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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

Characterization of lens GSH uptake from the anterior chamber. (A) Uptake buffer containing various concentrations of GSH-(glycine-13C2,15N) was heated to 37°C and injected into the anterior chambers of WT and LEGSKO mouse eyes after aqueous humor was removed. Lenses were taken for analysis after 30 minutes of incubation. Curves are best fits to the Michaelis-Menten equation. Both of the groups had approximately the same Vmax of 0.21 μM/min, but LEGSKO mice had a lower Km value of ∼50 μM compared to ∼250 μM for WT lenses. Values are means ± SEM; n = 4. (B) Uptake buffer containing 125 μM GSH-(glycine-13C2,15N) and 500 μM GSH, γ-EAG, γ-EAG or none of these was injected into the anterior chambers of WT mouse eyes after removal of the aqueous humor. Lenses were taken for analysis after 30 minutes of incubation. All three compounds significantly inhibited lens uptake of GSH-(glycine-13C2,15N) (∼75%; P < 0.005). There were no significant differences in degree of inhibition among the compounds. Values are means ± SD; n = 4. (C) Permeability of lens surfaces was tested by administering 100 μM lucifer yellow in uptake buffer to cultured lenses or injecting it into the anterior chambers of WT mouse eyes. Cultured lenses showed a significant increase in Pc (P < 0.05) compared to that in lenses treated with lucifer yellow only at the anterior surface. Values are means ± SD; n = 3.
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i1552-5783-57-8-3914-f04: Characterization of lens GSH uptake from the anterior chamber. (A) Uptake buffer containing various concentrations of GSH-(glycine-13C2,15N) was heated to 37°C and injected into the anterior chambers of WT and LEGSKO mouse eyes after aqueous humor was removed. Lenses were taken for analysis after 30 minutes of incubation. Curves are best fits to the Michaelis-Menten equation. Both of the groups had approximately the same Vmax of 0.21 μM/min, but LEGSKO mice had a lower Km value of ∼50 μM compared to ∼250 μM for WT lenses. Values are means ± SEM; n = 4. (B) Uptake buffer containing 125 μM GSH-(glycine-13C2,15N) and 500 μM GSH, γ-EAG, γ-EAG or none of these was injected into the anterior chambers of WT mouse eyes after removal of the aqueous humor. Lenses were taken for analysis after 30 minutes of incubation. All three compounds significantly inhibited lens uptake of GSH-(glycine-13C2,15N) (∼75%; P < 0.005). There were no significant differences in degree of inhibition among the compounds. Values are means ± SD; n = 4. (C) Permeability of lens surfaces was tested by administering 100 μM lucifer yellow in uptake buffer to cultured lenses or injecting it into the anterior chambers of WT mouse eyes. Cultured lenses showed a significant increase in Pc (P < 0.05) compared to that in lenses treated with lucifer yellow only at the anterior surface. Values are means ± SD; n = 3.

Mentions: Glutathione uptake at the anterior lens was characterized by replacing endogenous aqueous humor with uptake buffer containing various concentrations of GSH-(glycine-13C2,15N) (Fig. 4A). Anterior lens GSH uptake in both WT and LEGSKO appeared to be saturable and fit well to the Michaelis-Menten equation, with an apparent Vmax of ∼0.21 μM/min. LEGSKO lens uptake showed an enhanced affinity for GSH with an apparent Km of ∼50 μM compared to Km of ∼250 μM in WT.


Evidence of Dual Mechanisms of Glutathione Uptake in the Rodent Lens: A Novel Role for Vitreous Humor in Lens Glutathione Homeostasis
Characterization of lens GSH uptake from the anterior chamber. (A) Uptake buffer containing various concentrations of GSH-(glycine-13C2,15N) was heated to 37°C and injected into the anterior chambers of WT and LEGSKO mouse eyes after aqueous humor was removed. Lenses were taken for analysis after 30 minutes of incubation. Curves are best fits to the Michaelis-Menten equation. Both of the groups had approximately the same Vmax of 0.21 μM/min, but LEGSKO mice had a lower Km value of ∼50 μM compared to ∼250 μM for WT lenses. Values are means ± SEM; n = 4. (B) Uptake buffer containing 125 μM GSH-(glycine-13C2,15N) and 500 μM GSH, γ-EAG, γ-EAG or none of these was injected into the anterior chambers of WT mouse eyes after removal of the aqueous humor. Lenses were taken for analysis after 30 minutes of incubation. All three compounds significantly inhibited lens uptake of GSH-(glycine-13C2,15N) (∼75%; P < 0.005). There were no significant differences in degree of inhibition among the compounds. Values are means ± SD; n = 4. (C) Permeability of lens surfaces was tested by administering 100 μM lucifer yellow in uptake buffer to cultured lenses or injecting it into the anterior chambers of WT mouse eyes. Cultured lenses showed a significant increase in Pc (P < 0.05) compared to that in lenses treated with lucifer yellow only at the anterior surface. Values are means ± SD; n = 3.
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Related In: Results  -  Collection

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i1552-5783-57-8-3914-f04: Characterization of lens GSH uptake from the anterior chamber. (A) Uptake buffer containing various concentrations of GSH-(glycine-13C2,15N) was heated to 37°C and injected into the anterior chambers of WT and LEGSKO mouse eyes after aqueous humor was removed. Lenses were taken for analysis after 30 minutes of incubation. Curves are best fits to the Michaelis-Menten equation. Both of the groups had approximately the same Vmax of 0.21 μM/min, but LEGSKO mice had a lower Km value of ∼50 μM compared to ∼250 μM for WT lenses. Values are means ± SEM; n = 4. (B) Uptake buffer containing 125 μM GSH-(glycine-13C2,15N) and 500 μM GSH, γ-EAG, γ-EAG or none of these was injected into the anterior chambers of WT mouse eyes after removal of the aqueous humor. Lenses were taken for analysis after 30 minutes of incubation. All three compounds significantly inhibited lens uptake of GSH-(glycine-13C2,15N) (∼75%; P < 0.005). There were no significant differences in degree of inhibition among the compounds. Values are means ± SD; n = 4. (C) Permeability of lens surfaces was tested by administering 100 μM lucifer yellow in uptake buffer to cultured lenses or injecting it into the anterior chambers of WT mouse eyes. Cultured lenses showed a significant increase in Pc (P < 0.05) compared to that in lenses treated with lucifer yellow only at the anterior surface. Values are means ± SD; n = 3.
Mentions: Glutathione uptake at the anterior lens was characterized by replacing endogenous aqueous humor with uptake buffer containing various concentrations of GSH-(glycine-13C2,15N) (Fig. 4A). Anterior lens GSH uptake in both WT and LEGSKO appeared to be saturable and fit well to the Michaelis-Menten equation, with an apparent Vmax of ∼0.21 μM/min. LEGSKO lens uptake showed an enhanced affinity for GSH with an apparent Km of ∼50 μM compared to Km of ∼250 μM in WT.

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