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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 GSH transport in cultured lenses. All lenses were preincubated with 1 mM BSO and 500 μM acivicin for 1 hour to prevent any breakdown or turnover of GSH-(glycine-13C2,15N). (A) Wild-type and LEGSKO lenses were incubated with 500 μM GSH-(glycine-13C2,15N) at 37°C in 5% CO2 for 1 hour. No significant differences were observed between uptake rates of LEGSKO and those of WT lenses. (B) Wild-type mouse lenses were incubated with 2 mM of each substrate and taken at various time points. GSH-(glycine-13C2,15N) had the lowest initial uptake rate, followed by 15N2-arginine and 13C3-alanine (P < 0.005). (C) Lenses were incubated in various concentrations of substrates and taken for analysis within the initial rate period of uptake. Curves are best fits of the data to the Michaelis-Menten equation. GSH-(glycine-13C2,15N) showed a significantly lower rate of uptake than 13C3-alanine and 15N2-arginine at all concentrations (P < 0.01). (D, E) Lenses were incubated with 2 mM of each substrate at 37°C or 4°C. Lowering temperature had no effect on GSH-(glycine-13C2,15N) uptake but did significantly inhibit uptake of 13C3-alanine, and 15N2-arginine (P < 0.05 and P < 0.001, respectively). 13C3-alanine, and 15N2-arginine were taken up at significantly higher rates than GSH-(glycine-13C2,15N) at 37°C (P < 0.05), but all compounds were taken up at approximately the same rate at 4°C. (F) Lenses were incubated with 1 mM of GSH-(glycine-13C2,15N) at 37°C in 5% CO2 and 5 mM of GSH, γ-EAG, or γ-ESG. There were no significant differences between the uptake rates of GSH-(glycine-13C2,15N) between these groups. (G) To assess how tightly bound to lenses GSH-(glycine-13C2,15N) was, lenses were incubated with 5 mM GSH-(glycine-13C2,15N) for 30 minutes and then washed 6 times in 100 μL of PBS. Each wash fraction was saved and analyzed for GSH-(glycine-13C2,15N) content by LC-MS/MS. (H) The efflux of GSH from lenses was tested by measuring the GSH content of buffer after 1 hour of incubation. This efflux rate was significantly lower than the uptake rate at the GSH concentration found within lenses (P < 0.05), was unaffected by ouabain or Na+-free medium, and increased to match the rate of uptake when GSH-(glycine-13C2,15N) was present in the medium at a higher concentrations than within the lens (10 mM) but not when it was present at a lower concentration (2.5 mM). The increased efflux induced by excess GSH-(glycine-13C2,15N) was inhibited by the connexin inhibitors 18β-glycyrrhetinic acid and octanol at concentrations of 10 μM and 100 μM, respectively. Values are means ± SD for bar graphs and ± SEM for line graphs; n = 4.
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i1552-5783-57-8-3914-f02: Characterization of GSH transport in cultured lenses. All lenses were preincubated with 1 mM BSO and 500 μM acivicin for 1 hour to prevent any breakdown or turnover of GSH-(glycine-13C2,15N). (A) Wild-type and LEGSKO lenses were incubated with 500 μM GSH-(glycine-13C2,15N) at 37°C in 5% CO2 for 1 hour. No significant differences were observed between uptake rates of LEGSKO and those of WT lenses. (B) Wild-type mouse lenses were incubated with 2 mM of each substrate and taken at various time points. GSH-(glycine-13C2,15N) had the lowest initial uptake rate, followed by 15N2-arginine and 13C3-alanine (P < 0.005). (C) Lenses were incubated in various concentrations of substrates and taken for analysis within the initial rate period of uptake. Curves are best fits of the data to the Michaelis-Menten equation. GSH-(glycine-13C2,15N) showed a significantly lower rate of uptake than 13C3-alanine and 15N2-arginine at all concentrations (P < 0.01). (D, E) Lenses were incubated with 2 mM of each substrate at 37°C or 4°C. Lowering temperature had no effect on GSH-(glycine-13C2,15N) uptake but did significantly inhibit uptake of 13C3-alanine, and 15N2-arginine (P < 0.05 and P < 0.001, respectively). 