Ascorbic acid (AA), the reduced form of vitamin C, is taken up by most cell types via a high affinity/low capacity mechanism through Na+-AA cotransporters (SVCT1 and 2) (1, 2). Under these conditions, cells accumulate millimolar concentrations of the vitamin even when exposed to low micromolar concentrations of AA, that is, comparable to those detected in most biological fluids (30–50 μM) (3, 4). There are some cells, however, which poorly express SVCTs and therefore potentially accumulate lower amounts of vitamin C. In this perspective, different levels of expression of SVCTs might reflect different cellular requirements of vitamin C and indeed SVCT expression is critically regulated both at the transcriptional and translational levels (5).
An alternative strategy for vitamin C accumulation is based on the uptake of dehydroascorbic acid (DHA) through hexose transporters, however hampered by the very low availability of extracellular DHA, normally present at concentrations below 2 μM (6). On the other hand, under conditions associated with extracellular oxidation of AA, DHA concentrations may locally arise, thereby promoting a potentially effective mechanism for vitamin C accumulation (7).
In summary, while it appears clear that most cell types take up vitamin C as AA, via high affinity/low capacity mechanisms, some cells might under specific conditions take up DHA via a low affinity/high capacity mechanism, however with the important limitation of the low extracellular concentrations of DHA.
There has been some interest (8, 9) on the possibility that AA might accumulate in the mitochondria, as its antioxidant activity would be expected to be beneficial for these organelles characterized by the extensive formation of reactive oxygen species. In this perspective, the intracellular environment further polarizes some of the differences noted for the extracellular milieu, with potentially very high concentrations of AA and extremely low levels of DHA.
Despite this important limitation, most studies have primarily focused on the mitochondrial uptake of DHA, made possible by the expression of hexose transporters in the mitochondrial membranes (9). Based on the above considerations, however, mitochondrial accumulation of DHA would be restricted to conditions associated with extensive oxidation of cellular AA.
The present study was carried out with the purpose of investigating the existence of Na+-dependent transport of AA in the mitochondria of cultured mammalian cells.
tetrabutylammonium hydrogen sulfate
Materials and Methods
AA, DHA, dithiothreitol (DTT), tetrabutylammonium hydrogen sulfate (TBA), ethylenediaminetetraacetic acid (EDTA), cytochalasin B, choline chloride, 4-hydroxymercuribenzoic acid (pCMB), sulfinpyrazone (S-pyr) as well most reagent-grade chemicals were purchased from Sigma-Aldrich (Milan, Italy). Perkin–Elmer Life and Analytical Sciences (Boston, MA) supplied L-[1-14C]AA (specific activity 5.35 mCi/mmol), which was dissolved in deionized water containing 0.1 mM acetic acid and stored in multiple aliquots at −20 °C until use (10).
Cell Culture and Treatment Conditions
U937 human myeloid leukemia cells were cultured in suspension in RPMI 1640 medium (Sigma-Aldrich, Milan, Italy) supplemented with 10% foetal bovine serum (Euroclone, Celbio Biotecnologie, Milan, Italy), penicillin (100 units/mL), and streptomycin (100 μg/mL) (Euroclone), at 37 °C in T-75 tissue culture flasks (Corning, Corning, NY) gassed with an atmosphere of 95% air-5% CO2. A 10 mM AA stock solution was prepared in incubation buffer, IB (15 mM Hepes, 135 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 0.8 mM MgCl2, pH 7.4) immediately before utilization. Where indicated, NaCl was replaced with choline-chloride. Cells (1 × 106 cells/mL) were treated with AA for 15 min at 37 °C in IB supplemented with 0.1 mM DTT. Stability of AA in IB was assessed by monitoring the absorbance at 267 nm for 15 min (ε267 = 14,600 M−1 cm−1).
Purification of Mitochondria
Mitochondria were isolated as detailed by Fiorani et al. (11). Isolated mitochondria were exposed to AA or 14C-AA in mitochondrial buffer, MB (5 mM Hepes, 210 mM mannitol, 70 mM sucrose, 1 mM Na-EGTA, pH 7.4) and processed as described below. Mitochondria obtained from cells previously exposed to AA were also lysed and processed as indicated for isolated mitochondria.
Measurement of AA Content by High Performance Liquid Chromatography (HPLC)
Cells were washed twice with cold saline solution (8.182 g/L NaCl, 0.372 g/L KCl, 0.336 g/L NaHCO3, 0.9 g/L glucose, pH 7.4) and mitochondria with cold MB. The final pellets were extracted with ice-cold 70% (vol/vol) methanol/30% HPLC solution A (10 mM TBA, 10 mM KH2PO4, 0.5% methanol, pH 6.0) containing 1 mM EDTA. After 10 min at ice bath temperature, 10 mM DTT was added to the samples and centrifuged at 10,000g for 20 min at 4 °C. Where indicated, DTT was omitted. Samples were filtered through a 0.22-μm filter (Millipore, Milan, Italy) and analyzed immediately or frozen at −80 °C for later analysis. The intracellular AA content was measured by HPLC with the UV detection wavelength set at 265 nm, as described in refs.4, 12. Cellular and mitochondrial concentrations of AA were calculated using published values for cell (13) and mitochondrial (14) volumes.
Measurement of 14C-AA Mitochondrial Uptake
Isolated mitochondria were incubated at 37 °C in MB supplemented with 30 μM L-[1-14C]-AA. Uptake was stopped by rinsing the mitochondria twice with ice-cold MB. Mitochondria were then dissolved in 1 mL of 1 M NaOH and the incorporated radioactivity was measured by liquid scintillation spectrometry.
