Else Marie Fykse

Uptake of γ-Aminobutyric Acid by a Synaptic Vesicle Fraction Isolated from Rat Brain

Abstract: γ‐Aminobutyric acid (GABA) was taken up by a MgATP‐dependent mechanism into synaptic vesicles isolated by hypoosmotic shock and density gradient centrifugation. The properties of the vesicular uptake differed clearly from those of synaptosomal and glial uptake, both with respect to Na+, Mg2+, and ATP dependence and with respect to response to general GABA uptake inhibitors such as nipecotic acid, diaminobutyric acid, and β‐alanine. The uptake showed a Km of 5.6 mM and a net uptake rate of 1,500 pmol/min/mg of protein. It is suggested that the vesicular uptake of GABA is driven by an electrochemical proton gradient generated by a Mg2+‐ATPase.

Uptake of L-glutamate into rat brain synaptic vesicles : effect of inhibitors that bind specifically to the glutamate transporter

Abstract: In this study we have described a series of new and potent inhibitors of the vesicular uptake of glutamate. The two most efficient inhibitors were the dyes Evans blue and Chicago Skye Blue 6B, which are structurally related to glutamate and were competitive inhibitors in the nanomolar range. The anion channel blocker 4,4′‐diisothiocyanostilbene‐2,2′‐disulfonic acid (SITS) and the diuretics furosemide and bumetanide are inhibitors of chloride transport in other organs but were competitive inhibitors of glutamate and noncompetitive with respect to chloride ions. Evans blue, Chicago Skye Blue 6B, SITS, furosemide, and bumetanide are all large organic acids with two centers of negative charge and an electron‐donating group at close vicinity of the negative charge at physiological pH. The inhibition of the glutamate uptake with these inhibitors was noncompetitive with respect to Cl−. The inhibitors, therefore, probably interact directly with the glutamate carrier. Bafilomycin A1, which is a specific vacuolar ATPase inhibitor, was used as a control and inhibited the vesicular dopamine, glutamate, and GABA uptake to the same extent. None of the inhibitors had any effect on the plasma membrane carrier, which is therefore clearly different from the vesicular carrier.

Amino acid neurotransmission : Dynamics of vesicular uptake

Glutamate, GABA and glycine, the major neurotransmitters in CNS, are taken up and stored in synaptic vesicles by a Mg2+-ATP dependent process. The main driving force for vesicular glutamate uptake is the membrane potential, whereas both the membrane potential and the proton gradient contribute to the uptake of GABA and glycine. Glutamate is taken up by a specific transporter with no affinity for aspartate. Evans blue and related dyes are competitive inhibitors of the uptake of glutamate. GABA, β-alanine, and glycine are taken up by the same family of transporter molecules. Aspartate, taurine, and proline are not taken up by any synaptic vesicle preparations. It is suggested that vesicular uptake and release are characteristics that identify these amino acids as neurotransmitters. We also discuss that “quanta” in the brain are not necessarily related the content of neurotransmitter in the synaptic vesicles, but rather to postsynaptic events.

Comparison of the properties of γ-aminobutyric acid and L-glutamate uptake into synaptic vesicles isolated from rat brain

Abstract: Rat brain synaptic vesicles exhibit ATP‐dependent uptake of γ‐[3H]amino‐n‐butyric acid ([3H]GABA) and l‐[3H]glutamate. After hypotonic shock, the highest specific activities of uptake of both l‐glutamate and GABA were recovered in the 0.4 M fraction of a sucrose gradient. The uptakes of l‐glutamate and GABA were inhibited by similar, but not identical, concentrations of the mitochondrial uncoupler carbonyl cyanide m‐chlorophenylhydrazone and the ionophores nigericin and gramicidin, but they were not inhibited by the K+ carrier valinomycin. N, N′‐Dicyclohexyl‐carbodiimide and N‐ethylmaleimide, Mg2+‐ATPase inhibitors, inhibited the GABA and L‐glutamate uptakes similarly. Low concentrations of CI− stimulated the vesicular uptake of l‐glutamate but not that of GABA. The uptakes of both l‐glutamate and GABA were inhibited by high concentrations of CI−. These results indicate that the vesicular GABA and l‐glutamate uptakes are driven by an electrochemical proton gradient generated by a similar Mg2+‐ATPase. The vesicular uptake mechanisms are discussed in relation to other vesicle uptake systems.