13C3-alanine, and 15N2-arginine were taken up at significantly higher rates than GSH-(glycine-13C2,15N) at 37°C (P < 0.05), but all compounds were taken up at approximately the same rate at 4°C. (F) Lenses were incubated with 1 mM of GSH-(glycine-13C2,15N) at 37°C in 5% CO2 and 5 mM of GSH, γ-EAG, or γ-ESG. There were no significant differences between the uptake rates of GSH-(glycine-13C2,15N) between these groups. (G) To assess how tightly bound to lenses GSH-(glycine-13C2,15N) was, lenses were incubated with 5 mM GSH-(glycine-13C2,15N) for 30 minutes and then washed 6 times in 100 μL of PBS. Each wash fraction was saved and analyzed for GSH-(glycine-13C2,15N) content by LC-MS/MS. (H) The efflux of GSH from lenses was tested by measuring the GSH content of buffer after 1 hour of incubation. This efflux rate was significantly lower than the uptake rate at the GSH concentration found within lenses (P < 0.05), was unaffected by ouabain or Na+-free medium, and increased to match the rate of uptake when GSH-(glycine-13C2,15N) was present in the medium at a higher concentrations than within the lens (10 mM) but not when it was present at a lower concentration (2.5 mM). The increased efflux induced by excess GSH-(glycine-13C2,15N) was inhibited by the connexin inhibitors 18β-glycyrrhetinic acid and octanol at concentrations of 10 μM and 100 μM, respectively. Values are means ± SD for bar graphs and ± SEM for line graphs; n = 4.

Mentions: To obtain detailed kinetic measurements of lens GSH uptake, whole lenses were taken from mice and cultured ex vivo. Lenses were pretreated with BSO, an inhibitor of GCLC, and acivicin, an inhibitor of GGT, to prevent any potential breakdown or synthesis of GSH. These lenses were incubated with isotopically labeled compounds for analysis of uptake by LC-MS/MS Cultured LEGSKO and WT lenses showed no differences in GSH-(glycine-13C2,15N) uptake rates (Fig. 2A). Because this indicates that LEGSKO lenses take up GSH by the same mechanism as WT lenses, WT lenses were used for subsequent experiments.


Evidence of Dual Mechanisms of Glutathione Uptake in the Rodent Lens: A Novel Role for Vitreous Humor in Lens Glutathione Homeostasis
Characterization of GSH transport in cultured lenses. All lenses were preincubated with 1 mM BSO and 500 μM acivicin for 1 hour to prevent any breakdown or turnover of GSH-(glycine-13C2,15N). (A) Wild-type and LEGSKO lenses were incubated with 500 μM GSH-(glycine-13C2,15N) at 37°C in 5% CO2 for 1 hour. No significant differences were observed between uptake rates of LEGSKO and those of WT lenses. (B) Wild-type mouse lenses were incubated with 2 mM of each substrate and taken at various time points. GSH-(glycine-13C2,15N) had the lowest initial uptake rate, followed by 15N2-arginine and 13C3-alanine (P < 0.005). (C) Lenses were incubated in various concentrations of substrates and taken for analysis within the initial rate period of uptake. Curves are best fits of the data to the Michaelis-Menten equation. GSH-(glycine-13C2,15N) showed a significantly lower rate of uptake than 13C3-alanine and 15N2-arginine at all concentrations (P < 0.01). (D, E) Lenses were incubated with 2 mM of each substrate at 37°C or 4°C. Lowering temperature had no effect on GSH-(glycine-13C2,15N) uptake but did significantly inhibit uptake of 13C3-alanine, and 15N2-arginine (P < 0.05 and P < 0.001, respectively). 13C3-alanine, and 15N2-arginine were taken up at significantly higher rates than GSH-(glycine-13C2,15N) at 37°C (P < 0.05), but all compounds were taken up at approximately the same rate at 4°C. (F) Lenses were incubated with 1 mM of GSH-(glycine-13C2,15N) at 37°C in 5% CO2 and 5 mM of GSH, γ-EAG, or γ-ESG. There were no significant differences between the uptake rates of GSH-(glycine-13C2,15N) between these groups. (G) To assess how tightly bound to lenses GSH-(glycine-13C2,15N) was, lenses were incubated with 5 mM GSH-(glycine-13C2,15N) for 30 minutes and then washed 6 times in 100 μL of PBS. Each wash fraction was saved and analyzed for GSH-(glycine-13C2,15N) content by LC-MS/MS. (H) The efflux of GSH from lenses was tested by measuring the GSH content of buffer after 1 hour of incubation. This efflux rate was significantly lower than the uptake rate at the GSH concentration found within lenses (P < 0.05), was unaffected by ouabain or Na+-free medium, and increased to match the rate of uptake when GSH-(glycine-13C2,15N) was present in the medium at a higher concentrations than within the lens (10 mM) but not when it was present at a lower concentration (2.5 mM). The increased efflux induced by excess GSH-(glycine-13C2,15N) was inhibited by the connexin inhibitors 18β-glycyrrhetinic acid and octanol at concentrations of 10 μM and 100 μM, respectively. Values are means ± SD for bar graphs and ± SEM for line graphs; n = 4.
