Identificazione molecolare e caratterizzazione funzionale del trasportatore SVCT2 mitocondriale in cellule leucemiche e nel muscolo scheletrico

Ascorbic acid (AA), the reduced form of vitamin C, is transported within many cell types through high affinity Na+ dependent transporters belonging to the SVCTs family (SVCT1 and SVCT2). Although highly homologous to each other, these isoforms have different functional characteristics, with SVCT2 exhibiting a higher affinity/lower capacity for AA transport than SVCT1. Cells expressing these transporters, at the vitamin concentrations normally present in biological fluids, accumulate high concentrations of AA in the cytosol and eventually in other sub-cellular compartments, as the mitochondria. We recently reported the expression of a functional SVCT2 in the mitochondria (mtSVCT2) of pro-monocytic U937 cells [Azzolini C. et al., IUBMB Life. 2013]. Interestingly, U937 cell mtSVCT2 surprisingly works with high affinity despite the very low intracellular concentrations of Na+ and Ca2+, since fully active at Na+ concentrations lower than 1 mM and virtually Ca2+ independent [Fiorani M. et al., Biochim Biophys Acta. 2015]. This is not the case of the plasma membrane SVCT2 (pmSVCT2) requiring at least 100 mM Na+ and about 1 mM Ca2+ for its optimal activity. We then found that the activities of both the pm and mtSVCT2 are critically regulated by the concentrations of the oxidised form of vitamin C, dehydroascorbic acid (DHA) [Fiorani M. et al., Pharmacol Res. 2014; Fiorani M. et al., Pharmacol Res. 2015]. It appears that conditions associated with the oxidation of the vitamin transiently reduce cellular and mitochondrial AA transport, and promote a parallel switch in the mechanism of uptake of the vitamin. We also investigated the impact of the differentiation process in the expression and activity of U937 cell mtSVCT2. In cells differentiated by a 4 days growth in DMSO to monocytes, we observed an early SVCT2 mRNA down-regulation and an unmodified expression of SVCT2 immunoreactivity. Under these conditions the cellular uptake of the vitamin remained unchanged, however in parallel with a progressive decrease in AA mitochondrial accumulation surprisingly associated with an increased expression of mtSVCT2. Importantly, the reduced transport activity was due to a reduced Vmax with no change in affinity of mtSVCT2. We previously reported that preloading with as a low as 3 μM AA causes a significant mitochondrial accumulation of AA in undifferentiated cells and that this response is associated with an enhanced response to otherwise inactive concentrations of peroxynitrite, in terms of mitochondrial superoxide formation, DNA strand scission and cytotoxicity. The differentiated cells were much less responsive to these enhancing effects, and required exposure to 10-20 times greater AA concentrations to elicit effects comparable with those observed in the undifferentiated cells. We were also interested in the assessment of the relevance of the mitochondrial transporter of AA in the skeletal muscle, that contains the largest proportion of total body vitamin C and also represents a critical site of ROS production. In these studies, we employed C2C12 myoblasts and C2C12 myotubes, obtained by growth of the myoblasts in serum deprived medium. Expression of SVCT2 was confirmed both at the mRNA and protein levels, and turned out to be substantially similar in the two cell types. A similar outcome was obtained in studies measuring the kinetics and rates of AA uptake. Analysis by the Eadie-Hofstee method was indicative of the presence of a single high-affinity transporter, with characteristics compatible with SVCT2. We therefore moved to the more complex part of the study associated with a similar analysis in the mitochondrial compartment, keeping well in mind the enormous problems encountered in various laboratories during the process of isolation of mitochondria in the skeletal muscle. We had some problems when the analysis was performed in myoblasts, but nevertheless obtained evidence of mtSVCT2 expression with Western blot studies. This observation was confirmed by the results obtained in confocal microscopy and uptake studies. Interestingly, in contrast with our previous results obtained in U937 cell mitochondria, we found that mtSVCT2 operative in undifferentiated C2C12 cells is characterised by a low affinity. The problems encountered for mitochondrial isolation from myotubes were remarkably more significant. Despite numerous attempts, our only conclusion on the putative expression of mtSVCT2 is uniquely based on the results obtained by confocal microscopy studies. Although still expressed in some myotubes, a general decrease in mtSVCT2 expression was found in this cell population, in particular in myotubes presenting three or more nuclei. We also performed initial, explorative experiments in the tibialis anterior skeletal muscle, freshly dissected from CD1 mice. Quite surprisingly, our mitochondrial preparations were rather pure, in particular those obtained with the Percoll isolation procedure. Unfortunately, however, our experiments failed to produce conclusive results because of the poor expression of SVCT2 in the total lysates. We also failed to detect SVCT2 in the mitochondrial fractions. Clearly these results are inconclusive and we need to approach the problem with a more sensitive technique. In conclusion, we have provided evidence for the expression and functional activity characterised by a high affinity of mtSVCT2 in promonocytic U937 cells. We then demonstrated that the differentiation of these cells to monocytes causes SVCT2 mRNA down-regulation and decreased Vmax, with no change in affinity, of mtSVCT2. We also provided evidence of mtSVCT2 expression and activity, however characterised by a low affinity in C2C12 myoblasts. Finally, the differentiation of these cells to myotubes was paralleled by decreased SVCT2 mRNA expression, transiently associated with maintenance of unchanged SVCT2 protein expression and cellular uptake. Under these conditions, results uniquely based on confocal microscopy studies are indicative of a general decline in SVCT2 expression, particular in myotubes with more than 3 nuclei.