-60
neutrophils. Longer uptake studies showed that fMetLeuPhe had a small,
but consistent, stimulatory effect on the cellular trapping of
deoxyglucose (Fig 7I), an observation consistent with fMetLeuPhe
exerting its effect at a step downstream of the transport of
deoxyglucose and therefore not directly related to the glucose
transporters. In further studies, we showed that fMetLeuPhe did not
affect the initial rate or the Km for the transport of methylglucose by
HL-60 neutrophils (Fig 7J). The Km and the Vmax for methylglucose
transport were 6.7 mmol/L and 2.4 nmol/min/106 cells for
untreated HL-60 neutrophils, and 7.0 mmol/L and 2.6 nmol/min/106 cells for activated HL-60 neutrophils.
Overall, the data are consistent with the concept that fMetLeuPhe
increases cellular ascorbic acid accumulation in HL-60 neutrophils by
inducing the generation of the transported substrate dehydroascorbic
acid and does not directly affect the cellular trapping of ascorbic
acid or the activity of the glucose transporters.
| |
DISCUSSION |
We found that G- and GM-CSF increase the uptake of dehydroascorbic acid
in normal human neutrophils, monocytes, and HL-60 neutrophils. Although
the molecular details of the regulation are still undefined, the data
indicate that under conditions of a nonlimiting supply of
dehydroascorbic acid, the cellular uptake of vitamin C can be regulated
at the transport step (Fig 8). The increased transport of
dehydroascorbic acid in cells treated with GM-CSF is related to the
participation of hexose transporters of the facilitative type in the
transport of dehydroascorbic acid by normal human neutrophils,
monocytes, and HL-60 neutrophils. Growth factors increase the ability
of cells to take up hexoses through the glucose transporters by
mechanisms involving the recruitment of new transporters to the cell
surface and increased intrinsic activity of the
transporters.44-46 The human GM-CSF receptor is composed of
two subunits, and  , that together form a high-affinity receptor.
Although the subunit alone is unable to bind GM-CSF, the isolated
subunit binds GM-CSF with low affinity and is referred to as the
low-affinity GM-CSF receptor. We recently showed that in human
neutrophils and HL-60 cells expressing high-affinity GM-CSF receptors,
GM-CSF induces an increased uptake of hexoses in a
phosphorylation-independent manner.30 Moreover, glucose uptake experiments in Xenopus laevis oocytes expressing the
isolated subunit of the human GM-CSF receptor, and expressing a
low-affinity GM-CSF receptor, indicated that signaling for increased
transport is mediated through the isolated subunit and does not
require the presence of the subunit.30 Also, glucose
uptake was activated by GM-CSF in melanoma cell lines that express an
isolated subunit of the GM-CSF receptor.31 Given that
the cellular accumulation and trapping of deoxyglucose and ascorbate
are not related (phosphorylation v reduction), these
observations are consistent with the concept that GM-CSF may signal for
increased uptake by regulating the transport process at the level of
the glucose transporters. Direct support for this hypothesis was
obtained here in experiments showing that GM-CSF induced a decrease in
the Km for the transport of dehydroascorbic acid, deoxyglucose, and
methylglucose in HL-60 neutrophils, without changes in the Vmax of
transport. The most simple explanation consistent with these results is
that GM-CSF affects the functional activity of the glucose transporters
that mediate the cellular transport of dehydroascorbic acid in these cells. A similar effect, namely a reduction in the transport Km, was
described for the effect of IL-3 on glucose transport in the murine
cell line 32D.47 Based on the inhibitory effect on
transport of protein tyrosine kinase inhibitors such as genistein, the
investigators proposed that altered phosphorylation of plasma membrane
proteins associated with the glucose transporters may regulate the
affinity of the transporter for glucose.47 We have shown,
however, that genistein directly inhibits the activity of the glucose
transporter in a manner unrelated to its capacity to inhibit tyrosine
kinase function.48
View larger version (27K):
[in this window]
[in a new window]
| Fig 8.
