The metabolism of γ-aminobutyrate and glucose in potassium ion-stimulated brain tissue in vitro

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Abstract
1. The metabolism of γ-aminobutyrate (GABA) was investigated in cerebral-cortex slices incubated in glucose–saline medium with [1-14C]GABA and [U-14C]-glucose as labelled substrates. 2. A rapid release of GABA from the tissue, amounting to 25–30% of the total, was observed on addition of 66m-equiv. of K+/1 to the medium; the liberation of other amino acids was relatively small. The effect was apparently specific for K+; GABA was not released on addition of equivalent amounts of Na+ or on increasing the respiration rate with 10mm-ammonium chloride. The results show that GABA behaves like the transmitter compounds (acetylcholine, catecholamines) on K+ stimulation, and therefore now satisfies certain of the criteria required for a transmitter in mammalian brain. 3. The release of GABA from the tissue on addition of K+ was followed by a slow re-uptake. The rate of uptake of GABA in a medium containing 5.9m-equiv. of K+/1 was more than four times that in a medium containing 66m-equiv. of K+/1. 4. The concentration of GABA in brain tissue incubated for 1h in a medium containing 66m-equiv. of K+/1 was about 50% higher than that observed under normal conditions. 5. There was evidence that exogenous [14C]GABA mixed with the endogenous pool(s), since the proportion of the total GABA released on K+ stimulation was the same, and the specific radioactivity of the liberated GABA was close to that remaining in the tissue, whether the GABA was labelled by [1-14C]GABA from the medium or generated in the tissue from [14C]glucose. 6. On the basis of these findings and the observations outlined in the preceding papers it was possible to calculate the kinetic constants of GABA metabolism by computer simulation of the results. K+ stimulation led to a 2.5-fold increase in the flux through the tricarboxylic acid cycle, whereas the flux in the GABA bypath was little affected; as a result the flux through the GABA bypath, which under normal conditions was 8% of that through the tricarboxylic acid cycle, decreased to 3–5%. 7. The metabolism of glutamine was greatly affected by K+-stimulation. The ratio of the concentration of glutamine in the slices to that in the medium, which under normal conditions was the smallest among the amino acids investigated, increased from about 17 to 63 in 1h. This effect was attributable partly to an uptake of glutamine from the medium (1.8μmol/h per g) and partly to a net increase in the total amount of glutamine (2.6μmol/h per g). At 1h after the addition of K+ the net gain of glutamine could be accounted for by the decrease of glutamate. 8. Metabolic compartmentation was evident when brain-cortex slices were incubated in glucose–saline medium and the labelled substrate was [14C]GABA, since the specific radioactivity of glutamine exceeded that of glutamate. On addition of K+ the signs of metabolic compartmentation promptly disappeared: this effect was apparently associated with an increase in the permeability of the compartments containing labelled metabolites derived from [14C]GABA. The change in the permeability, however, did not affect all the compartments; when the labelled substrate was [14C]glucose the equilibration of labelled amino acids between tissue and medium was similar under normal conditions and in the presence of high concentrations of K+. 9. The metabolism of [14C]glucose was followed by measuring oxygen uptake, respiratory 14CO2, and incorporation of 14C into amino acids. The results showed that K+ stimulation increased the flux of glucose carbon, both in the glycolytic pathway and in the tricarboxylic acid cycle.