Calcium, mitochondria and oxidative stress in neuronal pathology. Novel aspects of an enduring theme

The interplay among reactive oxygen species (ROS) formation, elevated intracellular calcium concentration and mitochondrial demise is a recurring theme in research focusing on brain pathology, both for acute and chronic neurodegenerative states. However, causality, extent of contribution or the sequence of these events prior to cell death is not yet firmly established. Here we review the role of the alpha-ketoglutarate dehydrogenase complex as a newly identified source of mitochondrial ROS production. Furthermore, based on contemporary reports we examine novel concepts as potential mediators of neuronal injury connecting mitochondria, increased [Ca2+]c and ROS/reactive nitrogen species (RNS) formation; specifically: (a) the possibility that plasmalemmal nonselective cationic channels contribute to the latent [Ca2+]c rise in the context of glutamate-induced delayed calcium deregulation; (b) the likelihood of the involvement of the channels in the phenomenon of 'Ca2+ paradox' that might be implicated in ischemia/reperfusion injury; and (c) how ROS/RNS and mitochondrial status could influence the activity of these channels leading to loss of ionic homeostasis and cell death.     

Isoliquiritigenin isolated from Glycyrrhiza uralensis protects neuronal cells against glutamate-induced mitochondrial dysfunction

Glutamate-mediated excitotoxicity, which is associated with reactive oxygen species (ROS), is hypothesized to be a major contributor to pathological cell death in the mammalian central nervous system, and to be involved in many acute and chronic brain diseases. Here, we showed that isoliquiritigenin (ISL) isolated from Glycyrrhiza uralensis (Gu), one of the most frequently prescribed oriental herbal medicines, protected HT22 hippocampal neuronal cells from glutamate-induced oxidative stress. In addition, we clarified the molecular mechanisms by which it protects against glutamate-induced neuronal cell death. ISL reversed glutamate-induced ROS production and mitochondrial depolarization, as well as glutamate-induced changes in expression of the apoptotic regulators Bcl-2 and Bax. Pretreatment of HT22 cells with ISL suppresses the release of apoptosis-inducing factor from mitochondria into the cytosol. Taken together, our results suggest that ISL may protect against mitochondrial dysfunction by limiting glutamate-induced oxidative stress. In conclusion, our results demonstrated that ISL isolated from Gu has protective effects against glutamate-induced mitochondrial damage and hippocampal neuronal cell death. We expect ISL to be useful in the development of drugs to prevent or treat neurodegenerative diseases.     

Bradykinin enhances reactive oxygen species generation, mitochondrial injury, and cell death induced by ATP depletion--a role of the phospholipase C-Ca(2+) pathway

This study aimed to study the effect of bradykinin on reactive oxygen species (ROS) generation, mitochondrial injury, and cell death induced by ATP depletion in cell culture. Renal tubular cells were subjected to ATP depletion. Cell death was evaluated with LDH release, sub-G0/G1 fraction, Hoechst staining, and annexin V binding assay. ROS generation, mitochondrial membrane potential (DeltaPsi(m)), and intramitochondrial calcium were evaluated with flow cytometry. Translocation of cytochrome c and activation of apoptotic protein were analyzed with cell fractionating and Western blotting. Intracellular calcium was measured with a spectrofluorometer. Bradykinin enhanced cellular LDH release, apoptosis, generation of superoxide, and hydrogen peroxide induced by ATP depletion. Bradykinin also enhanced the loss of DeltaPsi(m), translocation of cytochrome c into cytosol, and activation of apoptotic protein. The intracellular/mitochondrial calcium was higher in bradykinin-treated cells. All these effects were reversed by coadministration with bradykinin B2 receptor (B2R) antagonist. Besides, blocking the phospholipase C (PLC) could reverse the synergistic effect of bradykinin with ATP depletion on ROS generation, mitochondrial damage, accumulation of intracellular/mitochondrial calcium, and apoptosis. Activation of B2R aggravates ROS generation, mitochondrial damage, and cell death induced by ATP depletion. These effects may act through the PLC-Ca(2+) signaling pathway.     

