SVCT2 transporter expression is post‐natally induced in cortical neurons and its function is regulated by its short isoform

Different studies have demonstrated the importance of micronutrients, such as vitamins, for normal adult brain function and development. Vitamin C is not synthesized in the brain, but high levels are detected in this organ because of the existence of specific uptake mechanisms, which concentrate ascorbic acid from the bloodstream to the cerebrospinal fluid and then into neurons and glial cells. Two different isoforms of sodium–vitamin C cotransporters (SVCT1 and SVCT2) have been cloned. SVCT2 expression has been observed in the adult hippocampus and cortical neurons by in situ hybridization. In addition, the localization of SVCT2 in the rat fetal brain has been studied by immunohistochemistry and in situ hybridization, demonstrating that SVCT2 is highly expressed in the ventricular and subventricular areas of the brain cortex. However, there are currently no immunohistochemical data regarding SVCT2 expression and function in the post‐natal brain. Therefore, we analyzed SVCT2 expression in the developing brain cortex of mice, and demonstrated an increase in SVCT2 mRNA in mice at 1–15 days of age. The expression of a short isoform, SVCT2sh, was also detected within the same period. SVCT2 expression was concentrated in neurons within the inner layer of the brain cortex. Both SVCT2 isoforms were coexpressed in N2a cells to obtain functional data. Fluorescence resonance energy transfer analysis revealed a molecular interaction between SVCT2wt and SVCT2sh. Finally, differences in transport ratios suggested that SVCT2sh expression inhibited ascorbic acid uptake in N2a cells when both isoforms were coexpressed.

     

High-affinity sodium-vitamin C co-transporters (SVCT) expression in embryonic mouse neurons

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 microM and 8 microM. A K(m) of 103 microM corresponds to a similar affinity constant reported for SVCT2, while the K(m) of 8 microM 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.     

Sodium-dependent vitamin C transporter isoforms in skin: Distribution, kinetics, and effect of UVB-induced oxidative stress

Two sodium-dependent vitamin C transporter isoforms (SVCT1 and SVCT2) were identified as ascorbic acid transporters, but their roles in skin have, as yet, not been elucidated. Here we analyze the expression and function of SVCTs in healthy human skin cells and skin tissues, and in UVB-induced cutaneous tissue injury. SVCT1 was primarily found in the epidermis expressed by keratinocytes, whereas SVCT2 expression was in the epidermis and dermis in keratinocytes, fibroblasts, and endothelial cells. Uptake experiments revealed that ascorbic acid affinity of SVCT1 was lower than SVCT2 (K(m)=75 muM and K(m)=44 muM, respectively), but maximal velocity was 9-times higher (36 nmol/min/well). In keratinocytes, SVCT1 was found to be responsible for vitamin C transport, although SVCT2 gene expression was higher. On UVB irradiation, SVCT1 mRNA expression in murine skin declined significantly in a time- and dose-dependent manner, whereas SVCT2 mRNA levels were unchanged. Furthermore, UVB irradiation of keratinocytes in vitro was accompanied by reduced ascorbic acid transport. In summary, these data indicate that the two vitamin C transporter isoforms fulfill specific functions in skin: SVCT1 is responsible for epidermal ascorbic acid supply, whereas SVCT2 mainly facilitates ascorbic acid transport in the dermal compartment. UVB-induced oxidative stress in mice resulted in depletion of SVCT1 mRNA levels and led to significantly decreased ascorbic acid uptake in keratinocytes, providing evidence on why ascorbic acid levels are decreased on UVB irradiation in vivo.     

The Na+-dependent l-ascorbic acid transporter SVCT2 expressed in brainstem cells, neurons, and neuroblastoma cells is inhibited by flavonoids

