BioFactors 34 (2009) 191200 191DOI 10.3233/BIO-2009-1072IOS Press
Transplasma membrane electron transportcomes in two flavors
Darius J. R. Lane and Alfons LawenDepartment of Biochemistry and Molecular Biology, School of Biomedical Sciences, MonashUniversity VIC 3800, Australia
Abstract. All tested cells possess transplasma membrane electron transfer (tPMET) systems that are capable of reducingextracellular electron acceptors at the cost of cytosolic electron donors. In mammals, classically NAD(P)H- and NADH-dependent systems have been distinguished. The NADH-dependent system has been suggested to be involved in non-transferrin-bound iron (NTBI) reduction and uptake. Recently we reported that transplasma membrane ascorbate/dehydroascorbatecycling can promote NTBI reduction and uptake by human erythroleukemia (K562) cells (D.J.R. Lane and A. Lawen, J BiolChem 283 (2008), 1270112708). This system, involves i) cellular import of dehydroascorbate, ii) intracellular reduction ofdehydroascorbate to ascorbate using metabolically-derived reducing equivalents, iii) export of ascorbate down its concentrationgradient, iv) direct reduction of low molecular weight iron chelates by ascorbate, and v) uptake of iron (II) into the cell. We herepropose the consideration of this system as a novel form of tPMET which shares with classical enzyme-mediated tPMET systemsthe net transfer of reducing equivalents from the cytoplasmic compartment to the extracellular space, but lacks the involvementof the plasma membrane oxidoreductases responsible for the latter. Thus, transplasma membrane electron transfer can anddoes occur at two mechanistically distinct levels: i) enzyme-mediated transmembrane electron transfer and ii) transmembranemetabolite shuttling/cycling.
Keywords: Astrocytes, dehydroascorbate, K562 cells, non-transferrin-bound iron, Vitamin C
Abbreviations: AFR, ascorbate free radical; DHA, dehydroascorbate; GLUT, facilitative glucose transporter; NTBI, non-transferrin-bound iron; tPMET, transplasma membrane electron transport; SVCT, sodium-ascorbate co-transporter; VSOAC,volume-sensitive osmolyte and anion channel
Transplasma membrane electron transport (tPMET) in eukaryotes is now well established [8,10,15,44,59,61,68]. The concept of tPMET arose from the observation that cell-impermeant dyes [43,87] can bereduced by tissue slices . tPMET activities have since been related to the regulation of vital cellularprocesses including cellular bioenergetics [53,86], growth control and differentiation [8,15,68], apopto-sis [54,55,70,101], pH control and mitogenesis [8,68], cell signal transduction , antioxidation [61,85], and iron/copper metabolism [8,15,67,68,102]. Accordingly, deregulation of tPMET has been linkedto various human conditions including aging and neurodegeneration [36,37], macrophage-mediated LDLoxidation in atherogenesis , diabetic nephropathy  and glycolytic cancer progression .
Address for correspondence: Alfons Lawen, Department of Biochemistry and Molecular Biology, School of BiomedicalSciences, Monash University, VIC 3800, Australia. Tel.: +61 3 9905 3711; Fax: +61 3 9905 3726; E-mail: email@example.com.
0951-6433/09/$17.00 2009 IUBMB/IOS Press and the authors. All rights reserved
192 D.J.R. Lane and A. Lawen / Transplasma membrane electron transport comes in two flavors
Classically a distinction was made between NAD(P)H- and NADH-dependent systems, the former ofwhich includes the members of the Nox and Duox families , while the latter often referred to as theplasma membrane NADH:oxidoreductase system or PMOR is suggested to include at least an NADHoxidase and an NADH:ferricyanide reductase activity [8,15,59].
Several enzymes have been suggested to be responsible for the plasma membrane NADH:ferricyanidereductase activity: a 57 kDa NADH-quinone oxidoreductase from rat liver plasma membranes ; aplasma membrane localized voltage-dependent anion channel (VDAC) isoform 1 ; and a membrane-bound form of cytochrome b5 reductase in neuronal plasma membranes . Moreover, a multi-component, quinone-dependent tPMET system is also well-described  that is capable of reducingcell-impermeant water-soluble tetrazolium salts (e.g. WST-1) or extracellular dioxygen at the expenseof intracellular NADH [10,31].
Iron is vital for cellular survival: without it, every cell will die. Iron is a cofactor for oxidative phos-phorylation, neurotransmitter and nucleotide synthesis, nitric oxide metabolism and oxygen transport.However, iron can also catalyze the formation of reactive oxygen species [21,75,103]. Since too muchand too little iron can compromise cell viability, cellular iron homeostasis has to be tightly controlled.
In its physiological form, extracellular iron is complexed by biological chelators, the most importantof which are transferrin and citrate. Whereas the uptake of iron from transferrin is reasonably wellunderstood, the mechanism of non-transferrin-bound iron (NTBI) uptake by mammalian cells remainselusive. In order for iron citrate to be taken up by a cell, iron has to be first reduced from iron (III) toiron (II) as almost all cellular iron uptake can be inhibited by iron (II) chelators [18,30,38,39,41,76,91].Ferrous iron is then taken up by ferrous-specific transporters (e.g., DMT1 [5,104] and Zip14 ) in theplasma membrane.
