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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

Visualization of GS-B uptake in cultured lenses. (A) Purity of synthesized GS-B was tested by LC-MS/MS analysis. The GS-B product was found to be 99.75% pure, with trace amounts of GSH and monobromobimane present. (B) The uptake rates of GS-B and GSH-(glycine-13C2,15N) were compared by incubating WT lenses with 2 mM of each compound in uptake buffer for 4 minutes and analyzing the intralenticular accumulation of the compound by LC-MS/MS. There were no significant differences in uptake rates. Values are means ± SD; n = 4. (C) Representative images are shown of GS-B uptake by cultured lenses after a 0, 5, 15, or 30 minutes' incubation with 1 mM GS-B.
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i1552-5783-57-8-3914-f03: Visualization of GS-B uptake in cultured lenses. (A) Purity of synthesized GS-B was tested by LC-MS/MS analysis. The GS-B product was found to be 99.75% pure, with trace amounts of GSH and monobromobimane present. (B) The uptake rates of GS-B and GSH-(glycine-13C2,15N) were compared by incubating WT lenses with 2 mM of each compound in uptake buffer for 4 minutes and analyzing the intralenticular accumulation of the compound by LC-MS/MS. There were no significant differences in uptake rates. Values are means ± SD; n = 4. (C) Representative images are shown of GS-B uptake by cultured lenses after a 0, 5, 15, or 30 minutes' incubation with 1 mM GS-B.

Mentions: Glutathione was reacted with monobromobimane to generate a glutathione-bimane conjugate (GS-B) by adding GSH to a final concentration of 10 mM to a solution of 40 mM monobromobimane, 200 mM N-ethylmorpholine, 20 mM KOH, pH 8.0, and reacting at room temperature in the dark for 30 minutes. GS-B was isolated from the reaction by phase separation after the addition of methylene chloride. The extraction procedure was repeated four times, after which the aqueous phase maintained a bright yellow coloration, indicating the presence of the bimane conjugate, and the organic phase was completely clear. Purity of the GS-B product was assessed by LC-MS/MS (see Fig. 3A).


Evidence of Dual Mechanisms of Glutathione Uptake in the Rodent Lens: A Novel Role for Vitreous Humor in Lens Glutathione Homeostasis
Visualization of GS-B uptake in cultured lenses. (A) Purity of synthesized GS-B was tested by LC-MS/MS analysis. The GS-B product was found to be 99.75% pure, with trace amounts of GSH and monobromobimane present. (B) The uptake rates of GS-B and GSH-(glycine-13C2,15N) were compared by incubating WT lenses with 2 mM of each compound in uptake buffer for 4 minutes and analyzing the intralenticular accumulation of the compound by LC-MS/MS. There were no significant differences in uptake rates. Values are means ± SD; n = 4. (C) Representative images are shown of GS-B uptake by cultured lenses after a 0, 5, 15, or 30 minutes' incubation with 1 mM GS-B.
© 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-f03: Visualization of GS-B uptake in cultured lenses. (A) Purity of synthesized GS-B was tested by LC-MS/MS analysis. The GS-B product was found to be 99.75% pure, with trace amounts of GSH and monobromobimane present. (B) The uptake rates of GS-B and GSH-(glycine-13C2,15N) were compared by incubating WT lenses with 2 mM of each compound in uptake buffer for 4 minutes and analyzing the intralenticular accumulation of the compound by LC-MS/MS. There were no significant differences in uptake rates. Values are means ± SD; n = 4. (C) Representative images are shown of GS-B uptake by cultured lenses after a 0, 5, 15, or 30 minutes' incubation with 1 mM GS-B.
Mentions: Glutathione was reacted with monobromobimane to generate a glutathione-bimane conjugate (GS-B) by adding GSH to a final concentration of 10 mM to a solution of 40 mM monobromobimane, 200 mM N-ethylmorpholine, 20 mM KOH, pH 8.0, and reacting at room temperature in the dark for 30 minutes. GS-B was isolated from the reaction by phase separation after the addition of methylene chloride. The extraction procedure was repeated four times, after which the aqueous phase maintained a bright yellow coloration, indicating the presence of the bimane conjugate, and the organic phase was completely clear. Purity of the GS-B product was assessed by LC-MS/MS (see Fig. 3A).

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