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Departments of Medicine and Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-6303, USA
1Correspondence: 736 Medical Research Building II, Vanderbilt University School of Medicine, 2220 Pierce Ave., Nashville, TN 37232-6303, USA. E-mail: james.may{at}mcmail.vanderbilt.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRECENT RESULTS: THE ROLE...CONTROVERSIES AND UNRESOLVED...FUTURE DIRECTIONS: ELUCIDATION...REFERENCES |
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-tocopheroxyl radical and a trans-plasma
membrane oxidoreductase activity. Ascorbate-derived reducing capacity
is thus transmitted both into and across the plasma membrane. Recycling
of -tocopherol by ascorbate helps to protect membrane lipids from
peroxidation. However, neither the mechanism nor function of the
ascorbate-dependent oxidoreductase activity is known. This activity has
typically been studied using extracellular ferricyanide as an electron
acceptor. Whereas an NADH:ferricyanide reductase activity is evident in
open membranes, ascorbate is the preferred electron donor within cells.
The oxidoreductase may be a single membrane-spanning protein or may
only partially span the membrane as part of a trans-membrane electron
transport chain composed of a cytochrome or even hydrophobic
antioxidants such as -tocopherol or ubiquinol-10. Further studies
are needed to elucidate the structural components, mechanism, and
physiological significance of this activity. Proposed functions for the
oxidoreductase include stimulation of cell growth, reduction of the
ascorbate free radical outside cells, recycling of -tocopherol,
reduction of lipid hydroperoxides, and reduction of ferric iron prior
to iron uptake by a transferrin-independent pathway.—May, J. M.
Is ascorbic acid an antioxidant for the plasma membrane?
Key Words: ascorbate free radical • dehydroascorbic acid • antioxidant • ferricyanide • oxidoreductase
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRECENT RESULTS: THE ROLE...CONTROVERSIES AND UNRESOLVED...FUTURE DIRECTIONS: ELUCIDATION...REFERENCES |
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-tocopheroxyl radical to
-tocopherol. Recycling of -tocopherol by ascorbate has been
demonstrated in liposomes and cellular organelles (1, 
2)
. Recent
evidence suggests that ascorbate also spares and probably recycles
-tocopherol in erythrocyte membranes (3)
and intact erythrocytes
(4)
. The erythrocyte results indicate that ascorbate can interact
directly with the plasma membrane as an antioxidant. It can also donate
electrons to a trans-plasma membrane electron transfer activity in
erythrocytes (5,
6)
and nucleated cells (7)
. The scope of ascorbate
interaction with both plant and mammalian plasma membranes has been
reviewed (8)
, so this discussion will focus primarily on the ability of
the vitamin to contribute electrons for transfer into and across the
plasma membrane of mammalian cells.
Ascorbate can donate either one or two electrons in redox reactions
(Fig. 1
). At physiological pH, more than 99% of ascorbate is in the monoanion
form (carbon-3 hydroxyl pKa = 4.2). Loss of the
first electron results in the ascorbate free radical
(AFR)2
(1)
. The AFR, although a stronger reducing agent than ascorbate (9)
, is
stabilized by resonance on the three-ring oxygens and is not very
reactive (10)
. Mild oxidants such as ferricyanide (11)
and
EDTA(Fe3+) (12)
can remove a second electron and
convert the AFR to dehydroascorbic acid (DHA). However, in the absence
of such oxidants, the predominant reaction of the AFR is
disproportionate to ascorbate and DHA, with a second order rate
constant of 105 M-1
s-1 at pH 7.0 (10)
. DHA is unstable at
physiological pH, with a half-life of ~6 min (13)
. Unless reduced
back to ascorbate, it undergoes irreversible ring opening to form
2,3-diketo-1-gulonic acid (Fig. 1)
. Based on these considerations,
recycling at the AFR stage may have advantages for the ascorbate
economy of the cell.
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The tendency of ascorbate to participate in one-electron interactions
also fits well with its ability to transfer electrons into and across
the plasma membrane. For example, a one-electron transfer has been
proposed to explain reduction of the -tocopheroxyl radical by
ascorbate (14)
. Electron transfer across the plasma membrane has
typically been measured by tracking the reduction of ferricyanide (15)
.
Both ferricyanide and its one-electron reduced form, ferrocyanide
(Fig. 2
), are large, negatively charged molecules that do not cross the plasma
membrane (16)
. The rate of extracellular ferricyanide reduction is
readily measured spectrophotometrically by following either the loss of
ferricyanide (15)
or the appearance of ferrocyanide (17)
. Although
documented in all cells in which it has been sought (18)
, ferricyanide
reduction has probably been most studied in human erythrocytes. These
cells lack intracellular membranes and organelles, and thus offer a
simple one-compartment system for study.
