Activity of a sodium-dependent vitamin C transporter (SVCT) in MDCK-MDR1 cells and mechanism of ascorbate uptake

Abstract

The objective of this research was to functionally characterize sodium-dependent vitamin C transporter (SVCT) in MDCK-MDR1 cells and to study the effect of substituted benzene derivatives on the intracellular accumulation of ascorbic acid (AA). Mechanism of AA uptake and transport was delineated. Uptake of [(14)C]ascorbic acid ([(14)C]AA) was studied in the absence and presence of excess unlabelled AA, anion transporter inhibitors, and a series of mono- and di-substituted benzenes. Transepithelial transport of [(14)C]AA across polarized cell membrane has been studied for the first time. Role of cellular protein kinase-mediated pathways on the regulation of AA uptake has been investigated. The cellular localizations of SVCTs were observed using confocal microscopy. Uptake of AA was found to be saturable with a K(m) of 83.2muM and V(max) of 94.2pmol/min/mg protein for SVCT1. The process was pH, sodium, temperature, and energy-dependent. It was under the regulation of cellular protein kinase C (PKC) and Ca(2+)/CaM mediated pathways. [(14)C]AA uptake was significantly inhibited in the presence of excess unlabelled AA and a series of electron-withdrawing group, i.e., halogen- and nitro-substituted benzene derivatives. AA appears to translocate across polarized cell membrane from apical to basal side (A-B) as well as basal to apical side (B-A) at a similar permeability. It appears that SVCT1 was mainly expressed on the apical side and SVCT2 may be located on both apical and basal sides. In conclusion, SVCT has been functionally characterized in MDCK-MDR1 cells. The interference of a series of electrophile-substituted benzenes on the AA uptake process may be explained by their structural similarity. SVCT may be targeted to facilitate the delivery of drugs with low bioavailability by conjugating with AA and its structural analogs. MDCK-MDR1 cell line may be utilized as an in vitro model to study the permeability of AA conjugated prodrugs.

Molecular expression and functional activity of vitamin C specific transport system (SVCT2) in human breast cancer cells

“…The presence of SVCT on corneal cells and Madin-Darby canine kidney cells has been previously reported from our laboratory (Luo et al, 2008; Talluri et al, 2006). Also, SVCT expression was shown on breast cancer epithelial cells (MDA-MB231 and T47D cells).…”

“…Therefore, AA is usually obtained from exogenous sources through the dietary intake (Luo et al, 2008). AA uptake via specific transport system has already been reported in intestine (Maulen et al, 2003), brain (Castro et al, 2001), kidney (Bowers-Komro and McCormick, 1991), skin (Padh and Aleo, 1987), eye (Garland, 1991; Talluri et al, 2006) and bone (Dixon et al, 1991).…”

“…Both SVCT1 and SVCT2 vary in distribution but express close sequence homology and functional similarity (Talluri et al, 2006). Structural and functional studies reveal that transport of AA across epithelial cells is primarily facilitated via SVCT (Luo et al, 2008; Talluri et al, 2006). …”

A fluorophore-conjugated ascorbic acid functions for the visualization of sodium vitamin C transporters in living cells

“…Under the physiological pH conditions (pH 7.2), RB-A-Vc became negatively-charged and difficult to permeate the cell membrane without the aid of transporters. 28 To further conrm the RB-A-Vc was transported by SVCTs, we examined the specicity of RB-A-Vc against SVCTs by Western blot assay. The results indicate there is only SVCT2 identied at $50 kDa, but no SVCT1 (at 65 kDa) was found in HEK-293T cells (Fig.…”

5-O-(4-[125I]Iodobenzyl)-L-ascorbic Acid: Electrophilic Radioiodination and Biodistribution in Mice

“…There are some literature reports on the substrate specificity of the AsA transporter using biological tissues or cells. [28][29][30][31][32] As most AsA exists as a monovalent anion at physiological pH and SVCT has a high affinity for the ionic form, it has been suggested that the ionic interactions between SVCT and its substrates are the predominant driving forces for the binding of SVCT. 31,32) Studies by Rumsey et al 33) demonstrated that one of the structural requisites of AsA and its analogs for transport in cells is an S-absolute configuration at the C-4 position in a five-membered reduced ring with no substitution on the C-2 and C-3 positions, and that 6-deoxy-6-halogeno-Lascorbic acid is an effective compound for inhibiting AsA uptake.…”