NMDA and non-NMDA receptors stimulation causes differential oxidative stress in rat cortical slices

Glutamate receptor activated neuronal cell death is attributed to a massive influx of Ca(2+) and subsequent formation of reactive oxygen species (ROS) but the relative contribution of NMDA and non-NMDA sub-types of glutamate receptors in excitotoxicity is not known. In the present study, we have examined the role of NMDA and non-NMDA receptors in glutamate-induced neuronal injury in cortical slices from young (20+/-2 day) and adult (80+/-5 day) rats. Treatment of slices with glutamate receptor agonists NMDA, AMPA and KA elicited the formation of reactive oxygen species (ROS) and neuronal cell death. In young slices, NMDA receptor stimulation caused a higher ROS formation and neurotoxicity, but KA was more effective in producing ROS and cell death in adult slices. AMPA exhibited an intermediate effect on ROS formation and toxicity in both the age groups. A significant protection in glutamate mediated ROS formation and neurotoxicity was observed in presence of NMDA or/and non-NMDA receptors antagonists APV and NBQX, respectively. This further confirms the involvement of both NMDA and non-NMDA receptors in glutamate mediated neurotoxicity. In adult slices, we did not find positive correlation between ligand induced neurotoxicity and mitochondrial depolarization. Though, NMDA and KA stimulation produced differential effect on ROS formation and neurotoxicity in young and adult slices, the mitochondrial depolarization was higher and comparable on NMDA stimulation in both the age groups as compared to KA, suggesting that the mitochondrial depolarization may not be a good indicator for neurotoxicity. Our results demonstrate that both NMDA and non-NMDA sub-types of glutamate receptors are involved in glutamate mediated neurotoxicity but their relative contribution is highly dependent on the age of the animal.     

Role of mitochondria in reactive oxygen species generation and removal; relevance to signaling and programmed cell death

Reactive oxygen species (ROS) are universal products of aerobic metabolism, which can be also produced in stress conditions. In eukaryotic cells, mitochondria are the main source of ROS. The main mitochondrial sites of ROS formation are electron carriers of respiratory chain. However, there are also other enzymatic sites capable of ROS generation in different mitochondrial compartments. Reactive oxygen species can cause serious damage to many biological macromolecules, such as proteins, lipids and nucleic acids, which oxidation leads to a lost of their biological properties and eventually to a cell death. Mitochondria, which are also exposed to harmful ROS action, have a defense system that decreases ROS production (first line of defense) or removes generated ROS (second line of defense). Mitochondrial antioxidant system involves proteins that decrease ROS formation, enzymes that directly react with ROS, and non-enzymatic antioxidants that also remove ROS and other oxygen derivatives. Mitochondrial ROS can also act as signal messengers and modify operation of many routes in different cell compartments. Mitochondrial ROS are also important in execution of programmed cell death.     

Cofilin1 oxidation links oxidative distress to mitochondrial demise and neuronal cell death

Many cell death pathways, including apoptosis, regulated necrosis, and ferroptosis, are relevant for neuronal cell death and share common mechanisms such as the formation of reactive oxygen species (ROS) and mitochondrial damage. Here, we present the role of the actin-regulating protein cofilin1 in regulating mitochondrial pathways in oxidative neuronal death. Cofilin1 deletion in neuronal HT22 cells exerted increased mitochondrial resilience, assessed by quantification of mitochondrial ROS production, mitochondrial membrane potential, and ATP levels. Further, cofilin1-deficient cells met their energy demand through enhanced glycolysis, whereas control cells were metabolically impaired when challenged by ferroptosis. Further, cofilin1 was confirmed as a key player in glutamate-mediated excitotoxicity and associated mitochondrial damage in primary cortical neurons. Using isolated mitochondria and recombinant cofilin1, we provide a further link to toxicity-related mitochondrial impairment mediated by oxidized cofilin1. Our data revealed that the detrimental impact of cofilin1 on mitochondria depends on the oxidation of cysteine residues at positions 139 and 147. Overall, our findings show that cofilin1 acts as a redox sensor in oxidative cell death pathways of ferroptosis, and also promotes glutamate excitotoxicity. Protective effects by cofilin1 inhibition are particularly attributed to preserved mitochondrial integrity and function. Thus, interfering with the oxidation and pathological activation of cofilin1 may offer an effective therapeutic strategy in neurodegenerative diseases.Subject terms: Apoptosis, Cell death in the nervous system, Neurodegeneration     

