From the Departamento de Histología
y Embriología, § Departamento de
Fisiopatología, Facultad de Ciencias Biológicas,
Universidad de Concepción, Barrio Universitario S/H,
Concepción, Chile, and the ¶ Program in Molecular
Pharmacology and Chemistry, Memorial Sloan-Kettering Cancer Center,
New York, New York 10021
Received for publication, October 18, 2002
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ABSTRACT |
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Human cells transport dehydroascorbic acid
through facilitative glucose transporters, in apparent contradiction
with evidence indicating that vitamin C is present in human blood only
as ascorbic acid. On the other hand, activated host defense cells
undergoing the oxidative burst show increased vitamin C accumulation.
We analyzed the role of the oxidative burst and the glucose
transporters on vitamin C recycling in an in vitro system
consisting of activated host-defense cells co-cultured with human cell
lines and primary cells. We asked whether human cells can acquire
vitamin C by a "bystander effect" by taking up dehydroascorbic acid
generated from extracellular ascorbic acid by neighboring cells
undergoing the oxidative burst. As activated cells, we used HL-60
neutrophils and normal human neutrophils activated with phorbol 12 myristate 13-acetate. As bystander cells, we used immortalized
cell lines and primary cultures of human epithelial and endothelial
cells. Activated cells produced superoxide anions that oxidized
extracellular ascorbic acid to dehydroascorbic acid. At the same time,
there was a marked increase in vitamin C uptake by the bystander cells that was blocked by superoxide dismutase but not by catalase and was
inhibited by the glucose transporter inhibitor cytochalasin B. Only
ascorbic acid was accumulated intracellularly by the bystander cells.
Glucose partially blocked vitamin C uptake by the bystander cells,
although it increased superoxide production by the activated cells. We
conclude that the local production of superoxide anions by activated
cells causes the oxidation of extracellular ascorbic acid to
dehydroascorbic acid, which is then transported by neighboring cells
through the glucose transporters and immediately reduced to ascorbic
acid intracellularly. In addition to causing increased intracellular
concentrations of ascorbic acid with likely associated enhanced
antioxidant defense mechanisms, the bystander effect may allow the
recycling of vitamin C in vivo, which may contribute to the
low daily requirements of the vitamin in humans.
Vitamin C is essential to human physiology and, because
humans are unable to synthesize the vitamin, there is an absolute dependence on dietary vitamin C (1). Only reduced vitamin C, ascorbic
acid, is present in human plasma and in cells and tissues, and the
cellular content of vitamin C can exceed by several orders of magnitude
the plasma levels of the vitamin (2). Human cells transport ascorbic
acid in a concentrative manner through sodium-ascorbic acid
co-transporters (3-8); they also transport oxidized vitamin C,
dehydroascorbic acid, down its concentration gradient through facilitative glucose transporters (9-18). Although all cells express facilitative glucose transporters, the absolute specificity of these
transporters for dehydroascorbic acid is in apparent contradiction to
evidence indicating that vitamin C is present in human plasma only as
ascorbic acid. Human host defense cells, however, have potent oxidative
properties that are greatly enhanced by triggering of the oxidative
burst, which leads to increased accumulation of vitamin C by the
activated cells (15, 19-22). It is believed that the increased vitamin
C uptake observed in the activated cells is caused by the oxidation of
ascorbic acid to dehydroascorbic acid, which is then rapidly
transported intracellularly by the glucose transporters. An important
implication of this concept is that the dehydroascorbic acid generated
by cells secreting oxidants can be transported intracellularly by any
cell present in the immediate area because glucose transporters are
present in all cells and tissues. Thus, activated neutrophils
participating in inflammatory reactions may provide other cell types
with increased antioxidant protection. Using phorbol 12-myristate
13-acetate (PMA)1-activated
cells (HL-60 neutrophils and normal human neutrophils), we validated
the concept that superoxide produced by activated cells does oxidize
extracellular ascorbic acid to dehydroascorbic acid. We used co-culture
assays to demonstrate that human cells can acquire vitamin C by a
"bystander effect," taking up dehydroascorbic acid generated by
superoxide anions produced by activated neighboring cells and
subsequently accumulating ascorbic acid. We propose that the bystander
effect may allow the cellular recycling of vitamin C by cells.
