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(Received for publication, April 4,
1995; and in revised form, July 31, 1995) From the
We performed a detailed kinetic analysis of the uptake of
dehydroascorbic acid by HL-60 cells under experimental conditions that
enabled the differentiation of dehydroascorbic acid transport from the
intracellular reduction/accumulation of ascorbic acid. Immunoblotting
and immunolocalization experiments identified GLUT1 as the main glucose
transporter expressed in the HL-60 cells. Kinetic analysis allowed the
identification of a single functional activity involved in the
transport of dehydroascorbic acid in the HL-60 cells. Transport was
inhibited in a competitive manner by both
3-O-methyl-D-glucose and 2-deoxy-D-glucose.
In turn, dehydroascorbic acid competitively inhibited the transport of
both sugars. A second functional component identified in experiments
measuring the accumulation of ascorbic acid appears to be associated
with the intracellular reduction of dehydroascorbic acid to ascorbic
acid and is not directly involved in the transport of dehydroascorbic
acid via GLUT1. Transport of dehydroascorbic acid by HL-60 cells was
independent of the presence of external Na
Vitamin C is fundamental to human
physiology(1, 2, 3) . Since humans cannot
synthesize vitamin C, it must be provided exogenously in the diet and
transported intracellularly(4, 5) . Vitamin C is
present in human blood at an average concentration of about
50-100 µM, and at least 95% is in the reduced form
(ascorbic acid) with the remaining 5% in the oxidized form
(dehydroascorbic acid)(5) . The observation that cells and
tissues accumulate characteristic intracellular concentrations of the
reduced form of vitamin C, both in vivo and in vitro,
suggests that the transport and cellular accumulation of vitamin C is a
highly regulated process(5) . Data accumulated over a number
of years pointed to the participation of glucose transporters in the
cellular transport of vitamin
C(6, 7, 8, 9, 10, 11, 12) ,
although functional evidence for the existence of additional
transporters is also
available(13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24) .
We recently demonstrated directly, by expression in Xenopus laevis oocytes, that the glucose transporters GLUT1, GLUT2, and GLUT4 are
efficient transporters of dehydroascorbic acid(25) . We
extended these studies to show that glucose transporters are also the
main pathway mediating the transport of dehydroascorbic acid in normal
human neutrophils and HL-60 myeloid leukemia
cells(25, 26) . There has been controversy regarding
the issue of the identity of the form of vitamin C, reduced or
oxidized, transported by human neutrophils. Freshly isolated human
neutrophils contain millimolar concentrations of reduced ascorbic acid,
and in vitro they accumulate high concentrations of ascorbic
acid when incubated with ascorbic or dehydroascorbic
acid(5, 27) . Current evidence indicates, however,
that dehydroascorbic acid, and not ascorbic acid, is preferentially
transported into
cells(9, 18, 25, 26, 28, 29, 30, 31, 32, 33, 34, 35, 36) .
We, and others, have shown that under the incubation conditions used by
most laboratories, ascorbic acid undergoes oxidation to dehydroascorbic
acid which is then transported
intracellularly(25, 26, 37, 38) .
The transported dehydroascorbic acid is reduced back to ascorbic acid,
providing the mechanism for its cellular accumulation. The oxidation of
ascorbic acid to dehydroascorbic acid appears to be part of the
mechanism by which cells of the host defense system, such as
neutrophils, increase their uptake of dehydroascorbic acid when
activated by physiological stimuli. Kinetic analysis of the uptake
of ascorbic acid in human neutrophils has revealed the presence of two
functional activities with different affinities for ascorbate, an
observation that was interpreted as suggesting the existence of at
least two separate transport systems involved in the cellular uptake of
ascorbic acid(27) . We detected two functional activities, one
with high affinity and one with low affinity, involved in the uptake of
dehydroascorbic acid in X. laevis oocytes expressing GLUT1 (25) and in human neutrophils and HL-60
cells(25, 26) . The X. laevis expression
experiments suggested the association of both functional activities
with the expression of a single transport protein. The studies
mentioned above were performed under conditions leading to the
accumulation of ascorbic acid in cells incubated with dehydroascorbic
acid. Uptake under these conditions is a function of both transport and
intracellular trapping. No clear distinction between transport of
dehydroascorbic acid and accumulation of ascorbic acid has been
presented, and, therefore, it has been impossible to assign the
functional activities detected to the steps of transport or
accumulation. An understanding of the mechanisms that regulate the
cellular content of ascorbic acid requires the identification of the
events involved in the transport of dehydroascorbic acid as well as in
the cellular accumulation of ascorbic acid. We studied this issue by
analyzing the accumulation of ascorbic acid in HL-60 cells under
experimental conditions that allowed us to dissociate the transport of
dehydroascorbic acid from its intracellular accumulation as ascorbic
acid. We identified GLUT1 as the glucose transporter expressed in HL-60
cells, characterized it functionally, established the conditions to
measure transport as distinct from accumulation, and applied these
conditions to measure the transport of dehydroascorbic acid. We now
provide evidence indicating that only one functional system,
corresponding to the glucose transporter GLUT1, is directly involved in
the facilitated transport of dehydroascorbic acid by HL-60 cells. A
second functional activity apparently associated with the trapping and
accumulation of the reduced form of ascorbic acid is not directly
involved in the transport of dehydroascorbic acid via the glucose
transporters.
