Hydrogen sulfide protects neurons from oxidative ... - Semantic Scholar
The FASEB Journal express article 10.1096/fj.04-1815fje. Published online May 20, 2004.
Hydrogen sulfide protects neurons from oxidative stress Yuka Kimura and Hideo Kimura National Institute of Neuroscience, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187-8502, Japan Corresponding author: Hideo Kimura, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187-8502, Japan. E-mail: [email protected] ABSTRACT Hydrogen sulfide (H2S), which is a well-known toxic gas, is found in relatively high concentrations in the brain. Although a neuromodulatory role of H2S has been demonstrated, little is known of its other biological functions. Here we show that H2S protects primary cultures of neurons from death in a well-studied model of oxidative stress caused by glutamate, a process called oxidative glutamate toxicity—or oxytosis. We found that H2S increases the glutathione levels, which normally decrease during the cell death cascade, by enhancing the activity of γglutamylcysteine synthetase and up-regulating cystine transport. Cystine (cysteine) is the ratelimiting substrate of glutathione synthesis. These observations reveal that H2S protects neurons from oxytosis by increasing the production of the antioxidant glutathione. Key words: glutamate toxicity • oxytosis • cystine transport • glutathione • neuroprotection
E
ndogenous levels of hydrogen sulfide (H2S) between 50 and 160 µM are found in the brains of rats, humans, and bovine (1–3). Physiological concentrations of H2S specifically potentiate the activity of the N-methyl-D-aspartate (NMDA) receptor and enhance the induction of hippocampal long-term potentiation (LTP), a synaptic model of learning and memory (4). H2S also increases intracellular concentrations of Ca2+ and induces Ca2+ waves in astrocytes (5). Based upon these observations it has been proposed that H2S may function as a neuromodulator in the brain (4, 5). Two forms of glutamate toxicity exist: receptor-initiated excitotoxicity (6) and non-receptormediated oxidative glutamate toxicity (7). Oxidative glutamate toxicity, recently renamed oxytosis (8), is a well-studied programmed cell death pathway that is independent of ionotropic glutamate receptors (7–9). It has been observed in primary cultures of neuronal cells (10), neuronal cell lines (7, 11, 12), and brain slices (13). Oxytosis is initiated by high concentrations of extracellular glutamate in newly plated neuronal primary cultures that have not yet expressed ionotropic glutamate receptors (14, 15) and is not suppressed by the antagonists of ionotropic glutamate receptors (15). Glutamate shares the same amino acid transporter with cystine, and it competes with cystine for transport into cells (16). Therefore, elevated extracellular glutamate inhibits the transport of cystine. Cystine is the primary source of intracellular cysteine necessary for glutathione synthesis. Glutathione, the ubiquitous tripeptide γ-glutamylcysteinyl glycine, exists in the thiol-reduced (GSH) and disulfide-oxidized (GSSG) forms and has several functions, including detoxifying Page 1 of 16 (page number not for citation purposes)
electrophiles, reducing disulfide bonds induced by oxidative stress, and scavenging free radicals (17). GSH synthesis is catalyzed by a rate-limiting enzyme, γ-glutamylcysteine synthetase (γGCS), and GSH synthetase (18, 19). GSH is also generated by the reduction of GSSG by GSSG reductase (17). Severe oxidative stress may overcome the ability of cells to reduce GSSG to GSH, resulting in the transport of GSSG into the extracellular space and the depletion of intracellular GSH (20). Oxidative stress is responsible for neuronal damage and degeneration in brain disorders, including stroke, epilepsy, and Alzheimer’s disease (21, 22). It is not known whether or not H2S has the ability to mediate the redox status of cells. The present study demonstrates that H2S increases the activity of γ-GCS and causes the recovery of cystine transport suppressed by glutamate, resulting in an increase in the levels of glutathione in neurons. These observations suggest that H2S may function as a neuroprotectant against oxidative stress. MATERIALS AND METHODS Cell culture and toxicity assay Primary cortical neurons were prepared from embryonic Day 17 Sprague Dawley rats as described (10). Cells were dissociated from the cortex and maintained in modified Eagles medium (MEM) supplemented with 30 mM glucose, 2 mM glutamine, 1 mM pyruvate, and 10% fetal calf serum. For toxicity studies, cells were plated on poly-D-lysine-coated 96-well microtiter dishes at 50,000 cells/100 µl in each well and exposed to glutamate or BSO in the presence or absence of NaHS (Aldrich, Milwaukee, WI) 24 h after the initial plating. The WST-8 (a tetrazolium salt, [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2Htetrazolium, monosodium salt]) viability assay was performed with the cell counting kit-8 (Dojindo, Kumamoto, Japan) 24 h after the application of glutamate. WST-8 (10 µl of 10 mM) was added to each well, cells were incubated at 37°C, and absorption values at 450 nm were measured. The results obtained by WST assay were confirmed by LDH assays and visual counting. The measurement of glutathione Total intracellular reduced (GSH) and oxidized (GSSG) glutathione were measured by a glutathione assay kit (Cayman chemical, Ann Arbor, MI) according to the manufacture’s instructions. Cells were plated at 9 × 106 cells per 10cm poly-D-lysine coated dish. After 16 h of incubation, cells were exposed to glutamate in the presence or absence of NaHS and harvested at 2, 4, 6, and 8 h after the treatment. The cell pellet was sonicated in cold buffer (50 mM MES, pH 7; 1 mM EDTA) and deproteinated with 7% 5-sulfosalicylic acid. After centrifugation at 10,000 × g for 15 min at 4°C, the supernatant was used for the assay. The amount of GSH plus GSSG in the sample was estimated by measuring the absorbance at 405 nm by using a microplate reader model 3550 (Bio-Rad Laboratories, Hercules, CA). Pure GSSG was used to obtain a standard curve. GSH and GSSG was measured by reverse-phase ion exchange high-performance liquid chromatography (HPLC; 23). Briefly, cells were harvested in 10% perchloric acid with 1 mM bathophenanthroline disulfonic acid and sonicated. After centrifugation at 15, 000 × g for 3 min, 10 µl of 100 mM iodoacetic acid in 0.2 mM cresol purple was added to 100 µl of supernatant.
