Vladimir J. Balcar

HIGH AFFINITY UPTAKE OF L-GLUTAMATE AND L-ASPARTATE BY GLIAL CELLS

The presence of an efficient, high affinity uptake system specific for L‐glutamate and L‐aspartate has been demonstrated in cultured glial cells originating from syrian hamster astroblasts (line NN). The system was found to be temperature dependent, to require sodium ions and its structural specificity is similar to systems studied previously in brain slices and synaptosomes. Co‐culturing of the glial cells with neuroblastoma cells brought about only minor changes in structural specificity while values of kinetic parameters remained virtually unchanged. The temperature dependence and the susceptibility to metabolic inhibitors suggest that the uptake is mediated by an active transport system.

Inhibition of glutamine transport depletes glutamate and GABA neurotransmitter pools: further evidence for metabolic compartmentation

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 13C label supplied as [3‐13C]pyruvate, [2‐13C]acetate, l‐[3‐13C]lactate, or d‐[1‐13C]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. This uptake was inhibited by MeAIB. When [1‐13C]glucose was used as substrate, glutamate/glutamine cycle turnover was inhibited by histidine but not MeAIB, suggesting that neuronal system A may not play a prominent role in neurotransmitter cycling. When transport was blocked by histidine under depolarising conditions, neurotransmitter pools were depleted, showing that glutamine transport is essential for maintenance of glutamate, GABA and alanine pools. Alanine labelling and release were decreased by histidine, showing that alanine was released from neurons and returned to astrocytes. The resultant implications for metabolic compartmentation and regulation of metabolism by transport processes are discussed.

P2X (Purinergic) receptor distributions in rat blood vessels

The distribution of purinergic (P2X1 and P2X2) receptors on smooth muscle cells in relation to autonomic nerve varicosities in rat blood vessels has been determined using immunofluorescence and confocal microscopy. P2X1 and P2X2 receptors were visualised using rabbit polyclonal antibodies against the extracellular domain of the receptors and varicosities visualised using a mouse monoclonal antibody against the ubiquitous synaptic vesicle proteoglycan SV2. Two size classes of P2X1 receptor clusters were observed on the smooth muscle cells of mesenteric, renal, and pulmonary arteries as well as in the aorta and in veins: a large approximately elliptical cluster 1.32+/-0.21 microm long and 0.96+/-0.10 microm in diameter; and a smaller spherical cluster with a diameter of 0.32+/-0.05 microm. The latter occurred throughout the media of arteries of all sizes, whereas the former were restricted to the adventitial surface of the media and to endothelial cells, except for the pulmonary artery, in which large receptor clusters were found throughout the media of the vessel. At the adventitial surface, the large clusters are in general located beneath SV2 labelled varicosities. None of the small clusters was associated with varicosities. Three-dimensional reconstruction of the P2X and SV2 labelling at individual varicosities showed that the varicosities were immediately apposed to the P2X receptor clusters. P2X2 receptors were located on nerves and on endothelial cells. They were also found in low density on the smooth muscle cells in the media. These observations are discussed in relation to the mechanism of purinergic transmission to the smooth muscle cells of blood vessels.

Heterogeneity of high affinity uptake of L-glutamate and L-aspartate in the mammalian central nervous system

