TY - JOUR
T1 - Functional characterization of folate transport proteins in staten's seruminstitut rabbit corneal epithelial cell line
AU - Jwala, J.
AU - Boddu, S. H.S.
AU - Paturi, D. K.
AU - Shah, S.
AU - Smith, S. B.
AU - Pal, D.
AU - Mitra, A. K.
N1 - Funding Information:
This research was supported by Missouri Life Sciences Grant and NIH/NEI—2R01 EY 10659 and NIH/NEI— 2R01 012830 grants.
PY - 2011/5
Y1 - 2011/5
N2 - 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, 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. Ex vivo corneal permeability studies were carried out with [3H]folic acid in the 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±0.2nM, Vmax of (1.5±0.1)*10-5 micro.moles/min/mg protein, and Kd of (2.1±0.2)*10-6 min-1. The 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, methyltetrahydro folate, and methotrexate), 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-αα (FR-alpha) and proton-coupled folate transporter (PCFT). Permeability of [3H]folic acid across the rabbit cornea was (1.48±0.13)*10-05 cm/sec, and in the presence of cold folic acid it was (1.08±0.10) *10-05 cm/sec. Conclusions: This work demonstrated the functional and molecular presence of FR-alpha and PCFT in SIRC epithelial cell line.
AB - 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, 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. Ex vivo corneal permeability studies were carried out with [3H]folic acid in the 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±0.2nM, Vmax of (1.5±0.1)*10-5 micro.moles/min/mg protein, and Kd of (2.1±0.2)*10-6 min-1. The 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, methyltetrahydro folate, and methotrexate), 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-αα (FR-alpha) and proton-coupled folate transporter (PCFT). Permeability of [3H]folic acid across the rabbit cornea was (1.48±0.13)*10-05 cm/sec, and in the presence of cold folic acid it was (1.08±0.10) *10-05 cm/sec. Conclusions: This work demonstrated the functional and molecular presence of FR-alpha and PCFT in SIRC epithelial cell line.
KW - Cornea
KW - Folate receptor-alpha
KW - Proton coupled folate transporter
KW - Reduced folate carrier
KW - SIRC cells
UR - http://www.scopus.com/inward/record.url?scp=79955041489&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=79955041489&partnerID=8YFLogxK
U2 - 10.3109/02713683.2011.566411
DO - 10.3109/02713683.2011.566411
M3 - Article
C2 - 21501073
AN - SCOPUS:79955041489
SN - 0271-3683
VL - 36
SP - 404
EP - 416
JO - Current Eye Research
JF - Current Eye Research
IS - 5
ER -