Ascorbate and dehydroascorbate transport was
investigated in rat liver microsomal vesicles using radiolabeled
compounds and a rapid filtration method. The uptake of both compounds
was time- and temperature-dependent, and saturable.
Ascorbate uptake did not reach complete equilibrium, it had low
affinity and high capacity. Ascorbate influx could not be inhibited by
glucose, dehydroascorbate, or glucose transport inhibitors (phloretin,
cytochalasin B) but it was reduced by the anion transport inhibitor
4,4
-diisothiocyanostilbene-2,2
-disulfonic acid and by the alkylating
agent N-ethylmaleimide. Ascorbate uptake could be
stimulated by ferric iron and could be diminished by reducing agents
(dithiothreitol, reduced glutathione). In contrast, dehydroascorbate
uptake exceeded the level of passive equilibrium, it had high affinity
and low capacity. Glucose cis inhibited and trans stimulated the uptake. Glucose transport inhibitors
were also effective. The presence of intravesicular reducing compounds increased, while extravesicular reducing environment decreased dehydroascorbate influx. Our results suggest that dehydroascorbate transport is preferred in hepatic endoplasmic reticulum and it is
mediated by a GLUT-type transporter. The intravesicular reduction of
dehydroascorbate leads to the accumulation of ascorbate and contributes
to the low intraluminal reduced/oxidized glutathione ratio.
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INTRODUCTION |
Ascorbate producing and utilizing pathways are connected to the
endomembrane system of the cell. The final enzymatic steps of ascorbate
synthesis are located in the endoplasmic reticulum of hepatocytes or
kidney cells; enzymes utilizing ascorbate (prolyl-3-hydroxylase, prolyl-4-hydroxylase, and lysyl hydroxylase) or its oxidized form dehydroascorbate (protein disulfide isomerase) are characteristic proteins of the lumen (1-3). Their presence in the lumen is necessary for the post-translational modification and folding of many proteins. Since ascorbate and dehydroascorbate are charged water-soluble compounds, transporter(s) should exist for their permeation through biological membranes. Such transporters have been thoroughly
investigated in plasma membrane of different cells (4-9) and in
chromaffin granula (10), but the transport of ascorbate and
dehydroascorbate in microsomes has not been described in detail. The
aim of the present study was to detect and characterize the activity of
the possible ascorbate and/or dehydroascorbate transporter(s) in the endoplasmic reticulum.
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EXPERIMENTAL PROCEDURES |
Preparation of Rat Liver Microsomes--
Microsomes were
prepared from 24-h fasted male Sprague-Dawley rats (180-230 g) as
reported (11). Microsomal fractions were resuspended in buffer A (100 mM KCl, 20 mM NaCl, 1 mM
MgCl2, 20 mM
MOPS,1 pH 7.2). The
suspensions (60-80 mg of protein/ml) were rapidly frozen and
maintained under liquid N2 until used. Intactness of microsomal vesicles was checked by measuring the latency of
mannose-6-phosphatase (12) and p-nitrophenol
UDP-glucuronosyltransferase activity (13), they were greater than 95%
in all the preparations employed. Microsomal protein concentrations
were determined by the biuret reaction using bovine serum albumin as
standard. To measure microsomal intravesicular water space, microsomes
were diluted (10 mg of protein/ml) in buffer A containing
[3H]H2O (0.2 µCi/ml) or
[3H(C)]inulin (0.17 µCi/ml) and centrifuged
(100,000 × g, 60 min), and the radioactivity
associated with pellets was measured to enable calculation of
extravesicular and intravesicular water spaces (14).
Uptake Measurements--
Liver microsomes (1 mg of protein/ml)
were incubated in buffer A containing the indicated amount of
ascorbate, dehydroascorbate, or glucose and their radiolabeled
analogues (1, 1, and 9 µCi/ml, respectively) at 22 °C. At the
indicated time intervals, samples (0.1 ml) were rapidly filtered
through cellulose acetate/nitrate filter membranes (pore size 0.22 µm) and filters were washed with 1 ml of Hepes (20 mM)
buffer (pH 7.2) containing 300 mM sucrose and 0.5 mM DIDS. The total radioactivity retained by filters was measured by liquid scintillation counting. In each experiment, the
pore-forming agent alamethicin (Ref. 15; 0.1 mg/mg protein) was added
to parallel incubates to distinguish the intravesicular and the bound
radioactivity. The alamethicin-permeabilized microsomes were
filtered and washed as above; that portion of radioactivity so released
was regarded as intravesicular (16).
