Proline, glutamate and glutamine metabolism in mouse brain synaptosomes

Abstract

In nerve terminals, glutamate (Glu) may serve as precursor of the inhibitory neurotransmitter, GABA, and the putative excitatory transmitter, aspartate (Asp), in addition to exerting its own excitatory neurotransmitter role in brain. Glu carbon can originate from glucose through glycolysis and the Krebs cycle, from glutamine (Gln) subsequent to uptake, and from proline (Pro) and ornithine (Orn). Orn, but not Glu, is an effective precursor in nerve terminals of Pro, a putative inhibitory neurotransmitter 32,44–46. [ 3H]Arg can be converted in mouse brain nerve terminals to Orn, which in turn gives rise to Glu, Pro and GABA. In the present study, the conversion subsequent to uptake of labeled Glu, Gln, and Pro to other amino acids was studied in unfractionated and subfractionated synaptosomal particles which layered, respectively, on 1.0 M, 1.2 M, 1.3 M and 1.5 M sucrose after centrifugation in a discontinuous gradient (fractions 1–4, respectively). Fraction 1 contained small synaptosomal fragments with vesicles and almost no mitochondria. Fractions 2 and 3 showed numerous normal-appearing mitochondria-containing synaptosomes, and fraction 4 contained large synaptosomes and more free mitochondrial than the other fractions. Glu was readily taken up in all fractions and converted to Asp, Gln and GABA, the greatest formation of Asp from Glu occuring in fractions 2 and 3 and of Gln in fraction 4. In contrast, Gln was taken up poorly in fraction 1 and not metabolized, converted extensively to Glu and GABA in fractions 2–4, giving rise only to very small amounts of Asp in fractions 2 and 3. Although Pro was taken up to the greatest extent in fraction 2, it was by far most readily converted to Glu, Gln and GABA in fraction, showing only small amounts of Asp formation in fractions 1–3 and none in 4. There was no significant production of Pro from Glu or Gln or Arg and Orn from any of the 3 precursors studied. The above results suggest that Glu, Gln and Pro may be taken up largely in different classes of synaptosomes which are distributed among the centrifugally separated fractions and which posses differing transport and metabolic characteristics. Determination of glutamate decar☐ylase activity (GAD) indicated that GABA-forming nerve terminals were present in all synaptosomal fractions studied. Amino acid determinations by HPLC in the subfractionated synaptosomes showed a similar distribution for Glu, Asp and GABA contents, peaking in fraction 2, and an inverse relationship of the latter 3 with Arg contents. Since reanalysis of data from another study 21 showed there to be linear relationships between Arg contents and Asp, GABA, and the sum of Asp and GABA contents in unfractionated synaptosomal particles from seven regions of bovine brain, it is likely that theArg → Glu → Asp and Arg → Glu → GABA pathways are operative in synaptosomes under the conditions of the rather lengthy procedure employed by us for subfractionation, depleting Arg and increasing the contents of the amino acids to which it ultimately is converted.