Limits...
Glutathione disulfide induces neural cell death via a 12-lipoxygenase pathway.

Park HA, Khanna S, Rink C, Gnyawali S, Roy S, Sen CK - Cell Death Differ. (2009)

Bottom Line: Inhibition of GSSG reductase by BCNU is known to result in GSSG accumulation and caused cell death in a 12-Lox-sensitive manner.GSSG S-glutathionylated purified 12-Lox as well as in a model of glutamate-induced HT4 cell death in vitro where V5-tagged 12-Lox was expressed in cells.Countering glutamate-induced 12-Lox S-glutathionylation by glutaredoxin-1 overexpression protected against cell death.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Davis Heart and Lung Research Institute, The Ohio State University Medical Center, Columbus, OH 43210, USA.

ABSTRACT
Oxidized glutathione (GSSG) is commonly viewed as a byproduct of GSH metabolism. The pathophysiological significance of GSSG per se remains poorly understood. Adopting a microinjection approach to isolate GSSG elevation within the cell, this work identifies that GSSG can trigger neural HT4 cell death via a 12-lipoxygenase (12-Lox)-dependent mechanism. In vivo, stereotaxic injection of GSSG into the brain caused lesion in wild-type mice but less so in 12-Lox knockout mice. Microinjection of graded amounts identified 0.5 mM as the lethal [GSSG]i in resting cells. Interestingly, this threshold was shifted to the left by 20-fold (0.025 mM) in GSH-deficient cells. This is important because tissue GSH lowering is commonly noted in the context of several diseases as well as in aging. Inhibition of GSSG reductase by BCNU is known to result in GSSG accumulation and caused cell death in a 12-Lox-sensitive manner. GSSG S-glutathionylated purified 12-Lox as well as in a model of glutamate-induced HT4 cell death in vitro where V5-tagged 12-Lox was expressed in cells. Countering glutamate-induced 12-Lox S-glutathionylation by glutaredoxin-1 overexpression protected against cell death. Strategies directed at improving or arresting cellular GSSG clearance may be effective in minimizing oxidative stress-related tissue injury or potentiating the killing of tumor cells, respectively.

Show MeSH

Related in: MedlinePlus

Cytosolic injection of GSSG compromised mitochondrial membrane potentialHT4 cells were injected with either PBS (A, Dextran alexa fluor 488; B, 8nM TMRM; C, 0.5 μl/ml PMPI; D, merged image) or 500 attomole GSSG (E, Dextran alexa fluor 488; F, 8nM TMRM; G, 0.5 μl/ml PMPI; H, merged image). Dextran alexa fluor 488 was co-injected to visualize injected cells. After 2h incubation, 8nM TMRM and 0.5 μl/ml PMPI were treated to cell culture medium. I, GSSG injected cells showed lower mitochondrial membrane potential (F) compared to sham injected cells (B) while plasma membrane potential was not affected (C and G). attomoles injected [micromolar]i, n=3, Bar, 30 μm. Results are mean ± S.D. *, p < 0.05.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2990696&req=5

Figure 3: Cytosolic injection of GSSG compromised mitochondrial membrane potentialHT4 cells were injected with either PBS (A, Dextran alexa fluor 488; B, 8nM TMRM; C, 0.5 μl/ml PMPI; D, merged image) or 500 attomole GSSG (E, Dextran alexa fluor 488; F, 8nM TMRM; G, 0.5 μl/ml PMPI; H, merged image). Dextran alexa fluor 488 was co-injected to visualize injected cells. After 2h incubation, 8nM TMRM and 0.5 μl/ml PMPI were treated to cell culture medium. I, GSSG injected cells showed lower mitochondrial membrane potential (F) compared to sham injected cells (B) while plasma membrane potential was not affected (C and G). attomoles injected [micromolar]i, n=3, Bar, 30 μm. Results are mean ± S.D. *, p < 0.05.

