L-alpha-aminoadipate reduces glutamate release from brain tissue exposed to combined oxygen and glucose deprivation.

The effect of the glutamate analogue L-alpha-aminoadipate (alpha AA) on the release of glutamate and gamma-aminobutyric acid (GABA) from rat hippocampal slices was investigated in vitro. Oxygen/glucose deprivation caused a large release of glutamate and GABA. alpha AA added during energy deprivation reduced the glutamate release in a dose-dependent manner (56% reduction at 5 mM), whereas GABA release was unchanged. We speculate that ischemic glutamate release from the brain is mediated by a low affinity transport mechanism that is blocked by alpha AA.

Calcium-dependent release of glutamate from human cerebral cortex.

The release of the excitatory amino acids glutamate and aspartate from human neocortex was investigated in vitro by utilizing brain tissue removed during anterior temporal lobectomies for tumor or epilepsy. Depolarization (50 mM K+) increased the glutamate release to 291% of control (809 pmol/mg/min) during blocked synaptic transmission and to 669% (1859 pmol/mg/min) when synaptic transmission was not blocked. Aspartate release increased to 141% (326 pmol/mg/min) and 178% (412 pmol/mg/min) respectively. The difference between release with and without blocked synaptic transmission was statistically significant only for glutamate (P less than 0.01). These data provides evidence for a Ca(2+)-dependent release of glutamate, supporting a possible role of this amino acid as a neurotransmitter in human neocortex.

The effect of the volatile anesthetic isoflurane on Ca(2+)-dependent glutamate release from rat cerebral cortex.

A major effect of volatile anesthetics is to reduce excitatory synaptic transmission. In the present study the stimulated release of glutamate under the influence of increasing concentrations of isoflurane was studied in vitro by utilizing hippocampal slices from Wistar rats. Ca(2+)-dependent release was calculated by subtracting stimulated release with blocked synaptic transmission (50 mM K+, 0 mM Ca2+ and 4 mM Mg2+) from total evoked release (50 mM K+, 2 mM Ca2+ and 1 mM Mg2+). Isoflurane 0.5, 1.5 and 3% reduced Ca(2+)-dependent release of glutamate to 69, 58 and 49%, respectively (P < 0.05 for all related to control). These results are in agreement with the possibility of reduced release of transmitter as a mechanism of action of volatile anesthetics.

Calcium dependent release of gamma-aminobutyric acid (GABA) from human cerebral cortex.

The release of the amino acids GABA, taurine, glycine, glutamine and leucine from human neocortex was investigated in vitro by utilizing brain tissue removed during 8 standard temporal lobectomies for epilepsy or tumor. Slices (0.5 mm thick) were cut from each biopsy and randomly placed in three different chambers. After 90 min preincubation, the three sets of slices were incubated for 60 s in wells containing, respectively, (A) regular ACSF (control), (B) ACSF with 50 mM K+ (to depolarize the cell membrane) and (C) ACSF with 50 mM K+, 0 mM Ca2+ and 4 mM Mg2+ (depolarization during blocked synaptic transmission). The content of amino acids in the wells was determined by high-performance liquid chromatography after pre-column derivatization of the amino acids with o-phthalaldehyde. Membrane depolarization (well B) increased the GABA release to 650% (620 pmol/mg) of control (well A, 95 pmol/mg). Blocking synaptic transmission (well C) reduced the evoked release by 50% (360 pmol/mg). The release of glycine, taurine, glutamine and leucine during membrane depolarization was not significantly different from the control values. The data provide evidence for a Ca(2+)-dependent release of GABA, supporting a possible role of this amino acid as a neurotransmitter in human neocortex.

Changes in brain amino acid content induced by hyposmolar stress and energy deprivation.