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Samples were neutralized by adding 96 µl of 2M KOH/2.4 M KHCO3 and incubated in the dark at room temperature for 10 min. Then 200 µl of 1% fluorodinitrobenzene was added and stored at 4°C overnight. After centrifugation at 15, 000 × g for 3 min, 50 µl of supernatant was injected into the HPLC with UV detection at 365 nm (Waters 2690 separations module and 2487 dual λ absorbance detector, Waters, Milford, MA) and separated by Waters Spherisorb NH2 column (4.6 × 250 mm: 5 µm). Measurement of the levels of γ-GC and cysteine The amount of γ-GC and cysteine was measured by modifying the method described in Herschbach et al. (38). Briefly, primary cultures of cortical neurons were treated with 1 mM glutamate, 100 µM NaHS, or both for 2, 4, 6, and 8 h. Cells were washed twice with ice-cold PBS and harvested in phosphate buffer (0.1 M NaH2PO4, pH 5.8; 2 mM EDTA; with 0.05 mg/ml acivicin for the measurement of γ-GC; or without acivicin for cysteine). After sonication, cell lysates were centrifuged at 16,000 × g for 10 min, and supernatants were derivatized for HPLC. The supernatant (75 µl) was mixed with 0.5 M CHES (2-[cyclohexylamino]-ethanesulfonic acid), pH 8.4, then derivatized with 4 µl of 50 mM monobromobimane (mBBr) for 15 min in the dark. The reaction was terminated by adding 10 µl of 30% (v/v) acetic acid. Samples were analyzed with a Beckman Ultrasphere ODS (250 × 4.6-mm ID) column. The mBBr adduct was monitored by scanning fluorescence detector (Waters 474) with an excitation wavelength at 370 nm and an emission wavelength at 485 nm. Measurement of γ-GCS mRNA levels The amount of mRNA was measured by real-time PCR using SYBR green dye (Applied Biosystems, Foster City, CA). Rat primary cortical cultures were treated with 1 mM glutamate for 2 h in the presence or absence of 100 µM NaHS. RNA was isolated by Trizol (Invitrogen, Carlsbad, CA) and used to synthesize the first-strand cDNA using SuperScript First-strand synthesis system for RT-PCR (Invitrogen, Carlsbad, CA). Three sets of primers were designed for catalytic and modifier subunits of γ-GCS using Primer Express 1.5 (Applied Biosystems). PCR was performed with SYBR Green PCR master mix (Applied Biosystems) and detected by ABI PRISM 7700 sequence detection system (Applied Biosystems). One set of primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control. Cystine uptake Cells were exposed to 1 mM glutamate in the presence or absence of NaHS. After 0, 2, 4, 6, and 8 h, cells were washed once with prewarmed (37°C) HEPES (N-[2-hydroxyethyl]piperazine-N′ethanesulfonic acid)-buffered medium and incubated with 400 µl HEPES-buffered medium containing 50 µM L-[35S] cystine (40–250 mCi/mmol, Amersham, Pharmacia Biotech, Cleveland, OH) and 50 µM cold L-cystine in 12-well plates. After incubation at 37°C for 20 min, the medium was aspirated. Cells were then washed three times with ice-cold PBS and lysed with 250 µl of 1% Triton/PBS. Radioactivity was counted with 100 µl, and the remaining was used for protein assays. Results were expressed as the amount of 35S incorporated per milligram of protein and then converted to the percentage of cystine uptake by comparison with the mocktreated samples in each experiment.
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Measurement of the activity of GSSG reductase Cells were washed twice with ice-cold PBS and harvested in ice-cold assay buffer (50 mM potassium phosphate, pH 7.5; 1 mM EDTA). After sonication, cell lysates were centrifuged at 10,000 × g for 15 min at 4°C. The activity of GSSG reductase in the supernatant was measured using a glutathione reductase assay kit (Cayman Chemical, Ann Arbor, MI). Briefly, 20 µl supernatants, 100 µl assay buffer, and 20 µl 9.5 mM GSSG solution were mixed. The enzyme reaction was initiated by adding 50 µl NADPH. GSSG reductase activity was determined as a decrease in absorbance at 340 nm using a microplate reader (Benchmark plus, BioRad). Statistics The data were analyzed by using StatView software (Abacus concepts, Berkeley, CA) for oneway ANOVA with post-hoc testing using the Fisher’s PLSD multiple comparison test. RESULTS Because H2S, an endogenous reducing agent, is produced by oxidative stress (24), it is possible that H2S functions as an antioxidant. To investigate this possibility, the effect of H2S on oxytosis was examined using primary cultures of neurons. Primary cultures of cortical immature neurons, which lack ionotropic glutamate receptors during their first few days in culture (10), were prepared from 17-day-old embryonic rat brains and cultured for 1 day. Most of neurons die within 24 h after the application of 1 mM glutamate, because glutamate inhibits cystine uptake causing oxidative stress-induced cell death, a process called oxytosis. Neurons simultaneously treated with 100 µM NaHS are viable (Fig. 1A). H2S protects cells from glutamate toxicity in a dose-dependent manner, and ED50 values of NaHS against 1 mM and 5 mM glutamate toxicity are 47 ± 11 µM and 93 ± 19 µM, respectively (Fig. 1B). H2S alone caused a significant increase in survival following plating, protecting cells from the spontaneous cell death that occurs in primary cultures (25). Because the application of glutamate reduces intracellular glutathione and glutathione is an endogenous antioxidant (26), the effect of H2S on the accumulation of glutathione was examined. NaHS (100 µM) alone increases the levels of GSH + GSSG 2 h after its application. The maximum is reached after 4 h, and then the level gradually decreases at 8 h (Fig. 2A). The effect of H2S on intracellular glutathione lowered by glutamate was also examined. The amount of glutathione is decreased in a time-dependent manner by 1 mM glutamate and reaches ~30% of the control at 8 h (Fig. 2A). In the presence of 100 µM NaHS and 1 mM glutamate, the levels of glutathione increase to more than the controls and almost reach the levels achieved by the application of NaHS alone at later times (Fig. 2A). These observations show that H2S causes the recovery of intracellular glutathione that is lowered by glutamate. Because the reduced form of glutathione can protect cells from oxidative stress, it is necessary to examine the redox state of glutathione in the nerve cell cultures. The amounts of GSH and GSSG in the presence or absence of H2S were measured by HPLC. In primary cultures GSH was 93% of GSH + GSSG (Fig. 2B). Eight hours after the addition of H2S, the amount of GSH increases to 193 ± 3% of the control, while the concentration of GSSG is not significantly changed. In contrast, the amounts of both GSH and GSSG are decreased by glutamate. When the glutamatetreated cells are simultaneously exposed to H2S, the amounts of both GSH and GSSG are largely
