Up-regulation and polarized expression of the sodium-ascorbic acid transporter SVCT1 in post-confluent differentiated CaCo-2 cells.
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 steady-state 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.
Maulén
Nancy P
NP
Departamento de Fisiopatologia, Facultad de Ciencias Biológicas, Universidad de Concepción, Barrio Universitario S/N, Concepción, Chile.
Henríquez
Esther A
EA
Kempe
Sybille
S
Cárcamo
Juan G
JG
Schmid-Kotsas
Alexandra
A
Bachem
Max
M
Grünert
Adolph
A
Bustamante
Marcelo E
ME
Nualart
Francisco
F
Vera
Juan Carlos
JC
D001205:Ascorbic Acid / Q000378:metabolism / Q000494:pharmacology; D001692:Biological Transport; D002454:Cell Differentiation; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D003683:Dehydroascorbic Acid / Q000378:metabolism; D003847:Deoxyglucose / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D051272:Glucose Transporter Type 1; D051296:Glucose Transporter Type 5; D006601:Hexoses / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009004:Monosaccharide Transport Proteins / Q000378:metabolism; D029363:Organic Anion Transporters, Sodium-Dependent / Q000096:biosynthesis* / Q000378:metabolism; D011487:Protein Conformation; D021381:Protein Transport; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D060347:Sodium-Coupled Vitamin C Transporters; D027981:Symporters / Q000096:biosynthesis* / Q000378:metabolism; D013997:Time Factors; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
D051272:Glucose Transporter Type 1; D051296:Glucose Transporter Type 5; D006601:Hexoses; D009004:Monosaccharide Transport Proteins; D029363:Organic Anion Transporters, Sodium-Dependent; D012333:RNA, Messenger; C555162:SLC23A1 protein, human; C555163:SLC23A2 protein, human; C495677:SLC2A1 protein, human; D060347:Sodium-Coupled Vitamin C Transporters; D027981:Symporters; D004247:DNA; D003847:Deoxyglucose; D001205:Ascorbic Acid; D003683:Dehydroascorbic Acid