- Top of page
- Materials and methods
Glutamate is the major excitatory amino acid of the mammalian brain but can be toxic to neurones if its extracellular levels are not tightly controlled. Astrocytes have a key role in the protection of neurones from glutamate toxicity, through regulation of extracellular glutamate levels via glutamate transporters and metabolic and antioxidant support. In this study, we report that cultures of rat astrocytes incubated with high extracellular glutamate (5 mM) exhibit a twofold increase in the extracellular concentration of the tripeptide antioxidant glutathione (GSH) over 4 h. Incubation with glutamate did not result in an increased release of lactate dehydrogenase, indicating that the rise in GSH was not because of membrane damage and leakage of intracellular pools. Glutamate-induced increase in extracellular GSH was also independent of de novo GSH synthesis, activation of NMDA and non-NMDA glutamate receptors or inhibition of extracellular GSH breakdown. Dose–response curves indicate that GSH release from rat astrocytes is significantly stimulated even at 0.1 mM glutamate. The ability of astrocytes to increase GSH release in the presence of extracellular glutamate could be an important neuroprotective mechanism enabling neurones to maintain levels of the key antioxidant, GSH, under conditions of glutamate toxicity.
Glutamate is the major excitatory amino acid of the mammalian brain (Danbolt 2001). It acts through a variety of ionotropic and metabotropic receptors: the first exert their effects via ligand-gated ion channels, whereas the second act through coupling to G proteins and activation of intracellular secondary messengers (Greenamyre and Porter 1994; Meldrum 2000). Although glutamate is an important excitatory neurotransmitter it can be toxic if its extracellular levels are not tightly controlled. In conditions where release and/or uptake of glutamate are altered, extracellular glutamate can accumulate causing a persistent or excessive activation of glutamate-gated ion channels (excitotoxicity) (Mark et al. 2001; Coyle and Puttfarcken 1993). A number of pathways have been implicated in glutamate excitotoxicity, namely calcium deregulation, loss of membrane potential, mitochondrial impairment and production of reactive nitrogen/oxygen species (RNOS), which can lead to oxidative/nitrosative stress and ultimately cell death (Coyle and Puttfarcken 1993; Massieu and Garcia 1998; Pitt et al. 2000).
The extracellular levels of glutamate have been measured in various in vivo disease models by microdialysis and have been shown to reach concentrations of > 500 μM following spinal cord injury (McAdoo et al. 1999) and be maintained at concentrations of > 50 μM for 1–2 h during and following ischaemic insult (Orwar et al. 1994; Ritz et al. 2004; Homola et al. 2006). As extracellular glutamate derives from intracellular vesicles (whose glutamate concentrations are between 0.24 and 11 mM; Harris and Sultan 1995), the local concentration of glutamate in these conditions is likely to be even higher. Prolonged exposure to such concentrations of glutamate is likely to result in significant neurotoxicity (Liu et al. 1999). Astrocytes have a fundamental role in the regulation of extracellular glutamate levels and in the protection of neurones from glutamate toxicity (Hertz and Zielke 2004). In normal synaptic transmission, glutamate released into the synaptic cleft by neurones is accumulated in astrocytes (Hertz et al. 1978) by means of glutamate transporters such as glutamate transporter 1 and glutamate aspartate transporter (Gadea and Lopez-Colome 2001), after which it is returned to neurones in the form of glutamine.
Astrocytes also protect neurones in other ways such as through metabolic and antioxidant support. One of the most important molecules in this respect is the antioxidant glutathione (GSH) (Schulz et al. 2000). The trafficking of GSH between astrocytes and neurones is particularly important in conditions of oxidative stress (Dringen 2000). Astrocytes are able to increase neuronal GSH levels by secreting GSH into the extracellular environment (Sagara et al. 1996; Dringen et al. 1999; Stewart et al. 2002). Neurones are unable to take up GSH directly but can make use of cysteinyl glycine and cysteine, which are produced from GSH by the consecutive action of γ-glutamyl transferase (γGT) and aminopeptidase N, two enzymes expressed on the surface of astrocytes and neurones respectively (Dringen et al. 1997, 2001). Cysteine is the rate-limiting substrate for GSH synthesis in neurones, so the supply of this substrate by astrocytes is essential for the maintenance of GSH levels in neurones (Dringen et al. 1999). Previous studies have shown that astrocytes increase GSH release in response to increases in RNOS, such as nitric oxide (NO) (Gegg et al. 2003) and hydrogen peroxide (Sagara et al. 1996). This increase in GSH release is hypothesised to be a neuroprotective mechanism which maintains and/or increases neuronal GSH levels to counteract the damaging effects of RNOS. As oxidative stress is considered to be a key component of glutamate toxicity it was the aim of this study to investigate whether high concentrations of extracellular glutamate also had an effect on GSH release from astrocytes.