Regulatory steps involved in the cellular uptake of
vitamin C. We identified three possible regulatory sites at which HL-60 neutrophils regulate their uptake and content of vitamin C. These steps
are also likely active in cells such as human neutrophils and monocytes
that transport dehydroascorbic acid, but lack the capacity to transport
ascorbic acid. (1) The first regulatory site relates directly to the
availability of the substrate that is transported. Activation of HL-60
neutrophils with the chemotactic peptide fMetLeuPhe triggers the
respiratory burst, with the subsequent generation of reactive oxygen
species that can oxidize locally available ascorbic acid to
dehydroascorbic acid. As a result, the locally generated
dehydroascorbic acid will be rapidly transported intracellularly. (2)
The second point of regulation is related to the transport step, and
its regulatory potential is determined by changes in the number,
molecular identity, or functional status of the transporters of vitamin
C. GM-CSF treatment increases the affinity of GLUT1 for the transport
of dehydroascorbic acid. As a result, increased transport of
dehydroascorbic acid is observed. Growth factors and cytokines that
affect the level of expression, subcellular localization, or the
intrinsic functional activity of the glucose transporters can
potentially modulate the cellular transport of dehydroascorbic acid.
(3) A third potential regulatory step is defined by the existence of
intracellular mechanisms that determine the capacity of cells to
accumulate characteristic intracellular levels of vitamin C. At least
two general enzymatic systems with dehydroascorbic acid reductase
activity, one GSH dependent and one GSH independent, exist in mammalian
cells. No data regarding their regulatory potential are currently
available.
|
|
We have also identified a second site of functional regulation involved
in the uptake of vitamin C by human host defense cells (Fig 8). The second site involves enhanced
generation of the transportable moiety of vitamin C, dehydroascorbic
acid, in the pericellular milieu. We have shown that neutrophils and
monocytes transport only the oxidized form of vitamin C,
dehydroascorbic acid, and are incapable of directly transporting
ascorbic acid. Detailed analysis showed no evidence of a
sodium-dependent ascorbate cotransporter. These considerations lead to
the conclusion that the generation of dehydroascorbic acid outside of
the cell is essential to the cell's ability to accumulate vitamin C. The human neutrophil has potent oxidative properties and these are
greatly enhanced by triggering of the oxidative burst. GM-CSF primes
neutrophils for an oxidative response to fMetLeuPhe due to its ability
to modulate the fMetLeuPhe receptor.33 We show here that
treatment of cells with fMetLeuPhe leads to increased accumulation of
vitamin C and that this accumulation is due to the transport of
dehydroascorbic acid through the facilitative glucose transporters.
Support for this conclusion comes from experiments in which the effect
of fMetLeuPhe was markedly decreased in the presence of the enzyme superoxide dismutase, which specifically destroys the superoxide anion
by catalyzing its conversion to oxygen and hydrogen peroxide. This
interpretation is consistent with the results of blocking experiments
in which glucose blocked the effect of fMetLeuPhe by competing with
dehydroascorbic acid for the glucose transporter. Further
substantiation for this mechanism was provided by showing that
fMetLeuPhe did not directly affect the glucose transporter, as there
were no changes in the transport of dehydroascorbic acid, deoxyglucose,
or methylglucose. Overall, the data indicate that the increased uptake
observed in cells treated with fMetLeuPhe and exposed to ascorbic acid
was due to the generation of the transported moiety, dehydroascorbic
acid, which is then rapidly transported intracellularly through the
facilitative glucose transporters and trapped inside the cell by
reduction. GM-CSF increased fMetLeuPhe-mediated uptake of vitamin C by
increasing the oxidative burst through its priming effect.
The implication of these results is that host defense cells such as
neutrophils will take up dehydroascorbic acid generated in situ and
recycle it back to ascorbic acid. Dehydroascorbic acid generated by
neutrophil oxidative potential can be transported intracellularly by
any cell present in the immediate area because glucose transporters are
universally present on all cells and tissues. Thus, activated
neutrophils participating in inflammatory reactions may provide other
cell types with increased antioxidant protection by making the
transportable moiety of vitamin C more available. Thus, there appears
to be a certain balance between oxidative and antioxidative actions in
inflammatory reactions with extracellular oxidation itself permitting
increased intracellular concentrations of ascorbic acid.