Soon after its discovery, the transplasma ferricyanide reductase activity was suggested to be responsiblefor the reduction of NTBI prior to uptake as Fe2+ through the plasma membrane by divalent metal iontransporters [3,15,17,39,41]. Several enzymes have been suggested to be responsible for NTBI reductionbefore uptake, including duodenal cytochrome b561 (Dcytb ) and voltage dependent anion-selectivechannel, isoform 2 (VDAC2 ). The involvement of Dcytb in NTBI uptake, however, has since beenquestioned  and work in our own laboratory was not successful in linking either VDAC1 or VDAC2to NTBI uptake.
Ascorbate is known to promote the bioavailability of iron from numerous food sources in vivo andin vitro [22,29,30]. An ascorbate-stimulatede ferricyanide reductase has been described, but remains tobe molecularly identified. Moreover, ascorbate supplementation was shown to stimulate extracellularferricyanide reduction by several cell types, including K562 cells [51,82,83], HL-60 cells  and humanerythrocytes [44,61]. These data prompted us to ask the question of whether the ascorbate-stimulatedferricyanide reductase is involved in iron reduction for the uptake of iron from iron citrate.
2. The biochemistry of ascorbate
The physiologically prevalent monovalent ascorbate anion can undergo sequential one-electron oxi-dations under physiological conditions of pH, temperature and oxygen tension [61,78] (Fig. 1). The firstoxidation yields the relatively long-lived and electrochemically stable ascorbate free radical (AFR; alsoknown as semi- or mono-dehydroascorbate; E 0 = + 330 mV) . This first oxidation of ascorbate isenhanced by low levels of circulating redox-active transition metals, such as iron and copper [78,84,88,89]. As AFR is relatively unreactive with dioxygen  unlike many other free radicals [13,57] andtends to decay mainly by disproportionation , the formation of AFR by reaction of ascorbate with
D.J.R. Lane and A. Lawen / Transplasma membrane electron transport comes in two flavors 193
Fig. 1. The oxidation products of vitamin C. The undissociated, fully reduced form of vitamin C (ascorbic acid) undergoes amonoprotic ionization at the carbon-3 hydroxyl with a pKa of 4.2, so that the ascorbate monoanion is the predominant species atphysiological pH. Ascorbate can undergo a thermodynamically favorable and reversible one-electron oxidation to the ascorbatefree radical (AFR). AFR is stabilized by resonant distribution of the resultant unpaired electron over the ring structure. AFRcan undergo a subsequent reversible one-electron oxidation to form the two-electron oxidized form, dehydroascorbate (DHA).DHA is highly unstable under physiological conditions and undergoes an essentially irreversible hydrolytic ring opening to2,3-diketogulonic acid (DKG) with a half-life of several minutes.
reactive radical species tends to inhibit free radical-induced oxidative chain reactions [13,57]; especiallyin the face of rapid AFR reduction back to ascorbate. AFR can undergo a further monoelectronic ox-idation to DHA (E0 = 210 mV)  in the presence of mild oxidants such as ferricyanide [61,96]and/or NTBI species [14,88,89]. In the absence of such oxidants, however, two AFR molecules willrapidly disproportionate to one ascorbate and one DHA molecule [11,63] (Fig. 1). Though oxidation(or disproportionation) of AFR to DHA effectively allows utilization of the two-electron reducing ca-pacity of ascorbate, DHA is a structurally labile species that rapidly undergoes an irreversible hydrolyticring-opening to 2,3-diketogulonic acid under conditions found in plasma with a half-life of severalminutes [48,49].
Degradation of DHA results in an irrevocable loss of the vitamin from mammalian systems [42,48,49] a point that is particularly pertinent in the case of species lacking gulono--lactone oxidaseactivity . In order to cope with this tendency for ascorbate loss, the vitamin must be maintainedpredominantly in the two-electron reduced form (i.e. ascorbate) in both intra- and extracellular biologicalfluids . Consistent with this observation, mammalian cells possess a variety of conservative reductivemechanisms for maintaining both intra- and extracellular ascorbate [61,66,78,99]. Even cultured cells which may be chronically ascorbate-deficient due to lack of supplementation under standard cultureconditions [35,90] still maintain an extraordinary ability for ascorbate regeneration.