The cell lines were maintained in culture at a cell viability
greater than 95% as assessed by trypan blue exclusion. The following established cells and cell lines were used: a neutrophilic subline of
HL-60 cells (15, 23); normal mammary epithelial cells and the breast
cancer cell lines MCF-7 and MDA-468 (24-26); the human melanoma cell
line GMEL (27); and the human prostate adenocarcinoma cell line DU-145
(28). Strains of normal human mammary epithelial cells (normal breast
cells) were obtained from Clonetics (San Diego, CA) and were cultured
using their media and supplements. Fresh normal human neutrophils were
isolated by fractionation in Ficoll-Hypaque (15). Human umbilical vein
endothelial cells were obtained from umbilical cords by collagenase
digestion, cultured in medium 199, and used within the first three
passages (29). Discarded umbilical cords from full-term normal
pregnancies were donated by the Regional Hospital in
Concepción.
Uptake assays were performed in incubation buffer (15 mM
Hepes pH 7.6, 135 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 0.8 mM MgCl2) containing 0.1 µCi of L-[14C]ascorbic acid
(8.2 mCi/mmol; PerkinElmer Life Sciences) and 50 µM ascorbic acid. For adherent cells, uptake assays were
performed in six-well plates (1-2 × 106 cells/well) (27). For
nonadherent cells, uptake assays were performed in suspension (0.1 to
20 × 106 cells/assay) (11). Dehydroascorbic acid was generated by treatment with ascorbic acid oxidase (Sigma) (11). When appropriate, enzymes and inhibitors were added to the uptake assays or the cells
were preincubated with them.
For the co-culture experiments, the adherent cells were grown in
six-well plates until sub-confluent (2 × 105 cells/well). On the
day of the experiment, the HL-60 neutrophils or freshly isolated normal
human neutrophils were added to plates containing the adherent cells
previously washed with incubation buffer, and PMA and radiolabeled
ascorbic acid or dehydroascorbic acid were added to the wells for the
uptake assays. After uptake, the supernatant was saved and the HL-60
neutrophils (or normal human neutrophils) were recovered by
centrifugation and washed with cold phosphate-buffered saline. The
adherent cells were also washed, and both sets of samples were
processed for scintillation counting (11).
For HPLC, the cells were lysed in a solution of 60% methanol-1
mM EDTA (pH 8.0) at 4° C, and the extracts were stored
at Production of superoxide was quantitated by monitoring the
superoxide-dismutase inhibitable reduction of ferricytochrome
c. Cells were suspended in incubation buffer containing
ferricytochrome c at 1 mg/ml and activated by the addition
of PMA. The reduction of ferricytochrome c was monitored by
determining the change in the absorbance at 550 nm and using an
extinction coefficient of 21,000 M Production of Extracellular Dehydroascorbic Acid by Activated HL-60
Neutrophils--
Compared with untreated cells, HL-60 neutrophils
treated with PMA and incubated with ascorbic acid showed a markedly
increased uptake of vitamin C. At 60 min, PMA-treated cells showed a
60-fold increase in vitamin C accumulation (500 pmol/106
cells) compared with control cells (8 pmol/106 cells) (Fig.
1A). Ten µM
cytochalasin B, but not Cytochalasin E, blocked vitamin C uptake by the
activated cells (Fig. 1A). Superoxide dismutase blocked the
uptake of vitamin C by the PMA-treated HL-60 neutrophils (Fig.
1A), whereas catalase had no effect (Fig. 1A).
Thus, the full effect of PMA on vitamin C uptake by the HL-60 neutrophils can be blocked by a specific inhibitor of the glucose transporters and an enzyme that dismutates superoxide anions.
Consistent with the above observations, PMA-treated cells generated
substantial amounts of superoxide anions. At 60 min, PMA-treated cells
generated 19 pmol/106 cells compared with less than 0.5 pmol/106 cells in control cells (Fig. 1B).
Parallel experiments revealed that neither cytochalasin B nor E, at 10 µM, affected the production of superoxide by the
activated cells (Fig. 1B), thereby confirming the
specificity of the inhibitory effect on the glucose transporter. Superoxide dismutase blocked the generation of superoxide by the PMA-treated HL-60 neutrophils, whereas catalase showed no effect (Fig.
1B). Thus, the full effect of PMA on vitamin C uptake by the
HL-60 neutrophils can be directly linked to enhanced production of
superoxide anions. When the HL-60 neutrophils were treated with PMA in
buffers in which the NaCl was replaced with choline chloride, the rate
of vitamin C uptake increased in a similar manner and approached a
similar plateau in the presence or in the absence of sodium ions, and
uptake was again specifically inhibited by cytochalasin B (data not shown).
