High Glutamate Attenuates S100B and LDH Outputs from Rat Cortical Slices Enhanced by Either Oxygen–Glucose Deprivation or Menadione

One hour incubation of rat cortical slices in a medium without oxygen and glucose (oxygen–glucose deprivation, OGD) increased S100B release to 6.53 ± 0.3 ng/ml/mg protein from its control value of 3.61 ± 0.2 ng/ml/mg protein. When these slices were then transferred to a medium containing oxygen and glucose (reoxygenation, REO), S100B release rose to 344 % of its control value. REO also caused 192 % increase in lactate dehydrogenase (LDH) leakage. Glutamate added at millimolar concentration into the medium decreased OGD or REO-induced S100B release and REO-induced LDH leakage. Alpha-ketoglutarate, a metabolic product of glutamate, was found to be as effective as glutamate in decreasing the S100B and LDH outputs. Similarly lactate, 2-ketobutyrate and ethyl pyruvate, a lipophilic derivative of pyruvate, also exerted a glutamate-like effect on S100B and LDH outputs. Preincubation with menadione, which produces H₂O₂ intracellularly, significantly increased S100B and LDH levels in normoxic medium. All drugs tested in the present study, with the exception of pyruvate, showed a complete protection against menadione preincubation. Additionally, each OGD–REO, menadione or H₂O₂-induced mitochondrial energy impairments determined by 2,3,5-triphenyltetrazolium chloride (TTC) staining and OGD–REO or menadione-induced increases in reactive oxygen substances (ROS) determined by 2,7-dichlorofluorescin diacetate (DCFH-DA) were also recovered by glutamate. Interestingly, H₂O₂-induced increase in fluorescence intensity derived from DCFH-DA in a slice-free physiological medium was attenuated significantly by glutamate and alpha-keto acids. All these drug actions support the conclusion that high glutamate, such as alpha-ketoglutarate and other keto acids, protects the slices against OGD- and REO-induced S100B and LDH outputs probably by scavenging ROS in addition to its energy substrate metabolite property.     

Protein S100B release from rat brain slices during and after ischemia: comparison with lactate dehydrogenase leakage

One hour of ischemia significantly increased protein S100B release from rat brain slices without altering lactate dehydrogenase leakage. Reoxygenation of the ischemic slices, however, increased the levels of these biochemical markers in the medium. Although removal of extracellular Ca⁺² ions from the medium did not alter the basal lactate dehydrogenase leakage from cortical slices, an excessive increase in basal protein S100B release was seen under this condition. Ischemia and/or reoxygenation induced enhancements in these markers were attenuated by removal of Ca⁺² ions from the medium. Ischemia significantly increased glutamate release, but neither ischemia nor reoxygenation induced rises in protein S100B and lactate dehydrogenase levels were altered by glutamate receptor antagonists. Rising the glutamate levels in the medium by each ouabain or exogenous glutamate, moreover, failed in exerting an ischemia like effect on protein S100B and LDH outputs. In contrast, exogenous glutamate added into the medium protected the slices against reoxygenation induced increments in protein S100B and lactate dehydrogenase levels.

These results indicate that protein S100B has a greater sensitivity against ischemia than lactate dehydrogenase in in vitro brain slice preparations. Since neither exogenous glutamate nor enhancements of the extracellular glutamate levels by ouabain had an ischemia like effect, and since glutamate receptor antagonists were also unsuccessful, it seems unlikely that ischemia-induced increase in glutamate release is directly involved in protein S100B release or lactate dehydrogenase leakage determined in the present study.     

Mechanism of S100b Release from Rat Cortical Slices Determined Under Basal and Stimulated Conditions

Incubation of rat cortical slices in a medium that was not containing oxygen and glucose (oxygen–glucose deprivation, OGD) caused a 200% increase in the release of S100B. However, when slices were transferred to a medium containing oxygen and glucose (reoxygenation conditions, or REO), S100B release reached 500% of its control value. Neither inhibition of nitric oxide (NO) synthase by L-NAME nor addition of the NO donors sodium nitroprussid (SNP) or hydroxylamine (HA) to the medium altered basal S100B release. Similarly, the presence of SNP, HA or NO precursor l-arginine in the medium, or inhibition of NO synthase by L-NAME also failed to alter OGD- and REO-induced S100B outputs. Moreover, individual inhibition of PKC, PLA₂ or PLC all failed to attenuate the S100B release determined under control condition or enhanced by either OGD or REO. Blockade of calcium channels with verapamil, chelating the Ca⁺² ions with BAPTA or blockade of sodium channels with tetrodotoxin (TTX) did not alter OGD- and REO-induced S100B release. In contrast to the pharmacologic manipulations mentioned above, glutamate and α-ketoglutarate added at high concentrations to the medium prevented both OGD- and REO-induced S100B outputs. These results indicate that neither NO nor the activation of PKC, PLA₂ or PLC seem to be involved in basal or OGD- and REO-induced S100B outputs. Additionally, calcium and sodium currents that are sensitive to verapamil and TTX, respectively, are unlikely to contribute to the enhanced S100B release observed under these conditions.     