3. The ascorbate-stimulated plasma membrane ferricyanide reductase
Human erythrocytes possess a tPMET activity that utilizes intracellular ascorbate as the major electrondonor to reduce extracellular ferricyanide [44,61]. It is not clear at this stage whether ascorbate is a
194 D.J.R. Lane and A. Lawen / Transplasma membrane electron transport comes in two flavors
Fig. 2. DHA uptake by K562 cells occurs by GLUT-mediated transport. To assess the involvement of facilitative glucosetransporters in DHA uptake by K562 human erythroleukemia cells, cells previously grown to 68 106 cells/ml in RPMI +10% fetal bovine serum at 37C, 5% CO2 and 95% air were initially washed three times with MOPS-buffered saline (MBS,137 mM NaCl, 2.7 mM KCl, 15 mM MOPS-Na+, pH 7.3). (a) Washed cells were then exposed to increasing concentrations ofthe GLUT-inhibitor cytochalasin B (CB) or the non-GLUT inhibiting structural analog, dihydrocytochalasin B (H2CB) 10 minprior to, and during incubation with 400 M dehydroascorbate (DHA) for 30 min at 37C. Cells were then washed three timesin 100 volumes in cold MBS and their intracellular ascorbate determined essentially according to Lane and Lawen . AsDHA uptake is inhibitable by CB, but not H2CB, GLUT-mediated DHA uptake is implied. (b) Alternatively, washed cells wereexposed to increasing concentrations of the GLUT-transportable, but non-metabolizable glucose analog 3-O-methyl-D-glucose(3-OMG) or the non-GLUT-transportable glucose stereoisomer L-glucose prior to incubation with DHA as in panel A. Againthe results indicate GLUT involvement in DHA import as inhibition of intracellular ascorbate accumulation occurs only in thepresence of the GLUT-transportable glucose analog.
significant electron donor for tPMET systems in other cells. When we tested human chronic myeloidleukemia (K562) cells, we observed a significant stimulation of the plasma membrane ferricyanide re-ductase activity after increasing intracellular ascorbate by preloading with dehydroascorbate . Thisstimulation is not affected by addition of ascorbate oxidase (i.e. at a concentration that oxidizes allextracellular ascorbate to DHA and consequently inhibits direct reduction of ferricyanide by ascorbate),indicating that intracellular ascorbate can serve as an electron donor for extracellular ferricyanide reduc-tion (Lane et al., data not shown). The stimulation of the reductase activity could not be reproduced whenascorbate was directly added to the cells, suggesting that these cells do not express significant levels ofsodium-ascorbate co-transporters (SVCTs) . Similar results were obtained with primary mouse andrat astrocytes (Lane et al., data not shown), indicating that these phenomena are not restricted to K562cells.
4. Cellular DHA uptake
The majority of mammalian cells maintain intracellular ascorbate concentrations that are markedlyhigher (e.g. up to 30-fold in some cases ) than those in the extracellular fluid or plasma [61,78,99]. Though many cells maintain this outward-facing concentration gradient by SVCT-mediatedascorbate import [81,99], the facilitated diffusion of DHA through low-affinity, high-capacity GLUTsis also a significant contributor . With respect to DHA uptake by cells, an inward-facing DHAgradient is maintained by the rapid reduction of imported DHA back to ascorbate, the latter of whichis a poor substrate for GLUT-mediated transport [34,77,99]. K562 cells showed elevated intracellularascorbate levels after loading with DHA  that was inhibitable by cytochalasin B, suggesting response-dependence on DHA uptake via GLUTs, as previously suggested [77,78,93,99]. The involvement ofGLUTs in DHA uptake by K562 cells is further supported by two pharmacological observations thatintracellular ascorbate accumulation in response to extracellular DHA is inhibited by: i) low micromolar
D.J.R. Lane and A. Lawen / Transplasma membrane electron transport comes in two flavors 195
Fig. 3. Ascorbate/DHA shuttling in mammalian NTBI uptake. Recent evidence suggests that NTBI ferrireduction may occurby transplasma membrane Asc cycling in which i) extracellular Asc reacts directly with NTBI, forming both DHA and Fe2+.The latter is then imported into the cell putatively via ferrous-selective transporters (e.g. DMT1 and/or Zip 14). ExtracellularAsc is subsequently regenerated for further ferric reduction events by ii) DHA import via glucose transporters (GLUTs), iii)intracellular reduction of DHA to Asc by an unspecified redox couple (R/O; e.g. GSH/GSSG or NADPH/NADP+), followedby release of Asc through as yet unidentified Asc transporters (Anion Channel) in the plasma membrane (PM).
concentrations of cytochalasin B, but not the structural analog dihydrocytochalasin B (Fig. 2a), the latterof which shares with cytochalasin B its inhibition of cellular motile processes but not that of facilitatedglucose transport ; and ii) millimolar concentrations of the transportable (but non-metabolizable)D-glucose analog 3-O-methyl-D-glucose, but not the non-transportable glucose stereoisomer L-glucose(Fig. 2b). Again, primary astrocytes demonstrate similar behavior (Lane et al., data not shown).
5. Iron uptake and the ascorbate/DHA shuttle
Cellular uptake of NTBI is well documented, but less well understood than the classical transferrin-dependent iron import pathway [2,16,19,28,38,39,41,50,64,71]. The former may be particularly relevantin iron overload diseases such as hereditary hemochromatosis, hypotransferrinemia, and thalassemia [4,12,18,23], in which plasma iron presents in excess of transferrin-binding capacity . When we analyzedthe ascorbate-mediated stimulation of NTBI reduction and uptake by human erythroleukemia (K562)cells we found that DHA loading of cells stimulated both processes (viz. 12- and 2-fold, respectively),yet unlike the reduction of ferricyanide remained inhibitable by extracellular ascorbate oxidase .Furthermore, as cells were able to import iron in a manner inhibitable by cell-impermeant ferrous ionchelators, the ascorbate-stimulated iron uptake is clearly dependent on the initial adoption of the ferrousstate, as previously observed [18,30,38,39,41,69,76,91].