From the above data, we concluded that the PMA-activated HL-60
neutrophils directly transport locally generated dehydroascorbic acid
through the glucose transporters. We used HPLC to directly verify the
generation of dehydroascorbic acid by the activated HL-60 neutrophils.
Treatment of HL-60 neutrophils with PMA resulted in the
time-dependent oxidation of extracellular ascorbic acid (Fig. 1C). At 60 min, ~40% of the radioactivity eluted in
the position corresponding to ascorbic acid, compared with greater than
95% in control samples treated with superoxide dismutase. These
experiments revealed the time-dependent generation of two radioactive peaks, a first one eluting at 4.6 min and corresponding to
dehydroascorbic acid, and a second one eluting at 11 min, which corresponded to dehydroascorbic acid hydrolysis products (data not
shown). We estimated the total amount of dehydroascorbic acid generated
in the presence of PMA-treated cells as the sum of the material
contained in the HPLC peaks eluting at 4.6 and 11 min, which revealed
that more than 60% of the original ascorbic acid present in solution
was oxidized to dehydroascorbic acid in 60 min (Fig.
1C).
Bystander Effect--
We tested the validity of the "bystander
effect" concept in an in vitro model consisting of
activated HL-60 neutrophils co-cultured with the human prostate cancer
cells DU-145 (the bystander cells). The prostate cells grow in
monolayers and adhere to the culture plates and therefore are easily
separated from the HL-60 neutrophils that grow in suspension,
permitting the determination of the content of vitamin C in each cell
type separately. There was some uptake of vitamin C in untreated
prostate cells incubated alone, probably because of the presence of low
levels of dehydroascorbic acid in the sample of ascorbic acid, and that
uptake was not increased by treatment with PMA (Fig.
2A). On the other hand, DU-145
cells incubated in the presence of activated HL-60 neutrophils showed increased uptake of vitamin C (Fig. 2A). At 60 min, they
contained 6 nmol of vitamin C per million cells, an amount 15-fold
higher than that accumulated by PMA-treated prostate cells incubated alone. Parallel experiments revealed a similar
time-dependent production of superoxide anions (rate: 0.17 nmol/106 HL-60 neutrophils × min) by co-cultures of
PMA-treated HL-60 neutrophils and prostate tumor cells compared with
cultures of PMA-treated HL-60 neutrophils alone (Fig. 2B).
The DU-145 cells did not produce superoxide anions when cultured under
similar conditions (Fig. 2B).
Experiments in which a fixed number of DU-145 cells were co-cultured
with variable numbers of HL-60 neutrophils revealed increased uptake of
vitamin C by the DU-145 cells as the number of HL-60 neutrophils
present in the assay increased (Fig. 2C). There was a
13-fold increase in vitamin C uptake, from 0.4 to 5.2 nmol per million
prostate cells when the number of HL-60 neutrophils in the assay was
increased from 0.1 to 5 million cells, but no further increase in
uptake was observed when the HL-60 neutrophils were increased up to 30 million per assay. Parallel experiments revealed a similar correlation
between the number of activated HL-60 neutrophils added to the
co-culture assay and the amount of superoxide anions generated (Fig.
2D). Superoxide dismutase abolished at least 75% of vitamin
C uptake by the DU-145 cells co-cultured with activated HL-60
neutrophils, indicating that superoxide is central to vitamin C uptake
by the bystander cells (Fig. 2E). Moreover, cytochalasin B,
but not cytochalasin E, inhibited in a dose-dependent
manner the uptake of vitamin C by the DU-145 cells co-cultured with
activated HL-60 neutrophils, with greater than 95% inhibition observed
at 10 µM cytochalasin B (Fig. 2E). Replacing
the NaCl in the medium with choline chloride failed to affect the
uptake of vitamin C by the DU-145 cells (data not shown).
Both the inhibitory effect of cytochalasin B and the sodium
independence of the overall uptake process are consistent with the
bystander cells taking up dehydroascorbic acid through glucose transporters. It is therefore possible that the capacity of the DU-145
cells to take up dehydroascorbic acid could be subjected to regulation
by the co-culture with HL-60 neutrophils or by activation with PMA.