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Related In: Results  -  Collection

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i1552-5783-57-8-3914-f02: Characterization of GSH transport in cultured lenses. All lenses were preincubated with 1 mM BSO and 500 μM acivicin for 1 hour to prevent any breakdown or turnover of GSH-(glycine-13C2,15N). (A) Wild-type and LEGSKO lenses were incubated with 500 μM GSH-(glycine-13C2,15N) at 37°C in 5% CO2 for 1 hour. No significant differences were observed between uptake rates of LEGSKO and those of WT lenses. (B) Wild-type mouse lenses were incubated with 2 mM of each substrate and taken at various time points. GSH-(glycine-13C2,15N) had the lowest initial uptake rate, followed by 15N2-arginine and 13C3-alanine (P < 0.005). (C) Lenses were incubated in various concentrations of substrates and taken for analysis within the initial rate period of uptake. Curves are best fits of the data to the Michaelis-Menten equation. GSH-(glycine-13C2,15N) showed a significantly lower rate of uptake than 13C3-alanine and 15N2-arginine at all concentrations (P < 0.01). (D, E) Lenses were incubated with 2 mM of each substrate at 37°C or 4°C. Lowering temperature had no effect on GSH-(glycine-13C2,15N) uptake but did significantly inhibit uptake of 13C3-alanine, and 15N2-arginine (P < 0.05 and P < 0.001, respectively). 13C3-alanine, and 15N2-arginine were taken up at significantly higher rates than GSH-(glycine-13C2,15N) at 37°C (P < 0.05), but all compounds were taken up at approximately the same rate at 4°C. (F) Lenses were incubated with 1 mM of GSH-(glycine-13C2,15N) at 37°C in 5% CO2 and 5 mM of GSH, γ-EAG, or γ-ESG. There were no significant differences between the uptake rates of GSH-(glycine-13C2,15N) between these groups. (G) To assess how tightly bound to lenses GSH-(glycine-13C2,15N) was, lenses were incubated with 5 mM GSH-(glycine-13C2,15N) for 30 minutes and then washed 6 times in 100 μL of PBS. Each wash fraction was saved and analyzed for GSH-(glycine-13C2,15N) content by LC-MS/MS. (H) The efflux of GSH from lenses was tested by measuring the GSH content of buffer after 1 hour of incubation. This efflux rate was significantly lower than the uptake rate at the GSH concentration found within lenses (P < 0.05), was unaffected by ouabain or Na+-free medium, and increased to match the rate of uptake when GSH-(glycine-13C2,15N) was present in the medium at a higher concentrations than within the lens (10 mM) but not when it was present at a lower concentration (2.5 mM). The increased efflux induced by excess GSH-(glycine-13C2,15N) was inhibited by the connexin inhibitors 18β-glycyrrhetinic acid and octanol at concentrations of 10 μM and 100 μM, respectively. Values are means ± SD for bar graphs and ± SEM for line graphs; n = 4.
Mentions: To obtain detailed kinetic measurements of lens GSH uptake, whole lenses were taken from mice and cultured ex vivo. Lenses were pretreated with BSO, an inhibitor of GCLC, and acivicin, an inhibitor of GGT, to prevent any potential breakdown or synthesis of GSH. These lenses were incubated with isotopically labeled compounds for analysis of uptake by LC-MS/MS Cultured LEGSKO and WT lenses showed no differences in GSH-(glycine-13C2,15N) uptake rates (Fig. 2A). Because this indicates that LEGSKO lenses take up GSH by the same mechanism as WT lenses, WT lenses were used for subsequent experiments.

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