Figure 1:
Expression of GLUT1 in HL-60 cells. A, for immunoblotting, equal amounts of protein were loaded on
each lane of a sodium dodecyl sulfate-containing polyacrylamide gel,
electrophoresed, transferred to Immobilion, reacted with the different
anti-GLUT antibodies, and visualized using horseradish peroxidase
anti-rabbit IgG and enhanced chemiluminescence. B and C, for immunofluorescence, cytospin preparations were reacted
with preimmune serum (panel B) or anti-GLUT1 antibodies (panel C), and visualized using fluorescein-coupled secondary
antibodies. No reactivity was observed with anti-GLUT2, -3, -4, or -5
antibodies.
Figure 2:
Kinetics of the uptake of methylglucose by
HL-60 cells and the effect of competition with different sugars. A, time course of the uptake of 1 mM methylglucose. B, dose-response of the transport of methylglucose using 30-s
uptake assays. C, double-reciprocal plot of the substrate
dependence for methylglucose transport. D, semi-log plot of
the concentration dependence for inhibition of methylglucose transport
in HL-60 cells by different sugars. Measurements were performed at 1
mM methylglucose using 30-s uptake assays. DOG,
deoxyglucose; OMG, methylglucose;
Dose-response experiments examining uptake at 30 s indicated that
transport of methylglucose approached saturation at millimolar
concentrations of methylglucose (Fig. 2B), with an
apparent K
Figure 3:
Kinetics of the uptake of deoxyglucose by
HL-60 cells and the effect of competition with different sugars. A, time course of the uptake of 2 and 5 mM deoxyglucose. B, time course of the uptake of 5 mM deoxyglucose. C, dose-response of the transport of
deoxyglucose using 30-s uptake assays. D, double-reciprocal
plot of the substrate dependence for deoxyglucose transport. Uptake
assay, 30 s. E, semi-log plot of the concentration dependence
for inhibition of deoxyglucose transport in HL-60 cells by different
sugars. Measurements were performed at 0.2 mM deoxyglucose
using 30-s uptake assays. DOG, deoxyglucose; OMG,
methylglucose.
Figure 4:
Kinetics of the uptake of dehydroascorbic
acid by HL-60 cells and the effect of competition with different
sugars. A, time course of the uptake of 50 µM dehydroascorbic acid. B, short time course of the uptake
of 50 µM dehydroascorbic acid. C, dose-response
of the transport of dehydroascorbic acid.
Dose-response studies using 30-s uptake assays revealed that
the transport of dehydroascorbic acid by the HL-60 cells saturated at
about 4 mM (Fig. 4C). These studies also
revealed the presence of a single component with an apparent K Competition
experiments using 30-s transport assays indicated that deoxyglucose,
methylglucose, and maltose inhibited the transport of dehydroascorbic
acid by the HL-60 cells with IC
Figure 5:
Competitive inhibition of methylglucose
uptake by dehydroascorbic acid. A, semi-log plot of the
concentration dependence for inhibition of methylglucose transport in
HL-60 cells by dehydroascorbic acid (
Figure 6:
Sodium-independence of the uptake of
dehydroascorbic acid by HL-60 cells. A, time course of the
uptake of dehydroascorbic acid in the presence of NaCl (
We further analyzed this
issue by measuring uptake in HL-60 cells incubated with ascorbic acid
in the absence of Na Our data indicate that the accumulation of ascorbic acid in
HL-60 cells is a process that can be dissociated into at least two
components with characteristic kinetics. The initial step consists of
the facilitated transport of dehydroascobate, followed by its reduction
and intracellular accumulation as ascorbic acid. The transport
component of uptake was very rapid and could be detected only by using
very short uptake assays. At extended incubation periods, the
rate-limiting step of uptake was the cellular trapping/reduction and
accumulation of ascorbic acid, and it was no longer possible to measure
transport separately and as distinct from accumulation. Transport was
mediated by GLUT1, the member of the family of facilitative glucose
transporters expressed in the HL-60 cells. Although six different
facilitative glucose transporters have been identified in mammalian
cells(39) , we identified GLUT1 as the main facilitative
glucose transporter expressed by HL-60 cells. The HL-60 cells were able
to transport deoxyglucose, a substrate specific for the facilitated
glucose transporters that is not transported by the sodium-glucose
cotransporters(40, 41) . In addition,
Our transport
data are consistent with the models presented in Fig. 7. Short
uptake assays enabled us to carry out a detailed kinetic analysis of
the transport of dehydroascorbic acid as distinct from the accumulation
of ascorbic acid. Only one functional component, with an apparent K
Figure 7:
Models for differentiating transport from
accumulation during uptake experiments. A, the initial rate of
transport of a substrate, such as methylglucose, that is not
