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Jeremy Whitson, Catherine Doller, David Sell, Vincent M Monnier, Xingjun Fan; The Source of Mouse Lens Glutathione in the Absence of De Novo Synthesis. Invest. Ophthalmol. Vis. Sci. 201657(12):.
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© ARVO (1962-2015); The Authors (2016-present)
LEGSKO mouse lenses lack the enzyme GCLC and are unable to synthesize glutathione (GSH) but still to maintain millimolar concentrations of GSH through a yet undescribed transport mechanism. We generated new methods to study GSH transport ex vivo and in vivo to determine the path that GSH takes from the blood to the lens and the mechanisms involved in lens GSH uptake.
Endogenous GSH content of mammalian lenses, aqueous humor, and vitreous humor, as well as ex vivo and in vivo GSH-(glycine-13C2,15N) transport were measured using LC-MS/MS. In vivo GSH transport was determined by catheterizing the carotid artery of mice and perfusing with labeled-GSH. The fluorescent conjugate GS-bimane was used to image GSH transport in vivo.
Mice were found to have 1.1 +/- 0.3 mM GSH and 19.9 +/- 6 uM GSH in their vitreous and aqueous humors, respectively. Ex vivo lens GSH uptake measurements revealed that uptake could be measured from the lens anterior, but only at substrate concentrations above 5-fold those found physiologically. Conversely, uptake did occur rapidly when GSH-(glycine-13C2,15N) was given at concentrations of 1 mM or above in cultured lenses. LEGSKO lens and GSH concentration was determined to be 1.1 +/- 0.2 mM, implying an equilibration between the vitreous and lens GSH pools. This was further indicated by measurements which show that lens and vitreous GSH content decrease equally when LEGSKO mice are treated with BSO, a specific inhibitor of GCLC. In vivo perfusion experiments revealed that GSH-(glycine-13C2,15N), circulating at a concentration of 200 uM, accumulates at an initial rate of ~1.5 uM/min in vitreous but enters the aqueous at a rate of ~80 nM/min. Furthermore, aqueous GSH-(glycine-13C2,15N) was only detectable after >2 uM had already accumulated in the lens. Preliminary in vivo imaging of GS-B transport in mouse eyes further confirms that circulating GSH travels to, and accumulates in, the lens and will be used to determine the tissues involved in the transport.
Mouse vitreous has high GSH content that appears to equilibrate with LEGSKO lenses and provide their high residual GSH concentration. In vivo GSH transport experiments indicate that circulating GSH rapidly enters the vitreous. This is being analyzed in greater detail by imaging the transport of GS-bimane.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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