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Ferricyanide reduction by intact erythrocytes is associated with
increased intracellular ATP generation (15)
, no change in oxygen
consumption (15)
, and proton movement out of the cells (19)
. The latter
likely reflects an attempt of the cell to avoid ferricyanide-induced pH
gradients across the cell membrane (20)
. The identity of the natural
electron donor(s) for this activity has long been a subject of interest
(18)
. Early studies in ghost membranes suggested that NADH is the
electron donor to this activity (21,
22)
. The possibility that
ascorbate might also serve as a donor derives from the studies of
Orringer and Roer (23)
, who first showed that addition of DHA to human
erythrocytes enhances reduction of extracellular ferricyanide. In their
experiments, ferricyanide reduction in erythrocytes was saturable with
increasing concentrations of added DHA and was evident in resealed
ghosts containing an ascorbate regeneration system. These authors
presented a model involving uptake of DHA, intracellular reduction to
ascorbate, efflux of ascorbate out of the cells, and direct reduction
of extracellular ferricyanide by ascorbate. The ability of DHA to
enhance extracellular ferricyanide reduction was confirmed in liver
(24)
and erythrocytes (5)
. In the erythrocyte studies, Schipfer et al.
(5)
also reported a saturable process with respect to both DHA and
ferricyanide, and provided evidence that ferricyanide is reduced by
intracellular rather than extracellular ascorbate. The latter derives
from the finding that medium obtained from cells incubated with
increasing amounts of DHA supports little or no ferricyanide reduction.
They also found DHA-induced ferricyanide reduction in intact cells to
be inhibited 25% by p-chloromercuribenzene sulfonic acid
(PCMBS), a slowly penetrating sulfhydryl reagent. These results
indicate that ascorbate-dependent ferricyanide reduction is mediated by
a trans-membrane process that likely involves a protein. Subsequent
reports in the last several years have extended the initial studies and
generated additional questions and controversies.
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RECENT RESULTS: THE ROLE OF INTRACELLULAR ASCORBATE IN EXTRACELLULAR FERRICYANIDE REDUCTION |
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TOPABSTRACTINTRODUCTIONRECENT RESULTS: THE ROLE...CONTROVERSIES AND UNRESOLVED...FUTURE DIRECTIONS: ELUCIDATION...REFERENCES |
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. The first step involves uptake of DHA across the plasma membrane.
Despite the term `acid' in its common name, DHA is uncharged at
physiological pH (Fig. 1)
. This lack of charge increases its ability to
diffuse across the plasma membrane into cells and allows it serve as a
substrate for the glucose transporter. Schweinzer and Goldenberg (25)
showed by direct ascorbate measurements that K562 erythroleukemic cells
were able to convert added DHA to ascorbate. However, because
ascorbate-dependent ferricyanide reduction was insensitive to
cytochalasin B, a specific inhibitor of the glucose transport protein
(26)
, they concluded that DHA entered the cells by diffusion. It is
clear from more recent studies that DHA does enter cells by facilitated
diffusion on the glucose transporter. Vera and colleagues reported that
Xenopus laevis oocytes, which lack glucose
transporters, do not transport DHA (27)
. When mRNA from any of several
transporter subtypes is microinjected into oocytes, they develop the
ability to transport DHA, but not ascorbate. Two subsequent studies in
HL-60 cells have confirmed that DHA enters on the glucose transporter
(28,
29)
. Dependence on the glucose transporter is more difficult to
demonstrate in a cell such as the human erythrocyte, which has a high
density of transporter protein (30)
. For example, Himmelreich and
Kuchel (31)
failed to detect inhibition of ferricyanide reduction by
the transport inhibitors phloretin and cytochalasin B or glucose
itself. However, their experiments were carried out for as long as an
hour at 37°C, conditions under which glucose is equilibrated across
the membrane even in the presence of strong transport inhibition. We
were able to show that cytochalasin B inhibits
[14C]DHA uptake by erythrocytes (6)
, but it was
necessary to measure transport at 23°C and use short time points to
assess initial rates of uptake (i.e., 30 s or less). There may be
an additional route of DHA uptake and reduction by erythrocytes not
depicted in Fig. 3
. In their nuclear magnetic resonance (NMR) studies
of 13C-labeled DHA uptake by erythrocytes,
Himmelreich et al. (32)
found DHA reduction to be so rapid and
sensitive to metabolic interference that they hypothesized a novel
transport mechanism in which DHA is reduced by NADH during
its transfer across the membrane. Once inside cells, DHA is very
rapidly reduced to ascorbate (Fig. 3)
, either directly by GSH (33)
or
in reactions catalyzed by thioredoxin reductase (4,
34)
or glutaredoxin
(35)
. NADH-dependent mechanisms may also contribute (23,
28)
.
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Ascorbate itself, probably because of its negative charge, is a very
poor substrate for the glucose transporter and exits cells slowly (36)
.
Ascorbate concentrations in many cells are in the low millimolar range
(37)
, despite plasma concentrations of 40–60 µM in unsupplemented
individuals (38)
. Such accumulation of ascorbate against a
concentration gradient appears to be due to the action of an energy-
and sodium-dependent transport protein (37)
. Human erythrocytes appear
to lack such a transporter, since plasma and intracellular ascorbate
concentrations are the same (38,
39)
. Nonetheless, through uptake of
DHA and conversion to ascorbate, even erythrocytes can generate
intracellular ascorbate concentrations as high as 1–2 mM and maintain
them for more than an hour of incubation at 37°C (39)
.
