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Molecular and Cellular Biochemistry 173: 4350, 1997.

1997 Kluwer Academic Publishers. Printed in the Netherlands.

43

Moderately controlled transport of ascorbate into


aortic endothelial cells against slowdown of the cell
cycle, decreasing of the concentration or increasing
of coexistent glucose as compared with
dehydroascorbate
Yasukazu Saitoh,1* Norio Nagao,1* Rika OUchida,1 Takashi Yamane,1
Katsuhiro Kageyama,2 Norio Muto3 and Nobuhiko Miwa1
1

Department of Cell Biochemistry, Hiroshima Prefectural University School of BioScience, Shobara, Hiroshima;
Radioisotope Centre, Osaka City University Medical School; 3Faculty of Pharmaceutics, Osaka University, Suita, Osaka,
Japan

Received 27 August 1996; accepted 8 January 1997

Abstract
Uptake of L-[1-14C]ascorbic acid (Asc) of 12.5200 M for 1 h into bovine aortic endothelial BAE-2 cells grown to confluence was as low as 4364% (per cell) of uptake into the cells grown to nearly one-fourth confluence. [ 14C]Asc undergoing
transmembrane uptake was concentrated and accumulated in the cell less efficiently ([Asc]in/ex = 813) at confluence than at
subconfluence ([Asc]in/ex = 1524). The declined Asc uptake at confluence is attributable to slowdown of the cell cycle, because a similar decrease in [Asc]in/ex was shown by subconfluent cells precultured in serum-insufficient medium, resulting in
an increase in G1 phase and concurrent decreases in S and G2 + M phase distributions as determined by flow cytometry. [114
C]Dehydroascorbic acid (DehAsc) was taken up and accumulated as Asc, after metabolic reduction, without detectable DehAsc.
The [Asc]in/ex values for DehAsc at confluence were as low as 1569% of those at subconfluence in contrast to the values as retentive as 6275% for Asc, suggesting the moderate control of Asc uptake against slowdown of the cell cycle. At either confluence
or subconfluence, dose-dependence for DehAsc uptake was more marked than for Asc uptake as shown by an uphill slope in a
curve of doses versus [Asc]in/ex for DehAsc in contrast to a downhill slope for Asc, suggesting the moderate control for Asc uptake
against fluctuation of the dose. Increasing of coexistent glucose of 5 mM to 2040 mM, plasma concentrations in diabetic patients, declined DehAsc uptake to 4648%, which was less moderately controlled than Asc uptake retained to 5973%. Asc uptake did not compete with DehAsc uptake, suggesting different transporter proteins for Asc and DehAsc. Thus, Asc uptake into
the aortic endothelial cells is more moderately controlled against slowdown of the cell cycle, decreasing of the extracellular concentrations or increasing of coexistent glucose than DehAsc uptake, suggesting a homeostatic advantage of Asc over DehAsc in
terms of retention of intracellular Asc contents within a definite range. (Mol Cell Biochem 173: 4350, 1997)
Key words: ascorbic acid, dehydroascorbic acid, intracellular transport, endothelial cells, cell cycle quiescence, glucose transport

Introduction
Vascular endothelial cells are receiving much attention not
only as the major initial target out of diverse tissues under-

going reactive oxygen intermediates (ROI) injury due to


ischemia and successive reperfusion, but also as a source of
xanthine oxidase [1] which generates superoxide anion (O2)
abruptly due to resumption of oxygen supply upon reperfusion

Address for offprints: N. Miwa, Department of Cell Biochemistry, Hiroshima Prefectural University School of BioScience, Shobara, Hiroshima 727, Japan
*The two top authors equally contributed to the present study.

