title: Biotin uptake by rabbit corneal epithelial cells: role of sodium-dependent multivitamin transporter (SMVT).
abstract: OBJECTIVE
  The objective of this research was to investigate the presence of sodium-dependent multivitamin transporter (SMVT) on rabbit corneal epithelial cells.
  
  
  METHODS
  Primary cultured rabbit corneal epithelial cells (rPCECs)and freshly excised rabbit corneas were used for characterization of biotin uptake and transport, respectively. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to confirm the molecular identity of SMVT. Liquid chromatography/tandem mass spectrometry (LC-MS/MS) analysis was performed to examine the presence of biotin in rabbit tears.
  
  
  RESULTS
  Uptake of biotin by rPCECs was found to be time and concentration dependent with Km of 32.52 microM and Vmax of 10.43 pmol min- 1 mg protein- 1. Biotin was significantly inhibited in the presence of pantothenic acid and lipoic acid. Biotin uptake was found to be energy and Na+ dependent but H+ and Cl- independent. The uptake was inhibited by valeric acid in a concentration-dependent manner but not much affected in the presence of biotin methyl ester and biocytin with no free carboxyl group. Modulators of both PKC- and PKA-mediated pathways had no effect on biotin uptake, but calcium-calmodulin inhibitor significantly inhibited its uptake. Sodium-dependent multivitamin transporter was identified by RT-PCR in rPCECs. Transport experiments across the rabbit corneas revealed the functional localization of SMVT on the apical side of the cornea, and thereby corroborating with in vitro results with cultured corneal cells. Finally, LC-MS/MS analysis showed the presence of biotin in rabbit tears.
  
  
  CONCLUSIONS
  Results obtained from both in vitro and exvivo studies suggest the possible role of SMVT expressed on corneal epithelial cells for the uptake of biotin, which co-transports pantothenic acid and lipoic acid. Further, the presence of biotin in tears suggests the physiological significance of this transporter in rabbit corneal epithelium.
authors.affiliation: Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, Kansas City, Missouri 64110-2499, USA.
authors.forename: Kumar G
authors.initials: KG
authors.lastname: Janoria
authors.forename: Sudharshan
authors.initials: S
authors.lastname: Hariharan
authors.forename: Durga
authors.initials: D
authors.lastname: Paturi
authors.forename: Dhananjay
authors.initials: D
authors.lastname: Pal
authors.forename: Ashim K
authors.initials: AK
authors.lastname: Mitra
mesh_terms: D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D001710:Biotin / Q000037:antagonists & inhibitors / Q000493:pharmacokinetics*; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D019573:Epithelium, Corneal / Q000166:cytology / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D006863:Hydrogen-Ion Concentration; D008297:Male; D010205:Pantothenic Acid / Q000494:pharmacology; D010421:Pentanoic Acids / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012964:Sodium / Q000494:pharmacology; D027981:Symporters / Q000502:physiology*; D013666:Tears / Q000378:metabolism; D008063:Thioctic Acid / Q000494:pharmacology
chemical_list: D010421:Pentanoic Acids; D027981:Symporters; C119981:biotin transporter; D010205:Pantothenic Acid; D001710:Biotin; D008063:Thioctic Acid; D012964:Sodium; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; C038780:n-pentanoic acid