Introduction
It is well documented that hypoxia and/or ischemia can elicit the release of several neurotransmitters (1,2), including glutamate (3). Such elevated levels of glutamate, and the subsequent activation of ionotropic NMDA receptors, can trigger the neuronal damage during hypoxia and/or ischemia (4,5). Glutamate homeostasis is therefore crucial to prevent neuronal death after a hypoxic/ischemic episode. Glutamate transport is the only mechanism for the removal of glutamate from the extracellular fluid in the brain (6,7), and it is essential for maintaining extracellular glutamate below neurotoxic levels in the normal brain (8). Therefore, glutamate transporters are considered to play a key role in the process of increase in extracellular glutamate during hypoxia/ischemia.
To date, 5 distinguishing high-affinity, Na+-dependent glutamate transporters have been identified: excitatory amino acid transporter (EAAT)1, glutamate-aspartate transporter (GLAST), EAAT2, glutamate transporter-1 (GLT-1), EAAT3, excitatory amino acid carrier 1 (EAAC1), EAAT4 and EAAT5. These transporters are present throughout the central nervous system (CNS), with GLT-1 being highly abundant in astroglial cells, whereas GLAST exists at higher levels in Bergmann glia in the cerebellum (9,10). GLT-1 plays a critical role in CNS homeostasis, accounting for up to 70% of glutamate clearance (10,11).
The roles of GLT-1 in hypoxia/ischemia-induced injury and neuroprotection have attracted extensive attention. However, the findings are controversial. A pharmacological study indicated that the GLT-1 blocker reduces the ischemia-induced glutamate release in rat cortical superfusates (12), revealing that GLT-1 releases glutamate during ischemia. By contrast, Rao et al (13) reported that the antisense knockdown of GLT-1 exacerbates ischemia-triggered neuronal damage in the rat brain, suggesting that GLT-1 takes up glutamate to protect neurons during ischemia. In addition, ischemic preconditioning upregulates the GLT-1 protein which may play a role in the neuroprotective mechanism of preconditioning (14). In neonatal rats, it was shown that the neuroprotection of ceftriaxone preconditioning against hypoxia/ischemia-induced neuronal injury is associated with upregulation of GLT-1 expression (15). On the other hand, an association between change in GLT-1 expression and hypoxia/ischemia has been reported by several studies (16,17). Raghavendra et al (16) observed that the expression of GLT-1 is reduced following transient global ischemia. Conversely, chronic hypoxia upregulates the expression of EAAC1 and GLT-1, but not GLAST (17). These findings support the theory that GLT-1 has a complicated function (cytoprotective vs. cytotoxic effects) after hypoxic/ischemic episodes. Thus, it is necessary to explore the roles of GLT-1 in neuronal injury or the neuroprotective effects in different hypoxic/ischemic models.
Hydrogen sulfide (H2S), recently considered a novel neuro-modulator in the CNS, has been shown to protect astrocytes against H2O2-induced neural damage by enhancing glutamate uptake (18), suggesting an impact of H2S.on..lutamate.trans. on glutamate transporters. We have also demonstrated that H2S protects PC12 cells against chemical hypoxia-induced injury by inhibiting reactive oxygen species (ROS) overproduction, extracellular signal-regulated kinase 1/2 (ERK1/2) and the p38 mitogen-activated protein kinase (MAPK) signaling pathways (19,20). Since it is reported that ROS and the activation of the ERK1/2 pathway are involved in the downregulation of GLT-1 protein expression induced by H2O2 or amyloid-ß (Aß) in astrocytes (19,21), we hypothesized that ROS and ERK1/2-mediated downregulation of GLT-1 might be implicated in chemical hypoxia-induced neuronal injury and that H2S might confer neuroprotection by enhancing GLT-1 expression. To test this hypothesis, PC12 cells, which are derived from chromafin cells of the adrenal medulla, were exposed to cobalt chloride (CoCl2), a well-known hypoxia mimetic agent, to establish a model of chemical hypoxia injury. The effects of CoCl2 and pretreatment with NaHS (a donor of H2S) on GLT-1 expression were observed. We found that: i) CoCl2 significantly inhibits the expression of GLT-1, ROS and the ERK1/2 pathway contribute to this inhibitory effect; ii) NaHS pretreatment clearly attenuates the inhibitory effect of CoCl2 on GLT-1 expression; iii) DHK, a selective inhibitor of GLT-1, blocks the neuroprotection of H2S against CoCl2-induced injury in PC12 cells.