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[2]. ROI generated upon post-ischemic reperfusion may be
efficiently scavenged by L-ascorbic acid (Asc), one of the
major water-soluble antioxidative intercellular components
[3], because plasma lipoproteins exposed to aqueous peroxy
radicals begins to undergo no hydroperoxidation until depletion of endogenous Asc, which is depleted more promptly and
earlier than other plasma ROI-scavengers such as SH groups,
alpha-tocopherol, bilirubin and urate, suggesting that Asc
efficiently protects biomolecules including lipids from
oxidative damage [4]. One of the key factors preventing
reperfusion injury may be therefore the vascular active transport of Asc [5] against a steep concentration gradient across
the cell membrane [68] into endothelial cells which may
play a central role in distribution of Asc to diverse tissues of
the body [9]. However, vascular transport of Asc has been
scarcely analyzed whereas characteristics of Asc transport
have been studied for some other tissues including oocytes
[10], hepatocyte [11], eye epithelium [1214], osteoblasts
[15] and fibroblasts [16].
Asc is so labile in the aqueous solution as to be reversibly
oxidized to dehydroascorbic acid (DehAsc), which is irreversibly decomposed via 2,3-diketogulonic acid into diverse
reductones. DehAsc transport [1719] should be also taken
into consideration for cytoprotective effects of Asc. In contrast to antioxidant effects of Asc, its prooxidant and/or cytotoxic effects are observed for coexistent ferrous ions [20],
for sparsely seeded cells [21, 22] or for lipophilic microenvironments [20, 23, 24]. DehAsc is less efficient than Asc
in terms of such an amphoteric function as favoring mitosis
at the low doses and inhibiting it at the high doses, suggesting differences of Asc and DehAsc in terms of stability in the
extracellular space and/or modes of the transport into the cell.
The present study was performed to elucidate differences of
Asc and DehAsc in modes of transport into aortic endothelial cells.

Materials and methods


Cell culture
Vascular endothelial BAE-2 cells obtained from bovine aorta
were generously gifted by Dr. Kiyotaka Yamamoto of Tokyo
Metropolitan Institute of Gerontology, and were used at population doubling levels of 2959. Cells were seeded on a 60 mm
dish at densities of 2.02.4 104 cells/cm2, and were cultured
in Eagles minimum essential medium (MEM, Nissui Pharm.,
Tokyo) supplemented with 10% dialyzed and heat-inactivated
fetal bovine serum (FBS, Gibco). The subconfluent cells were
treated for subculture with 0.25% trypsin-0.02% EDTA in
PBS(-), neutralized with an equivoluminal MEM-10% FBS
and detached with a scraper. The cell number was determined
with a Coulter electric particle counter ZM.

Transport of [1-14C]ascorbic acid (Asc) and


[1-14C]dehydroascorbic acid (DehAsc) into the cell
Cells were seeded on 2 cm2 wells of a 24-well microplate,
precultured for 18 h and administered for 1 h with L-[carboxyl14
C]Asc (Amersham, 80113 Ci/mg) or [1-14C]DehAsc,
which was prepared by ascorbate oxidase (from cucumber;
Oriental Yeast, Osaka) reaction of [1-14C]Asc in the presence
of 0.1 mM dithiothreitol and spectrophotometric determination [17]. Then cells underwent aspiration, rinsing twice with
PBS and cytolysis with 0.2% SDS. The cell lysate was measured with an Aloka liquid scintillation counter LC-3600. The
control wells containing no cells were similarly operated, and
the radioactivity value was subtracted from values of cellcontaining wells.

Flow cytometric analysis


The cell precultured in MEM supplemented with 10 or 1%
PBS for 2 days were treated with the propidium iodide staining kit, CycleTEST PLUS (Becton Dickinson). The stained
cells were analyzed with a Coulter flow cytometer Epics Elite.

Ion paired HPLC with coulometric electrochemical


detection (ECD) and UV
After uptake of Asc, dehydroascorbic acid (DehAsc, Aldrich)
or Asc2P for 2224 h, the cells collected underwent aspiration, were rinsed twice, and crushed with a pencil-type or
Potter-type homogenizer. After removal of proteinic ingredients with a Millipore filter Molcut II, the homogenate was
injected on a Shodex octadecyl ODSpak F-411A collumn
(4.6 150 mm) built in a Gilson HPLC system 305 and developed at 40C and 1.5 ml/min with 0.1 M sodium acetate,
200 mg/l EDTA-2Na and 0.17 ml/l n-octylamine essentially
as previously described [32]. Asc/derivatives were detected
with an ESA (Bedford, USA) coulometric ECD Coulochem
II at 200 mV/150 mV or with a Gilson UV detector 115 at
254/265 nm.