Inhibitors used in the experiments (cytochalasin B, phloretin, DIDS,
and N-ethylmaleimide) were added to the microsomes 30 min
before the uptake measurement. The putative competitive inhibitors (ascorbate, dehydroascorbate, and glucose) and FeCl3 were
added at the beginning of the uptake measurement. Loading of microsomes (10 mg of protein/ml) was accomplished by incubating them in the presence of the indicated compound for 30 min at 22 °C, then
incubates were diluted 10-fold with buffer A containing ascorbate or
dehydroascorbate.
Light-scattering Measurements--
Osmotically induced changes
in the size and shape of microsomal vesicles (17) after the addition of
ascorbate or dehydroascorbate (12.5-12.5 mM) were
monitored at 550 nm excitation and emission wavelenght by the
light-scattering technique as described in detail in an earlier paper
(18).
Microsomal Metabolism of Ascorbate and Dehydroascorbate--
The
ascorbic acid content (reduced and total) of microsomal incubates was
measured by high performance liquid chromatography after specific
sample preparation as described earlier (19, 20).
Materials--
Ascorbate, alamethicin, DIDS, cytochalasin B,
N-ethylmaleimide, phloretin, and
D-[1-3H]glucose (15.5 Ci/mmol) were obtained
from Sigma. Dehydroascorbate was produced by the bromine oxidation
method according to Ref. 21.
L-[carboxyl-14C]Ascorbic acid
(13.7 mCi/mmol) was from Amersham, Buckinghamshire, United Kingdom.
Cellulose acetate/nitrate filter membranes were from Millipore. All
other chemicals were of analytical grade.
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RESULTS |
The uptake of dehydroascorbate and ascorbate exhibited different
kinetic characteristics with Vmax values of 3.1 and 37 nmol/min/mg protein and Km values of 0.7 and
45 mM, respectively (Fig. 1).
The time course of the uptake processes showed that dehydroascorbate
uptake exceeded the level of the passive equilibrium (3.5 nmol/mg
protein; calculated from the intravesicular water space of microsomal
vesicles: 3.5 µl/mg protein). The uptake of ascorbate reached only
one-third of the level of equilibrium within 10 min (Fig.
2) and did not reach a complete
equilibrium even after 1 h incubation (data not shown). Both
uptake processes were temperature-dependent (Fig.
3).

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Fig. 1.
Dependence of dehydroascorbate and ascorbate
uptake on ligand concentration. Microsomes (1 mg/ml protein) were
incubated in the presence of various concentrations of dehydroascorbate (a) and ascorbate (b) plus the radioactive tracer
for 1 min. After the incubation, 0.1 ml of sample was filtered as
described under "Experimental Procedures." Parallel samples were
incubated in the presence of the pore-forming alamethicin; the
radioactivity associated with these samples were regarded as binding
and were subtracted from the total radioactivity associated with the
microsomes. Incubations were performed at room temperature (22 °C).
Means of four to ten experiments are shown on a double-reciprocal
plot.
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Fig. 2.
Time course of dehydroascorbate and ascorbate
uptake in rat liver microsomal vesicles. Microsomes (1 mg/ml
protein) were incubated in the presence of 1 mM
dehydroascorbate or ascorbate at 22 °C. The alamethicin-releasable
portion of dehydroascorbate ( ) or ascorbate ( ) associated with
microsomes is shown. The effect of reducing compounds (2 mM
dithiothreitol, ; and 3 mM reduced glutathione, ) on
ascorbate uptake is also indicated. To modify the intravesicular
environment, microsomes (10 mg/ml protein) were loaded with 1 mM glutathione ( ) or 1 mM glucose ( ) by a
30-min preincubation in the presence of the indicated compound; then
they were diluted 10-fold simultaneously with the addition of ascorbate
or dehydroascorbate. Data are mean ± S.D. of four to ten or mean
of two experiments.
