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Evidence of Dual Mechanisms of Glutathione Uptake in the Rodent Lens: A Novel Role for Vitreous Humor in Lens Glutathione Homeostasis

View Article: PubMed Central - PubMed

ABSTRACT

Purpose: Lens glutathione synthesis knockout (LEGSKO) mouse lenses lack de novo glutathione (GSH) synthesis but still maintain >1 mM GSH. We sought to determine the source of this residual GSH and the mechanism by which it accumulates in the lens.

Methods: Levels of GSH, glutathione disulfide (GSSG), and GSH-related compounds were measured in vitro and in vivo using isotope standards and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.

Results: Wild-type (WT) lenses could accumulate GSH from γ-glutamylcysteine and glycine or from intact GSH, but LEGSKO lenses could only accumulate GSH from intact GSH, indicating that LEGSKO lens GSH content is not due to synthesis by a salvage pathway. Uptake of GSH in cultured lenses occurred at the same rate for LEGSKO and WT lenses, could not be inhibited, and occurred primarily through cortical fiber cells. In contrast, uptake of GSH from aqueous humor could be competitively inhibited and showed an enhanced Km in LEGSKO lenses. Mouse vitreous had >1 mM GSH, whereas aqueous had <20 μM GSH. Testing physiologically relevant GSH concentrations for uptake in vivo, we found that both LEGSKO and WT lenses could obtain GSH from the vitreous but not from the aqueous. Vitreous rapidly accumulated GSH from the circulation, and depletion of circulating GSH reduced vitreous but not aqueous GSH.

Conclusions: The above data provide, for the first time, evidence for the existence of dual mechanisms of GSH uptake into the lens, one mechanism being a passive, high-flux transport through the vitreous exposed side of the lens versus an active, carrier-mediated uptake mechanism at the anterior of the lens.

No MeSH data available.


Related in: MedlinePlus

Comparison of concentrations of aqueous, vitreous, and lens GSH in LEGSKO and WT eyes. Aqueous, vitreous, and lenses were dissected from WT and LEGSKO mice and analyzed for total glutathione content. Wild-type and LEGSKO aqueous and vitreous had similar glutathione content, but LEGSKO lenses showed a significant loss of glutathione (P < 0.01). LEGSKO lens glutathione concentration matches LEGSKO vitreous glutathione concentration. Values are means ± SD; n = 4.
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i1552-5783-57-8-3914-f05: Comparison of concentrations of aqueous, vitreous, and lens GSH in LEGSKO and WT eyes. Aqueous, vitreous, and lenses were dissected from WT and LEGSKO mice and analyzed for total glutathione content. Wild-type and LEGSKO aqueous and vitreous had similar glutathione content, but LEGSKO lenses showed a significant loss of glutathione (P < 0.01). LEGSKO lens glutathione concentration matches LEGSKO vitreous glutathione concentration. Values are means ± SD; n = 4.

Mentions: Glutathione content in aqueous, vitreous, and lenses from WT and LEGSKO mice was analyzed by LC-MS/MS (Fig. 5). These data revealed surprisingly high (>1 mM) vitreous GSH content and very low (<20 μM) aqueous GSH content in both LEGSKO and WT lenses. LEGSKO vitreous and lens GSH concentrations were robustly the same, strongly implying an equilibration between the tissues.


Evidence of Dual Mechanisms of Glutathione Uptake in the Rodent Lens: A Novel Role for Vitreous Humor in Lens Glutathione Homeostasis
Comparison of concentrations of aqueous, vitreous, and lens GSH in LEGSKO and WT eyes. Aqueous, vitreous, and lenses were dissected from WT and LEGSKO mice and analyzed for total glutathione content. Wild-type and LEGSKO aqueous and vitreous had similar glutathione content, but LEGSKO lenses showed a significant loss of glutathione (P < 0.01). LEGSKO lens glutathione concentration matches LEGSKO vitreous glutathione concentration. Values are means ± SD; n = 4.
© Copyright Policy - cc-by-nc-nd
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4998144&req=5

i1552-5783-57-8-3914-f05: Comparison of concentrations of aqueous, vitreous, and lens GSH in LEGSKO and WT eyes. Aqueous, vitreous, and lenses were dissected from WT and LEGSKO mice and analyzed for total glutathione content. Wild-type and LEGSKO aqueous and vitreous had similar glutathione content, but LEGSKO lenses showed a significant loss of glutathione (P < 0.01). LEGSKO lens glutathione concentration matches LEGSKO vitreous glutathione concentration. Values are means ± SD; n = 4.
Mentions: Glutathione content in aqueous, vitreous, and lenses from WT and LEGSKO mice was analyzed by LC-MS/MS (Fig. 5). These data revealed surprisingly high (>1 mM) vitreous GSH content and very low (<20 μM) aqueous GSH content in both LEGSKO and WT lenses. LEGSKO vitreous and lens GSH concentrations were robustly the same, strongly implying an equilibration between the tissues.

View Article: PubMed Central - PubMed

ABSTRACT

Purpose: Lens glutathione synthesis knockout (LEGSKO) mouse lenses lack de novo glutathione (GSH) synthesis but still maintain &gt;1 mM GSH. We sought to determine the source of this residual GSH and the mechanism by which it accumulates in the lens.

Methods: Levels of GSH, glutathione disulfide (GSSG), and GSH-related compounds were measured in vitro and in vivo using isotope standards and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.

Results: Wild-type (WT) lenses could accumulate GSH from &gamma;-glutamylcysteine and glycine or from intact GSH, but LEGSKO lenses could only accumulate GSH from intact GSH, indicating that LEGSKO lens GSH content is not due to synthesis by a salvage pathway. Uptake of GSH in cultured lenses occurred at the same rate for LEGSKO and WT lenses, could not be inhibited, and occurred primarily through cortical fiber cells. In contrast, uptake of GSH from aqueous humor could be competitively inhibited and showed an enhanced Km in LEGSKO lenses. Mouse vitreous had &gt;1 mM GSH, whereas aqueous had &lt;20 &mu;M GSH. Testing physiologically relevant GSH concentrations for uptake in vivo, we found that both LEGSKO and WT lenses could obtain GSH from the vitreous but not from the aqueous. Vitreous rapidly accumulated GSH from the circulation, and depletion of circulating GSH reduced vitreous but not aqueous GSH.

Conclusions: The above data provide, for the first time, evidence for the existence of dual mechanisms of GSH uptake into the lens, one mechanism being a passive, high-flux transport through the vitreous exposed side of the lens versus an active, carrier-mediated uptake mechanism at the anterior of the lens.

No MeSH data available.


Related in: MedlinePlus