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Keywords:

  • alanine;
  • cycle;
  • GABA;
  • glutamate;
  • glutamine;
  • leucine

Abstract

  1. Top of page
  2. Abstract
  3. The glutamate/GABA-glutamine cycle
  4. Model systems to study the glutamate/ GABA-glutamine cycle in vitro and in vivo
  5. Transporters of the glutamate/GABA-glutamine cycle
  6. Metabolism of the glutamate/GABA-glutamine cycle
  7. Glutamate/GABA-glutamine cycling and intercellular ammonia homeostasis
  8. Glutamatergic vs. GABAergic synapses: relative significance of neurotransmitter cycling
  9. Glutamate/GABA-glutamine cycling in pathological conditions
  10. Potential drug targets of the neurotransmitter cycling system
  11. Conclusion
  12. Acknowledgements
  13. References

Neurons are metabolically handicapped in the sense that they are not able to perform de novo synthesis of neurotransmitter glutamate and γ-aminobutyric acid (GABA) from glucose. A metabolite shuttle known as the glutamate/GABA-glutamine cycle describes the release of neurotransmitter glutamate or GABA from neurons and subsequent uptake into astrocytes. In return, astrocytes release glutamine to be taken up into neurons for use as neurotransmitter precursor. In this review, the basic properties of the glutamate/GABA-glutamine cycle will be discussed, including aspects of transport and metabolism. Discussions of stoichiometry, the relative role of glutamate vs. GABA and pathological conditions affecting the glutamate/GABA-glutamine cycling are presented. Furthermore, a section is devoted to the accompanying ammonia homeostasis of the glutamate/GABA-glutamine cycle, examining the possible means of intercellular transfer of ammonia produced in neurons (when glutamine is deamidated to glutamate) and utilized in astrocytes (for amidation of glutamate) when the glutamate/GABA-glutamine cycle is operating. A main objective of this review is to endorse the view that the glutamate/GABA-glutamine cycle must be seen as a bi-directional transfer of not only carbon units but also nitrogen units.

Abbreviations used
AAT

aspartate aminotransferase

ALAT

alanine aminotransferase

BCAT

branched-chain amino acid transaminase

GABA

γ-aminobutyric acid

GAD

glutamate decarboxylase

GDH

glutamate dehydrogenase

GS

glutamine synthetase

MeAIB

2-(methylamino)isobutyric acid

NMR

nuclear magnetic resonance

PAG

phosphate-activated glutaminase

PC

pyruvate carboxylase

TBOA

DL-threo-β-benzyloxyaspartate

TCA

tricarboxylic acid

The amino acid glutamate serves a multitude of roles in the mammalian brain; it is an excitatory neurotransmitter as well as the immediate precursor of the inhibitory neurotransmitter γ-aminobutyric acid (GABA); an essential component of intermediary metabolism; a building block of proteins; an energy substrate; and, paradoxically, a potent neurotoxin. Perhaps as a result of this, glutamate homeostasis is quite complex involving several cell specific elements including membrane transporters and enzymes in both neurons and astrocytes. One of the most striking elements regarding the enzymatic machinery is the lack of pyruvate carboxylase (PC) in neurons, making them incapable of de novo synthesis of glutamate and GABA from glucose (Patel 1974; Yu et al. 1983; Shank et al. 1985; Schousboe et al. 1997; Hertz et al. 1999). Another remarkable element regarding cell membrane transport is the generally accepted view of a predominant astrocytic glutamate uptake (Schousboe 1981; Rothstein et al. 1996; Gegelashvili and Schousboe 1998; Danbolt 2001). The latter results in a drain of glutamate from neurons to astrocytes which due to the incapability of de novo synthesis in the neuronal compartment is compensated for by a supply of a glutamate precursor formed in the astrocytic compartment.

The glutamate/GABA-glutamine cycle

  1. Top of page
  2. Abstract
  3. The glutamate/GABA-glutamine cycle
  4. Model systems to study the glutamate/ GABA-glutamine cycle in vitro and in vivo
  5. Transporters of the glutamate/GABA-glutamine cycle
  6. Metabolism of the glutamate/GABA-glutamine cycle
  7. Glutamate/GABA-glutamine cycling and intercellular ammonia homeostasis
  8. Glutamatergic vs. GABAergic synapses: relative significance of neurotransmitter cycling
  9. Glutamate/GABA-glutamine cycling in pathological conditions
  10. Potential drug targets of the neurotransmitter cycling system
  11. Conclusion
  12. Acknowledgements
  13. References

Among others, discoveries of intercellular compartmentation of glutamine and glutamate pools, related to astrocytes and neurons, respectively, led to the suggestion of a glutamate-glutamine cycle working between (glutamatergic) neurons and astrocytes (van den Berg and Garfinkel 1971; Benjamin and Quastel 1972; Berl and Clarke 1983; Ottersen et al. 1992). The cycle in a glutamatergic synapse is outlined in Fig. 1a in which released neurotransmitter is taken up into surrounding astrocytes, transformed into glutamine by the astrocyte-specific enzyme glutamine synthetase (GS; Norenberg & Martinez-Hernandez 1979) and released into the extracellular space from which it is taken up into neurons and transformed back to glutamate by phosphate-activated glutaminase (PAG; Kvamme et al. 2001). In the GABAergic synapse (Fig. 1b), GABA is taken up into astrocytes and catabolized to the TCA cycle intermediate succinate via the concerted action of GABA transaminase and succinate semialdehyde dehydrogenase. Glutamine may be synthesized from succinate via the TCA cycle including condensation of oxaloacetate and acetyl-CoA forming citrate and subsequently synthesis of α-ketoglutarate and glutamate. Glutamate formed by PAG activity in the GABAergic neurons is converted by glutamate decarboxylase (GAD) to GABA. The supply of glutamine to GABAergic neurons might be quantitatively less significant, as these neurons most likely exhibit a larger proportion of reuptake of the released neurotransmitter compared to their glutamatergic counterparts (e.g. Schousboe et al. 2004). These seemingly simple mechanisms are supported by cell-specific transport and metabolism, as will be discussed in the following sections. In addition, an emphasis will be put on the inherent dilemma of the ammonia homeostasis linked to this cycle. As can be seen in Fig. 1a and b, for each molecule of glutamate or GABA cycled, one molecule of ammonia will be produced in the neurons and one molecule of ammonia assimilated in the astrocytes. This ammonia will obviously have to be transported out of the neurons and back into the astrocytes for detoxification, as an elevated ammonia concentration has detrimental effects on a number of cellular functions (e.g. Felipo and Butterworth 2002).

