Decrease in GABA synthesis rate in rat cortex following GABA-transaminase inhibition correlates with the decrease in GAD(67) protein

gamma -Aminobutyric acid (GABA) synthesis in the brain is mediated by two m ajor isoforms of glutamic acid decarboxylase, GAD(65) and GAD(67). The cont ribution of these isoforms to GABA synthesis flux (V-GAD) is not known quan titatively. In the present study we compared V-GAD in cortex of control and vigabatrin-treated rats under alpha -chloralose/70% nitrous oxide anesthes ia, with total GAD activity and GAD isoform composition (GAD(67) and GAD(67 )) measured by enzymatic assay and quantitative immunoblotting. V-GAD was d etermined by re-analysis of C-13 NMR data obtained ex vivo and in vivo duri ng infusions of [1-(13)]glucose using an extension of a model of glutamate- glutamine cycling that included a discrete GABAergic neuronal compartment w ith relevant interconnecting fluxes' V A was significantly lower in vigabat rin-treated rats (0.030-0.05 V mol/min per g, P<0.003) compared to the non- treated control group (0.10-0.15 <mu>mol/min per g), The 67-70% decrease in V-GAD was associated with a 13% decrease in total GAD activity (P=0.01) an d a selective 44 +/- 15% decrease in GAD(67) protein (from 0.63 +/-0.10 to 0.35 +/-0.08 mug protein/mg tissue, P <0.05); GAD(65) protein was unchanged . The reduction in GAD(67) protein could account for a maximum of similar t o 65% of the decrease in V-GAD in vigabatrin-treated animals G suggesting t hat inhibition of GAD(65) must have also occurred in these experiments, alt hough product inhibition of GAD(67) by increased GABA could play a role. GA D(67) could account for 56-85% of cortical GABA synthesis flux under basal conditions and the entire flux after vigabatrin treatment. (C) 2001 Elsevie r Science B.V. All rights reserved.