The changes in endogenous amino acids in brain extracellular and intracellular compartments evoked by hyposmotic stress and energy deprivation were compared. Tissue content and release of ten amino acids were measured simultaneously in rat hippocampal slices by means of high performance liquid chromatography. Hyposmotic stress induced a large release of taurine (25568 pmol mg-1 protein), and a smaller release of glutamate, accompanied by an inverse change in tissue content. Adding mannitol to correct osmolarity, blocked these changes. Energy deprivation caused an increase in the release of all amino acids except glutamine. The release was particularly large for glutamate and GABA (31141 and 13282 pmol mg-1, respectively). The intracellular concentrations were generally reduced, but the total amount of the released amino acids increased In contrast to the effect seen during hyposmolar stress, mannitol enhanced the changes due to energy deprivation. The results show that hyposmolar stress and energy deprivation cause different content and release profiles, suggesting that the mechanisms involved in the two situations are either different or modulated in different ways. The intracellular amino acid depletion seen during energy deprivation shows that increased outward transport is probably a primary event, and increased amino acid formation likely secondary to this release.

Changes in amino acid release and membrane potential during cerebral hypoxia and glucose deprivation.

Excessive release of glutamate is believed to play a major role in the susceptibility of neurons to ischaemia. Whether the glutamate release is the primary event or occurs in response to electrophysiologic alterations has not been clarified. In the present study, the amino acid release was therefore correlated to changes in electrophysiological parameters and energy status during conditions of low oxygen tension and varying glucose concentrations in rat hippocampal slices. Plain hypoxia failed to produce glutamate release. All neurons underwent, however, a slow depolarization causing most of the neurons to lose their membrane potential within 10 minutes. By restoring the membrane potential to resting level by current injection, the neurons could still be activated synaptically and respond to transmitter application. Following reoxygenation most of the cells regained their resting membrane potential, but showed reduced excitability. When the slices were exposed to hypoxia combined with glucose deprivation (simulated ischaemia), there was a pronounced increase in the glutamate release. This glutamate release was always preceded by a fast anoxic depolarization. Whereas hypoxia reduced the ATP content only to approximately 50%, ATP was depleted in slices exposed to simulated ischaemia. The results demonstrate that although the neurons lose their membrane potential completely during hypoxia, there is no glutamate release. A fast anoxic depolarization provoked by simulated ischaemia, however, is always followed by glutamate release, probably due to a more severe ATP depletion.

Release of brain amino acids during hyposmolar stress and energy deprivation.

The release of 10 amino acids from rat hippocampal slices during exposure to hyposmotic stress or energy deprivation was measured by high-performance liquid chromatography. Exposing the slices to hyposmotic stress by lowering extracellular NaCl caused a 10-fold release of taurine (p < 0.01) and over a twofold increase of gamma-aminobutyric acid (GABA) and glutamate (p < 0.01). These changes were reversed by mannitol. Exposure to combined glucose and oxygen deprivation (energy deprivation) caused a 50-fold increase in the release of GABA, a 40-fold increase in glutamate release (p < 0.01), and a twofold to sixfold increase in taurine, aspartate, glycine, asparagine, serine, and alanine release (p < 0.05) but no change in glutamine. Energy deprivation increased the water content by 21%. Mannitol blocked this increase and further enhanced the release of glutamate and aspartate (p < 0.01) but not of GABA. The permissivity of the amino acids was plotted against the pI (pH at isoelectric point) and hydropathy indexes. Energy deprivation increased the permissivity in the following order: acidic > neutral > basic. Among neutral amino acids, permissivity increased with increasing hydrophobicity. These results indicate that the mechanisms of amino acid release are different during cerebral ischemia and hyposmotic stress.

The effect of isoflurane on brain amino acid release and tissue content induced by energy deprivation.

This article describes the effect of isoflurane on amino acid release and tissue content induced by energy deprivation in slices of rat hippocampus. Energy deprivation (95% N2 / 5% CO2 and glucose free medium) (ED) induced an increase in the release of all amino acids measured, with the exception of glutamine. The tissue content of all amino acids except gamma-aminobutyric acid (GABA) and arginine was concomitantly reduced. Isoflurane (1.5% and 3.0%) reduced glutamate release during ED by 27% and 28% (p < 0.05 as compared with release without isoflurane), respectively, whereas the tissue content was slightly increased. Similarly, GABA release was reduced by 25% and 25% (p < 0.05 as compared with release without isoflurane) accompanied by an insignificant enhancement in tissue content as compared with ED without isoflurane. Isoflurane reduced the release of taurine and most of the other amino acids. The total amount of all amino acids (both released and retained) was not significantly altered by the anesthetic. These observations demonstrate that isoflurane can modify the changes in amino acid handling induced by energy deprivation.