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Fig. 3.
Temperature dependence of dehydroascorbate
and ascorbate uptake in rat liver microsomal vesicles. Microsomes
(1 mg/ml protein) were preincubated at various temperature for 30 min. Uptake measurements were initiated by the addition of 1 mM
ascorbate or dehydroascorbate (plus the radioactive tracer). After 0.5 min incubation 0.1 ml of sample was filtered as described under
"Experimental Procedures." The alamethicin-releasable portion of
dehydroascorbate ( ) or ascorbate ( ) associated with microsomes is
shown. Data are expressed as mean ± S.D., n = 3-4.
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The intravesicular accumulation of radioactivity upon dehydroascorbate
addition in the absence of any source of energy in the incubation
medium, indicated that microsomal metabolism of dehydroascorbate and
ascorbate may have affected their transport. Indeed, a slow metabolism
of ascorbate (4.0 ± 1.6 nmol/min/mg protein, mean ± S.D.,
n = 4; predominantly oxidation) and a more evident
disappearance of dehydroascorbate due to its instability at neutral pH
(17 ± 5 nmol/min/mg protein, mean ± S.D., n = 4) could be observed. A minor fraction of dehydroascorbate was
reduced to ascorbate (0.4 ± 0.1 nmol/min/mg protein, mean ± S.D., n = 4.). In accordance with our assumption,
reducing compounds (dithiothreitol, reduced glutathione) decreased
ascorbate uptake, more evidently after longer incubation (Fig. 2). On
the other hand, oxidation of ascorbate by ferric iron stimulated the
uptake (Table I). Reduction of
dehydroascorbate by extravesicular reducing compounds inhibited its
uptake, while reduced glutathione present intraluminally in preloaded
vesicles stimulated the influx (Fig. 2). These findings suggest that
dehydroascorbate transported into the lumen could be reduced at the
expense of reduced glutathione and/or protein thiols; the reduction led
to the intravesicular accumulation of ascorbate.
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Table I
Ascorbate and dehydroascorbate uptake in rat liver microsomal
vesicles
Microsomes (1 mg/ml protein) were preincubated in the presence of
various compounds for 30 min except for FeCl3 that was given together with dehydroascorbate or ascorbate. Uptake measurements were
initiated by the addition of 1 mM ascorbate or
dehydroascorbate (plus the radioactive tracer). After 1 min incubation,
0.1 ml of sample was filtered as described under "Experimental
Procedures." Parallel samples were incubated in the presence of the
pore-forming alamethicin; the radioactivity associated with these
samples were regarded as binding and were subtracted from the uptake.
All preincubations and incubations were performed at room temperature
(22 °C). Data are expressed as means ± S.D. (n).
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Since GLUT transporters have been reported to mediate dehydroascorbate
transport through the plasma membrane, we checked the effect of GLUT
inhibitors on ascorbate and dehydroascorbate transport. Phloretin and
cytochalasin B inhibited dehydroascorbate but not ascorbate uptake. On
the other hand, the anion transport inhibitor DIDS and the alkylating
agent N-ethylmaleimide inhibited the uptake of ascorbate
more effectively (Table I). Accordingly with the effect of GLUT
inhibitors, glucose cis inhibited dehydroascorbate uptake
(Fig. 4a), while from the
trans side (i.e. in glucose-loaded vesicles) it
was stimulatory (Fig. 2) suggesting that glucose and dehydroascorbate
use the same microsomal transporter. Dehydroascorbate also
cis inhibited the microsomal glucose uptake (Fig.
4c), supporting this assumption.

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Fig. 4.
Effect of ascorbate, dehydroascorbate, and
glucose on the uptake of each other in rat liver microsomal
vesicles. Transport measurements were performed at 1 mM concentration for 1 min. The alamethicin-releasable
portion of dehydroascorbate (a), ascorbate (b),
or glucose (c) associated with microsomes is shown.
