Folic acid transport via high affinity carrier-mediated system in human retinoblastoma cells

Abstract

The primary objective of this study was to investigate the expression of a specialized carrier-mediated system for folic acid and to delineate its uptake mechanism and intracellular trafficking in a human derived retinoblastoma cell line (Y-79). Uptake of [ 3H]Folic acid was determined at various concentrations, pH, temperatures, in the absence of sodium and chloride ions and in the presence of structural analogs, methyltetrahydro folate (MTF) and methotrexate (MTX), vitamins, membrane transport and metabolic inhibitors to delineate the mechanism of uptake. Kinetics of uptake was studied in the presence of various intracellular regulatory pathways; protein kinases A and C (PKA and PKC), protein tyrosine kinase (PTK) and calcium–calmodulin modulators. Reverse transcription polymerase chain reaction (RT-PCR) was performed to confirm the molecular identity of folate carrier systems. The uptake was found to be linear up to 30 min. The rate of uptake followed saturation kinetics with apparent K m of 8.29 ± 0.74 nM, 17.03 ± 1.98 nM and 563.23 ± 115.2 nM and V max of 393.47 ± 9.33, 757.58 ± 26.21 and 653.17 ± 31.7 fmol/(min mg) protein for folic acid, MTF and MTX, respectively. The process was chloride, temperature and energy dependent but sodium and pH independent; inhibited by the structural analogs MTF and MTX but not by structurally unrelated vitamins. Membrane transport inhibitors did not affect the uptake of [ 3H]Folic acid, however endocytic inhibitor, colchicine, significantly inhibited the [ 3H]Folic acid uptake indicating the involvement of receptor mediated endocytosis process. PKC, PTK and Ca 2+/calmodulin pathways appeared to play important roles in the regulation of folic acid uptake. Molecular evidence of the presence of folate receptor (FR) precursor was identified by RT-PCR analysis. This research work demonstrated, for the first time, the functional and molecular existence of a specialized high affinity carrier-mediated system for folic acid uptake, in human retinoblastoma cells.