NEW INSIGHTS INTO THE COMPARTMENTATION OF GLUTAMATE AND GLUTAMINE IN CULTURED RAT-BRAIN ASTROCYTES

Studies from several groups have provided evidence that glutamate and glutamine are metabolized in different compartments in astrocytes. In the present study we measured the rates of (CO2)-C-14 production from U-[C-14]glutamate and U-[C-14]glutamine, and utilized both substrate c ompetition experiments and the transaminase inhibitor aminooxyacetic a cid (AOAA) to obtain more information about the compartmentation of th ese substrates in cultured rat brain astrocytes, The rates of oxidatio n of 1 mM glutamine and glutamate were 26.4+/-1.4 and 63.0+/-7.4 nmol/ h/mg protein, respectively. The addition of 1 mM glutamate decreased t he rate of oxidation of glutamine to 26.3% of the control rate, demons trating that glutamate can effectively compete with the oxidation of g lutamine by astrocytes, In contrast, the addition of 1 mM glutamine ha d little or no effect on the rate of oxidation of glutamate by astrocy tes, demonstrating that the glutamate produced intracellularly from ex ogenous glutamine does not dilute the glutamate taken up from the medi a. The addition of 5 mM AOAA decreased the rate of (CO2)-C-14 producti on from glutamine to 29.2% of the control rate, consistent with earlie r studies by our group. The addition of 5 mM AOAA decreased the rate o f oxidation of concentrations of glutamate less than or equal to 0.1 m M by similar to 50%, but decreased the oxidation of 0.5-1 mM glutamate by only similar to 20%, demonstrating that a substantial portion of g lutamate enters the tricarboxylic acid (TCA) cycle via glutamate dehyd rogenase (GDH) rather than transamination, and that as the concentrati on of glutamate increases the relative proportion entering the TCA cyc le via GDH also increases. To determine if the presence of an amino gr oup acceptor (i.e. a ketoacid) would increase the rate of metabolism o f glutamate, pyruvate was added in some experiments. Addition of 1 mM pyruvate increased the rate of oxidation of glutamate, and the increas e was inhibited by AOAA, consistent with enhanced entry of glutamate i nto the TCA cycle via transamination in the presence of pyruvate, Enzy matic studies showed that pyruvate increased the activity of mitochond rial aspartate aminotransferase (AAT), Overall, the data demonstrate t hat glutamate formed intracellularly from glutamine enters the TCA cyc le primarily via transamination, but does not enter the same TCA cycle compartment as glutamate taken up from the extracellular milieu. In c ontrast, extracellular glutamate enters the TCA cycle in astrocytes vi a both transamination and GDH, and can compete with, or dilute, the ox idation of glutamate produced intracellularly from glutamine.