Glutathione metabolism and transport pathways in the anterior rat eye: Is the lens a reservoir of glutathione?

Abstract

Maintaining glutathione (GSH) homeostasis is critical for the proper functioning of ocular tissues. Despite the important roles of GSH as an antioxidant and in detoxification of compounds by conjugation, relatively little is known regarding the molecular identity of pathways utilised by anterior ocular tissues to regulate GSH levels. Therefore, in this thesis, I have mapped GSH metabolism and transport pathways in the rat ciliary body, trabecular meshwork, and cornea and revealed that these tissues express similar mechanisms for the synthesis, uptake, efflux, and degradation of GSH. Molecular screening for GSH efflux transporters in the rat lens identified the multidrug resistance-associated proteins (Mrp) 1, 4, 5, and organic anion transporting polypeptide (Oatp) 1a4 at the transcript level. Only Mrp1, 4, and 5 were detected at the protein level with all three isoforms localised to the lens epithelial membrane, positioning these transporters in the ideal location for mediating GSH conjugate and/or GSH/GSSG efflux from the lens. Functional assays confirmed that the rat lens was capable of mediating GSH conjugate release and pharmacological experiments revealed that the Mrps were the predominant transporters in mediating this process. This indicates that the lens contains the molecular machinery to detoxify and remove unwanted compounds to minimise oxidative stress. Under isosmotic conditions, the rat lens was also shown to be able to release GSH levels comparable to those previously measured in the aqueous humour. This suggests that the rat lens can release basal levels of GSH and, in addition to the ciliary body, may also contribute to setting GSH levels in the aqueous humour. Culturing lenses in physiological levels of H2O2 resulted in the predominant release of oxidised glutathione (GSSG), rather than GSH. In addition, exposure to higher levels of H2O2 resulted in increased GSSG release. This implies that, under normal physiological conditions, the lens predominantly exports GSSG into the aqueous humour. This, in turn, would minimise oxidative stress in the lens as well as supply GSSG to the corneal endothelium, which preferentially accumulates GSSG and subsequently regenerates GSH to afford protection against oxidative stress. Taken together, my findings reveal that anterior ocular tissues depend on each other to regulate and maintain GSH homeostasis and, in particular, highlights the central role of the lens in mediating the removal of GSH conjugates and the export of GSH/GSSG into the aqueous humour to protect tissues against oxidative stress and thus, preserve overall ocular health.