A third potential site of regulation of vitamin C uptake in host
defense cells is at the level of reduction of dehydroascorbic acid to
ascorbic acid intracellularly. Our evidence for an effect of GM-CSF on
this step is tenuous, although we did find enhanced kinetics of the
reduction process. The precise mechanism whereby dehydroascorbic acid
is reduced intracellularly is unknown.35 Although
accumulation kinetics provide only a crude approach to measuring this
function, the data are consistent with the concept that the HL-60
neutrophils possess a remarkable capacity to reduce the recently
transported dehydroascorbic acid, which is then trapped as ascorbic
acid. Overall, the data suggest that transport of dehydroascorbic acid
through facilitative glucose transporters, followed by the
intracellular trapping of ascorbic acid, is the only mechanism by which
the host defense cells acquire vitamin C.
Vitamin C is fundamental to human physiology and, because humans cannot
synthesize it, it must be provided externally and transported
intracellularly.1-6 The cellular mechanisms that define the
normal or pharmacologic requirements for vitamin C are mostly unknown.
Studies with experimental scurvy have shown a critical role for vitamin
C in the maintenance of a normally functioning host defense
system,1 suggesting that a tight regulation of the cellular
content of vitamin C is central for normal host defense. This concept
is consistent with our observations that the cellular content of
vitamin C in host defense cells can be modulated by cytokines that are
key modulators of the production, maturation, and function of these
cells. These results establish a role for the CSFs in the regulation of
the uptake of vitamin C in host defense cells and thereby implicate
positive cellular vitamin C flux to enhanced host defense.
| |
FOOTNOTES |
Submitted June 4, 1997;
accepted November 19, 1997.
Supported by Grants No. CA30388, HL42107, and CA08748 from the National
Institutes of Health, Bethesda, MD, the Schultz
Foundation, the DeWitt Wallace Clinical Research Foundation, and
Memorial Sloan-Kettering Cancer Center Institutional funds.
Address reprint requests to Juan Carlos Vera, PhD, Program in Molecular
Pharmacology and Therapeutics, Box 451, Memorial Sloan-Kettering Cancer
Center, 1275 York Ave, New York, NY 10021.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
REFERENCES |
1.
Hodges RE,
Hood J,
Canham JE,
Sauberlich HE,
Baker EM:
Clinical manifestations of ascorbic acid deficiency in man.
Am J Clin Nutr
24:432,
1971[Abstract]
2.
Englard S,
Seifter S:
The biochemical function of ascorbic acid.
Ann Rev Nutr
6:365,
1986[Medline]
[Order article via Infotrieve]
3.
Padh H:
Cellular functions of ascorbic acid.
Biochem Cell Biol
68:1166,
1990[Medline]
[Order article via Infotrieve]
4.
Jacob RA,
Kelley DS,
Pianalto FS,
Swendseid ME,
Henning SM,
Zhang JZ,
Ames BN,
Fraga CG,
Peters JH:
Immunocompetence and oxidant defense during ascorbate depletion of healthy men.
Am J Clin Nutr
54:1302S,
1991[Abstract/Free Full Text]
5.
Nishikimi M,
Yagi K:
Molecular basis for the deficiency in humans of gulonolactone oxidase, a key enzyme for ascorbic acid biosynthesis.
Am J Clin Nutr
54:1203S,
1991[Abstract/Free Full Text]
6.
Rose RC:
Transport of ascorbic acid and other water-soluble vitamins.
Biochim Biophys Acta
947:335,
1988[Medline]
[Order article via Infotrieve]
7.
Washko P,
Rotrosen D,
Levine M:
Ascorbic acid transport and accumulation in human neutrophils.
J Biol Chem
264:18996,
1989[Abstract/Free Full Text]
8.
Winkler BS:
In vitro oxidization of ascorbic acid and its prevention by GSH.
Biochim Biophys Acta
925:258,
1987[Medline]
[Order article via Infotrieve]
9.
Bode AM,
Cunningham L,
Rose RC:
Spontaneous decay of oxidized ascorbic acid (dehydro-L-ascorbic acid) evaluated by high-pressure liquid chromatography.