Short uptake experiments revealed that DU-145 cells cultured alone, in
the absence of PMA, transported dehydroascorbic acid at a rate of 0.83 nmol/106 cells × min, rate was unchanged in cells
treated with PMA and also in cells co-cultured with HL-60 cells with or
without PMA (data not shown), indicating that the capacity of the
DU-145 cells to transport dehydroascorbic acid was not affected by PMA
or the co-culture with HL-60 neutrophils. Long uptake studies to
measure accumulation of vitamin C revealed that for all conditions
tested, uptake increased in a linear manner for the first 30 min of
incubation, with an average uptake rate of 0.55 nmol/106 cells × min and approached saturation at
about 60 min (data not shown). Thus, PMA treatment or co-culture with
the HL-60 cells did not affect the accumulation of vitamin C by the
DU-145 cells. We next identified the chemical form of vitamin C
accumulated intracellularly by the DU-145 cells. HPLC analysis revealed
the presence of only ascorbic acid accumulated intracellularly, with more than 97% of the radioactivity accumulated by the cells eluting in
the position of ascorbic acid (data not shown). We conclude that human
prostate tumor cells transport the dehydroascorbic acid that is
generated by locally activated cells through the glucose transporters
and subsequently accumulate ascorbic acid intracellularly.
We tested the general applicability of the bystander effect by
co-culturing activated HL-60 neutrophils with a number of human primary
cells and cell lines. This group of cells included normal mammary
epithelial cells and the breast cancer cell lines MCF-7 and
MDA-468. These cells do not express functional ascorbic acid transporters and take up dehydroascorbic acid through glucose transporters (data not shown). When incubated in the presence of
activated HL-60 neutrophils, the breast cells showed increased uptake
of vitamin C; at 60 min, they contained an amount of vitamin C that was
6- to 20-fold higher than that accumulated by PMA-treated cells
incubated alone (Fig. 3A).
Moreover, cytochalasin B, but not cytochalasin E, completely blocked
the uptake of vitamin C by the co-cultured bystander cells, with
greater than 90% inhibition observed at 10 µM
cytochalasin B (Fig. 3A and data not shown). The bystander
effect was also observed when PMA-activated fresh human neutrophils
were co-cultured with the human endothelial-like cell line ECV304, the
human melanoma cell line GMEL, or human umbilical vein endothelial
cells (Fig. 3B). These cells express both ascorbic acid and
glucose transporters and therefore transport both oxidized and reduced
vitamin C (Ref. 27, and data not shown). With both cell lines, there
was a marked increase in the uptake of vitamin C by the bystander
cells, an increase that was blocked by cytochalsin B (Fig.
3B). The fraction of uptake that was not blocked by
cytochalasin B was absent when the cells were incubated in medium
lacking sodium ions (data not shown) and therefore corresponds to transport of ascorbic acid through sodium-ascorbate
cotransporters.
Because the bystander effects involves the transport of locally
generated dehydroascorbic acid through facilitative glucose transporters, we tested whether glucose, at concentrations within the
expected ranges found in vivo, could block the bystander
effect by directly competing for the glucose transporters. These
experiments revealed that 15 mM glucose decreased by less
than 40% the uptake of dehydroascorbic acid by the bystander cells
(Fig. 3C), indicating that the bystander effect occurs
efficiently in the presence of physiological concentrations of glucose.
This less-than-expected effect of glucose on vitamin C uptake can be
explained by the observation that glucose increases the production of
superoxide anions by the activated cells in a
dose-dependent manner, with a maximal activation of near
100% at 15 mM glucose (Fig. 3D).
The capacity of human neutrophils and mononuclear phagocytes to
generate oxidants has been widely documented (19, 30), and these cells
accumulate increased amounts of ascorbic acid when incubated under
conditions that lead to the activation of the oxidative burst (15,
20-22). The data have been previously analyzed in the context of the
recycling of vitamin C by activated cells, and based on the evidence
that the glucose transporters are highly efficient transporters of
dehydroascorbic acid, it has been suggested that the increased uptake
probably represents the transport of locally generated dehydroascorbic
acid (15, 22). The detailed analysis presented here is consistent with this interpretation and furthermore, we argue for a more general role
of the oxidative burst in modulating vitamin C availability. Our
central proposal is that the dehydroascorbic acid generated by
activated cells producing superoxide can be transported by other cells
present in the immediate area. We tested this hypothesis using an
experimental system consisting of activated cells (HL-60 neutrophils
and normal human neutrophils) co-cultured with adherent cells
(bystander cells). The co-culture experiments confirmed that activated
cells are able to provide vitamin C, as dehydroascorbic acid, to
adjacent bystander cells by oxidizing ascorbic acid to dehydroascorbic acid, which is then taken up by the adjacent cells and immediately reduced back to ascorbic acid intracellularly.
