Introduction

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High extracellular concentrations of the amino acid neurotransmitter, glutamate, can be neurotoxic. Its effects are terminated by re-uptake into neurons or astrocytes, with astrocytes responsible for a major part of glutamate uptake in the brain. Glutamate released into the synaptic cleft can depolarize neurons via specific receptors. Its action is terminated by its uptake from the synaptic cleft, mostly by Na⁺-dependent uptake systems that are located on both astrocytes and neurons. Anion conductance is also associated with activation of the glutamate transporters, but it is not coupled to glutamate movement and varies widely for the different transporters. The generated electrogenic gradient translocates glutamate against a several thousand-fold concentration gradient, maintaining optimal glutamate concentrations in the extracellular fluid.

Although essential for normal functioning of the central nervous system (CNS), increased extracellular glutamate levels are neurotoxic (Choi, 1992). Over-activation of neuronal N-methyl-D-aspartate receptors (NMDAR) by exogenous glutamate is associated with increased influx of Na⁺ and Ca²⁺ ions and the ensuing neuronal death (Choi, 1992). Increased intracellular Na⁺ alters membrane potentials, produces neuronal swelling due to osmotically obliged water movement, and eventually causes cellular lysis. Intracellular Ca²⁺ overload, in addition to the osmotic stress and cell swelling, leads to stimulation of numerous Ca²⁺-activated enzymes, such as the protease calpain I that degrade cellular structural proteins (Siman et al., 1989). Additionally, Ca²⁺ overload activates phospholipases leading to breakdown of the cellular membrane and subsequent release of endonucleases that degrade DNA. The Ca²⁺-mediated activation of phospholipase A₂ releases arachidonic acid, increasing production of reactive oxygen species (ROS) (Lafon-Cazal et al., 1993). These ROS, in combination with peroxynitrate and other free radicals that are generated by the activity of nitric oxide synthetase (Dawson et al., 1991), lead to peroxidation of lipid, cellular lysis, and eventual cell demise. Arachidonic acid and ROS (Volterra et al., 1994) inhibit excitatory amino acid (EAA) transporter function limiting removal of extracellular glutamate, thus producing increased NMDAR stimulation, further production of arachidonic acid and ROS, and greater inhibition of EAA transport. This feed-forward NMDAR- and Ca²⁺-mediated cycle eventually leads to neuronal death (Choi, 1992).

In addition to Ca²⁺-dependent ROS-mediated neuronal lysis, elevated levels of extracellular glutamate inhibit the uptake of cystine, a precursor of glutathione (GSH) (Murphy et al., 1990), the major intracellular antioxidant in brain cells. This inhibition of precursor uptake decreases neuronal GSH levels (Murphy et al., 1990) and thus increases neuronal susceptibility to pro-oxidant events, such as those seen following exposure to the neurotoxic heavy metal, methylmercury (MeHg) (Aschner et al., 1994).

Glutamate transporters have been identified on neuronal and astrocytic membranes for removal of extracellular glutamate (Pines et al., 1992; Storck et al., 1992; Rothstein et al., 1994). One of these transporters, commonly referred to as glutamate/aspartate transporter (GLAST), was shown to be the predominant one in cultured astrocytes (Gegelashvili et al., 1996).

Disruption in glutamate homeostasis is thought to be a factor in the pathogenesis of certain neurological and psychiatric diseases. Glutamate uptake decreases with normal brain aging and can thus contribute to age-related neurodegenerative disorders (Danbolt, 2001). During ischemia, glutamate accumulates in the extracellular space, which may contribute to cell death. The main cause of such accumulation is presumably the reversal or failure of glutamate uptake transporters, rather than synaptic release (Attwell et al., 1993).

The present review underscores the role of glutamate transporters in maintaining optimal CNS function, with specific emphasis on the cycling of glutamate between astrocytes and neurons. Neuropathologic conditions that might be associated with aberrant glutamate transporter function are highlighted. Finally, the roles of astrocytes and neurons in glutamate cycling are discussed within the context of anesthetic treatment with pentobarbital and thiopental. Because of the broad nature of this subject and the limited scope of this review, we have been unable to cite many relevant articles. The curious reader will find additional information in a scholarly review by Danbolt (2001).

