Functional and Molecular Aspects of Biotin Uptake via SMVT in Human Corneal Epithelial (HCEC) and Retinal Pigment Epithelial (D407) Cells

ABSTRACTSodium-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 [3H] 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 Km of 296.2 ± 25.9 and 863.8 ± 66.9 μM and Vmax 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.

[1]  H. Tähti,et al.  Effects of mercuric chloride exposure on the glutamate uptake by cultured retinal pigment epithelial cells. , 2001, Toxicology in vitro : an international journal published in association with BIBRA.

[2]  S. Rubin,et al.  Biotin uptake by human colonic epithelial NCM460 cells: a carrier-mediated process shared with pantothenic acid. , 1998, American journal of physiology. Cell physiology.

[3]  V. Ganapathy,et al.  Molecular and functional characterization of the intestinal Na+-dependent multivitamin transporter. , 1999, Archives of biochemistry and biophysics.

[4]  L. Machlin Handbook of vitamins: nutritional, biochemical, and clinical aspects. , 1984 .

[5]  L. Salminen,et al.  Glutamate uptake is inhibited by tamoxifen and toremifene in cultured retinal pigment epithelial cells. , 2002, Pharmacology & toxicology.

[6]  A. Mitra,et al.  Sodium dependent multivitamin transporter (SMVT): a potential target for drug delivery. , 2012, Current drug targets.

[7]  A. Mitra,et al.  Targeted lipid based drug conjugates: a novel strategy for drug delivery. , 2012, International journal of pharmaceutics.

[8]  A. Mitra,et al.  Molecular Expression and Functional Evidence of a Drug Efflux Pump (BCRP) in Human Corneal Epithelial Cells , 2009, Current eye research.

[9]  A. Urtti,et al.  Ocular Pharmacokinetic Modeling Using Corneal Absorption and Desorption Rates from in Vitro Permeation Experiments with Cultured Corneal Epithelial Cells , 2003, Pharmaceutical Research.

[10]  E. Toropainen,et al.  Paracellular and passive transcellular permeability in immortalized human corneal epithelial cell culture model. , 2003, European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences.

[11]  M. Häkli,et al.  Monocarboxylate transport in human corneal epithelium and cell lines. , 2010, European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences.

[12]  H. Said Cellular uptake of biotin: mechanisms and regulation. , 1999, The Journal of nutrition.

[13]  U. Pleyer,et al.  Ocular drug permeation following experimental excimer laser treatment on the isolated pig eye. , 2002, Journal of ocular pharmacology and therapeutics : the official journal of the Association for Ocular Pharmacology and Therapeutics.

[14]  P. Bernstein,et al.  A human retinal pigment epithelial cell line that retains epithelial characteristics after prolonged culture. , 1995, Investigative ophthalmology & visual science.

[15]  V. Ganapathy,et al.  Human Placental Na+-dependent Multivitamin Transporter , 1999, The Journal of Biological Chemistry.

[16]  K. Ruprecht,et al.  Expression of ABC-transporters in human corneal tissue and the transformed cell line, HCE-T. , 2007, Journal of ocular pharmacology and therapeutics : the official journal of the Association for Ocular Pharmacology and Therapeutics.

[17]  A. Mitra,et al.  Vitreal pharmacokinetics of biotinylated ganciclovir: role of sodium-dependent multivitamin transporter expressed on retina. , 2009, Journal of ocular pharmacology and therapeutics : the official journal of the Association for Ocular Pharmacology and Therapeutics.

[18]  M. Häkli,et al.  Effluxing ABC transporters in human corneal epithelium. , 2010, Journal of pharmaceutical sciences.

[19]  V. Ganapathy,et al.  Cloning and functional expression of a cDNA encoding a mammalian sodium-dependent vitamin transporter mediating the uptake of pantothenate, biotin, and lipoate. , 1998, The Journal of biological chemistry.

[20]  J. Zempleni,et al.  Uptake and metabolism of biotin by human peripheral blood mononuclear cells. , 1998, American journal of physiology. Cell physiology.

[21]  A. Urtti,et al.  Culture model of human corneal epithelium for prediction of ocular drug absorption. , 2001, Investigative ophthalmology & visual science.

[22]  A. Mitra,et al.  Mechanism of L-Ascorbic Acid Uptake by Rabbit Corneal Epithelial Cells: Evidence for the Involvement of Sodium-Dependent Vitamin C Transporter 2 , 2006, Current eye research.

[23]  A. Urtti,et al.  Evaluation of Cytotoxicity of Various Ophthalmic Drugs, Eye Drop Excipients and Cyclodextrins in an Immortalized Human Corneal Epithelial Cell Line , 1998, Pharmaceutical Research.

[24]  V. H. Lee,et al.  Topical ocular drug delivery: recent developments and future challenges. , 1986, Journal of ocular pharmacology.

[25]  A. Mitra,et al.  Functional characterization of sodium-dependent multivitamin transporter in MDCK-MDR1 cells and its utilization as a target for drug delivery. , 2006, Molecular pharmaceutics.

[26]  G. Chader,et al.  PHARMACOLOGY OF THE EYE , 1984, Handbook of Experimental Pharmacology.