Characteristics of high affinity uptake of L-glutamate are examined in order to evaluate the possible use of the uptake of [3H]L-glutamate, [3H]L-aspartate or any other suitable [3H]-labelled substrate as a marker for glutamatergic and aspartergic synapses in autoradiographic studies in the mammalian brain. Review of data on substrate specificity indicates the presence of at least two high affinity uptake systems specific for acidic amino acids in the central nervous tissue; one which takes up L-glutamate and L-aspartate and the other which is selective for L-glutamate only. Studies on ionic requirements, too, point to the existence of at least two distinct uptake systems with high affinity for L-glutamate. The Na(+)-dependent uptake system(s) handle(s) both L-glutamate and L-aspartate whereas the Na(+)-independent uptake system(s) show(s) selectivity for L-glutamate only. Available data do not favour the Na(+)-dependent binding of [3H]D-aspartate to thaw-mounted sections of frozen brain tissue as a suitable marker for glutamatergic/aspartergic synaptic nerve endings. However, there are reasons--such as the results of lesion studies and the existence of uptake sites which have a higher affinity for L-aspartate than for D-aspartate--to suggest that Na(+)-dependent binding of [3H]L-aspartate, rather than that of [3H]D-aspartate, should be further investigated as a possible marker for the glutamatergic/aspartergic synapses in the autoradiographic studies using sections of frozen brain.

THE DISTRIBUTION OF SINGLE P2X1-RECEPTOR CLUSTERS ON SMOOTH MUSCLE CELLS IN RELATION TO NERVE VARICOSITIES IN THE RAT URINARY BLADDER

The distribution of purinergic (P2x1) receptors on smooth muscle cells in relation to autonomic nerve varicosities in the rat urinary bladder has been determined using immunofluorescence and confocal microscopy. P2x1 receptors were visualized using rabbit polyclonal antibodies against the extracellular domain of the P2x1 receptor, and varicosities were visualized using a mouse monoclonal antibody against the ubiquitous synaptic vesicle proteoglycan SV2. Two size classes of P2x1 receptor clusters were observed on the smooth muscle cells of the detrusor, namely, a large ellipse of mean long axis 1.23 ± 0.21 μm and short axis 0.92 ± 0.17 μm and a smaller spherical cluster with a mean diameter of 0.40 ± 0.04 μm. The latter occured in much greater numbers than the former in selected areas, with a density as high as 0.8 per μm2 or two orders of magnitude more than the larger-sized clusters. The large clusters are in general located beneath varicosities, with only 4.5% of P2x1 clusters not possessing an overlying varicosity. None of the small clusters was associated with varicosities. Three-dimensional reconstruction of the P2x1 and SV2 labelling at individual varicosities showed that the varicosities were immediately apposed to the P2x1 receptor clusters. On occasions, two or more small SV2-labelled varicosities about 0.7 μm in diameter each with a receptor patch were found juxtaposed to each other; these might represent the splitting up of a single large varicosity. These observations are discussed in relation to the identity of the autonomic neuromuscular junction.

Autoradiography of P2X ATP receptors in the rat brain

1 Binding of a P2, receptor specific radioligand, [3H]‐α,β‐methylene adenosine triphosphate ([3H]‐m43‐MeATP) to sections of rat brain was reversible and association/dissociation parameters indicated that it consisted of two saturable components. Non‐specific binding was very low (<7% at 10 nM ligand concentration). 2 The binding was completely inhibited by suramin (IC50∼ 14–26 μm) but none of the ligands specific for P2y receptors such as 2‐methylthio‐adenosine triphosphate (2‐methyl‐S‐ATP) and 2‐chloro‐adenosine triphosphate (2‐C1‐ATP) nor 2‐methylthio‐adenosine diphosphate (2‐methyl‐S‐ADP) a ligand for the P2 receptor on blood platelets (′P2T′ type) produced strong inhibitions except for P1, P4‐di(adenosine‐5′)tetraphosphate (Ap4A). 3 Inhibitors of Na+, K+‐dependent adenosine triphosphatase (ATPase) ouabain, P1‐ligand adenosine and an inhibitor of transport of, respectively, adenosine and cyclic nucleotides, dilazep, had no effect. 4 The highest density of P2x binding sites was found to be in the cerebellar cortex but the binding sites were present in all major brain regions, especially in areas known to receive strong excitatory innervation.

