Elsevier

Neurochemistry International

Volume 43, Issues 4–5, September–October 2003, Pages 411-416
Neurochemistry International

Effect of methylmercury on glutamate metabolism in cerebellar astrocytes in culture

https://doi.org/10.1016/S0197-0186(03)00029-9Get rights and content

Abstract

The effect of methylmercury (MeHg) on [U-13C]glutamate metabolism was studied in cerebellar astrocytes using 13C nuclear magnetic resonance spectroscopy. The cells were preincubated in medium containing 25 or 50 μM MeHg and 10% fetal calf serum for 4 h and then in medium with [U-13C]glutamate (0.5 mM) for 2 h. Labeled glutamate, glutamine and aspartate were observed both in the cell extracts and media, labeled glutathione in the cell extracts and labeled lactate and alanine in the media. The amount of glutamate removed from the media was decreased in the 50 μM MeHg group, furthermore, the levels of both labeled and unlabeled glutamine were decreased. This might indicate a decreased synthesis and/or increased degradation. An increase was observed for glutathione in the 25 μM group, which might be due to an upregulated synthesis of glutathione in response to the toxic effects of MeHg. The percentage of [U-13C]glutamate used for the synthesis of metabolites via the tricarboxylic acid cycle was increased in the presence of 50 μM MeHg. However, the percentage used for energy production was decreased in both groups, indicating selective mitochondrial vulnerability due to the inhibitory effect of MeHg.

Introduction

Methylmercury (MeHg) is an organic form of mercury with toxic effects in multiple organs. In the brain, MeHg poisoning is characterized by damage to discrete anatomical areas and is the cause of the “so called” Minamata disease (for review, see Castoldi et al., 2001). The main route for human exposure involves the consumption of MeHg-adulterated fish from water polluted with organic mercury. Due to the high binding affinity of MeHg to sulfur, proteins and peptides bearing thiol groups are susceptible to structural and functional modifications by MeHg.

The mechanism of action for MeHg is not fully understood (Aschner, 2000). Several targets, such as blood–brain barrier, axonal transport, neurotransmisson, and synthesis of protein, DNA and RNA, have been proposed as sensitive sites for MeHg’s toxic effects (for review, see Castoldi et al., 2001). Numerous observations have established the role of astrocytes in mediating MeHg neurotoxicity. (a) MeHg selectively inhibits astrocytic uptake systems, such as cystine, and cysteine transport (Shanker et al., 2001, Shanker and Aschner, 2001, Allen et al., 2001b), compromising the redox potential and attenuating glutathione content. (b) Notably, astrocytes represent a preferential cellular site for MeHg accumulation (Aschner, 1996, Charleston et al., 1996, Garman et al., 1975). (c) MeHg inhibits astrocytic glutamate (and aspartate) uptake and stimulates its efflux (Mullaney et al., 1994, Allen et al., 2001c), increasing glutamate concentrations in the extracellular fluid and sensitizing neurons to excitotoxic injury (Coyle and Puttfarken, 1993, Rothstein et al., 1996). (d) MeHg-induced neuronal dysfunction is secondary to disturbances in astrocytes (Brookes, 1992), and the in vitro co-application of non-toxic concentrations of MeHg with glutamate results in the appearance of typical neuronal lesions associated with excitotoxic stimulation (Matyja and Albrecht, 1993).

Glutamate is the main excitatory neurotransmitter (Fonnum, 1984) and is released during neuronal excitation. The effect of glutamate is terminated by uptake into the surrounding cells, and astrocytes represent a major site for glutamate uptake (for review, see Gegelashvili and Schousboe, 1998). Intracellular glutamate is metabolized in astrocytes and its metabolites, such as glutamine, aspartate, and lactate, can be formed and released for uptake by neurons (for review, see Sonnewald et al., 1997). Such interaction is exquisitely important for normal neuronal function due to the drain of tricarboxylic acid (TCA) cycle intermediates for neurotransmitter synthesis.

MeHg has been shown to preferentially accumulate in astrocytes, where it induces cell swelling and specifically inhibits excitatory amino acid uptake (Aschner et al., 2000). In a previous study cortical astrocytes were exposed to MeHg and subsequently incubated with [U-13C]glutamate (Allen et al., 2001a), showing that MeHg affected mitochondria. Given these earlier observations, the present study was designed to examine the effects of MeHg on cerebellar astrocytes using [U-13C]glutamate and 13C NMR spectroscopy.

Section snippets

Materials

Plastic tissue culture dishes were purchased from Nunc A/S (Roskilde, Denmark), fetal calf serum from Seralab Ltd. (Sussex, UK) and culture medium from GIBCO BRL, Life Technologies (Roskilde, Denmark). NMRI mice were purchased from Møllegaard Breeding Center (Copenhagen, Denmark). [U-13C]glutamate (98% enriched) and 99.9% D2O were from Cambridge Isotopes Laboratories (Woburn, MA, USA), and ethylene glycol from Merck (Darmstadt, Germany). All other chemicals were of the purest grade available

Results

Fig. 1 shows the transformation of [U-13C]glutamate in astrocytes. For detailed description of [U-13C]glutamate metabolism in cerebellar astrocytes see Qu et al. (2001). Briefly, [U-13C]glutamate enters the astrocytes and can be converted to glutamine and glutathione (GSH) directly. It can also enter the TCA cycle via 2-oxoglutarate for the synthesis of metabolites and energy production. [U-13C]aspartate and [U-13C]lactate can be formed from intermediates derived via the first turn of the TCA

Discussion

Several high-affinity glutamate transporters on the astrocytic membrane maintain optimal extracellular concentrations of glutamate (for review, see Gegelashvili and Schousboe, 1998). In the present study the amount of [U-13C]glutamate removed from the extracellular medium was unchanged when astrocytes were exposed to 25 μM MeHg for 2 h. The same was found in a study by Allen et al., 2001a using cortical astrocytes. However, the amount of [U-13C]glutamate removed from the media was decreased in

Acknowledgements

This research was supported by the Research Council of Norway, the Blix Foundation. The excellent technical assistance of Bente Urfjell is greatly appreciated.

References (33)

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    Neuronal dysfunction has been proposed to be secondary to disturbances in astrocytes [164]. As referred to by Aschner and Syversen [165], astrocytes accumulate MeHg, where among other effects it potently inhibits astrocytic glutamate uptake and stimulates its efflux [166,167]. This causes an increase in glutamate levels in the extracellular fluid, which may cause exitotoxic injury towards neurons.

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