	Glutamate Transporters in Neurotoxicology

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To prevent initiation of glutamate-induced neurotoxic cascades, a family of high-affinity Na⁺-dependent transporters has evolved to keep extracellular levels of glutamate below toxic concentrations. These transporters maintain a 10,000-fold gradient of astrocytic intracellular glutamate (3-10 mM) to extracellular glutamate (0.3-1 µM) (Schousboe and Divac, 1979) that is driven by the ionic gradients generated by ion-exchanging pumps such as Na⁺/K⁺-ATPase. Originally termed system X_AG, at present there are five distinct Na⁺- and K⁺-dependent EAA transporters that have been identified and cloned. They are referred to as EAAT1 (also designated as GLAST in rodents) (Storck et al., 1992), EAAT2 (also designated as GLT1 in rodents) (Pines et al., 1992), EAAT3 (also designated as EAAC1 in rodents) (Kanai et al., 1995b), EAAT4 (Fairman et al., 1995), and EAAT5 (Attwell and Mobbs, 1994). These transporters display heterogeneous regional and cellular expression. EAAT1 and EAAT2 are localized to astrocytes, with EAAT1 predominating in the cerebellum and EAAT2 predominating in the cortex and forebrain (Furuta et al., 1997). EAAT3 is localized to neurons throughout the CNS (Kanai et al., 1995a), whereas EAAT4 localization is largely restricted to cerebellar Purkinje cells (Fairman et al., 1995). EAAT5 has been localized exclusively in the retina (Arriza et al., 1997) and has been hypothesized to function as a photoreceptor on bipolar rod and cone cells in the rat (Pow and Barnett, 2000). Studies with antisense knockdown of specific transporter subtypes have revealed that the astrocytic transporters (EAAT1 and EAAT2) are responsible for removing the majority of the extracellular glutamate (Rothstein et al., 1996).

Interaction between astrocytes and neurons is a prerequisite for the expression and maintenance of glutamate transporter function. Astroglial cultures express only GLAST, whereas astrocytes, which are cocultured with neurons, express both GLAST and GLT1 (Gegelashvili et al., 1997). Soluble factors derived from neurons are important for the induction of GLT1 protein and its mRNA in astrocytes, given that pure cortical astroglial cultures supplemented with conditioned medium from cortical neuronal cultures or from mixed neuron-glia cultures (Gegelashvili et al., 1997; Swanson et al., 1997) express this protein (GLT1). Treatment of astrocyte cultures with dibutyryl cyclic AMP (dB-cAMP) led to expression of GLT1 and increased expression of GLAST (Swanson et al., 1997) with an increase in glutamate uptake V_max but no change in the glutamate K_m and no increased sensitivity to inhibition by dihydrokainate. These results suggest that soluble neuronal factors differentially regulate the expression of GLT1 and GLAST in cultured astroglia (Gegelashvili et al., 1997) and that dB-cAMP can partially mimic this influence (Swanson et al., 1997). In addition to transporting L-glutamate, these proteins also have an approximately equal affinity for D-aspartate and L-aspartate, although D-glutamate is a poor substrate (Fairman and Amara, 1999). For each EAA molecule that is transported, there is also cotransport of two or three Na⁺ ions, counter-transport of one K⁺ ion (Wadiche et al., 1995), and either the cotransport of a proton (Zerangue and Kavanaugh, 1996) or the counter-transport of a hydroxyl ion (or HCO₃) (Attwell and Mobbs, 1994). In addition, there is a glutamate-activated Cl flux distinct from EAA transport (Wadiche et al., 1995; Arriza et al., 1997; Wadiche and Kavanaugh, 1998) that is not blocked by compounds that inhibit endogenous Cl channels (Wadiche et al., 1995). The existence of channel-like, substrate-mediated ion flux is not limited to EAA transporters but has been described for numerous neurotransmitter transporters (Wadiche et al., 1995). The function of this Cl flux in EAA transporters has been hypothesized to counteract Na⁺-induced cellular depolarization that would otherwise decrease EAA transport (Eliasof and Jahr, 1996).

The importance of astrocytic EAA transporters in controlling extracellular levels of glutamate has been demonstrated by anti-sense knockdown (Rothstein et al., 1996) and genomic disruption (Tanaka et al., 1997; Watase et al., 1998). Inactivation of the astrocytic transporters EAAT1 (GLAST) or EAAT2 (GLT1) in rats by chronic antisense oligonucleotide infusion produced increased extracellular glutamate and excitotoxic neurodegeneration (Rothstein et al., 1996). In transgenic mice, knockout of EAAT1 (Watase et al., 1998) or EAAT2 (Tanaka et al., 1997) produced increased susceptibility to traumatic injury and increased brain edema. Antisense knockdown of the neuronal EAA transporter EAAT3 (EAAC1) did not elevate glutamate levels and produced only mild neurotoxicity (Storck et al., 1992). Loss of EAAT3 by genomic disruption in mice produced no neurodegeneration (Peghiniet al., 1997). These data clearly attest to the vital role of astrocytic EAA transporters in preventing glutamate-mediated neurotoxicity.