Ascorbic acid (AA) is best known for its role as an essential nutrient in humans and other species. As the brain does not synthesize AA, high levels are achieved in this organ by specific uptake mechanisms, which concentrate AA from the bloodstream to the CSF and from the CSF to the intracellular compartment. Two different isoforms of sodium–vitamin C co-transporters (SVCT1 and SVCT2) have been cloned. Both SVCT proteins mediate high affinity Na⁺-dependent l -AA transport and are necessary for the uptake of vitamin C in many tissues. In the adult brain the expression of SVCT2 was observed in the hippocampus and cortical neurons by in situ hybridization; however, there is no data regarding the expression and distribution of this transporter in the fetal brain. The expression of SVCT2 in embryonal mesencephalic neurons has been shown by RT-PCR suggesting an important role for vitamin C in dopaminergic neuronal differentiation. We analyze SVCT2 expression in human and rat developing brain by RT-PCR. Additionally, we study the normal localization of SVCT2 in rat fetal brain by immunohistochemistry and in situ hybridization demonstrating that SVCT2 is highly expressed in the ventricular and subventricular area of the rat brain. SVCT2 expression and function was also confirmed in neurons isolated from brain cortex and cerebellum. The kinetic parameters associated with the transport of AA in cultured neurons and neuroblastoma cell lines were also studied. We demonstrate two different affinity transport components for AA in these cells. Finally, we show the ability of different flavonoids to inhibit AA uptake in normal or immortalized neurons. Our data demonstrates that brain cortex and cerebellar stem cells, neurons and neuroblastoma cells express SVCT2. Dose-dependent inhibition analysis showed that quercetin inhibited AA transport in cortical neurons and Neuro2a cells.     

Sodium-dependent vitamin C transporter SVCT2: Expression and function in bone marrow stromal cells and in osteogenesis

Ascorbic acid (Vitamin C) has a critical role in bone formation and osteoblast differentiation, but very little is known about the molecular mechanisms of ascorbic acid entry into bone marrow stromal cells (BMSCs). To address this gap in knowledge, we investigated the identity of the transport system that is responsible for the uptake of ascorbic acid into bone marrow stromal cells (BMSCs). First, we examined the expression of the two known isoforms of the sodium-coupled ascorbic acid transporter, namely SVCT1 and SVCT2, in BMSCs (Lin ? ve Sca1 + ve) and bone at the mRNA level. Only SVCT2 mRNA was detected in BMSCs and bone. Uptake of ascorbic acid in BMSCs was Na⁺-dependent and saturable. In order to define the role of SVCT2 in BMSC differentiation into osteoblasts, BMSCs were stimulated with osteogenic media for different time intervals, and the activity of SVCT2 was monitored by ascorbic acid uptake. SVCT2 expression was up-regulated during the osteogenic differentiation of BMSCs; the expression was maximal at the earliest phase of differentiation. Subsequently, osteogenesis was inhibited in BMSCs upon knock-down of SVCT2 by lentivirus shRNA. We also found that the expression of the SVCT2 could be negatively or positively modulated by the presence of oxidant (Sin-1) or antioxidant (Ascorbic acid) compounds, respectively, in BMSCs. Furthermore, we found that this transporter is also regulated with age in mouse bone. These data show that SVCT2 plays a vital role in the osteogenic differentiation of BMSCs and that its expression is altered under conditions associated with redox reaction. Our findings could be relevant to bone tissue engineering and bone related diseases such as osteoporosis in which oxidative stress and aging plays important role.     

Post-ischemic transcriptional and translational responses of EC-SOD in mouse brain and serum

Extracellular superoxide dismutase (EC-SOD) is neuroprotective, but its role in cerebral ischemia remains to be determined. We herein describe the topographical localization and quantitative changes in EC-SOD and its mRNA expression following cerebral ischemia in mice. Mice were subjected to transient forebrain ischemia and varied intervals of reperfusion. The measurements of EC-SOD using ELISA showed increased brain EC-SOD after 24 h of reperfusion and an increase in EC-SOD brain/serum ratio after 3 h. The immunohistochemical examination in normal mice showed strong EC-SOD immunoreactivity in the choroid plexus, pia mater, and ventral tuberal area of the hypothalamus. EC-SOD immunoreactivity in cortical and striatal capillary wall was conspicuous after 3 h. EC-SOD immunoreactivity was also noted in cortical neurons after 24 h. Northern blot analysis showed an increased EC-SOD mRNA expression in the brain after 24 h. An in situ hybridization study in normal mice demonstrated the mRNA expression of EC-SOD in choroid plexus and neurons through the brain especially in the cortex or ventral tuberal area of the hypothalamus, but demonstrated no mRNA expression of EC-SOD in the capillary wall. These findings suggest that EC-SOD accumulates on endothelial cells in response to this injury by an unknown mechanism, while cortical neurons produce EC-SOD themselves after cerebral ischemia with reperfusion.     

