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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

Analysis of the source and mechanism of LEGSKO lens GSH. (A) Treated mice were supplied exclusively with water containing 10 mM BSO for 2 months before analysis by LC-MS/MS. Wild-type mouse lenses were ∼15% depleted of GSH by BSO treatment (P < 0.01). LEGSKO mouse lens GSH content was depleted ∼70% compared to WT before BSO treatment (P < 0.01) and ∼90% depleted from WT after BSO treatment (P < 5E-10). (B) To test for GSH salvage pathway activity, we injected 250 μM of GSH-(glycine-13C2,15N) or glycine-(13C2,15N) and γ-GC into the anterior chambers of WT and LEGSKO mouse eyes. After 30 minutes, lenses were removed and analyzed by LC-MS/MS. Wild-type lenses accumulated equal amounts of GSH-(glycine-13C2,15N) from both treatments. Conversely, LEGSKO mice only accumulated GSH-(glycine-13C2,15N) when it was administered in its intact form (P < 0.05). Values are means ± SD; n = 4.
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i1552-5783-57-8-3914-f01: Analysis of the source and mechanism of LEGSKO lens GSH. (A) Treated mice were supplied exclusively with water containing 10 mM BSO for 2 months before analysis by LC-MS/MS. Wild-type mouse lenses were ∼15% depleted of GSH by BSO treatment (P < 0.01). LEGSKO mouse lens GSH content was depleted ∼70% compared to WT before BSO treatment (P < 0.01) and ∼90% depleted from WT after BSO treatment (P < 5E-10). (B) To test for GSH salvage pathway activity, we injected 250 μM of GSH-(glycine-13C2,15N) or glycine-(13C2,15N) and γ-GC into the anterior chambers of WT and LEGSKO mouse eyes. After 30 minutes, lenses were removed and analyzed by LC-MS/MS. Wild-type lenses accumulated equal amounts of GSH-(glycine-13C2,15N) from both treatments. Conversely, LEGSKO mice only accumulated GSH-(glycine-13C2,15N) when it was administered in its intact form (P < 0.05). Values are means ± SD; n = 4.

Mentions: Using LC-MS/MS methodology, we first confirmed that lens GSH levels were suppressed by >60% (P < 0.01) in LEGSKO mouse lenses and that these levels could be further depleted by treating mice with BSO, an irreversible inhibitor of GCLC (Fig. 1A). LEGSKO lenses were depleted of GSH by >65% (P < 0.005) by using BSO treatment, although WT lenses lost only ∼15% of their GSH (P < 0.01). It has been reported that BSO has very poor penetration through the blood–brain and blood–eye barriers in adult mice20 and will only largely affect GSH levels in circulation and organs not protected by such barriers. These experiments suggest that the residual GSH levels in the LEGSKO lens are linked to a GSH pool that is rapidly replenished from systemic circulation.


Evidence of Dual Mechanisms of Glutathione Uptake in the Rodent Lens: A Novel Role for Vitreous Humor in Lens Glutathione Homeostasis
Analysis of the source and mechanism of LEGSKO lens GSH. (A) Treated mice were supplied exclusively with water containing 10 mM BSO for 2 months before analysis by LC-MS/MS. Wild-type mouse lenses were ∼15% depleted of GSH by BSO treatment (P < 0.01). LEGSKO mouse lens GSH content was depleted ∼70% compared to WT before BSO treatment (P < 0.01) and ∼90% depleted from WT after BSO treatment (P < 5E-10). (B) To test for GSH salvage pathway activity, we injected 250 μM of GSH-(glycine-13C2,15N) or glycine-(13C2,15N) and γ-GC into the anterior chambers of WT and LEGSKO mouse eyes. After 30 minutes, lenses were removed and analyzed by LC-MS/MS. Wild-type lenses accumulated equal amounts of GSH-(glycine-13C2,15N) from both treatments. Conversely, LEGSKO mice only accumulated GSH-(glycine-13C2,15N) when it was administered in its intact form (P < 0.05). Values are means ± SD; n = 4.
© Copyright Policy - cc-by-nc-nd
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4998144&req=5

i1552-5783-57-8-3914-f01: Analysis of the source and mechanism of LEGSKO lens GSH. (A) Treated mice were supplied exclusively with water containing 10 mM BSO for 2 months before analysis by LC-MS/MS. Wild-type mouse lenses were ∼15% depleted of GSH by BSO treatment (P < 0.01). LEGSKO mouse lens GSH content was depleted ∼70% compared to WT before BSO treatment (P < 0.01) and ∼90% depleted from WT after BSO treatment (P < 5E-10). (B) To test for GSH salvage pathway activity, we injected 250 μM of GSH-(glycine-13C2,15N) or glycine-(13C2,15N) and γ-GC into the anterior chambers of WT and LEGSKO mouse eyes. After 30 minutes, lenses were removed and analyzed by LC-MS/MS. Wild-type lenses accumulated equal amounts of GSH-(glycine-13C2,15N) from both treatments. Conversely, LEGSKO mice only accumulated GSH-(glycine-13C2,15N) when it was administered in its intact form (P < 0.05). Values are means ± SD; n = 4.
Mentions: Using LC-MS/MS methodology, we first confirmed that lens GSH levels were suppressed by >60% (P < 0.01) in LEGSKO mouse lenses and that these levels could be further depleted by treating mice with BSO, an irreversible inhibitor of GCLC (Fig. 1A). LEGSKO lenses were depleted of GSH by >65% (P < 0.005) by using BSO treatment, although WT lenses lost only ∼15% of their GSH (P < 0.01). It has been reported that BSO has very poor penetration through the blood–brain and blood–eye barriers in adult mice20 and will only largely affect GSH levels in circulation and organs not protected by such barriers. These experiments suggest that the residual GSH levels in the LEGSKO lens are linked to a GSH pool that is rapidly replenished from systemic circulation.

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