Purchase this article with an account.
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.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
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, XC– was distributed throughout the lens in a pattern that correlated with the cysteine uptake profile. Changes in the subcellular distribution of XC– occurred as a function of fiber cell differentiation. In the outer cortex, XC– labelling was predominantly cytoplasmic, but progressively became more membraneous with distance into the lens due to XC– insertion into the broad sides of fiber cells. In the core, XC– labelling was localised around the entire membrane of inner fiber cells suggesting a redistribution of the exchanger. Conclusions: Our findings suggest the EAATs and XC– to 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 XC– in 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.
This PDF is available to Subscribers Only