Release of Endogenous Glutamate,Aspartate, GABA,and Taurine from Hippocampal Slices from Adult and Developing Mice under Cell-Damaging Conditions

The releases of endogenous glutamate, aspartate, GABA and taurine from hippocampal slices from 7-day-, 3-, 12-, and 18-month-old mice were investigated under cell-damaging conditions using a superfusion system. The slices were superfused under hypoxic conditions in the presence and absence of glucose and exposed to hydrogen peroxide. In the adult hippocampus under normal conditions the basal release of taurine was highest, with a response only about 2-fold to potassium stimulation (50 mM). The low basal releases of glutamate, aspartate, and GABA were markedly potentiated by K⁺ ions. In general, the release of the four amino acids was enhanced under all above cell-damaging conditions. In hypoxia and ischemia (i.e., hypoxia in the absence of glucose) the release of glutamate, aspartate and GABA increased relatively more than that of taurine, and membrane depolarization by K⁺ markedly potentiated the release processes. Taurine release was doubled in hypoxia and tripled in ischemia but K⁺ stimulation was abolished. In both the mature and immature hippocampus the release of glutamate and aspartate was greatly enhanced in the presence of H₂O₂, that of aspartate particularly in developing mice. In the immature hippocampus the increase in taurine release was 10-fold in hypoxia and 30-fold in ischemia, and potassium stimulation was partly preserved. The release processes of the four amino acids in ischemia were all partially Ca²⁺-dependent. High concentrations of excitatory amino acids released under cell-damaging conditions are neurotoxic and contribute to neuronal death during ischemia. The substantial amounts of the inhibitory amino acids GABA and taurine released simultaneously may constitute an important protective mechanism against excitatory amino acids in excess, counteracting their harmful effects. In the immature hippocampus in particular, the massive release of taurine under cell-damaging conditions may have a significant function in protecting neural cells and aiding in preserving their viability.     

Involvement of Metabotropic Glutamate Receptors in Ischemia-Induced Taurine Release in the Developing and Adult Hippocampus

Metabotropic glutamate receptors have recently been envisaged as involved in both potentiation and prevention of ischemic and excitotoxic neuronal damage. The release of the inhibitory amino acid taurine is markedly enhanced in ischemia in both the immature and mature mouse hippocampus. The modulation of [³H]taurine release by metabotropic receptor agonists and antagonists was studied in hippocampal slices from developing (7-day-old) and adult (3-month-old) mice using a superfusion system. Agonists of group I, II and III metabotropic glutamate receptors generally reduced the ischemia-induced release in adult animals. In the immature hippocampus the group I agonists (S)-3,5-dihydroxyphenylglycine and (1±)-1-aminocyclopentane-trans-1,3-dicarboxylate, which mainly enhance neuronal excitation, potentiated initial taurine release in ischemia. Ionotropic glutamate receptor agonists also enhance the ischemia-induced taurine release in developing mice. This glutamate-activated taurine release may thus constitute an important protective mechanism against excitotoxicity in the immature hippocampus.     

Elevation of the Extracellular Concentrations of Glutamate and Aspartate in Rat Hippocampus During Transient Cerebral Ischemia Monitored by Intracerebral Microdialysis

Rats were implanted with 0.3-mm-diameter dialysis tubing through the hippocampus and subsequently perfused with Ringer's solution at a flow rate of 2 microliter/min. Samples of the perfusate representing the extracellular fluid were collected over 5-min periods and subsequently analyzed for contents of the amino acids glutamate, aspartate, glutamine, taurine, alanine, and serine. Samples were collected before, during, and after a 10-min period of transient complete cerebral ischemia. The extracellular contents of glutamate and aspartate were increased, respectively, eight- and threefold during the ischemic period; the taurine concentration also was increased 2.6-fold. During the same period the extracellular content of glutamine was significantly decreased (to 68% of the control value), whereas the concentrations of alanine and serine did not change significantly during the ischemic period. The concentrations of gamma-aminobutyric acid (GABA) were too low to be measured reliably. It is suggested that the large increase in the content of extracellular glutamate and aspartate in the hippocampus induced by the ischemia may be one of the causal factors in the damage to certain neurons observed after ischemia.     