Our data suggest that ascorbate released from cells following uptake and reduction of DHA mediatesdirect reduction of ferric to ferrous iron, the latter of which is then imported (Fig. 3). Subsequent additionof DHA to control or loaded cells resulted in a dose-dependent stimulation of both iron reduction and
196 D.J.R. Lane and A. Lawen / Transplasma membrane electron transport comes in two flavors
uptake that was inhibitable by cytochalasin B, suggesting response-dependence on DHA uptake viaGLUTs (see Section 4). Again, these results are basically reproducible with primary astrocyte cultures(Lane et al., data not shown).
Several plausible candidates for the cellular export of ascorbate have been proposed , includ-ing exocytosis of ascorbate-containing vesicles [98,99], ascorbate-ascorbate homeoexchangers [24,45],connexin hemi-channels  and volume-sensitive osmolyte and anion channels (VSOACs) [47,62,99,100]. VSOAC permeability  and ascorbate efflux from cells [47,99] can be inhibited by generic anionchannel inhibitors, such as 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) and 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (SITS), suggesting that a significant proportion of ascorbaterelease occurs via this pathway. The observation that DIDS inhibits ascorbate release, ferrireduction andiron uptake to a similar degree in K562 cells  supports this conclusion.
6. Shuttle-based tPMET systems conclusions
Historically, research focused on enzymatic tPMET systems; however several examples of shuttle-based tPMET systems have been documented as well, including ascorbate/DHA [30,52,64], dihy-drolipoic acid/-lipoic acid [40,65], reduced glutathione/cysteine [20,25] and superoxide/dioxygen [26,73] shuttles. As with classical enzyme-mediated tPMET systems, these shuttle-based systems resultin the net transfer of metabolically-derived reducing equivalents from the cytoplasmic compartment tothe extracellular space. Once in the extracellular space, however, the fate of these reducing equivalentswill depend on the particular redox couple involved. A canonical example of shuttle-based tPMET istransplasma membrane ascorbate/DHA cycling, which was the focus of this brief review. Our sugges-tion is that tPMET can and does come in two flavors: i) enzyme-mediated transmembrane electrontransfer and ii) transmembrane metabolite shuttling/cycling. In the latter reducing equivalents derivedfrom cellular metabolism are transferred via transmembrane metabolite shuttling to the extracellularspace for participation in extracellular redox events. Transplasma membrane ascorbate/DHA cyclingmay contribute significantly to NTBI ferric reduction prior to ferrous uptake.
 S. Ahmad and W.H. Evans, Post-translational integration and oligomerization of connexin 26 in plasma membranesand evidence of formation of membrane pores: implications for the assembly of gap junctions, Biochem J 365 (2002),693699.
 T. Akompong, R.S. Inman and M. Wessling-Resnick, Phorbol esters stimulate non-transferrin iron uptake by K562cells, J Biol Chem 270 (1995), 2093720941.
 F.J. Alcain, H. Low and F.L. Crane, Iron at the cell surface controls DNA synthesis in CCI 39 cells, Biochem BiophysRes Commun 203 (1994), 1621.
 G.J. Anderson, Ironing out disease: inherited disorders of iron homeostasis, IUBMB Life 51 (2001), 1117. C. Au, A. Benedetto and M. Aschner, Manganese transport in eukaryotes: The role of DMT1, Neurotoxicology 29
(2008), 569576. E. Baker, S.M. Baker and E.H. Morgan, Characterisation of non-transferrin-bound iron (ferric citrate) uptake by rat
hepatocytes in culture, Biochim Biophys Acta 1380 (1998), 2130. M.A. Baker, D.J.R. Lane, J.D. Ly, V. De Pinto and A. Lawen, VDAC1 is a transplasma membrane NADH-ferricyanide
reductase, J Biol Chem 279 (2004), 48114819. M.A. Baker and A. Lawen, Plasma membrane NADH-oxidoreductase system: a critical review of the structural and
functional data, Antioxid Redox Signal 2 (2000), 197212. A. Baoutina, R.T. Dean and W. Jessup, Trans-plasma membrane electron transport induces macrophage-mediated low
density lipoprotein oxidation, FASEB J 15 (2001), 15801582.
D.J.R. Lane and A. Lawen / Transplasma membrane electron transport comes in two flavors 197
 M.V. Berridge and A.S. Tan, High-capacity redox control at the plasma membrane of mammalian cells: trans-membrane,cell surface, and serum NADH-oxidases, Antioxid Redox Signal 2 (2000), 231242.
 B.H.J. Bielski, H.W. Richter and P.C. Chan, Some properties of the ascorbate free radical, Ann NY Acad Sci 258 (1975),231237.
 W. Breuer, C. Hershko and Z.I. Cabantchik, The importance of non-transferrin bound iron in disorders of iron metabolism,Transfus Sci 23 (2000), 185192.