metabolized in the interior of the cell, corresponds to the early
linear phase of the uptake curve. As the intracellular concentration of
the recently transported substrate increases, efflux becomes
significant until its rate equals the rate of influx. Under steady
state conditions, the intracellular and extracellular concentrations of
the transported substrate are identical, and the net rate of transport
in any direction is zero. The kinetic analysis is done using data
obtained at short uptake times in which the rate-limiting step of
uptake is transport. OMG, methylglucose. B, the
situation is more complex for a substrate such as dehydroascorbic acid
that is transported intracellularly and then trapped by reduction to
the non-transported species ascorbic acid. The early linear phase of
the uptake curve represents the initial rate of transport, but a second
linear phase that represents the trapping component and may last for a
long period of time is also evident. Trapping is the rate-limiting step
when using long uptake assays because the rate of transport exceeds the
rate of trapping, and part of the transported dehydroascorbic acid will
be transported back out of the cell. At steady state, the intracellular
concentration of the trapped, non-transported species, may exceed by
severalfold the extracellular concentration of the transported
substrate. In a typical uptake experiment using radioactive
dehydroascorbic acid, this result may give the false impression that
the transported substrate, dehydroascorbic acid, is accumulating
against a concentration gradient. Thus, long uptake experiments measure
the accumulation of the non-transported species and its respective
associated kinetic constants, rather than the transport of the
substrate handled by the transporter. Similar considerations apply to
the uptake of deoxyglucose. AA, ascorbic acid; DHA,
dehydroascorbic acid.
The existence of several components involved in the
uptake of dehydroascorbic acid by HL-60 cells was evident only in long
uptake experiments that are a complex function of the transport of
dehydroascorbic acid and the intracellular trapping of ascorbic acid.
We failed to detect the presence of a second, high-affinity component
involved in the transport of dehydroascorbic acid by HL-60 cells. We
observed, however, a high-affinity component when measuring the
accumulation of ascorbic acid in cells incubated with dehydroascorbic
acid for periods of time equal to or longer than 10 min. Under these
conditions, the intracellular trapping of ascorbic acid rather than
transport is the rate-limiting step for uptake. An additional
low-affinity component was also observed in these studies, adding
another level of complexity to the overall process of accumulation of
ascorbic acid. Competition experiments showed that the transport of
dehydroascorbic acid was inhibited by methylglucose and deoxyglucose,
and the specificity of this inhibition was demonstrated by the results
indicating that other sugars unable to interact with the glucose
transporters had no effect on the transport of dehydroascorbic acid.
Competition experiments, using 10-min incubation assays to measure
accumulation, indicated that hexoses completely blocked the
accumulation of ascorbic acid in HL-60 cells by a noncompetitive
mechanism. We interpreted these results as indicating that glucose acts
directly on the primary event responsible for the entry of
dehydroascorbic acid, that is GLUT1-mediated transport, not on the
functional component(s) involved in the intracellular accumulation of
ascorbic acid. Therefore, the high- and low-affinity components
detected in the 10-min uptakes are likely associated with the reduction
of dehydroascorbic acid permitting the intracellular accumulation of
ascorbic acid. Support for the interpretation that the high-affinity
component is not related to the glucose transporter is provided by the
observation that low concentrations of dehydroascorbic acid (<50
µM) did not affect the transport of methylglucose or
deoxyglucose. A high-affinity component has been identified in
experiments measuring the accumulation of ascorbic acid in different
cellular systems and the hypothesis has been advanced that it
corresponds to a high-affinity transporter of reduced ascorbic acid (27) . These studies were performed, however, under
experimental conditions that led to the oxidation of ascorbic acid with
generation of the transported form, dehydroascorbic acid. Of major
concern is the fact that in such studies the kinetic constants were
derived from long uptake experiments (90 min), raising the question of
the identity of the rate-limiting step under those
conditions(27) . Our data indicating that deoxyglucose was able
to completely inhibit the cellular uptake of dehydroascorbic acid in a
range of experimental conditions, including different concentrations of
