Decreased glutamate metabolism in cultured astrocytes in the presence of thiopental

doi:10.1016/S0006-2952(99)00175-6

Biochemical Pharmacology

Volume 58, Issue 6, 15 September 1999, Pages 1075-1080

https://doi.org/10.1016/S0006-2952(99)00175-6 Get rights and content

Abstract

The effect of thiopental on glutamate metabolism was studied by ¹³C magnetic resonance spectroscopy. Cerebral cortical astrocytes were incubated with 0.5 mM [U-¹³C]glutamate for 2 hr in the presence of 0.5 or 1 mM thiopental. Labeled glutamate, glutamine, aspartate, and glutathione were observed in cell extracts, and glutamine, aspartate, and lactate in the medium. Not only present in the medium was uniformly labeled glutamate, but also glutamate derived from the tricarboxylic acid (TCA) cycle, and thus glutamate release could be detected. The amounts of [U-¹³C]glutamate and unlabeled glucose taken up by astrocytes were unchanged in the presence of 0.5 mM thiopental and decreased to about 50% and 80%, respectively when the concentration was increased to 1 mM. The amounts of most metabolites synthesized from [U-¹³C]glutamate were unchanged in the presence of 0.5 mM thiopental, but decreased [U-¹³C]glutamine, [U-¹³C]aspartate, and [U-¹³C]lactate were observed in the 1 mM group. Surprisingly, the amounts of [1,2,3-¹³C]glutamate, [2,3-¹³C]aspartate, and [3,4-¹³C]aspartate (2nd turn via the TCA cycle) were unchanged. However, this was not the case for [1,2-¹³C]lactate and [2,3-¹³C]lactate. Such variations indicate cellular compartmentation, possibly caused by a heterogeneous glutamate concentration within the cells affecting TCA cycle turnover rates differently.

Section snippets

Materials

Plastic tissue culture dishes were purchased from Nunc A/S, FBS from Seralab Ltd., and culture medium from GIBCO BRL, Life Technologies. NMRI mice were purchased from Møllegaard Breeding Center. [U-¹³C]glutamate (99% enriched) and 99.9% D₂O (deuterium oxide) were from Cambridge Isotopes Laboratories, sodium thiopental from Abbott, and ethyleneglycol from Merck. All other chemicals were of the purest grade available from regular commercial sources.

Cell cultures

All animal procedures were conducted according

Results

Typical spectra from cultured cortical astrocytes after incubation with [U-¹³C]glutamate in the presence of thiopental are shown in Fig. 1 (cell extract, bottom; cell culture medium, top). As seen from the spectra, glutamate was metabolized in cultured astrocytes to a great extent. Labeled glutamine, aspartate, and glutathione synthesized from [U-¹³C]glutamate are clearly seen in the spectrum from cell extract, whereas in the spectrum of medium, in addition to the added [U-¹³C]glutamate,

Effect of thiopental on glucose

Unlabeled glucose (3 mM) was present in the medium under all experimental conditions, and the amount of glucose and lactate in the medium could be quantified due to natural abundance of ¹³C (1.1%). Thus, information concerning the effects of thiopental on glucose metabolism was obtained. The amount of glucose removed from the medium and lactate synthesized from glucose was unchanged with 0.5 mM thiopental, but decreased more than 50% in the 1-mM group. This agrees with results reported by

Acknowledgements

This research was supported by the Research Council of Norway, the Special Medical Application (RiT), Blix, and SINTEF UNIMED Foundations, and the Department of Physics, Norwegian University of Science and Technology (NTNU). The excellent technical assistance of Inger Beate Følstad is greatly appreciated.

References (27)

T Ito et al.
Pharmacology of barbiturate tolerance/dependenceGABA_A receptors and molecular aspects
Life Sci
(1996)
L Hertz
Inhibition by barbiturates of an intense net uptake of potassium into astrocytes
Neuropharmacology
(1979)
W Marszalec et al.
Use-dependent pentobarbital block of kainate and quisqualate currents
Brain Res
(1993)
A.C.H Yu et al.
Effects of barbiturates on energy and intermediary metabolism in cultured astrocytes
Prog Neuropsychopharmacol Biol Psychiatry
(1983)
E Hertz et al.
Effects of barbiturates on energy metabolism by cultured astrocytes and neurons in the presence of normal and elevated concentrations of potassium
Neuropharmacology
(1986)
G Gegelashvili et al.
Cellular distribution and kinetic properties of high-affinity glutamate transporters
Brain Res Bull
(1998)
K Kondo et al.
Expression of glutamate transporters in cultured glial cells
Neurosci Lett
(1995)
N Zarchin et al.
Thiopental induced cerebral protection during ischemia in gerbils
Brain Res
(1998)
N.P Franks et al.
Molecular and cellular mechanisms of general anaesthesia
Nature
(1994)
H McIlwain et al.
Biochemistry and the Central Nervous System
(1985)

C Frenkel et al.

Molecular actions of pentobarbital isomers on sodium channels from human brain cortex

Anesthesiology

(1990)

H Miyazaki et al.

Increase of glutamate uptake in astrocytes

Anesthesiology

(1997)

Y Kanai et al.

Primary structure and functional characterization of a high-affinity glutamate transporter

Nature

(1992)

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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-¹³C]glutamate, 3 mM glucose, 50 μM 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-¹³C]glutamate and unlabeled glucose was seen under all conditions. The amounts of most metabolites synthesized from [U-¹³C]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 [1,2,3-¹³C]glutamate and [U-¹³C]aspartate were found in the medium. In the cell extracts increased [U-¹³C]glutamate, [1,2,3-¹³C]glutamate, labeled glutathione and [U-¹³C]aspartate were observed in the 1 mM thiopental groups. Glutamate efflux and uptake were studied using [³H]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-¹³C]glutamate but at the same time increased the detectable metabolites derived from the TCA cycle. These latter effects were also exerted by GABA.

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NeuroscienceDecreased glutamate metabolism in cultured astrocytes in the presence of thiopental

Abstract

Section snippets

Materials

Cell cultures

Results

Effect of thiopental on glucose

Acknowledgements

Life Sci

Neuropharmacology

Brain Res

Prog Neuropsychopharmacol Biol Psychiatry

Neuropharmacology

Brain Res Bull

Neurosci Lett

Brain Res

Molecular and cellular mechanisms of general anaesthesia

Nature

Biochemistry and the Central Nervous System

Molecular actions of pentobarbital isomers on sodium channels from human brain cortex

Anesthesiology

Increase of glutamate uptake in astrocytes

Anesthesiology

Primary structure and functional characterization of a high-affinity glutamate transporter

Nature

Neuroscience
Decreased glutamate metabolism in cultured astrocytes in the presence of thiopental