Address correspondence and reprint requests to F. Nualart, Departamento de Histología y Embriología, Facultad de Ciencias Biológicas, Casilla 160-C, Universidad de Concepción, Concepción, Chile. E-mail: email@example.com
The sodium–vitamin C co-transporters SVCT1 and SVCT2 transport the reduced form of vitamin C, ascorbic acid. High expression of the SVCT2 has been demonstrated in adult neurons and choroid plexus cells by in situ hybridization. Additionally, embryonic mesencephalic dopaminergic neurons express the SVCT2 transporter. However, there have not been molecular and kinetic analyses addressing the expression of SVCTs in cortical embryonic neurons. In this work, we confirmed the expression of a SVCT2-like transporter in different regions of the fetal mouse brain and in primary cultures of neurons by RT-PCR. Kinetic analysis of the ascorbic acid uptake demonstrated the presence of two affinity constants, 103 µm and 8 µm. A Km of 103 µm corresponds to a similar affinity constant reported for SVCT2, while the Km of 8 µm might suggest the expression of a very high affinity transporter for ascorbic acid. Our uptake analyses also suggest that neurons take up dehydroascorbic acid, the oxidized form of vitamin C, through the glucose transporters. We consider that the early expression of SVCTs transporters in neurons is important in the uptake of vitamin C, an essential molecule for the fetal brain physiology. Vitamin C that is found at high concentration in fetal brain may function in preventing oxidative free radical damage, because antioxidant radical enzymes mature only late in the developing brain.
Relatively high concentrations of AA are found in the fetal brain. Recently, Yan et al. (2001) reported the expression of SVCT2 in embryonal mesencephalic neurons and suggested an important role of the vitamin C in dopaminergic neurons differentiation. However, there have not been molecular and kinetic analyses addressing the expression of SVCT transporters in embryonic cortical neurons.
We examined vitamin C transport in primary cultures of mouse embryonic cortical neurons and demonstrated the expression of high affinity AA–sodium co-transporters. RT-PCR analysis confirmed the early expression of SVCT2-like transporters in cultured mouse neurons and in different areas of the brain tissue. Finally, we observed that neurons transport dehydroascorbic acid through glucose transporters.
Primary cultures of neurons
Cultured neurons were obtained from C57BL/J6 mice. The forebrains were removed from 16-day-old embryos. Tissues were digested for 10 min with trypsin 0.12% (wt/vol) in phosphate buffer 0.1 m (pH 7.4, 320 mOsm) and triturated to homogeneity with a fire-polished Pasteur pipet (Tapia et al. 2000). The cells were plated at 300 000 cells/mL onto glass coverslips coated with poly l-lysine (mol. Wt > 350 kDa, Sigma, St Louis, MO, USA). After 20 min, the floating cells were removed and the attached cells were incubated and fed every 3 days with minimal essential medium (MEM, Gibco Co., Rockville, MD, USA) containing 10% bovine fetal serum (Gibco), 30 mm glucose, 293 mg/mL l-glutamine (Nalgene, Rochester, NY, USA), 25 mm KCl, 0.1% bovine serum albumin (BSA), 20 µg/mL transferrin, 100 µm putrescine, 30 nm sodium selenite, 0.2 mm insulin (Sigma), 2 µg/mL triiodothyronine (Sigma), 20 mm progesterone (Sigma), 4 µg/mL corticosterone (Sigma). The cultures were incubated in 5% CO2 in a humidified environment at 37°C. After 3 days the cells were treated for 24 h with 40 µm cytoscine arabinose (Sigma) to inhibit astrocyte proliferation.
For immunocytochemistry, neurons were grown on coverslip, fixed with 4% p-formaldehyde in phosphate-buffered saline (PBS) for 30 min at 4°C, washed with PBS and incubated in PBS containing 1% BSA and 0.2% Triton X-100 for 5 min at room temperature. The cells were incubated overnight at room temperature with the following antibodies: anti-neuron-specific enolase (1 : 500, Dako, Campintene, CA, USA), anti-Tau (5 µg/mL, Boehringer Mannheim, Mannheim, Germany), anti-neurofilament (high molecular weight subunit, 1 : 500) (Nualart et al. 1991, 1999), and anti-GFAP (glial fibrillary acidic protein; 1 : 200, Dako). Cells were incubated with goat anti-rabbit IgG or rabbit anti-mouse IgG (1 : 100, Dako) for 2 h and them for 30 min with peroxidase anti-peroxidase complex (PAP, 1 : 100, Dako). The enzymatic activity was developed using diaminobenzidine and hydrogen peroxide (Nualart et al. 1999; García et al. 2001).