Stimulation of differentiation in sodium-dependent vitamin C transporter 2 overexpressing MC3T3-E1 osteoblasts

Sodium-dependent vitamin C transporter (SVCT) 2 facilitates reduced ascorbic acid (AA) transport in MC3T3-E1 osteoblasts. Our previous studies suggested that Zn-induced osteoblast differentiation and Ca2+-, PO4(3-)-stimulated osteopontin (OPN) expression might result from their up-regulation effect on SVCT2 expression and AA uptake. Here, we investigated the role of SVCT2 on osteoblast differentiation by using SVCT2-overexpressing cells. Two clones of SVCT2-introduced cells overexpressed SVCT2 mRNA by 2.8- and 3.1-fold those of control cells, which resulted in obvious increase of AA uptake by 2.1- and 2.4-fold in Vmax with no change in Km. Alkaline phosphatase activity, hydroxyproline content significantly increased in SVCT2-overexpressing cells, and the induction of OPN mRNA was through up-regulation of OPN promoter activity by SVCT2 overexpression. Moreover, SVCT2-overexpressing cells exhibited more ability to promote mineralization and increase calcium deposition under the stimulation of 5 mM beta-glycerophosphate. These findings indicate that SVCT2 stimulates osteoblast differentiation and mineralization.     

Effect of middle cerebral artery occlusion on mRNA expression for the sodium-coupled vitamin C transporter SVCT2 in rat brain

The sodium-vitamin C co-transporter SVCT2 is primarily responsible for the accumulation of the important antioxidant ascorbate into brain cells. In vitro studies have demonstrated strong expression of this transporter in cultured astrocytes, whereas in situ hybridization analysis has so far detected SVCT2 only in neurons. In the present study, we examined the response of SVCT2 mRNA expression in the brain to focal ischemia induced for 2 h by unilateral middle cerebral artery occlusion. The mRNA expression patterns of SVCT2 and the glutamate-activated immediate early gene Arc were investigated at 2 and 22 h after ischemia. SVCT2 and Arc mRNA expression was lost in the ischemic core at both time points. In areas outside the core, Arc was strongly up-regulated, primarily at 2 h, whereas SVCT2 showed an increase at 2 and 22 h. SVCT2 expression was increased in neurons as well as in astrocytes, providing the first evidence for SVCT2 expression in astrocytes in situ. These findings underscore the importance of ascorbate as a neuroprotective agent and may have implications for therapeutic strategies. In addition, the increase of SVCT2 in astrocytes after ischemia suggests that cultured astrocytes are exposed to chronic oxidative stress.     

Increased expression of SVCT2 in a new mouse model raises ascorbic acid in tissues and protects against paraquat-induced oxidative damage in lung

A new transgenic mouse model for global increases in the Sodium Dependent Vitamin C transporter 2 (SVCT2) has been generated. The SVCT2-Tg mouse shows increased SVCT2 mRNA levels in all organs tested and correspondingly increased ascorbic acid (ASC) levels in all organs except liver. The extent of the increase in transporter mRNA expression differed among mice and among organs. The increased ASC levels did not have any adverse effects on behavior in the SVCT2-Tg mice, which did not differ from wild-type mice on tests of locomotor activity, anxiety, sensorimotor or cognitive ability. High levels of SVCT2 and ASC were found in the kidneys of SVCT2-Tg mice and urinary albumin excretion was lower in these mice than in wild-types. No gross pathological changes were noted in kidneys from SVCT2-Tg mice. SVCT2 immunoreactivity was detected in both SVCT2 and wild-type mice, and a stronger signal was seen in tubules than in glomeruli. Six treatments with Paraquat (3x10 and 3x15 mg/kg i.p.) were used to induce oxidative stress in mice. SVCT2-Tg mice showed a clear attenuation of Paraquat-induced oxidative stress in lung, as measured by F(2)-isoprostanes. Paraquat also decreased SVCT2 mRNA signal in liver, lung and kidney in SVCT2-Tg mice.     

