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Hydrogen sulfide protects neurons from oxidative stress

National Institute of Neuroscience, Kodaira, Tokyo, Japan

¹Correspondence: National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187-8551, Japan. E-mail: kimura{at}ncnp.go.jp

SPECIFIC AIM

Hydrogen sulfide (H₂S), 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 about its other biological functions. In the present study we determined 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 specifically examined the neuroprotective role of H₂S and its mechanism.

PRINCIPAL FINDINGS

1. H₂S protects neurons from oxidative stress
Because H₂S, an endogenous reducing agent, is produced in response to oxidative stress in yeast, it is possible that H₂S functions as an antioxidant. 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, were prepared from 17-day-old embryonic rat brains and cultured for 1 day. Most neurons die within 24 h after application of 1 mM glutamate: glutamate inhibits cystine uptake, causing oxidative stress-induced cell death (oxytosis). Neurons treated simultaneously with 100 µM NaHS are viable (Fig. 1 A). H₂S protects cells from glutamate toxicity in a dose-dependent manner; ED₅₀ values of NaHS against 1 and 5 mM glutamate toxicity are 47 ± 11 µM and 93 ± 19 µM, respectively (Fig. 1B ). H₂S alone caused a significant increase in survival after plating, protecting cells from the spontaneous cell death that occurs in primary cultures.

View larger version (49K): [in this window] [in a new window]   Figure 1. H2S protects neurons from oxytosis. A) Protection of neurons by H2S from glutamate toxicity. Primary cultures of neurons 24 h after application of 1 mM glutamate in the presence or absence of 100 µM NaHS are shown. Bar represents 100 µm. B) 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; (•) 5 mM glutamate.

2. H₂S enhances the production of glutathione
The effect of H₂S on glutathione accumulation was examined. NaHS (100 µM) alone increases GSH + GSSG levels 2 h after application. The maximum level is reached after 4 h, then gradually decreases at 8 h (Fig. 2 A). 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, glutathione levels exceed those of controls and almost reach levels achieved by applying NaHS alone at later times (Fig. 2A ). These observations show that H₂S causes the recovery of intracellular glutathione that is lowered by glutamate.

View larger version (36K): [in this window] [in a new window]   Figure 2. H2S causes recovery of the glutathione levels decreased by glutamate. A) Time course of the recovery of total glutathione levels caused by H2S. The effect of 100 µM NaHS on glutathione levels in neurons was determined 2, 4, 6, and 8 h after application of 1 mM glutamate. **P<0.01 and *P<0.05 by ANOVA. B) Recovery of GSH by H2S. 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, NaHS, and glutamate. C) BSO decreases the levels of glutathione. D) BSO decreases cell survival. 10 or 30 µM BSO was applied to cells in the presence or absence of 100 µM H2S and the glutathione levels determined at 8 h (C) and cell survival at 24 h (D). All data represent mean ±SE of at least 4 experiments.

Since the reduced form of glutathione can protect cells from oxidative stress, it is necessary to examine the redox state of glutathione in nerve cell cultures. 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 addition of H₂S, the amount of GSH increases to 193 ± 3% of the control although the concentration of GSSG is not significantly changed. In contrast, GSH and GSSG are decreased by glutamate. When glutamate-treated cells are simultaneously exposed to H₂S, GSH and GSSG amounts are largely restored and reach the levels achieved by application of H₂S alone (Fig. 2B ). These observations indicate that H₂S increases GSH levels 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 -glutamylcysteine synthetase (-GCS), buthionine sulfoximine (BSO), in the presence or absence of H₂S was examined. BSO dose-dependently suppressed levels of both glutathione and cell survival even in the presence of H₂S (Fig. 2C, D ), suggesting that protection of neuronal cells by H₂S requires an increase in glutathione levels.

3. H₂S increases the levels of -glutamylcysteine
Because H₂S increases GSH levels, H₂S may enhance the activity of -GCS and increase production of -glutamylcysteine (-GC), changes in -GC levels after application of glutamate in the presence or absence of H₂S were examined. In the presence of H₂S, -GC in cells is increased by >2-fold that of cells in the absence of H₂S 2 h after application and decreases thereafter. At 2 h, even in the presence of glutamate, H₂S increases levels of -GC in cells by 2-fold. At 4 h, -GC levels start to decrease. These observations show that H₂S increases -GC levels, leading to the increase in glutathione levels.

To investigate whether the increase in -GC induced by H₂S is caused by the transcriptional regulation of -GCS, levels of -GCS mRNA were measured by quantitative PCR. -GCS mRNA levels were not changed 2 h after application of H₂S, suggesting the increase in -GC levels induced by H₂S is not caused by transcriptional regulation of -GCS mRNA (data not shown).

