title: Functional and molecular aspects of biotin uptake via SMVT in human corneal epithelial (HCEC) and retinal pigment epithelial (D407) cells.
abstract: Sodium-dependent multivitamin transporter (SMVT) is a vital transmembrane protein responsible for translocating biotin and other essential cofactors such as pantothenate and lipoate. Unlike primary cultures of corneal and retinal pigment epithelial (RPE) cells, immortalized cells can be subcultured many times, yet maintain their physiological properties. Hence, the purpose of this study was to delineate the functional and molecular aspects of biotin uptake via SMVT on immortalized human corneal epithelial (HCEC) and RPE (D407) cells. Functional aspects of [(3)H] biotin uptake were studied in the presence of different concentrations of unlabeled biotin, pH, temperature, metabolic inhibitors, ions, substrates, structural analogs and biotinylated prodrug (Biotin-Acyclovir (B-ACV)). Molecular identity of SMVT was examined with reverse transcription-polymerase chain reaction. Biotin uptake was found to be saturable in HCEC and D407 cells with K                 (m) of 296.2 ± 25.9 and 863.8 ± 66.9 μM and V                 (max) of 77.2 ± 2.2 and 308.3 ± 10.7 pmol/mg protein/min, respectively. Uptake was found to be pH, temperature, energy, and sodium-dependent. Inhibition of biotin uptake was observed in the presence of structural analogs and specific substrates. Further, uptake was lowered in the presence of B-ACV indicating the translocation of biotinylated prodrug by SMVT. A distinct band at 774 bp confirmed the molecular existence of SMVT in both the cells. This study shows for the first time the functional and molecular presence of SMVT in HCEC and D407 cells. Therefore, these cell lines may be utilized as in vitro models to study the cellular translocation of biotin-conjugated prodrugs.
authors.affiliation: Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, 2464 Charlotte Street, Kansas City, Missouri 64108-2718, USA.
authors.forename: Aswani Dutt
authors.initials: AD
authors.lastname: Vadlapudi
authors.forename: Ramya Krishna
authors.initials: RK
authors.lastname: Vadlapatla
authors.forename: Dhananjay
authors.initials: D
authors.lastname: Pal
authors.forename: Ashim K
authors.initials: AK
authors.lastname: Mitra
mesh_terms: D000212:Acyclovir / Q000008:administration & dosage / Q000737:chemistry / Q000493:pharmacokinetics; D001710:Biotin / Q000008:administration & dosage / Q000737:chemistry / Q000493:pharmacokinetics*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D019573:Epithelium, Corneal / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D011355:Prodrugs; D055213:Retinal Pigment Epithelium / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012964:Sodium / Q000378:metabolism; D027981:Symporters / Q000378:metabolism*; D013696:Temperature
chemical_list: D011355:Prodrugs; D027981:Symporters; C119981:biotin transporter; D001710:Biotin; D012964:Sodium; D000212:Acyclovir