Ascorbate as a substrate for a trans-plasma membrane oxidoreductase
If intracellular ascorbate can donate electrons to a trans-plasma
membrane oxidoreductase activity, the question arises as to the
relative importance of ascorbate compared with other potential electron
donors. In intact erythrocytes, we found physiological intracellular
concentrations of ascorbate to contribute as much as two-thirds of the
basal rate of ferricyanide reduction (6)
. To determine the response to
decreased intracellular ascorbate, endogenous erythrocyte ascorbate was
depleted using the nitroxide free radical Tempol
(2,2,6,6,-tetramethyl-4-hydroxypiperidine-N-oxyl), which
enters cells and oxidizes ascorbate to DHA (33)
. Some of this DHA
effluxes from the cells and can be removed by centrifugation washes.
Three such Tempol treatments of erythrocytes deplete ascorbate by
~80%, without affecting GSH or -tocopherol (40)
. Cells depleted
of ascorbate in this manner had only about one-third the basal rate of
ferricyanide reduction seen in control cells (6)
. A preference for
ascorbate over NADH was also evident in both resealed and open
erythrocyte ghosts. In erythrocyte ghosts that were resealed to contain
400 µM concentrations of either NADH or ascorbate, ascorbate-loaded
ghosts had about twice the ferricyanide-reducing capacity as the
NADH-loaded ghosts (41)
. Further, open erythrocyte ghost membranes were
able to catalyze reduction of nitroblue tetrazolium with ascorbate as
the electron donor at almost twice the rate observed with NADH (41)
.
Cells that can concentrate ascorbate may depend even more on ascorbate
as a donor for trans-plasma membrane electron transfer. Human HL-60
cells (28)
and U-937 cells (J. M. May, unpublished data), when
cultured in ascorbate-deficient media, contain no endogenous ascorbate.
Loading of these cells with DHA to achieve intracellular ascorbate
concentrations of 4–6 mM increases ferricyanide reduction by five- to
eightfold. Freshly prepared human monocytes, from which U-937 cells are
thought to derive (42)
, have intracellular ascorbate concentrations of
~6–8 mM (43)
. Therefore, ascorbate may be even more important as an
electron donor to this trans-plasma membrane electron transfer activity
in nucleated cells than in erythrocytes.
As noted previously, early studies showed saturable ferricyanide
reduction in erythrocytes loaded with increasing concentrations of DHA.
Although kinetic values were not reported, half-maximal ferricyanide
reduction occurred at initial extracellular DHA concentrations of less
than 100 µM (5,
23)
. This contrasts with ascorbate accumulation in
erythrocytes, which is half-maximal after loading with 400 µM
extracellular DHA (39)
. Together, these results suggest that the
trans-plasma membrane reductase and not the intracellular ascorbate
concentration is rate-limiting for ferricyanide reduction. In other
cells, direct ascorbate measurements prior to assessment of
ferricyanide reduction have confirmed this hypothesis. In HL-60 cells
(28)
and in U-937 cells (J. M. May, unpublished data), rates
of extracellular ferricyanide reduction were half-maximal at
intracellular ascorbate concentrations of 0.6 and 0.8 mM, respectively.
These values are well below the ascorbate concentration measured in
freshly prepared monocytes. However, decreases in intracellular
ascorbate due to oxidant stress, ascorbate secretion (44
45
46)
, or loss
during culture in ascorbate-deficient media might limit the capacity of
the oxidoreductase.
Ferricyanide, the usual artificial electron acceptor for the
trans-plasma membrane oxidoreductase, necessarily participates in
one-electron transfers. Thus the question arises as to whether the
oxidoreductase activity removes one electron at a time from the
intracellular electron donor. This can be tested with ascorbate, which
is readily detected in its one-electron oxidized form as the AFR. We
found that extracellular ferricyanide does generate the AFR in human
erythrocytes (Fig. 3)
, whereas ascorbate oxidase fails to do so under
the same conditions (47)
. Ascorbate oxidase produces the AFR in a
direct reaction, with subsequent dismutation of two AFR molecules to
one molecule each of DHA and ascorbate (48)
. Since ascorbate oxidase is
restricted to the extracellular space, its failure to generate an AFR
signal in intact erythrocytes suggests that the signal observed with
ferricyanide was from intracellular ascorbate and not from oxidation of
ascorbate that had leaked out of the cells. The ferricyanide-induced
AFR signal in intact erythrocytes is also not due to participation of
the AFR as an intermediate in intracellular DHA reduction, since DHA
added to erythrocytes does not generate an AFR signal (47)
. Rather, as
depicted in Fig. 3
, DHA reduction likely involves a two-electron
substitution mechanism for GSH alone (49)
and for the reactions
catalyzed by glutaredoxin (35)
and thioredoxin reductase (34)
.