The triggering of the oxidative burst has been classically linked to
the physiological mechanism of host defense that has as its central
purpose the elimination of microorganisms by strong oxidants generated
by cells of the host defense system (30). Our present findings indicate
that the oxidative burst leads not only to increased accumulation of
vitamin C by the activated cells but also by neighboring cells via a
novel bystander effect. Because of the ubiquitous expression of glucose
transporters, all cells have potentially the capacity to acquire
vitamin C through the bystander mechanism described here. Thus, our
present findings add an additional level of complexity to previous
interpretations by revealing a certain balance between oxidative and
anti-oxidative actions in inflammatory reactions whereby extracellular
oxidation itself causes increased intracellular concentrations of
ascorbic acid. The importance of this concept lies in the fact that
oxidants produced by activated phagocytes to kill microorganisms also
damage adjacent tissues and cells. Similarly, oxidant damage to tissues resulting from autoimmune reactions is due in part to host defense cell
activation. On the other hand, the antioxidative properties of vitamin
C make it likely that an increased intracellular content of vitamin C
in the adjacent bystander cells enhances their antioxidant defense
mechanisms. Thus, the bystander effect links antioxidant cellular
defense with extracellular oxidation.
Our data directly address the apparent paradox that although all human
cells have the capacity to transport oxidized vitamin C,
dehydroascorbic acid represents a very small fraction of total ascorbate in vivo (2, 31). Moreover, in vitro
transport studies with isolated cells have revealed the existence of
sodium-ascorbic acid co-transporters in a number of cells of animal
origin (4, 6, 18, 27, 32-34). Two isoforms with the expected
properties for sodium-ascorbic acid co-transporters were recently
cloned from rat and human RNA, and Northern and in situ
hybridization analysis revealed the presence of the mRNA in small
intestine, liver, kidney, adrenal glands, brain, retina, and other
tissues (35-37). Using in vitro transport assays under
strictly controlled conditions to avoid the uncontrolled oxidation of
ascorbic acid, we have shown absence of functional ascorbic acid
transporters in a number of cells of human origin (10-12, 15). We have
recently extended this analysis to normal and neoplastic cells and
cells lines derived from human prostate and breast (this
study)2 and found that they
transport dehydroascorbic acid efficiently through facilitative glucose
transporters. Furthermore, our studies in vivo
suggest that oxidized vitamin C may cross the blood-brain barrier (16).
Why, then, if all human cells express dehydroascorbic acid
transporters, is there no detectable dehydroascorbic acid in blood? We
reason that this may be related to the chemical properties of the
different forms of vitamin C. Although ascorbic acid appears to be
stable in vivo, in solution dehydroascorbic acid undergoes hydrolysis with an estimated half-life of less than 1 min (10, 38-41).
The hydrolysis is an irreversible process, which if it where to occur
in vivo, would imply a vast consumption of vitamin C. This
is an issue of physiological significance because humans are unable to
synthesize vitamin C and must obtain the vitamin from external sources
in the diet. The absence of vitamin C in the diet leads to the
development of scurvy with its associated problems and ultimately
death. Although there has been some controversy regarding the
definition of the optimal daily requirements of vitamin C in humans in
exact physiological terms (42, 43), it is clear that small daily
amounts of vitamin C in the diet are sufficient for the maintenance of
a "normal" human physiology. It is therefore evident that the
salvage of vitamin C in vivo through continuous recycling
could be central to the maintenance of low daily requirements of the
vitamin. This is an issue that has been addressed in the case of the
recycling of vitamin C by human erythrocytes and its effect on the
antioxidant reserve of whole blood (44, 45). Our present data extend
these previous observations by indicating that the dehydroascorbic acid
generated locally by oxidation of ascorbic acid can be immediately
transported intracellularly and reduced back to ascorbic acid by
neighboring cells. Moreover, our in vitro data indicate that
the bystander effect is operative in the presence of physiological
concentrations of glucose, and we have preliminary evidence indicating
that the bystander effect may be functional in vivo (46).
Thus, the bystander effect may provide cells with an efficient system
for the recycling and salvage of vitamin C.