 G.R. Buettner, The pecking order of free radicals and antioxidants: lipid peroxidation, -tocopherol, and ascorbate,Arch Biochem Biophys 300 (1993), 535543.
 G.R. Buettner and B.A. Jurkiewicz, Catalytic metals, ascorbate and free radicals: combinations to avoid, Radiat Res145 (1996), 532541.
 F.L. Crane, I.L. Sun, M.G. Clark, C. Grebing and H. Low, Transplasma-membrane redox systems in growth anddevelopment, Biochim Biophys Acta 811 (1985), 233264.
 C.M. Craven, J. Alexander, M. Eldridge, J.P. Kushner, S. Bernstein and J. Kaplan, Tissue distribution and clearancekinetics of non-transferrin-bound iron in the hypotransferrinemic mouse: a rodent model for hemochromatosis, ProcNatl Acad Sci USA 84 (1987), 34573461.
 R.R. Crichton, A role for ferritin in the regulation of iron metabolism, FEBS Lett, 34 (1973), 125128. D.M. De Silva, C.C. Askwith and J. Kaplan, Molecular mechanisms of iron uptake in eukaryotes, Physiol Rev 76 (1996),
3147. R. Dringen, G.M. Bishop, M. Koeppe, T.N. Dang and S.R. Robinson, The pivotal role of astrocytes in the metabolism
of iron in the brain, Neurochem Res 32 (2007), 18841890. R. Dringen, B. Pfeiffer and B. Hamprecht, Synthesis of the antioxidant glutathione in neurons: supply by astrocytes of
CysGly as precursor for neuronal glutathione, J Neurosci 19 (1999), 562569. L.L. Dunn, Y.S. Rahmanto and D.R. Richardson, Iron uptake and metabolism in the new millennium, Trends Cell Biol
17 (2007), 93100. R. Engle-Stone, A. Yeung, R. Welch and R. Glahn, Meat and ascorbic acid can promote Fe availability from Fe-phytate
but not from Fe-tannic acid complexes, J Agric Food Chem 53 (2005), 1027610284. B.P. Esposito, W. Breuer, P. Sirankapracha, P. Pootrakul, C. Hershko and Z.I. Cabantchik, Labile plasma iron in iron
overload: redox activity and susceptibility to chelation, Blood 102 (2003), 26702677. F.M. Finn and P.A. Johns, Ascorbic acid transport by isolated bovine adrenal cortical cells, Endocrinology 106 (1980),
811817. J. Frade, S. Pope, M. Schmidt, R. Dringen, R. Barbosa, J. Pocock, J. Laranjinha and S. Heales, Glutamate induces
release of glutathione from cultured rat astrocytes a possible neuroprotective mechanism?, J Neurochem 105 (2008),11441152.
 A.J. Ghio, E. Nozik-Grayck, J. Turi, I. Jaspers, D.R. Mercatante, R. Kole and C.A. Piantadosi, Superoxide-dependentiron uptake: a new role for anion exchange protein 2, Am J Respir Cell Mol Biol 29 (2003), 653660.
 H. Gunshin, C.N. Starr, C. DiRenzo, M.D. Fleming, J. Jin, E.L. Greer, V.M. Sellers, S.M. Galica and N.C. Andrews,Cybrd1 (duodenal cytochrome b) is not necessary for dietary iron absorption in mice, Blood 106 (2005), 28792883.
 J.A. Gutierrez, R.S. Inman, T. Akompong, J. Yu and M. Wessling-Resnick, Metabolic depletion inhibits the uptake ofnontransferrin-bound iron by K562 cells, J Cell Physiol 177 (1998), 585592.
 L. Hallberg, M. Brune and L. Rossander, Iron absorption in man: ascorbic acid and dose-dependent inhibition byphytate, Am J Clin Nutr 49 (1989), 140144.
 O. Han, M.L. Failla, A.D. Hill, E.R. Morris and J.C. Smith, Jr., Reduction of Fe(III) is required for uptake of nonhemeiron by Caco-2 cells, J Nutr 125 (1995), 12911299.
 P.M. Herst and M.V. Berridge, Plasma membrane electron transport: a new target for cancer drug development, CurrMol Med 6 (2006), 895904.
 P.M. Herst and M.V. Berridge, Cell surface oxygen consumption: a major contributor to cellular oxygen consumptionin glycolytic cancer cell lines, Biochim Biophys Acta 1767 (2007), 170177.
 P.M. Herst, T. Petersen, P. Jerram, J. Baty and M.V. Berridge, The antiproliferative effects of phenoxodiol are associatedwith inhibition of plasma membrane electron transport in tumour cell lines and primary immune cells, BiochemPharmacol 74 (2007), 15871595.
 U. Himmelreich, K.N. Drew, A.S. Serianni and P.W. Kuchel, 13C NMR studies of vitamin C transport and its redoxcycling in human erythrocytes, Biochemistry 37 (1998), 75787588.
 K.E. Hoffman, K. Yanelli and K.R. Bridges, Ascorbic acid and iron metabolism: alterations in lysosomal function, AmJ Clin Nutr 54 (1991), 1188S1192S.