4. H₂S enhances the cystine transport
Glutathione is synthesized from cysteine produced from cystine that is transported into cells from the outside. Because oxytosis is caused by blockade of the cystine/glutamate antiporter that couples the import of cystine and export of glutamate, the effect of H₂S on cystine transport was examined. Transport of cystine into primary neurons is increased by 200 µM NaHS 2 h after application. 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. NaHS (200 µM) significantly reversed the inhibition of cystine transport by glutamate for up to 4 h; with the diminished effect of glutamate at 6 and 8 h, there was no difference at these times. H₂S-induced recovery of glutamate-suppressed cystine transport therefore may be involved in the increased production of glutathione and neuroprotection.

Cystine uptake by the cystine/glutamate antiporter x_c^– mediates oxytosis. The specific inhibitor for x_c^–, glutamate, significantly suppresses cystine uptake, and this inhibition is significantly reduced by NaHS. Because H₂S is a reducing agent, it is possible that H₂S reduces cystine to cysteine and enhances the transport of cysteine. To investigate this, the effect of H₂S on ASC (alanine, serine, and cysteine) transporter was examined. Inhibitors for ASC transporters alanine and serine do not significantly inhibit basal cysteine uptake. Alanine and serine do not significantly inhibit cysteine uptake in the presence of NaHS, abrogating the notion that H₂S increases the transport of cysteine by enhancing ASC transporter activity. These observations suggest that the antiporter x_c^– may be involved in the cystine transport recovered by H₂S.

In the presence of H₂S, levels of cysteine are increased 6-fold relative to those in the absence of H₂S 2 h after application, then gradually decrease. At 2 h, H₂S increases cysteine levels in cells 2-fold even in the presence of glutamate. At 4 h, cysteine levels start to decrease but are still 2-fold those in the absence of H₂S. These observations show that H₂S increases levels of cysteine. Since cells synthesize little cysteine themselves, H₂S must function by enhancing cystine transport, leading to the increase in -GC and glutathione.

At each concentration of extracellular cystine investigated, glutathione levels are increased by >2-fold in the presence of H₂S relative to those in its absence after 2 h of application. When extracellular concentrations of cystine are decreased, glutathione levels are decreased in the presence or absence of H₂S, indicating that the enhancing effect of H₂S on glutathione levels depends on extracellular concentrations of cystine. These observations confirm that H₂S enhances cystine transport to increase the levels of glutathione.

CONCLUSIONS AND SIGNIFICANCE

Sulfur-containing dimethylsulfoniopropionate and its enzymatic cleavage product dimethylsulfide have recently been identified as endogenous scavengers for hydroxyl radicals and other reactive oxygen species in marine algae. Since H₂S is a reducing agent that readily reacts with hydrogen peroxide, 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. Endogenous levels of glutathione (1–8 mM) are much greater than those of H₂S (50–160 µM). Therefore, H₂S itself does not 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 either dependent on or independent of glutathione metabolism. For example, antioxidants such as vitamin E protect neuronal cells from oxytosis by acting directly as antioxidants even when intracellular glutathione levels are decreased. In contrast, dihydroxyphenylglycine, an agonist of group I metabotropic glutamate receptors, protects neurons by up-regulating glutathione. Because H₂S rescues neurons by increasing the accumulation of glutathione, protection from oxytosis by H₂S belongs to the latter class of mechanisms.

Levels of -GC and cysteine in cells treated with H₂S reach a peak 2 h after application, whereas glutathione levels start to increase 2 h after application and last for 6 h (Fig. 2A ). A similar observation was made in plants. The level of -GC is increased and reaches a peak 1 h after application of cysteine whereas glutathione levels are increased for several hours, suggesting a general mechanism for regulating the glutathione levels.

H₂S is an active molecule with a strong effect on several targets. H₂S potentiates induction of LTP by enhancing NMDA receptor activity in neurons and activates calcium channels to induce calcium waves in astrocytes. In smooth muscle, H₂S activates ATP-dependent potassium channels. The present study shows that H₂S enhances -GCS activity and increases -GC levels. Although the contribution is less critical than -GCS, the activity of cystine/glutamate antiporter x_c^– is also enhanced by H₂S. The combined enhancement of activity of these different targets may engender an integrated effect that results in the increased levels of glutathione. Although not well understood, the uptake of atmospheric H₂S by leaves also increases levels of glutathione in plants, suggesting that H₂S activates a common pathway in plants and animals to accumulate glutathione.

In conclusion, H₂S protects neurons against glutamate-mediated oxidative stress, or oxytosis, through the pleiotropic effects of maintaining the activities of -GCS and cystine transport, leading to an increase in glutathione levels (Fig. 3 ). H₂S may therefore have a significant neuroprotective role in the nervous system.

View larger version (28K): [in this window] [in a new window]   Figure 3. H2S protects neurons from oxidative stress. H2S increases cysteine levels by enhancing xc– glutamate/cystine antiporter and -GCS activity, which produces glutathione. H2S protects neurons from oxidative stress by increasing levels of glutathione, a major intracellular antioxidant.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1815fje; doi: 10.1096/fj.04-1815fje

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