dehydroascorbic acid and different times of incubation, are consistent
with the concept that GLUT1 is the only means by which the HL-60 cells
transport dehydroascorbic acid. Our results also indicate that HL-60
cells do not express a Na Taken together,
our data indicate that the transport of dehydroascorbic acid via the
glucose transporters is kinetically and biologically separable from the
reduction of dehydroascorbic acid to ascorbic acid and its subsequent
intracellular accumulation. These findings have major significance for
our understanding of the biological control mechanisms involved in the
homeostasis of the cellular content of ascorbic acid in human cells. We
can envision the existence of separate mechanisms that regulate the
ability of a cell to transport dehydroascorbic acid and its capacity to
reduce the transported substrate intracellularly and accumulate
ascorbic acid. The issue of the mechanisms involved in the
intracellular reduction of dehydroascorbic acid and the concomitant
accumulation of ascorbic acid has been controversial. No
dehydroascorbic acid reductase activity has been consistently detected
in cells or human tissues that accumulate intracellularly millimolar
concentrations of ascorbic acid when incubated in the presence of
dehydroascorbic acid(45, 46) . The existence of a
dehydroascorbate reductase has been clearly demonstrated in plants that
use reduced glutathione to catalyze the reduction of dehydroascorbate
to ascorbate, and a similar activity has been partially characterized
in bovine iris-ciliary body(47) . In humans, it has been
proposed that the tripeptide glutathione, which is present at
millimolar concentrations in cells, may play a central role in
maintaining extracellular and intracellular ascorbic acid in its
reduced state(48) . Experimental observations in animal models
have revealed a close interrelationship between the cellular content of
ascorbate and glutathione. Guinea pigs and newborn rats cannot
synthesize ascorbic acid and it must be provided in the
diet(4) . Newborn rats, made glutathione deficient by treatment
with L-buthionine-(S,R)-sulfoximine, showed
a decreased content of ascorbic acid in tissues, and supplementation
with ascorbic acid in the diet induced increased tissue levels of
ascorbic acid and glutathione(49) . In guinea pigs fed an
ascorbic acid-free diet, supplementation with glutathione monoester, a
compound that increases the cellular levels of glutathione, delayed the
onset and the development of the pathologic complications of scurvy (50) . These observations established a close functional
correlation between the respective in vivo levels of
glutathione and ascorbic acid, but did not provide information about
the mechanisms involved. On the other hand, in vitro observations have established that glutathione is able to reduce
dehydroascorbic acid to ascorbic acid in the absence of any enzymatic
activity through a direct chemical reaction(37, 51) .
No direct evidence is available, however, indicating that glutathione
is the physiological reducer of dehydroascorbic acid in cells or that
it is directly involved in the ability of cells to take up
dehydroascorbic acid and accumulate ascorbic acid(52) .
Recently, it was described that two well known and widely expressed
enzymes, protein disulfide isomerase and glutaredoxin, possess
dehydroascorbate reductase activity(53) , raising the
possibility that they could be involved in the reduction-dependent
intracellular accumulation of ascorbic acid in human cells. In fact,
the characterization and purification of two different dehydroascorbate
reductase activities present in rat liver led to the identification of
glutaredoxin as one of the proteins (54) . The ability to
differentiate the transport of dehydroascorbic acid from its
accumulation as ascorbic acid offers an opportunity to address these
and related issues in a controlled cellular system amenable to
experimentation.
Volume 270,
Number 40,
Issue of October 06, pp. 23706-23712, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
, whereas
the intracellular accumulation of ascorbic acid was found to be a
Na
-sensitive process. Thus, the transport of
dehydroascorbic acid via glucose transporters is a
Na
-independent process which is kinetically and
biologically separable from the reduction of dehydroascorbic acid to
ascorbic acid and its subsequent intracellular accumulation.
Cells
Human myeloid HL-60 cells were cultured in
Iscove's modified Dulbecco's medium supplemented with 10%
fetal bovine serum, 1% penicillin/streptomycin, and 2 mML-glutamine. Cell viability was greater than 95% as assessed
by trypan blue exclusion.Uptake Studies
For uptake assays, the cells were
suspended in incubation buffer (15 mM Hepes pH 7.6, 135 mM NaCl, 5 mM KCl, 1.8 mM CaCl
, 0.8
mM MgCl), washed by centrifugation in the same
buffer and resuspended at 0.5-2 
10
cells/ml.