Reverse transcription-polymerase chain reaction
The following samples were used for RT-PCR analysis: (i) hypothalamic area, brain cortical tissue, hippocampal region and cerebellum from 16-day-old embryos of C57BL/J6 mice; (ii) 7-day cultured neurons isolated from 16-day-old embryos of C57BL/J6 mice; (iii) fetal and adult mRNA isolated from human brain (Clontech, Palo Alto, CA, USA). The poly (A) RNA of the neuron and brain tissues was isolated using the Oligotex direct kit (Qiagen, Valencia, CA, USA). For RT-PCR, 0.5–1 µg of RNA was incubated in 20 µL reaction volume containing 10 mm Tris pH 8.3, 50 mm KCl, 5 mm MgCl2, RNase inhibitor 20 U, 1 mm dNTPs, 2.5 µm of random hexanucleotides, and 50 units of MuLV reverse transcriptase (Perkin Elmer, Branchburg, NJ, USA) for 10 min at 23°C followed by 30 min at 42°C and 5 min at 94°C. Parallel reactions were performed in the absence of reverse transcriptase to control for the presence of contaminant DNA. For amplification, a cDNA aliquot in a volume of 12 µL containing 20 mm Tris, pH 8.4, 50 mm KCl, 1.6 mm MgCl2, 0.4 mm dNTPs, 0. 04 units of Taq DNA polymerase (Gibco-BRL, Rockville, MD, USA), and 0.4 µm primers was incubated at 94°C for 4 min, 94°C for 50 s, 55°C for 50 s, and 72°C for 135 s for 35 cycles. PCR products were separated by 1.2% agarose gel electrophoresis and visualized by staining with ethidium bromide. The following primers (based on human sequence F164142) were used to analyze the expression of SVCT2-like transporters: (i) set one, forward primer 5′-TTCTGTGTGGGAATCACTAC-3′ and reverse primer 5′-ACCAGAGAGGCCAATTAGGG-3′ (expected product 339 bp); (ii) set two, forward primer 5′-AGTATGGCTTCTATGCTCGC-3′ and reverse primer 5′-TCCCAGCACAGGATCCGGAA-3′ (expected product 440 bp). The following primers (based on human sequence AF170911) were used to analyze the expression of SVCT1-like transporters: forward primer 5′-GCCCCTGAA CACCTCTCATA-3′ and reverse primer 5′-ATGGCCAGCATG ATAGGAAA-3′ (expected product 360 bp).
Vitamin C uptake analysis
After 7 days in culture the neurons were used for kinetic analysis. Cultures were carefully selected under the microscope to ensure that only plates showing uniformly growing cells were used. In each experiment, cells from 12 wells incubated with buffer were removed and used to estimate the average total cell number present in each well. We did not observe a great variation in cell number between the wells after buffer incubation (Spielholz et al. 1997). The cells were washed with incubation buffer (15 mm HEPES, 135 mm NaCl, 5 mm KCl, 1.8 mm CaCl2, 0.8 mm MgCl2) and incubated in the same medium for 30 min at room temperature. Uptake assays were performed in 400 µL of incubation buffer containing 0.1–0.4 µCi of 1–14C-l-ascorbic acid (specific activity 8.2 mCi/mmol), to a final concentration of 5–300 µm. Different preparations of ascorbic acid were oxidized to dehydroascorbic acid by the addition of 0.02 unit of ascorbate oxidase per ml of 1 µm ascorbic acid and used to a final concentration of 100 µm. The uptake was stopped by washing the cells with ice-cold PBS. Cells were dissolved in 0.5 mL of lysis buffer (10 mm Tris–HCl, pH 8.0, 0.2% SDS), and the incorporated radioactivity was assayed by liquid scintillation spectrometry. The Michaelis constant, Km, was calculated using the Eadie–Hofstee analysis. Data represent means ± SD of three experiments with each determination done in duplicate. In inhibition experiments statistical comparison between two or more groups of data was carried out using analysis of variance (anova, followed by Bonferroni post-test). p < 0.05 was considered to be statistically significant.
