GABA METABOLISM IN MAMMALIAN CENTRAL NERVOUS SYSTEM
(gamma)-Aminobutyric Acid (GABA) has been found to be a major inhibitory neurotransmitter in the mammalian CNS. In addition there has been a quantity of work implicating abnormalities in GABA activity in a variety of CNS disorders. Studies directed toward understanding the metabolism of GABA and the effects of various compounds on that metabolism may aid in the understanding and treatment of these CNS disorders. Previous work had been done on the isolated enzymes involved in GABA metabolism. However, this system by definition is artificial. It was believed that a more accurate reflection of the real system could be gotten by studying the metabolism of GABA by the organelles responsible for its metabolism in the living system, namely, the non-synaptosomal mitochondria. For this reason the oxidation of 4-aminobutyric acid (GABA) by nonsynaptosomal mitochondria isolated from rat forebrain and the inhibition of this metabolism by the branched-chain fatty acids 2-methyl-2-ethyl caproate (MEC) and 2,2-dimethyl valerate (DMV) were studied. The rate of GABA oxidation, as measured by O(,2) uptake, was determined in medium containing either 5 or 100 mM-(K('+)). The apparent K(,m) for GABA was 1.16 (+OR-) 0.19 mM and the V(,max) in state 3 was 23.8 (+OR-) 5.5 ng-atoms O(,2) (.) min('-1) (.) mg protein('-1) in 5 mM-(K('+)). In a medium with 100 mM-(K('+)) the apparent K(,m) was 1.11 (+OR-) 0.17 mM and V(,max) was 47.4 (+OR-) 5.7 ng-atoms O(,2) (.) min('-1) (.) mg protein('-1). The K(,i) for MEC was determined to be 0.58 (+OR-) 0.24 or 0.32 (+OR-) 0.08 mM, in 5 or 100 mM-(K('+)), respectively. For DMV, the K(,i) was 0.28 (+OR-) 0.05 or 0.34 (+OR-) 0.06 mM, in 5 or 100 mM-(K('+)) medium, respectively. The O(,2) uptake of the mitochondria in the presence of GABA was coupled to the formation of glutamate and aspartate; the ratio of oxygen uptake to the rate of amino acid formation was close to the theoretical value of 3. Neither the (K('+)) nor any of the above inhibitors had any effect on this ratio. The metabolism of exogenous succinic semialdehyde (SSA) by these same mitochondria was also examined. The V(,max) for utilization of oxygen in the presence of SSA was much greater than that found with exogenously added GABA, indicating that the capacity for GABA oxidation by these mitochondria is not limited by SSA dehydrogenase. In addition, the branched-chain fatty acids did not inhibit the metabolism of exogenously added SSA. Thus, the inhibitors examined apparently act by competitively inhibiting the GABA transaminase system of the mitochondria. The mitochondrial system is extremely useful for many types of biochemical studies. However, for more complex tissue systems it was necessary to develop a methodology for measuring GABA metabolism in tissue and whole animal. The method chosen was to use the GCMS in the SIM mode to detect the TAB derivative of GABA, as well as its metabolites, aspartate and glutamate. A standard curve using 5-Aminovalerate as an internal standard was prepared and GABA was found to vary (+OR-) 8.5 ng/ml, less than 10% of the determined value of the CSF samples later analysed. These CSF samples were purified on an ion exchange resin and then subjected to TAB derivatization. The result was a remarkable degree of consistency of reading done on the same samples analysed on different days after separate derivatization. The results presented in here indicate this methodology should be quite useful in future metabolic studies involving GABA.
CUNNINGHAM, JAMES PATRICK, "GABA METABOLISM IN MAMMALIAN CENTRAL NERVOUS SYSTEM" (1980). ETD Collection for Fordham University. AAI8020055.