Although the study presented here refers to human cells, its
conclusions are generally applicable to cells that express glucose transporters and are involved in oxidative events of the kind described
here. A mechanism for the acquisition of vitamin C based in the local
oxidation of ascorbic acid is not necessarily restricted to cells of
the host defense system. Although the NADPH oxidase of phagocytes has
been the most thoroughly studied, there is evidence indicating that
non-phagocytic human cells express a membrane oxidase with the capacity
to generate superoxide. Endothelial cells, smooth muscle cells, retinal
epithelial cells, and fibroblasts generate low levels of superoxide,
and the activity of the oxidases expressed in these cells is regulated
in a cell-dependent manner by growth factors and other
cell-specific stimuli (47-49). It has been proposed that the
generation of superoxide by non-phagocytic cells may be central to the
delivery of signals important for cell function. Based on the results
presented here, we propose that the NADPH oxidase present in
non-phagocyte cells may play a role in regulating not only their own
cellular uptake of vitamin C, but may also potentially modulate the
uptake of vitamin C in other cells.
Our data may provide useful insights into the underlying role of
vitamin C deficiency in human disease. Patients with
insulin-dependent and non-insulin-dependent
diabetes mellitus show an impairment of
endothelium-dependent vasodilation that can be restored
with vitamin C administration (50), and it has been proposed that hyperglycemia may contribute to impaired vascular function through production of superoxide anions (51). Moreover, diabetes and inflammatory disease are often accompanied by a decrease in plasma and
intracellular ascorbate levels and by alterations in plasma ascorbate-dehydroascorbate ratios (52). In this context, our data are
compatible with the concept that hyperglycemia may lead to decreased
intracellular regeneration of ascorbic acid because of irreversible
loss of extracellular dehydroascorbic acid.
In conclusion, we propose a model for vitamin C recycling that resolves
the apparent contradiction that although dehydroascorbic acid is only a
minor fraction of the total content of ascorbate in vivo,
all human cells express dehydroascorbic acid transporters. In this
model, superoxide generated by activated cells undergoing the oxidative
burst oxidize extracellular ascorbic acid to dehydroascorbic acid which
is then transported through glucose transporters by the activated cells
themselves as well as other cells present in the immediate area. The
transported dehydroascorbic acid is immediately reduced back to
ascorbic acid, which accumulates at high concentrations
intracellularly. This recycling model may work as an efficient system
for the salvage of vitamin C by avoiding the irreversible hydrolysis of
the dehydroascorbic acid produced in inflammatory reactions. Moreover,
it provides cells with increased intracellular concentrations of
ascorbic acid with its associated antioxidant properties, therefore
linking normal oxidative events to the regulation of antioxidant
protection of cells and tissues.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C. Samples containing vitamin C in solution were adjusted
to 60% methanol and 1 mM EDTA and stored at
70 °C.
HPLC analyses were performed using a Whatman strong anion exchange
reverse-phase column (Partisil 10 SAX, 4.6 mm × 25 cm, 10-µm
particle) (15). Dehydroascorbic acid eluted at 4.6 min, and ascorbic
acid eluted at 9.4 min. The elution of dehydroascorbic acid was
monitored by radioactivity, whereas ascorbic acid was monitored by both radioactivity and absorbance at 266 nm.
1·cm
1.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Vitamin C uptake and superoxide production in
HL-60 neutrophils. A, vitamin C uptake by HL-60
neutrophils treated with (+) or without ( ) PMA. HL-60 neutrophils
were resuspended in medium containing 50 µm ascorbic acid and 50 µm
PMA and incubated with 20 µM cytochalasin B
(CytB) or cytochalasin E (CytE), or with 100 enzyme units of superoxide dismutase (SOD) or catalase
(Cat). Uptake lasted for 60 min. Enzymes were added to the
assay 30 min prior to, although cytochalasin B and E were added at the
start of the uptake assay. Data are the mean ± standard deviation
of one experiment performed in quadruplicate. B, superoxide
generation by HL-60 neutrophils treated with (+) or without (
) 0.5 µM PMA. HL-60 neutrophils were resuspended in media
containing PMA and incubated, for 60 min, with 20 µM
cytochalasin B (CytB) or cytochalasin E (CytE) or
with 100 enzyme units of superoxide dismutase (SOD) or
catalase (Cat). Data are the mean ± standard deviation
of one experiment performed in quadruplicate. C, generation
of dehydroascorbic acid from extracellular ascorbic acid in cultures of
activated HL-60 neutrophils. Cell were incubated for up to 60 min with
50 µM ascorbic acid in the presence of 0.5 µM PMA (+PMA), or simultaneously with 0.5 µM PMA and 100 units of superoxide dismutase
(+SOD). Aliquots of the culture media obtained at different
times after starting the incubation were extracted with 3 volumes of
70%methanol-1 mM EDTA. Samples were fractionated by HPLC
to determine the content of ascorbic acid (
,
) and
dehydroascorbic acid (
). Data represent the results of one
experiment of two performed. Abbreviations: AA, ascorbic acid; DHA,
dehydroascorbic acid; SOD, superoxide dismutase; Cat, catalase; CytB,
cytochalasin B; Cyt E, cytochalasin E.
