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Varun Khurana, Aswani Dutt Vadlapudi, Ramya Krishna Vadlapatla, Dhananjay Pal, Ashim Mitra; MOLECULAR IDENTIFICATION AND FUNCTIONAL CHARACTERIZATION OF THE VITAMIN C TRANSPORTERS EXPRESSED BY HUMAN CORNEAL EPITHELIAL CELLS (HCEC). Invest. Ophthalmol. Vis. Sci. 2013;54(15):2574.
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© ARVO (1962-2015); The Authors (2016-present)
The objective of this research was to functionally characterize the Sodium Dependent Vitamin C Transporter (SVCT) in human corneal epithelial cells (HCEC). This nutrient transporter can be targeted to improve the delivery of drugs that get effluxed out by the efflux transporters (P-gp, MRP and BCRP).
Uptake was performed with human corneal epithelial cells (HCEC). Uptake of [14C] ascorbic acid ([14C] AA) was studied in the absence and presence of excess unlabelled AA, anion transporter inhibitors. Effect of pH, sodium and temperature on the uptake of [14C] AA was also studied. Concentration dependency studies (0.1µM-1000µM) were performed. Role of cellular protein kinase mediated pathways and Ca2+/CaM mediated pathways on the regulation of AA uptake has also been studied. Reverse transcription polymerase chain reaction (RT-PCR) for SVCT was carried out on RNA isolated from HCEC.
Uptake of [14C] AA was found to be saturable with a K¬m of 144.87 µM and Vmax of 38.33 pmol/mg protein/min. Uptake of [14C] AA was pH, sodium, energy and temperature dependent. The membrane transporter was also under the regulation of cellular protein kinase C (PKC), protein kinase A (PKA), Ca2+/CaM mediated pathways and metabolic inhibitors. Uptake of [14C] AA was significantly inhibited in the presence of different concentrations of cold L- ascorbic acid and D-Iso ascorbic acid. The observed permeability values for transepithelial transport were 2.29±0.56×10-4, 2.07±0.45×10-4, 1.49±0.38×10-4, 1.8±0.24×10-4 and 1.57±0.66×10-4 cm/s for [14C] AA, L-AA (100µM), L-AA(500µM), D-Iso AA(100µM) and D-Iso AA (500µM) respectively. Ethidium bromide staining of the gel showed a major 626-bp band corresponding with the amplified SVCT2 cDNA.
Sodium Dependent Vitamin C Transporter (SVCT) which plays an important role in the uptake of ascorbic acid has been identified and functionally characterized in HCE cell line. This nutrient transporter can be targeted to improve the delivery of drugs that acts as substrate for the efflux transporters (P-gp, MRP and BCRP). The study also provides useful information on the substrate specificity of this carrier system. This study suggests that HCE cells express the hSVCT2 system and may serve as a useful in vitro experimental model to study the permeability of ascorbic acid conjugated prodrugs.
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