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restored and reach the levels achieved by the application of H2S alone (Fig. 2B). These observations indicate that H2S increases the GSH levels both in untreated cells and in cells where GSH is normally depleted by glutamate. To investigate the requirement of glutathione for cell survival induced by H2S, the effect of a specific inhibitor of γ-glutamylcysteine synthetase (γ-GCS), buthionine sulfoximine (BSO; 26) on glutathione levels, and cell survival in the presence or absence of H2S was examined. BSO dose-dependently suppressed both the levels of glutathione and cell survival even in the presence of H2S (Fig. 2C, D). These observations suggest that protection of neuronal cells by H2S requires the increase in glutathione levels. Because H2S increases the GSH levels, it is possible that H2S enhances the activity of γ-GCS and increases the production of γ-glutamylcysteine (γ-GC). To examine this possibility, changes in the levels of γ-GC after the application of glutamate in the presence or absence of H2S were examined. In the presence of H2S the levels of γ-GC in cells are increased more than twofold of those in cells in the absence of H2S 2 h after the application and decreased thereafter (Fig. 3). At two hours, even in the presence of glutamate, H2S increases the levels of γ-GC in cells approximately twofold. At four hours, the levels of γ-GC start decreasing (Fig. 3). These observations show that H2S increases the levels of γ-GC that lead to the increase in the levels of glutathione. To investigate whether the increase in the levels of γ-GC induced by H2S is caused by the transcriptional regulation of γ-GCS, the levels of γ-GCS mRNA were measured by the quantitative polymerase chain reaction (PCR). The levels of γ-GCS mRNA were not changed 2 h after the application of H2S, which suggests that the increase in the levels of γ-GC induced by H2S is not caused by the transcriptional regulation of γ-GCS mRNA (data not shown). Glutathione is synthesized from cysteine that is produced from cystine transported into cells from the outside (16). Because oxytosis is caused by the blockade of the cystine/glutamate antiporter that couples the import of cystine and the export of glutamate (7, 27), the effect of H2S on cystine transport was examined. The transport of cystine into primary neurons is significantly increased by 200 µM NaHS 2 h after its application (Fig. 4A). The effect of H2S on cystine transport suppressed by 1 mM glutamate was also examined. In the presence of glutamate, cystine transport is reduced to 12 ± 3% of control at 20 min and 33 ± 4% at 2 h. 200 µM NaHS significantly reversed the inhibition of cystine transport by glutamate at times up to 4 h, but with the diminished effect of glutamate at 6 and 8 h, there was no difference at these time points (Fig. 4B). The H2S induced-recovery of glutamate-suppressed cystine transport may therefore be involved in the increased production of glutathione and neuroprotection. The cystine uptake by the cystine/glutamate antiporter xc– mediates oxytosis (28). To examine the effect of H2S on antiporter xc–, the effects of inhibitors for xc– were examined. The specific inhibitor for xc–, glutamate, significantly suppresses the cystine uptake, and this inhibition is significantly reduced by NaHS (Fig. 5). Because H2S is a reducing agent, it is possible that H2S reduces cystine to cysteine and enhances the transport of cysteine. To investigate this possibility, the effect of H2S on ASC (alanine, serine, and cysteine) transporter was examined. The inhibitors for ASC transporter, alanine and serine, do not significantly inhibit the cysteine uptake. In addition, alanine and serine do not significantly inhibit the cysteine uptake in the presence of NaHS, which suggests that the possibility that H2S increases the transport of cysteine by
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enhancing the activity of the ASC transporter is excluded. These observations suggest that the antiporter xc– may be involved in the cystine transport recovered by H2S. Because H2S enhances cystine transport, changes in the endogenous levels of cysteine in the presence of H2S were examined. In the presence of H2S, the levels of cysteine in cells are increased approximately sixfold relative to those in cells in the absence of H2S 2 h after application and they gradually decreased thereafter (Fig. 6A). At 2 h, even in the presence of glutamate, H2S increases the levels of cysteine in cells approximately twofold. At 4 h, the levels of cysteine start decreasing, but are still twofold of those in the absence of H2S (Fig. 6A). These observations show that H2S increases the levels of cysteine. Because cells synthesize little cysteine themselves (29), H2S must function by enhancing cystine transport, which leads to the increase in the levels of γ-GC and glutathione. To further examine the effect of H2S on cystine transport, the effect of extracellular concentrations of cystine on the glutathione levels in the presence or absence of H2S was investigated. At each concentration of extracellular cystine, the glutathione levels are increased by greater than twofold in the presence of H2S relative to those in the absence of H2S after 2 h of application. When the extracellular concentrations of cystine are decreased, glutathione levels are decreased both in the presence or absence of H2S, indicating that the enhancing effect of H2S on the glutathione levels is dependent on the extracellular concentrations of cystine (Fig. 6B). These observations confirm that H2S enhances cystine transport to increase the levels of glutathione. DISCUSSION Sulfur containing substances, dimethylsulphoniopropionate (DMSP) and