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Fig. 2.
Vitamin C uptake and superoxide production in
co-cultures of HL-60 neutrophils and prostate DU-145 cells.
A, time course of the effect of PMA on vitamin C uptake by
DU-145 cells cultured alone ( ), or co-cultured with HL-60
neutrophils (
). B, time course of the effect of PMA on
superoxide production by DU-145 cells cultured alone (
), HL-60
neutrophils cultured alone (
), or co-cultures of HL-60 neutrophils
and DU-145 cells (
). C, vitamin C uptake by DU-145 cells
co-cultured with increasing numbers of HL-60 neutrophils. D,
superoxide production in co-cultures of DU-145 cells and increasing
numbers of activated HL-60 neutrophils. E, effect of
superoxide dismutase (
), or catalase (
) on vitamin C uptake by
DU-145 cells co-cultured with HL-60 neutrophils in the presence of PMA.
F, effect of cytochalasin B (
) or cytochalasin E (
) on
vitamin C uptake by DU-145 cells co-cultured with HL-60 neutrophils in
the presence of PMA. PMA was used at 0.5 µM and ascorbic
acid was used at 50 µM. Data are the mean ± standard deviation of one experiment performed in
quadruplicate.
View larger version (21K):
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Fig. 3.
The bystander effect for the recycling of
vitamin C. A, vitamin C uptake by normal mammary
epithelial cells (HMEC) and the breast cancer cell lines
MCF-7 and MDA-468, co-cultured with (+) or without ( ) HL-60
neutrophils. B, vitamin C uptake by the endothelial cell
line ECV-304, the melanoma cell line GMEL and human umbilical vein
endothelial cells (HUVEC), co-cultured with (+) or without (
) normal
human neutrophils. Uptake of 50 µM ascorbic acid was
tested in the absence (
) or in the presence (+) of 0.5 µM PMA, and with (+) or without (
) 20 µM
cytochalasin B. Cells cultured alone in medium containing ascorbic acid
without PMA were used as controls. Uptake was measured at 60 min.
C, effect of glucose on vitamin C uptake by DU-145 cells
co-cultured with HL-60 cells. The incubation medium contained 50 µM ascorbic acid, 0.5 µM PMA and the
indicated concentrations of glucose. Uptake was measured at 60 min.
D, effect of glucose on superoxide production in co-cultures
of DU-145 cells and HL-60 cells. PMA was used at 0.5 µM,
and superoxide production was determined at 60 min. Data are the
mean ± standard deviation of one experiment performed in
quadruplicate. Abbreviations: PMN, normal human neutrophils; HMEC,
normal human breast epithelial cells; CytB, cytochalasin B.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
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FOOTNOTES |
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* This work was supported by Grants 1990333, 1010843, and 1020451 from Fondo Nacional de Investigación Científica y Tecnólogica, Chile, National Institutes of Health Grant CA30388, and Grant DIUC 201034006-1.4 from the Dirección de Investigación, Universidad de Concepción, Concepción, Chile.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. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Departamento de
Fisiopatología, Facultad de Ciencias Biológicas,
Universidad de Concepción, Barrio Universitario S/N,
Concepción, Chile. Tel.: 41-203817; Fax: 41-216558; E-mail:
juvera@udec.cl.
Published, JBC Papers in Press, November 14, 2002, DOI 10.1074/jbc.M210686200
2 C. I. Rivas, F. J. Nualart, and J. C. Vera, unpublished results.
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ABBREVIATIONS |
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The abbreviation used is: PMA, phorbol 12-myristate 13-acetate.
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