Reduced calcium-dependent mitochondrial damage underlies the reduced vulnerability of excitotoxicity-tolerant hippocampal neurons

In central neurons, over-stimulation of NMDA receptors leads to excessive mitochondrial calcium accumulation and damage, which is a critical step in excitotoxic death. This raises the possibility that low susceptibility to calcium overload-induced mitochondrial damage might characterize excitotoxicity-resistant neurons. In this study, we have exploited two complementary models of preconditioning-induced excitotoxicity resistance to demonstrate reduced calcium-dependent mitochondrial damage in NMDA-tolerant hippocampal neurons. We have further identified adaptations in mitochondrial calcium handling that account for enhanced mitochondrial integrity. In both models, enhanced tolerance was associated with improved preservation of mitochondrial membrane potential and structure. In the first model, which exhibited modest neuroprotection, mitochondria-dependent calcium deregulation was delayed, even though cytosolic and mitochondrial calcium loads were quantitatively unchanged, indicating that enhanced mitochondrial calcium capacity accounts for reduced injury. In contrast, the second model, which exhibited strong neuroprotection, displayed further delayed calcium deregulation and reduced mitochondrial damage because downregulation of NMDA receptor surface expression depressed calcium loading. Reducing calcium entry also modified the chemical composition of the calcium-buffering precipitates that form in calcium-loaded mitochondria. It thus appears that reduced mitochondrial calcium loading is a major factor underlying the robust neuroprotection seen in highly tolerant cells.     

Downregualtion of dynamin-related protein 1 attenuates glutamate-induced excitotoxicity via regulating mitochondrial function in a calcium dependent manner in HT22 cells

Glutamate-mediated excitotoxicity is involved in many acute and chronic brain diseases. Dynamin related protein 1 (Drp-1), one of the GTPase family of proteins that regulate mitochondrial fission and fusion balance, is associated with apoptotic cell death in cancer and neurodegenerative diseases. Here we investigated the effect of downregulating Drp-1 on glutamate excitotoxicity-induced neuronal injury in HT22 cells. We found that downregulation of Drp-1 with specific small interfering RNA (siRNA) increased cell viability and inhibited lactate dehydrogenase (LDH) release after glutamate treatment. Downregulation of Drp-1 also inhibited an increase in the Bax/Bcl-2 ratio and cleavage of caspase-9 and caspase-3. Drp-1 siRNA transfection preserved the mitochondrial membrane potential (MMP), reduced cytochrome c release, enhanced ATP production, and partly prevented mitochondrial swelling. In addition, Drp-1 knockdown attenuated glutamate-induced increases of cytoplasmic and mitochondrial Ca²⁺, and preserved the mitochondrial Ca²⁺ buffering capacity after excitotoxicity. Taken together, these results suggest that downregulation of Drp-1 protects HT22 cells against glutamate-induced excitatory damage, and this neuroprotection may be dependent at least in part on the preservation of mitochondrial function through regulating intracellular calcium homeostasis.     

