May 2005
Volume 46, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2005
Molecular Mechanisms for Amino Acid Uptake in the Rat Lens
Author Affiliations & Notes
  • J. Lim
    Physiology,
    University of Auckland, Auckland, New Zealand
  • Y. Lam
    Physiology,
    University of Auckland, Auckland, New Zealand
  • J. Kistler
    School of Biological Sciences,
    University of Auckland, Auckland, New Zealand
  • P. Donaldson
    Physiology,
    University of Auckland, Auckland, New Zealand
  • Footnotes
    Commercial Relationships  J. Lim, None; Y. Lam, None; J. Kistler, None; P. Donaldson, None.
  • Footnotes
    Support  Health Research Council of New Zealand Project Grant
Investigative Ophthalmology & Visual Science May 2005, Vol.46, 1128. doi:
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      J. Lim, Y. Lam, J. Kistler, P. Donaldson; Molecular Mechanisms for Amino Acid Uptake in the Rat Lens . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1128.

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

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Abstract

Abstract: : Purpose: To map regions of glutamate and cysteine uptake in the lens and determine whether the excitatory amino acid transporters (EAAT1–5) and the cystine/glutamate exchanger (XC) are responsible for mediating this uptake. Methods: To map areas of amino acid uptake in the lens, immunocytochemistry using antibodies that detect free glutamate and cysteine were used. RT–PCR was used to produce a profile of EAAT1–5 and XC transcript expression and Western blotting utilized to verify protein expression. The localisation of transporters was determined in different regions of the lens by immunocytochemistry. Results: Amino acid profiling showed that glutamate and cysteine levels were high in the outer cortex. In the core, cysteine levels were high but glutamate levels were low suggesting regional differences in amino acid uptake. Transcripts for EAAT1–5 and XC were detected by RT–PCR. Western blotting confirmed expression of all the EAAT isoforms and XC at the protein level. Immunocytochemistry showed the EAATs to be specifically localised to the outer cortex region that coincided with the glutamate uptake pattern. In contrast, XCwas distributed throughout the lens in a pattern that correlated with the cysteine uptake profile. Changes in the subcellular distribution of XCoccurred as a function of fiber cell differentiation. In the outer cortex, XClabelling was predominantly cytoplasmic, but progressively became more membraneous with distance into the lens due to XCinsertion into the broad sides of fiber cells. In the core, XClabelling was localised around the entire membrane of inner fiber cells suggesting a redistribution of the exchanger. Conclusions: Our findings suggest the EAATs and XCto work together to accumulate glutamate and cysteine uptake in the outer cortex of the lens; two precursor amino acids necessary for the synthesis of glutathione; the key lenticular antioxidant. The presence of XCin the centre of the lens suggests this exchanger to work with an alternative glutamate uptake pathway to accumulate cysteine; a known small molecular weight antioxidant. Overall, these studies have provided an emerging molecular model for amino acid uptake in the lens.

Keywords: ion transporters • cell membrane/membrane specializations • immunohistochemistry 
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