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Expression and Developmental Regulation of the Cystine/Glutamate Exchanger (x −c ) in the Rat

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Abstract

The cystine/glutamate exchanger (antiporter x −c ) is a membrane transporter involved in the uptake of cystine, the rate-limiting amino acid in the synthesis of glutathione. Recent studies suggest that the antiporter plays a role in the slow oxidative excitotoxity and in the pathological effects of β-N-oxalylamino-l-alanine, the molecule responsible for neurolathyrism, a neurotoxic upper motor neuron disease. The mouse cystine/glutamate exchanger has been cloned and showed to be composed of two distinct proteins, one of which being a novel protein, named xCT, of 502 amino acids and 12 putative trans-membrane domains. We have generated and purified a polyclonal antibody to mouse xCT and studied its expression in rat brain and in different cultured cells (astrocytes, fibroblasts and neurons) using Western blot and immunocytochemical techniques. Expression of xCT was also studied in rat brain and muscle at different developmental stages. Parallel experiments were carried out with antibodies to the heavy chain of 4F2 surface antigen, the non-specific subunit of the antiporter x −c . xCT antibody detected in all cell and tissue extracts a specific band of about 40 kDa. Subcellular fractionation demonstrated that xCT is concentrated mainly in the microsomal-mitochondrial fraction, in accord with its structure as transmembrane protein. Immunocytochemical analysis showed a strong staining in all cells examined, included neurons. Furthermore, both xCT and the heavy chain of 4F2 surface antigen increased in the brain during development, reaching the highest expression in adulthood. The study of the expression and developmental profile of xCT represents a first step towards a better characterization of its biochemical properties and function, which in turn may help to understand the relative contribution of the x −c antiporter in the pathogenesis of certain neurodegenerative diseases.

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Abbreviations

GSH:

Glutathione

ROS:

Reactive oxygen species

BOAA:

β-N-oxalylamino-l-alanine

4F2hc:

Heavy chain of 4F2 surface antigen

CSF:

Cerebrospinal fluid

PVDF:

Polyvinylidene difluoride

KLH:

Keyhole Limphet Hemocyanin

SDS:

Sodium dodecyl sulphate

PAGE:

Polyacrilamide gel electrophoresis

PMSF:

Phenylmethylsulphonyl fluoride

DMEM:

Dulbecco’s modified Eagle’s medium

FCS:

Foetal calf serum

BSA:

Bovine serum albumine

M/M:

Microsomal/mitochondrial fraction

Cyt:

Cytoplasmic fraction

PBS-T:

phosphate-buffered saline—0.2% Tween-20

HRP:

Horseradish peroxidase

IF:

Immunofluorescence

RER:

Rough endoplasmic reticulum

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Acknowledgments

We thank Pasquale and Ernesto Russiello who generously supported the cost of the antibodies used. This work was also supported by a grant from the Italian Ministry of University and Scientific Research (ex 60%).

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Authors and Affiliations

  1. Department of Clinical Neurosciences, Laboratory of Neurochemistry, University of Palermo, 90129 , Palermo, Italy

    Vincenzo La Bella, Francesca Valentino, Tommaso Piccoli & Federico Piccoli

Authors
  1. Vincenzo La Bella
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  2. Francesca Valentino
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  3. Tommaso Piccoli
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  4. Federico Piccoli
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Correspondence to Vincenzo La Bella.

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La Bella, V., Valentino, F., Piccoli, T. et al. Expression and Developmental Regulation of the Cystine/Glutamate Exchanger (x −c ) in the Rat. Neurochem Res 32, 1081–1090 (2007). https://doi.org/10.1007/s11064-006-9277-6

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  • DOI: https://doi.org/10.1007/s11064-006-9277-6

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