Studies on the effects of lactate transport inhibition, pyruvate, glucose and glutamine on amino acid, lactate and glucose release from the ischemic rat cerebral cortex

A rat four vessel occlusion model was utilized to examine the effects of ischemia/reperfusion on cortical window superfusate levels of amino acids, glucose, and lactate. Superfusate aspartate, glutamate, phosphoethanolamine, taurine, and GABA were significantly elevated by cerebral ischemia, then declined during reperfusion. Other amino acids were affected to a lesser degree. Superfusate lactate rose slightly during the initial ischemic period, declined during continued cerebral ischemia and then was greatly elevated during reperfusion. Superfusate glucose levels declined to near zero levels during ischemia and then rebounded beyond basal levels during the reperfusion period. Inhibition of neuronal lactate uptake with alpha-cyano-4-hydroxycinnamate dramatically elevated superfusate lactate levels, enhanced the ischemia/reperfusion evoked release of aspartate but reduced glutamine levels. Topical application of an alternative metabolic fuel, glutamine, had a dose dependent effect. Glutamine (1 mM) elevated basal superfusate glucose levels, diminished the decline in glucose during ischemia, and accelerated its recovery during reperfusion. Lactate levels were elevated during ischemia and reperfusion. These effects were not evident at 5 mM glutamine. At both concentrations, glutamine significantly elevated the superfusate levels of glutamate. Topical application of sodium pyruvate (20 mM) significantly attenuated the decline in superfusate glucose during ischemia and enhanced the levels of both glucose and lactate during reperfusion. However, it had little effect on the ischemia-evoked accumulation of amino acids. Topical application of glucose (450 mg/dL) significantly elevated basal superfusate levels of lactate, which continued to be elevated during both ischemia and reperfusion. The ischemia-evoked accumulations of aspartate, glutamate, taurine and GABA were all significantly depressed by glucose, while phosphoethanolamine levels were elevated. These results support the role of lactate in neuronal metabolism during ischemia/reperfusion. Both glucose and glutamine were also used as energy substrates. In contrast, sodium pyruvate does not appear to be as effectively utilized by the ischemic/reperfused rat brain since it did not reduce ischemia-evoked amino acid efflux.     

Anoxia-induced dopamine release from rat striatal slices: involvement of reverse transport mechanism

Incubation of rat striatal slices in the absence of oxygen (anoxia), glucose (aglycemia), or oxygen plus glucose (ischemia) caused significant increases in dopamine (DA) release. Whereas anoxia decreased extracellular 3,4-dihydroxyphenylacetic acid levels by 50%, aglycemia doubled it, and ischemia returned this aglycemia-induced enhancement to its control level. Although nomifensine, a DA uptake blocker, completely protected the slices against anoxia-induced DA depletion, aglycemia- and ischemia-induced increases were not altered. Moreover, hypothermia differentially affected DA release stimulated by anoxia, aglycemia, and ischemia. Involvement of glutamate in DA release induced by each experimental condition was tested by using MK-801 and also by comparing the glutamate-induced DA release with that during anoxia, aglycemia, or ischemia. MK-801 decreased the anoxia-induced DA depletion in a dose-dependent manner. This treatment, however, showed a partial protection in aglycemic conditions but failed to improve ischemia-induced DA depletion. Like anoxia, DA release induced by exogenous glutamate was also sensitive to nomifensine and hypothermia. These results indicate that anoxia enhances DA release by a mechanism involving both the reversed DA transporter and endogenous glutamate. Partial or complete lack of effect of nomifensine, hypothermia, or MK-801 in the absence of glucose or oxygen plus glucose also suggests that experimental conditions, such as the degree of anoxia/ischemia, may alter the mechanism(s) involved in DA depletion.     