Inhibition of l-glutamate uptake into synaptic vesicles

The effects of different agents similar in structure to glutamate were tested for inhibition of the vesicular uptake of L-glutamate. Kainate and L-homocysteate turned out to be non-competitive inhibitors of the L-glutamate uptake. Kainate was not taken up by the vesicle fraction. The vesicular uptake of gamma-aminobutyric acid (GABA) was also inhibited by kainate and L-homocysteate. Kynurenate, on the other hand, strongly inhibited the uptake of L-glutamate, whereas the uptake of GABA was hardly affected. L-alpha-Aminoadipate and D-glutamate inhibited the uptake of L-glutamate, whereas L- and D-aspartate and L-cysteate only weakly inhibited the uptake of L-glutamate. GABA, glycine, L-serine and taurine did not inhibit the uptake of L-glutamate.

Application of sonication to release DNA from Bacillus cereus for quantitative detection by real-time PCR

A rapid sonication method for lysis of Gram-positive bacteria was evaluated for use in combination with quantitative real-time polymerase chain reaction (PCR) analyses for detection. Other criteria used for evaluation of lysis were microscopic cell count, colony forming units (cfu), optical density at 600 nm and total yield of DNA measured by PicoGreen fluorescence. The aim of this study was complete disruption of cellular structures and release of DNA without the need for lysing reagents and time-consuming sample preparation. The Gram-positive bacterium Bacillus cereus was used as a model organism for Gram-positive bacteria. It was demonstrated by real-time PCR that maximum yield of DNA was obtained after 3 to 5 min of sonication. The yield of DNA was affected by culture age and the cells from a 4-h-old culture in the exponential phase of growth gave a higher yield of DNA after 5 min of sonication than a 24-h-old culture in the stationary phase of growth. The 4-h-old culture was also more sensitive for lysis caused by heating. The maximum yield of DNA, evaluated by real-time PCR, from a culture of the Gram-negative bacterium Escherichia coli, was obtained after 20 s of sonication. However, the yield of target DNA from E. coli rapidly decreased after 50 s of sonication due to degradation of DNA. Plate counting (cfu), microscopic counting and absorbance at 600 nm showed that the number of viable and structurally intact B. cereus cells decreased rapidly with sonication time, whereas the yield of DNA increased as shown by PicoGreen fluorescence and real-time PCR. The present results indicate that 3-5 min of sonication is sufficient for lysis and release of DNA from samples of Gram-positive bacteria.

Uptake of L-glutamate into synaptic vesicles : Competitive inhibition by dyes with biphenyl and amino- and sulphonic acid-substituted naphthyl groups

The specificity of the vesicular L-glutamate carrier was characterized using dyes with biphenyl and amino- and sulphonic acid substituted naphthyl groups, structurally similar to the specific vesicular L-glutamate inhibitor Evans Blue. The dye Trypan Blue was the most potent inhibitor; the IC50 value was determined to be 49 nM. Naphthol Blue Black, Reactive Blue 2, Benzopurpurin 4B, Ponceau SS, Direct Blue 71 and Acid red 114 were also highly potent inhibitors with IC50 values from 330 to 1670 nM (series 1). The dyes were competitive inhibitors of vesicular glutamate uptake, and acted therefore on the glutamate transporter. Their IC50 values for the vesicular uptake of gamma-aminobutyric acid (GABA) were all higher than 20 microM. They had no effect on synaptosomal uptake of glutamate. Furthermore, we have also found several other dyes with IC50 values for the vesicular uptake of glutamate ranging between 1 and 30 microM and for gamma-aminobutyric acid higher than 50 microM (series 2). The most potent inhibitor Trypan Blue contains a biphenyl group, linked by azo groups to side chains containing sulphonic, amino and/or hydroxyl groups coupled to a naphthalene ring system. Trypan Blue and Evans Blue are by molecular mechanics, shown to have planar structures with conjugated double bonds throughout the structure. The other dyes, which were less effective, had phenyl and/or naphthalene groups linked by an azo group. We have also tested a series of amino and/or hydroxyl naphthalene di-/sulphonic acids that correspond to the side chains of the most potent dyes, but they had no effect on glutamate nor on gamma-aminobutyric acid uptake. We conclude that the inhibitory action of these compounds is strictly dependent of the complete molecule.

