Glucose deprivation neuronal injury in vitro is modified by withdrawal of extracellular glutamine

Cultured cortical neurons deprived of glucose in a defined solution containing 2 mM glutamine became acutely swollen and went on to degenerate over the next day; this neuronal loss could be substantially attenuated by an N-methyl-D-aspartate (NMDA) antagonist. Removal of extracellular glutamine produced two effects: an increase in overall neuronal injury and a decrease in the protective effect of an NMDA antagonist. Both effects of glutamine removal were glutamine concentration dependent (EC50 for both approximately 300 microM) and not reversed by substitution of equimolar concentrations of alanine or arginine. These observations suggest that glucose deprivation neuronal injury may be tonically regulated by the presence of extracellular glutamine. We speculate that glutamine may reduce overall injury by serving as an energy substrate in the absence of glucose, but may increase NMDA receptor-mediated injury by serving as a precursor for transmitter excitatory amino acids.     

Structure-activity relationships for amino acid transmitter candidates acting at N-methyl-D-aspartate and quisqualate receptors

Dose-response curves for activation of excitatory amino acid receptors on mouse embryonic hippocampal neurons in culture were recorded for 15 excitatory amino acids, including the L-isomers of glutamate, aspartate, and a family of endogenous sulfur amino acids. In the presence of 3 microM glycine, with no extracellular Mg, micromolar concentrations of 11 of these amino acids produced selective activation of N-methyl-D-aspartate (NMDA) receptors. L-Glutamate was the most potent NMDA agonist (EC50 2.3 microM) and quinolinic acid the least potent (EC50 2.3 mM). Dose-response curves were well fit by the logistic equation, or by a model with 2 independent agonist binding sites. The mean limiting slope of log-log plots of NMDA receptor current versus agonist concentration (1.93) suggests that a 2-site model is appropriate. There was excellent correlation between agonist EC50S determined in voltage clamp experiments and KdS determined for NMDA receptor binding (Olverman et al., 1988). With no added glycine, and 1 mM extracellular Mg, responses to NMDA were completely blocked; responses to kainate and quisqualate were unchanged. Under these conditions, glutamate and the sulfur amino acids activated a rapidly desensitizing response, similar to that evoked by micromolar concentrations of quisqualate and AMPA, but mM concentrations of L-aspartate, homoquinolinic acid, and quinolinic acid failed to elicit a non-NMDA receptor-mediated response. Except for L-glutamate (EC50 480 microM), the low potency of the sulfur amino acids prevented the study of complete dose-response curves for the rapidly desensitizing response at quisqualate receptors. Small-amplitude nondesensitizing quisqualate receptor responses were activated by much lower concentrations of all quisqualate receptor agonists. Full dose-response curves for the nondesensitizing response were obtained for 9 amino acids; L-glutamate was the most potent endogenous agonist (EC50 19 microM). Domoate (EC50 13 microM) and kainate (EC50 143 microM) activated large-amplitude, nondesensitizing responses.     

Evidence for a direct action of N-methylaspartate on non-neuronal cells

The effects of N-methylaspartate (NMA) on extracellular amino acids and purine catabolites in the hippocampus were studied with brain dialysis in rats with unilateral hippocampal NMA lesions. In the lesioned side, an increased basal output of glutamine was observed while glutamate was significantly decreased. NMA evoked a drop in extracellular glutamine. The effect was not observed in the lesioned hippocampus. NMA markedly enhanced the release of taurine and phosphoethanolamine (PEA). This response was unchanged in NMA-lesioned hippocampus. Analysis of the tissue content of endogenous amino acids revealed decrements in glutamate and GABA whereas other amino acids were not significantly altered. The resting and NMA-stimulated efflux of inosine was higher in the intact hippocampus. However, the extracellular concentrations of the inosine break-down products hypoxanthine and xanthine were not influenced by a prior NMA lesion, neither before nor after NMA administration. The present findings indicate that NMA releases amino acids (mainly taurine and PEA) from non-neuronal cells. The depression of extracellular glutamine elicited by NMA is probably a neuronal event. A direct stimulation of the energy metabolism of non-neuronal cells by NMA appears to exist as measured by the efflux of purine catabolites. I propose that non-neuronal cells, possibly glia, possess NMA receptors which, upon stimulation, initiate biochemical changes. The physiological significance of these responses remains to be elucidated.     