The potential involvement of altered glutamate homeostasis in both acute and chronic neurodegenerative diseases has been reviewed in a seminal paper by Danbolt (2001). Given the comprehensive nature of the review, we will only briefly address the role of glutamate transporters in the etiology of neurodegenerative disorders, encouraging those who are interested in the topic to seek additional details in Danbolt's review (2001). In patients with amyotrophic lateral sclerosis (ALS), abnormality in the glutamate transport system has been implicated as potentially playing an etiologic role. Synaptic preparations from the motor and sensory cortex of these patients demonstrate decreased glutamate transporter activity (Rothstein et al., 1992), reflecting reduced activity of the glutamate transporter isoform, GLT1 (Rothstein et al., 1995). An abnormal RNA editing process was invoked as a probable cause for loss of GLT1 in the sporadic form of ALS. More recent studies, in which the functional impact of a naturally occurring mutation of human GLT1 in a patient with sporadic ALS suggests that the mutation involves a substitution of the putative N-linked glycosylation site, asparagine 206, by a serine residue (N206S), resulting in reduced glycosylation of the transporter and attenuated uptake activity (Trotti et al., 2001). Selective, focal loss of GLT1 and GLAST transporter proteins was also invoked as a potential explanation for the increase in interstitial glutamate levels and the selective vulnerability of thalamic structures to thiamine deficiency-induced cell death (Hazell et al., 2001).

Given evidence that in patients with various epilepsies the level of extracellular glutamate is increased (Ferrie et al., 1999), recent studies have also examined whether changes in glutamate transporter function and expression in the medial temporal cortex and hippocampus could be invoked in the development or maintenance of seizures. Studies by Tessler et al. (1999) have demonstrated that there is no reduction in the level of GLT1 encoding messenger RNA in the temporal lobe of epilepsy patients compared with controls, suggesting that major changes in the level of expression of GLT1 do not play an important role in the development of human temporal lobe epilepsy. It has yet to be determined whether GLT1 plays a role in the etiology of other types of epilepsy (Danbolt, 2001). Similarly, a definitive role for altered expression or activity of GLAST (EAAT1) and GLT1 (EAAT2) in Alzheimer's disease has, so far, not been established (Danbolt, 2001). Studies by Beckstrom et al. (1999) showed a negative correlation between Alzheimer's disease and glutamate transporters with both normal and reduced levels of GLAST and GLT1. Although reports (Danbolt, 2001) suggest that glutamate uptake inhibition might aggravate the progression of Alzheimer's disease, direct evidence for altered GLAST and GLT1 expression and activity has yet to be established.

	Glutamate and Glucose Metabolism

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The metabolic fate of glutamate has been studied in cultured cortical astrocytes (Sonnewald et al., 1993), cerebellar astrocytes (Qu et al., 2001b), and cerebellar granule neurons (Sonnewald et al., 1996) using [U-¹³C]glutamate and magnetic resonance spectroscopy. In cortical and cerebellar astrocytes, it could be shown that glutamate was not only converted to glutamine but, to a large extent, entered the tricarboxylic acid (TCA) cycle. The carbon skeleton was found in aspartate and in newly synthesized glutamate and glutamine (Fig. 1, Table 1). Surprisingly, labeled lactate signifying glutamate metabolism in the TCA cycle was detected in the medium from these cells (Table 1; Sonnewald et al., 1993, Qu et al., 2001b). In cerebellar neurons [U-¹³C]glutamate was converted to aspartate and glutathione (Sonnewald et al., 1996). It should be noted that [U-¹³C]glutamate is handled differently in astrocytes and cerebellar granule neurons (Table 1). Pyruvate recycling as indicated by the labeling pattern of aspartate and lactate in astrocytes (Håberg et al., 1998) was not observed in these neurons (Sonnewald et al., 1996). The physiological role of pyruvate recycling is at present not clear.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Schematic representation of possible isotopomers arising from [U-13C]glutamate.  represents 13C; lac, lactate; Mal, malate; OAA, oxaloacetate.