its enzymatic cleavage product dimethylsulphide (DMS), have recently been identified as endogenous scavengers for hydroxyl radicals and other reactive oxygen species in marine algae (30). Because H2S is a reducing agent that readily reacts with hydrogen peroxide (H2O2; 31), it is possible that endogenous H2S can scavenge oxygen species. The present study, however, shows that H2S protects neurons from oxidative stress by increasing glutathione levels instead of functioning directly as an antioxidant. The endogenous levels of glutathione (1–8 mM; 32) are much greater than those of H2S (50–160 µM; 33). Therefore, H2S does not itself rescue cells from oxidative stress, but H2S induces the production of a major and potent antioxidant, glutathione. Cells can be rescued from oxidative stress by mechanisms that are either dependent upon or independent of glutathione metabolism. For example, antioxidants such as vitamin E protect neuronal cells from oxytosis by acting directly as antioxidants even when the intracellular glutathione levels are decreased (7, 34). In contrast, dihydroxyphenylglycine, an agonist of group I metabotropic glutamate receptors, protects neurons by up-regulating glutathione (35). Because H2S rescues neurons by increasing the accumulation of glutathione, the protection from oxytosis by H2S belongs to the latter class of mechanisms. The levels of γ-GC and cysteine in cells treated with H2S reach a peak 2 h after the application of H2S, whereas the glutathione levels start increasing 2 h after the application of H2S and last 6 h (Figs. 2A, 3, and 6A). A similar observation was made in plants (36). The level of γ-GC is increased and reaches a peak 1 h after the application of cysteine in plants, whereas glutathione
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levels are increased for several hours, which suggests a general mechanism for the regulation of the glutathione levels. H2S is an active molecule and has a strong effect on several targets. For example, H2S potentiates the induction of LTP by enhancing the activity of NMDA receptors in neurons, and it activates calcium channels to induce calcium waves in astrocytes (4, 5). In smooth muscle, H2S activates ATP-dependent potassium channels (37). The present study shows that H2S enhances the activity of γ-GCS and increases the levels of γ-GC (Fig. 3). Although the contribution is less critical than γ-GCS, the activity of cystine/glutamate antiporter xc– is also enhanced by H2S. The combined enhancement of the activity of these different targets may cause an integrated effect that results in the increase in the levels of glutathione. Although the function is not well understood, the uptake of atmospheric H2S by leaves also increases the levels of glutathione in plants (38), which suggests that H2S activates a common pathway in plants and animals to accumulate glutathione. In conclusion, H2S protects neurons against glutamate-mediated oxidative stress, oxytosis, through the pleiotropic effects of maintaining the activities of γ-GCS and cystine transport, which leads to the increase in glutathione levels. H2S may therefore have a significant neuroprotective role in the nervous system. ACKNOWLEDGMENT We thank Drs. Y. Enokido, and S. Aoki, for technical advice. This work was supported by a grant from the National Institute of Neuroscience to H. K. REFERENCES 1.
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Fig. 1
Figure 1. H2S protects neurons from oxytosis. A) Protection of neurons by H2S from glutamate toxicity. Primary
cultures of neurons 24 h after the application of 1 mM glutamate in the presence or absence of 100 µM NaHS are shown. The bar represents 100 µm. B) The dose-dependent protection of H2S against oxytosis. Relative survival of neurons 24 h after the simultaneous application of glutamate and NaHS was measured with the WST assay and confirmed by visual counting: –|-, without glutamate; --, 1 mM glutamate; -z-, 5 mM glutamate.
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Fig. 2
Figure 2. H2S causes the recovery of the glutathione levels decreased by glutamate. A) The time course of the recovery of the total glutathione levels caused by H2S. The effect of 100 µM NaHS on the levels of glutathione in neurons was determined 2, 4, 6, and 8 h after the application of 1 mM glutamate. **P < 0.01 and *P < 0.05 by ANOVA. B) Recovery of GSH by H2S. The amounts of GSH (filled bars) and GSSG (shaded bars) 8 h after the application of 1 mM glutamate, 100 µM NaHS, or both were measured by HPLC. H,100 µM NaHS; G, 1 mM glutamate; HG, both NaHS and glutamate. C) BSO decreases the levels of glutathione. D) BSO decreases cell survival. BSO (10 or 30 µM) was applied to cells in the presence or absence of 100 µM NaHS and the glutathione levels determined at 8 h (C) and cell survival at 24 h (D). All data represent the mean ± SEM of at least four experiments.
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Fig. 3
Figure 3. H2S increases the levels of γ–GC. The endogenous levels of γ–GC were measured by HPLC in the presence
or absence of H2S after 2, 4, 6, and 8 h after the application. H, 100 µM NaHS; G, 1 mM glutamate; HG, both NaHS and glutamate. **P < 0.01 by ANOVA. All the data represent the mean ± SEM of at least four experiments.
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Fig. 4
Figure 4. H2S enhances the cystine uptake. A) Increase in uptake of cystine into neurons induced by H2S. The
incorporation of [35S]-cystine after the application of NaHS in the absence of glutamate was measured. B) H2S-induced recovery of cystine uptake suppressed by glutamate. The effect of NaHS on the incorporation of [35S]-cystine into primary cultures of neurons was measured in the presence of 1 mM glutamate. **P < 0.01 and *P < 0.05 by ANOVA (A, B). All the data represent the mean ± SEM of at least four experiments.
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Fig. 5
Figure 5. The effect of inhibitors on the cystine uptake. The effect of 1 mM glutamate, alanine, or serine on the incorporation of [35S]-cystine into primary cultures of neurons in the presence or absence of NaHS was measured. **P < 0.01 and *P < 0.05 by ANOVA. All the data represent the mean ± SEM of at least four experiments.