Amino acids in the neuronal microenvironment of focal human epileptic lesions

Extracellular fluid was topically sampled with a dialysis probe during electrocorticography from the exposed cerebral cortex in 23 patients undergoing epilepsy surgery. Sampling was done in parallel from epileptiform regions and from non-epileptic areas. The former were classified according to the histopathology, into neoplastic, non-tumoral or 'special cases'. The epileptiform regions had significantly higher extracellular concentrations of alanine, glycine and phosphoethanolamine in the majority of the cases. The excised epileptic lesions were analyzed to provide the corresponding intracellular concentrations of amino acids. Several of the non-tumoral group showed high concentrations of GABA, ethanolamine and alanine. The intra- to extracellular concentration ratio for amino acids was low for phosphoethanolamine, glycine, serine and glutamine in most of the samples of epileptiform cortex, while the intracellular accumulative ability for ethanolamine apparently was stronger in epileptiform than in normal cortex.     

GABA concentrations in forebrain areas of suicide victims

Concentrations of GABA and seven other amino acids, including the neurotransmitters or neuromodulators taurine, glycine, aspartate, and glutamate, were determined in postmortem brain samples from suicide victims and normal controls. The five brain areas (caudate nucleus, nucleus accumbens, frontal cortex, amygdala, and hypothalamus) contained very similar concentrations of the amino acids in both groups. The only significant difference between the groups was a low glutamine concentration in the hypothalamus of suicide victims. Even when the data were adjusted for differences in tryptophan concentration, a putative biochemical index for agonal and postmortem changes of brain tissue, no new differences emerged in the concentrations of neuroactive amino acids between suicide victims and control subjects.     

Differences in ischemia-induced accumulation of amino acids in the cat cortex

It is well established that excitatory amino acid neurotransmitters are extensively liberated during ischemia and that they have neurotoxic properties contributing to neuronal injury. To study changes in the liberation of excitatory and other amino acids during cerebral ischemia, we measured their extracellular concentrations and related them to blood flow levels and electrophysiologic activity (electrocorticogram and auditory evoked potentials) before and for up to 2 hours after multiple cerebral vessel occlusion in 14 anesthetized cats. Blood flow levels between 0 and 43 ml/100 g/min were reached. Concentrations of the excitatory amino acid neurotransmitters increased most (aspartate 10-fold, glutamate 30-fold, and gamma-aminobutyric acid 300-fold compared with control values) below a blood flow threshold of 20 ml/100 g/min. The total power of the electrocorticogram and the amplitude of the auditory evoked potentials were affected below the same blood flow threshold. In contrast, concentrations of the nontransmitter amino acids taurine, alanine, asparagine, serine, and glutamine increased 1.5-5-fold as blood flow decreased, while concentrations of the essential amino acids phenylalanine, valine, leucine, and isoleucine did not change during cerebral ischemia. The great increases in concentrations of the excitatory amino acid neurotransmitters below a blood flow threshold close to that for functional disturbance is in accordance with the role of these amino acids in ischemic cell damage. Their release at blood flow levels compatible with cell survival and the increase in their concentrations with severity and duration of cerebral ischemia imply that excitotoxic antagonists may have potential as therapeutic agents.     