Taurine and neural cell damage

Summary. The inhibitory amino acid taurine is an osmoregulator and neuromodulator, also exerting neuroprotective actions in neural tissue. We review now the involvement of taurine in neuron-damaging conditions, including hypoxia, hypoglycemia, ischemia, oxidative stress, and the presence of free radicals, metabolic poisons and an excess of ammonia. The brain concentration of taurine is increased in several models of ischemic injury in vivo. Cell-damaging conditions which perturb the oxidative metabolism needed for active transport across cell membranes generally reduce taurine uptake in vitro, immature brain tissue being more tolerant to the lack of oxygen. In ischemia nonsaturable diffusion increases considerably. Both basal and K⁺-stimulated release of taurine in the hippocampus in vitro is markedly enhanced under cell-damaging conditions, ischemia, free radicals and metabolic poisons being the most potent. Hypoxia, hypoglycemia, ischemia, free radicals and oxidative stress also increase the initial basal release of taurine in cerebellar granule neurons, while the release is only moderately enhanced in hypoxia and ischemia in cerebral cortical astrocytes. The taurine release induced by ischemia is for the most part Ca²⁺-independent, a Ca²⁺-dependent mechanism being discernible only in hippocampal slices from developing mice. Moreover, a considerable portion of hippocampal taurine release in ischemia is mediated by the reversal of Na⁺-dependent transporters. The enhanced release in adults may comprise a swelling-induced component through Cl⁻ channels, which is not discernible in developing mice. Excitotoxic concentrations of glutamate also potentiate taurine release in mouse hippocampal slices. The ability of ionotropic glutamate receptor agonists to evoke taurine release varies under different cell-damaging conditions, the N-methyl-D-aspartate-evoked release being clearly receptor-mediated in ischemia. Neurotoxic ammonia has been shown to provoke taurine release from different brain preparations, indicating that the ammonia-induced release may modify neuronal excitability in hyperammonic conditions. Taurine released simultaneously with an excess of excitatory amino acids in the hippocampus under ischemic and other neuron-damaging conditions may constitute an important protective mechanism against excitotoxicity, counteracting the harmful effects which lead to neuronal death. The release of taurine may prevent excitation from reaching neurotoxic levels. Received January 25, 2000/Accepted January 31, 2000     

Changes in the Extracellular Concentrations of Amino Acids in the Rat Striatum During Transient Focal Cerebral Ischemia

Abstract: Although considerable evidence supports a role for amino acids in transient global cerebral ischemia and permanent focal cerebral ischemia, effects of transient focal cerebral ischemia on the extracellular concentrations of amino acids have not been reported. Accordingly, our study was undertaken to examine the patterns of changes of extracellular glutamate, aspartate, GABA, taurine, glutamine, alanine, and phosphoethanolamine in the striatum of transient focal cerebral ischemia, as evidence to support their pathogenic roles. Focal ischemia was induced using the middle cerebral artery occlusion model, with no need for craniotomy. Microdialysis was used to sample the brain's extracellular space before, during, and after the ischemic period. One hour of middle cerebral artery occlusion followed by recirculation caused neuronal damage that was common in the frontoparietal cortex and the lateral segment of the caudate nucleus. During 1 h of ischemia, the largest increase occurred for GABA and moderate increases were observed for taurine, glutamate, and aspartate. Alanine, which is a nonneuroactive amino acid, increased little. After recirculation, the levels of glutamate and aspartate reverted to normal baseline values right after reperfusion. Despite these rapid normalizations, neuronal damage occurred. Therefore, uptake of excitatory amino acids can still be restored after 1 h of middle cerebral artery occlusion, and tissue damage occurs even though high extracellular levels of glutamate are not maintained.     

Taurine reduces caspase-8 and caspase-9 expression induced by ischemia in the mouse hypothalamic nuclei

Summary. Taurine is a sulphur-containing amino acid abundant in the nervous system. It protects cells from ischemia-induced apoptosis, but the mechanism underlying this is not well established. The aim of our study was to explore the effects of taurine on two main pathways of apoptosis induced by ischemia: receptor-mediated and mitochondrial cell death. Brain slices containing the supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus were incubated in vitro under control and simulated ischemic (oxygen-glucose deprivation for 30 min) conditions in the absence and presence of 20 mM taurine. Brain slices were harvested after the 180-min “postischemic” period and fixed in 4% paraformaldehyde. To estimate apoptosis, immunostaining was done for caspase-8 and caspase-9 in paraffin-embedded sections. Immunoreactive caspase-8 and caspase-9 cells were observed in SON and PVN in all experimental groups, but in the “ischemic” group the expression of caspase-8 and caspase-9 and the number of immunoreactive cells was significantly increased in both hypothalamic nuclei. Addition of taurine (20 mM) to the incubation medium induced a marked decrease in caspase-8 and caspase-9 immunoreactivity after ischemia in SON and PVN when compared with the taurine-untreated “ischemic” group. Taurine reduces ischemia-induced caspase-8 and caspase-9 expression, the key inductors of apoptosis in SON and PVN. Authors’ address: Dr. Andrey Taranukhin, Tampere Brain Research Center, Medical School, University of Tampere, FI-33014 Finland     

