Decrease in GABA synthesis rate in rat cortex following GABA-transaminase inhibition correlates with the decrease in GAD67 protein

Abstract

γ-Aminobutyric acid (GABA) synthesis in the brain is mediated by two major isoforms of glutamic acid decarboxylase, GAD65 and GAD67. The contribution of these isoforms to GABA synthesis flux (VGAD) is not known quantitatively. In the present study we compared VGAD in cortex of control and vigabatrin-treated rats under α-chloralose/70% nitrous oxide anesthesia, with total GAD activity and GAD isoform composition (GAD65 and GAD67) measured by enzymatic assay and quantitative immunoblotting. VGAD was determined by re-analysis of 13C NMR data obtained ex vivo and in vivo during infusions of [1-13C]glucose using an extension of a model of glutamate–glutamine cycling that included a discrete GABAergic neuronal compartment with relevant interconnecting fluxes. VGAD was significantly lower in vigabatrin-treated rats (0.030–0.05 μmol/min per g, P<0.003) compared to the non-treated control group (0.10–0.15 μmol/min per g). The 67–70% decrease in VGAD was associated with a 13% decrease in total GAD activity (P=0.01) and a selective 44±15% decrease in GAD67 protein (from 0.63±0.10 to 0.35±0.08 μg protein/mg tissue, P<0.05); GAD65 protein was unchanged. The reduction in GAD67 protein could account for a maximum of ∼65% of the decrease in VGAD in vigabatrin-treated animals suggesting that inhibition of GAD65 must have also occurred in these experiments, although product inhibition of GAD67 by increased GABA could play a role. GAD67 could account for 56–85% of cortical GABA synthesis flux under basal conditions and the entire flux after vigabatrin treatment.