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Fig. 6
Figure 6. H2S increases the levels of cysteine and glutathione. A) The endogenous levels of cysteine were measured by HPLC in the presence or absence of H2S after 2, 4, 6, and 8 h after the application. H: 100 µM NaHS; G: 1 mM glutamate; HG: both NaHS and glutamate. **P < 0.001 and *P < 0.05 by ANOVA. B) The effect of H2S on increasing glutathione levels is dependent on extracellular concentrations of cystine. The glutathione levels were measured in the presence or absence of 100 µM NaHS. Cys 100, 10, and 0 represent 100 or 10 µM cystine or cystine-free medium, respectively. All the data represent the mean ± SEM of at least three experiments. Page 16 of 16 (page number not for citation purposes)
Hydrogen sulfide protects neurons from oxidative stress Yuka Kimura and Hideo Kimura National Institute of Neuroscience, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187-8502, Japan Corresponding author: Hideo Kimura, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187-8502, Japan. E-mail: [email protected] ABSTRACT Hydrogen sulfide (H2S), which is a well-known toxic gas, is found in relatively high concentrations in the brain. Although a neuromodulatory role of H2S has been demonstrated, little is known of its other biological functions. Here we show that H2S protects primary cultures of neurons from death in a well-studied model of oxidative stress caused by glutamate, a process called oxidative glutamate toxicity—or oxytosis. We found that H2S increases the glutathione levels, which normally decrease during the cell death cascade, by enhancing the activity of γglutamylcysteine synthetase and up-regulating cystine transport. Cystine (cysteine) is the ratelimiting substrate of glutathione synthesis. These observations reveal that H2S protects neurons from oxytosis by increasing the production of the antioxidant glutathione. Key words: glutamate toxicity • oxytosis • cystine transport • glutathione • neuroprotection
E
ndogenous levels of hydrogen sulfide (H2S) between 50 and 160 µM are found in the brains of rats, humans, and bovine (1–3). Physiological concentrations of H2S specifically potentiate the activity of the N-methyl-D-aspartate (NMDA) receptor and enhance the induction of hippocampal long-term potentiation (LTP), a synaptic model of learning and memory (4). H2S also increases intracellular concentrations of Ca2+ and induces Ca2+ waves in astrocytes (5). Based upon these observations it has been proposed that H2S may function as a neuromodulator in the brain (4, 5). Two forms of glutamate toxicity exist: receptor-initiated excitotoxicity (6) and non-receptormediated oxidative glutamate toxicity (7). Oxidative glutamate toxicity, recently renamed oxytosis (8), is a well-studied programmed cell death pathway that is independent of ionotropic glutamate receptors (7–9). It has been observed in primary cultures of neuronal cells (10), neuronal cell lines (7, 11, 12), and brain slices (13). Oxytosis is initiated by high concentrations of extracellular glutamate in newly plated neuronal primary cultures that have not yet expressed ionotropic glutamate receptors (14, 15) and is not suppressed by the antagonists of ionotropic glutamate receptors (15). Glutamate shares the same amino acid transporter with cystine, and it competes with cystine for transport into cells (16). Therefore, elevated extracellular glutamate inhibits the transport of cystine. Cystine is the primary source of intracellular cysteine necessary for glutathione synthesis. Glutathione, the ubiquitous tripeptide γ-glutamylcysteinyl glycine, exists in the thiol-reduced (GSH) and disulfide-oxidized (GSSG) forms and has several functions, including detoxifying Page 1 of 16 (page number not for citation purposes)
electrophiles, reducing disulfide bonds induced by oxidative stress, and scavenging free radicals (17). GSH synthesis is catalyzed by a rate-limiting enzyme, γ-glutamylcysteine synthetase (γGCS), and GSH synthetase (18, 19). GSH is also generated by the reduction of GSSG by GSSG reductase (17). Severe oxidative stress may overcome the ability of cells to reduce GSSG to GSH, resulting in the transport of GSSG into the extracellular space and the depletion of intracellular GSH (20). Oxidative stress is responsible for neuronal damage and degeneration in brain disorders, including stroke, epilepsy, and Alzheimer’s disease (21, 22). It is not known whether or not H2S has the ability to mediate the redox status of cells. The present study demonstrates that H2S increases the activity of γ-GCS and causes the recovery of cystine transport suppressed by glutamate, resulting in an increase in the levels of glutathione in neurons. These observations suggest that H2S may function as a neuroprotectant against oxidative stress. MATERIALS AND METHODS Cell culture and toxicity assay Primary cortical neurons were prepared from embryonic Day 17 Sprague Dawley rats as described (10). Cells were dissociated from the cortex and maintained in modified Eagles medium (MEM) supplemented with 30 mM glucose, 2 mM glutamine, 1 mM pyruvate, and 10% fetal calf serum. For toxicity studies, cells were plated on poly-D-lysine-coated 96-well microtiter dishes at 50,000 cells/100 µl in each well and exposed to glutamate or BSO in the presence or absence of NaHS (Aldrich, Milwaukee, WI) 24 h after the initial plating. The WST-8 (a tetrazolium salt, [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2Htetrazolium, monosodium salt]) viability assay was performed with the cell counting kit-8 (Dojindo, Kumamoto, Japan) 24 h after the application of glutamate. WST-8 (10 µl of 10 mM) was added to each well, cells were incubated at 37°C, and absorption values at 450 nm were measured. The results obtained by WST assay were confirmed by LDH assays and visual counting. The measurement of glutathione Total intracellular reduced (GSH) and oxidized (GSSG) glutathione were measured by a glutathione assay kit (Cayman chemical, Ann Arbor, MI) according to the manufacture’s instructions. Cells were plated at 9 × 106 cells per 10cm poly-D-lysine coated dish. After 16 h of incubation, cells were exposed to glutamate in the presence or absence of NaHS and harvested at 2, 4, 6, and 8 h after the treatment. The cell pellet was sonicated in cold buffer (50 mM MES, pH 7; 1 mM EDTA) and deproteinated with 7% 5-sulfosalicylic acid. After centrifugation at 10,000 × g for 15 min at 4°C, the supernatant was used for the assay. The amount of GSH plus GSSG in the sample was estimated by measuring the absorbance at 405 nm by using a microplate reader model 3550 (Bio-Rad Laboratories, Hercules, CA). Pure GSSG was used to obtain a standard curve. GSH and GSSG was measured by reverse-phase ion exchange high-performance liquid chromatography (HPLC; 23). Briefly, cells were harvested in 10% perchloric acid with 1 mM bathophenanthroline disulfonic acid and sonicated. After centrifugation at 15, 000 × g for 3 min, 10 µl of 100 mM iodoacetic acid in 0.2 mM cresol purple was added to 100 µl of supernatant.
