Glutathione release from the rat lens: implications for overall ocular health

Abstract

Purpose : To demonstrate glutathione (GSH) efflux from the rat lens and to identify and functionally characterise the molecular pathways involved.

Methods : Rat lenses were cultured in isosmotic AAH for varying amounts of time and GSH and GSSG (oxidised GSH) released into the media and retained in the lens measured. Lenses were cultured in the absence or presence of acivicin, a γ-glutamyl transpeptidase inhibitor, and GSH/GSSG measured. RT-PCR, western blotting and immunohistochemistry was used to screen and localise GSH efflux transporters which included members of the Multidrug Resistance Associated Proteins (Mrps). To test the involvement of Mrps in GSH release, lenses were cultured in the presence or absence of MK571, an Mrp-specific inhibitor, and GSH/GSSG measured. Finally, lenses were cultured in the presence of low (~10µM) or high (~70µM) levels of H₂O₂ and GSH/GSSG measured in the media and lenses.

Results : The lens is able to release basal levels of GSH (~125mM) under isosmotic conditions. GSH efflux was elevated in the presence of acivicin (~56% increase) indicating that GSH released from the lens undergoes some breakdown into precursor amino acids. GSH efflux was inhibited in the presence of MK571 (~73% inhibition), suggesting GSH release to be primarily mediated by Mrp transporters. Molecular screening identified Mrp1 and Mrp5 to be the most likely isoforms to mediate GSH release from the lens. In the presence of H₂O₂, the release of total GSH was increased (~2 fold increase) and this increase was driven by an increase in GSSG rather than GSH release.

Conclusions : The lens is able to release basal levels of GSH suggesting that it may contribute to maintaining GSH levels in the aqueous humour as well as providing amino acids for subsequent GSH synthesis to nearby tissues. During periods of oxidative stress, the lens releases GSSG which inadvertently may provide protection against oxidative stress to the corneal endothelium, which can utilise GSSG to regenerate GSH. Removal of the lens may reduce the availability of GSH, GSH precursor amino acids and GSSG to other anterior ocular tissues increasing their susceptibility to oxidative damage and the development of secondary ocular pathologies.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.