Essential role of intracellular glutathione in controlling ascorbic acid transporter expression and function in rat hepatocytes and hepatoma cells

Although there is in vivo evidence suggesting a role for glutathione in the metabolism and tissue distribution of vitamin C, no connection with the vitamin C transport systems has been reported. We show here that disruption of glutathione metabolism with buthionine-(S,R)-sulfoximine (BSO) produced a sustained blockade of ascorbic acid transport in rat hepatocytes and rat hepatoma cells. Rat hepatocytes expressed the Na(+)-coupled ascorbic acid transporter-1 (SVCT1), while hepatoma cells expressed the transporters SVCT1 and SVCT2. BSO-treated rat hepatoma cells showed a two order of magnitude decrease in SVCT1 and SVCT2 mRNA levels, undetectable SVCT1 and SVCT2 protein expression, and lacked the capacity to transport ascorbic acid, effects that were fully reversible on glutathione repletion. Interestingly, although SVCT1 mRNA levels remained unchanged in rat hepatocytes made glutathione deficient by in vivo BSO treatment, SVCT1 protein was absent from the plasma membrane and the cells lacked the capacity to transport ascorbic acid. The specificity of the BSO treatment was indicated by the finding that transport of oxidized vitamin C (dehydroascorbic acid) and glucose transporter expression were unaffected by BSO treatment. Moreover, glutathione depletion failed to affect ascorbic acid transport, and SVCT1 and SVCT2 expression in human hepatoma cells. Therefore, our data indicate an essential role for glutathione in controlling vitamin C metabolism in rat hepatocytes and rat hepatoma cells, two cell types capable of synthesizing ascorbic acid, by regulating the expression and subcellular localization of the transporters involved in the acquisition of ascorbic acid from extracellular sources, an effect not observed in human cells incapable of synthesizing ascorbic acid.     

Poster Sessions CP10: Blood–Brain Barrier. Expression of vitamin C transporter SVCT2 in hypothalamic glial cells. An immunohistochemical and kinetic analysis

Kinetic analysis of vitamin C uptake has demonstrated that specialized cells take up ascorbic acid (AA), the reduced form of vitamin C, through sodium‐AA cotransporters. Recently, two different isoforms of sodium‐vitamin C cotransporters (SVCT 1, 2) that mediate high affinity Na⁺‐dependent l ‐ascorbic acid have been cloned. SVCT2 was detected mainly in choroid plexus cells and neurons, however, there are no evidences of SVCT2 expression in glial cells. High concentrations of vitamin C has been demonstrated in brain hypothalamic area. The hypothalamic glial cells, known as alpha and beta tanycytes, are specialized ependymal cells that bridge the cerebrospinal fluid and the portal blood of the median eminence. Our hypothesis postulates that tanycytes take up reduced vitamin C from the portal blood and cerebrospinal fluid generating an high concentration of this vitamin in brain hypothalamic area. In situ immunohistochemical analyses demonstrated that SVCT2 transporter is selectively expressed in apical region of tanycytes. A newly developed primary culture of mouse hypothalamic tanycytes was used to confirm the expression and function of SVCT2 isoform in these cells. Reduced vitamin C uptake was temperature and sodium dependent. Kinetic analysis showed an apparent Km of 20 μm and a Vmax of 45 pmol/min per million cells for the transport of ascorbic acid. The expression of SVCT2 was confirmed by immunoblots and RT–PCR. Tanycytes may perform a neuroprotective role concentrating the vitamin C in the hypothalamic area. Acknowledgements: Supported by Grands FONDECYT 1010843 and DIUC‐GIA 201.034.006‐1.4 from Concepción University.     

Hypothalamic ependymal cells express the glucose transporter GLUT2, a protein involved in glucose sensing

Kinetic analysis of vitamin C uptake has demonstrated that specialized cells take up ascorbic acid (AA), the reduced form of vitamin C, through sodium-AA cotransporters. Recently, two different isoforms of sodium-vitamin C cotransporters (SVCT 1, 2) that mediate high affinity Na⁺-dependent l -ascorbic acid have been cloned. SVCT2 was detected mainly in choroid plexus cells and neurons, however, there are no evidences of SVCT2 expression in glial cells. High concentrations of vitamin C has been demonstrated in brain hypothalamic area. The hypothalamic glial cells, known as alpha and beta tanycytes, are specialized ependymal cells that bridge the cerebrospinal fluid and the portal blood of the median eminence. Our hypothesis postulates that tanycytes take up reduced vitamin C from the portal blood and cerebrospinal fluid generating an high concentration of this vitamin in brain hypothalamic area. In situ immunohistochemical analyses demonstrated that SVCT2 transporter is selectively expressed in apical region of tanycytes. A newly developed primary culture of mouse hypothalamic tanycytes was used to confirm the expression and function of SVCT2 isoform in these cells. Reduced vitamin C uptake was temperature and sodium dependent. Kinetic analysis showed an apparent Km of 20 μ m and a Vmax of 45 pmol/min per million cells for the transport of ascorbic acid. The expression of SVCT2 was confirmed by immunoblots and RT–PCR. Tanycytes may perform a neuroprotective role concentrating the vitamin C in the hypothalamic area.
Acknowledgements:   Supported by Grands FONDECYT 1010843 and DIUC-GIA 201.034.006-1.4 from Concepción University.     