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Samples were neutralized by adding 96 µl of 2M KOH/2.4 M KHCO3 and incubated in the dark at room temperature for 10 min. Then 200 µl of 1% fluorodinitrobenzene was added and stored at 4°C overnight. After centrifugation at 15, 000 × g for 3 min, 50 µl of supernatant was injected into the HPLC with UV detection at 365 nm (Waters 2690 separations module and 2487 dual λ absorbance detector, Waters, Milford, MA) and separated by Waters Spherisorb NH2 column (4.6 × 250 mm: 5 µm). Measurement of the levels of γ-GC and cysteine The amount of γ-GC and cysteine was measured by modifying the method described in Herschbach et al. (38). Briefly, primary cultures of cortical neurons were treated with 1 mM glutamate, 100 µM NaHS, or both for 2, 4, 6, and 8 h. Cells were washed twice with ice-cold PBS and harvested in phosphate buffer (0.1 M NaH2PO4, pH 5.8; 2 mM EDTA; with 0.05 mg/ml acivicin for the measurement of γ-GC; or without acivicin for cysteine). After sonication, cell lysates were centrifuged at 16,000 × g for 10 min, and supernatants were derivatized for HPLC. The supernatant (75 µl) was mixed with 0.5 M CHES (2-[cyclohexylamino]-ethanesulfonic acid), pH 8.4, then derivatized with 4 µl of 50 mM monobromobimane (mBBr) for 15 min in the dark. The reaction was terminated by adding 10 µl of 30% (v/v) acetic acid. Samples were analyzed with a Beckman Ultrasphere ODS (250 × 4.6-mm ID) column. The mBBr adduct was monitored by scanning fluorescence detector (Waters 474) with an excitation wavelength at 370 nm and an emission wavelength at 485 nm. Measurement of γ-GCS mRNA levels The amount of mRNA was measured by real-time PCR using SYBR green dye (Applied Biosystems, Foster City, CA). Rat primary cortical cultures were treated with 1 mM glutamate for 2 h in the presence or absence of 100 µM NaHS. RNA was isolated by Trizol (Invitrogen, Carlsbad, CA) and used to synthesize the first-strand cDNA using SuperScript First-strand synthesis system for RT-PCR (Invitrogen, Carlsbad, CA). Three sets of primers were designed for catalytic and modifier subunits of γ-GCS using Primer Express 1.5 (Applied Biosystems). PCR was performed with SYBR Green PCR master mix (Applied Biosystems) and detected by ABI PRISM 7700 sequence detection system (Applied Biosystems). One set of primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control. Cystine uptake Cells were exposed to 1 mM glutamate in the presence or absence of NaHS. After 0, 2, 4, 6, and 8 h, cells were washed once with prewarmed (37°C) HEPES (N-[2-hydroxyethyl]piperazine-N′ethanesulfonic acid)-buffered medium and incubated with 400 µl HEPES-buffered medium containing 50 µM L-[35S] cystine (40–250 mCi/mmol, Amersham, Pharmacia Biotech, Cleveland, OH) and 50 µM cold L-cystine in 12-well plates. After incubation at 37°C for 20 min, the medium was aspirated. Cells were then washed three times with ice-cold PBS and lysed with 250 µl of 1% Triton/PBS. Radioactivity was counted with 100 µl, and the remaining was used for protein assays. Results were expressed as the amount of 35S incorporated per milligram of protein and then converted to the percentage of cystine uptake by comparison with the mocktreated samples in each experiment.
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Measurement of the activity of GSSG reductase Cells were washed twice with ice-cold PBS and harvested in ice-cold assay buffer (50 mM potassium phosphate, pH 7.5; 1 mM EDTA). After sonication, cell lysates were centrifuged at 10,000 × g for 15 min at 4°C. The activity of GSSG reductase in the supernatant was measured using a glutathione reductase assay kit (Cayman Chemical, Ann Arbor, MI). Briefly, 20 µl supernatants, 100 µl assay buffer, and 20 µl 9.5 mM GSSG solution were mixed. The enzyme reaction was initiated by adding 50 µl NADPH. GSSG reductase activity was determined as a decrease in absorbance at 340 nm using a microplate reader (Benchmark plus, BioRad). Statistics The data were analyzed by using StatView software (Abacus concepts, Berkeley, CA) for oneway ANOVA with post-hoc testing using the Fisher’s PLSD multiple comparison test. RESULTS Because H2S, an endogenous reducing agent, is produced by oxidative stress (24), it is possible that H2S functions as an antioxidant. To investigate this possibility, the effect of H2S on oxytosis was examined using primary cultures of neurons. Primary cultures of cortical immature neurons, which lack ionotropic glutamate receptors during their first few days in culture (10), were prepared from 17-day-old embryonic rat brains and cultured for 1 day. Most of neurons die within 24 h after the application of 1 mM glutamate, because glutamate inhibits cystine uptake causing oxidative stress-induced cell death, a process called oxytosis. Neurons simultaneously treated with 100 µM NaHS are viable (Fig. 1A). H2S protects cells from glutamate toxicity in a dose-dependent manner, and ED50 values of NaHS against 1 mM and 5 mM glutamate toxicity are 47 ± 11 µM and 93 ± 19 µM, respectively (Fig. 1B). H2S alone caused a significant increase in survival following plating, protecting cells from the spontaneous cell death that occurs in primary cultures (25). Because the application of glutamate reduces intracellular glutathione and glutathione is an endogenous antioxidant (26), the effect of H2S on the accumulation of glutathione was examined. NaHS (100 µM) alone increases the levels of GSH + GSSG 2 h after its application. The maximum is reached after 4 h, and then the level gradually decreases at 8 h (Fig. 2A). The effect of H2S on intracellular glutathione lowered by glutamate was also examined. The amount of glutathione is decreased in a time-dependent manner by 1 mM glutamate and reaches ~30% of the control at 8 h (Fig. 2A). In the presence of 100 µM NaHS and 1 mM glutamate, the levels of glutathione increase to more than the controls and almost reach the levels achieved by the application of NaHS alone at later times (Fig. 2A). These observations show that H2S causes the recovery of intracellular glutathione that is lowered by glutamate. Because the reduced form of glutathione can protect cells from oxidative stress, it is necessary to examine the redox state of glutathione in the nerve cell cultures. The amounts of GSH and GSSG in the presence or absence of H2S were measured by HPLC. In primary cultures GSH was 93% of GSH + GSSG (Fig. 2B). Eight hours after the addition of H2S, the amount of GSH increases to 193 ± 3% of the control, while the concentration of GSSG is not significantly changed. In contrast, the amounts of both GSH and GSSG are decreased by glutamate. When the glutamatetreated cells are simultaneously exposed to H2S, the amounts of both GSH and GSSG are largely