Intracellular water contents


An intracellular water content of BAE-2 cells was determined
by gas chrosatographic method using [14C]polyethylene glycol (PEG) with minor modification [25]. The cells of 2.84
2.96 107 were centrifuged, received [14C]PEG of 6 Ci.
weighed and extracted with methanol containing 4% butanol, which was added as an internal standard to correct errors as slight as an inner volume of the needle of injection
volumes. Gas chromatography was performed with the use

45
of a 10% PEG 6000 Shimalite F 3 m stainless column built
in a Shimazu gas chromatograph GC-4A equipped with a
thermal conductivity detector with He gas carrier of 60 ml/
min at 120C. The cell samples were dried up, received
Instagel (Packard) and were determined for the radioactivity with an Aloka liquid scintillation counter LC-3600.

Determination of intracellular Asc concentration


The cells were collected and counted with a Coulter electric
particle counter ZM equipped with a channelyzer type 256.
Based on values determined as intracellular water contents,
intracellular Asc concentrations were calculated from Asc
amounts determined by coulometric ECD- and UV-HPLC
methods.

Results
Dose-dependence of ascorbic acid (Asc) uptake into aortic
endothelial cells
Bovine aortic endothelial cells BAE-2 at confluence or
subconfluence (an approximately one-fourth cell density of
the confluence) were administered for 1 h with [14C]Asc of
12.5200 M which corresponds to an Asc concentration range
in the normal human plasma, followed by scintillascopic quantification of intracellular radioactivity. Asc uptake into the cells
was shown to increase in a dose-dependent manner at either
confluence or subconfluence (Fig. 1). It is notable that Asc uptake amount was 1.82.2 fold larger at subconfluence than at
confluence. The ratio ([Asc]in/ex) of an intracellularly accumulated Asc concentration versus an extracellular Asc concentration nearly equal to the dose was 1524 for the subconfluent
cells and 8.513 for the confluent cells (Fig. 1) as estimated
based on the intracellular water content (0.587 0.008 pL/cell,
independently of the cell density) and the intracellular water
rate (84.2 1.2 wt%) of BAE-2 cells determined as the average and standard deviation of two independent experiments
performed in quardruplicate by gas chromatographic method
using [l4C]polyethyleneglycol 6000 [25].

Fig. 1. Dose-dependence of [14C]ascorbic acid (Asc) uptake into bovine


aortic endothelial cells BAE-2 at confluence or subconfluence and intracellular Asc accumulation: Cells at a density of 3.6 (subconfluence) or 13.9
(confluence) 104 cells/2 cm2 well were exposed to [14C]Asc of 12.5200
M for 1 h, and were subjected to be determined for intracellular Asc contents by liquid scintillascopy. Asc accumulation ratio ([Asc] in/ex) is defined
as a proportion of the intracellular Asc concentration versus the extracellular Asc concentration nearly equal to the Asc dose. The data were typical
of three independent experiments with wells in triplicate, the SD of which
is represented by the bar.

or 100 M in serum-sufficient culture medium, followed by


quantification of the intracellular radioactivity. The subconfluent cells precultured under serum-starved condition
exhibited no greater uptake of Asc than the serum-sufficient
confluent cells at an Asc dose of either 20 or 100 M (Fig.
2). The results were obtained under the condition where a sufficient amount of serum was provided upon Asc uptake but
not upon preculture. Thus more efficient Asc uptake is executed by serum-sufficient subconfluent cells than by serumsufficient confluent cells or serum-starved subconfluent cells.