image

Figure 1.  Schematic representations outlining the glutamate-glutamine cycle in a glutamatergic synapse (a) and the GABA-glutamine cycle in a GABAergic synapse (b) In the glutamatergic synapse (a) the released neurotransmitter glutamate is predominantly taken up into astrocytes, where it is amidated to glutamine by GS using free ammonia and returned to the neurons. In the neurons, the PAG reaction regenerates the glutamate and produces ammonia. As indicated by the dashed line, this ammonia will have to be transported back to the astrocytes for detoxification. As discussed in the text and shown in Fig. 3, this might take place via an amino acid shuttle. Glutamate might to some extent be metabolized within the TCA cycle of both neurons and astrocytes. In the GABAergic synapse (b) the released neurotransmitter GABA is taken up into both the surrounding astrocytes and the pre-synaptic terminal. In the astrocytes, GABA is metabolized in two steps to succinate, which is further metabolized in the TCA cycle to α-ketoglutarate and then glutamate. From here on, the steps are similar to the glutamatergic synapse. As discussed in the text, the GABAergic synapse also needs to shuttle ammonia back to the astrocytes. The role of reuptake of glutamate and GABA into the pre-synaptic neuron is at present not known in detail, although the present dogma is that GABAergic neurons rely more on reuptake than glutamatergic neurons. Abbreviations: GABA, γ-aminobutyric acid; GAD, glutamate decarboxylase; Glu, glutamate; Gln, glutamine; GS, glutamine synthetase; α-KG, α-ketoglutarate; PAG, phosphate-activated glutaminase; Suc, succinate; TCA, tricarboxylic acid.

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Model systems to study the glutamate/ GABA-glutamine cycle in vitro and in vivo

  1. Top of page
  2. Abstract
  3. The glutamate/GABA-glutamine cycle
  4. Model systems to study the glutamate/ GABA-glutamine cycle in vitro and in vivo
  5. Transporters of the glutamate/GABA-glutamine cycle
  6. Metabolism of the glutamate/GABA-glutamine cycle
  7. Glutamate/GABA-glutamine cycling and intercellular ammonia homeostasis
  8. Glutamatergic vs. GABAergic synapses: relative significance of neurotransmitter cycling
  9. Glutamate/GABA-glutamine cycling in pathological conditions
  10. Potential drug targets of the neurotransmitter cycling system
  11. Conclusion
  12. Acknowledgements
  13. References

The individual properties of the amino acid transport systems involved in the glutamate/GABA-glutamine cycle have been studied extensively in expression systems such as Xenopus laevis oocytes. This, however, only offers clues to the function of individual transporters and consequently investigations based on in vitro, ex vivo and in vivo models of the brain must be employed to delineate the roles of these transporters in systems with an operating glutamate/GABA-glutamine cycle.

To study the glutamate/GABA-glutamine cycle in vitro, a system in which intact neuronal–astrocytic interactions are present is needed. Over the years, acute slices as well as slice cultures of different brain regions from different species have been used to study neuronal–astrocytic interaction. While these preparations might constitute a system more closely related to the in vivo environment compared to primary cell cultures, the latter offer the advantage of preparations enriched in neurons of specific phenotype, i.e. glutamatergic or GABAergic neurons (Drejer and Schousboe 1989; Hertz et al. 1989; Schousboe et al. 1989; Sonnewald et al. 2004). When cerebellar and neocortical neurons are cultured together with astrocytes from the corresponding brain regions (Westergaard et al. 1991; Schousboe et al. 1993), these co-cultures represent a valuable tool for studying the importance of the glutamate-glutamine and the GABA-glutamine cycle in glutamatergic and GABAergic synapses, respectively.

In vivo nuclear magnetic resonance (NMR) spectroscopy represents an especially well-suited method for studying the glutamate/GABA-glutamine cycle in intact animals and humans. Such studies have confirmed the existence of the glutamate/GABA-glutamine cycle in vivo and significant progress has been made to quantify the cycling both during normal and pathological conditions (Sibson et al. 1997; Lebon et al. 2002; Petroff et al. 2002; Oz et al. 2004; Patel et al. 2005Hyder et al. 2006), as discussed in the following sections.