 D.-H. Hyun, S.S. Emerson, D.-G. Jo, M.P. Mattson and R. de Cabo, Calorie restriction up-regulates the plasmamembrane redox system in brain cells and suppresses oxidative stress during aging, Proc Natl Acad Sci USA 103 (2006),1990819912.
198 D.J.R. Lane and A. Lawen / Transplasma membrane electron transport comes in two flavors
 D.-H. Hyun, J.O. Hernandez, M.P. Mattson and R. de Cabo, The plasma membrane redox system in aging, Ageing ResRev 5 (2006), 209220.
 R.S. Inman, M.M. Coughlan and M. Wessling-Resnick, Extracellular ferrireductase activity of K562 cells is coupled totransferrin-independent iron transport, Biochemistry 33 (1994), 1185011857.
 R.S. Inman and M. Wessling-Resnick, Characterization of transferrin-independent iron transport in K562 cells. Uniqueproperties provide evidence for multiple pathways of iron uptake, J Biol Chem 268 (1993), 85218528.
 W. Jones, X. Li, Z.-C. Qu, L. Perriott, R.R. Whitesell and J.M. May, Uptake, recycling, and antioxidant actions of-lipoic acid in endothelial cells, Free Radic Biol Med 33 (2002), 8393.
 I. Jordan and J. Kaplan, The mammalian transferrin-independent iron transport system may involve a surface ferrire-ductase activity, Biochem J 302 (1994), 875879.
 C.-H. Jung and W.W. Wells, Spontaneous conversion of L-dehydroascorbic acid to L-ascorbic acid and L-erythroascorbicacid, Arch Biochem Biophys 355 (1998), 914.
 D. Keilin and E.F. Hsrtee, Spectroscopic study of the permeability and lysis of red blood corpuscles, Nature 157 (1946),210213.
 E.C. Kennett and P.W. Kuchel, Plasma membrane oxidoreductases: effects on erythrocyte metabolism and redoxhomeostasis, Antioxid Redox Signal 8 (2006), 12411247.
 M. Khatami, L.E. Stramm and J.H. Rockey, Ascorbate transport in cultured cat retinal pigment epithelial cells, Exp EyeRes 43 (1986), 607615.
 C. Kim, F.L. Crane, W.P. Faulk and D.J. Morre, Purification and characterization of a doxorubicin-inhibited NADH-quinone (NADH-ferricyanide) reductase from rat liver plasma membranes, J Biol Chem 277 (2002), 1644116447.
 C. Konya and P. Ferdinandy, Vitamin C: new role of the old vitamin in the cardiovascular system?, Br J Pharmacol 147(2006), 125127.
 I. Koshiishi, Y. Mamura and T. Imanari, Bicarbonate promotes a cleavage of lactone ring of dehydroascorbate, BiochimBiophys Acta 1379 (1998), 257263.
 I. Koshiishi, Y. Mamura, J. Liu and T. Imanari, Degradation of dehydroascorbate to 2,3-diketogulonate in bloodcirculation, Biochim Biophys Acta 1425 (1998), 209214.
 J. Kovar, J. Neubauerova, M. Cimburova, J. Truksa, K. Balusikova and J. Horak, Stimulation of non-transferrin ironuptake by iron deprivation in K562 cells, Blood Cells Mol Dis 37 (2006), 9599.
 D.J.R. Lane and A. Lawen, A highly sensitive colorimetric microplate ferrocyanide assay applied to ascorbate-stimulatedtransplasma membrane ferricyanide reduction and mitochondrial succinate oxidation, Anal Biochem 373 (2008), 287295.
 D.J.R. Lane and A. Lawen, Non-transferrin iron reduction and uptake are regulated by transmembrane ascorbate cyclingin K562 cells, J Biol Chem 283 (2008), 1270112708.
 J.A. Larm, F. Vaillant, A.W. Linnane and A. Lawen, Up-regulation of the plasma membrane oxidoreductase as aprerequisite for the viability of human Namalwa 0 cells, J Biol Chem 269 (1994), 3009730100.
 A. Lawen, M.A. Baker and S. Malik, Apoptosis and redox homeostasis: on a possible mechanism of action of Bcl-2,Protoplasma 205 (1998), 1020.
 A. Lawen, R.D. Martinus, G.L. McMullen, P. Nagley, F. Vaillant, E.J. Wolvetang and A.W. Linnane, The universalityof bioenergetic disease: the role of mitochondrial mutation and the putative inter-relationship between mitochondriaand plasma membrane oxidoreductase, Molec Asp Med 15(Supplement) (1994), s13s27.
 S. Lin, D.C. Lin and M.D. Flanagan, Specificity of the effects of cytochalasin B on transport and motile processes, ProcNatl Acad Sci USA 75 (1978), 329333.
 C.L. Linster and E. Van Schaftingen, Vitamin C. Biosynthesis, recycling and degradation in mammals, FEBS J, 274(2007), 122.
 J.P. Liuzzi, F. Aydemir, H. Nam, M.D. Knutson and R.J. Cousins, Zip14 (Slc39a14) mediates non-transferrin-boundiron uptake into cells, Proc Natl Acad Sci USA 103 (2006), 1361213617.