Release of amino acids by zinc in the hippocampus

Zinc exists in the synaptic vesicles of hippocampal mossy fibers in high concentrations. On the basis of inhibitory zinc action against glutamate release in the hippocampus, the role of zinc in release of several amino acids were studied in rat hippocampus by using in vivo microdialysis. When the hippocampal CA3 region was perfused with 10 microM ZnCl(2), the concentrations of glutamine, serine, arginine, aspartate, and glycine in the perfusate were significantly increased, whereas the concentrations of amino acids except for glycine were not increased by perfusion with 30 microM ZnCl(2). Chelation of endogenous zinc with 50 microM CaEDTA significantly decreased the concentrations of amino acids in the perfusate except for glycine. In the CA1 region, on the other hand, the concentrations of these five amino acids were not increased by perfusion with 10 microM ZnCl(2) and the concentrations of glutamine and glycine were decreased significantly. The present study suggests that zinc enhances release of glutamine, serine, arginine, and aspartate in the CA3 region and attenuates release of glutamine and glycine in the CA1 region. Zinc seems to modulate glutamatergic synapses multifunctionally in the hippocampus, because glutamine, serine, aspartate, and glycine are involved in synaptic neurotransmission.     

Free amino acids in the isolated cortex of the rat determined by the Dns microassay

Changes in free amino acids were studied in a rat-isolated cortex preparation using the micro-Dns technique with thin-layer chromatography on polyamide plates. For some amino acids that are putative neurotransmitters in the mammalian central nervous system, the following was observed: Gamma-aminobutyric acid and taurine concentrations, after a pronounced initial decrease, returned to control values after 6 weeks. Glutamic and aspartic acid also exhibited a postoperative decrease. However this was not fully reversed at 6 weeks. On the other hand, glycine and glutamine values were elevated in rat-isolated cortex. The results are compatible with the assumption, for gamma-aminobutyric acid, and tentatively for taurine, of acting as transmitter substances in cerebral cortical interneurons.     

Abnormalities in the levels of extracellular and tissue amino acids in the brain of the seizure-susceptible rat

Basal and high potassium-stimulated release of endogenous amino acids was measured using brain dialysis in the hippocampus of urethane-anesthetized seizure-resistant (SR) and seizure-susceptible (SS) rats. Moreover, the tissue level of amino acids was determined in the hippocampus, sensorimotor cortex, cerebellum and corpus striatum. The basal extracellular concentration of amino acids did not differ between SR and SS rats. However, aspartate release was higher, and taurine and phosphoethanolamine release was lower in SS rats during stimulation with 100 mM K+. Several strain differences were observed with regard to regional tissue levels of amino acids. Aspartate was significantly elevated in the hippocampus, cortex and cerebellum of SS animals, and the catecholamine precursor tyrosine was diminished in all regions examined. Other disparities included a depressed gamma-aminobutyrate concentration in the hippocampus and cortex, slightly increased levels of phosphoethanolamine in the cerebellum and minor decreases in striatal and cortical taurine. Glutamate, glutamine, serine and alanine concentrations were not significantly altered in any brain area of the SS rat. The results confirm and extend previous findings on abnormalities in aspartate, taurine and phosphoethanolamine regulation in this model. In addition, decreased availability of tyrosine may provide a partial explanation for the well-documented deficiency in cerebral norepinephrine in the SS strain.     

HPLC-EC determination of free primary amino acid concentrations in cat cisternal cerebrospinal fluid.

This paper describes an HPLC-EC method for measuring the concentrations of 9 free primary amino acids in cerebrospinal fluid (CSF) withdrawn from the cisterna magna of Nembutal-anesthetized adult cats. Amino acid derivatives were formed with o-phthalaldehyde and beta-mercaptoethanol; subsequently, excess thiol reagent was removed with iodoacetamide. During elution through a C18 5-micron column, the electrochemical detector's sensitivity was switched to accommodate the wide ranges of CSF amino acid concentrations. The analysis was acceptably precise and linear at and above the CSF levels and did not require CSF deproteinization. During the 23 min elution, the concentrations of 8 CSF amino acids were determined: alanine, asparagine, glutamate, glutamine, glycine, serine, taurine, and tyrosine; measurable concentrations were between 1 and 800 microM. The concentration of GABA was below its detection limit (0.5 microM). To assess the ability to detect small concentration increases which might occur due to experimental manipulations, the minimum detectable increments in CSF amino acid concentrations above endogenous levels were determined.