Uptake of Glycine into Synaptic Vesicles Isolated from Rat Spinal Cord

Glycine was taken up by a synaptic vesicle fraction from spinal cord in a Mg‐ATP‐dependent manner. The accumulation of glycine was inhibited by carbonyl cyanide‐m‐chlorophenylhydrazone (CCCP) and nigericin, agents known to destroy the proton gradient across the vesicle membrane. Vesicular uptake of glycine was clearly different from synaptosomal uptake, with respect to both the affinity constant and the effect of Na+, ATP, CCCP, and temperature. Oligomycin and strychnine did not inhibit the vesicular uptake, showing that neither mitochondrial H+‐ATPase nor binding to strychnine‐sensitive glycine receptors was involved. It is suggested that the vesicular uptake of glycine is driven by a proton gradient generated by a Mg2+‐ATPase. A low concentration of Cl‐ had little effect on the uptake of glycine, whereas the uptake of glutamate in the same experiment was highly stimulated. High concentrations of γ‐amino‐n‐butyric acid and β‐alanine inhibited vesicular glycine uptake, but glutamate did not. Accumulation of glycine was found to be fourfold higher in a spinal cord synaptic vesicle fraction than in a vesicle fraction from cerebral cortex.

Inhibition of γ-aminobutyrate and glycine uptake into synaptic vesicles

The substrate specificity of vesicular GABA and glycine uptake was studied in vesicle fractions from brain and spinal cord, respectively. Glycine, β -alanine and γ -vinyl-GABA were competitive inhibitors of the GABA uptake by synaptic vesicles in brain. Likewise GABA and β -alanine turned out to be competitive inhibitors of vesicular uptake of glycine in spinal cord. The apparent K i values were in the same range as the respective K m values for the transport systems. Accumulation of different amino acids were examined, and the structurally related amino acids GABA, β -alanine and glycine were all taken up by both vesicle fractions. In the present study, we suggest that there are similarities in the vesicular transporters for GABA and glycine, and the two amino acids are probably taken up into the same vesicle population. The key factor in differentiating between GABA and glycine as transmitters in the terminals could be the synthesis and the high-affinity synaptosomal uptake.

Chapter 7 Uptake of glutamate into synaptic vesicles

Publisher Summary The most important excitatory neurotransmitter is glutamate, of which about 10 mM is present in the brain. In glutamatergic nerve terminals, the concentration of glutamate is estimated to be about 40 mM. Glutamate has other functions in the brain besides neurotransmission such as general metabolism and protein synthesis. Neurotransmitters are stored in specialized organelles called synaptic vesicles in the brain, as well as in the peripheral nervous system. Several different forms and sizes of synaptic vesicles have been described in fixed brain tissue. These different forms may indicate differences in both protein content and mechanical stability toward the fixation treatment. In general, the excitatory neurotransmitters glutamate and acetylcholine are stored in nerve terminals with small clear round vesicles, the catecholamines in terminals with granule vesicles, and the inhibitory amino acid transmitters in terminals with pleomorphic vesicles. In the giant reticulo-spinal terminals of lamprey, the density of glutamate immuno-staining correlates well with the packing density of the synaptic vesicle. The synthesis of neurotransmitters takes place extravesicularly necessitating an uptake of neurotransmitters into synaptic vesicles. The catecholamines, acetylcholine, and transmitter amino acids are taken up into vesicles by an energy dependent uptake system.