 J.D. Ly and A. Lawen, Transplasma membrane electron transport: enzymes involved and biological function, RedoxRep 8 (2003), 321.
 E. Matteucci and O. Giampietro, Transmembrane electron transfer in diabetic nephropathy, Diabetes Care 23 (2000),994999.
 J.M. May, Is ascorbic acid an antioxidant for the plasma membrane? FASEB J 13 (1999), 9951006. J.M. May, L. Li, K. Hayslett and Z.-c. Qu, Ascorbate transport and recycling by SH-SY5Y neuroblastoma cells: response
to glutamate toxicity, Neurochem Res 31 (2006), 785794. J.M. May, Z.-c. Qu and C.E. Cobb, Human erythrocyte recycling of ascorbic acid: relative contributions from the
ascorbate free radical and dehydroascorbic acid, J Biol Chem 279 (2004), 1497514982. J.M. May, Z.-c. Qu and S. Mendiratta, Role of ascorbic acid in transferrinindependent reduction and uptake of iron by
U-937 cells, Biochem Pharmacol 57 (1999), 12751282.
D.J.R. Lane and A. Lawen / Transplasma membrane electron transport comes in two flavors 199
 J.M. May, Z.-c. Qu and D.J. Nelson, Uptake and reduction of -lipoic acid by human erythrocytes, Clin Biochem 40(2007), 11351142.
 J.M. May, Z.-c. Qu and R.R. Whitesell, Ascorbic acid recycling enhances the antioxidant reserve of human erythrocytes,Biochemistry 34 (1995), 1272112728.
 A.T. McKie, D. Barrow, G.O. Latunde-Dada, A. Rolfs, G. Sager, E. Mudaly, M. Mudaly, C. Richardson, D. Barlow,A. Bomford, T.J. Peters, K.B. Raja, S. Shirali, M.A. Hediger, F. Farzaneh and R.J. Simpson, An iron-regulated ferricreductase associated with the absorption of dietary iron, Science 291 (2001), 17551759.
 M. A. Medina, A. del Castillo-Olivares and I. Nunez de Castro, Multifunctional plasma membrane redox systems,Bioessays 19 (1997), 977984.
 E.H. Morgan, Mechanisms of iron transport into rat erythroid cells, J Cell Physiol 186 (2001), 193200. D.J. Morre, P.-J. Chueh and D.M. Morre, Capsaicin inhibits preferentially the NADH oxidase and growth of transformed
cells in culture, Proc Natl Acad Sci USA 92 (1995), 18311835. J. Muslkova and J. Kovar, Additive stimulatory effect of extracellular calcium and potassium on non-transferrin ferric
iron uptake by HeLa and K562 cells, Biochim Biophys Acta 1514 (2001), 117126. D. Njus and P.M. Kelley, The secretory-vesicle ascorbate-regenerating system: a chain of concerted H+/e-transfer
reactions, Biochim Biophys Acta 1144 (1993), 235248. E. Nozik-Grayck, C.A. Piantadosi, J. van Adelsberg, S.L. Alper and Y.-C.T. Huang, Protection of perfused lung from
oxidant injury by inhibitors of anion exchange, Am J Physiol Lung Cell Mol Physiol 273 (1997), L296L304. F.J. Nualart, C.I. Rivas, V.P. Montecinos, A.S. Godoy, V.H. Guaiquil, D.W. Golde and J.C. Vera, Recycling of vitamin
C by a bystander effect, J Biol Chem 278 (2003), 1012810133. P. Ponka, Cellular iron metabolism, Kidney Int 55(Suppl 69s) (1999), S2S11. E.W. Randell, J.G. Parkes, N.F. Olivieri and D.M. Templeton, Uptake of nontransferrin-bound iron by both reductive
and nonreductive processes is modulated by intracellular iron, J Biol Chem 269 (1994), 1604616053. S.C. Rumsey, O. Kwon, G.W. Xu, C.F. Burant, I. Simpson and M. Levine, Glucose transporter isoforms GLUT 1 and
GLUT3 transport dehydroascorbic acid, J Biol Chem 272 (1997), 1898218989. S.C. Rumsey and M. Levine, Absorption, transport and disposition of ascorbic acid in humans, J Nutr Biochem 9 (1998),
116130. A.K. Samhan-Arias, M.A. Garcia-Bereguiain, F.J. Martin-Romero and C. Gutierrez-Merino, Clustering of plasma
membrane-bound cytochrome b5 reductase within lipid raft microdomains of the neuronal plasma membrane, MolCell Neurosci 40 (2009), 1426.
 R. Sanchez-Olea, C. Pena, J. Moran and H. Pasantes-Morales, Inhibition of volume regulation and efflux of osmoregu-latory amino acids by blockers of Cl-transport in cultured astrocytes, Neurosci Lett 156 (1993), 141144.
 I. Savini, A. Rossi, C. Pierro, L. Avigliano and M.V. Catani, SVCT1 and SVCT2: key proteins for vitamin C uptake,Amino Acids 34 (2008), 347355.