Effects of serum starvation on Asc uptake into the cell


To clarify whether less efficient Asc uptake into confluent cell
(Fig. 1) is attributed to a possibility that some proportions of
putative Asc transporters, located at the cell surface, may be
insulated from the extracellular fluid through intercellular
contact, or attributed to quiescence of the cell cycle, BAE-2
cells at subconfluence or confluence were forced into serum
starvation and then were administered with [14C]Asc of 20

Cell cycle and Asc uptake efficiency


To clarify a cause for lowered efficiency in Asc uptake into
serum-starved subconfluent cells, frequency DNA histograms of BAE-2 cells cultured under conditions similar to
those for Asc uptake were determined by flow cytometry. A
G1-phase cell fraction was increased from 3964%, and con-

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Fig. 2. Effects of previous serum starvation on Asc uptake into BAE-2 cells in serum-sufficient medium: [14C]Asc of 20 or 100 M was administered for 1
h to subconfluent (an approximately 1/4 confluent cell density) or confluent cells in MEM-10% FBS as described in Fig. 1 except preculture in MEM-1% or
10% FBS for 2 days. The data were typical of three independent experiments with wells in triplicate, the SD of which is represented by the bar.

currently G2+M and S cell fractions were decreased for the


serum-starved subconfluent cells than the serum-sufficient
cells of the approximate cell density (Fig. 3). The results
suggest that lowered efficiency of Asc uptake into the confluent cells (Fig. 1) may be attributed primarily to slowdown
of the cell cycle rather than to veiling of Asc transporters from
the extracellular fluid.

Dose-dependence of Asc and dehydroascorbic acid


(DehAsc) uptake
Uptake of [14C]Asc into BAE-2 cells in comparison with that
of [14C]DehAsc was examined in terms of dependence on the
dose and cell density. DehAsc uptake showed more marked
dose-dependence than Asc uptake into either confluent or
subconfluent cells (Fig. 4). At both the cell densities, DehAsc
underwent less efficient uptake than Asc at a dose as low as
25 M. At increased doses of 50100 M, however, more
markedly efficient uptake was observed for DehAsc than for

Asc at either cell density. Ratios of uptake at 25 or 100 M


versus uptake at 50 uM was as fluctuant as 0.354.7 and 0.09
3.4 for DehAsc, but was within a range as narrow as 0.65
1.17 and 0.591.27 for Asc at subconfluence and confluence,
respectively, showing less variability of Asc uptake against
a change in the dose as compared with DehAsc. In addition,
dose-dependence of [Asc]in/ex, an index for intracellular Asc
accumulation, was of an uphill slope for DehAsc in contrast
to a downhill slope for Asc (the upper part of Fig. 4). The
results suggest that Asc uptake is more steadily retained over
the definite amount even at low doses than DehAsc uptake,
whereas Asc uptake is more markedly repressed than DehAsc
uptake at a dose as high as 100 M corresponding nearly to
the upper limit of an Asc concentration range in normal human plasma. In addition to the marked dose-dependence,
DehAsc uptake showed a more marked dependence on the cell
density than Asc uptake. DehAsc uptake was shown to be more
markedly repressed than Asc uptake at confluent cells at all
the doses examined. Ratios of uptake amounts at confluence
versus those at subconfluence were 1569% for DehAsc, and

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Fig. 3. Frequency distribution DNA histograms of BAE-2 cells which were cultured in MEM-10% or 1% FBS for 2 days and grown to 3.33.9 104 cells/
2 cm2 well (subconfluence) as determined by propidium iodide staining and flow cytometry: Cell fractions of G1, S and G2 + M phases were 39 3%, 29
2% and 32 2% for 10% FBS, and 64 2%, 22 2% and 14 1% for 1% FBS, respectively. The data were typical of three independent experiments.

6275% for Asc, respectively, showing that Asc is superior to


DehAsc in terms of efficiency of uptake into the confluent cells.
Thus, Asc uptake into the aortic endothelial cells is shown to
be more homeostatic against either lowering of the dose or increasing of the cell density than DehAsc uptake.