[27]  H. Said,et al.  A carrier-mediated, Na+ gradient-dependent transport for biotin in human intestinal brush-border membrane vesicles. , 1987, The American journal of physiology.

[28]  H. Said,et al.  Uptake of biotin by human hepatoma cell line, Hep G2: A carrier‐mediated process similar to that of normal liver , 1994, Journal of cellular physiology.

[29]  A. Mitra,et al.  Expression of multidrug resistance associated protein 5 (MRP5) on cornea and its role in drug efflux. , 2009, Journal of ocular pharmacology and therapeutics : the official journal of the Association for Ocular Pharmacology and Therapeutics.

[30]  H. Handa,et al.  An SV40-immortalized human corneal epithelial cell line and its characterization. , 1995, Investigative ophthalmology & visual science.

[31]  K. Dakshinamurti,et al.  Liver glucokinase of the biotin deficient rat. , 1968, Canadian journal of biochemistry.

[32]  N. Mangini,et al.  P-glycoprotein expression in human retinal pigment epithelium. , 2002, Molecular vision.

[33]  A. Mitra,et al.  Molecular evidence and functional expression of a novel drug efflux pump (ABCC2) in human corneal epithelium and rabbit cornea and its role in ocular drug efflux. , 2007, International journal of pharmaceutics.

[34]  S. Akanuma,et al.  Blood-to-retina transport of biotin via Na+-dependent multivitamin transporter (SMVT) at the inner blood-retinal barrier. , 2010, Experimental eye research.

[35]  W. Nyhan,et al.  Inheritable biotin-treatable disorders and associated phenomena. , 1986, Annual review of nutrition.

[36]  A. Mitra,et al.  Biotin Uptake by Rabbit Corneal Epithelial Cells: Role of Sodium-Dependent Multivitamin Transporter (SMVT) , 2006, Current eye research.

[37]  H. Said,et al.  Biotin transport in the human intestine: site of maximum transport and effect of pH. , 1988, Gastroenterology.

[38]  K. Kaarniranta,et al.  Efflux Protein Expression in Human Retinal Pigment Epithelium Cell Lines , 2009, Pharmaceutical Research.

[39]  M. Prausnitz,et al.  Permeability of cornea, sclera, and conjunctiva: a literature analysis for drug delivery to the eye. , 1998, Journal of pharmaceutical sciences.

[40]  V. Ganapathy,et al.  Structure and function of mammalian sodium-dependent multivitamin transporter. , 2000, Current opinion in clinical nutrition and metabolic care.

[41]  A. Mitra,et al.  Differential expression of folate receptor-alpha, sodium-dependent multivitamin transporter, and amino acid transporter (B (0, +)) in human retinoblastoma (Y-79) and retinal pigment epithelial (ARPE-19) cell lines. , 2012, Journal of ocular pharmacology and therapeutics : the official journal of the Association for Ocular Pharmacology and Therapeutics.

[42]  S Reichl,et al.  Human corneal equivalent as cell culture model for in vitro drug permeation studies , 2004, British Journal of Ophthalmology.

[43]  R. Hunt,et al.  Altered expression of keratin and vimentin in human retinal pigment epithelial cells in vivo and in vitro , 1990, Journal of cellular physiology.

[44]  M. Pfaffl,et al.  A new mathematical model for relative quantification in real-time RT-PCR. , 2001, Nucleic acids research.

[45]  K. Balamurugan,et al.  Biotin uptake by human intestinal and liver epithelial cells: role of the SMVT system. , 2003, American journal of physiology. Gastrointestinal and liver physiology.

[46]  D. Cui,et al.  Expression of adenosine receptors in human retinal pigment epithelium cells in vitro. , 2011, Chinese medical journal.

[47]  H. Tähti,et al.  Expression of glutamate transporter subtypes in cultured retinal pigment epithelial and retinoblastoma cells , 2004, Current eye research.

[48]  G. Arden,et al.  P-Glycoprotein expression in human retinal pigment epithelium cell lines. , 2006, Experimental eye research.

[49]  J. Daniels,et al.  Characterisation and functional features of a spontaneously immortalised human corneal epithelial cell line with progenitor-like characteristics , 2010, Brain Research Bulletin.

[50]  Stephan Reichl,et al.  Cell culture models of the human cornea — a comparative evaluation of their usefulness to determine ocular drug absorption in‐vitro , 2008, The Journal of pharmacy and pharmacology.

[51]  H. Said,et al.  Human intestinal cell line Caco-2: a useful model for studying cellular and molecular regulation of biotin uptake. , 1994, Biochimica et biophysica acta.

[52]  E. Toropainen,et al.  Cell culture models of the ocular barriers. , 2005, European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V.

[53]  Y. Omidi,et al.  Ocular Drug Delivery; Impact of in vitro Cell Culture Models , 2009, Journal of ophthalmic & vision research.

[54]  J. Zempleni,et al.  Biotin requirements are lower in human Jurkat lymphoid cells but homeostatic mechanisms are similar to those of HepG2 liver cells. , 2010, The Journal of nutrition.