Mentions: Characterization of GSSG-induced death of HT4 neural cells was started by testing the involvement of mitochondrial dysfunction as is commonly associated with cell death. Cytosolic injection of GSSG was observed to selectively compromise mitochondrial membrane potential while not affecting plasma membrane potential (Fig. 3). Previously, we had reported that GSH-depletion in glutamate-challenged HT4 neural cells leads to the activation of 12-lipoxygenase which is central in executing glutamate-induced neural cell death. Inhibitors of 12-lipoxygenase, including BL-15 and α-tocotrienol, prevent glutamate-induced HT4 cell death (5, 6). We therefore sought to examine whether GSSG-induced death of HT4 neural cells is mediated by 12-lipoxygenae. Both inhibitors of 12-lipoxygenase, BL-15 as well as α-tocotrienol, significantly protected against GSSG-induced loss of cell viability suggesting the involvement of 12-lipoxygenase in this death pathway (Fig. 4). Next, we utilized the advantages of the microinjection approach to test the significance of 12-lipoxygenase and its substrate arachidonic acid in the death of GSH-deficient HT4 cells. While depletion of cellular GSH reserves by arresting GSH synthesis using BSO does not cause cell death, such GSH-deficient cells are known to be highly sensitive to extracellular arachidonic acid treatment (5). Consistently, in this study we observed that microinjection of small amounts of free arachidonic acid to GSH-deficient HT4 cells caused cell death (Fig. 5). Both BL-15 as well as α-tocotrienol protected against such death suggesting the involvement of 12-lipoxygenase in intracellular free arachidonic acid induced cell death. Supporting this conclusion is our observation that direct administration of active 12-lipoxygenase into GSH-deficient cells causes cell death in a BL-15 and α-tocotrienol sensitive manner (Fig. 5). Next, we sought to test whether endogenous GSSG may kill cells by a 12-lipoxygenase dependent mechanism. BCNU, a well-characterized GSSG reductase inhibitor, was chosen as a pharmacological tool to increase cellular GSSG:GSH ratio (8, 9). Indeed, BCNU-induced elevation of cellular GSSG caused cell death. BCNU-induced cell death was significantly lowered by BL-15 as well as α-tocotrienol suggesting the involvement of 12-lipoxygenase (Fig. 6A–F). Inhibition of GSSG efflux by MK-571, an inhibitor for multidrug resistance protein-1 (MRP1)(10, 11), significantly increased loss of HT4 viability (Fig. 6G).


Glutathione disulfide induces neural cell death via a 12-lipoxygenase pathway.

Park HA, Khanna S, Rink C, Gnyawali S, Roy S, Sen CK - Cell Death Differ. (2009)

Cytosolic injection of GSSG compromised mitochondrial membrane potentialHT4 cells were injected with either PBS (A, Dextran alexa fluor 488; B, 8nM TMRM; C, 0.5 μl/ml PMPI; D, merged image) or 500 attomole GSSG (E, Dextran alexa fluor 488; F, 8nM TMRM; G, 0.5 μl/ml PMPI; H, merged image). Dextran alexa fluor 488 was co-injected to visualize injected cells. After 2h incubation, 8nM TMRM and 0.5 μl/ml PMPI were treated to cell culture medium. I, GSSG injected cells showed lower mitochondrial membrane potential (F) compared to sham injected cells (B) while plasma membrane potential was not affected (C and G). attomoles injected [micromolar]i, n=3, Bar, 30 μm. Results are mean ± S.D. *, p < 0.05.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2990696&req=5