Materials and methods
Materials
NaHS, CoCl2, N-acetyl-L-cysteine (NAC), Hoechst 33258, propidium iodide (PI), RNase and Rhodamine 123 (Rh123) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The cell counter kit-8 (CCK-8) was purchased from Dojindo Laboratories (Kumamoto, Japan). The DMEM medium and fetal bovine serum (FBS) were supplied by Gibco-BRL (Grand Island, NY, USA). Anti-GLT-1 antibody was purchased from Abcam (Cambridge, UK). DHK was purchased from Merck Co. Anti-β-actin antibody, horseradish peroxidase (HRP)-conjugated secondary antibody and the BCA protein assay kit were purchased from KangChen Bio-tech, Inc. (Shanghai, China). Enhanced chemiluminescence (ECL) solution was purchased from Nanjing KeyGen Biotech Co., Inc. (Nanjing, China).
Cell culture and treatments
The rat pheochromocytoma cell line PC12 cells were purchased from the Sun Yat-Sen University Experimental Animal Center, and were grown in DMEM medium supplemented with 10% FBS at 37°C under an atmosphere of 5% CO2 and 95% air. According to our previous study (20), chemical hypoxia was achieved by adding CoCl2 at 600 μM into the medium and cells were incubated in the presence of CoCl2 for the indicated times. The cytoprotective effects of H2S were observed by administering 400 μM NaHS (a donor of H2S) for 30 min prior to exposure to CoCl2 for 24 h. NAC (a scavenger of ROS) or U0126 (a MEK1/2 inhibitor) was administered 60 or 120 min prior to exposure of the PC12 cells to 600 μM CoCl2 for 24 h.
Cell viability assay
The CCK-8 assay was employed to investigate the cell viability of PC12 cells cultured in 96-well plates. After the indicated treatments, 10 μl CCK-8 solution was added to each well of the plate and the cells in the plate were incubated for 4 h in the incubator. The absorbance at 450 nm was measured with a microplate reader (Molecular Devices, Sunnyvale, CA, USA). Means of 4 well optical density (OD) in the indicated groups were used to calculate the percentage of cell viability according to the formula below: Percentage of cell viability (%) = (ODtreatment group/ODcontrol group) × 100%. The experiment was repeated 3 times.
Nuclear staining for assessment of apoptosis with Hoechst 33258
Morphological changes, such as chromosomal condensation and fragmentation in the nuclei of PC12 cells, were observed by Hoechst 33258 staining followed by photo-fluorography. Cells were plated at a density of 1×106 cells/well in 35 mm dishes. Cells were preconditioned with 400 μM NaHS for 30 min, and subsequently exposed to 600 μM CoCl2 for 48 h. To test the role of GLT-1 in H2S-induced cytoprotection against chemical hypoxia-induced apoptosis, cells were treated with the GLT-1 inhibitor DHK for 30 min prior to preconditioning with NaHS. At the end of the indicated treatments, cells were harvested and fixed with 4% paraformaldehyde in 0.1 mol/l phosphate-buffered saline (PBS, pH 7.4) for 10 min. After rinsing with PBS, the nuclear DNA was stained with 5 mg/ml Hoechst 33258 solution for 10 min before being rinsed briefly with PBS and then visualized under a fluorescence microscope (Bx50-FLA; Olympus, Tokyo, Japan). Viable cells displayed a uniform blue fluorescence throughout the nucleus, whereas apoptotic cells showed condensed and fragmented nuclei.
Flow cytometric analysis of apoptosis
Treated PC12 cells were digested with trypsin (2.5 mg/ml), centrifuged at 350 × g for 10 min and the supernatant was removed. Cells were washed twice with PBS and fixed with 70% ice-cold ethanol. Cells were then centrifuged at 350 × g for 10 min, washed twice with PBS and adjusted to a concentration of 1×106 cells/ml. Subsequently, 0.5 ml RNase (1 mg/ml in PBS) was added to a 0.5 ml cell sample. After gentle mixing with PI (at a terminal concentration of 50 mg/l), mixed cells were filtered and incubated in the dark at 4°C for 30 min before flow cytometric analysis (FCM). The PI fluorescence of individual nuclei was measured by a flow cytometer (Beckman-Coulter, Los Angeles, CA, USA). Excitation, 488 nm; emission, 615 nm. The research software matched with FCM was used to analyze all the data of DNA labeling. In the DNA histogram, the amplitude of the sub-G1 DNA peak, which is lower than the G1 DNA peak, represents the number of apoptotic cells. The experiment was repeated 3 times.