There are several possible reactions of the AFR generated by
ferricyanide in erythrocytes, which are schematized in Fig. 4
. Since the AFR carries a negative charge at physiological pH (50)
, it
is unlikely to efflux from the cells at an appreciable rate. The AFR
could 1) donate another electron to the trans-membrane
reductase, 2) dismutate to DHA and ascorbate, or
3) be reduced back to ascorbate by an AFR reductase. The
first two reactions would generate DHA, which could either efflux from
cells or undergo reduction back to ascorbate, as depicted in Fig. 3
.
Ferricyanide does generate intracellular DHA from ascorbate, which we
have detected as efflux of [14C]DHA on the
glucose transporter (6)
and which Himmelreich and Kuchel (32)
have
measured directly in ferricyanide-treated erythrocytes. Nonetheless,
direct recycling of the AFR may also occur. Open erythrocyte membranes
show NADH-dependent AFR reductase activity (51)
. Such ghosts contain
both cytochrome b5 and cytochrome
b5 reductase (52,
53)
, and the latter
appears to be identical to microsomal cytochrome
b5 reductase in other cell types (54)
.
The microsomal cytochrome b5 reductase
system has long been known to carry out NADH-dependent reduction of the
AFR (55)
. Therefore, reduction of the AFR immediately after its
generation (Fig. 4)
is plausible and makes sense in the ascorbate
economy of the cell, since there would be less flux down the pathway
toward irreversible ring opening of DHA. The question then arises as to
the relationship, if any, between the trans-plasma membrane
oxidoreductase and the putative AFR reductase (indicated by the
question mark in Fig. 4
). This question will be addressed next.
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CONTROVERSIES AND UNRESOLVED QUESTIONS: MECHANISM AND FUNCTION OF THE OXIDOREDUCTASE |
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TOPABSTRACTINTRODUCTIONRECENT RESULTS: THE ROLE...CONTROVERSIES AND UNRESOLVED...FUTURE DIRECTIONS: ELUCIDATION...REFERENCES |
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18)
or ascorbate (6)
as
electron donors (Fig. 5
, mechanism 1). A trans-plasma membrane orientation of the erythrocyte
ascorbate-dependent enzyme is also supported by the observations that
its activity is inhibited by the poorly permeant protein reagents (5,
56)
and that external ferricyanide is reduced by internal ascorbate in
tightly resealed ghosts (23,
41,
57)
. Such a trans-plasma membrane
activity requires an exofacial site reactive with ferricyanide and an
endofacial site available to accept electrons from cytoplasmic donors
(18)
. Whereas erythrocyte cytochrome
b5 reductase may be a major
ferricyanide reductase in open membranes, it has a cytoplasmic
orientation (18,
58)
. The same argument holds for cytochrome
b5 itself (18,
59)
and for cytochrome
c reductase activity (60)
. Much of the latter activity may
be due to the cytochrome b5 system
(18)
, since cytochrome c can be reduced by the combined
action of cytochrome b5 and cytochrome
b5 reductase (53)
. A cytoplasmic
orientation does not necessarily rule out participation in a
trans-plasma membrane process, but it does require involvement of other
membrane proteins or transfer agents, as discussed below.
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A presumed membrane-spanning NADH-dependent ferricyanide reductase has
been purified from erythrocyte ghosts by Wang and Alaupovic (21)
. The
monomeric form of this protein differs from cytochrome
b5 reductase with respect to size,
kinetic behavior, cofactor dependence, and membrane orientation. With
an apparent molecular mass of 40 kDa, it is ~10% larger than the
erythrocyte cytochrome b5 reductase
(53)
. It has a lower affinity for NADH (20 µM) than the
membrane-bound cytochrome b5 reductase
(0.6 µM) (53)
and also differs from the latter enzyme in showing both
substrate and product inhibition. A trans-plasma membrane orientation
is suggested by the presence of ~6% by weight carbohydrate. However,
the location of the NADH binding site for this enzyme has not been
established, so that it could correspond to a cell surface
NADH:ferricyanide reductase activity that has been reported in
erythrocytes (56)
and other cells (61)
. It is also not known whether
this enzyme can use ascorbate as an electron acceptor. Nonetheless, it
is plausible that the enzyme purified by Wang and Alaupovic corresponds
to the trans-plasma membrane NADH-dependent ferricyanide reductase
activity later characterized by Grebing et al. (56)
.
Is it an AFR reductase?