Transporters of the glutamate/GABA-glutamine cycle

  1. Top of page
  2. Abstract
  3. The glutamate/GABA-glutamine cycle
  4. Model systems to study the glutamate/ GABA-glutamine cycle in vitro and in vivo
  5. Transporters of the glutamate/GABA-glutamine cycle
  6. Metabolism of the glutamate/GABA-glutamine cycle
  7. Glutamate/GABA-glutamine cycling and intercellular ammonia homeostasis
  8. Glutamatergic vs. GABAergic synapses: relative significance of neurotransmitter cycling
  9. Glutamate/GABA-glutamine cycling in pathological conditions
  10. Potential drug targets of the neurotransmitter cycling system
  11. Conclusion
  12. Acknowledgements
  13. References

Underlining the important role of glutamate and GABA as neurotransmitters, highly efficient and specific transport systems for these two amino acids are found throughout the brain. For glutamate, five high-affinity transporters have been identified and cloned, designated EAAT1(GLAST), EAAT2(GLT-1), EAAT3(EAAC1), EAAT4 and EAAT5 (Kanai and Hediger 1992; Pines et al. 1992; Storck et al. 1992; Fairman et al. 1995; Arriza et al. 1997). EAAT1 and 2 are primarily astroglial and EAAT3 is a neuronal transporter (Danbolt 2001). EAAT4 and 5 are less abundant and seem to be primarily expressed by Purkinje cells in the cerebellar molecular layer and retinal Müller cells, respectively (Danbolt 2001). For the present discussion the astroglial transporters EAAT1 and 2 are the most important, as they seem to be the predominant ones responsible for glutamate uptake throughout the brain, although with some regional variation (Danbolt 2001; Schousboe et al. 2004). The inward transport of one glutamate is coupled to co-transport of three Na+ and one H+, with one K+ being counter-transported (Zerangue and Kavanaugh 1996; Levy et al. 1998). This provides the uptake with a huge driving force, capable of maintaining an inside-out concentration gradient of 106 (Bröer 2005). A neuronal pre-synaptic glutamate transporter still eludes certain identification, although attempts have been made to assign this role to a second isoform of EAAT2, called GLT-1b (Chen et al. 2002). Neuronal reuptake of glutamate has been shown to play a role in sustaining glutamate homeostasis in cultured cerebellar (glutamatergic) neurons even in the presence of exogenous glutamine (Waagepetersen et al. 2005), an aspect to be further discussed in a subsequent section. However, uptake of released glutamate in cerebellar neuronal-astrocytic co-cultures is substantial compared to reuptake into neurons in mono-culture (Bak et al. 2004), supporting the generally accepted view that astrocytes are the cells predominantly responsible for neurotransmitter glutamate uptake.

Regarding GABA uptake, four transporters have been cloned and characterized (Guastella et al. 1990; Liu et al. 1992, 1993; Lopez-Corcuera et al. 1992). Unfortunately, the nomenclature applied to the mouse, rat and human homologues is rather confusing. The four cloned mouse transporters are products of the slc6a1 (mouse, GAT1; rat and human, GAT-1), slc6a12 (mouse, GAT2; rat and human, BGT-1), slc6a13 (mouse, GAT3; rat, GAT-2; human not cloned) and slc6a11 genes (mouse, GAT4; rat and human, GAT-3). The rat and human homologues of GAT2 are in fact a betaine transporter (BGT-1) with a lower affinity for GABA than GAT1, 3 and 4 (Bolvig et al. 1999). Transport of GABA is electrogenic and coupled to two Na+ and one Cl (Larsson et al. 1980; Radian and Kanner 1983). GAT-1 seems to be the predominant neuronal GABA transporter whereas the others show a more ubiquitous distribution (Minelli et al. 1996, 2003; Ribak et al. 1996a,b; De Biasi et al. 1998; Gadea and Lopez-Colome 2001; Schousboe and Kanner 2002).

Compared to transport of glutamate and GABA, the transport mechanisms of glutamine involved in the glutamate/GABA-glutamine cycle are more complex, as transport-mediated efflux from astrocytes must be met by transport-mediated influx in neurons. Several identified amino acid transporters might be able to fulfil this need and Fig. 2 shows the transport systems suggested to be involved, as discussed below. In general one could imagine that the transporters for glutamine on the neuronal side of the glutamate/GABA-glutamine cycle should be highly concentrative. Glutamine might otherwise leave the brain, since this is the most important way for the brain to dispose of an excess of ammonia, as discussed by Hawkins et al. (2002). The astrocytic side, on the other hand, could suffice with facilitated diffusion controlled by the intracellular concentration of glutamine. This is based on the notion that the glutamine concentration in astrocytes reflects the amount of glutamate taken up, providing a simple regulatory mechanism for efflux.

image

Figure 2.  Schematic representation showing the amino acid transport systems with suggested involvement in glutamine transport related to the glutamate/GABA-glutamine cycle. Details are discussed in the text. Abbreviations: aa, amino acid; Ala, alanine; Gln, glutamine; Leu, leucine.