Uptake assays were performed in a final volume of 0.2 ml of incubation
buffer containing 0.5-2 10
cells,
0.1-0.4 µCi of L-[
C]ascorbic
acid (specific activity 8.2 mCi/mmol, DuPont NEN), a final
concentration of 0.05-15 mM ascorbic acid, and
1-100 units of ascorbic acid oxidase (50 units/mg protein,
Sigma). The mixture was incubated for 30 s or 10 min at room
temperature, and uptake was stopped by adding 10 volumes of cold
phosphate-buffered saline (4 °C) containing 100 µM phloretin (stopping solution). The cells were collected and washed
twice by centrifugation in cold stopping solution. Samples were
processed for scintillation spectrometry or high performance liquid
chromatography as described (26) . Hexose uptake assays were
similarly performed using 1 µCi of D-3-O-[methyl-
H]glucose
(specific activity 10 Ci/mmol, DuPont NEN) (methylglucose) (
)and 0.3-20 mM methylglucose or 1 µCi of
2-[1,2-H]deoxy-D-glucose (specific
activity 26.2 Ci/mmol, DuPont NEN) (deoxyglucose) and 0.3-20
mM deoxyglucose. When appropriate, competitors and inhibitors
were added to the uptake assays, or the cells were preincubated in
their presence.Western Blot Analysis
HL-60 cells were homogenized
in buffered Tris-HCl (pH 7.4) containing 0.5% deoxycholic acid, 0.5%
Triton X-100, 1 mM EDTA, 1 mM EGTA, 5 µg/ml each
of aprotinin, leupeptin, and soybean trypsin inhibitor, and 1
mM phenylmethylsulfonyl fluoride. Cell debris was removed by
centrifugation at 10,000 g for 10 min at 4 °C,
membranes were collected on a 45% sucrose cushion by centrifugation at
105,000 g for 2 h, and 100 µg of protein were
loaded on each lane of a sodium dodecyl sulfate-containing 10%
polyacrylamide gel and electrophoresed at 20 mA for 6 h. After transfer
to a polyvinylidene difluoride membrane (Immobilion, Millipore), blots
were stained with anti-GLUT or with preimmune serum followed by
incubation with horseradish peroxidase-anti-rabbit antibody. Proteins
were visualized by enhanced chemiluminescence (Amersham).Immunolocalization
For immunocytochemistry, the
cells were collected by centrifugation (Cytospin, Shandon) onto
microscope slides, fixed in buffered formaldehyde-acetone for 30 s, and
washed with phosphate-buffered saline (pH 7.4). Fixed cells were
incubated in a humid chamber for 60 min in phosphate-buffered saline
containing 5% bovine serum albumin, followed by incubation for 2 h at
room temperature in the same buffer containing 1% bovine serum albumin,
0.3% Triton X-100 and the respective anti-GLUT antiserum, or rabbit
preimmune serum, diluted 1:100. The cells were then washed and
incubated with anti-rabbit IgG-fluorescein (Life Technologies, Inc.,
1:50) for 1 h, washed, and mounted.
Expression of GLUT1 in HL-60 Cells
Six
facilitative glucose transporter isoforms have been described in
mammalian cells of which five, GLUT1-GLUT5, appear to be expressed on
the cell membrane, and one, GLUT7, is restricted in expression to the
internal membranes of the endoplasmic reticulum(39) . We used a
panel of antibodies specific for each of five members of the family of
glucose transporters (GLUT1-GLUT5) to identify the isoforms expressed
in the HL-60 cells and responsible for the uptake of hexoses in these
cells. By immunoblotting, the anti-GLUT1 antibodies reacted with a
protein of M
50,000 present in a plasma
membrane-enriched fraction prepared from HL-60 cells (Fig. 1A) or in a preparation of total cellular
proteins (data not shown). No reactivity was observed with anti-GLUT2,
-GLUT3, -GLUT4, or -GLUT5 antibodies (Fig. 1A),
although longer exposures of the blot revealed a weak reactivity with
anti-GLUT5 antibodies. Control experiments using membranes or total
cellular proteins prepared from cells expressing the different glucose
transporter isoforms indicated that the antibodies recognized their
respective antigens under identical experimental conditions (data not
shown). Parallel immunolocalization experiments using
fluorescein-labeled secondary antibodies indicated abundant expression
of GLUT1 in the HL-60 cells, but failed to detect the presence of the
other transporter isoforms (Fig. 1, B-C, and data not
shown). Similar results were obtained using silver enhanced colloidal
gold or alkaline phosphatase (data not shown). The specificity and the
reactivity of each antibody were tested, with positive results, in
cells expressing the different transporter isoforms.
Facilitated Transport of Methylglucose
Having
established the presence of GLUT1 in the HL-60 cells, we carried out a
detailed characterization of the uptake of hexoses in these cells. The
aim of these studies was to develop experimental conditions that would
allow differentiation between the transport of dehydroascorbic acid by
the glucose transporters and the intracellular accumulation of ascorbic
acid. In initial experiments, we analyzed the transport of the
nonmetabolizable glucose analog methylglucose by HL-60 cells. Time
course experiments revealed that transport of methylglucose by HL-60
cells occurred rapidly, with half the equilibrium concentration reached
in about 5 min (Fig. 2A). The rate of transport was
linear for the first 30 s, and equilibrium was reached in about 20 min.
The intracellular concentration of methylglucose never exceeded the
respective extracellular concentrations even in samples incubated with
the hexose for several hours. Similar results were obtained using
concentrations of methylglucose from 1 to 20 mM.
