A Glutamate/Cystine Antiporter Is Present in the Lens and Its Activity Is Regulated by Iron

Abstract

Purpose: : Our recent findings that lens epithelial cells (LEC) secrete glutamate and that iron regulates this process led us to question the physiological significance of glutamate secretion by LEC. We determined the presence of a vesicular glutamate transporter, and a recent study described the presence of a glutamate/cystine antiporter (X_c^–) in the lens. These findings suggest that both systems participate in the secretion of glutamate. It was the purpose of this study to characterize the activity and localization of X_c^–, and to determine if iron regulates its function.

Methods: : Primary cultures of canine LEC were used in these studies. The expression of X_c^– by LEC was investigated using RT–PCR and Western blot analysis. Immunohistochemistry using vibratome sections of canine lenses was performed to determine the distribution of the antiporter in situ. The activity of X_c^– was determined by measuring ³⁵S–cystine uptake by LEC under various incubation conditions.

Results: : LEC express the glutamate/cystine antiporter X_c^–, as shown by the presence of its mRNA by RT–PCR and protein by Western analysis. X_c^– was localized prominently at the apical region epithelial/fiber cell interface and in equatorial epithelial cells and fiber cells of the cortex. Cystine can be taken up by a number of different transport systems. In LEC, cystine uptake was Na⁺–independent and inhibited by quisqualate (40%), indicating that at least some of the cystine uptake was mediated by X_c^–. Furthermore, the addition of iron significantly increased cystine uptake by 30%, and this increase was completely inhibited by quisqualate. Therefore, it is likely that the iron–induced increase in cystine uptake was due completely to an increase in X_c^– activity.

Conclusions: : In many tissues, the availability of cystine (cysteine) is rate limiting for the production of glutathione, an essential intracellular reducing agent. Therefore, the secretion of glutamate and uptake of cystine by X_c^– would provide cysteine for glutathione synthesis. Particularly important is the finding that iron increases the uptake of cystine. This would provide reducing power to protect against the potential of iron to catalyze damaging oxidative reactions. The presence of high levels of X_c^– in cells at the equatorial region, where epithelial cells divide and differentiate into fiber cells, may provide more protection for these metabolically active cells. X_c^– labeling at the apical epithelial/fiber interface also suggests that glutamate can be transported further into the lens, thus supporting an intralenticular mechanism for glutamate/cystine exchange.

Keywords: metabolism • microscopy: light/fluorescence/immunohistochemistry • antioxidants