Intracellular forms after uptake of exogenous Asc or


DehAsc
Some informations need to be acquired concerning what
forms Asc or DehAsc undergoing uptake is metabolized into.
Extracts from BAE-2 cells administered with Asc or DehAsc
of 50200 M for 624 h were assessed by ion-paired HPLC,
which separated Asc and DehAsc at retention times of 1.68
and 2.l0 min, respectively, under the employed conditions.
Intracellular Asc was abundantly detected in the Asc-administered cells, scarcely detected in DehAsc-administered cells
but not in the unadministered cells (Fig. 5). Intracellular
DehAsc was not detectable in any cells examined, even in
cells administered with DehAsc in the presence of 50 M
dithiothreitol, suggesting that DehAsc was rapidly reduced
to Asc within the cell which may be retained to a preferable
reduced potential.

Fig. 4. Dose-dependence of uptake of [14C]Asc or [14C]dehydroascorbic


acid (DehAsc) into subconfluent or confluent BAE-2 cells in complete
medium for 1 h as described in Fig. 1: Note that the Asc accumulation ratio
([Asc]in/ex) increased along with the dose for DehAsc, but decreased for
Asc at either cell density. Ratios (not shown) of uptake amounts at confluence versus those at subconfluence at doses of 25, 50 and 100 M were
0.15, 0.69 and 0.50 for DehAsc, and 0.62, 0.59 and 0.75 for Asc, respectively. The data were typical of three independent experiments with wells
in triplicate, the SD of which is represented by the bar.

Dependence of Asc or DehAsc uptake on coexistent


glucose
The above-mentioned uptake mode of Asc or DehAsc was
examined using culture media containing glucose of 5 mM
nearly equal to an average concentration in normal human
blood. However, [ 14]Asc or [14C]DehAsc uptake into the confluent cells needs to be examined for the dependence on coexistent glucose, which elevates in normal plasma after a meal

48

Fig. 5. Typical profiles of extracts from BAE-2 cells administered with


Asc or DehAsc of 200 M for 6 h as assessed by ion-paired HPLC on an
octadecylsilica gel column with detection by absorbance at 265 nm. The
arrow indicates a retention time (tR) of authentic Asc or DehAsc. The similar results were obtained by coulometric ECD.

as well as in some diseases such as diabetes, resulting in the


direct contact of abundant glucose to the aortic endothelium.
Coexistence of glucose of 2040 mM, concentrations in the
plasma of cases of diabetes, diminished DehAsc uptake into
BAE-2 cells more markedly than Asc uptake (Fig. 6), showing that Asc uptake was not so susceptibly repressed by
cooexistent glucose as DehAsc uptake in the same tendency
as observed for dependence on the dose and cell density (Fig.
S). Dithiothreitol of 50 M was added to prevent oxidative
degradation of exogenous Asc and DehAsc, whereas depletion of the reducing agent induced an apparent decrease in
DehAsc uptake but not in Asc uptake, suggesting the oxidative lability of DehAsc in physiological aqueous solution.

Uptake competition of Asc with DehAsc


Uptake of 50 uM [14C]Asc into BAE-2 cells was inhibited by
concurrent addition of non-radioisotopic Asc of 200 M for
either l or 22 h uptake (Fig. 7). Theoretically, the [14C]Asc
uptake amounted to approximately one-fifth of uptake for administration with 250 M [14C]Asc without unlabeled Asc for
either uptake time. In contrast, uptake of 500 M [14C]Asc
was not inhibited by unlabeled DehAsc of the same dose for

Fig. 6. Effects of coexistent glucose on uptake of [14C]Asc or [14C]DehAsc


into confluent BAE-2 cells in complete medium for 1 h as described in Fig.
1: Glucose was further added to MEM originally containing 5 mM glucose
so as to a final concentration of 20 or 40 mM. Dithiothreitol (DTT) of 50
M were added to prevent oxidative degradation of exogenous Asc and,
especially, DehAsc.

either uptake time, suggesting different transporters between


Asc and DehAsc in the cell in accord with some above-mentioned differences in Asc and DehAsc uptake.

