Up-regulation and Polarized Expression of the Sodium-Ascorbic Acid Transporter SVCT1 in Post-confluent Differentiated CaCo-2 Cells

Abstract

Human cells acquire vitamin C using two different transporter systems, the sodium-ascorbic acid co-transporters with specificity for ascorbic acid, and the facilitative glucose transporters with specificity for dehydroascorbic acid. There is no information on the mechanism of vitamin C transport across the intestinal barrier, a step that determines the bioavailability of vitamin C in humans. We used the colon carcinoma cell line CaCo-2 as an in vitro model for vitamin C transport in enterocyte-like cells. The results of transport kinetics, sodium dependence, inhibition studies, and reverse transcriptase-PCR analysis indicated that CaCo-2 cells express the sodium-ascorbate co-transporters SVCT1 and SVCT2, the dehydroascorbic acid transporters GLUT1 and GLUT3, and a third dehydroascorbic acid transporter with properties expected for GLUT2. Analysis by real time quantitative PCR revealed that the post-confluent differentiation of CaCo-2 cells was accompanied by a marked increase (4-fold) in the steadystate level of SVCT1 mRNA, without changes in SVCT2 mRNA levels. Functional studies revealed that the differentiated cells expressed only one functional ascorbic acid transporter having properties expected for SVCT1, and transported ascorbic acid with a V max that was increased at least 2-fold compared with pre-confluent cells. Moreover, post-confluent Caco-2 cells growing as monolayers in permeable filter inserts showed selective sorting of SVCT1 to the apical membrane compartment, without functional evidence for the expression of SVCT2. The identification of SVCT1 as the transporter that allows vectorial uptake of ascorbic acid in differentiated CaCo-2 cells has a direct impact on our understanding of the mechanism for vitamin C transport across the intestinal barrier.

Mechanistic Insights and Functional Determinants of the Transport Cycle of the Ascorbic Acid Transporter SVCT2

“…1A, lanes 3 and 4, and data not shown). These results are in agreement with studies of the presence of SVCT1 and SVCT2-specific transcripts in mammalian tissues and cells (9,11,12). Expression of SVCT2 at the protein level was confirmed by immunoblotting (Fig.…”

“…Rat SVCT1 and SVCT2 expressed in X. laevis oocytes showed transport K m values of 19 -30 M (SVCT1) and 9.4 M (SVCT2), whereas for the human isoforms the K m values were 75-250 M (SVCT1) and 22-69 M (SVCT2) (11-14, 18, 19). In comparison, in human CaCo-2 cells, SVCT1 and SVCT2 have transport K m values of 113 and 15 M, respectively (9). The reported differences may be attributable to aspects of the experimental procedures.…”

“…RT-PCR-mRNA was prepared using the Micro poly(A) Pure kit (Ambion Inc.), and reverse transcription was performed using the rapid RT-PCR kit (Clontech Inc.) (9). Approximately 10 ng of cDNA synthesis product was used as template in a reaction mix containing 40 pmol of the primer pair (13), with the program: 2 min at 94°C; 35 cycles of 2 min each at 94°C, 1 min at 55°C, and 2 min at 72°C; and 7 min at 72°C.…”

Vitamin C controls the cystic fibrosis transmembrane conductance regulator chloride channel

“…Whether knockout of SVCT2 results in defective activation of CFTR by vitamin C has not been considered and will need further investigation. Selective sorting to the apical membrane compartment has been recently demonstrated for SVCT1 in Caco-2 intestinal cells (43). Here we show the localization of recombinant SVCT2-EGFP fusion protein to the apical membrane pole of nasal epithelial cells (Fig.…”

Transport model of the human Na+-coupled l-ascorbic acid (vitamin C) transporter SVCT1

“…32 and 35), the product of the SLC23A1 gene. Selective sorting of SVCT1 protein to the apical membrane has been demonstrated in cultured cell lines (Caco2 and MDCK) of intestinal and renal origin (2,23).…”