The trans-plasma membrane ferricyanide reductase could also
function by a mechanism analogous to that of the chromaffin granule
system in adrenomedullary cells. These granules contain high
concentrations of ascorbate (22 mM) (62)
, which serves as a cofactor
for hydroxylation of dopamine by dopamine ß-hydroxylase to generate
norepinephrine (46)
. The hydroxylation step is a one-electron transfer
that generates the AFR. The AFR is reduced back to ascorbate by a
trans-membrane electron carrier that has been identified as cytochrome
b561 (63,
64)
. This reaction is driven
by an inward proton gradient that is generated by an
H+-ATPase in the granule membrane (46)
. The
protons are required for reduction of the AFR and serve to balance the
influx of electrons via the cytochrome. This system, which is shown in
Fig. 6
, has the same orientation as the plasma membrane system if one
considers that the granule interior corresponds to the extracellular
space in the model of Fig. 3
. Although erythrocyte membranes do not
contain cytochrome b561, the
cytochrome b5/cytochrome
b5 reductase pair could perform this
function, with the caveat that other membrane elements are also
required. Erythrocyte membranes do contain an additional cytochrome,
cytochrome P-420, which presumably is derived from cytochrome P-450
(52,
65)
. Whether this cytochrome is involved in trans-plasma membrane
electron transfer is unknown. Another issue raised by consideration of
the chromaffin granule AFR reductase is the necessary coupling between
electron transfer by the trans-plasma membrane system and proton
export. Since the initial results of Dormandy and Zarday (19)
, which
documented proton export from human erythrocytes during ferricyanide
reduction, there has been little consideration of whether intracellular
pH changes occur, whether they limit ferricyanide reduction, or whether
electron and proton efflux are coupled. Again, by analogy to the
chromaffin granule system, a pH gradient maintained by an
H+-ATPase may be essential to drive any
trans-membrane reduction of the AFR by intracellular ascorbate.
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An AFR reductase activity has been reported for human erythrocyte
membranes that appears to span the cell membrane, since it was latent
in both resealed ghosts and inside-out resealed ghosts (51)
. Schweinzer
and Goldenberg (7,
25)
later described a cell surface AFR reductase
activity in K562 and U-937 cells that also presumably spans the cell
membrane. In this system, ferricyanide is used to generate the AFR from
low concentrations of extracellular ascorbate. The AFR is then reduced
at the cell surface by an AFR reductase, which obtains electrons from
as yet unidentified intracellular donor (Fig. 5
, mechanism 2). The AFR
reductase has a high affinity for the AFR, with an apparent
Km in the low picomolar range (7)
. It is dependent
on the membrane potential (25)
, which may reflect the need for proton
export (Fig. 6)
. It is also inhibited by thenoyltrifluoroacetone (25)
,
a known inhibitor of microsomal NADH-dependent AFR reductase activity
(66)
. In the natural setting, the AFR would be generated outside cells
by oxidants such as superoxide and traces of ferric iron; intracellular
NADH or ascorbate would serve as intracellular electron donors.
Interpretation of these studies is complicated because ferricyanide is
used both to oxidize extracellular ascorbate to the AFR and quantify
AFR reduction. Any DHA generated outside the cells will be taken up and
converted to ascorbate. Intracellular ascorbate can then serve as a
substrate for the trans-plasma membrane system.
The question of whether the trans-plasma membrane oxidoreductase is an
AFR reductase has been addressed by Villalba et al. (67)
, with negative
results. They reported that purified liver plasma membranes have
ferricyanide and AFR reductase activities, but that these differ with
regard to sensitivity to detergent extraction, clathrin depletion or
inactivation, and to inhibition by wheat germ agglutinin. They
concluded that the ferricyanide and AFR reductase activities are not
mediated by the same protein in these membranes, although these
proteins could form part of an electron transport chain across the
plasma membrane.
Is it mediated by hydrophobic antioxidants?
Another possibility is that lipid-soluble antioxidants such as
ubiquinol-10, -tocopherol, or -tocopheryl hydroquinone contribute
to the trans-membrane electron transfer from intracellular ascorbate to
extracellular ferricyanide (or to the AFR). In such a mechanism,
one-electron oxidation of the phenolic group of the quinones or
-tocopherol generates a phenoxyl radical on the head group; the
latter then migrates to the cytoplasmic leaflet of the lipid bilayer
and reacts with a reductase or, in the case of -tocopherol, with
ascorbate itself (Fig. 5
, mechanisms 3, 4). Sun et al. (68)
have shown
that the ferricyanide reductase activity of erythrocyte ghosts or
hepatocyte plasma membranes is proportional to the content of
ubiquinone-10 and -tocopherylquinone, and that this reductase
activity is inhibited by ubiquinone analogs. These results prompted the
suggestion that ubiquinol/one-10 is involved in trans-plasma membrane
electron transfer processes (68)
.
More recently, an ubiquinone-10-dependent NADH:AFR reductase activity
has been described in purified K562 cell plasma membranes (69)
. This
activity also parallels the ubiquinone-10 content of the membranes and
is inhibited by capsaicin and dicumarol, which are inhibitors of
ubiquinone-10-dependent electron transport (69)
. Significantly, this
NADH:ubiquinone-10 reductase has an internal protein sequence identical
to that of cytochrome b5 reductase
(70,
71)
. It has been suggested that ubiquinol/one-10 serves as an
electron transfer agent between the external cell surface and the
inward-facing cytochrome b5 reductase
and that this could account for stabilization of extracellular
ascorbate by K562 cells (69)
. In this mechanism, ubiquinol-10 donates
one or two electrons at the cell surface to reduce the AFR to
ascorbate. Either the quinone or radical form then migrates across the
bilayer to be reduced by cytochrome b5
reductase, using NADH-derived electrons. However, the notion that a
long-chain hydrophobic quinone such as ubiquione/ol-10 can move
efficiently across the membrane bilayer is controversial. Initial
studies using dithionite reduction of ferricyanide trapped within
liposomes showed that long-chain quinones were more effective electron
transfer agents than were short-chain forms (72,
73)
. However, when
less hydrophobic reducing agents such as ascorbate (73,
74)
and sodium
borohydride (75)
are used, there is very little tendency for
trans-bilayer electron transfer by long-chain quinones or semiquinones.
