title: Glutamine and aspartate loading of synaptosomes: a reevaluation of effects on calcium-dependent excitatory amino acid release.
abstract: Guinea-pig cerebral cortical synaptosomes were preincubated for 60 min with 100 microM D-aspartate, L-aspartate, or L-glutamate. The total D- plus L-aspartate content of the synaptosomal fraction increased to 235%, 195%, or 164%, respectively, of the control. Despite this no increase was seen in the very low KCl evoked, Ca2+-dependent release of aspartate. Preincubation with the three amino acids changed the synaptosomal glutamate content to 78% (D-aspartate), 149% (L-aspartate), or 168% (L-glutamate) of control. However there was no statistically significant effect of these preincubations on the extent of Ca2+-dependent glutamate release. Thus the Ca2+-dependent release of aspartate and glutamate is not determined by the total synaptosomal content of these amino acids. The addition of 0.1-0.5 mM glutamine to the incubation caused a massive appearance of glutamate in the extrasynaptosomal medium. Analysis of specific activities showed that glutamine was hydrolysed directly by an extrasynaptosomal glutaminase, and that intrasynaptosomal glutamate was predominantly labelled by uptake of this glutaminase-derived glutamate. No increase was seen in the extent of Ca2+-dependent release of glutamate (by fluorimetry) either after preincubation with glutamine or in the continued presence of glutamine. Thus we are unable to confirm reports that glutamine expands the transmitter pool of glutamate. The extrasynaptosomal glutaminase activity in the synaptosomal preparation was inhibited by Ca2+ and activated by phosphate. Identical kinetics were obtained with "free" brain mitochondria, confirming the origin of the glutamine-derived glutamate.
authors.affiliation: Department of Biochemistry, University of Dundee, Scotland.
authors.forename: H T
authors.initials: HT
authors.lastname: McMahon
authors.forename: D G
authors.initials: DG
authors.lastname: Nicholls
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism* / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology*; D005971:Glutamates / Q000378:metabolism / Q000494:pharmacology; D018698:Glutamic Acid; D005973:Glutamine / Q000378:metabolism* / Q000494:pharmacology; D006168:Guinea Pigs; D013574:Synaptosomes / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D005971:Glutamates; D005973:Glutamine; D001224:Aspartic Acid; D018698:Glutamic Acid; D002118:Calcium