Antioxidant activity of phenylethanoid glycosides on glutamate-induced neurotoxicity

ABSTRACT

Exposure of PC12 cells to 10 mM glutamate caused significant viability loss, cell apoptosis, decreased activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) as well as increased levels of malondialdehyde (MDA). In parallel, glutamate significantly increased the intracellular levels of ROS and intracellular calcium. However, pretreatment of the cells with acteoside and isoacteoside significantly suppressed glutamate-induced cellular events. Moreover, acteoside and isoacteoside reduced the glutamate-induced increase of caspase-3 activity and also ameliorated the glutamate-induced Bcl-2/Bax ratio reduction in PC12 cells. Furthermore, acteoside and isoacteoside significantly inhibited glutamate-induced DNA damage. In the mouse model, acteoside significantly attenuated cognitive deficits in the Y maze test and attenuated neuronal damage of the hippocampal CA1 regions induced by glutamate. These data indicated that acteoside and isoacteoside play neuroprotective effects through anti-oxidative stress, anti-apoptosis, and maintenance of steady intracellular calcium.     

Gartanin Protects Neurons against Glutamate-Induced Cell Death in HT22 Cells: Independence of Nrf-2 but Involvement of HO-1 and AMPK

Oxidative stress mediates the pathogenesis of neurodegenerative disorders. Gartanin, a natural xanthone of mangosteen, possesses multipharmacological activities. Herein, the neuroprotection capacity of gartanin against glutamate-induced damage in HT22 cells and its possible mechanism(s) were investigated for the first time. Glutamate resulted in cell death in a dose-dependent manner and supplementation of 1–10 µM gartanin prevented the detrimental effects of glutamate on cell survival. Additional investigations on the underlying mechanisms suggested that gartanin could effectively reduce glutamate-induced intracellular ROS generation and mitochondrial depolarization. We further found that gartanin induced HO-1 expression independent of nuclear factor erythroid-derived 2-like 2 (Nrf2). Subsequent studies revealed that the inhibitory effects of gartanin on glutamate-induced apoptosis were partially blocked by small interfering RNA-mediated knockdown of HO-1. Finally, the protein expression of phosphorylation of AMP-activated protein kinase (AMPK) and its downstream signal molecules, Sirtuin activator (SIRT1) and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), increased after gartanin treatment. Taken together, these findings suggest gartanin is a potential neuroprotective agent against glutamate-induced oxidative injury partially through increasing Nrf-2-independed HO-1 and AMPK/SIRT1/PGC-1α signaling pathways.     

Calcium-induced generation of reactive oxygen species in brain mitochondria is mediated by permeability transition

Mitochondrial uptake of calcium in excitotoxicity is associated with subsequent increase in reactive oxygen species (ROS) generation and delayed cellular calcium deregulation in ischemic and neurodegenerative insults. The mechanisms linking mitochondrial calcium uptake and ROS production remain unknown but activation of the mitochondrial permeability transition (mPT) may be one such mechanism. In the present study, calcium increased ROS generation in isolated rodent brain and human liver mitochondria undergoing mPT despite an associated loss of membrane potential, NADH and respiration. Unspecific permeabilization of the inner mitochondrial membrane by alamethicin likewise increased ROS independently of calcium, and the ROS increase was further potentiated if NAD(H) was added to the system. Importantly, calcium per se did not induce a ROS increase unless mPT was triggered. Twenty-one cyclosporin A analogs were evaluated for inhibition of calcium-induced ROS and their efficacy clearly paralleled their potency of inhibiting mPT-mediated mitochondrial swelling. We conclude that while intact respiring mitochondria possess powerful antioxidant capability, mPT induces a dysregulated oxidative state with loss of GSH- and NADPH-dependent ROS detoxification. We propose that mPT is a significant cause of pathological ROS generation in excitotoxic cell death.     

Reactive oxygen species induce different cell death mechanisms in cultured neurons

