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Y. Hao, V. Kansara, D. Paturi, A. K. Mitra; Uptake Mechanism and Intracellular Regulation of Folic Acid in Human Retinoblastoma Cells: Role of High Affinity Folate Carrier Mediated System. Invest. Ophthalmol. Vis. Sci. 2007;48(13):47.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the presence of a specialized carrier-mediated system for folic acid and delineate uptake mechanism and intracellular trafficking of folic acid in the human derived retinoblastoma cell line (Y-79).
Human derived retinoblastoma cell line, Y-79, was used for uptake studies. Uptake of [3H]Folic acid was determined at various concentrations, pH, temperatures, in the absence of sodium and chloride, in the presence of other vitamins and metabolic inhibitors to delineate the mechanism of uptake. Uptake was determined in the presence of various intracellular regulatory pathways (protein kinase A & C, protein tyrosine kinase and calcium-calmodulin) modulators. Reverse transcription polymerase chain reaction (RT-PCR) was performed to confirm the molecular identity of folate receptor (FR) and reduced folate carrier (RFC).
Uptake of [3H]Folic acid were found to be saturable at nanomolar concentration range, with apparent Km of 8.29 nM and Vmax of 393.47 fmol/min/mg protein. Uptake was temperature, energy, chlordie dependent but sodium and pH independent; inhibited by the structural analogue methyl methyltetrahydrofolate (MTF) and methotrexate (MTX). PKA, PKT and Ca2+/calmodulin pathways appeared to play important roles in the regulation of folic acid uptake in Y-79 cells. A folate receptor alpha and reduced folate carrier (RFC) were identified by RT-PCR.
These studies demonstrated for the first time the existence of a high affinity carrier system for folic acid uptake in human derived retinoblastoma cells.
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