title: 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.
authors.affiliation: Division of Pharmaceutical Science, School of Pharmacy, University of Missouri-Kansas City, 5005 Rockhill Road, Kansas City, MO 64110-2499, USA.
authors.forename: Shuanghui
authors.initials: S
authors.lastname: Luo
authors.forename: Zhiying
authors.initials: Z
authors.lastname: Wang
authors.forename: Viral
authors.initials: V
authors.lastname: Kansara
authors.forename: Dhananjay
authors.initials: D
authors.lastname: Pal
authors.forename: Ashim K
authors.initials: AK
authors.lastname: Mitra
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism; D000818:Animals; D001205:Ascorbic Acid / Q000378:metabolism*; D001693:Biological Transport, Active; D002460:Cell Line; D002463:Cell Membrane Permeability; D003627:Data Interpretation, Statistical; D004285:Dogs; D004734:Energy Metabolism / Q000502:physiology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D018613:Microscopy, Confocal; D029363:Organic Anion Transporters, Sodium-Dependent / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D012964:Sodium / Q000502:physiology; D060347:Sodium-Coupled Vitamin C Transporters; D013379:Substrate Specificity; D027981:Symporters / Q000378:metabolism*
chemical_list: C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D029363:Organic Anion Transporters, Sodium-Dependent; C555162:SLC23A1 protein, human; C555163:SLC23A2 protein, human; D060347:Sodium-Coupled Vitamin C Transporters; D027981:Symporters; D012964:Sodium; D001205:Ascorbic Acid