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 Km of 0.6mM. 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.
•We describe a general mechanism for the mitochondrial acquisition of ascorbic acid.•Mitochondria express a low-affinity form of the ascorbic acid transporter SVCT2.•SVCT2 transport kinetics is altered by the intracellular ionic microenvironment.•Mitochondrial SVCT2 shows an apparent transport Km of 0.6mM.•Mitochondrial SVCT2 is fully functional under normal high ascorbate concentrations.
The data presented in this article are related to the research paper entitled “Increased expression of mitochondrial sodium-coupled ascorbic acid transporter-2 (mitSVCT2) as a central feature in ...breast cancer”, available in Free Radical Biology and Medicine Journal 1. In this article, we examined the SVCT2 transporter expression in various breast cancer cell lines using RT-PCR and Western blot assays. In addition, we analyzed the subcellular localization of SVCT2 by immunofluorescence colocalization assays and cellular fractionation experiments. Finally, an analysis of different cancer tissue microarrays immunostained for SVCT2 and imaged by The Human Protein Atlas (https://www.proteinatlas.org) is presented.
N-methyl-D-aspartate receptors (NMDARs) are known to regulate axonal refinement and dendritic branching. However, because NMDARs are abundantly present as tri-heteromers (e.g., NR1/NR2A/NR2B) during ...development, the precise role of the individual subunits NR2A and NR2B in these processes has not been elucidated. Ventral spinal cord neurons (VSCNs) provide a unique opportunity to address this problem, because the expression of both NR2A and NR2B (but not NR1) is downregulated in culture. Exogenous NR2A or NR2B were introduced into these naturally NR2-null neurons at 4 DIV, and electrophysiological recordings at 11 DIV confirmed that synaptic NR1NR2A receptors and NR1NR2B receptors were formed, respectively. Analysis of the dendritic architecture showed that introduction of NR2B, but not NR2A, dramatically increased the number of secondary and tertiary dendritic branches of VSCNs. Whole cell patch-clamp recordings further indicated that the newly formed branches in NR2B-expressing neurons were able to establish functional synapses because the frequency of miniature AMPA-receptor synaptic currents was increased. Using previously described mutants, we also found that disruption of the interaction between NR2B and RasGRF1 dramatically impaired dendritic branch formation in VSCNs. The differential role of the NR2A and NR2B subunits and the requirement for RasGRF1 in regulating branch formation was corroborated in hippocampal cultures. We conclude that the association between NR1NR2B-receptors and RasGRF1 is needed for dendritic branch formation in VSCNs and hippocampal neurons in vitro. The dominated NR2A expression and the limited interactions of this subunit with the signaling protein RasGRF1 may contribute to the restricted dendritic arbor development in the adult CNS.
The mammary gland increases energy requirements during pregnancy and lactation to support epithelial proliferation and milk nutrients synthesis. Lactose, the principal carbohydrate of the milk, is ...synthetized in the Golgi of mammary epithelial cells by lactose synthase from glucose and UPD galactose. We studied the temporal changes in the expression of GLUT1 and GLUT8 in mammary gland and their association with lactose synthesis and proliferation in BALB/c mice. Six groups were used: virgin, pregnant at 2 and 17 days, lactating at 2 and 10 days, and weaning at 2 days. Temporal expression of GLUT1 and GLUT8 transporters by qPCR, western blot and immunohistochemistry, and its association with lactalbumin, Ki67, and cytokeratin 18 within mammary tissue was studied, along with subcellular localization. GLUT1 and GLUT8 transporters increased their expression during mammary gland progression, reaching 20-fold increasing in GLUT1 mRNA at lactation (
p
< 0.05) and 2-fold at protein level for GLUT1 and GLUT8 (
p
< 0.05 and 0.01, respectively). The temporal expression pattern was shared with cytokeratin 18 and Ki67 (
p
< 0.01). Endogenous GLUT8 partially co-localized with 58 K protein and α-lactalbumin in mammary tissue and with Golgi membrane–associated protein 130 in isolated epithelial cells. The spatial-temporal synchrony between expression of GLUT8/GLUT1 and alveolar cell proliferation, and its localization in
cis-
Golgi associated to lactose synthase complex, suggest that both transporters are involved in glucose uptake into this organelle, supporting lactose synthesis.
The liver has an extraordinary regenerative capacity in response to partial hepatectomy (PHx), which develops with neither tissue inflammation response nor alterations in the whole organism. This ...process is highly coordinated and it has been associated with changes in glutathione (GSH) metabolism. However, there are no reports indicating ascorbic acid (AA) levels after partial hepatectomy. AA and GSH act integrally as an antioxidant system that protects cells and tissues from oxidative damage and imbalance observed in a variety of diseases that affect the liver. Although rat hepatocytes are able to synthesize AA and GSH, which are the providers of AA for the whole organism, they also acquire AA from extracellular sources through the sodium-coupled ascorbic acid transporter-1 (SVCT1). Here, we show that hepatocytes from rat livers subjected to PHx increase their GSH and AA levels from 1 to 7 days post hepatectomy, whose peaks precede the peak in cell proliferation observed at 3 days post-hepatectomy. The increase in both antioxidants was associated with higher expression of the enzymes involved in their synthesis, such as the modifier subunit of enzyme glutamine cysteine ligase (GCLM), glutathione synthetase (GS), gulonolactonase (GLN) and gulonolactone oxidase (GULO). Importantly, rat hepatocytes, that normally exhibit kinetic evidence indicating only SVCT1-mediated transport of AA, lost more than 90% of their capacity to transport it at day 1 after PHx without evidence of recovery at day 7. This observation was in agreement with loss of SVCT1 protein expression, which was undetectable in hepatocytes as early as 2h after PHx, with partial recovery at day 7, when the regenerated liver weight returns to normal.
We conclude that after PHx, rat hepatocytes enhance their antioxidant capacity by increasing GSH and AA levels prior to the proliferative peak. GSH and AA are increased by de novo synthesis, however paradoxically hepatocytes from rat subjected to PHx also suppress their capacity to acquire AA from extracellular sources through SVCT1.
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•Ascorbic acid and glutathione levels increased in rat hepatocytes after partial hepatectomy.•The increase of ascorbic acid and glutathione anteceded the peak in cell proliferation.•SVCT1 mediates ascorbic acid transport in rat hepatocytes.•SVCT1 protein is decreased after partial hepatectomy without changes in mRNA expression.•Liver regeneration after partial hepatectomy was associated with a loss of ascorbic acid transport by hepatocytes.
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