New Insights into the Compartmentation of Glutamate and Glutamine in Cultured Rat Brain Astrocytes

Abstract

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 ¹⁴C0₂ production from U-[¹⁴C]glutamate and U-[¹⁴C]glutamine, and utilized both substrate competition experiments and the transaminase inhibitor aminooxyacetic acid (AOAA) to obtain more information about the compartmentation of these substrates in cultured rat brain astrocytes. The rates of oxidation 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 the rate of oxidation of glutamine to 26.3 % of the control rate, demonstrating that glutamate can effectively compete with the oxidation of glutamine by astrocytes. In contrast, the addition of 1 mM glutamine had little or no effect on the rate of oxidation of glutamate by astrocytes, demonstrating that the glutamate produced intracellularly from exogenous glutamine does not dilute the glutamate taken up from the media. The addition of 5 mM AOAA decreased the rate of ¹⁴C0₂ production from glutamine to 29.2% of the control rate, consistent with earlier studies by our group. The addition of 5 mM AOAA decreased the rate of oxidation of concentrations of glutamate ≤0.1 mM by ∼50%, but decreased the oxidation of 0.5–1 mM glutamate by only ∼20%, demonstrating that a substantial portion of glutamate enters the tricarboxylic acid (TCA) cycle via glutamate dehydrogenase (GDH) rather than transamination, and that as the concentration of glutamate increases the relative proportion entering the TCA cycle via GDH also increases. To determine if the presence of an amino group acceptor (i.e. a ketoacid) would increase the rate of metabolism of glutamate, pyruvate was added in some experiments. Addition of 1 mM pyruvate increased the rate of oxidation of glutamate, and the increase was inhibited by AOAA, consistent with enhanced entry of glutamate into the TCA cycle via transamination in the presence of pyruvate. Enzymatic studies showed that pyruvate increased the activity of mitochondrial aspartate aminotransferase (AAT). Overall, the data demonstrate that glutamate formed intracellularly from glutamine enters the TCA cycle primarily via transamination, but does not enter the same TCA cycle compartment as glutamate taken up from the extracellular milieu. In contrast, extracellular glutamate enters the TCA cycle in astrocytes via both transamination and GDH, and can compete with, or dilute, the oxidation of glutamate produced intracellularly from glutamine.

Human GLUD2 Glutamate Dehydrogenase Is Expressed in Neural and Testicular Supporting Cells

“…These observations are consistent with existing evidence that GDH in brain functions mainly toward the oxidative deamination of glutamate (31). On the other hand, there is little evidence that GDH is involved in the synthesis of glutamate in brain, as the enzyme has a high K m for ammonia (10 -20 mM) and the NAD ϩ /NADH ratio is high in this organ (31). However, local increases in ammonia concentration could facilitate the reductive amination of ␣-ketoglutarate, as has been suggested for the "glutamate/glutaminebranched chain amino acid shuttle" that is thought to be involved in the cycling of ammonia between neurons and astro-FIGURE 7.…”

“…This requires conversion of glutamate to ␣-ketoglutarate catalyzed partly by GDH. These observations are consistent with existing evidence that GDH in brain functions mainly toward the oxidative deamination of glutamate (31). On the other hand, there is little evidence that GDH is involved in the synthesis of glutamate in brain, as the enzyme has a high K m for ammonia (10 -20 mM) and the NAD ϩ /NADH ratio is high in this organ (31).…”

Structure, function and regulation of pyruvate carboxylase

“…Subsequently, glutamate is converted into glutamine by astrocytic glutamine synthetase and secreted into the extracellular fluid, from which it is taken up by neurons for conversion into glutamate, aspartate and γ-aminobutyric acid [36,38,39]. Oxaloacetate, produced by PC, can also participate in this glutamate\glutamine cycle [40], and a recent study has shown that PC can alter the rate of de no o astrocytic synthesis of glutamate by increasing the amount of tricarboxylic acid cycle intermediates [39].…”

Human T-Cell Lymphotropic Virus Type 1-Infected T Lymphocytes Impair Catabolism and Uptake of Glutamate by Astrocytes via Tax-1 and Tumor Necrosis Factor Alpha

“…These are the excitatory amino acid transporters 1 and 2 (EAAT1 and EAAT2 in humans, the rat counterparts being GLAST and GLT-1, respectively) (6,29). Glutamate taken up by astrocytes is converted to glutamine by glutamine synthetase (GS; EC 6.3.1.2), and also passes into the astrocytic tricarboxylic (TCA) cycle (39) by conversion into ␣-ketoglutarate by glutamate dehydrogenase (GDH; EC.1.4.1.3). If glutamate management is impaired within astrocytes, this will compromise the functional integrity of the CNS in general and that of neurons and oligodendrocytes in particular, as these cells de-pend on metabolic precursors provided by astrocytes (48) and are highly sensitive to excessive concentrations of extracellular glutamate (7,37,54).…”