- Top of page
- Materials and methods
In the present report, we demonstrate that prolonged exposure to glutamate induces an increase in the concentration of extracellular GSH in three different types of cultured astrocytes. These cells are known to release GSH (Sagara et al. 1996), and when cultured with 5 mM glutamate we observed a significant increase in the amount of extracellular GSH over 240 min (Fig. 1), without evidence of cellular damage. At least a twofold increase in extracellular GSH was observed in both cortical and hippocampal astrocytes after 240 min treatment with glutamate, suggesting this to be a feature common to astrocytes from different brain regions. Dose–response curves also indicated that glutamate induces GSH release from astrocytes at concentrations as low as 0.1 mM (Fig. 4). A number of possible causes for this increase in extracellular GSH have been investigated in this study and are discussed in more detail below.
Glutamate is one of the precursors of GSH (Kranich et al. 1996), and an increase in the synthesis of GSH could result in its increased release into the media. However, under our experimental conditions, glutamate did not cause a significant increase in intracellular GSH (Table 1). This is not surprising as it has been shown previously that addition of 1 mM glutamate to astrocytes only results in an increase in intracellular GSH concentration if cystine/cysteine and glycine are also added (Dringen and Hamprecht 1996). The absence of these substrates in our media suggests that de novo GSH synthesis does not explain the increase in extracellular levels. Support for this argument also come from our experiments with BSO, a potent and specific inhibitor of glutamate-cysteine ligase (the rate-limiting step in GSH synthesis) (Griffith and Meister, 1979). Presence of BSO had no significant effect on GSH release in the time frame of the experiment (Fig. 1). A longer BSO incubation would be expected to lower intracellular GSH to a larger extent, and possibly have an effect on glutamate-induced release if critical intracellular GSH levels were reached. Altogether, these results are in agreement with reports showing that astrocytes rely on stored GSH to resist otherwise harmful conditions, failing to survive only when these pools are depleted (Chen et al. 2000), and emphasise the capacity of astrocytes to release GSH when exposed to glutamate.
High concentrations of glutamate can be toxic to some cell types, leading to necrotic cell death with membrane rupture and leakage of intracellular content (Coyle and Puttfarcken 1993). As intracellular GSH concentrations are about 1000-times extracellular concentrations (mM vs. μM respectively; Dringen 2000), an increase in membrane leakage could explain the significant increase in extracellular GSH in the current study. However, no significant differences could be detected between control and glutamate-treated cells in terms of LDH release, suggesting that increased extracellular GSH detection was not a result of membrane rupture induced by glutamate. Our results are consistent with those of others in terms of the gliotoxic action of glutamate. Chen et al. (2000) demonstrated that 10 mM L-glutamate leads to LDH release only after a very prolonged incubation period (16 h), during which changes in cell morphology and oxidative stress occurs. These changes could be terminated by removal of glutamate before the onset of cell damage (estimated to occur at 4–6 h), indicating that the glutamate effect was reversible and that continuous exposure was required for astrocyte death. As glutamate did not appear to cause release of GSH through non-specific cell leakage other mechanisms were investigated.