Sub-Cellular Fractionation and Western Blot Analysis
The cells were processed to obtain the mitochondrial and membrane fractions, as described in ref.15. Equal amounts (30 μg) of the mitochondrial and membrane fractions were resolved in 15% sodium dodecyl sulfate polyacrylamide gel and electrotransferred to polyvinyldiene difluoride membranes. Western blot analyses were performed using antibodies against SVCT1 (N-20: sc-9924) and 2 (S-19: sc-9926), cytochrome c and actin (Santa Cruz, Santa Cruz, CA). Details on Western blotting apparatus and conditions are reported elsewhere (15). Antibodies against cytochrome c and actin were used to assess the purity of the fractions.
The results are expressed as means ± SD. Statistical differences were analyzed by one-way ANOVA followed by Dunnett's test for multiple comparison or two-way ANOVA followed by Bonferroni's test for multiple comparison. A value of P < 0.05 was considered significant.
Results and Discussion
The results illustrated in Fig. 1A are from experiments in which the cells were exposed for 15 min to 0–30 μM AA in IB supplemented with 0.1 mM DTT to prevent AA oxidation during treatments. There was a linear relationship between the initial AA concentration and the fraction of vitamin C associated with the cells, unaffected by addition of 10 mM DTT to the lysates prior to analysis. As indicated in the inset to Fig. 1A, AA uptake was both insensitive to cytochalasin B (25 μM), a well established inhibitor of hexose transporters (12), and abolished by Na+ omission (replaced with coline) (12).
These results indicate that vitamin C accumulation resulting from exposure to low micromolar concentrations of AA is largely dependent on AA uptake through Na+-dependent transporters, with hardly any contribution of DHA uptake, which might take place as a consequence of extracellular oxidation of AA. A second important consideration is that intracellular vitamin C is kept in its reduced form under the specific conditions employed.
Results illustrated in Fig. 1B provide evidence for mitochondrial accumulation of vitamin C, that appears to be both proportional to the extracellular concentrations of AA and insensitive to incubation of the lysates with 10 mM DTT prior to analysis. Interestingly, the putative intramitochondrial concentrations of the vitamin were significantly greater than those found in the cytosol (Fig. 1C). As an example, exposure of U937 cells to 10 μM AA resulted in the accumulation of about 150 μM AA and this concentration increased to more than 10 mM in the mitochondrial compartment.
We next performed experiments with isolated mitochondria. These organelles effectively took up AA and, in analogy with the results obtained with intact cells, mitochondrial accumulation of vitamin C was (i) unaffected by prior incubation of the lysates with 10 mM DTT (Fig. 2A); (ii) insensitive to cytochalasin B (Fig. 2B); and (iii) abolished by Na+ omission (Fig. 2B). As a final note, mitochondrial accumulation of vitamin C was also suppressed by two well established inhibitors of SVCTs, that is, pCMB (16) and S-Pyr (10) (Fig. 2B).
These results provide functional evidence for Na+-dependent transport of AA in mitochondria, similar to that found in plasma membrane (Fig. 1A and (1, 2)). The interdependence between these events is emphasized by the existence of a strong correlation between the intramitochondrial and extramitochondrial concentrations of AA, in both intact cells (Fig. 1B) and isolated mitochondria (Fig. 2A). The linear relationship clearly detected (R2 = 0.9679) despite the different conditions and over a wide range of extramitochondrial concentrations of AA is illustrated in Fig. 1D. These results imply the use of the same transporters, most likely highly expressed in the mitochondrial membranes. The elevated intramitocondrial concentrations of AA appear therefore uniquely dependent on the high cytosolic concentrations of the vitamin achieved during the exposure to low micromolar concentrations of AA.
Figure 2C provides evidence for expression of SVCT2 in both compartments, whereas SVCT1 was uniquely expressed in the plasma membrane. Lack of expression of SVCT1 in mitochondria provides an excellent internal control to rule out the possibility that the observed expression of SVCT2 is in fact attributable to the presence of plasma membrane contaminants. The mitochondrial fraction was associated with the expression of cytochrome c in the absence of actin, whereas none of these proteins was detectable in plasma membrane.
Mitochondria are the major source of reactive oxygen species, generated as byproducts of the electron transport chain and producing deleterious effects to various biological targets (17). Not surprisingly, these organelles accumulate various lipid soluble antioxidants and it is still unclear whether the same is true for vitamin C.
Previous studies (9) addressed the possibility of a mitochondrial uptake of the oxidized form of vitamin C and demonstrated that hexose transporters are indeed expressed also in the mitochondrial membranes. We now extend these findings with the demonstration of SVCT2-dependent transport of physiological concentrations of AA in U937 cell mitochondria. While cells grown in culture medium containing very low concentrations of vitamin C likely upregulate high affinity transporters, their overexpression in the mitochondrial membrane argues in favor of an important role of vitamin C in these organelles.
At present, we are unable to predict whether mitochondrial transport of AA is a general phenomenon or is rather restricted to some cell types, as U937 cells. We have indeed to remind that the general view is that DHA -and not AA- is taken up by mitochondria. While this notion is based on a surprisingly limited information (14), failure to detect mitochondrial AA uptake in these studies might relate to existing high mitochondrial AA concentrations.
In conclusion, more work is needed to establish the generality and relevance of the mechanism described in this study. At present, it can only be suggested that, as extensively discussed for plasma membrane transport (4, 5, 18), mitochondrial uptake of vitamin C may also significantly vary (AA vs. DHA) in different cell types and in different pathological conditions.