Amino-acid release from human cerebral cortex during simulated ischaemia in vitro.

The aim of the present study was to investigate the release of amino-acids in human cerebral cortex during membrane depolarization and simulated ischaemia (energy deprivation). Superfluous tissue from temporal Iobe resections for epilepsy was cut into 500 microns thick slices and incubated in vitro. Membrane depolarization with 50 mM K+ caused a release of glutamate, aspartate, GABA and glycine, but not glutamine or leucine. The release of glutamate and GABA was Ca(++)-dependent. Slices were exposed to simulated ischaemia (energy deprivation; ED) by combined glucose/oxygen deprivation. This caused a Ca(++)-independent release of glutamate, aspartate, GABA, glycine, and taurine which started after 8 min, peaked at the end or shortly after the 27 min period of ED, and returned to control levels within 11 min following termination of ED. Preloaded D-[3H]aspartate was released both during K(+)-stimulation and ED. Release of D-[3H]aspartate during ED was delayed compared to glutamate supporting an initial phase of synaptic glutamate release. Uptake of L-[3H]glutamate was increased during the period of glutamate release, suggesting passive diffusion across the cell membrane or enhanced transport efficacy in cellular elements with functioning uptake mechanisms.

Effect of isoflurane on release and uptake of gamma-aminobutyric acid from rat cortical synaptosomes.

We have studied the effect of isoflurane on potassium-evoked release and high-affinity uptake of gamma-aminobutyric acid (GABA) in rat cortical synaptosomes. Isoflurane 1.5% and 3% increased calcium-dependent release by 38% and 36% of control values, respectively (P < 0.05). Calcium-independent release was reduced correspondingly by 24% and 26% (P < 0.05). High-affinity uptake of GABA was not affected by isoflurane. The findings of increased synaptic GABA release combined with unaltered uptake suggest that isoflurane increases GABA in the synaptic cleft and thus may enhance inhibition.

Efflux of gamma-aminobutyric acid caused by changes in ion concentrations and cell swelling simulating the effect of cerebral ischaemia.

The relationships among ischaemic GABA efflux from brain tissue and extracellular and intracellular concentrations of sodium, chloride and potassium ions were investigated by means of 1) transverse hippocampal slices from rat and 2) functional expression of a high affinity GABA transporter in Xenopus oocytes. Brain slices were incubated for 20 min in medium where extracellular sodium and chloride were substituted with impermeant ions. Isethionate (Iseth) substitution for chloride generated a 7-fold increase in GABA efflux. Choline (Chol) but not N-methyl-D-glucamine (NMDG) substitution for sodium likewise increased GABA efflux. Reducing the osmolarity of the medium by decreasing both sodium and chloride concentrations (Hyp) increased GABA efflux 3-fold. This release was blocked by mannitol (Man). Blocking sodium channels with 1 microM of tetrodotoxin (TTX) also increased the release 3-fold. Energy deprivation (ED) increased the GABA release 50-fold. ED/Iseth left the release unchanged, ED/Chol increased the GABA efflux by 23%, whereas ED/NMDG reduced the release by 41%. Adding mannitol did not block the ED-evoked release, whereas TTX reduced it by 52%. Release of preloaded [3H]-GABA from oocytes expressing the GAT-1 GABA transporter was then examined. Depolarisation by current injection or 100 mM extracellular K+ did not increase GABA release. Sodium chloride injection, however, caused membrane depolarisation and a 100-fold increased GABA efflux from the oocytes. This release was blocked when the osmolarity was increased extracellularly by adding mannitol. These results show that 1) TTX releases GABA from brain tissue but blocks release during ED, 2) the high affinity GABA carrier must be altered in order to reverse, 3) ischaemic GABA release is sodium independent, and is modulated by large cations, 4) mannitol blocks the reversal of high affinity carriers in oocytes, but the release from brain slices during ED is unaffected. Taken together, the results suggest that ischaemic release of GABA from brain tissue does not occur by means of reversed high affinity carriers alone, but rather that it is controlled by more complex mechanisms.