May 2006
Volume 47, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2006
Identification and Functional Characterization of a Riboflavin Transporter in Rabbit Corneal Epithelium and Intact Rabbit Cornea
Author Affiliations & Notes
  • S. Agarwal
    Division of Pharmaceutical Sciences, School of Pharmacy and Vision Research Center, University of Missouri–Kansas City, Kansas City, MO
  • S. Hariharan
    Division of Pharmaceutical Sciences, School of Pharmacy and Vision Research Center, University of Missouri–Kansas City, Kansas City, MO
  • D. Pal
    Division of Pharmaceutical Sciences, School of Pharmacy and Vision Research Center, University of Missouri–Kansas City, Kansas City, MO
  • A. Mitra
    Division of Pharmaceutical Sciences, School of Pharmacy and Vision Research Center, University of Missouri–Kansas City, Kansas City, MO
  • Footnotes
    Commercial Relationships  S. Agarwal, None; S. Hariharan, None; D. Pal, None; A. Mitra, None.
  • Footnotes
    Support  NIH Grant RO1 EY09171
Investigative Ophthalmology & Visual Science May 2006, Vol.47, 5098. doi:
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      S. Agarwal, S. Hariharan, D. Pal, A. Mitra; Identification and Functional Characterization of a Riboflavin Transporter in Rabbit Corneal Epithelium and Intact Rabbit Cornea . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5098.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : The primary objective of the study was to investigate the functional expression of a carrier mediated transport mechanism for riboflavin (Vit. B2) across cultured rabbit primary corneal epithelial cells (rPCEC) and intact rabbit cornea.

Methods: : rPCEC and freshly excised rabbit corneas were selected as in vitro and ex vivo models respectively. Transport and uptake characteristics of [3H] riboflavin were determined at various time points, concentrations, temperatures and pH. Competitive inhibition of [3H] riboflavin uptake was performed in the presence of unlabeled riboflavin, its structural analogues (lumiflavin and lumichrome) and unrelated vitamins (biotin, thiamine, ascorbic acid, folic acid, pyridoxine and nicotinic acid) to delineate the specificity of the transport system. Sodium azide and 2, 4 DNP were used to study the energy dependence of this process. Sodium and chloride free incubation medium were used to examine specific ion dependency of riboflavin transport.

Results: : The uptake process was found to be saturable with Km of 2.05 µM and Vmax of 3.99 pmol/min per mg protein in rPCEC cells. However, riboflavin uptake was independent of pH, Na+ and Cl, but dependent on energy and temperature. Unlabeled riboflavin and its structural analogues caused significant inhibition; whereas unrelated vitamins did not interfere with the process. Transport of [3H] riboflavin across rabbit cornea was also saturable with a Km of 3.27 µM and Vmax of 2.04 pmol/min per cm2. Riboflavin transport was also found to be energy dependent but Na+ independent. Substrate specificity studies produced results similar to the uptake studies in cultured rPCEC.

Conclusions: : Results presented in this article suggest the presence of a specialized, high affinity transport mechanism for riboflavin in rPCEC and intact rabbit corneal epithelium. This transporter may in turn be utilized to deliver topically applied drugs for which the corneal epithelium constitutes a major barrier.

Keywords: cornea: epithelium • protein structure/function • anterior chamber 
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