The data in Table 1 show that glutamate increases the proportion of GSH that is extracellular in astrocyte cultures. Two possible explanations for this rise in extracellular GSH have been discounted in this study – namely glutamate inhibition of extracellular processing of GSH by γGT (Fig. 2) and glutamate affecting the extracellular GSH/GSSG ratio (Determination of cellular GSH). Therefore, the most likely explanation for the increase in extracellular GSH in astrocyte cultures upon exposure to glutamate is stimulation of GSH release (Fig. 5). This increased release of GSH from rat astrocytes could result from the activation of glutamate receptors and/or activation of downstream signalling pathways by glutamate. Glutamate receptors are considered to be expressed mainly on neurones but are also present on astrocytes (Porter and McCarthy 1996, 1997), where they have been increasingly implicated in a number of important pathways, including e.g. regulation of intracellular Ca2+ levels, stimulation of protein kinase C and inhibition of adenylate cyclase (Porter and McCarthy 1996, 1997; Winder and Conn 1996). Neurotransmitter(s) released from pre-synaptic terminals could therefore activate receptors located on astrocytes, leading to GSH release. However, data from experiments using agonists for ionotropic glutamate receptors suggests that neither NMDA nor AMPA/kainate receptors are involved in GSH release, as we were unable to detect elevated extracellular levels of GSH after incubation with NMDA or AMPA. Preliminary experiments have also so far failed to show a role of metabotropic receptors in GSH release (data not shown). Glutamate is also able to induce various changes in astrocytes which are not mediated via glutamate receptors. These changes include a switch of astrocytic metabolism from glycolytic to oxidative, via decreased glucose utilisation and increased mitochondrial activity (Liao and Chen 2003). Such changes to astrocyte energy metabolism may also affect GSH metabolism and export, although this remains to be elucidated.
Figure 5. Proposed neuroprotective role of glutamate-induced up-regulation of GSH release from astrocytes. (1) Extracellular glutamate is toxic to neurones via excessive stimulation of glutamate receptors (GluR) (Coyle and Puttfarcken 1993), which may result in increased oxidative stress and an increase in GSH consumption (black full arrows). (2) In the present study, we have demonstrated that extracellular glutamate also increases the release of GSH from astrocytes (grey full arrows) by an unknown mechanism(s), possibly via the transporter Mrp1 (Minich et al. 2006) and/or modifications at gene level (Shih et al. 2003) (grey dashed arrows). (3) This extracellular GSH can be used by neurones to increase their intracellular GSH levels (Dringen 2000), making them more resistant to glutamate-induced oxidative stress (Gegg et al. 2005). Increased extracellular GSH may also counteract glutamate toxicity by competing with glutamate for binding sites on glutamate receptors (Oja et al. 2000).
Download figure to PowerPoint
Reactive nitrogen/oxygen species such as hydrogen peroxide and NO have also been implicated in the increase of GSH in cultured astrocytes (Sagara et al. 1996; Gegg et al. 2003), and oxidative stress was shown to result in the over-expression of Nrf2, a transcription factor implicated in GSH use, production and efflux pathways in astrocytes, via antioxidant-response element activation (Shih et al. 2003). Hypothetically, such transcription factor regulated changes could also be induced by glutamate and increase GSH efflux. However, significant changes to gene expression are likely to take hours rather than minutes and are therefore unlikely to contribute to the initial glutamate-induced GSH release observed in this study. Several of the transporters reported to transport GSH are expressed in cultured astrocytes (Minich et al. 2006). However, so far only multidrug resistance protein 1 has been identified in astrocytes to participate in GSH transport under basal conditions (Minich et al. 2006). Whether this transporter, other multidrug resistance proteins, organic anion transporters or the cystic fibrosis transmembrane conductance regulator protein contribute to the elevated GSH release from astrocytes in the presence of glutamate remains to be elucidated.
This increased release of GSH in response to high extracellular glutamate can be regarded as a candidate antioxidant defence mechanism preventing neuronal damage (Drukarch et al. 1997, 1998; Gegg et al. 2005), but GSH may also be implicated in other regulatory events. GSH has been described as candidate modulator of CNS excitability, through binding to the NMDA receptor complex as either an agonist or antagonist in particular circumstances (Ogita et al. 1995; Oja et al. 2000); has been shown to limit cell sensitivity to NO-mediated mitochondrial injury (Bolanos et al. 1996; Gegg et al. 2005); and GSH and other reductants have also been demonstrated to increase the glutamate uptake current of glutamate transporters, an event that could be reversed by the oxidative agent 5,5′-dithio-bis(2-nitrobenzoic) acid (Trotti et al. 1997). In light of this, the ability of astrocytes to release GSH may prove to be important in protecting neurons from glutamate toxicity in distinct brain structures by means other than its role as an antioxidant.
In conclusion, our experimental strategy models conditions where extracellular glutamate levels are raised for prolonged periods such as during ischaemia. Considering the range of glutamate-mediated mechanisms leading to neuronal death, including nitrosative and oxidative stress, the increased availability of GSH, an endogenous low-molecular weight antioxidant, may constitute an important protective mechanism in response to excitotoxic insults.