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The concentrative, sodium-dependent and electrogenic system A transporter has been suggested to be involved in neuronal glutamine uptake (Varoqui et al. 2000; Bröer and Brookes 2001; Chaudhry et al. 2002a,b). System A is comprised of three isoforms named SNAT1 (ATA1, GlnT, SA2, SAT1, NAT2), SNAT2 (ATA2, SA1, SAT2) and SNAT4 (ATA3, NAT3) of which SNAT1 and 2 are expressed in the brain, with SNAT1 being fairly brain- and neuron-specific and found in the membrane of both glutamatergic and GABAergic neurons (Varoqui et al. 2000; Mackenzie et al. 2003; Weiss et al. 2003). SNAT2 seems more widely distributed, and is found on both neurons and astrocytes (Reimer et al. 2000; Sugawara et al. 2000). SNAT1 shows the aforementioned requirements for efficient glutamine transport into neurons, being a Na+-glutamine symporter with a Km for glutamine of 230 µm compared to 1.65 mm for SNAT2 (Yao et al. 2000; Albers et al. 2001; Mackenzie et al. 2003;). For the astrocytic part, system N comprising SNAT3 (SN1, NAT1) and SNAT5 (SN2) has been suggested to mediate the glutamine efflux, as discussed by Bröer and Brookes (2001) and Chaudhry et al. (2002a). System N is a Na+-glutamine symporter and a H+ anti-porter, making it sodium-dependent, yet electroneutral (Chaudhry et al. 1999; Bröer et al. 2002). An interesting feature of this transporter is that it mediates glutamine flux in both directions under normal physiological conditions, probably governed by the intracellular Na+ concentration and pH as shown in Xenopus laevis oocytes expressing SNAT3 (Bröer et al. 2002). The Na+ concentration and pH are parameters which are likely to vary at synaptic/glial microdomains during neurotransmission. In line with this, system N has been suggested to constitute a novel regulatory site in the glutamate/GABA-glutamine cycle, as it was shown in rat brain that glutamine release from glia (assumed to be mediated by system N) was reduced when the extracellular glutamine concentration reached a level (approximately > 2.4 mm) at which the neuronal transport (assumed to be mediated by system A) is saturated (Kanamori and Ross 2005). This mechanism is probably also important during cerebral hyperammonemia (see below). The importance of system N (SNAT3) for astrocytic efflux of glutamine was substantiated by the demonstration that the Km of this transporter was lowered in cultured astrocytes by incubation in the presence of glutamate (30–60 min, 10–100 µm; Bröer et al. 2004). Besides system N, efflux of glutamine from astrocytes might be mediated by other transport systems, such as system L and ASC. In cultured rat astrocytes, Deitmer et al. (2003) found that glutamine efflux was mediated by four different transport systems, namely system N (SNAT3), system L (LAT2), system ASC (ASCT2) and a yet unidentified transport system mediating 40% of the efflux. System L is an amino acid antiporter (Kanai et al. 1998) comprising two isoforms, LAT1 and 2, of which LAT2 show affinity for glutamine. This system can mediate glutamine release in the presence of extracellular amino acid substrates such as alanine and leucine, which might be important for two proposed shuttles involving leucine and alanine, respectively, as noted by Deitmer et al. (2003) and discussed in a following section. System ASC is a sodium-dependent anti-porter, which exists as two different isoforms termed ASCT1 and 2 with ASCT2 having affinity for glutamine, although this transport activity is probably of minor importance in the adult mammalian brain (Bröer et al. 2000; Bröer 2005).

In support of an important role of system A in neurons, it has recently been shown that 2-(methylamino)isobutyric acid (MeAIB), a relatively specific competitive substrate of system A, increases the extracellular level of glutamine in vivo, a mechanism likely involving inhibition of neuronal glutamine uptake (Kanamori and Ross 2004). However, Rae et al. (2003) showed in Guinea pig cortical tissue slices that histidine, but not MeAIB, inhibited flux of 13C label from [1-13C]glucose into glutamate and GABA. These authors propose that a transport activity different from system A, but inhibitable by histidine, is involved on the neuronal side.

Metabolism of the glutamate/GABA-glutamine cycle

  1. Top of page
  2. Abstract
  3. The glutamate/GABA-glutamine cycle
  4. Model systems to study the glutamate/ GABA-glutamine cycle in vitro and in vivo
  5. Transporters of the glutamate/GABA-glutamine cycle
  6. Metabolism of the glutamate/GABA-glutamine cycle
  7. Glutamate/GABA-glutamine cycling and intercellular ammonia homeostasis
  8. Glutamatergic vs. GABAergic synapses: relative significance of neurotransmitter cycling
  9. Glutamate/GABA-glutamine cycling in pathological conditions
  10. Potential drug targets of the neurotransmitter cycling system
  11. Conclusion
  12. Acknowledgements
  13. References

Several enzymes necessary for sustaining glutamate and GABA homeostasis are heterogeneously distributed among neurons and astrocytes. The glutamine synthesizing enzyme, GS, is selectively localized in astrocytes (Norenberg and Martinez-Hernandez 1979) and the enzyme transforming glutamine to glutamate, PAG, seems preferentially expressed in neurons (Kvamme et al. 2001), although a low activity has been observed in cultured astrocytes (Schousboe et al. 1979; Kvamme et al. 1982). PAG has been suggested to be located on the outer surface of the inner mitochondrial membrane (Kvamme et al. 2001), although this is controversial (Zieminska et al. 2004). Finally, PC, the quantitatively most active anaplerotic enzyme in the brain (Patel 1974) is confined to the astrocytic compartment as shown by immunohistochemistry, cell culture studies as well as work using isolated non-synaptic and synaptic mitochondria (Yu et al. 1983; Shank et al. 1985; Faff-Michalak and Albrecht 1991). This, in turn, means that neurons must rely on astrocytes providing precursors for anaplerosis of the TCA cycle which is a prerequisite for synthesis of neurotransmitter glutamate and GABA (via glutamate) from α-ketoglutarate. In this context, it should be mentioned that several other substrates than glutamine have been suggested to be transported from astrocytes to neurons in order to replenish the neuronal TCA cycle. Thus, citrate, malate, succinate and α-ketoglutarate are released to a higher extent from cultured astrocytes than from cultured neurons (Sonnewald et al. 1991; Westergaard et al. 1994a,b). α-Ketoglutarate, but not citrate, has been shown to act as precursor for neurotransmitter glutamate, although not to the same extent as glutamine (Kihara and Kubo 1989; Shank et al. 1989; Peng et al. 1991; Westergaard et al. 1994b). The uptake of malate as well as the incorporation of 14C label into glutamate from 14C-labeled malate is limited when compared to the need for glutamate synthesis during neurotransmission (Shank and Campbell 1984; Hertz and Schousboe 1992). Additionally, the utilization of TCA cycle intermediates for glutamate synthesis requires an amino group donor which is not the case when glutamine acts as a glutamate precursor. In conclusion, none of the TCA cycle intermediates investigated appear to be efficient precursors for glutamate synthesis in neurons.

