—The stability of the GABA content of synaptosomal‐enriched fractions was evaluated by two approaches. Firstly, the addition of 10−3m‐aminooxyacetic acid to the homogenizing medium totally inhibited the GABA‐degrading enzyme in the fractions but did not affect the GABA levels. This indicated that GABA was not being metabolized during the normal preparation of the synaptosomal‐enriched fraction. Secondly, when synaptosomal‐enriched fractions were re‐fractionated by discontinuous density gradient centrifugation, the GABA contents of the fractions before and after the second fractionation were very similar provided they were expressed on a per mg protein basis. It was therefore concluded that the GABA content of the organelles was not subject to change during the fractionation procedures. On the basis of these findings and others it was suggested that the synaptosomal‐enriched fraction could be used as a model to evaluate drug‐induced changes in GABA levels in nerve endings. In vivo experimentation indicated that the convulsant agents hydrazine, isonicotinic acid hydrazide and aminooxyacetic acid brought about similar decreases in the GABA content of the synaptosomal‐enriched fractions prepared from tissue at the onset of seizures despite the fact that no correlation was observed between seizure activity and whole brain GABA levels.
This paper is dedicated to the latt Dr. G . Malcolm Brown Wood, J. B., KuryIo, E. & Newstead, J. B. (1978) Aminooxyacetic acid induced changes in y-aminobutyrate nletabolism at the subcellular level. Can. J . Biochern. 56,667-672Aminooxyacetic acid (AOAA) (0.1 or 0.23 mmolikg) was administered to mice which were killed 1.5 and 6 h after treatment. Synaptosomal-and mitochondrial-enriched fractions were obtained by conventional ultracentrifugation procedures. The y-aminobutyric acid (GABA) content of the synaptosomalfraction was elevated by previous treatment of the mice with AOAA, the increase being greater at 6 h than at 1.5 h posttreatment. This finding suggested that a transient elevation of the GABA content of nerve endings was not responsible for the fast-developing anticonvulsant action of AOAA. The activity of aminobutyrate aminotransferase (GABA-T) was inhibited after AOAA treatment, the degree of inhibition being greater in the mitochondrialenriched than in the synaptosomal-enriched fraction. Evidence for circadian changes in GABA-T activity was obtained. The limitations and advantages of subcel8ulru-fractionation techniques in determining the effects of drugs on GABA metabolism were assessed.
The intramuscular administration of L-cycloserine, gabaculine, and aminooxyacetic acid caused significant, time-dependent increases in the gamma-aminobutyric acid (GABA) content of both whole brain and synaptosomal-enriched preparations obtained from the tissue, a linear relationship being observed between the two parameters. In contrast, the administration of hydrazine resulted in a large increase in whole brain GABA level, with little change in the synaptosomal GABA content. The key factor in these different responses appeared to be the degree of inhibition of glutamic acid decarboxylase by the drugs. Pretreatment of mice with the GABA-elevating agents resulted in a delay in the onset of seizures, which was related directly to the increase in synaptosomal GABA content. Although the seizures were delayed, they occurred while the GABA content of nerve endings (synaptosomes) was above that in preparations from untreated animals. The decrease in GABA content at the onset of seizures, expressed as a percentage of the level at the time of injection of the convulsant agent, was, however, reasonably constant. A hypothesis to explain these results is proposed.
The potassium-stimulated release of gamma-aminobutyric acid (GABA) from synaptosomes was determined in preparations from control rats and from rats treated with a convulsant agent [isonicotinic acid hydrazide (INH)] and an anticonvulsant agent (gabaculine). INH treatment brought about a significant decrease in Ca2+-dependent release of GABA with no effect on Ca2+-independent release, whereas gabaculine caused an increase in Ca2+-independent release with no effect on Ca2+-dependent release of GABA. Thus, the anticonvulsant action of gabaculine was not a simple reversal of the effects of INH on GABA release. The results indicate that there are at least two pools of GABA in nerve endings and support the hypothesis that exogenous GABA is taken up first into a pool that supplies GABA for Ca2+-independent release and then is transferred to a second pool (Ca2+-dependent releasable), where it mixes with newly synthesized GABA.
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