Clin Chem
36:1807,
1990[Abstract/Free Full Text]
10.
Vera JC,
Rivas CI,
Zhang R-H,
Farber CM,
Golde DW:
Human HL-60 myeloid leukemia cells transport dehydroascorbic acid via the glucose transporters and accumulate reduced ascorbic acid.
Blood
84:1628,
1994[Abstract/Free Full Text]
11.
Liebes L,
Krigel R,
Kuo S,
Devrla D,
Pelle E,
Silber R:
Increased ascorbic acid content in chronic lymphocytic leukemia B lymphocytes.
Proc Natl Acad Sci USA
78:6481,
1981[Abstract/Free Full Text]
12.
Daniels AJ,
Dean G,
Viveros OH,
Diliberto EJ:
Secretion of newly taken-up ascorbic acid by adrenomedullary chromaffin cells.
Science
216:737,
1982[Abstract/Free Full Text]
13.
Bianchi J,
Rose RC:
Glucose-independent transport of dehydroascorbic acid in human erythrocytes.
Proc Soc Exp Biol Med
181:333,
1986[Medline]
[Order article via Infotrieve]
14.
Ingermann RL,
Stankova L,
Bigley RH:
Role of monosaccharide transporter in vitamin C uptake by placental membrane vesicles.
Am J Physiol
250:C637,
1986[Abstract/Free Full Text]
15.
Choi J-L,
Rose RC:
Transport and metabolism of ascorbic acid in human placenta.
Am J Physiol
257:C110,
1989[Abstract/Free Full Text]
16.
Padh H,
Aleo JJ:
Ascorbic acid transport by 3T6 fibroblasts.
J Biol Chem
264:6065,
1989[Abstract/Free Full Text]
17.
Wilson JX,
Dixon JS:
High-affinity sodium-dependent uptake of ascorbic acid by rat osteoblasts.
J Membr Biol
111:83,
1989[Medline]
[Order article via Infotrieve]
18.
Fay MJ,
Bush MJ,
Verlangier AJ:
Effects of cytochalasin B on the uptake of ascorbic acid and glucose by 3T3 fibroblasts: Mechanism of impaired ascorbate transport in diabetes.
Life Sci
46:619,
1990[Medline]
[Order article via Infotrieve]
19.
Washko P,
Levine M:
Inhibition of ascorbic acid transport in human neutrophils by glucose.
J Biol Chem
267:23568,
1992[Abstract/Free Full Text]
20.
Washko PW,
Wang YH,
Levine M:
Ascorbic acid recycling in human neutrophils.
J Biol Chem
268:15531,
1993[Abstract/Free Full Text]
21.
Vera JC,
Rivas CI,
Fischbarg J,
Golde DW:
Mammalian facilitative hexose transporters mediate the transport of dehydroascorbic acid.
Nature
364:79,
1993[Medline]
[Order article via Infotrieve]
22.
Vera JC,
Rivas CI,
Velásquez FV,
Zhang RH,
Concha II,
Golde DW:
Resolution of the facilitated transport of dehydroascorbic acid from its intracellular accumulation as ascorbic acid.
J Biol Chem
270:23706,
1995[Abstract/Free Full Text]
23.
Weisbart RH,
Golde DW,
Clark SC,
Wong GG,
Gasson JC:
Human granulocyte macrophage colony-stimulating factor is a neutrophil activator.
Nature
314:361,
1985[Medline]
[Order article via Infotrieve]
24.
Clark SC,
Kamen R:
The human hematopoietic colony-stimulating factors.
Science
236:1229,
1987[Abstract/Free Full Text]
25.
Groopman JE,
Molina J-M,
Scadden DT:
Hematopoietic growth factors. Biology and clinical applications.
N Engl J Med
321:1449,
1989[Abstract]
26.
Nicola NA:
Hematopoietic growth factors and their receptors.
Annu Rev Biochem
58:45,
1989[Medline]
[Order article via Infotrieve]
27. Dexter TM, Garland JM, Testa NG: Colony-Stimulating Factors.
Molecular and Cellular Biology. New York, NY, Marcel Dekker, 1990
28.
Gasson JC:
Molecular physiology of granulocyte-macrophage colony-stimulating factor.
