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 con®rmed 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 af®nity constants, 103 mM and 8 mM. A K m of 103 mM corresponds to a similar af®nity constant reported for SVCT2, while the K m of 8 mM might suggest the expression of a very high af®nity 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.

Specialized cells transport vitamin C in its reduced form using sodium-dependent cotransporters (SVCT1 and SVCT2). Additionally, different cells transport the oxidized form of vitamin C, dehydroascorbic acid, through glucose transporters (GLUTs). We have proposed recently a model for vitamin C uptake that resolves the apparent contradiction that although only ascorbic acid is detectable in vivo, there are cells that transport only dehydroascorbic acid. We carried out a detailed kinetic analysis to compare the mechanisms of vitamin C uptake in normal human melanocytes, neurons isolated from brain cortex, hypothalamic ependymal-glial cells, and astrocytes. Uptake of ascorbic acid was also analyzed in the human oligodendroglioma cell line TC620, in human choroid plexus papilloma cells (HCPPC-1), and in the neuroblastoma cell line Neuro-2a. Melanocytes were used to carry out a detailed analysis of vitamin C uptake. Analysis of the transport data by the Lineweaver-Burk plot revealed the presence of one functional component (K(m) 20 microM) involved in ascorbic acid transport by melanocytes. Vitamin C sodium-dependent saturable uptake was also observed in neurons and hypothalamic tanycytes. We confirmed SVCT2 expression in neurons by in situ hybridization; however, SVCT2 expression was not detected in astrocytes in situ. Functional data indicate that astrocytes transport mainly dehydroascorbic acid, using the glucose transporter GLUT1. Our functional uptake analyses support the hypothesis that astrocytes are involved in vitamin C recycling in the nervous system. This recycling model may work as an efficient system for the salvage of vitamin C by avoiding the hydrolysis of dehydroascorbic acid produced by antioxidative protection.

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.

Breast cancer incidence increases in women receiving combined estrogen and progesterone therapy. Breast tumors show increased expression of the glucose transporter GLUT1. We determined the effect of these hormones on GLUT1-4 expression and deoxyglucose transport in ZR-75-1 breast cancer cells. Immunoblotting, immunocytochemistry, flow cytometry, and RT-PCR showed that GLUT1 expression is up-regulated by progesterone and, to a greater degree, combined therapy. GLUT2 expression is unaffected by hormonal treatment. GLUT3 protein and RNA is up-regulated by progesterone and combined therapy, and GLUT4 protein expression is up-regulated by all hormonal treatments. Deoxyglucose transport studies revealed the presence of three transport components with characteristics corresponding to GLUT1/4, GLUT2, and GLUT3. 17beta-Estradiol produced a slight increase in transport at the Michaelis constant (Km) corresponding to GLUT3. Progesterone produced a small increase in transport at the Km corresponding to GLUT1/4, and combined 17beta-estradiol and progesterone produced a small increase in transport at the Km corresponding to GLUT3 and a large increase in transport at the Km corresponding to GLUT1/4. This indicates that 17beta-estradiol and progesterone differentially regulate GLUT1-4 expression and that these changes correlate to changes in glucose uptake. We postulate that combined hormone replacement therapy provides a survival advantage to developing ZR-75 breast cancer cells.

Ascorbic acid (AA), the reduced form of vitamin C, is incorporated into neurons via the sodium ascorbate co-transporter SVCT2. However, this transporter is not expressed in astrocytes, which take up the oxidized form of vitamin C, dehydroascorbic acid (DHA), via the facilitative hexose transporter GLUT1. Therefore, neuron and astrocyte interactions are thought to mediate vitamin C recycling in the nervous system. Although astrocytes are essential for the antioxidant defense of neurons under oxidative stress, a condition in which a large amount of ROS is generated that may favor the extracellular oxidation of AA and the subsequent neuronal uptake of DHA via GLUT3, potentially increasing oxidative stress in neurons. This study analyzed the effects of oxidative stress and DHA uptake on neuronal cell death in vitro. Different analyses revealed the presence of the DHA transporters GLUT1 and GLUT3 in Neuro2a and HN33.11 cells and in cortical neurons. Kinetic analyses confirmed that all cells analyzed in this study possess functional GLUTs that take up 2-deoxyglucose and DHA. Thus, DHA promotes the death of stressed neuronal cells, which is reversed by incubating the cells with cytochalasin B, an inhibitor of DHA uptake by GLUT1 and GLUT3. Additionally, the presence of glial cells (U87 and astrocytes), which promote DHA recycling, reverses the observed cell death of stressed neurons. Taken together, these results indicate that DHA promotes the death of stressed neurons and that astrocytes are essential for the antioxidative defense of neurons. Thus, the astrocyte-neuron interaction may function as an essential mechanism for vitamin C recycling, participating in the antioxidative defense of the brain.

No abstract

No abstract

Saxitoxin (STX) is a neurotoxin produced by dinoflagellates in diverse species, such as Alexandrium spp., and it causes paralytic shellfish poisoning (PSP) in humans after the ingestion of contaminated shellfish. Recent studies have suggested that the immune functions of bivalves could be affected by harmful algae and/or by their toxins. Herein, hemocytes are the main effector cells of the immune cellular response. In this study, we evaluated the response of hemocytes from the mussel Mytilus chilensis to STX exposure in a primary culture. Cell cultures were characterized according to size and complexity, while reactive oxygen species (ROS) production was evaluated using a dichlorofluorescein diacetate (DCFH-DA) assay. Finally, phagocytic activity was measured using both flow cytometry and fluorescence microscopy assays. Additionally, gene transcription of candidate genes was evaluated by qPCR assays. The results evidenced that exposures to different concentrations of STX (1–100 nM) for 24 h did not affect cell viability, as determined by an MTT assay. However, when hemocytes were exposed for 4 or 16 h to STX (1–100 nM), there was a modulation of phagocytic activity and ROS production. Moreover, hemocytes exposed to 100 nM of STX for 4 or 16 h showed a significant increase in transcript levels of genes encoding for antioxidant enzymes (SOD, CAT), mitochondrial enzymes (COI, COIII, CYTB, ATP6, ND1) and ion channels (K+, Ca2+). Meanwhile, C-type lectin and toll-like receptor genes revealed a bi-phase transcriptional response after 16 and 24–48 h of exposure to STX. These results suggest that STX can negatively affect the immunocompetence of M. chilensis hemocytes, which were capable of responding to STX exposure in vitro by increasing the mRNA levels of antioxidant enzymes.