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N. Mandava, K. Janoria, A.K. Mitra; Identification and Functional Characterization of Sodium–Dependent Multiple Vitamin Transporter (smvt) on Primary Cultured Rabbit Corneal Epithelial Cells (rpcec) . Invest. Ophthalmol. Vis. Sci. 2006;47(13):76.
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© ARVO (1962-2015); The Authors (2016-present)
The objective of this study is to investigate the functional expression of sodium–dependent multiple vitamin transporter (SMVT) on primary cultured rabbit corneal epithelial cells (rPCEC).
Primary cultured rabbit corneal epithelial cells (rPCEC) were employed as the model. Uptake kinetics of [3H] biotin was determined at various concentrations and time points. Competitive inhibition studies were conducted in the presence of unlabelled biotin, its analog (desthiobiotin), pantothenic acid, lipoic acid, B group vitamins, and vitamin C. To delineate the mechanism of uptake; metabolic inhibitors (ouabain, 2,4– dinitrophenol (2,4–DNP) and sodium azide); amiloride, ethionine were employed. Sodium and chloride ion dependency, pH dependency, requirement of valeric acid group and effect of pathway (PKC and PKA mediated pathway) modulators on the uptake of biotin on rPCEC were examined. To generate the molecular evidence of this transporter, RT–PCR studies were also performed on cultured rabbit primary corneal epithelial cells.
The uptake of [3H] biotin into rPCEC was linear over 5 minutes, and found to be saturable with a Km of 32.52 µM and a Vmax of 10.43 pmol/min /mg protein. The process was inhibited by unlabelled biotin and its analog (desthiobiotin) but was unaffected in the presence of vitamins (thiamine, riboflavin, pyridoxine and ascorbic acid). Pantothenic acid and lipoic acid causes about four fold decrease in biotin uptake. Biotin uptake in rPCEC was found to be energy and Na+ dependent but H+ and Cl– independent. Uptake process was inhibited by valeric acid in a concentration dependent manner and was not significantly altered in the presence of biotin conjugates (biotin methyl ester and biocytin) where the carboxyl group was utilized for conjugation. No effect of PKC and PKA mediated pathway modulators on the uptake of biotin was observed. Further, RT–PCR studies identified a band of 652 bp indicating the presence of SMVT on rPCEC.
Sodium–dependent multiple vitamin transporter responsible for the transport of biotin was identified and functionally characterized on primary cultured rabbit corneal epithelial cells.
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