Vitamin C uptake and recycling among normal and tumor cells from the central nervous system

Abstract

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.

Development of ascorbate transporters in brain cortical capillary endothelial cells in culture

“…In agreement with previous in situ hybridization (Astuya et al, 2005;García et al, 2005) and immunocytochemical studies, the SVCT2 was not present in endothelial cells lining cortical capillaries in the mouse, either by direct staining or by immunoblotting. However, when the cells were purified and placed into culture, the SVCT2 was readily detected after several days and was associated with ascorbate transport rates comparable to immortalized endothelial cell lines.…”

“…In agreement with previous in situ hybridization (Astuya et al, 2005;García et al, 2005) and immunocytochemical studies, the SVCT2 was not present in endothelial cells lining cortical capillaries in the mouse, either by direct staining or by immunoblotting.…”

“…Moreover, this phenomenon is not unique to CCECs, since cortical astrocytes have also been shown not to express SVCT2 mRNA (Astuya et al, 2005;Berger et al, 2000) or protein in the brain, but readily develop specific ascorbate transport in culture (Berger et al, 2000;Korcok et al, 2000). Exposure of cells to increased oxidant stress in culture seems a likely cause.…”

Brain Energy Metabolism in Health and Disease

“…We have demonstrated that neuronal intracellular ascorbic acid inhibits glucose utilization in neurons (Castro et al, 2009) through GLUT3 inhibition (Figure 8, Beltrán et al, 2011). This mechanism is supported by the idea of ascorbic acid recycling in brain (see above, Figure 10, Astuya et al, 2005). Using primary cultures of cortical neurons treated with a specific shRNA (to block GLUT3 expression) and a fluorescent glucose analogue, we have shown that this transporter is important, using real-time experiments (Figure 7).…”

“…Reduction is mainly an enzymatically catalyzed reaction, which may be glutathione-dependent (Ishikawa et al 1998). Astrocytes are thought to be involved in ascorbic acid recycling (Figure 10, Astuya et al 2005). During synaptic activity, ascorbic acid is released from intracellular reservoirs (O'Neill et al ,1984;Ghasemzadeh et al, 1991;Yusa, 2001).…”