title: Folate (pteroylglutamate) uptake in human red blood cells, erythroid precursors and KB cells at high extracellular folate concentrations. Evidence against a role for specific folate-binding and transport proteins.
abstract: Membrane-associated folate (pteroylglutamate, PteGlu)-binding proteins (FBPs) play an important role as PteGlu-transport proteins in malignant and normal human cells. Since high extracellular folate (PteGlu) concentrations (EFC) profoundly influenced uptake and toxicity of the anti-PteGlu methotrexate in malignant KB cells, we studied human cells to determine additional mechanisms for PteGlu uptake when the EFC was varied. At low EFC (less than 10 nM), the predominant mechanism for folate uptake in mature erythrocytes was through binding to externally oriented FBPs which were quantitatively insignificant (4-6 orders of magnitude lower) and of no apparent physiological relevance when compared with KB cells. However, the predominant mechanism of PteGlu accumulation at high EFC [10-250 nM] in intact erythrocytes and sealed right-side-out (RSO) ghosts was not FBP-mediated and non-specific. This conclusion was based on the findings that radiolabelled PteGlu uptake: (i) continued even in the presence of a 1000-fold excess of unlabelled PteGlu and was linear and not saturable up to 250 nM; (ii) was two-fold higher at pH 4.5 than 7.5; (iii) was less than 2-fold increased at 37 degrees C compared with 4 degrees C; and (iv) was unaffected after trypsin-mediated proteolysis of greater than 75% FBPs. The [3H]PteGlu and 125I-PteGlu (histamine derivative) accumulated intracellularly through the non-specific PteGlu-uptake mechanism was unaltered biochemically and in a soluble compartment. Raising the EFC 500-fold higher than controls during erythropoiesis in vitro resulted in reversal of the expected anti-(placental folate-receptor)-antiserum-induced megaloblastic changes in orthochromatic normoblasts derived from burst-forming unit-erythroid colonies. Furthermore, at EFC greater than 0.1 microM, KB-cell accumulation of [3H]PteGlu was also predominantly through a mechanism that did not involve specific FBPs. Thus, at high EFC, a major component of PteGlu transport in human cells is not mediated through FBPs and is likely to be a passive diffusion process.
authors.affiliation: Department of Medicine, Indiana University School of Medicine, Indianapolis 46223.
authors.forename: A C
authors.initials: AC
authors.lastname: Antony
authors.forename: M A
authors.initials: MA
authors.lastname: Kane
authors.forename: S R
authors.initials: SR
authors.lastname: Krishnan
authors.forename: R S
authors.initials: RS
authors.lastname: Kincade
authors.forename: R S
authors.initials: RS
authors.lastname: Verma
mesh_terms: D002352:Carrier Proteins / Q000097:blood / Q000378:metabolism*; D002478:Cells, Cultured; D004910:Erythrocyte Membrane / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism*; D004920:Erythropoiesis; D058973:Folate Receptors, GPI-Anchored; D005492:Folic Acid / Q000097:blood / Q000378:metabolism*; D006801:Humans; D009303:Nasopharyngeal Neoplasms / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D011956:Receptors, Cell Surface; D005492:Folic Acid