Long-chain quinones are viewed as restricted to the center of the
bilayer and incapable of movement to either membrane face (75)
. If
ubiquinol/one-10 is restricted to the center of the bilayer, it would
not be able to donate electrons to ascorbate. Reduction of the AFR
would then require a link to the membrane surface. Ubiquinol-10
efficiently reduces -tocopherol in lipid bilayers (76)
; since the
chromanol ring of -tocopherol does reside at the level of the
phospholipid polar headgroups (9)
, both hydrophobic antioxidants could
work together (Fig. 5
, mechanism 3).
It is also possible that -tocopherol could work independently of
ubiquinol/one-10 to transfer electrons across the membrane bilayer
(Fig. 5
, mechanism 4), especially since -tocopherol is much more
abundant than ubiquinol/one-10 in membranes (68)
. It has been shown
that -tocopherol can transfer electrons from ascorbate trapped
within liposomes to extravesicular ferricyanide (74)
. We recently
confirmed this reaction and found that it was associated with sparing
of -tocopherol and protection against lipid hydroperoxide formation
(77)
. Ilani and Krakover also demonstrated that the rate of reaction of
-tocopherol at the lipid–water interface was much slower than its
reaction in solution (74)
. Thus, trans-membrane electron transfer by
-tocopherol appears to be limited both by its shuttle frequency
across the bilayer and by the rates of redox reactions at the
lipid–water interface on either side of the bilayer. We have reported
that the ability of ascorbate in tightly sealed human erythrocyte
ghosts to reduce extravesicular ferricyanide parallels the membrane
content of -tocopherol (57)
. In the latter studies, ferricyanide
reduction by resealed ghosts was increased by addition of exogenous
-tocopherol during ghost preparation and decreased by removal of
endogenous -tocopherol. On the other hand, supplementation of
-tocopherol in the culture medium of HL-60 cells had no effect on
their ability to reduce ferricyanide nor did -tocopherol affect the
enhanced rates of ferricyanide reduction in ascorbate-supplemented
cells (28)
. Whereas -tocopherol-mediated electron transfer is
plausible from in vitro studies, it may not be present in
all cells or may require additional co-factors not present in cultured
cells.
Does it involve protein sulfhydryl groups?
Trans-membrane electron transfer by sulfhydryl groups on
thiol-rich trans-plasma membrane proteins has also been suggested (Fig. 5
, mechanism 5). Using the technique of spin-echo
1H-NMR spectroscopy to detect GSH in human
erythrocytes, Reglinski et al. (78)
found that incubation of intact
erythrocytes with 0.5–1 mM 5, 5'-dithiobis(2-nitrobenzoic acid) (DTNB)
caused depletion of intracellular GSH by as much as 30%. DTNB, which
does not penetrate the erythrocyte membrane, generates a mixed
disulfide between a surface-exposed protein sulfhydryl and
5-thio-2-nitrobenzoic acid. This oxidant stress is somehow transduced
across the cell membrane to form mixed disulfides between intracellular
GSH and protein sulfhydryls. Ciriolo et al. (79)
have used the same
technique to confirm the initial DTNB results and to show that 0.5 mM
GSH (but not cysteine) added externally to erythrocytes increases
intracellular GSH concentrations. The latter effect was considered to
be due to transfer of the reducing power of GSH across the plasma
membrane to reduce mixed disulfides of GSH with intracellular proteins.
However, external GSH did not reverse DTNB-induced depletion in
intracellular GSH. Since GSH can reduce mixed disulfides of
2-nitro-5-thiobenzoic acid and GSH (80)
, it may be able to do so for
mixed disulfides of 2-nitro-5-thiobenzoic acid and protein thiols. If
so, then either the 2-nitro-5-thiobenzoic acid-protein thiol conjugates
on the cell surface proteins are not accessible to GSH or the
mechanisms of DTNB and GSH effects are different. Whether either
phenomenon is related to ascorbate-dependent ferricyanide reduction is
unknown. By oxidizing cell surface thiols, ferricyanide could generate
a trans-membrane oxidant stress in a manner analogous to that produced
by DTNB. It has been shown that PCMBS decreases ascorbate-dependent
ferricyanide reduction by both erythrocytes (5)
and HL-60 cells (28)
.
On the other hand, we found that ferricyanide treatment of
glucose-depleted erythrocytes increased, rather than decreased, their
content of GSH by 17% (47)
. Despite the discrepancies, these results
suggest that membrane thiols should receive further study for
involvement in ascorbate-dependent, trans-membrane electron transfer.