Mitochondrial ascorbic acid transport is mediated by a low-affinity form of the sodium-coupled ascorbic acid transporter-2

Despite the fundamental importance of the redox metabolism of mitochondria under normal and pathological conditions, our knowledge regarding the transport of vitamin C across mitochondrial membranes remains far from complete. We report here that human HEK-293 cells express a mitochondrial low-affinity ascorbic acid transporter that molecularly corresponds to SVCT2, a member of the sodium-coupled ascorbic acid transporter family 2. The transporter SVCT1 is absent from HEK-293 cells. Confocal colocalization experiments with anti-SVCT2 and anti-organelle protein markers revealed that most of the SVCT2 immunoreactivity was associated with mitochondria, with minor colocalization at the endoplasmic reticulum and very low immunoreactivity at the plasma membrane. Immunoblotting of proteins extracted from highly purified mitochondrial fractions confirmed that SVCT2 protein was associated with mitochondria, and transport analysis revealed a sigmoidal ascorbic acid concentration curve with an apparent ascorbic acid transport K_m of 0.6 mM. Use of SVCT2 siRNA for silencing SVCT2 expression produced a major decrease in mitochondrial SVCT2 immunoreactivity, and immunoblotting revealed decreased SVCT2 protein expression by approximately 75%. Most importantly, the decreased protein expression was accompanied by a concomitant decrease in the mitochondrial ascorbic acid transport rate. Further studies using HEK-293 cells overexpressing SVCT2 at the plasma membrane revealed that the altered kinetic properties of mitochondrial SVCT2 are due to the ionic intracellular microenvironment (low in sodium and high in potassium), with potassium acting as a concentration-dependent inhibitor of SVCT2. We discarded the participation of two glucose transporters previously described as mitochondrial dehydroascorbic acid transporters; GLUT1 is absent from mitochondria and GLUT10 is not expressed in HEK-293 cells. Overall, our data indicate that intracellular SVCT2 is localized in mitochondria, is sensitive to an intracellular microenvironment low in sodium and high in potassium, and functions as a low-affinity ascorbic acid transporter. We propose that the mitochondrial localization of SVCT2 is a property shared across cells, tissues, and species.     

Ascorbic acid depletion enhances expression of the sodium-dependent vitamin C transporters, SVCT1 and SVCT2, and uptake of ascorbic acid in livers of SMP30/GNL knockout mice

In this study, we examined whether ascorbic acid (AA) and dehydroascorbic acid (DHA), the oxidized form of AA, levels in tissues regulate the AA transporters, sodium-dependent vitamin C transporters (SVCT) 1 and SVCT2 and DHA transporters, glucose transporter (GLUT) 1, GLUT3, GLUT4 mRNA by using senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice. These mice are incapable of synthesizing AA in vivo. AA depletion enhanced SVCT1 and SVCT2 mRNA expression in the liver and SVCT1 and GLUT4 mRNA expression in the small intestine, but not in the cerebrum or kidney. Next, we examined the actual impact of AA uptake by using primary cultured hepatocytes from SMP30/GNL KO mice. In the AA-depleted hepatocytes from SMP30/GNL KO mice, AA uptake was significantly greater than in matched cultures from wild-type mice. These results strongly affirm that intracellular AA is an important regulator of SVCT1 and SVCT2 expression in the liver.     

Differential regulation of the ascorbic acid transporter SVCT2 during development and in response to ascorbic acid depletion

The sodium-dependent vitamin C transporter-2 (SVCT2) is the only ascorbic acid (ASC) transporter significantly expressed in brain. It is required for life and is critical during brain development to supply adequate levels of ASC. To assess SVCT2 function in the developing brain, we studied time-dependent SVCT2 mRNA and protein expression in mouse brain, using liver as a comparison tissue because it is the site of ASC synthesis. We found that SVCT2 expression followed an inverse relationship with ASC levels in the developing brain. In cortex and cerebellum, ASC levels were high throughout late embryonic stages and early post-natal stages and decreased with age, whereas SVCT2 mRNA and protein levels were low in embryos and increased with age. A different response was observed for liver, in which ASC levels and SVCT2 expression were both low throughout embryogenesis and increased post-natally. To determine whether low intracellular ASC might be capable of driving SVCT2 expression, we depleted ASC by diet in adult mice unable to synthesize ASC. We observed that SVCT2 mRNA and protein were not affected by ASC depletion in brain cortex, but SVCT2 protein expression was increased by ASC depletion in the cerebellum and liver. The results suggest that expression of the SVCT2 is differentially regulated during embryonic development and in adulthood.     