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restored and reach the levels achieved by the application of H2S alone (Fig. 2B). These observations indicate that H2S increases the GSH levels both in untreated cells and in cells where GSH is normally depleted by glutamate. To investigate the requirement of glutathione for cell survival induced by H2S, the effect of a specific inhibitor of γ-glutamylcysteine synthetase (γ-GCS), buthionine sulfoximine (BSO; 26) on glutathione levels, and cell survival in the presence or absence of H2S was examined. BSO dose-dependently suppressed both the levels of glutathione and cell survival even in the presence of H2S (Fig. 2C, D). These observations suggest that protection of neuronal cells by H2S requires the increase in glutathione levels. Because H2S increases the GSH levels, it is possible that H2S enhances the activity of γ-GCS and increases the production of γ-glutamylcysteine (γ-GC). To examine this possibility, changes in the levels of γ-GC after the application of glutamate in the presence or absence of H2S were examined. In the presence of H2S the levels of γ-GC in cells are increased more than twofold of those in cells in the absence of H2S 2 h after the application and decreased thereafter (Fig. 3). At two hours, even in the presence of glutamate, H2S increases the levels of γ-GC in cells approximately twofold. At four hours, the levels of γ-GC start decreasing (Fig. 3). These observations show that H2S increases the levels of γ-GC that lead to the increase in the levels of glutathione. To investigate whether the increase in the levels of γ-GC induced by H2S is caused by the transcriptional regulation of γ-GCS, the levels of γ-GCS mRNA were measured by the quantitative polymerase chain reaction (PCR). The levels of γ-GCS mRNA were not changed 2 h after the application of H2S, which suggests that the increase in the levels of γ-GC induced by H2S is not caused by the transcriptional regulation of γ-GCS mRNA (data not shown). Glutathione is synthesized from cysteine that is produced from cystine transported into cells from the outside (16). Because oxytosis is caused by the blockade of the cystine/glutamate antiporter that couples the import of cystine and the export of glutamate (7, 27), the effect of H2S on cystine transport was examined. The transport of cystine into primary neurons is significantly increased by 200 µM NaHS 2 h after its application (Fig. 4A). The effect of H2S on cystine transport suppressed by 1 mM glutamate was also examined. In the presence of glutamate, cystine transport is reduced to 12 ± 3% of control at 20 min and 33 ± 4% at 2 h. 200 µM NaHS significantly reversed the inhibition of cystine transport by glutamate at times up to 4 h, but with the diminished effect of glutamate at 6 and 8 h, there was no difference at these time points (Fig. 4B). The H2S induced-recovery of glutamate-suppressed cystine transport may therefore be involved in the increased production of glutathione and neuroprotection. The cystine uptake by the cystine/glutamate antiporter xc– mediates oxytosis (28). To examine the effect of H2S on antiporter xc–, the effects of inhibitors for xc– were examined. The specific inhibitor for xc–, glutamate, significantly suppresses the cystine uptake, and this inhibition is significantly reduced by NaHS (Fig. 5). Because H2S is a reducing agent, it is possible that H2S reduces cystine to cysteine and enhances the transport of cysteine. To investigate this possibility, the effect of H2S on ASC (alanine, serine, and cysteine) transporter was examined. The inhibitors for ASC transporter, alanine and serine, do not significantly inhibit the cysteine uptake. In addition, alanine and serine do not significantly inhibit the cysteine uptake in the presence of NaHS, which suggests that the possibility that H2S increases the transport of cysteine by
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enhancing the activity of the ASC transporter is excluded. These observations suggest that the antiporter xc– may be involved in the cystine transport recovered by H2S. Because H2S enhances cystine transport, changes in the endogenous levels of cysteine in the presence of H2S were examined. In the presence of H2S, the levels of cysteine in cells are increased approximately sixfold relative to those in cells in the absence of H2S 2 h after application and they gradually decreased thereafter (Fig. 6A). At 2 h, even in the presence of glutamate, H2S increases the levels of cysteine in cells approximately twofold. At 4 h, the levels of cysteine start decreasing, but are still twofold of those in the absence of H2S (Fig. 6A). These observations show that H2S increases the levels of cysteine. Because cells synthesize little cysteine themselves (29), H2S must function by enhancing cystine transport, which leads to the increase in the levels of γ-GC and glutathione. To further examine the effect of H2S on cystine transport, the effect of extracellular concentrations of cystine on the glutathione levels in the presence or absence of H2S was investigated. At each concentration of extracellular cystine, the glutathione levels are increased by greater than twofold in the presence of H2S relative to those in the absence of H2S after 2 h of application. When the extracellular concentrations of cystine are decreased, glutathione levels are decreased both in the presence or absence of H2S, indicating that the enhancing effect of H2S on the glutathione levels is dependent on the extracellular concentrations of cystine (Fig. 6B). These observations confirm that H2S enhances cystine transport to increase the levels of glutathione. DISCUSSION Sulfur containing substances, dimethylsulphoniopropionate (DMSP) and its enzymatic cleavage product dimethylsulphide (DMS), have recently been identified as endogenous scavengers for hydroxyl radicals and other reactive oxygen species in marine algae (30). Because H2S is a reducing agent that readily reacts with hydrogen peroxide (H2O2; 31), it is possible that endogenous H2S can scavenge oxygen species. The present study, however, shows that H2S protects neurons from oxidative stress by increasing glutathione levels instead of functioning directly as an antioxidant. The endogenous levels of glutathione (1–8 mM; 32) are much greater than those of H2S (50–160 µM; 33). Therefore, H2S does not itself rescue cells from oxidative stress, but H2S induces the production of a major and potent antioxidant, glutathione. Cells can be rescued from oxidative stress by mechanisms that are either dependent upon or independent of glutathione metabolism. For example, antioxidants such as vitamin E protect neuronal cells from oxytosis by acting directly as antioxidants even when the intracellular glutathione levels are decreased (7, 34). In contrast, dihydroxyphenylglycine, an agonist of group I metabotropic glutamate receptors, protects neurons by up-regulating glutathione (35). Because H2S rescues neurons by increasing the accumulation of glutathione, the protection from oxytosis by H2S belongs to the latter class of mechanisms. The levels of γ-GC and cysteine in cells treated with H2S reach a peak 2 h after the application of H2S, whereas the glutathione levels start increasing 2 h after the application of H2S and last 6 h (Figs. 2A, 3, and 6A). A similar observation was made in plants (36). The level of γ-GC is increased and reaches a peak 1 h after the application of cysteine in plants, whereas glutathione
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levels are increased for several hours, which suggests a general mechanism for the regulation of the glutathione levels. H2S is an active molecule and has a strong effect on several targets. For example, H2S potentiates the induction of LTP by enhancing the activity of NMDA receptors in neurons, and it activates calcium channels to induce calcium waves in astrocytes (4, 5). In smooth muscle, H2S activates ATP-dependent potassium channels (37). The present study shows that H2S enhances the activity of γ-GCS and increases the levels of γ-GC (Fig. 3). Although the contribution is less critical than γ-GCS, the activity of cystine/glutamate antiporter xc– is also enhanced by H2S. The combined enhancement of the activity of these different targets may cause an integrated effect that results in the increase in the levels of glutathione. Although the function is not well understood, the uptake of atmospheric H2S by leaves also increases the levels of glutathione in plants (38), which suggests that H2S activates a common pathway in plants and animals to accumulate glutathione. In conclusion, H2S protects neurons against glutamate-mediated oxidative stress, oxytosis, through the pleiotropic effects of maintaining the activities of γ-GCS and cystine transport, which leads to the increase in glutathione levels. H2S may therefore have a significant neuroprotective role in the nervous system. ACKNOWLEDGMENT We thank Drs. Y. Enokido, and S. Aoki, for technical advice. This work was supported by a grant from the National Institute of Neuroscience to H. K. REFERENCES 1.