Measurement of MMP
Mitochondrial membrane potential (MMP) was monitored using the fluorescent dye Rh123, a cell-permeable cationic dye that preferentially enters into the mitochondria based on the highly negative MMP. Depolarization of MMP results in loss of Rh123 from the mitochondria and a decrease in intracellular fluorescence. In the present study, PC12 cells were cultured in 24-well plates and treated with 400 μM NaHS for 30 min prior to the administration of 600 μM CoCl2 for 24 h. DHK was administered 30 min prior to NaHS preconditioning. To evaluate MMP, Rh123 (100 μg/l) was added to cell cultures for 45 min at 37°C and fluorescence was measured over the entire field of vision using a fluorescent microscope connected to an imaging system (BX50-FLA; Olympus). The mean fluorescence intensity (MFI) of Rh123 from 5 random fields was analyzed using ImageJ 1.410 software (National Institutes of Health, Bethesda, MD, USA), and the MFI was taken as an index of the MMP. The experiment was repeated 3 times.
Western blot assay for protein expression
After the cells were subjected to the indicated treatments, they were harvested and lysed with cell lysis solution. Total protein in the cell lysate was quantified using the BCA protein assay kit. Sample buffer was added to cytosolic extracts, and after boiling for 5 min, equal amounts of supernatant from each sample were fractionated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Total protein in the gel was transferred into polyvinylidene difluoride (PVDF) membranes. Membranes were blocked for 1.5 h at room temperature in fresh blocking buffer [0.1% Tween-20 in Tris-buffered saline (TBS-T) containing 5% fat-free milk] and then incubated with either anti-GLT-1 (1:2,500 dilution), or anti-β-actin antibodies (1:5,000 dilution) in freshly prepared TBS-T with 3% free-fat milk overnight with gentle agitation at 4°C. After 3 washes with TBS-T, membranes were incubated with HRP-conjugated goat anti-rabbit secondary antibodies (1:3,000 dilution; KangChen Bio-tech, Inc.) in TBS-T with 3% fat-free milk for 1.5 h at room temperature. Membranes were washed 3 times with TBS-T, developed in ECL solution and visualized with X-ray film. Each experiment was repeated at least 3 times. For quantification, the film were scanned and analyzed using ImageJ 1.410 software. The density of specific bands was measured and normalized with the bands of Action. The experiment was repeated 3 times.
Statistical analysis
Data are representative of experiments performed in triplicate and are expressed as the mean ± SE. Differences between groups were analyzed by one-way analysis of variance (ANOVA) using SPSS 13.0 software, followed by the LSD post hoc comparison test. P<0.05 was considered to indicate statistically significant differences.
Results
CoCl<sub>2</sub> reduces the level of GLT-1 expression in PC12 cells
In order to explore the effect of CoCl2 on the GLT-1 expression level in PC12 cells, PC12 cells were exposed to 600 μM CoCl2 for the indicated times (i.e., 3, 6, 12 and 24 h). Western blot analysis revealed that treatment with 600 μM CoCl2 caused downregulation of GLT-1 expression in a time-dependent manner (Fig. 1). These data indicate that chemical hypoxia may reduce GLT-1 protein levels in PC12 cells.
H<sub>2</sub>S reverses CoCl<sub>2</sub>-induced downregulation of GLT-1 expression in PC12 cells
After PC12 cells were exposed to 600 μM CoCl2 for 24 h, the levels of GLT-1 protein expression were markedly decreased (Fig. 2). However, pretreatment of PC12 cells with 400 μM NaHS for 30 min before exposure to CoCl2 reversed this effect, suggesting that NaHS preconditioning may enhance GLT-1 protein expression level in CoCl2-treated PC12 cells.
