Biochemical and Biophysical Research Communications
Protein disulfide isomerase mediates glutathione depletion-induced cytotoxicity
Graphical abstract
Introduction
Glutathione (GSH) is a major endogenous reducing agent involved in phase II metabolic detoxification of various reactive chemical species [1]. GSH depletion is a common phenomenon underlying many forms of oxidative stress and cytotoxicity [2]. Acetaminophen overdose-induced fatal liver damage in humans is probably the best-known clinical example resulting from chemically-induced GSH depletion [3]. It is also widely believed that GSH depletion precedes the development of certain forms of neurodegeneration, such as Parkinson’s disease [4]. Studies have shown that artificial depletion of GSH induces dopaminergic neuronal death in vitro [5] and potentiates neuronal toxicity induced by treating the animals with 6-hydroxydopamine or 1-methyl-4-phenylpyridinium (MPP+) in vivo [6], [7]. Together, these data suggest that GSH depletion contributes to cell death under certain pathogenic conditions [8].
The immortalized HT22 mouse hippocampal neuronal cells lack glutamate receptor, and in recent years, this cell line has become a selective model for studying GSH depletion-induced oxidative cytotoxicity [9]. In this model system, the presence of high concentrations of extracellular glutamate selectively blocks the uptake of cystine (a precursor for GSH synthesis) via the glutamate/cystine antiporter system, and thereby depletes intracellular GSH in a time- and dose-dependent manner [10]. Earlier studies showed that glutamate-induced GSH depletion in HT22 cells leads to reactive oxygen species (ROS) accumulation [11]. Moreover, glutamate induces nitric oxide (NO) formation in cerebellar slices [12] and primary cortical neurons [8], thus leading to the suggestion that NO may be involved in glutamate-induced cytotoxicity. However, the precise mechanism by which glutamate-induced GSH depletion leads to ROS accumulation and cell death is not clear.
In the present study, we sought to use the glutamate-treated HT22 cells as a model system to investigate further the biochemical mechanism as to how GSH depletion leads to ROS formation and ultimately cell death.
Section snippets
Materials
DAF-FM-DA and anti-PDI antibody was purchased from SIGMA-Aldrich (St. Louis, MO). H2-DCF-DA was purchased from Molecular Probe (Eugene, OR). Anti-nNOS and anti-eNOS antibodies were obtained from BD Biosciences (San Jose, CA). Anti-iNOS antibody was from Cell Signaling Technology (Beverly, MA). l-[3H]arginine (Arginine monohydrochloride, [2,3,4-3H]-) was purchased from Perkin Elmer (Boston, MA). siRNA duplexes targeting the mouse PDI and scrambled non-targeting siRNA were purchased from Santa
NO accumulation precedes ROS accumulation and cytotoxicity
To probe whether NO accumulation is involved during GSH depletion-induced oxidative toxicity in HT22 cells, we determined changes in NO levels following glutamate treatment. NO was found to be accumulated by glutamate treatment in a dose- and time-dependent manner (Fig. 1a,b), and its accumulation occurs a little earlier than ROS accumulation (Fig. 1b,c).
To determine whether NO accumulation is involved in ROS accumulation and cell death, we tested the effect of
PDI catalyzes NOS dimerization
The formation of dimer NOS is stabilized by an intermolecular disulfide bond and the dimer NOS is apt for NO production [23]. We postulated that protein disulfide isomerase (PDI) may regulate nNOS dimerization in HT22 cells during glutamate-induced GSH depletion because PDI is a ubiquitous dithiol/disulfide oxidoreductase of the thioredoxin superfamily and is involved in protein processing and translocation by catalyzing intra- and inter-molecular disulfide bridges in proteins [24]. To test
Acknowledgements
The study described here is supported by an Endowment Fund from the University of Kansas Medical Center to Bao-Ting Zhu. We thank Dr. David Schubert (Salk Institute, La Jolla, CA) for providing the HT22 cells as a gift.
References (30)
Oxidative damage in neurodegenerative disease
Lancet
(1994)- et al.
Protein kinase C activation inhibits glutamate-induced cytotoxicity in a neuronal cell line
Brain Res.
(1994) - et al.
Mechanism of glutamate-induced neurotoxicity in HT22 mouse hippocampal cells
Eur. J. Pharmacol.
(2009) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays
J. Immunol. Methods
(1983)- et al.
Over-expression of protein disulfide isomerase reduces the release of growth hormone induced by bisphenol A and/or T3
Mol. Cell. Endocrinol.
(2007) - et al.
Mechanism of superoxide generation by neuronal nitric-oxide synthase
J. Biol. Chem.
(1999) - et al.
Protein disulfide isomerase
Biochim. Biophys. Acta
(2004) - et al.
Assessment and application of the biotin switch technique for examining protein S-nitrosylation under conditions of pharmacologically induced oxidative stress
J. Biol. Chem.
(2007) - et al.
The role of glutathione in the transport and catabolism of nitric oxide
FEBS Lett.
(1996) - et al.
Inhibition of mitochondrial respiratory complex I by nitric oxide, peroxynitrite and S-nitrosothiols
Biochim. Biophys. Acta
(2004)
Glutathione transferases
Annu. Rev. Pharmacol. Toxicol.
Glutathione
Annu. Rev. Biochem.
Metabolism and disposition of acetaminophen: recent advances in relation to hepatotoxicity and diagnosis
Pharm. Res.
Oxidative stress and the pathogenesis of Parkinson’s disease
Neurology
Reduction of brain glutathione by L-buthionine sulfoximine potentiates the dopamine-depleting action of 6-hydroxydopamine in rat striatum
J. Neurochem.
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Present address: Aomori University, Department of Pharmacy, Aomori, 030-0943, Japan.