Effects of thiopental on transport and metabolism of glutamate in cultured cerebellar granule neurons.

This study was performed to analyze the effects of the barbiturate thiopental on neuronal glutamate uptake, release and metabolism. Since barbiturates are known to bind to the GABA(A) receptor, some experiments were carried out in the presence of GABA. Cerebellar granule neurons were incubated for 2 h in medium containing 0.25 mM [U-(13)C]glutamate, 3 mM glucose, 50 microM GABA and 0.1 or 1 mM thiopental when indicated. When analyzing cell extracts, it was surprisingly found that in addition to glutamate, aspartate and glutathione, GABA was also labeled. In the medium, label was observed in glutamate, aspartate and lactate. Glutamate exhibited different labeling patterns, indicating metabolism in the tricarboxylic acid cycle, and subsequent release. A net uptake of [U-(13)C]glutamate and unlabeled glucose was seen under all conditions. The amounts of most metabolites synthesized from [U-(13)C]glutamate were unchanged in the presence of GABA with or without 0.1 mM thiopental. In the presence of 1 mM thiopental, regardless of the presence of GABA, decreased amounts of [1,2, 3-(13)C]glutamate and [U-(13)C]aspartate were found in the medium. In the cell extracts increased [U-(13)C]glutamate, [1,2, 3-(13)C]glutamate, labeled glutathione and [U-(13)C]aspartate were observed in the 1 mM thiopental groups. Glutamate efflux and uptake were studied using [(3)H]D-aspartate. While efflux was substantially reduced in the presence of 1 mM thiopental, this barbiturate only marginally inhibited uptake even at 3 mM. These results may suggest that the previously demonstrated neuroprotective action of thiopental could be related to its ability to reduce excessive glutamate outflow. Additionally, thiopental decreased the oxidative metabolism of [U-(13)C]glutamate but at the same time increased the detectable metabolites derived from the TCA cycle. These latter effects were also exerted by GABA.

Effect of glutamine and GABA on [U-(13)C]glutamate metabolism in cerebellar astrocytes and granule neurons.

To probe the effect of glutamine and GABA on metabolism of [U-(13)C]glutamate, cerebellar astrocytes were incubated with [U-(13)C]glutamate (0.5 mM) in the presence and absence of glutamine (2.5 mM) or GABA (0.2 mM). It could be shown that consumption of [U-(13)C]glutamate was decreased in the presence of glutamine and release of labeled aspartate and [1,2,3-(13)C]glutamate decreased as well, whereas the concentrations of these metabolites increased inside the cells. Glutamine decreased energy production from [U-(13)C]glutamate presumably by substituting for glutamate as an energy substrate. No additional effect was seen in the presence of both glutamine and GABA. When cerebellar granule neurons were incubated with [U-(13)C]glutamate (0.25 mM) and GABA (0.05 mM), less [U-(13)C]glutamate was used for energy production than in controls. Because the barbiturate thiopental did not elicit such response (Qu et al., 2000, Neurochem Int 37:207-215) it appears that GABA also has a metabolic function in the glutamatergic cerebellar granule neurons in contrast to the astrocytes.