GLT-1 is involved in the cytoprotection of H<sub>2</sub>S against CoCl<sub>2</sub>-induced injury
To explore whether GLT-1 is involved in the cytoprotection of H2S against CoCl2-induced injuries, PC12 cells were pretreated with DHK (a inhibitor of GLT-1) at 400 μM for 30 min prior to NaHS preconditioning followed by exposure to 600 μM CoCl2 for 24 h. As shown in Fig. 3, DHK pretreatment significantly blocked the protection of NaHS preconditioning against CoCl2-induced cytotoxicity, the cell viability was considerably decreased, from 62±2.3% to 50±2.1% (P<0.01) (Fig. 3). Moreover, pretreatment with 400 μM DHK also markedly inhibited H2S-induced anti- apoptotic effects, increasing the number of apoptotic cells with nuclear condensation and fragmentation (Fig. 4A) as well as the apoptotic percentage of PC12 cells compared with the NaHS pretreatment + CoCl2 group (P<0.01) (Fig. 4B). Additionally, pretreatment of PC12 cells with DHK for 30 min before 400 μM NaHS preconditioning clearly inhibited H2S-induced preservation of MMP (Fig. 5). These findings suggest that GLT-1 contributes to the cytoprotection of H2S against CoCl2-induced injuries.
ROS are involved in the CoCl<sub>2</sub>-induced downregulation of GLT-1 expression in PC12 cells
Since ROS generation inhibits glutamate uptake function (22), we examined whether ROS is involved in the CoCl2-induced downregulation of GLT-1 protein expression in PC12 cells. Pretreatment of cells with 500 μM NAC (a ROS scavenger) for 60 min prior to exposure to 600 μM CoCl2 for 24 h significantly blocked CoCl2-induced downregulation of GLT-1 expression (Fig. 6). These data indicate that the inhibitory effect of CoCl2 on GLT-1 expression may be associated with oxidative stress.
Activation of ERK1/2 contributes to the downregulation of GLT-1 expression induced by CoCl<sub>2</sub> in PC12 cells
Stimulation of ERK1/2MAPK also contributes to the inhibition of glutamate uptake (23). In order to investigate the effect of ERK1/2 activation on the downregulation of GLT-1 expression induced by CoCl2, PC12 cells were pretreated with 10 μM U0126 (a MEK1/2 inhibitor) for 120 min prior to treatment with 600 μM CoCl2 for 24 h. U0126 significantly reversed the inhibitory effect of CoCl2 on the expression of GLT-1 in PC12 cells, suggesting that activation of ERK1/2 contributes to the downregulation of GLT-1 expression induced by CoCl2 in PC12 cells (Fig. 2).
Discussion
GLT-1 has been classified as an astroglial transporter due to its predominant and widespread expression in astrocytes. In the present study, we found that PC12 cells expressed GLT-1, suggesting that GLT-1 may be involved in maintaining a normal level of glutamate in PC12 cells, which is consistent with a previous study (17). It is well known that GLT-1 plays a major role in glutamate re-uptake from the synaptic cleft after neuronal transmission (6,24,25). Lack of GLT-1 has indeed been shown to promote extracellular glutamate accumulation, excitotoxicity and, ultimately, cell death (26,27). GLT-1 has been estimated to represent up to 1% of total brain protein (6). The expression of GLT-1 is reduced in several animal models of neurodegenerative diseases, including traumatic brain injury (28) and hypoxic/ischemic insults (16,29). The levels of the GLT-1 and/or GLAST protein are also lower in the brain tissue from the patients with Alzheimer’s disease (AD) (30) and Huntington’s disease (31). The results of the present study showed that CoCl2, a well-known hypoxia mimetic agent, attenuates expression of GLT-1 in a time-dependent manner. Our findings are comparable with a study showing that transient global ischemia reduces GLT-1 expression (16). Similarly, it was reported that GLT-1 protein levels are reduced in the brain in various models of central hypoxia/ischemia (16,29,32). Under hypoxic conditions (2.5 and 1% O2 exposure for 24 h), glutamate uptake and GLT-1 protein levels are significantly decreased in astrocytes (33). These studies all support our results. By contrast, Kobayashi and Millhorn (17) indicated that exposure of PC12 cells to hypoxia (1% O2) for 6 to 24 h increases GLT-1 protein levels. Therefore, it is likely that the effects of hypoxia/ischemia on the expression of GLT-1 may be affected by many factors, including tissue or cell types, the level of hypoxia, manner of hypoxia induction and also the period of hypoxia/ischemia.