Discussion
Asc uptake is appreciably repressed at confluence relative to
at subconfluence of aortic endothelial BAE-2 cells under
serum-sufficient conditions (Fig. 1), which is attributable to
augmentaticn in the G1-phase cell fraction and concurrent
deminution in G2 + M and S fractions, but not to insulation
of effective Asc-transporter proteins from the extracellular
fluid (Figs 2 and 3). The results, obtained by 1 h uptake of
[1-14C]Asc, suggest that a necessary degree for Asc uptake
may be lowered at G1 phase, and heightened at S and/or M
phase when a greater deal of reactive oxygen intermediate

[14C]Asc Uptake (dpm/106 cells)

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Fig. 7. Competitive effects of non-radioisotopic (cold) Asc or DehAsc of


200 M on [14C]Asc uptake into confluent BAE-2 cells in complete medium for 1 or 22 h: Note the similar competitive effects in spite of the
difference in axis scales of 1 and 22 h uptake.

(ROI) generates assumedly upon DNA synthesis and/or mitosis amounting to 13% of the total consumption of cellular oxygen which is converted to O2 and H2O2 [26, 27].
Asc uptake into the aortic endothelial cells is more homeostatic against lowering of the dose, heightening of the G1phase cell fraction and increasing of coexistent glucose than
DehAsc uptake (Figs 4 and 6). The advantages of Asc over
DehAsc are in accordance with the existence of more abundant Asc than DehAsc in mammalian plasma which the aortic endothelium directly contacts, resulting in continuous and
invariable uptake of Asc and subsequent distribution to the
underlying tissues [9]. The endothelium constantly exposed
to oxidative stress [5] may demand homeostatic uptake of Asc
independently of extracellular concentrations of Asc and
glucose, and phases of the cell cycle, which is in accord to
the results in the present study.
DehAsc prevents oxidation-induced cataract in the eye
[28], and protects oxidative stress-induced peroxidation of low
density lipoproteins [29] assumedly through scavenging O2

via self-decarboxylation [30]. DehAsc but not Asc protects


UV-induced disruption of cell membrane integrity in keratinocytes [31]. In contrast to above-mentioned tissues, the physiological significance of DehAsc in the blood vessel may be
limited to its uptake which is more efficient than Asc at higher
doses (Fig. 4) assumedly due to scarce distribution and/or, low
affinity of putative DehAsc transporter proteins.
The modes of transport of Asc or DehAsc have been characterized in diverse cell lines other than vascular endothelial
cells [1019]. However, comparative studies on transport of
Asc and DehAsc have been scarcely reported. Human myeloid leuchemia cells HL-60 is shown to lack the capacity to
transport Asc, but to exhibit a marked activity to transport
DehAsc preferably [10], which is a transport mode inverse
to that of aortic endothelial cells determined in the present
study. DehAsc transport may be correlated with extent of cell
differentiation as suggested by a progressive increase in
DehAsc uptake into granurocyte-macrophage progenitors as
a function of advancement of cytodifferentiation to neutrophils [18], in contrast to an unaltered mode of Asc or DehAsc
uptake into aortic endothelial cells during successive subculture of population doubling levels examined in the present
study (not shown). Less susceptibility of Asc uptake to coexistent glucose than DehAsc uptake is demonstrated in the
present study to hold true for one and the same cell line,
whereas it has been separately demonstrated so far that glucose reduces uptake of DehAsc in one cell line [10, 17, 19]
but scarcely of Asc in another cell line [9, 15, 16]. In addition, Asc transport has been analyzed at confluence or unspecified cell densities in most of studies hitherto [1019],
but scarcely in terms of dependence on the cell cycle as determined by the present study.

Acknowledgements
The authors thank Ms. Yuko Yamada for her skillful technical assistance. This study was supported in part by Special
Coordinating Fund for Promoting Science, and Technology
from the Science and Technology Agency of Japan to N.M.,
by Grant-in-Aid for Exploratory Research from the Ministry of Education, Science, Sports and Culture of Japan to
N.M., and Grant-in-Aid for BioScientific Research from
Furukawa Technical Foundation of Japan to N.M.

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