An important question related to glutamate and GABA homeostasis is the stoichiometry of the glutamate/GABA-glutamine cycle. It seems clear that for the cycle to work, neurons must receive at least the same amount of carbon as it looses in a given time period, meaning that the transfer of glutamine from astrocytes (or another C4, C5 or C6 unit capable of replenishing the neuronal TCA cycle as discussed above) must at least equal that of the lost neurotransmitter glutamate or GABA.

The extent to which glutamate is completely oxidized is an unravelled issue. It is important because the oxidized glutamate must be replaced by de novo synthesis of TCA cycle constituents in order to form the α-ketoglutarate/glutamate for glutamine synthesis and export to neurons. Numerous studies in cultured astrocytes have shown that a large proportion of the glutamate taken up is metabolized in the TCA cycle and to some extent is oxidized completely by exiting the TCA cycle (via the malic enzyme or phosphoenolpyruvate carboxykinase reactions) to re-enter as acetyl-CoA, a process known as pyruvate recycling (Sonnewald et al. 1993; McKenna et al. 1996a,b; Bakken et al. 1997; Hertz and Hertz 2003). A recent report suggests that in rat brain, flux through PC is not only significant but activity-dependent, showing that the astrocytes to some extent rely on anaplerosis to maintain glutamine levels during neurotransmission (Oz et al. 2004). This favors the notion that astrocytes to some extent oxidize exogenous glutamate/GABA in situ as well, as also indicated by in vivo NMR spectroscopic studies in humans (Lebon et al. 2002; Patel et al. 2005).

Regulation of the GS pathway has been elegantly investigated in cultured neocortical astrocytes by Fonseca et al. (2005). A 9-fold increase in flux through the GS pathway was observed over a time period of up to 48 h by adding glutaminase to the culture medium to mimic the presence of neurons (producing glutamate from glutamine). Both the activity and the actual level of the enzyme increased, although the increase was fairly slow and therefore is probably not important for short-term regulation of glutamate-glutamine cycling in response to increased neurotransmission activity. Thus, it was rather interpreted as an up-regulation of GS activity to a level more comparable to the in vivo situation, as cultured astrocytes not challenged with a high concentration of glutamate during development in culture might down-regulate this pathway. At this higher level of GS activity, it constituted the main glutamate-metabolizing pathway, showing only a low flux of glutamate to the TCA cycle which was primarily mediated by transaminase activity, as opposed to GDH.

A novel receptor-mediated control of the glutamate/GABA-glutamine cycle at the metabolic level was recently reported by Rae et al. (2005). It was shown in Guinea pig slices that agonists and antagonists of metabotropic glutamate receptors of both group I and II, coupled to the phosphoinositide/Ca2+ and the cyclic AMP second messenger systems, respectively, affected TCA cycle activity as well as the glutamate-glutamine cycling rate. The general trend was that group I agonists/antagonists affected TCA cycle activity, whereas group II agonists/antagonists influenced the glutamate/GABA-glutamine cycling rate.

Glutamate/GABA-glutamine cycling and intercellular ammonia homeostasis

  1. Top of page
  2. Abstract
  3. The glutamate/GABA-glutamine cycle
  4. Model systems to study the glutamate/ GABA-glutamine cycle in vitro and in vivo
  5. Transporters of the glutamate/GABA-glutamine cycle
  6. Metabolism of the glutamate/GABA-glutamine cycle
  7. Glutamate/GABA-glutamine cycling and intercellular ammonia homeostasis
  8. Glutamatergic vs. GABAergic synapses: relative significance of neurotransmitter cycling
  9. Glutamate/GABA-glutamine cycling in pathological conditions
  10. Potential drug targets of the neurotransmitter cycling system
  11. Conclusion
  12. Acknowledgements
  13. References

Clearly, ammonia generated in the neuronal PAG reaction during neurotransmission activity must be disposed of, as elevated ammonia levels have detrimental effects. This can basically happen in two different ways; ammonia itself might simply diffuse (as NH3) or be transported (as NH4+) across the cell membranes in and out of the extracellular space or a shuttle system involving carrier molecules (amino acids) might be employed. Certainly, ammonia can diffuse across lipid membranes and it has been shown that ammonia can be transported by K+/CI co-transporters, as reviewed extensively by Marcaggi and Coles (2001). In addition, it should be noted that ammonia might diffuse through aquaporins (Nakhoul et al. 2001; Jahn et al. 2004). Aquaporins 1, 4 and 9 have been observed in brain cells (Nielsen et al. 1997; Badaut et al. 2001) and aquaporin 4 in cultured astrocytes is up-regulated as a consequence of ammonia treatment (Rama Rao et al. 2003).

As diffusion and transport of free ammonia across the cell membrane will necessitate a pH regulatory response from the cell, a seemingly more attractive and regulated way of transporting ammonia between the neuronal and the astrocytic compartment is via an amino acid shuttle. In such a shuttle, ammonia generated in neurons is incorporated into a non-neuroactive amino acid and carried across the extracellular space trapped within this amino acid. Two amino acid shuttles have been proposed to be operating in this manner between glutamatergic neurons and astrocytes (Fig. 3a and b). One is based on leucine (Yudkoff et al. 1996; Bixel et al. 1997, 2001; Lieth et al. 2001) and another is based on alanine (Waagepetersen et al. 2000; Zwingmann et al. 2000) as the amino acid translocated in the opposite direction of glutamine, i.e. from neurons to astrocytes. In the opposite direction of the amino acid (i.e. alanine or leucine), a corresponding molecule is translocated; for alanine this is proposed to be lactate, and for leucine, the keto acid corresponding to leucine, α-ketoisocaproate is supposed to play this role. The specific experimental details giving rise to these shuttles is beyond the scope of this review and the reader is referred to the original publications cited. The rest of this section will be concerned with two major questions. (i) The dilemma of the assimilation of ammonia into glutamate by glutamate dehydrogenase (GDH) in the neuronal mitochondrion, a prerequisite for both shuttles to work, as can be seen from Fig. 3a and b. (ii) The coupling between the individual shuttle and the glutamate-glutamine cycle. Both shuttles are based on a glutamatergic synapse, although GABAergic synapses in all likelihood will encounter a similar ammonia imbalance, albeit probably to a lesser extent (as discussed in previous and following sections).

