Elsevier

Brain Research Bulletin

Volume 65, Issue 6, 30 May 2005, Pages 521-528
Brain Research Bulletin

Release of arginine, glutamate and glutamine in the hippocampus of freely moving rats: Involvement of nitric oxide

https://doi.org/10.1016/j.brainresbull.2005.03.011Get rights and content

Abstract

Using in vivo microdialysis, we have monitored the release of three amino acids (arginine, glutamate and glutamine) in the hippocampus of freely moving rats in response to various drugs. In response to N-methyl-d-aspartate (NMDA) infusion, extracellular glutamate was increased, glutamine was decreased and arginine remained unchanged. By contrast, α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) elicited an increase in arginine release but had no effect on either glutamate or glutamine. When S-nitroso-N-acetylpenicillamine (SNAP), a nitric oxide (NO) donor, was infused into the hippocampus, an increase in glutamate, a decrease in glutamine and no change in arginine were recorded. The effect of SNAP on extracellular glutamine levels was reversed by prior infusion of the guanylate cyclase inhibitor [1], [2], [4] oxadiazolo[4,3-α]quinoxalin-1-one (ODQ), however its effect on glutamate release was unchanged. Interestingly, SNAP was found to promote the release of arginine in the presence of ODQ. We also assessed the effect of two nitric oxide synthase inhibitors, N-nitro-l-arginine methylester (l-NAME) and 7-nitroindazole (7-NI), on the release of these amino acids. l-NAME was found to increase arginine and glutamate levels but decrease those of glutamine. In contrast, 7-NI reduced the release of all three amino acids. The results presented here confirm some but not all of the findings previously obtained using in vitro preparations. In addition, they suggest that complex relationships exist between the release of these amino acids, and that endogenous NO plays an important role in regulating their release.

Introduction

It is now becoming increasingly clear that glia serve a variety of functions in the central nervous system. Their close apposition to neurons and their connections, their ability to respond to neurotransmitters released at synapses and to act as a source of molecules that, in turn, influence neuronal activity, suggests that these cells play an important role in the integrative functions of the brain [3], [17], [18]. Amongst the many biologically active molecules that are, at least in part, derived from glia, are the amino acids arginine, glutamate and glutamine.

In eukaryotic cells, arginine is the only physiological substrate for nitric oxide synthase (NOS), thus an adequate supply of the amino acid is an important consideration in ensuring that nitric oxide (NO)-mediated processes occur in an appropriate manner. Arginine cannot be synthesised de novo in the brain because the tissue lacks a complete urea cycle and the enzymes required to convert citrulline to arginine are not always co-localised with NOS [47]. Thus, neuronal NO production is dependent, to some extent, on the supply of arginine from another source. Immunocytochemical studies performed on various brain regions have revealed that arginine is located predominantly in astrocytes [2], [32]. Using brain slices and cultured cells, Grima et al. [15] have shown that the activation of non-N-methyl-d-aspartate (non-NMDA) ionotropic glutamate receptors promotes the release of radiolabelled arginine from astrocytes, and that this can be utilised by neurons to synthesise NO. In addition, Vega-Agapito et al. [44], [45] have reported arginine to be released from these cells in response to an NO donor and peroxynitrite. This would suggest that the supply of arginine in the brain may be controlled by both the main activator of NOS, glutamate and its product, NO.

Glutamate is the major excitatory neurotransmitter in the brain. It is becoming increasingly clear, however that neurons are not the only source of this amino acid and that glia-derived glutamate also plays an important role in synaptic activity [3], [18]. Studies on cultured and freshly isolated astrocytes have demonstrated that glutamate release from these cells is from vesicular stores [27] and can be promoted by NO [6], [37]. Another route by which glia might influence extracellular glutamate levels involves volume-regulated anion channels. Glutamate release via these channels has been demonstrated in the intact brain [40] and to be potentiated by peroxynitrite in cultured cells [19]. Interestingly, there is evidence that the bulk of the extracellular glutamate measured in vivo may be of glial origin. Baker et al. [5] have shown that extracellular glutamate levels in the striatum were reduced markedly by blockade of the cysteine–glutamate antiporter, which appears to be located primarily in glia [12], [33]. Once released at synapses, glutamate is recycled by the glutamate–glutamine cycle. Glutamate is rapidly taken up into surrounding astrocytes where it is converted to glutamine by glutamine synthetase. Glutamine is then released by astrocytes, taken up by neurons and converted back to glutamate via glutaminase [11]. This cycle has been shown to occur in the intact brain [36] and its importance is highlighted by studies showing that impaired glutamate–glutamine cycling might underlie pathological states such as epilepsy [14], [31] and that it may undergo adaptive changes in response to brain injury [34]. Evidence also suggests that glutamine synthetase is a target for acute regulation. McBean et al. [25] showed that NMDA receptor activation reduced glutamine synthetase activity in brain slices, and more recently, Kosenko et al. [21] reported that whole brain glutamine concentrations were increased upon administration of an NMDA receptor antagonist, suggesting that these receptors exert tonic inhibitory control over the enzyme. McBean et al. [25] also showed that the effect of NMDA was reversed by an inhibitor of NOS. These findings suggest that NO released from neurons may be responsible for regulating glutamine synthetase activity. Support for this has been provided by studies on cultured astrocytes, which show that the enzyme is inhibited by the application of an NO donor [26].

Despite the wealth of knowledge about the factors regulating the release of these amino acids, a drawback of the work discussed is that it is often difficult to extrapolate findings from cell culture or other in vitro preparations to the in vivo situation. To address this issue, we undertook an in vivo microdialysis study in freely moving animals to determine the effect of various drugs that stimulate glutamate receptors and/or influence NOS activity on the release of arginine, glutamate and glutamine into the extracellular space.

Section snippets

In vivo microdialysis

Experiments were performed under a project licence issued by the Home Office (UK) in accordance with the Animals (Scientific Procedures) Act 1986. Male Wistar rats (250–300 g) were housed individually in perspex cages with free access to food and water. Animals were anaesthetised with isoflurane and concentric microdialysis probes implanted into the left ventral hippocampus (co-ordinates: A-5.0 mm, L-5.0 mm from Bregma, 7.5 mm dura). Probes were constructed in the laboratory from fused silica

Effect of glutamate receptor agonists

Fig. 1 shows the effect of a brief (30 min) infusion of either NMDA (100 μM) or AMPA (100 μM) on the release of arginine, glutamate and glutamine in the ventral hippocampus. NMDA was found to elicit no change in arginine release when compared to controls whilst extracellular glutamate levels were increased approximately two-fold within the drug infusion period. In contrast, glutamine was reduced by up to 70%. A statistically significant reduction in glutamine was evident 30 min after the

Arginine

The results presented here show that the infusion of NMDA into the hippocampus had no effect on the release of arginine into the extracellular space whilst AMPA promoted its release. Our finding with AMPA clearly supports the work of Grima et al. [15] who reported that arginine release from cultured astrocytes was promoted by the activation of non-NMDA receptors. However, they also showed that arginine release was increased by the application of NMDA to cerebellar or cortical slices. This

Acknowledgement

J.W. was in receipt of a Medical Research Council (UK) research studentship.

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