To clarify the mechanisms underlying the inhibitory effect of chemical hypoxia on GLT-1 expression, we tested the possible involvement of ROS. Several previous studies have shown that oxidative stress is implicated in glutamate clearance impairment and reduction of GLT-1 expression (18,21,34). Our recent studies have demonstrated the promotive effects of CoCl2 on ROS production (19,20). In this study, we found that NAC, a ROS scavenger, can significantly block the inhibition of GLT-1 expression induced by CoCl2, revealing that ROS partly contribute to the inhibitory effect of chemical hypoxia on the expression of GLT-1 in PC12 cells. We provide novel evidence for the role of ROS in CoCl2-induced neuronal injury. Additionally, there is currently a lot of data demonstrating that oxidative stress may trigger and modulate the MAPK signaling pathways (21,35,36). We recently demonstrated that ROS can activate the MAPK pathway (20), linking to the possibility of an altered ERK1/2 activation that ultimately affects the expression of GLT-1. To confirm this possibility, we observed the effects of pretreatment of PC12 cells with UO126 (an inhibitor of MEK1/2) on the inhibition of GLT-1 expression by CoCl2. Our results showed that U0126 clearly suppressed the CoCl2-induced decrease in the expression of GLT-1, suggesting that the ERK1/2 pathway is involved in the inhibitory effect of CoCl2 on GLT-1 expression. This is also a novel finding showing that the ROS-activated ERK1/2 pathway plays a role in the inhibition of GLT-1 expression by CoCl2. Our findings are supported by previous studies (18,35). Lu et al (18) reported that PD98059, a specific ERK1/2 inhibitor, significantly reverses the reduction of trafficking of GLT-1 from cytoplasma to plasma membrane.
Although research on the regulatory mechanisms for GLT-1 expression has intensified, scarce data are available regarding the regulatory effect of gasotransmitter on the expression of GLT-1. H2S, recently recognized as the third gasotransmitter alongside nitric oxide (NO) and carbon monoxide (CO) (39), has attracted extensive attention due to its multiple physiological and pathophysiological roles in various body systems (18–20,37–41). Kimura and Kimura (38) demonstrated the nueroprotective effect of H2S against oxidative stress-induced injury in primary rat cortical neurons. H2S.also.protects.astro. also protects astrocytes from H2O2-induced neural injury (18). We recently found that H2S protects PC12 cells against CoCl2-induced damage by enhancing heat shock protein 90 (HSP90) (19), inhibiting the ROS-activated ERK1/2 and p38MAPK signaling pathways (20) and scavenging ROS (19,20). In the present study, we provide evidence for the first time that NaHS (a donor of H2S) pretreatment prevents the CoCl2-induced downregulation of GLT-1 expression in PC12 cells. Our results are in line with a recent study that H2S protects astrocytes against oxidative stress-induced neural damage by increasing glutamate uptake (18). Based on our recent results (19,20,39–41) and other studies (18,21,35,36,38,42), there are several possible mechanisms responsible for the regulatory effect of H2S on the expression of GLT-1: i) its antioxidation, by which H2S can protect PC12 cells from CoCl2-induced suppression of GLT-1 expression; ii) its inhibitory effect on the ERK1/2 pathway (20); and iii) H2S functions as an ATP-sensitive potassium (KATP) channel opener (43). Hu et al (42) indicated that iptakalim, a KATP channel opener, can reverse the inhibition of glutamate uptake induced by N-methyl-4-4-phenylpyridinium (MPP+) [used to stimulate Parkinson’s disease (PD)-like conditions], revealing a role of the KATP channel opener in the functional regulation of glutamate transporter. Further research is required to confirm these findings.
We further explored the role of GLT-1 in the neuroprotection of H2S against chemical hypoxia-induced injury. We found that pretreatment with DHK, a selective inhibitor of GLT-1, significantly reversed the protective effect of H2S against CoCl2-induced injuries, evidenced by a decrease in cell viability and an increase in apoptotic PC12 cells as well as MMP loss, suggesting that upregulation of GLT-1 expression may play an important role in the neuroprotective effects of H2S.
In summary, in the present study, we have demonstrated for the first time that: i) both ROS and the ERK1/2 pathway contribute to the downregulation of GLT-1 expression induced by CoCl2; ii) H2S, a novel gaseous neuromodulator, reverses CoCl2-induced downregulation of GLT-1 expression; and iii) upregulation of GLT-1 expression may play a crucial role in the neuroprotective effects of H2S against chemical hypoxia- induced neuronal injury in PC12 cells. The findings of the present study may provide a potential neuroprotective therapeutic approach for treatment of hypoxia/ischemia-related neuronal injury. In addition, based on the notable findings that both levels of endogenous H2S and GLT-1 are reduced in neurodegenerative diseases, such as AD and PD, we speculate that endogenous H2S may be an important modulator of GLT-1. These findings remain to be confirmed in future studies.