Astrocytic uptake of glutamate by specific transporters has also been invoked as potentially regulating signal-transducing properties that are distinct from their transporter activity (Pellerin and Magistretti, 1994). Astrocytic uptake of glutamate has been proposed to stimulate glycolysis (glucose utilization and lactate production) via the activation of a Na⁺-dependent uptake system that involves the Na⁺/K⁺-ATPase (resulting from increased intracellular Na⁺ concentration that is cotransported with glutamate by the electrogenic uptake system). It was suggested (Pellerin and Magistretti, 1994) that when glutamate is released from active synapses and taken up by astrocytes, this signaling pathway provides a simple and direct mechanism to tightly couple neuronal activity to glucose utilization. Glutamate-stimulated glycolysis and lactate production is also consistent with data obtained from functional brain imaging studies, which indicate that the mammalian CNS normally shifts to local nonoxidative glucose utilization during physiological activation (Pellerin and Magistretti, 1994). These observations point to a critical role of the astrocyte in coupling neuronal activity to glucose utilization. Indeed, it appears that in response to glutamate released by active neurons, glucose is predominantly taken up by specialized astrocytic processes, the end-feet. Subsequently, glucose is metabolized to lactate, which provides a preferred energy substrate for neurons (Pellerin et al., 1996). However there are conflicting results concerning glucose consumption in the presence of glutamate. Qu et al. (2001a) showed that the amount of [U-¹³C]glucose removed from the medium of cerebellar astrocytes was decreased in the presence of 0.5 mM glutamate. This was also observed by Swanson et al. (1997), using 1.0 mM glutamate in cultured cortical rat astrocytes and by Peng et al. (2001), who used up to 1.0 mM glutamate in cultured cortical mouse astrocytes. The distribution of metabolized [U-¹³C]glucose into different pathways in cerebellar astrocytes showed that 83.3% of labeled glucose was metabolized mainly for energy production, and this decreased to 77.9% when unlabeled glutamate was present (Qu et al., 2001a). Uptake of glutamate is an energy demanding process requiring one molecule of ATP for uptake of one molecule of glutamate. In addition, the direct conversion of glutamate to glutamine by glutaminase is also ATP-dependent. To enter the TCA cycle, glutamate must be transported into mitochondria and be converted to 2-oxoglutarate, which can be converted to oxaloacetate producing nine molecules of ATP (truncated TCA cycle). The activities of glutamate dehydrogenase and aminotransferases, the enzymes responsible for conversion of glutamate to 2-oxoglutarate and further metabolism in the TCA cycle, have been shown to be sufficient such that glutamate can replace glucose as an energy substrate in astrocytes (Hertz, 1982). It has been shown that glutamate metabolism is concentration-dependent, where more glutamate is consumed for direct formation of glutamine at low concentration (0.01-0.1 mM) and more is metabolized via the TCA cycle at high concentration (0.2-0.5 mM) (McKenna et al., 1996). Thus, the uptake and metabolism of glutamate is an energy demanding process at low glutamate concentrations; however, it becomes an energy producing process when glutamate is present in sufficient concentration. Qu et al. (2001a) showed that the total amount of aspartate was increased when 0.5 mM unlabeled glutamate was present, indicating that the "truncated TCA cycle" was indeed operative. Thus, the uptake and metabolism of glutamate can spare glucose as an energy substrate.

	Effects of Thiopental on Glutamate Transport and Metabolism

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Glutamate Uptake and Release. It has been shown that thiopental inhibits uptake of GABA, aspartate, glutamate, as well as biogenic amines in rat cortical synaptosomes (Pastuszko et al., 1984). In cortical astrocytes the amount of glutamate taken up was unchanged during incubation with 0.5 mM thiopental (Qu et al., 1999), in agreement with the study of Miyazaki et al. (1997) where unchanged glutamate uptake with pentobarbital concentrations ranging from 0.03 to 0.3 µM was observed. However, with 1 mM thiopental, glutamate uptake was decreased by more than 50% (Table 1). It is well known that barbiturates can affect cell membrane proteins, such as GABA_A receptors (for review, see Ito et al., 1996), and thus could eventually interfere with intracellular ion homeostasis and glutamate metabolism, thereby decreasing the Na⁺-dependent glutamate uptake. It should be noted that this reduction of glutamate uptake might exert a protective effect under conditions where the glutamate transporters are reversed, such as during ischemia. Decreased glutamate release was observed after ischemia in the presence of thiopental in gerbils (Amakawa et al., 1996), which is in agreement with this hypothesis. That thiopental, indeed, exerts protective effects has been shown by reduced clinical expression of cerebral emboli in cardiopulmonary bypass patients (Nussmeier et al., 1986) and improved energy metabolism during ischemia in gerbils (Zarchin et al., 1998). It should be noted that in cultured cerebellar astrocytes and neurons the uptake of glutamate was unaffected by thiopental (Table 1).

Glutamate Metabolism. After entering the cells, glutamate can be converted directly into peptides, such as GSH, or be metabolized via the TCA cycle for energy production and synthesis of various metabolites (Fig. 1; Table 1; for review, see Sonnewald et al., 1997). [U-¹³C]Glutamate was metabolized via the same pathways in cerebellar and cortical astrocytes. However, the amounts of lactate formed from [U-¹³C]glutamate, especially in the presence of 1 mM thiopental (Table 1) or unlabeled glucose, were higher in cerebellar than in cortical astrocytes, and alanine formation was only observed in cerebellar astrocytes (Qu et al., 1999, 2001). Such regional differences were also reported in an earlier study using glucose (Hassel et al., 1995) and underscore the importance of analyzing cells from different brain regions. Furthermore, the distribution of glutamate to different pathways was different in the astrocyte cultures from these two regions. Differences between cortical and cerebellar astrocytes were also reported (Hassel et al., 1995, Merle et al., 1996, Martin et al., 1997) and might be related to different cell maturation stages, as pointed out by Martin et al. (1997).