Release of Endogenous Amino Acids from the Striatum from Developing and Adult Mice in Ischemia

In most other studies the release of amino acid neurotransmitters and modulators in vitro has been studied mostly using labeled preloaded compounds. For several reasons the estimated release may not reliably reflect the release of endogenous compounds. The magnitudes of the release cannot thus be quite correctly estimated using radioactive labels. The basal and K⁺-evoked release of the neuroactive endogenous amino acids γ-aminobutyrate (GABA), glycine, taurine, glutamate and aspartate was now studied in slices from the striatum from 7-day-old to 3-month-old mice under control (normoxic) and ischemic conditions. The release of alanine, threonine and serine was assessed as control. GABA and glutamate release was much greater in 3-month-old than in 7-day-old mice, whereas with taurine the situation was the opposite. Ischemia markedly enhanced the release of all these three amino acids. The release of aspartate and glycine was markedly enhanced as well whereas no effects were discernible in the release of glutamine, alanine, serine and threonine. K⁺ stimulation (50 mM) enhanced the release of GABA, glutamate, taurine, aspartate and glycine in most cases, except with taurine in 3-month-old mice under the ischemic conditions and with aspartate in 7-day-old mice under the control conditions. K⁺ stimulation did not affect the release of glutamine, alanine, serine or threonine. The results on endogenous amino acids are qualitatively similar to those obtained in our earlier experiments with labeled preloaded amino acids. In conclusion, in developing mice only inhibitory taurine is released in such amounts that may counteract the harmful effects of excitatory amino acids in ischemia.     

Metabotropic Glutamate Receptors Modulate Ischemia-Induced GABA Release in Mouse Hippocampal Slices

The involvement of glutamate receptors in GABA release in ischemia was investigated in hippocampal slices from adult (3-month-old) and developing (7-day-old) mice. For in vitro ischemia, the slices were superfused in glucose-free media under nitrogen. Ionotropic glutamate receptor agonists failed to affect the ischemia-induced basal GABA release at either age. The K(+)-stimulated release in the immature hippocampus was potentiated by N-methyl-D-aspartate receptors, whereas in adults this release was reduced by both kainate and 2-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate receptor activation. The group I metabotropic receptor agonist (1+/-)-1-aminocyclopentane-trans-1,3-dicarboxylate enhanced the basal ischemic GABA release in a receptor-mediated manner in adults, this being concordant with the positive modulation of GABAergic neurotransmission by group I metabotropic glutamate receptors. (1 +/-)-1-Aminocyclopentane-trans-1,3-dicarboxylate and (S)-3,5-dihydroxyphenylglycine also enhanced the K(+)-stimulated release in the developing hippocampus in a receptor-mediated manner. Because group I receptors generally increase neuronal excitability, the enhanced GABA release may attenuate hyperexcitation or strengthen inhibition, being thus neuroprotective, particularly under ischemic conditions. Group III metabotropic glutamate receptors were not at all involved in ischemic GABA release in the immature mice, but in adults their activation by O-phospho-L-serine potentiated the basal release and reduced the K(+)-stimulated release. These opposite effects were abolished by the antagonist (RS)-2-cyclopropyl-4-phosphonophenylglycine. Metabotropic glutamate receptors, namely group I and III receptors, are able to modify the release of GABA from hippocampal slices under ischemic conditions, both positive and negative effects being discernible, depending on the age and type of receptor activated.     