image

Figure 3.  Schematic representations of the proposed amino acid shuttles at the glutamatergic synapse involved in the return of ammonia generated in neurons when the glutamate-glutamine cycle is running. In the lactate-alanine shuttle (a) the ammonia produced in neurons is fixed into α-ketoglutarate by the GDH reaction to form glutamate, then transaminated by ALAT into lactate-derived pyruvate to form alanine which is exported to astrocytes. In the astrocytes this process is then reversed, and lactate is transported in the other direction. It should be noted that the shuttle is not stoichiometric, i.e. alanine and lactate are not transported in the same amounts. The figure is adapted from Waagepetersen et al. (2000). The branched-chain amino acid shuttle (b) is depicted using leucine as an example. Here, the ammonia fixed in the GDH reaction in the neurons is transaminated into α-ketoisocaproate to form leucine, which is exported to astrocytes. In the astrocytes, the process is reversed. α-Ketoisocaproate is transported in the other direction. Notice that two cell-specific isoforms of BCAT are involved in the transaminations of α-ketoisocaproate/leucine. The figure is modified from Lieth et al. (2001). Abbreviations: Ace-CoA, acetyl-CoA; Ala, alanine; ALAT, alanine aminotransferase; α-KG, α-ketoglutarate; α-KIC, α-ketoisocaproate; BCAT, branched-chain aminotransferase (mit, mitochondrial and cyt, cytosolic isoforms); GDH, glutamate dehydrogenase; Glc, glucose; Gln, glutamine; Glu, glutamate; GS, glutamine synthetase; Lac, lactate; Leu, leucine; OAA, oxaloacetate; PAG, phosphate-activated glutaminase; PC, pyruvate carboxylase; Pyr, pyruvate; TCA, tricarboxylic acid.

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(i) GDH, which is localized in the mitochondrial matrix catalyzes the NAD(P)+-dependent oxidative deamination of glutamate to α-ketoglutarate. This enzyme is tightly regulated by the NAD+/NADH ratio and has a relatively high Km for ammonia (Chee et al. 1979; Colon et al. 1986; Zaganas et al. 2001) making the reductive amination of α-ketoglutarate to glutamate an unlikely process to occur under most conditions. However, as suggested by Waagepetersen et al. (2000), the local ammonia concentration in the mitochondrial microenvironment close to the PAG reaction might well be high enough to push the reaction in the direction of reductive amination. Incubating cultured cortical neurons in the presence of 0.3 mm[15N]ammonia resulted in a very low glutamate formation by GDH (Yudkoff et al. 1990), as did superfusion of cultured cerebellar neurons in the presence of 0.3 mm[15N]ammonia in combination with either 0.25 mm glutamine or [5–15N]glutamine (Bak et al. 2005). This might be explained by the fact that the glutamate pool generated via GDH in this high-ammonia environment might be very small with a rapid turn-over rate. One study, though, by Kanamori and Ross (1995) suggests, that under mild hyperammonemic conditions in the rat brain in vivo induced by infusion of [15N]ammonium acetate, the GDH reaction might be significant for de novo synthesis of glutamate. Interestingly, the activity of GDH in cultured cerebellar (glutamatergic) neurons was found to be 60% higher than in cultured (GABAergic) cortical neurons. In addition, the Km for ammonia was 1.7-fold higher in the latter cultures (Zaganas et al. 2001), suggesting different roles of GDH in glutamatergic and GABAergic neurons.

(ii) An important matter to explore experimentally is whether there is an activity-dependent coupling between the shuttles and the glutamate-glutamine cycle. So far, only one study has been directed towards this question, showing that in co-cultures of cerebellar (glutamatergic) neurons and astrocytes, no apparent coupling between the glutamate-glutamine cycle and the lactate-alanine shuttle could be found (Bak et al. 2005). This was evidenced by the finding that superfusing these co-cultures in the presence of [15N]alanine (0.2 mm) and lactate (1 mm) failed to label glutamine, glutamate and aspartate more extensively under conditions of repetitive neuronal vesicular glutamate release and increased activity of the glutamate-glutamine cycle. Although this seems to indicate that the lactate-alanine shuttle is not coupled to activity of the glutamate-glutamine cycle, it does not rule out a role for alanine as a nitrogen carrier between neurons and astrocytes.

Support for a role of both alanine and leucine for shuttling of ammonia in Guinea pig brain tissue preparations was provided by Rae et al. (2003). It was shown that inhibition of the glutamate-glutamine cycle (by blocking glutamine transport) reduced the flux of 13C label from [1-13C]glucose or [3-13C]lactate into alanine as well as the amount of leucine released to the incubation medium.

In conclusion, a scenario, in which both shuttles might be operating in the brain, at the same time and/or under different conditions of activity, seems to be emerging from the above considerations.

