title: 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 30min. The rate of uptake followed saturation kinetics with apparent Km of 8.29+/-0.74nM, 17.03+/-1.98nM and 563.23+/-115.2nM and Vmax of 393.47+/-9.33, 757.58+/-26.21 and 653.17+/-31.7fmol/(minmg) 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 Ca2+/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.
authors.affiliation: Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, 5005 Rockhill Road, Kansas City, MO 64110-2499, USA.
authors.forename: Viral
authors.initials: V
authors.lastname: Kansara
authors.forename: Durga
authors.initials: D
authors.lastname: Paturi
authors.forename: Shuanghui
authors.initials: S
authors.lastname: Luo
authors.forename: Ripal
authors.initials: R
authors.lastname: Gaudana
authors.forename: Ashim K
authors.initials: AK
authors.lastname: Mitra
mesh_terms: D000963:Antimetabolites / Q000494:pharmacology; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003627:Data Interpretation, Statistical; D004337:Drug Carriers; D004735:Energy Transfer; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005492:Folic Acid / Q000008:administration & dosage* / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D019572:Retinal Neoplasms / Q000378:metabolism*; D012175:Retinoblastoma / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013379:Substrate Specificity; D013696:Temperature; D014815:Vitamins / Q000008:administration & dosage* / Q000378:metabolism*
chemical_list: D000963:Antimetabolites; D004337:Drug Carriers; D004791:Enzyme Inhibitors; D026901:Membrane Transport Proteins; D014815:Vitamins; D005492:Folic Acid; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C