 E. Schweinzer and H. Goldenberg, Ascorbate-mediated transmembrane electron transport and ascorbate uptake inleukemic cell lines are two different processes, Eur J Biochem 206 (1992), 807812.
 E. Schweinzer and H. Goldenberg, Monodehydroascorbate reductase activity in the surface membrane of leukemiccells. Characterization by a ferricyanide-driven redox cycle, Eur J Biochem 218 (1993), 10571062.
 E. Schweinzer, G. Waeg, H. Esterbauer and H. Goldenberg, No enzymatic activities are necessary for the stabilizationof ascorbic acid by K-562 cells, FEBS Lett 334 (1993), 106108.
 D. Su, J.M. May, M.J. Koury and H. Asard, Human erythrocyte membranes contain a cytochrome b561 that may beinvolved in extracellular ascorbate recycling, J Biol Chem 281 (2006), 3985239859.
 I.L. Sun, F.L. Crane, C. Grebing and H. Low, Properties of a transplasma membrane electron transport system in HeLacells, J Bioenerg Biomembr 16 (1984), 583595.
 M. Szekely, S. Manyal and F.B. Straub, Uber den Mechanismus der osmotischen Hamolyse, Acta Physiol Acad SciHung 3 (1952), 571583.
 M.M. Taqui Khan and A.E. Martell, Metal ion and metal chelate catalyzed oxidation of ascorbic acid by molecularoxygen. I. Cupric and ferric ion catalyzed oxidation, J Am Chem Soc 89 (1967), 41764185.
 M.M. Taqui Khan and A.E. Martell, Metal ion and metal chelate catalyzed oxidation of ascorbic acid by molecularoxygen. II. Cupric and ferric chelate catalyzed oxidation, J Am Chem Soc 89 (1967), 71047111.
 I. Toth, J.T. Rogers, J.A. McPhee, S.M. Elliott, S.L. Abramson and K.R. Bridges, Ascorbic acid enhances iron-inducedferritin translation in human leukemia and hepatoma cells, J Biol Chem 270 (1995), 28462852.
 D. Trinder and E. Morgan, Mechanisms of ferric citrate uptake by human hepatoma cells, Am J Physiol GastrointestLiver Physiol 275 (1998), G279G286.
 M.-B. Troadec and J. Kaplan, Some vertebrates go with the GLO, Cell 132 (2008), 921922. J.M. Upston, A. Karjalainen, F.L. Bygrave and R. Stocker, Efflux of hepatic ascorbate: a potential contributor to the
maintenance of plasma vitamin C, Biochem J 342 (1999), 4956.
200 D.J.R. Lane and A. Lawen / Transplasma membrane electron transport comes in two flavors
 K. Valis, J. Neubauerova, P. Man, P. Pompach, J. Vohradsky and J. Kovar. VDAC2 and aldolase A identified as membraneproteins of K562 cells with increased expression under iron deprivation, Mol Cell Biochem 311 (2008), 225231.
 M.M. Van Duijn, J. Van der Zee, J. VanSteveninck and P.J.A. Van den Broek, Ascorbate stimulates ferricyanide reductionin HL-60 cells through a mechanism distinct from the NADH-dependent plasma membrane reductase, J Biol Chem 273(1998) 1341513420.
 M.M. Van Duijn, J. Van der Zee and P.J.A. Van den Broek, Electron spin resonance study on the formation of ascorbatefree radical from ascorbate: the effect of dehydroascorbic acid and ferricyanide, Protoplasma 205 (1998), 122128.
 C. Voegtlin, J.M. Johnson and H.A. Dyer, Quantitative estimation of the reducting power of normal and cancer tissue,J Pharmacol Exp Ther 24 (1924), 305334.
 M. von Zastrow, T.R. Tritton and J.D. Castle, Exocrine secretion granules contain peptide amidation activity, Proc NatlAcad Sci USA 83 (1986), 32973301.
 J.X. Wilson, Regulation of vitamin C transport, Annu Rev Nutr 25 (2005), 105125. J.X. Wilson, C.E. Peters, S.M. Sitar, P. Daoust and A.W. Gelb, Glutamate stimulates ascorbate transport by astrocytes,
Brain Res 858 (2000), 6166. E.J. Wolvetang, J.A. Larm, P. Moutsoulas and A. Lawen, Apoptosis induced by inhibitors of the plasma membrane
NADH-oxidase involves Bcl-2 and calcineurin, Cell Growth Differ 7 (1996), 13151325. S. Wyman, R.J. Simpson, A.T. McKie and P.A. Sharp, Dcytb (Cybrd1) functions as both a ferric and a cupric reductase
in vitro, FEBS Lett 582 (2008), 19011906. Y. Yu, Z. Kovacevic and D.R. Richardson, Tuning cell cycle regulation with.an iron key, Cell Cycle 6 (2007), 19821994. A.-S. Zhang, F. Canonne-Hergaux, S. Gruenheid, P. Gros and P. Ponka, Use of Nramp2-transfected Chinese hamster
ovary cells and reticulocytes from mk/mk mice to study iron transport mechanisms, Exp Hematol 36 (2008), 12271235.