The expression of AA transporters in embryonic brain tissue was analyzed by RT-PCR with primers specific for each sodium–AA co-transporter isoform (SVCT1 or SVCT2). The conditions were optimized using RNA from human brain tissue as a control for the expression of SVCT2 and RNA from human hepatocytes for SVCT1. As judged by their migration, the amplified DNA bands were approximately 339 or 440 bp, the expected sizes for the amplification products of SVCT2 using the set primers 1 or 2, respectively (data not shown and Fig. 1a, lane 6), and 360 bp, the expected size for the amplification product of SVCT1 (Fig. 1b, lane 6). No amplification product was observed in samples in which the cDNA synthesis step was performed in the absence of reverse transcriptase, indicating the absence of DNA contamination in the RNA preparation (Figs 1a and b, lane 7).
Using mRNA isolated from 15-day-old embryonic mouse, we amplified the SVCT2 isoform from the brain cortex (Fig. 1a, lane 2). We detected SVCT2 expression in the cerebellum, hippocampus and hypothalamus (Fig. 1a, lanes 3, 4, and 5, respectively). SVCT1 was not detected in these tissues (Fig. 1b, lanes 2, 3, 4, and 5, respectively). The 360 bp band was only detected in the human hepatocytes positive control sample (Fig. 1b, lane 6). The expression and concentration equivalent of the β-actin gene in reactions indicates the high quality of the RNAs used to perform the analysis (Fig. 1c). Our results indicate the expression of SVCT2-like transporters in embryonic mouse and adult human brain tissue.
In order to demonstrate the specific expression of SVCT2 in neurons, we isolated neurons from embryonic mouse brain cortex. The cells were cultured for 7 days and the purity of the culture was analyzed by phase-contrast microscopy and immunocytochemistry. Most of the cells in the culture were aggregated forming clusters of different sizes, a typical characteristic of cultures rich in neurons (Figs 2a and b; arrows). Anti-neural-specific enolase (NSE) and anti-TAU were positives in neuronal somas and processes (Figs 2a–d). Anti-TAU showed a clear staining in neuronal processes (Fig. 2d). The cells were also positives with anti-neurofilament (data not shown). Only a few cells were positive with anti-GFAP, an astrocytes marker (Fig. 2e,f). Using the primary culture of neurons we analyzed the expression of SVCT isoforms by RT-PCR using specific primer to SVCT2 and SVCT1. Bands either 339 or 440 bp were amplified using the different sets of primers to analyze the expression of SVCT2 (data not shown and Fig. 3, lane 1). The human brain mRNA, the positive control, showed a band of similar size (Fig. 3, lane 2). No amplification products were detected with specific primers for SVCT1 (data not shown). Similarly, no amplification product was observed in samples in which the cDNA synthesis step was performed in the absence of reverse transcriptase (Fig. 3, lane 4). To confirm the expression of SVCT2 in neurons, we sequenced the 440 pb band obtained from mouse brain and human brain mRNA, showing a highly homologous sequence (data not shown).
