No abstract

Primary cultures of cerebral cortical astrocytes were incubated with [U-13C]glutamate (0.5 mM) in modified Dulbecco's medium for 2 h. Perchloric acid (PCA) extracts of the cells as well as redissolved lyophilized media were subjected to NMR spectroscopy to identify 13C-labeled metabolites. NMR spectra of the PCA extracts exhibited distinct multiplets for glutamate, aspartate, glutamine, and malate. The culture medium showed peaks for a multitude of compounds released from the astrocytes, among which lactate, glutamine, alanine, and citrate were readily identifiable. For the first time incorporation of label into lactate from glutamate was clearly demonstrated by doublet formation in the C-3 position and two doublets in the C-2 position of lactate. This labeling pattern can only occur by incorporation from glutamate, because natural abundance will only produce singlets in proton-decoupled 13C spectra. Glutamine, released into the medium, was labeled uniformly to a large extent, but the C-3 position not only showed the expected apparent triplet but also a doublet due to 13C incorporation into the C-4 position of glutamine. The doublet accounted for 11% of the total label in the glutamine synthesized and released within the incubation period. The corresponding labeling pattern of [13C]glutamate in the PCA extracts showed that 19% of the glutamate contained 12C. Labeling of lactate, citrate, malate, and aspartate as well as incorporation of 12C into uniformly labeled glutamate and glutamine could only arise via the tricarboxylic acid cycle. The relative amount of glutamate metabolized via this route is at least 70% as calculated from the areas of the C-3 resonances of these compounds.(ABSTRACT TRUNCATED AT 250 WORDS)

Glutamate uptake and metabolism was studied in cerebral cortical astrocytes. The expression of the astrocytic glutamate transporter GLAST was found to be stimulated by extracellular glutamate through activation of kainate receptors on the astrocytes. Energy metabolism and ammonia homeostasis are two important aspects of glutamate handling in astrocytes. It is well known that glutamate transport into astrocytes and glutamine formation are energy consuming processes. Furthermore, ammonia is required for glutamine production. On the other hand, glutamate metabolism through the tricarboxylic acid cycle is an energy and ammonia producing pathway.In the present study it was shown that at an extracellular glutamate concentration of 0.5 mM, high energy phosphates were reduced, and more than 50% of the glutamate carbon skeleton entered the tricarboxylic acid cycle to yield products like lactate, aspartate, and additionally glutamate and glutamine derived from tricarboxylic acid cycle intermediates. Entry into the cycle was not affected by the transaminase inhibitor aminooxyacetic acid, indicating that deamination is the major route for 2-oxoglutarate formation from glutamate. Synthesis of glutamate from 2-oxoglutarate, however, proceeded via transamination. In an earlier study it was shown that at glutamate concentrations at and below 0.2 mM, glutamine appears to be the major product and entry of glutamate into the tricarboxylic acid cycle is decreased 70% by aminooxyacetic acid. In an attempt to unify the above mentioned results, it is suggested that availability of ammonia and energy demands are major factors determining the metabolic fate of glutamate in astrocytes.

Metabolism of glutamate and glutamine in cultured mouse cerebral cortical astrocytes has been investigated using either radioactively labelled (¹⁴C) amino acids or ¹³C-labelled amino acids combined with NMR spectroscopy of cell extracts and lyophilyzed incubation media. Using [U-¹³C]glutamate it has been shown that in astrocytes exogenously supplied glutamate is primarily (70%) metabolized oxidatively through the tricarboxylic acid (TCA) cycle and to a lesser jextent (30%) directly to glutamine. Glutamate metabolized in the TCA cycle is to a large extent recovered as lactate showing that the astrocyte-specific enzyme, malic enzyme is functionally active. Incubation with [U-¹⁴C]glutamine led to a higher specific radioactivity in glutamate than in glutamine. It could also be shown that glutamate and glutamine were metabolized differently to aspartate and alanine. These results taken together strongly suggest that glutamate/glutamine metabolism in astrocytes is compartmentalized and a model with multiple cytoplasmic and mitochondrial compartments of these amino acids is proposed.

Use of ¹³C-labeled precursors for the neuroactive amino acids glutamate and GABA as well as [U-¹³C]glutamate and glutamine combined with NMR spectroscopy has allowed detailed studies to be performed on cultured neurons and astrocytes yielding new information about synthesis and metabolism of these amino acids at the cellular level. Thus, it has become clear that astrocytes metabolize glutamate extensively through the tricarboxylic acid (TCA) cycle in a rather complex manner enabling the cells to generate lactate from malate. It has been shown that astrocytes can supply neurons not only with glutamine but also with TCA cycle constituents, lactate and alanine. Hence, an extended version of the glutamate/glutamine cycle is presented. Moreover, it has been demonstrated that citrate synthesized in astrocytes and released into the extracellular space can modify neuronal activity by regulating the Zn²⁺ concentration and thereby modulate N-methyl-D-aspartate receptor-mediated depolarization.