Possible cellular functions of the oxidoreductase
Several roles for the trans-plasma membrane
oxidoreductase activity have been proposed, including function as an
AFR reductase, an -tocopheroxyl or hydroperoxyl radical reductase, a
lipid hydroperoxide reductase, a stimulant of cell growth, and a ferric
iron reductase to facilitate transferrin-independent iron uptake.
Function as an AFR reductase
Function of the trans-plasma membrane oxidoreductase as a cell
surface AFR reductase would provide a mechanism for cells to
efficiently regenerate extracellular ascorbate from the AFR. Since most
cells maintain a steep concentration gradient of ascorbate across their
plasma membranes (37)
, release of ascorbate into plasma and the
interstitium is probably minimal. Exceptions are release of ascorbate
stored in secretory granules from chromaffin cells (45,
46)
and brain
cells (44)
. A cell surface AFR reductase that uses electrons from
either NADH or ascorbate might thus play a role in maintaining critical
extracellular stores of the vitamin. Extracellular recycling of
ascorbate by white and red cells would be especially important in areas
of inflammation or atherosclerosis in the vascular bed, since such
ascorbate could recycle -tocopherol and prevent lipid peroxidation
in LDL and lipids (81)
.
A cell surface NADH:AFR reductase activity has been proposed to
maintain ascorbate concentrations outside of cells in culture (69,
82)
.
Prevention of extracellular ascorbate loss is stimulated by growth
factors (83)
, addition of lactate (82)
, and increases in the cellular
content of cyclic AMP (84)
. It is inhibited by lectins that are known
to bind to cell surface carbohydrate (82)
. However, these studies rely
on an indirect assay, which does not measure either extracellular AFR
reduction or intracellular NADH oxidation. Goldenberg and colleagues
(85)
have pointed out that other mechanisms could also preserve
ascorbate outside cells. For example, a cell surface DHA reductase
activity has been reported in K562 cells (86)
, and chelation of trace
metals by proteins released from cells could account for the observed
preservation of ascorbate (85)
. Secretion of ascorbate from specialized
cells might also contribute. Additional studies are needed to document
the presence, capacity, and physiological significance of cell surface
AFR reduction. Nonetheless, a cell surface AFR reductase would provide
an efficient mechanism for regenerating extracellular ascorbate. If
coupled to an energy-generating step such as an
H+-ATPase, as in the chromaffin granule system
(Fig. 6)
, ascorbate could serve as a donor in addition to NADH.
Function as a lipid hydroperoxyl or -tocopheroxyl free radical
reductase
The trans-plasma membrane oxidoreductase could transfer
ascorbate-derived electrons into as well as across the plasma membrane.
In the lipid bilayer, electrons from ascorbate or even the AFR could
help to reduce lipid hydroperoxyl radicals or recycle the
one-electron-oxidized forms of lipid-soluble antioxidants. Based on
oxidation reduction potentials, neither ascorbate nor the AFR can
reduce ubiquinone-10 nor its semiquinone (9)
. Moreover, a ubiquinol-10
reductase has been proposed to reduce the AFR to ascorbate (69)
, and
not the converse. Whether ascorbate or the AFR can reduce lipid
hydroperoxyl radicals is unknown, although a consideration of the redox
potentials involved suggests that this reaction may be possible (9)
.
Ascorbate is well known to recycle the -tocopheroxyl radical. It is
also possible that an intervening enzyme or carrier in the cell
membrane enhances the ability of ascorbate at the cytoplasmic membrane
face to reduce the -tocopheroxyl radical within the lipid bilayer.
To implicate the ascorbate- or NADH-dependent oxidoreductase activity
in such recycling, additional studies are needed to show that
-tocopherol recycling depends on the enzyme activity.
An NADH-dependent reductase activity of human erythrocyte membranes has
been reported to reduce cumene and linoleoyl hydroperoxides to their
alcohol forms (87)
. It was suggested that this activity might
correspond to the NADH-dependent ferricyanide reductase activity (87)
,
which could implicate ascorbate as an electron donor. Ascorbate is
unable to directly reduce lipid hydroperoxides (88)
, but it might be
able to do so indirectly by donating electrons to an enzyme with that
capability. Such an activity could also serve to enhance the
antioxidant function of -tocopherol, which reduces lipid
hydroperoxyl radicals to hydroperoxides. Hydroperoxides generated by
-tocopherol will accumulate unless reduced or removed from the
membrane (89)
. In this mechanism, ascorbate donates electrons to the
hydroperoxide reductase, which then detoxifies hydroperoxides generated
by the action of -tocopherol. This would complement the ability of
ascorbate to reduce the -tocopheroxyl radical. However, the presence
of such an NADH-dependent membrane-bound lipid hydroperoxide reductase
has not been confirmed nor has ascorbate been studied as an electron
donor. To be of physiological relevance, this activity would also have
to reduce lipid hydroperoxides present in phospholipids, as has been
done for phospholipid hydroperoxide glutathione peroxidase (90)
.
