PURPOSE: 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.
PURPOSE: 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: Kumar G Janoria; Sai H S Boddu; Zhiying Wang; Durga K Paturi; Swapan Samanta; Dhananjay Pal; Ashim K Mitra Journal: J Ocul Pharmacol Ther Date: 2009-02 Impact factor: 2.671