Polarized localization of vitamin C transporters, SVCT1 and SVCT2, in epithelial cells

Messenger RNA of homologous sodium-vitamin C cotransporters, SVCT1 and SVCT2, were found in the intestine. Studies using cultured intestinal cells suggested an apical presence of SVCT1 but the function of SVCT2 was unknown. Here, we showed that enterocytes from heterozygous SVCT2-knockout mice had lower sodium-dependent vitamin C accumulation compared to those from the wildtype. Thus, SVCT2 appears to be functional in enterocytes. We then tested whether SVCT2 could have a redundant function as SVCT1 by constructing and expressing EGFP-tagged SVCTs in intestinal Caco-2 and kidney MDCK cells. In confluent epithelial cells, SVCT1 protein expressed predominantly on the apical membrane. SVCT2, in contrast, accumulated at the basolateral surface. Functionally, SVCT1 expression led to more transport activity from the apical membrane, while SVCT2 expression only increased the uptake under the condition when basolateral membrane was exposed. This differential epithelial membrane distribution and function suggests non-redundant functions of these two isoforms.     

Vitamin C transport systems of mammalian cells

Vitamin C is essential for many enzymatic reactions and also acts as a free radical scavenger. Specific non-overlapping transport proteins mediate the transport of the oxidized form of vitamin C, dehydroascorbic acid, and the reduced form, L-ascorbic acid, across biological membranes. Dehydroascorbic acid uptake is via the facilitated-diffusion glucose transporters, GLUT 1, 3 and 4, but under physiological conditions these transporters are unlikely to play a major role in the uptake of vitamin C due to the high concentrations of glucose that will effectively block influx. L-ascorbic acid enters cells via Na+-dependent systems, and two isoforms of these transporters (SVCT1 and SVCT2) have recently been cloned from humans and rats. Transport by both isoforms is stereospecific, with a pH optimum of approximately 7.5 and a Na+:ascorbic acid stoichiometry of 2:1. SVCT2 may exhibit a higher affinity for ascorbic acid than SVCT1 but with a lower maximum velocity. SVCT1 and SVCT2 are predicted to have 12 transmembrane domains, but they share no structural homology with other Na+ co-transporters. Potential sites for phosphorylation by protein kinase C exist on the cytoplasmic surface of both proteins, with an additional protein kinase A site in SVCT1. The two isoforms also differ in their tissue distribution: SVCT1 is present in epithelial tissues, whereas SVCT2 is present in most tissues with the exception of lung and skeletal muscle.     

Vitamin C transport systems of mammalian cells

Vitamin C is essential for many enzymatic reactions and also acts as a free radical scavenger. Specific non-overlapping transport proteins mediate the transport of the oxidized form of vitamin C, dehydroascorbic acid, and the reduced form, Lascorbic acid, across biological membranes. Dehydroascorbic acid uptake is via the facilitated-diffusion glucose transporters, GLUT 1, 3 and 4, but under physiological conditions these transporters are unlikely to play a major role in the uptake of vitamin C due to the high concentrations of glucose that will effectively block influx. L-ascorbic acid enters cells via Na⁺-dependent systems, and two isoforms of these transporters (SVCT1 and SVCT2) have recently been cloned from humans and rats. Transport by both isoforms is stereospecific, with a pH optimum of ~ 7.5 and a Na⁺: ascorbic acid stoichiometry of 2 : 1. SVCT2 may exhibit a higher affinity for ascorbic acid than SVCT1 but with a lower maximum velocity. SVCT1 and SVCT2 are predicted to have 12 transmembrane domains, but they share no structural homology with other Na⁺ co-transporters. Potential sites for phosphorylation by protein kinase C exist on the cytoplasmic surface of both proteins, with an additional protein kinase A site in SVCT1. The two isoforms also differ in their tissue distribution: SVCT1 is present in epithelial tissues, whereas SVCT2 is present in most tissues with the exception of lung and skeletal muscle.