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36. Noctor, G., Strohm, M., Jouanin, L., Kunert, K.-J., Foyer, C. H., and Rennenberg, H. (1996) Synthesis of glutathione in leaves of transgenic poplar overexpression γ–glutamylcysteine synthetase. Plant Physiol. 112, 1071–1078 37. Zhao, W., Zhang, J., Lu, Y., and Wang, R. (2001) The vasorelaxant effect of H2S as a novel endogenous gaseous KATP channel opener. EMBO J. 20, 6008–6016 38. Herschbach, C., Zalm, E., Schneider, A., Jouanin, L., De Kok, L. J., and Rennernberg, H. (2000) Regulation of sulfur nutrition in wild-type and transgenic poplar over-expressing γ– glutamylcysteine syntetase in the cytosol as affected by atmospheric H2S. Plant Physiol. 124, 461–473 Received March 2, 2004; accepted April 8, 2004.
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Fig. 1
Figure 1. H2S protects neurons from oxytosis. A) Protection of neurons by H2S from glutamate toxicity. Primary
cultures of neurons 24 h after the application of 1 mM glutamate in the presence or absence of 100 µM NaHS are shown. The bar represents 100 µm. B) The dose-dependent protection of H2S against oxytosis. Relative survival of neurons 24 h after the simultaneous application of glutamate and NaHS was measured with the WST assay and confirmed by visual counting: –|-, without glutamate; --, 1 mM glutamate; -z-, 5 mM glutamate.
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Fig. 2
Figure 2. H2S causes the recovery of the glutathione levels decreased by glutamate. A) The time course of the recovery of the total glutathione levels caused by H2S. The effect of 100 µM NaHS on the levels of glutathione in neurons was determined 2, 4, 6, and 8 h after the application of 1 mM glutamate. **P < 0.01 and *P < 0.05 by ANOVA. B) Recovery of GSH by H2S. The amounts of GSH (filled bars) and GSSG (shaded bars) 8 h after the application of 1 mM glutamate, 100 µM NaHS, or both were measured by HPLC. H,100 µM NaHS; G, 1 mM glutamate; HG, both NaHS and glutamate. C) BSO decreases the levels of glutathione. D) BSO decreases cell survival. BSO (10 or 30 µM) was applied to cells in the presence or absence of 100 µM NaHS and the glutathione levels determined at 8 h (C) and cell survival at 24 h (D). All data represent the mean ± SEM of at least four experiments.
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Fig. 3
Figure 3. H2S increases the levels of γ–GC. The endogenous levels of γ–GC were measured by HPLC in the presence
or absence of H2S after 2, 4, 6, and 8 h after the application. H, 100 µM NaHS; G, 1 mM glutamate; HG, both NaHS and glutamate. **P < 0.01 by ANOVA. All the data represent the mean ± SEM of at least four experiments.
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Fig. 4
Figure 4. H2S enhances the cystine uptake. A) Increase in uptake of cystine into neurons induced by H2S. The
incorporation of [35S]-cystine after the application of NaHS in the absence of glutamate was measured. B) H2S-induced recovery of cystine uptake suppressed by glutamate. The effect of NaHS on the incorporation of [35S]-cystine into primary cultures of neurons was measured in the presence of 1 mM glutamate. **P < 0.01 and *P < 0.05 by ANOVA (A, B). All the data represent the mean ± SEM of at least four experiments.
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Fig. 5
Figure 5. The effect of inhibitors on the cystine uptake. The effect of 1 mM glutamate, alanine, or serine on the incorporation of [35S]-cystine into primary cultures of neurons in the presence or absence of NaHS was measured. **P < 0.01 and *P < 0.05 by ANOVA. All the data represent the mean ± SEM of at least four experiments.
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Fig. 6
Figure 6. H2S increases the levels of cysteine and glutathione. A) The endogenous levels of cysteine were measured by HPLC in the presence or absence of H2S after 2, 4, 6, and 8 h after the application. H: 100 µM NaHS; G: 1 mM glutamate; HG: both NaHS and glutamate. **P < 0.001 and *P < 0.05 by ANOVA. B) The effect of H2S on increasing glutathione levels is dependent on extracellular concentrations of cystine. The glutathione levels were measured in the presence or absence of 100 µM NaHS. Cys 100, 10, and 0 represent 100 or 10 µM cystine or cystine-free medium, respectively. All the data represent the mean ± SEM of at least three experiments. Page 16 of 16 (page number not for citation purposes)
