Functional characterization of folate transport proteins in staten's seruminstitut rabbit corneal epithelial cell line

J. Jwala, S. H.S. Boddu, D. K. Paturi, S. Shah, Sylvia B Smith, D. Pal, A. K. Mitra

Research output: Contribution to journalArticle

6 Citations (Scopus)

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, 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.

Original languageEnglish (US)
Pages (from-to)404-416
Number of pages13
JournalCurrent Eye Research
Volume36
Issue number5
DOIs
StatePublished - Jan 1 2011

Fingerprint

Folic Acid Transporters
Folic Acid
Epithelial Cells
Rabbits
Cell Line
Proton-Coupled Folate Transporter
Calmodulin
Ions
Sodium Chloride
Vitamins
Protein-Tyrosine Kinases
Permeability
Folate Receptor 1
4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid
Probenecid
Temperature
Colchicine
Cyclic AMP-Dependent Protein Kinases
Methotrexate

Keywords

  • Cornea
  • Folate receptor-alpha
  • Proton coupled folate transporter
  • Reduced folate carrier
  • SIRC cells

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Functional characterization of folate transport proteins in staten's seruminstitut rabbit corneal epithelial cell line. / Jwala, J.; Boddu, S. H.S.; Paturi, D. K.; Shah, S.; Smith, Sylvia B; Pal, D.; Mitra, A. K.

In: Current Eye Research, Vol. 36, No. 5, 01.01.2011, p. 404-416.

Research output: Contribution to journalArticle

Jwala, J. ; Boddu, S. H.S. ; Paturi, D. K. ; Shah, S. ; Smith, Sylvia B ; Pal, D. ; Mitra, A. K. / Functional characterization of folate transport proteins in staten's seruminstitut rabbit corneal epithelial cell line. In: Current Eye Research. 2011 ; Vol. 36, No. 5. pp. 404-416.
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AU - Shah, S.

AU - Smith, Sylvia B

AU - Pal, D.

AU - Mitra, A. K.

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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.

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KW - Folate receptor-alpha

KW - Proton coupled folate transporter

KW - Reduced folate carrier

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