-MethylG,
-methyl-D-glucopyranoside.
for transport of 8.5 mM (Fig. 2C). Uptake was decreased in the presence of
deoxyglucose, a typical substrate of the facilitative glucose
transporters(40) . Deoxyglucose, at 3 mM, inhibited by
50% (IC = 3 mM) the transport of
methylglucose by the HL-60 cells (Fig. 2D). Maltose, a
disaccharide that binds to the glucose transporters but is not
transported(40) , inhibited the uptake of methylglucose with an
IC
of approximately 25 mM, but no effect on
transport was observed with fructose, sucrose,
-methyl-D-glucoside, or L-glucose (Fig. 2D). -Methyl-D-glucoside is a
substrate of the sodium-dependent glucose cotransporter (41) that is not transported by the facilitative glucose
transporters, and sucrose and L-glucose do not interact with
the glucose transporters(40) . Cytochalasin B, but not
cytochalasin E, inhibited the transport of methylglucose with an
IC of 200 nM (data not shown).
Facilitated Transport of Deoxyglucose
Deoxyglucose
is a substrate of facilitative glucose transporters, and the
transported deoxyglucose accumulates intracellularly in cells as
deoxyglucose-6-phosphate(40) . Discrimination between transport
and accumulation is usually accomplished experimentally by carrying out
very short uptake assays or by measuring the uptake of low micromolar
concentrations of deoxyglucose. Two components were observed when the
time course of the uptake of 5 mM deoxyglucose was measured in
the HL-60 cells (Fig. 3, A and B). The amount
of cell-associated radioactivity increased rapidly at short incubation
times, and the rate of increase was linear for the first minute of
incubation. Thereafter, the rate of incorporation decreased and a
second component was observed that was linear for at least 30 min. Only
one component, that was linear for at least 30 min, was observed in
experiments measuring the time course of uptake of 2 mM deoxyglucose (Fig. 3A). We interpreted these
results as indicating that the initial linear phase of incorporation of
radioactivity by the HL-60 cells represented the transport of
deoxyglucose, with the second linear component representing the
phosphorylation and intracellular accumulation of phosphorylated
deoxyglucose. Dose-response experiments examining uptake at 30 s
indicated that transport of deoxyglucose reached saturation at
millimolar concentrations of deoxyglucose (Fig. 3C)
with an apparent K for transport of 2.5 mM (Fig. 3D). Uptake was competed by methylglucose
and maltose, with IC of 10 and 30 mM,
respectively, but not by fructose, sucrose,
-methyl-D-glucoside, or L-glucose (Fig. 3E). No measurable uptake of fructose was
observed in cells incubated in the presence of radiolabeled fructose
(data not shown). Cytochalasin B, but not cytochalasin E, inhibited the
transport of deoxyglucose with an IC of 250 nM (data not shown).
Facilitated Transport of Dehydroascorbic Acid
We
analyzed the characteristics of facilitated transport of
dehydroascorbic acid in HL-60 cells distinct from the accumulation of
ascorbic acid. Uptake of dehydroascorbic acid proceeded in an
apparently linear fashion for at least 10 min, with no evidence of
saturation at 60 min (Fig. 4A). Two kinetic components
were observed, however, when the time course of the uptake of
dehydroascorbic acid by the HL-60 cells was measured at short
incubation periods (Fig. 4B). Initially, the amount of
cell-associated radioactivity increased very rapidly, and the rate of
increase was linear for the first minute of incubation. Afterwards, the
rate of incorporation decreased, and a second kinetic component that
was linear for at least 10 min was observed. The amount of
cell-associated radioactivity after 10 min of incubation with
radioactive dehydroascorbic acid greatly exceeded the amount expected
at equilibrium after facilitated transport. The two kinetic components
were observed in uptake experiments using concentrations of
dehydroascorbic acid ranging from 20 µM to 4 mM,
but only one component was observed at 10 µM dehydroascorbic acid (data not shown). We interpreted these
results as indicating that the initial linear phase of incorporation of
radioactivity by the HL-60 cells represented the transport of
dehydroascorbic acid, with the second, slower linear component
representing the reduction of the recently transported dehydroascorbic
acid to ascorbic acid leading to the intracellular accumulation of
reduced ascorbic acid. Thus, the reduction of dehydroascorbic acid to
ascorbic acid is the rate-limiting step when measuring uptake at
concentrations of ascorbic acid equal to or greater than 20 µM and when using uptake assays lasting longer than 1 min.
Alternatively, measuring uptake at incubation times of less than 30 s
gives information on transport at any concentration of dehydroascorbic
acid.
, 10-min uptake assay.