Blood
77:1131,
1991[Free Full Text]
29.
Baldwin GC:
The biology of granulocyte-macrophage colony-stimulating factor  effects on hematopoietic and nonhematopoietic cells.
Dev Biol
151:352,
1992[Medline]
[Order article via Infotrieve]
30.
Ding DX-H,
Rivas CI,
Heaney ML,
Raines MA,
Vera JC,
Golde DW:
The subunit of the human granulocyte-macrophage colony-stimulating factor receptor signals for glucose transport via a phosphorylation-independent pathway.
Proc Natl Acad Sci USA
91:2537,
1994[Abstract/Free Full Text]
31.
Spielholz C,
Heaney M,
Morrison ME,
Houghton AN,
Vera JC,
Golde DW:
Granulocyte-macrophage colony-stimulating factor signals for increased glucose uptake in human melanoma cells.
Blood
85:973,
1995[Abstract/Free Full Text]
32. McPhail LC, Strum SL, Leone PA, Sozzani S: The neutrophil
respiratory burst mechanism, in Coffey RG (ed): Granulocyte Response to
Cytokines. New York, NY, Marcel Dekker, 1992, p 47
33.
Weisbart RH,
Golde DW,
Gasson JC:
Biosynthetic human GM-CSF modulates the number and affinity of neutrophil f-Met-Leu-Phe receptors.
J Immunol
137:3584,
1986[Abstract]
34.
Tomonaga M,
Gasson JC,
Quan SG,
Golde DW:
Establishment of eosinophilic sublines from human promyelocytic leukemia (HL-60) cells: Demonstration of multipotentiality and single-lineage commitment of HL-60 stem cells.
Blood
67:1433,
1986[Abstract/Free Full Text]
35.
Guaiquil VH,
Farber CM,
Golde DW,
Vera JC:
Efficient transport and accumulation of vitamin C in HL-60 cells depleted of glutathione.
J Biol Chem
272:9915,
1997[Abstract/Free Full Text]
36.
Baldwin SA:
Mammalian passive glucose transporters: Members of an ubiquitous family of active and passive transport proteins.
Biochim Biophys Acta
1154:17,
1993[Medline]
[Order article via Infotrieve]
37.
Sogin DC,
Hinkle PC:
Binding of cytochalasin B to human erythrocyte glucose transporter.
Biochemistry
19:5417,
1980[Medline]
[Order article via Infotrieve]
38.
Griffin JF,
Rampal AL,
Jung CY:
Inhibition of glucose transport in human erythrocytes by cytochalasins: A model based on diffraction studies.
Proc Natl Acad Sci USA
79:3759,
1982[Abstract/Free Full Text]
39.
Shanahan MF:
Cytochalasin B. A natural photoaffinity ligand for labeling the human erythrocyte glucose transporter.
J Biol Chem
257:7290,
1982[Abstract/Free Full Text]
40.
Hediger MA,
Coady M,
Ikeda T,
Wright EM:
Expression cloning and cDNA sequencing of the Na+/glucose co-transporter.
Nature
330:379,
1987[Medline]
[Order article via Infotrieve]
41.
Gasson JC,
Kaufman SE,
Weisbart RH,
Tomonaga M,
Golde DW:
High-affinity binding of granulocyte-macrophage colony-stimulating factor to normal and leukemic human myeloid cells.
Proc Natl Acad Sci USA
83:669,
1986[Abstract/Free Full Text]
42.
DiPersio J,
Billing P,
Kaufman S,
Eghtesady P,
Williams RE,
Gasson JC:
Characterization of the human granulocyte-macrophage colony-stimulating factor receptor.
J Biol Chem
263:1834,
1988[Abstract/Free Full Text]
43.
Cannistra SA,
Groshek P,
Garlick R,
Miller J,
Griffen JD:
Regulation of surface expression of the granulocyte/macrophage colony-stimulating factor receptor in normal human myeloid cells.
Proc Natl Acad Sci USA
87:93,
1990[Abstract/Free Full Text]
44.
Czech MP,
Clancy BM,
Pessino A,
Woon CW,
Harrison SA:
Complex regulation of simple sugar transport in insulin-responsive cells.