Changes in regional levels of putative neurotransmitter amino acids in brain under unilateral forebrain ischemia

The levels of the neurotransmitter amino acids glutamate, aspartate, and GABA were determined in different brain regions during ischemia and post-ischemic recirculation periods using the unilateral carotid artery occlusion model of stroke in gerbils. The levels of glutamate, aspartate and GABA in ischemic hemisphere were increased significantly by 10 min of ischemia and later declined with time. Reperfusion for 30 min following 10 min. of ischemia further enhanced the levels of glutamate and aspartate. Increase in GABA levels were found during early periods of reperfusion. Regional variations in the changes of amino acids' levels were noticed following ischemia. Hippocampus showed the highest increase in glutamate levels followed by striatum and cerebral cortex. Aspartate levels in striatum and hippocampus increased during 10 min ischemia (46% and 30%) and recirculation (70% and 79%), whereas in cerebral cortex the levels were doubled only during recirculation. Ischemia induced elevations of GABA levels were observed in cerebral cortex (68%) and in hippocampus (30%), and the levels were normalized during recirculation. No changes in GABA levels were found in striatum. It is suggested that the large increase in the levels of excitatory neurotransmitter amino acids in brain regions specially in hippocampus during ischemia and recirculation may be one of the causal factors for ischemic brain damage.     

Characteristics of taurine release induced by free radicals in mouse hippocampal slices

Summary. The release of the inhibitory neuromodulator taurine in the hippocampus is markedly enhanced under various neural cell-damaging conditions, including ischemia and exposure to free radicals. The properties and regulation of the release evoked by a medium containing free radicals was investigated in hippocampal slices from adult (3-month-old) and developing (7-day-old) mice, using a superfusion system. The free radical damage was induced by applying 0.01% H₂O₂. The release of [³H]taurine was in both adult and developing hippocampus partly Ca²⁺-independent, mediated by Na⁺-dependent transporters and probably resulting from disruption of cell membranes and subsequent ion imbalance. The release in developing mice appeared to be more susceptible to regulation than that in adults, the stimulation by free radicals being in the latter already maximal. The release was reduced by adenosine A₁ receptor agonist R(–)N⁶-(2-phenylisopropyl)adenosine, which effect was, however, abolished by the antagonist 8-cyclopentyl-1,3-dipropylxanthine only in the immature hippocampus, indicating a receptor-mediated process. Moreover, the evoked taurine release in developing mice was potentiated by the ionotropic glutamate receptor agonists N-methyl-D-aspartate, kainate and 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate in a receptor-mediated manner, since the effects were abolished by their respective antagonists. The metabotropic glutamate receptors are of only minor significance in the release, the agonists of group I and II receptors slightly reducing the release. Furthermore, NO may also be involved in this release, the NO-generating compounds hydroxylamine and S-nitroso-N-acetylpenicillamine being able to enhance the free-radical-evoked release. It seems that the free-radical-stimulated release, potentiated by ionotropic glutamate receptor activation and NO production, could constitute part of the neuroprotective properties of taurine, being important particularly in the developing hippocampus and hence preventing excitotoxicity.     

Characteristics of basal taurine release in the rat striatum measured by microdialysis

Summary. Taurine is a sulfur-containing amino acid thought to be an osmoregulator, neurotransmitter or neuromodulator in the brain. Our objective was to establish how much taurine is released in the striatum and examine the mechanisms controlling extracellular taurine concentrations under resting conditions. The experiments were made on rats by microdialysis in vivo. Changes in taurine were compared with those in glutamate, glycine and the non-neuroactive amino acid threonine. Using the zero net flux approach we showed the extracellular concentration of taurine to be 25.2±5.1M. Glutamate was increased by tetrodotoxin and decreased by Ca²⁺ omission, glycine and threonine were not affected and both treatments increased extracellular taurine. The basal taurine release was increased by the taurine transport inhibitor guanidinoethanesulfonate and reduced by the anion channel blocker 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid.     

Validity of elevated interstitial levels of taurine as a predictor of myocardial ischemic injury

Summary. The microdialysis (MD) technique allows for continuous in vivo monitoring of dynamic changes in the interstitial levels of energy-related metabolites. The release of taurine from the myocyte has been suggested as a marker of ischemic injury. The relationship between (interstitial) taurine release and the degree of myocardial ischemic injury was evaluated following a 40min long ischemia in a porcine heart-infarct-model. Different protocols of ischemia and reperfusion were used in order to achieve a graded level of myocardial injury. Both interstitial peak levels and the area under curve of taurine obtained during ischemia and reperfusion correlated with the degree of ischemic injury (assessed by developed infarct size estimation). The release of taurine in the myocardium measured by the MD-technique correlated with the degree of ischemic injury during ongoing ischemic insult. Hence, taurine determination in the MD-setting represents a powerful tool to follow the development of myocardial ischemic injury over time.     