, 30-s uptake assay. D, double-reciprocal plot of the
substrate dependence for dehydroascorbic acid transport. , 10-min
uptake assay. , 30-s uptake assay. E, dose-response of
the transport of dehydroascorbic acid using 30-s uptake assays. F, dose-response of the transport of dehydroascorbic acid
using 10-min uptake assays. G, semi-log plot of the
concentration dependence for inhibition of dehydroascorbic acid
transport in HL-60 cells by different sugars. Measurements were
performed at 50 µM dehydroascorbic acid using 30-s uptake
assays. DHA, dehydroascorbic acid; DOG, deoxyglucose; OMG, methylglucose; MethylG,
-methyl-D-glucopyranoside.
of 0.85 ± 0.12 mM (n = 8), and a V
of 4 nmol/min/10 cells for the transport of dehydroascorbic acid by the HL-60
cells (Fig. 4D). Parallel studies measuring uptake of
dehydroascorbic acid using 10-min assays revealed the presence of one
component saturating at about 15 mM of dehydroascorbic acid. A
more detailed examination of the dose-response curve, however, revealed
the presence of two components, one saturating at about 4 mM
dehydroascorbic acid, and a second one saturating at about 15 mM dehydroascorbic acid (Fig. 4C). The
Lineweaver-Burk analysis revealed the presence of two components, with
apparent uncorrected K values of 0.9 and 3.5
mM and V of 1.6 and 2 nmol/min/10 cells, respectively (Fig. 4D). HL-60 cells
incubated for 10 min with dehydroascorbic acid showed the presence of
an additional high affinity component involved in uptake, but the
kinetic analysis failed to reveal the presence of the high affinity
component when measuring transport of dehydroascorbic acid using 30-s
assays (Fig. 4, E and F). of 2, 8, and 15
mM, respectively, but no effect of L-glucose,
fructose, sucrose, or
-methyl-D-glucoside was observed (Fig. 4G). Cytochalasin B, but not cytochalasin E,
inhibited transport with an IC of approximately 200 nM (data not shown). Dehydroascorbic acid, but not ascorbic acid,
inhibited the transport of methylglucose by the HL-60 cells in a
dose-dependent manner, with an IC
of about 1 mM (Fig. 5A). The inhibition of the transport of
methylglucose by dehydroascorbic acid was competitive, with a K
of 0.8 mM (Fig. 5, B and C). We also determined the effect of deoxyglucose on
the cellular uptake of concentrations of dehydroascorbic acid, from 20
µM to 5 mM, using incubation periods from 30 s to
10 min. Deoxyglucose completely blocked the uptake of dehydroascorbic
acid under every condition tested, confirming that GLUT1 is the only
pathway for the transport of dehydroascorbic acid by HL-60 cells (data
not shown).
) and reduced ascorbic acid
(). Measurements were performed at 1 mM methylglucose
using 30-s uptake assays. B, double-reciprocal plot of the
effect of different concentrations of dehydroascorbic acid on the
substrate dependence for methylglucose transport using 30-s assays.
Uptake was measured in the absence () or in the presence of 0.5
(), 1 (), or 3 mM (
) dehydroascorbic acid. C, secondary plot of the effect of dehydroascorbic acid on the
substrate dependence for methylglucose transport. DHA,
dehydroascorbic acid; OMG,
methylglucose.
Na
We explored the potential existence of
other transporters involved in the transport of dehydroascorbic acid or
reduced ascorbic acid in the HL-60 cells. It has been reported that the
transport of ascorbate into several tissues and cells is a
NaIndependent Transport of
Dehydroascorbic Acid
-sensitive
process(5, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 24, 42) ,
but no evidence for the presence of a Na
-dependent
ascorbate transporter in human blood cells has been provided. We
studied this issue in the HL-60 cells by measuring the uptake of
dehydroascorbic acid in the absence of Na
. We used
choline chloride, LiCl, and sucrose to replace NaCl in the incubation
medium. A substantial decrease in the uptake of dehydroascorbic acid by
the HL-60 cells was observed when uptake was measured in the absence of
Na
and in the presence of choline
(Fig. 6A). The uptake of dehydroascorbic acid in
the presence of Na
(controls) increased almost
linearly for the first 30 min of incubation, and approximated a plateau
at 120 min. In the presence of choline
, however,
uptake reached a plateau much earlier, and total uptake at 120 min was
only 30% of the uptake observed in the control cells (Fig. 6A). The effect of Na
was most
evident after the first 15 min of incubation, suggesting that it is
related to the accumulation of ascorbic acid rather than to the
transport of dehydroascorbic acid. Consistent with the previous
interpretation, short uptake experiments indicated that the transport
of dehydroascorbic acid, as defined by the first 30 s of the uptake
curve, was not affected by the absence of Na
in the
incubation medium, and decreased uptake was noticeable after the first
2 min of incubation (Fig. 6B). Similar results were
obtained in experiments in which sucrose (Fig. 6C) or
LiCl (Fig. 6D) were used to replace NaCl in the
incubation medium, confirming that the transport of dehydroascorbic
acid is Na
-independent.
) or
choline chloride (). B, time course of the uptake of
dehydroascorbic acid in the presence of NaCl () or choline
chloride (). C, time course of the uptake of
dehydroascorbic acid in the presence of NaCl () or sucrose
(). D, time course of the uptake of dehydroascorbic acid
in the presence of NaCl () or LiCl (). E, time
course of the uptake of ascorbic acid in the presence of Na (
) or choline (
). F, time
course of the uptake of ascorbic acid in the presence of Na (
) or choline (
). G, time
course of the uptake of ascorbic acid in the presence of Na (
) or choline (
). H, time
course of the uptake of ascorbic acid (AA, ) or
dehydroascorbic acid (DHA,
).