Trends Biochem Sci
17:197,
1992[Medline]
[Order article via Infotrieve]
45.
Kahn CR,
Goldfine AB:
Molecular determinants of insulin action.
J Diabetes Complications
7:92,
1993[Medline]
[Order article via Infotrieve]
46.
Kanai F,
Nishioka Y,
Hayashi H,
Kamohara S,
Todaka M,
Ebina Y:
Direct demonstration of insulin-induced GLUT4 translocation to the surface of intact cells by insertion of a c-myc epitope into an exofacial GLUT4 domain.
J Biol Chem
268:14523,
1993[Abstract/Free Full Text]
47.
Berridge MV,
Tan AS:
Interleukin-3 facilitates glucose transport in a myeloid cell line by regulating the affinity of the glucose transporter for glucose: Involvement of protein phosphorylation in transport activation.
Biochem J
305:843,
1995
48.
Vera JC,
Reyes AM,
Carcamo JG,
Velasquez FV,
Rivas CI,
Zhang RH,
Strobel P,
Iribarren R,
Scher HI,
Slebe JC,
Golde DW:
Genistein is a natural inhibitor of hexose and dehydroascorbic acid transport through the glucose transporter, Glut1.
J Biol Chem
271:8719,
1996[Abstract/Free Full Text]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
E. A. Lutsenko, J. M. Carcamo, and D. W. Golde
A Human Sodium-Dependent Vitamin C Transporter 2 Isoform Acts as a Dominant-Negative Inhibitor of Ascorbic Acid Transport
Mol. Cell. Biol.,
April 15, 2004;
24(8):
3150 - 3156.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
I. Perez-Cruz, J. M. Carcamo, and D. W. Golde
Vitamin C inhibits FAS-induced apoptosis in monocytes and U937 cells
Blood,
July 1, 2003;
102(1):
336 - 343.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Dhar-Mascareno, J. Chen, R. H. Zhang, J. M. Carcamo, and D. W. Golde
Granulocyte-Macrophage Colony-stimulating Factor Signals for Increased Glucose Transport via Phosphatidylinositol 3-Kinase- and Hydrogen Peroxide-dependent Mechanisms
J. Biol. Chem.,
March 21, 2003;
278(13):
11107 - 11114.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Leuzzi, G. Banhegyi, T. Kardon, P. Marcolongo, P.-L. Capecchi, H.-J. Burger, A. Benedetti, and R. Fulceri
Inhibition of microsomal glucose-6-phosphate transport in human neutrophils results in apoptosis: a potential explanation for neutrophil dysfunction in glycogen storage disease type 1b
Blood,
March 15, 2003;
101(6):
2381 - 2387.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F. J. Nualart, C. I. Rivas, V. P. Montecinos, A. S. Godoy, V. H. Guaiquil, D. W. Golde, and J. C. Vera
Recycling of Vitamin C by a Bystander Effect
J. Biol. Chem.,
March 14, 2003;
278(12):
10128 - 10133.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. M. Carcamo, O. Borquez-Ojeda, and D. W. Golde
Vitamin C inhibits granulocyte macrophage-colony-stimulating factor-induced signaling pathways
Blood,
May 1, 2002;
99(9):
3205 - 3212.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Niu, D. W. Golde, J. C. Vera, and M. L. Heaney
Kinetic Resolution of Two Mechanisms for High-Affinity Granulocyte-Macrophage Colony-Stimulating Factor Binding to Its Receptor
Blood,
December 1, 1999;
94(11):
3748 - 3753.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. B. Agus, J. C. Vera, and D. W. Golde
Stromal Cell Oxidation: A Mechanism by Which Tumors Obtain Vitamin C
Cancer Res.,
September 1, 1999;
59(18):
4555 - 4558.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
V. H. Guaiquil, J. C. Vera, and D. W. Golde
Mechanism of Vitamin C Inhibition of Cell Death Induced by Oxidative Stress in Glutathione-depleted HL-60 Cells
J. Biol. Chem.,
October 26, 2001;
276(44):
40955 - 40961.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Abstract
Introduction
Abstract