Functional Characterization of Folate Transport Proteins in Staten’s Seruminstitut Rabbit Corneal Epithelial Cell Line

Abstract

Purpose The overall objective of this study was to investigate and characterize the expression of folate transport proteins in Staten's Seruminstitut rabbit corneal (SIRC) epithelial cell line. Methods [3H]Folic acid uptake was studied with respect to time, pH, temperature, sodium and chloride ion dependency. Inhibition studies were conducted with structural analogs methyltetrahydro folate (MTF) and methotrexate (MTX), vitamins and metabolic inhibitors. [3H]Folic acid uptake was also determined with varying concentrations of cold folic acid. Uptake kinetics was studied in the presence of various modulators of 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) and Western blot analysis were performed to substantiate the expression of folate transport proteins. Ex vivo corneal permeability studies were carried out with [3H]Folic acid in presence and absence of 1mM cold folic acid. Results Linear increase in [3H]Folic acid uptake was observed over 30min. The process followed saturation kinetics with apparent Km of 14.2 nM, Vmax of 1.5*10-5 micro.moles/min/mg protein and Kd of 2.1*10-6 min.-1 Uptake process was found to be dependent on pH, sodium ions, chloride ions, temperature and energy. Uptake was inhibited in the presence of structural analogs (cold folic acid, MTF and MTX) but structurally unrelated vitamins did not show any effect. Membrane transport inhibitors SITS, DIDS, probenecid and endocytic inhibitor, colchicine, significantly inhibited the [3H]Folic acid uptake indicating the involvement of receptor/transporter mediated process. PKA, PTK and Ca2+/calmodulin pathways significantly regulate the process. RT-PCR and Western blot analysis confirmed the presence of folate receptor-α (FR-alpha) and proton-coupled folate transporter (PCFT). Permeability of [3H]Folic acid across rabbit cornea was1.48*10-05 cm/sec, and in the presence of cold folic acid it was 1.08*10-05 cm/sec. Conclusions This work demonstrated the functional and molecular presence of FR-alpha and PCFT in SIRC epithelial cell line. Permeability studies have indicated the existence of folate carrier mediated system across rabbit cornea.

Transporters and receptors in the anterior segment of the eye

“…A highaffi nity transport mechanism for ribofl avin was found to be expressed on the apical side of the cornea. Expression, functional activity of folate receptor α in Statens Seruminstitut rabbit corneal epithelial cells was identifi ed [96]. The uptake of [ 3 H] folic acid (vitamin B9) followed saturation kinetics with apparent K m and V max of 14.2 ± 0.2 n m , and 1.5 × 10 −5 ± 0.1 μ m /min/mg protein, respectively.…”

Differential Expression of Folate Receptor-Alpha, Sodium-Dependent Multivitamin Transporter, and Amino Acid Transporter (B(0, +)) in Human Retinoblastoma (Y-79) and Retinal Pigment Epithelial (ARPE-19) Cell Lines

“…Following aspiration of the culture medium, cell monolayers were washed with DPBS thrice and uptake was performed as mentioned previously. 16 Following the method just outlined, saturation kinetics of all the substrates was studied by performing the uptake in presence of various concentrations of unlabeled cold substrates.…”

“…15 Several transporter/receptor systems such as folate receptoralpha (FR-a), 16 sodium-dependent multivitamin transporter (SMVT), and neutral and cationic amino acid transporter (B (0, + ) ) play a critical role in the internalization of vitamins and amino acids. However, detailed understanding of the transporter/receptor overexpression in cancer cells can aid in designing an effective transporter/receptor-mediated drug delivery system.…”