Glutamate vesicular uptake

Roseth S, Fykse EM, Fonnum F (1998) The effect of arachidonic acid and free fatty adds on vesicular uptake of glutamate and gamma-aminobutyric acid. Eur J Pharmacol 341 281-288. 

Fig. 2. Acidification of small synaptic vesicles by glutamate and chloride in synap-tosomes. The acidification assay was performed as described in section 3.2 of this chapter. Two representative experiments with intact (upper trace) or SLO-permeabilized (lower trace) synaptosomes are shown. The ordinate gives the changes of absorbance obtained (A 492-530). Final concentrations of potassium glutamate (Glut), KCl, and ammonium sulfate (NH/) were 10 mM, 45 mM and 30 mM, respectively. The uptake of glutamate and chloride result in an acidification of the lumen of small synaptic vesicles, which increases the vesicular uptake of acridine orange, resulting in a decrease in the amount of extravesicular dye. This acidification can be only observed when the plasma membrane is permeabilized...

Bole DG, Ueda T (2005) Inhibition of vesicular glutamate uptake by Rose Bengal-related compounds structure-activity relationship. Neurochem Res 30 363-369. 

Carlson MD, Kish PE, Ueda T (1989) Characterization of the solubilized and reconstituted ATP-dependent vesicular glutamate uptake system. J Biol Chem 264 7369-7376. 

Szatkowski, M., Barbour, B., and Attwell, D. (1990). Non-vesicular release of glutamate from glial cells by reversed electrogenic glutamate uptake. Nature 348, 443—446. 

The neurotransmitter phenotype, (i.e., what type of neurotransmitter is stored and ultimately will be released from the synaptic bouton) is determined by the identity of the neurotransmitter transporter that resides on the synaptic vesicle membrane. Although some exceptions to the rule may exist all synaptic vesicles of a given neuron normally will express only one transporter type and thus will have a dehned neurotransmitter phenotype (this concept is enveloped in what is known as Dale s principle see also Reference 19). To date, four major vesicular transporter systems have been characterized that support synaptic vesicle uptake of glutamate (VGLUT 1-3), GABA and glycine (VGAT), acetylcholine (VAChT), and monoamines such as dopamine, norepinephrine, and serotonin (VMAT 1 and 2). Vesicles that store and release neuropeptides do not have specific transporters to load and concentrate the peptides but, instead, are formed with the peptides already contained within. 

Uptake of glutamate results in an acidification of the intravesicular lumen. This acidification can be indicated by the fluorescent dye acridine orange, which accumulates in acid compartments. Synapto-somes were prepared (McMahon etal., 1992) and stored as pellets of about 1 mg overlaid with 200 p.1 Krebs-Ringer-HEPES buffer on ice. Acidification was performed with either glutamate or chloride (Hell et al., 1992 Hartinger and Jahn, 1993). To get rid of the non-vesicular glutamate extensive washing of the permeabilized synaptosomes is required. 

Cu is normally found at relatively high levels in the brain (100-150 xM) with substantial variations at the cellular and subcellular level [55-57]. Ionic Cu is compartmentalized into a post-synaptic vesicle and released upon activation of the NMDA-R but not AMPA/kainate-type glutamate receptors [58]. The Menkes Cu7aATPase is the vesicular membrane Cu transporter, and upon NMDA-R activation, it traffics rapidly and reversibly to neuronal processes, independent of the intracellular Cu concentration [58]. Cu ions function to suppress NMDA activation and prevent excitotoxicity by catalyzing S-nitrosylation of specific cysteine residues on the extracellular domain of the NRl and NR2A subunits of the NMDA receptor [58]. The concentrations of Cu in the synaptic cleft can reach approximately 15 xM. Subsequently, Cu is cleared by uptake mechanisms from the synaptic cleft. Several studies have shown that Cu levels increase with age in the brains of mice [22-24]. 

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