. HL-60 cells incubated with
ascorbic acid accumulate lower levels of ascorbate (as compared to
cells incubated with dehydroascorbic acid) due to the oxidation of
ascorbic acid to the transported form, dehydroascorbic
acid(26) . Uptake in the presence of Na
proceeded in a linear fashion for the length of the incubation
period (120 min), but accounted for less than 5-7% of the uptake
of dehydroascorbic acid under similar experimental conditions (Fig. 6, E and A). A decrease of approximately
40% in uptake was observed in the absence of Na
at 120
min, but no effect was evident during the first 45 min of incubation (Fig. 6E). In the presence of choline
,
there was a small but reproducible stimulation of uptake during the
first 30 min of incubation, as compared to control cells (Fig. 6F). Short uptake experiments indicated that, in
the presence of Na
, the cells failed to accumulate any
radioactivity during the first 30 s of incubation, but uptake increased
rapidly afterwards, compatible with the time-dependent generation of
the transported substrate dehydroascorbic acid (Fig. 6G). Similar uptake kinetics were observed in
cells incubated in the presence of choline
, but uptake
was always greater than in the control cells (Fig. 6G).
Again, uptake under these conditions (cells incubated with ascorbic
acid) was only a fraction of the uptake observed in cells incubated
with dehydroascorbic acid (Fig. 6H). Similar results
were obtained when sucrose or LiCl were used to replace NaCl in the
incubation buffer (data not shown). Overall, these data are consistent
with the concept that GLUT1, a transporter whose functional activity is
Na
-independent, is the only pathway involved in the
cellular uptake of dehydroascorbic acid by the HL-60 cells.
-methyl-D-glucoside, the specific substrate of the
sodium-glucose cotransporter(41) , did not inhibit the
transport of deoxyglucose or methylglucose by the HL-60 cells. The
specificity of the transporter was further confirmed by the lack of
effect of L-glucose and sucrose and the inhibitory effect of
maltose on the transport of deoxyglucose and methylglucose. The HL-60
cells were unable to transport fructose, and transport of methylglucose
and deoxyglucose was not affected by fructose. These findings are
consistent with the immunological evidence indicating that the HL-60
cells do not express GLUT2 and GLUT5. The inhibitory effect of
nanomolar concentrations of cytochalasin B on the transport of
deoxyglucose and methylglucose confirmed the lack of expression of
GLUT2 and GLUT5 by HL-60 cells. GLUT5 transports fructose but not
deoxyglucose, and its functional activity is not affected by
cytochalasin B(43) . GLUT2 transports fructose and
deoxyglucose, and its functional activity is affected by micromolar but
not by nanomolar concentrations of cytochalasin B(44) .
Furthermore, the K for the transport of
deoxyglucose or methylglucose by GLUT2 is in the range of 15 to 30
mM, as opposed to 3-8 mM for the transporter
expressed by the HL-60 cells. Direct evidence for the presence of GLUT1
in HL-60 cells was provided by the results of immunoblotting and
immunolocalization experiments with specific antibodies that indicated
a clear reactivity with anti-GLUT1 and an absence of reactivity with
anti-GLUT2, -GLUT3, -GLUT4, and -GLUT5 antibodies. of 0.75 mM, was involved in the
transport of dehydroascorbic acid by HL-60 cells. The competition
studies indicated that dehydroascorbic acid was transported by a
facilitative mechanism which, together with the identification of GLUT1
as the main glucose transporter present in the HL-60 cells, strongly
supports the concept that GLUT1 mediates the transport of
dehydroascorbic acid in these cells. The competition studies,
especially the absence of effect of -methylglucoside on the
transport of dehydroascorbic acid, indicated the lack of participation
of a transporter with the functional characteristics of the
sodium-glucose cotransporter in the transport of dehydroascorbic acid
by HL-60 cells.
-sensitive ascorbate
transporter. Although we observed a major decrease in uptake of
dehydroascorbic acid by HL-60 cells in the absence of
Na
, the kinetic data clearly showed that the step
inhibited in the absence of Na
was the accumulation of
ascorbic acid, and not the transport of dehydroascorbic acid via GLUT1.
Na
-sensitive ascorbate transporters have been proposed
to exist in several tissues and cells such as adrenomedullary
chromaffin cells, retinal epithelial cells, osteoblasts, and the
brush-border membrane of reabsorbing renal
epithelia(5, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 42) .
In these studies, incubating the cells in the absence of external
Na
produced a marked decrease in the uptake of
ascorbic acid. Evidence for Na
-sensitive uptake of
ascorbate was recently provided by expression studies in X. laevis oocytes injected with poly(A)
RNA
extracted from rabbit kidney cortex(24) .
)
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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