Beruflich Dokumente
Kultur Dokumente
43
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
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-
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.
44
[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.
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.
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].
46
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.
47
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.
48
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
49
(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
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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