Stimulation of cell growth and differentiation
The trans-plasma membrane oxidoreductase that uses ferricyanide as
an external electron acceptor may be involved in stimulation of cell
growth and differentiation. As reviewed by Crane et al. (91)
, this
hypothesis is based on the finding that ferricyanide added to
serum-deprived tumor cells in culture enhances cell division and
differentiation (92)
. Involvement of an ascorbate-dependent,
trans-plasma membrane oxidoreductase in ferricyanide-induced cell
division has little support. As reviewed by Navas et al. (8)
,
ascorbate, probably through its facilitation of collagen synthesis, can
stimulate cell growth. However, since cultured cells generally lack
ascorbate (28)
, there seems to be little role for ascorbate in the
ferricyanide effect in cultured cells.
Function as part of a transferrin-independent iron uptake system
It is possible that ascorbate, through the activity of a
trans-plasma membrane enzyme, could provide electrons for
transferrin-independent reduction of ferric iron and thus facilitate
iron uptake by cells. This function was postulated many years ago for
both ascorbate (23)
and a trans-plasma membrane enzyme (5)
.
Transferrin-independent iron uptake has been documented in a variety of
cell types (93,
94)
and may complement transferrin-dependent
mechanisms. Transferrin-independent iron uptake is thought to involve
binding of ferric iron to the cell surface (95)
, reduction of the
ferric to ferrous iron, and uptake of the latter (96)
. Ferricyanide has
been shown to inhibit uptake of radiolabeled iron from ferric chelates
in K562 cells (97)
and Caco-2 intestinal cells (98)
. The effect of
ferricyanide brings up the question of whether the trans-membrane
oxidoreductase is involved and whether intracellular reducing
equivalents from ascorbate can reduce ferric to ferrous iron before
uptake of the latter. Han et al. (98)
found that increasing the
intracellular concentration of ascorbate by loading Caco-2 cells with
DHA enhanced radiolabled iron uptake. However, since this effect was
abolished by inclusion of ascorbate oxidase, it may have been due to
direct reduction of ferric iron by ascorbate that had leaked from the
cells. Our recent results in U-937 cells favor the latter explanation
(99)
. Whereas ascorbate-loaded cells had enhanced uptake of
55Fe and reduction of extracellular ferric
citrate, both activities were abolished by ascorbate oxidase. Further,
ascorbate oxidase was without effect on ascorbate-dependent
ferricyanide reduction, which strongly argues against involvement
trans-plasma membrane oxidoreductase in iron uptake.
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FUTURE DIRECTIONS: ELUCIDATION OF THE STRUCTURE, FUNCTION, AND REGULATION OF A TRANS-PLASMA MEMBRANE OXIDOREDUCTASE |
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TOPABSTRACTINTRODUCTIONRECENT RESULTS: THE ROLE...CONTROVERSIES AND UNRESOLVED...FUTURE DIRECTIONS: ELUCIDATION...REFERENCES |
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have not been
confirmed or extended with molecular techniques. Complicating
purification of such a protein is its hydrophobicity, and whether it
might require a cofactor, such as a cytochrome or a lipid-soluble
antioxidant. Ferricyanide reductase activity from Ehrlich ascites cell
plasma membranes, which appears to be trans-membrane in orientation,
has been reconstituted into phospholipid vesicles (100)
. However, this
activity was not shown to span the lipid bilayer and its ascorbate
dependence was not tested, since NADH was used as substrate. Neither the function of the trans-membrane oxidoreductase nor the role that intracellular ascorbate might play in that function has been established. Available evidence narrows the function of this activity to two major areas: enhancement of cell growth and differentiation and an antioxidant role to protect the cell membrane or to recycle extracellular ascorbate. Regarding the latter, further studies are needed to define the role of intracellular ascorbate as a natural substrate, to establish whether intracellular ascorbate can reduce extracellular AFR, and to determine whether the system can serve in some manner to protect the cell membrane from extracellular oxidant stress in areas of inflammation.
There is no evidence for either pre- or posttranslational regulation of
the trans-plasma membrane oxidoreductase. On the other hand, it is
intriguing that ferricyanide reduction varies greatly between blood
cells obtained from different donors (31)
. Whereas much of this
variance appears to relate simply to different intracellular ascorbate
concentrations (31)
, the possibility of genetic variation must be
considered. Defining the identity, mechanism, and function of the
oxidoreductase will provide clues as to how it might be
regulated.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRECENT RESULTS: THE ROLE...CONTROVERSIES AND UNRESOLVED...FUTURE DIRECTIONS: ELUCIDATION...REFERENCES |
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-tocopherol radical and recycling of -tocopherol by ascorbate during peroxidation of phosphatidylcholine liposomes. An electron paramagnetic resonance study. Biochim. Biophys. Acta 801,215-219[Medline]
-tocopherol in human erythrocytes by intracellular ascorbic acid. Arch. Biochem. Biophys. 349,281-289[Medline]
-tocopherol in resealed human erythrocyte ghosts - Transmembrane electron transfer and protection from lipid peroxidation. J. Biol. Chem. 271,10577-10582