Figure 3: Cytosolic injection of GSSG compromised mitochondrial membrane potentialHT4 cells were injected with either PBS (A, Dextran alexa fluor 488; B, 8nM TMRM; C, 0.5 μl/ml PMPI; D, merged image) or 500 attomole GSSG (E, Dextran alexa fluor 488; F, 8nM TMRM; G, 0.5 μl/ml PMPI; H, merged image). Dextran alexa fluor 488 was co-injected to visualize injected cells. After 2h incubation, 8nM TMRM and 0.5 μl/ml PMPI were treated to cell culture medium. I, GSSG injected cells showed lower mitochondrial membrane potential (F) compared to sham injected cells (B) while plasma membrane potential was not affected (C and G). attomoles injected [micromolar]i, n=3, Bar, 30 μm. Results are mean ± S.D. *, p < 0.05.
Mentions: Characterization of GSSG-induced death of HT4 neural cells was started by testing the involvement of mitochondrial dysfunction as is commonly associated with cell death. Cytosolic injection of GSSG was observed to selectively compromise mitochondrial membrane potential while not affecting plasma membrane potential (Fig. 3). Previously, we had reported that GSH-depletion in glutamate-challenged HT4 neural cells leads to the activation of 12-lipoxygenase which is central in executing glutamate-induced neural cell death. Inhibitors of 12-lipoxygenase, including BL-15 and α-tocotrienol, prevent glutamate-induced HT4 cell death (5, 6). We therefore sought to examine whether GSSG-induced death of HT4 neural cells is mediated by 12-lipoxygenae. Both inhibitors of 12-lipoxygenase, BL-15 as well as α-tocotrienol, significantly protected against GSSG-induced loss of cell viability suggesting the involvement of 12-lipoxygenase in this death pathway (Fig. 4). Next, we utilized the advantages of the microinjection approach to test the significance of 12-lipoxygenase and its substrate arachidonic acid in the death of GSH-deficient HT4 cells. While depletion of cellular GSH reserves by arresting GSH synthesis using BSO does not cause cell death, such GSH-deficient cells are known to be highly sensitive to extracellular arachidonic acid treatment (5). Consistently, in this study we observed that microinjection of small amounts of free arachidonic acid to GSH-deficient HT4 cells caused cell death (Fig. 5). Both BL-15 as well as α-tocotrienol protected against such death suggesting the involvement of 12-lipoxygenase in intracellular free arachidonic acid induced cell death. Supporting this conclusion is our observation that direct administration of active 12-lipoxygenase into GSH-deficient cells causes cell death in a BL-15 and α-tocotrienol sensitive manner (Fig. 5). Next, we sought to test whether endogenous GSSG may kill cells by a 12-lipoxygenase dependent mechanism. BCNU, a well-characterized GSSG reductase inhibitor, was chosen as a pharmacological tool to increase cellular GSSG:GSH ratio (8, 9). Indeed, BCNU-induced elevation of cellular GSSG caused cell death. BCNU-induced cell death was significantly lowered by BL-15 as well as α-tocotrienol suggesting the involvement of 12-lipoxygenase (Fig. 6A–F). Inhibition of GSSG efflux by MK-571, an inhibitor for multidrug resistance protein-1 (MRP1)(10, 11), significantly increased loss of HT4 viability (Fig. 6G).

Bottom Line: Inhibition of GSSG reductase by BCNU is known to result in GSSG accumulation and caused cell death in a 12-Lox-sensitive manner.GSSG S-glutathionylated purified 12-Lox as well as in a model of glutamate-induced HT4 cell death in vitro where V5-tagged 12-Lox was expressed in cells.Countering glutamate-induced 12-Lox S-glutathionylation by glutaredoxin-1 overexpression protected against cell death.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Davis Heart and Lung Research Institute, The Ohio State University Medical Center, Columbus, OH 43210, USA.

ABSTRACT
Oxidized glutathione (GSSG) is commonly viewed as a byproduct of GSH metabolism. The pathophysiological significance of GSSG per se remains poorly understood. Adopting a microinjection approach to isolate GSSG elevation within the cell, this work identifies that GSSG can trigger neural HT4 cell death via a 12-lipoxygenase (12-Lox)-dependent mechanism. In vivo, stereotaxic injection of GSSG into the brain caused lesion in wild-type mice but less so in 12-Lox knockout mice. Microinjection of graded amounts identified 0.5 mM as the lethal [GSSG]i in resting cells. Interestingly, this threshold was shifted to the left by 20-fold (0.025 mM) in GSH-deficient cells. This is important because tissue GSH lowering is commonly noted in the context of several diseases as well as in aging. Inhibition of GSSG reductase by BCNU is known to result in GSSG accumulation and caused cell death in a 12-Lox-sensitive manner. GSSG S-glutathionylated purified 12-Lox as well as in a model of glutamate-induced HT4 cell death in vitro where V5-tagged 12-Lox was expressed in cells. Countering glutamate-induced 12-Lox S-glutathionylation by glutaredoxin-1 overexpression protected against cell death. Strategies directed at improving or arresting cellular GSSG clearance may be effective in minimizing oxidative stress-related tissue injury or potentiating the killing of tumor cells, respectively.

Show MeSH
Related in: MedlinePlus