Ascorbate ( ), dehydroascorbate ( ), and glucose ( ) were added
in the indicated concentrations. Data are mean ± S.D.,
n = 4-6, or mean of two measurements.
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Ascorbate and dehydroascorbate did not influence the transport of each
other (Fig. 4, a and b). Ascorbate inhibited
glucose transport only at high concentrations presumably due to the
shrinkage of vesicles (Fig. 4c), while glucose did not alter
ascorbate influx (Fig. 4b).
Light-scattering experiments performed at high (12.5 mM)
ligand concentration revealed that ascorbate was taken up by microsomes (for details see Ref. 16) and the permeabilization of vesicles by
alamethicin resulted in a further influx. By contrast, dehydroascorbate rapidly entered the vesicles and after alamethicin addition only a
minor further influx could be observed (Fig.
5).

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Fig. 5.
Osmotically induced changes in
light-scattering intensity of rat liver microsomal vesicles caused by
dehydroascorbate or ascorbate. Light-scattering measurements were
performed as described under "Experimental Procedures."
Concentrated solutions (0.5 M) of dehydroascorbate
(DHA) and ascorbate (AA) were added resulting in
12.5 mM final concentration. When indicated (A), alamethicin (0.1 mg/mg protein) was added. A typical set of experiments out of six is shown.
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DISCUSSION |
Ascorbate and dehydroascorbate uptake in the endoplasmic
reticulum, similarly to plasma membrane, appears to involve different transporters. Both processes are temperature, time, and microsomal protein dependent, saturable, and inhibitable, indicating that they are
mediated by membrane proteins. The uptake of dehydroascorbate is
preferred, it has higher affinity and higher velocity in the physiologic range of concentrations. Moreover, ascorbate transport(er) is present only in one-third of the hepatic microsomal vesicles. Since
dehydroascorbate can enter virtually all of the vesicles, intravesicular ascorbate accumulation upon dehydroascorbate reduction can occur. A similar mechanism is operative at plasma membrane level;
the concerted action of these mechanisms may result in a high ascorbate
concentration in the endoplasmic reticulum. The accumulated ascorbate
serves as a cofactor of several intraluminal enzymes
(prolyl-3-hydroxylase, prolyl-4-hydroxylase, and lysyl hydroxylase),
while the reduced glutathione dependent reduction of dehydroascorbate
may produce an excess of oxidized glutathione necessary for the
oxidation of protein thiols and for protein folding in the lumen (22,
23).
The microsomal metabolism of ascorbate and its influx which can be
inhibited by reducing compounds and stimulated by oxidizing agent
indicate that the oxidation of ascorbate to dehydroascorbate helps the
uptake process. It is an open question whether this oxidation is
mediated enzymatically (by a putative ascorbate oxidase) which could
generate a high local dehydroascorbate concentration.
The results indicate that glucose and dehydroascorbate share the
transporter not only in the plasma membrane, but also in the
endoplasmic reticulum. GLUT1, GLUT2, and GLUT4 are efficient transporters of dehydroascorbate in the plasma membrane (4). These
glucose transporters may also be present in the endomembranes due to
vesicular transport and recycling (24); additionally, GLUT7, a
microsomal glucose transporter (25, 26) can also mediate the transport
of dehydroascorbate in the endoplasmic reticulum. Therefore,
dehydroascorbate can be used as a surrogate or as a competitive
inhibitor in the investigation of microsomal glucose transport, which
is an important question in respect of the topology and mechanism of
the glucose-6-phosphatase system (27). Moreover, high glucose levels
(e.g. in diabetes) can efficiently inhibit the
dehydroascorbate transport in the endoplasmic reticulum of hepatocyte
and pancreatic
-cell. This effect worsens the intracellular shortage
of ascorbate due to the decreased transport through the plasma membrane
(6), may contribute to the inhibition of insulin secretion (28), and
leads to latent scurvy (29) and elevation of plasma dehydroascorbate
level (30).