Brain gene expression, metabolism, and bioenergetics: interrelationships in murine models of cerebral and noncerebral malaria

Malaria infection can cause cerebral symptoms without parasite invasion of brain tissue. We examined the relationships between brain biochemis¬try, bioenergetics, and gene expression in murine mod¬els of cerebral (Plasmodium berghei ANKA) and nonce¬rebral (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 endoge¬nous cytokines in malaria cerebral pathology, there was significant up‐regulation of mRNAs for TNF‐α, inter¬feron‐γ, and lymphotoxin. These changes are consis¬tent 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.—Rae, C., McQuillan, J. A., Parekh, S. B., Bubb, W. A., Weiser, S., Balcar, V. J., Hansen, A., Ball, H., Hunt, N. H. Brain gene expression, metabolism, and bioenergetics: interrela¬tionships in murine models of cerebral and noncerebral malaria.

Uptake of l‐Glutamate and Taurine in Neuroblastoma Cells with Altered Fatty Acid Composition of Membrane Phospholipids

Abstract: Effects of altered composition of membrane lipids on high‐affinity uptake of l‐glutamate and taurine were studied in an established neuroblastoma cell line M1. Increase in participation of certain fatty acids (20: 5ω3 and 22: 5ω3) as constituents of membrane lipids resulted in a fourfold increase in the maximum initial rate of l‐glutamate uptake (Vmax) while increase in Vmax of taurine uptake was much smaller. Neither structural requirements of l‐glutamate uptake nor passive permeability of the membrane to l‐glutamate or taurine seemed to be influenced by the alterations in the lipid composition. Since increased proportions of 20: 5ω3 and 22: 5ω3 in the membrane phosphatides caused no dramatic changes in kinetic parameters of taurine uptake and incorporation of either linoleic or linolenic acid alone into the phosphatides had only a relatively small effect on some of the measured parameters, the possibility of a specific relationship between 22: 5ω3 and/or 20: 5ω3 and l‐glutamate uptake is discussed. Unlike l‐glutamate uptake systems in other preparations, the high‐affinity uptake system of l‐glutamate in neuroblastoma cells did not readily accept l‐aspartate as a substrate.

Strategies for studies of neurotoxic mechanisms involving deficient transport of L-glutamate: antisense knockout in rat brain in vivo and changes in the neurotransmitter metabolism following inhibition of glutamate transport in guinea pig brain slices

This communication briefly reviews characteristics of glutamate transport in the central nervous system and is involved in the aetiology of slow neurodegenerative diseases. Data in the literature suggest that antisense oligonucleotides targeted against glutamate transporters and administered in vivo over a period of days could be used to test the hypothesis. Data from our laboratory have indicated that single intraventricular doses of antisense oligonucleotides can also results in significant reductions in the numbers of substrate binding sites associated with glutamate transporters and may even cause subtle changes in their characteristics. In order to study metabolism in brain tissue, we have used 13C-nuclear magnetic resonance spectroscopy to analyse extracts of slices of guinea pig cerebral cortex exposed to glutamate transport inhibitor L-anti,endo-methanopyrrolidine dicarboxylate (L-a,e-MPDC). The results have shown-for the first time in an experimental model that preserves the relationship between glia and neurones within the context of brain tissue-that inhibition of L-glutamate transport can exert a significant influence on neurotransmitter-related metabolism. These findings suggest that metabolic disturbances caused by deficient glutamate transport could play a significant role in the death of neurones under pathological conditions in vivo.

Forty years of amino acid transmission in the brain.

This article is concerned with the discovery that amino acids, particularly L-glutamate and gamma-aminobutyrate (GABA), are central neurotransmitters. The crucial observations that lead to the conclusion that these two amino acids produce most of the synaptic excitation and inhibition in the central nervous system, were made in late 1950s. The combination of neurochemical knowledge and improved electrophysiological techniques was paramount in making these discoveries possible. In particular, the use of specific antagonists in microiontophoretic experiments provided the most decisive evidence. The relationship is also explored between these early findings and those of the present era characterised by extensive use of techniques of molecular biology and the development of drugs against targets identified 30 to 40 years ago.