Since there were no previous studies addressing the kinetic properties of SVCT2 transporter in neuronal cultures, we examined AA transporter present in neurons. The transport of ascorbic acid was linear for the first 10 min and reached a plateau in about 20 min (Fig. 4a and data not shown). The initial velocity of the AA uptake was 130 pmol × 106 cells/min. The total uptake in 5 min was 520 pmol × 106 cells (Fig. 4a). To test the sodium dependence of AA transport we replaced the NaCl in the incubation buffer with choline chloride. Transport of AA by neurons was decreased by at least 75% in the absence of sodium, showing a initial velocity of 50 pmol × 106 cells/min and the uptake reached a plateau of 150 pmol × 106 cells in 4 min (Fig. 4a). To further characterize the transport of AA in neurons we studied the effect of the temperature (Fig. 4b). The transport was almost basal at 4°C. However, when the cells were incubated at 25°C, the velocity of the AA transport was 50 pmol × 106 cells/min. The AA transport was almost three times higher when the uptake was done at 37°C (125 pmol × 106 cells/min). These results strongly suggested the participation of sodium co-transporter in the transport of AA by neurons. Next, we performed a preliminary uptake analysis to determine if neurons also transport dehydroascorbic acid (DHA) in vitro. It has been previously demonstrated that the facilitative glucose transporters (GLUTs) are efficient transporters of DHA (Vera et al. 1993; Rumsey et al. 1997), and that astrocytes take up DHA by using the GLUT1 isoform (Siushansian et al. 1997). Therefore, it was expected that neurons would transport DHA, due to the fact that they express GLUT3 and GLUT1 (Mueckler 1994). Our uptake analysis showed that the transport of DHA by neurons was linear for 5 min (Fig. 4c) and reached a plateau at 10 min (data not shown). The initial velocity was 180 pmol × 106 cells/min, and after 5 min the uptake reached 900 pmol × 106 cells. It is known that the transport of DHA by glucose transporters is not sodium-dependent. To test the sodium independence in the DHA transport we replaced the NaCl in the incubation buffer with choline chloride. Transport of DHA by neurons was not affected, showing a total uptake of 800 pmol × 106 cells in 5 min (Fig. 4c). These data indicate that the DHA uptake by neurons might be an alternative form to take up vitamin C.
Different inhibitors have been used to characterize the vitamin C transport through sodium–AA co-transporters or hexose transporters (Bloch 1973; Spielholz et al. 1997). Because the sodium co-transport process requires that sodium transported into the cells eventually be transported back out of the cell, we tested the dependence of AA transport on the activity of the sodium–potassium ATPase using ouabain, a specific inhibitor of the enzyme. When neurons were incubated in the presence of ouabain, the AA transport was decreased by 50% (Fig. 5). Additionally, we did not observe any effect of AA transport when either cytochalasin B (a typical glucose transporter inhibitor) or cytochalisin E (a control for cytochalasin B effect) was used (Fig. 5). Thus, the transport of AA in neurons is only mediated by the SVCT isoforms. On the other hand, we detected an 85% inhibition of DHA transport when neurons were incubated with cytochalasin B (Fig. 5) but not cytochalasin E (Fig. 5). These data confirm that the DHA transport occurs by glucose transporters.
We then performed a detailed kinetic analysis of the AA transport by neurons. This study was difficult because of the low levels of AA uptake at short incubation times. Dose–response studies using 5-min assays revealed that the transport of AA by neurons is saturated at concentrations higher than 150 µm(Fig. 6a). A different analysis of the data suggested two kinetic components of ascorbate transport. The Eadie–Hofstee transformation showed an apparent Km of 8 and 103 µm and a Vmax of 39 and 125 pmol × 106/min, respectively (Fig. 6b).
Our data suggest two mechanisms of vitamin C transport in mouse embryonic neurons. One mechanism involves the efficient transport of the oxidized form of vitamin C, DHA, through glucose transporters by an energy-independent facilitative process, and the other involves the sodium-dependent co-transport of AA, the reduced form of vitamin C.
DHA is not detectable in normal brain but appears after ischemia, because vitamin C contributes to the scavenging of reactive oxygen species and it is oxidized to DHA. However, high concentrations of DHA might induce brain toxicity. In response to this, it is believed that brain cells may take up the oxidized form of vitamin C and reduce the molecule to AA in order to minimize its deleterious effects (Patterson and Mastin 1951; Rose et al. 1992). For example, it was demonstrated that astrocytes take up DHA through the glucose transporter GLUT1 (Siushansian et al. 1997), and that the oxidized vitamin C is reduced inside the cell and stored as AA, at levels that might reach millimolar concentration (Rice 2000). Furthermore, astrocytes can release AA to the extracellular fluid under swelling conditions (Wilson 1997; Korkok et al. 2000). This mechanism may represent an important process involved in the recycling of vitamin C within the brain.
