Abc act-The high affinity uptake system for L-glutamate and L-aspartate in rs cerebral cortex may not be specific for these likely excitatory synaptic transmitters, as threo-3hydroxy-DL-aspartate, L-cysteinesulphinate, L-cysteate and D-aspartate strongly inhibit the observed high affinity uptake of ~-[~H]glutamate by rat brain slices in a manner consistent with linear competitive inhibition. These substances should therefore be considered as possible substrates for the transport system. Each of these four acidic amino acids excites central neurones in a manner similar to excitation induced by L-glutamate, and as each might occur in brain tissue, their possible synaptic role should be investigated.L-Glutamate high affinity uptake was shown to be sodium-dependent, but under certain conditions appeared to be less sensitive than GABA uptake to changes in the external sodium ion concentration, and to drugs which modify sodium ion movements. This may be relevant to the efficiency of the glutamate uptake process during synaptic depolarization induced by glutamate.L-Glutamate high affinity uptake was inhibited in a relatively nonspecific manner by a variety of drugs including mercurials and some electron transport inhibitors.L-GLUTAMATE and L-aspartate are likely to be excitatory synaptic transmitters in the mammalian central nervous system (CURTIS and JOHNSTON, 1970). These acidic amino acids depolarize feline spinal neurones when administered extracellularly by electrophoresis from multi-barelled micropipettes (CURTIS, PHILLIS and WATKINS, 1960) and this depolarizing action is potentiated by the simultaneous administration of p-chloromercuriphenylsulphonate (CURTIS, DUGGAN and JOHNSTON, 1970). These observations, together with the lack of effect of substances known to inhibit Lglutamate-metabolizing enzymes on L-glutamate-induced depolarization (CURTIS et al., 1960), have been interpreted on the basis of active transport being partially responsible for the removal of L-glutamate from the synaptic environment (CURTIS et al., 1970). L-Glutamate is taken up actively into brain slices (STERN, EGGLESTON, HEMS and KREBS, 1949; BLASBERG and LAJTHA, 1966), by at least two kinetically distinct transport systems (LOGAN and SNYDER, 1971). Both these transport systems can be inhibited by p-chloromercuriphenylsulphonate (BALCAR and JOHNSTON, 1972).The system of higher affinity for L-glutamate, but not that of lower affinity, appears to be associated with a unique population of nerve terminals which can be separated from other terminals that concentrate other synaptic transmitters, such as GABA and noradrenaline (WOFSEY, KUHAR and SNYDER, 1971). We have examined a variety of amino acids, other putative transmitters, drugs and inorganic ions, for their ability to inhibit the high affinity uptake of ~-[~H]glutamate (and ~-['H]aspartate) in slices of rat cerebral cortex. This study was aimed at finding (i) possible substrates, which should strongly inhibit the observed uptake of ~-f~H]glutamate in a competi-Abbreviation used ICso, i...

The role of glutamine and alanine transport in the recycling of neurotransmitter glutamate was investigated in Guinea pig brain cortical tissue slices and prisms, and in cultured neuroblastoma and astrocyte cell lines. The ability of exogenous (2 mM) glutamine to displace 13 C label sup-13 C]glucose was investigated using NMR spectroscopy.Glutamine transport was inhibited in slices under quiescent or depolarising conditions using histidine, which shares most transport routes with glutamine, or 2-(methylamino)isobutyric acid (MeAIB), a specific inhibitor of the neuronal system A. Glutamine mainly entered a large, slow turnover pool, probably located in neurons, which did not interact with the glutamate/glutamine neurotransmitter cycle. Abbreviations used: ASCT-2, system ASC amino acid transporter; ATA1 and ATA2, system A amino acid transporters; EAAT, excitatory amino acid transporter; HBSS, HEPES-buffered salt solution; MeAIB, 2-(methylamino)isobutyric acid; RPMI, Roswell Park Memorial Institute Medium 1640.

SUMMARYWe have investigated the mechanism of action of a series of glutamate derivatives on the cloned excitatory amino acid transporters 1 and 2 (EAAT1 and EAAT2), expressed in Xenopus laevis oocytes. The compounds were tested as substrates and competitive blockers of the glutamate transporters. A number of compounds showed contrasting mechanisms of action on EAAT1 compared with EAAT2. In particular, (2S,4R)-4-methylglutamate and 4-methylene-glutamate were transported by EAAT1, with K m values of 54 M and 391 M, respectively, but potently blocked glutamate transport by EAAT2, with K b values of 3.4 M and 39 M, respectively. Indeed, (2S,4R)-4-methylglutamate is the most potent blocker of EAAT2 yet described. (Ϯ)-Threo-3-methylglutamate also potently blocked glutamate transport by EAAT2 (K b ϭ 18 M), but was inactive on EAAT1 as either a substrate or a blocker at concentrations up to 300 M. In contrast to (2S,4R)-4-methylglutamate, L-threo-4-hydroxyglutamate was a substrate for both EAAT1 and EAAT2, with K m values of 61 M and 48 M, respectively. It seems that the chemical nature and also the orientation of the group at the 4-position of the carbon backbone of glutamate is crucial in determining the pharmacological activity. The conformations of these molecules have been modeled to understand the structural differences between, firstly, compounds that are blockers versus substrates of EAAT2 and, secondly, the pharmacological differences between EAAT1 and EAAT2.

No abstract

Malaria infection can cause cerebral symptoms without parasite invasion of brain tissue. We examined the relationships between brain biochemistry, bioenergetics, and gene expression in murine models of cerebral (Plasmodium berghei ANKA) and noncerebral (P. berghei K173) malaria using multinuclear NMR spectroscopy, neuropharmacological approaches, and real-time RT-PCR. In cerebral malaria caused by P. berghei ANKA infection, we found biochemical changes consistent with increased glutamatergic activity and decreased flux through the Krebs cycle, followed by increased production of the hypoxia markers lactate and alanine. This was accompanied by compromised brain bioenergetics. There were few significant changes in expression of mRNA for metabolic enzymes or transporters or in the rate of transport of glutamate or glucose. However, in keeping with a role for endogenous cytokines in malaria cerebral pathology, there was significant up-regulation of mRNAs for TNF-alpha, interferon-gamma, and lymphotoxin. These changes are consistent with a state of cytopathic hypoxia. By contrast, in P. berghei K173 infection the brain showed increased metabolic rate, with no deleterious effect on bioenergetics. This was accompanied by mild up-regulation of expression of metabolic enzymes. These changes are consistent with benign hypermetabolism whose cause remains a subject of speculation.