Glutamatergic vs. GABAergic synapses: relative significance of neurotransmitter cycling

  1. Top of page
  2. Abstract
  3. The glutamate/GABA-glutamine cycle
  4. Model systems to study the glutamate/ GABA-glutamine cycle in vitro and in vivo
  5. Transporters of the glutamate/GABA-glutamine cycle
  6. Metabolism of the glutamate/GABA-glutamine cycle
  7. Glutamate/GABA-glutamine cycling and intercellular ammonia homeostasis
  8. Glutamatergic vs. GABAergic synapses: relative significance of neurotransmitter cycling
  9. Glutamate/GABA-glutamine cycling in pathological conditions
  10. Potential drug targets of the neurotransmitter cycling system
  11. Conclusion
  12. Acknowledgements
  13. References

As mentioned previously, Rae et al. (2003) have provided evidence that inhibition of glutamine transport in Guinea pig brain tissue slices reduces the flux of 13C label from [1-13C]glucose into both glutamate and GABA, indicating that both glutamatergic and GABAergic neurons rely on glutamine from astrocytes to maintain neurotransmitter homeostasis. A prevalent viewpoint is that reuptake of GABA plays a significant role for GABAergic neurons, whereas glutamatergic neurons rely primarily on glutamine from astrocytes as glutamate precursor (Schousboe 2003; Schousboe and Waagepetersen 2003; Schousboe et al. 2004). A recent report, however, indicates that reuptake of glutamate into cultured cerebellar (glutamatergic) neurons is necessary to sustain glutamate homeostasis (Waagepetersen et al. 2005). It is well known that cultured neurons express glutamate transporters (Drejer et al. 1982), however, evidence for the existence of a presynaptic glutamate transporter in vivo is scarce (as already discussed in a previous section). One study based on immunogold labeling of exogenously applied d-aspartate and glutamate, showed uptake into presynaptic structures in rat hippocampal slices (Gundersen et al. 1993). Recently, an in vivo13C NMR study using infusion of [1,6–13C]glucose and [2–13C]acetate during halothane anesthesia attempted to shed light on the relative importance of GABA and glutamate in glutamate/GABA-glutamine cycling in the rat brain cortex (Patel et al. 2005). This report concluded that the contribution of GABA-glutamine cycling to the total glutamate/GABA-glutamine cycling was 23 %, indicating a relatively smaller, but pronounced role of GABA-glutamine cycling. It is worth noting that the number of inhibitory synapses in general is lower than that of excitatory synapses (Waldvogel et al. 2000 and references herein). However, even though this implies that the GABA-glutamine cycling makes up one quarter of the total glutamate/GABA-glutamine cycling, the relative importance of neuronal reuptake vs. astroglial uptake of released GABA and glutamate at the individual synapse still awaits a satisfactory description.

Glutamate/GABA-glutamine cycling in pathological conditions

  1. Top of page
  2. Abstract
  3. The glutamate/GABA-glutamine cycle
  4. Model systems to study the glutamate/ GABA-glutamine cycle in vitro and in vivo
  5. Transporters of the glutamate/GABA-glutamine cycle
  6. Metabolism of the glutamate/GABA-glutamine cycle
  7. Glutamate/GABA-glutamine cycling and intercellular ammonia homeostasis
  8. Glutamatergic vs. GABAergic synapses: relative significance of neurotransmitter cycling
  9. Glutamate/GABA-glutamine cycling in pathological conditions
  10. Potential drug targets of the neurotransmitter cycling system
  11. Conclusion
  12. Acknowledgements
  13. References

Numerous reports have been published indicating that the glutamate/GABA-glutamine cycle is affected in a variety of neurological disorders and conditions (e.g. review by Cruz and Cerdan 1999). A complete overview of all such pathologies is beyond the scope of this review, and thus only a few recent papers on representative pathological conditions will be discussed.

Biopsies of sclerotic hippocampal tissue from human subjects suffering from epilepsy have shown decreased glutamate-glutamine cycling (Petroff et al. 2002). In cocultures of hippocampal neurons and astrocytes inhibition of glutamine synthesis by methionine sulfoximine led to reduced epileptiform activity, as did block of glutamine transport by MeAIB in a corresponding neuronal culture (Bacci et al. 2002). A recent study in a rat model of lithium-pilocarpine induced temporal lobe epilepsy showed the same extent of [1,2–13C]acetate metabolism in controls as in epileptic animals, implying that astrocytic metabolism is not compromised in these animals (Meløet al. 2005). However, glutamate labeling from [1-13C]glucose was reduced suggesting preferential metabolic malfunction in the neuronal compartment. In general, animal models of different epileptiform activity show metabolic disturbances of both neurons and astrocytes, however, the initial or primary changes might take place in only one cell type (Sonnewald and Kondziella 2003).

Another pathology in which the glutamate/GABA-glutamine cycle might be compromised is Alzheimer's disease, as aberrant expression of GS has been reported (Robinson 2000, 2001). In addition, a study employing in vivo NMR spectroscopy showed decreased glutamate neurotransmission activity and TCA cycling rate in patients suffering from Alzheimer's disease, as evidenced by labeling patterns in glutamate and glutamine from infused [1-13C]glucose (Lin et al. 2003).

Hyperammonemia in the brain, typically occurring as a secondary complication of primary liver disease and known as hepatic encephalopathy, is a condition that has an impact on glutamate/GABA-glutamine cycling in the brain. Several extensive reviews on this subject already exist (e.g. Felipo and Butterworth 2002), and so this discussion will focus on a recent paper by Kanamori and Ross (2005), showing that elevated ammonia affects glutamine transport and thereby glutamate-glutamine cycling (a novel regulation of system N transport was also suggested in this paper, as discussed elsewhere in this review). As the brain lacks a urea cycle, the astrocytic GS reaction and efflux of glutamine from brain to blood constitutes the most important mechanism for excreting excess ammonia. As part of an attempt to develop an in vivo rat model to study hyperammonemic conditions by microdialysis and in vivo NMR spectroscopy, Kanamori and Ross (2005) showed a complex pattern of events linked to continuous infusion of [15N]ammonium acetate. One of the major points made was that when the extracellular concentration of glutamine rises to a level at which the neuronal uptake is saturated, then the efflux from astrocytes is actually inhibited by reversal of system N transport (as discussed in a previous section).