Transmitter amino acid release from rat neocortex: Complete versus incomplete ischemia models

Release of the excitotoxic amino acids, glutamate and aspartate, from the ischemic rat cerebral cortex was compared in two models; the seven vessel occlusion model (7VO) of complete cerebral ischemia and the four vessel occlusion model (4VO) of incomplete cerebral ischemia. Amino acid efflux into cortical superfusates was measured using cortical cups placed on both hemispheres. Whereas a 20 min period of ischemia causes a pronounced release of glutamate and aspartate from the 4VO model, efflux was significantly reduced in the 7VO model. Release of the inhibitory transmitter GABA, was similar in the two models. This result suggests that excitotoxic amino acid efflux into the extracellular spaces of the cerebral cortex may be enhanced by the residual blood flow in an incomplete ischemia.Special issue dedicated to Dr. Sidney Ochs.     

Ischemia-Induced Taurine Release Is Modified by Nitric Oxide-Generating Compounds in Slices from the Developing and Adult Mouse Hippocampus

The novel neurotransmitter/neuromodulator nitric oxide (NO), which is linked to the activation of the N-methyl-D-aspartate class of glutamate receptors, has been shown to modify transmitter release in brain tissue. Release of the inhibitory amino acid taurine is also markedly enhanced by N-methyl-D-aspartate and NO-producing agents under normal conditions in the mouse hippocampus. The release of preloaded [³H]taurine from hippocampal slices from adult (3-month-old) and developing (7-day-old) mice was characterized under ischemic conditions in the presence of different NO-generating compounds, hydroxylamine, sodium nitroprusside, and S-nitroso-N-acetylpenicillamine (SNAP), using a superfusion system. The ischemia-induced taurine release at both ages was markedly enhanced by 1.0 mM nitroprusside and 1.0 mM SNAP, whereas 5.0 mM hydroxylamine was effective only in adults. The nitroprusside- and SNAP-induced releases were reduced by the inhibitors of NO synthase (nitroarginine and 7-nitroindazole) and NO-sensitive soluble guanylyl cyclase [1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one], suggesting involvement of the NO/cGMP pathway. The release in ischemia in the absence of Na⁺ was modified by NO compounds only in adults; the 0.1 mM N-methyl-D-aspartate stimulated taurine release at both ages. The enhanced release of taurine associated with NO production could be beneficial to brain tissue under cell-damaging conditions and corroborates the neuroprotective role of this amino acid, particularly in the immature brain.     

Taurine and GABA release from mouse cerebral cortex slices: Effects of structural analogues and drugs

The effects of structural analogues, excitatory amino acids and certain drugs on spontaneous and potassium-stimulated exogenous taurine and GABA release were investigated in mouse cerebral cortex slices using a superfusion system. Spontaneous efflux of both amino acids was rather slow but could be enhanced by their uptake inhibitors. Taurine efflux was facilitated by exogenous taurine, hypotaurine, -alanine and GABA, whereas GABA, nipecotic acid and homotaurine effectively enhanced GABA release. The stimulatory potency of the analogues closely corresponded to their ability to inhibit taurine and GABA uptake, respectively, indicating that these efflux processes could be mediated by the carriers operating outwards. Glutamate induced GABA release, whereas taurine efflux was potentiated by aspartate, glutamate, cysteate, homocysteate and kainate. The centrally acting drugs, including GABA agonists and antagonists, as well as the proposed taurine antagonist TAG (6-aminomethyl-3-methyl-4H-1,2,4-benzothiadiazine-1,1-dioxide), had no marked effects on spontaneous taurine and GABA release. Potassium ions stimulated dosedependently both taurine and GABA release from the slices, the responses of taurine being strikingly slow but sustained. Exogenous GABA and nipecotic acid accelerated the potassium-stimulated GABA release, whereas picrotoxin and bicuculline were ineffective. The potassium-stimulated taurine release was unaltered or suppressed by exogenous taurine and analogues, differing in this respect from GABA release. The apparent magnitude of the depolarization-induced GABA release is thus influenced by the function of membrane transport sites, but the same conclusion cannot be drawn with regard to taurine. Haloperidol and imipramine were able to affect the evoked release of both taurine and GABA.