Neurons express two different glucose transporters (GLUT1 and GLUT3) in fetal and adult brain (Mueckler 1994; Aller et al. 1997; Nualart et al. 1999), which take up DHA (Rumsey et al. 1997). Because the transport of DHA through the glucose transporters can occur in either direction (Rose 1988; Vera et al. 1995; Rumsey et al. 1997), the cell must trap the vitamin C molecule in its reduced form, ascorbate. The eventual mechanism by which neurons might reduce DHA to AA is not known. A potential mechanism involving glutathione and other different reducing enzymes has been proposed for the interconvertion of oxidized and reduced form of vitamin C (Winkler 1992; Del Bello et al. 1994). Astrocytes have four times more glutathione than the neurons, and potentially a higher capacity to reduce the vitamin C (Rice 2000). Thus, the ability to transport DHA by different cells may be regulated by the expression level of glucose transporters and by the differential capacity to reduce the oxidized vitamin C. For instance, by performing a comparative analysis in human melanocytes and melanoma cells we demonstrated that even though melanocytes express glucose transporters, these transporters are not primarily involved in the uptake of vitamin C. However, melanoma cells showed a preferential uptake of DHA (Spielholz et al. 1997), because the tumoral cells express glucose transporters at a higher level, and may have a greater reducing capability.
The second transport mechanism of vitamin C by neurons involves the sodium-dependent co-transport of reduced ascorbate. In addition to its dependency of sodium, the uptake of ascorbate differs from the DHA uptake because uptake of ascorbate is inhibited by ouabain (whereas the uptake of DHA is not affected) and is not inhibited by cytochalasin B. The transport inhibition by ouabain indicates a role for a functional sodium–potassium ATPase in the uptake of ascorbate through the sodium–ascorbate co-transporter.
Transport of ascorbate through a sodium-dependent co-transporter has been described kinetically in osteoclasts (Wilson and Dixon 1989), adrenomedullary chromaffin cells (Diliberto et al. 1983), astrocytes (Wilson 1989; Korkok et al. 2000), ependymal cells (Nualart et al. 2000) and human fibroblasts (Welch et al. 1993; Rumsey et al. 1999). In all cells, only one kinetic constant associated to the AA transport has been demonstrated. We found in melanocytes a constant of 47 µm for the AA transport that correlates with the expression of SVCT2-like transporter; however, in melanoma cells a Km of 13 µm was determined by the uptake of AA (Spielholz et al. 1997), suggesting the expression of a sodium–AA co-transporter of very high affinity (SVCTh) (Fig. 6). Welch et al. (1993) have shown in fibroblasts a very high affinity constant of 6 µm for AA transport. Even though, a SVCTh-like transporter has not been molecularly characterized, the expression of SVCTh transporters in melanoma cells, fibroblasts and neurons might suggest the synthesis of a new sodium–vitamin C co-transporter in some specialized cells.
From our data we are able to extrapolate that neurons potentially use two sodium–AA co-transporters in vivo. A central question raised by these observations is why neurons develop the ability to express two transporters with different affinities to take up AA. The normal extracellular concentration of AA in brain is 200–400 µm (Rice 2000), indicating that SVCT2 transporter (apparent Km of 103 µm) should be responsible for the transport and accumulation of near millimolar concentration of vitamin C. Therefore, the expression of transporters with very high affinity to transport AA might suggest a specific mechanism present in neurons, and maybe in other cell types, to take up vitamin C under pathological conditions, so extracellular vitamin C may be maintained at a very low concentration (Chauhan et al. 1987; Kallner 1987).
Recently, it has been found by RT-PCR that embryonic mesencephalic neurons express the SVCT2 transporter. Additionally, functional data suggested a novel role for AA on dopamine neuron differentiation independent of its antioxidative properties (Yan et al. 2001). Our RT-PCR results have demonstrated the expression of the SVCT2 transporter in brain cortical area, hippocampus, hypothalamus and cerebellum. The kinetic analysis and the inhibition data consistently indicate the expression of SVCT2 transporter in primary cultures of cortical neurons. However, we cannot exclude the existence of another yet to be identified SVCT isoform for the high affinity Km.
The authors thank Ms Maria de la Luz Pascal and Juan Carlos Tapia for the expert technical support. The encouragement of Dr David Golde from Memorial Sloan-Kettering Cancer Center is kindly appreciated. Supported by Grand FONDECYT 1010843 and DIUC-GIA 201.034.006–1.4, Universidad de Concepción, Chile.