Potential drug targets of the neurotransmitter cycling system

  1. Top of page
  2. Abstract
  3. The glutamate/GABA-glutamine cycle
  4. Model systems to study the glutamate/ GABA-glutamine cycle in vitro and in vivo
  5. Transporters of the glutamate/GABA-glutamine cycle
  6. Metabolism of the glutamate/GABA-glutamine cycle
  7. Glutamate/GABA-glutamine cycling and intercellular ammonia homeostasis
  8. Glutamatergic vs. GABAergic synapses: relative significance of neurotransmitter cycling
  9. Glutamate/GABA-glutamine cycling in pathological conditions
  10. Potential drug targets of the neurotransmitter cycling system
  11. Conclusion
  12. Acknowledgements
  13. References

In the treatment of epilepsy, drugs targeting both GABA transporters (tiagabin) and the GABA metabolizing enzyme GABA-transaminase (vigabatrin) have been marketed, providing proof of principle for the neurotransmitter cycling systems as pharmacological targets (Sarup et al. 2003). However, with regard to glutamate transport and metabolism no such drugs have been developed, although malfunctions in the glutamate-glutamine cycle clearly are present (see previous section). This probably reflects that glutamatergic synapses are so abundant and that glutamate is an important metabolite in intermediary metabolism, making interference with glutamate homeostasis a potential nightmare of adverse effects. So far, most of the drug development directed at the glutamatergic system seems to have been focused on ionotropic glutamate receptors as pharmacological targets, although G-protein coupled receptors have been attracting increased attention over the years. The work by Rae et al. (2005) shows that metabotropic glutamate receptors might constitute attractive drug targets for regulating the metabolism associated with the glutamate/GABA-glutamine cycle.

Conclusion

  1. Top of page
  2. Abstract
  3. The glutamate/GABA-glutamine cycle
  4. Model systems to study the glutamate/ GABA-glutamine cycle in vitro and in vivo
  5. Transporters of the glutamate/GABA-glutamine cycle
  6. Metabolism of the glutamate/GABA-glutamine cycle
  7. Glutamate/GABA-glutamine cycling and intercellular ammonia homeostasis
  8. Glutamatergic vs. GABAergic synapses: relative significance of neurotransmitter cycling
  9. Glutamate/GABA-glutamine cycling in pathological conditions
  10. Potential drug targets of the neurotransmitter cycling system
  11. Conclusion
  12. Acknowledgements
  13. References

The glutamate/GABA-glutamine cycle cannot be seen in isolation, but rather must be seen as a route of transferring not only carbon units between neurons and astrocytes, but also nitrogen units. The latter must for reasons discussed predominantly take place in the form of an amino acid shuttle, rather than solely as simple diffusion or facilitated transport of ammonia/ammonium. Further, to understand the cycle in relation to neurotransmitter homeostasis it appears particularly pertinent to elucidate the importance of the glutamate/GABA-glutamine cycle relative to neuronal reuptake in the individual glutamatergic and GABAergic synapses. Additionally, it should be emphasized that de novo synthesis of glutamate depending on anaplerosis should be quantitatively closely related to the net loss of glutamate caused by oxidative metabolism via pyruvate recycling. Currently, specific knowledge about this interrelationship between pyruvate carboxylation and recycling is incomplete. Insight into these aspects might be accomplished in vitro using co-cultures of astrocytes and glutamatergic or GA-BAergic neurons, respectively. However, to confirm in vitro findings in intact animals significant refinement of in vivo techniques is required. In this respect, improvements of in vivo NMR spectroscopy methods will probably be of instrumental significance.

Acknowledgements

  1. Top of page
  2. Abstract
  3. The glutamate/GABA-glutamine cycle
  4. Model systems to study the glutamate/ GABA-glutamine cycle in vitro and in vivo
  5. Transporters of the glutamate/GABA-glutamine cycle
  6. Metabolism of the glutamate/GABA-glutamine cycle
  7. Glutamate/GABA-glutamine cycling and intercellular ammonia homeostasis
  8. Glutamatergic vs. GABAergic synapses: relative significance of neurotransmitter cycling
  9. Glutamate/GABA-glutamine cycling in pathological conditions
  10. Potential drug targets of the neurotransmitter cycling system
  11. Conclusion
  12. Acknowledgements
  13. References

The authors would like to extend their gratitude to Dr Stefan Bröer and Angelika Bröer (Australian National University, Canberra, Australia) for providing critical comments and engaging in fruitful discussions regarding this manuscript. The Danish Medical Research Council (22-03-0250; 22-04-0314) and the Lundbeck and Benzon Foundations are cordially acknowledged for financial support.

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  1. Top of page
  2. Abstract
  3. The glutamate/GABA-glutamine cycle
  4. Model systems to study the glutamate/ GABA-glutamine cycle in vitro and in vivo
  5. Transporters of the glutamate/GABA-glutamine cycle
  6. Metabolism of the glutamate/GABA-glutamine cycle
  7. Glutamate/GABA-glutamine cycling and intercellular ammonia homeostasis
  8. Glutamatergic vs. GABAergic synapses: relative significance of neurotransmitter cycling
  9. Glutamate/GABA-glutamine cycling in pathological conditions
  10. Potential drug targets of the neurotransmitter cycling system
  11. Conclusion
  12. Acknowledgements
  13. References
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