Apoptosis is characterized by chromatin condensation, phosphatidylserine translocation, and caspase activation. Neuronal apoptotic death involves the participation of reactive oxygen species (ROS), which have also been implicated in necrotic cell death. In this study we evaluated the role of different ROS in neuronal death. Superoxide anion was produced by incubating cells with xanthine and xanthine oxidase plus catalase, singlet oxygen was generated with rose Bengal and luminic stimuli, and hydrogen peroxide was induced with the glucose and glucose oxidase. Cultured cerebellar granule neurons died with the characteristics of apoptotic death in the presence of superoxide anion or singlet oxygen. These two conditions induced caspase activation, nuclear condensation, phosphatidylserine translocation, and a decrease in intracellular calcium levels. On the other hand, hydrogen peroxide led to a necrosis-like cell death that did not induce caspase activation, phosphatidylserine translocation, or changes in calcium levels. Cell death produced by both singlet oxygen and superoxide anion, but not hydrogen peroxide, was partially reduced by an increase in intracellular calcium levels. These results suggest that formation of specific ROS can lead to different molecular cell death mechanisms (necrosis and apoptosis) and that ROS formed under different conditions could act as initiators or executioners on neuronal death.     

Serine-70 phosphorylated Bcl-2 prevents oxidative stress-induced DNA damage by modulating the mitochondrial redox metabolism

Bcl-2 phosphorylation at serine-70 (S70pBcl2) confers resistance against drug-induced apoptosis. Nevertheless, its specific mechanism in driving drug-resistance remains unclear. We present evidence that S70pBcl2 promotes cancer cell survival by acting as a redox sensor and modulator to prevent oxidative stress-induced DNA damage and execution. Increased S70pBcl2 levels are inversely correlated with DNA damage in chronic lymphocytic leukemia (CLL) and lymphoma patient-derived primary cells as well as in reactive oxygen species (ROS)- or chemotherapeutic drug-treated cell lines. Bioinformatic analyses suggest that S70pBcl2 is associated with lower median overall survival in lymphoma patients. Empirically, sustained expression of the redox-sensitive S70pBcl2 prevents oxidative stress-induced DNA damage and cell death by suppressing mitochondrial ROS production. Using cell lines and lymphoma primary cells, we further demonstrate that S70pBcl2 reduces the interaction of Bcl-2 with the mitochondrial complex-IV subunit-5A, thereby reducing mitochondrial complex-IV activity, respiration and ROS production. Notably, targeting S70pBcl2 with the phosphatase activator, FTY720, is accompanied by an enhanced drug-induced DNA damage and cell death in CLL primary cells. Collectively, we provide a novel facet of the anti-apoptotic Bcl-2 by demonstrating that its phosphorylation at serine-70 functions as a redox sensor to prevent drug-induced oxidative stress-mediated DNA damage and execution with potential therapeutic implications.     

Constitutive Reactive Oxygen Species Generation from Autophagosome/Lysosome in Neuronal Oxidative Toxicity

Reactive oxygen species (ROS) are involved in several cell death processes, including cerebral ischemic injury. We found that glutamate-induced ROS accumulation and the associated cell death in mouse hippocampal cell lines were delayed by pharmacological inhibition of autophagy or lysosomal activity. Glutamate, however, did not stimulate autophagy, which was assessed by a protein marker, LC3, and neither changes in organization of mitochondria nor lysosomal membrane permeabilization were observed. Fluorescent analyses by a redox probe PF-H₂TMRos revealed that autophagosomes and/or lysosomes are the major sites for basal ROS generation in addition to mitochondria. Treatments with inhibitors for autophagy and lysosomes decreased their basal ROS production and caused a burst of mitochondrial ROS to be delayed. On the other hand, attenuation of mitochondrial activity by serum depletion or by high cell density culture resulted in the loss of both constitutive ROS production and an ROS burst in mitochondria. Thus, constitutive ROS production within mitochondria and lysosomes enables cells to be susceptible to glutamate-induced oxidative cytotoxicity. Likewise, inhibitors for autophagy and lysosomes reduced neural cell death in an ischemia model in rats. We suggest that cell injury during periods of ischemia is regulated by ROS-generating activity in autophagosomes and/or lysosomes as well as in mitochondria.     

Potentiation of Fas-mediated apoptosis by attenuated production of mitochondria-derived reactive oxygen species

The role of reactive oxygen species (ROS) production in death receptor-mediated apoptosis is ill-defined. Here, we show that ROS levels play a role in moderating Fas-dependent apoptosis. Treatment of Jurkat T cells with oligomycin (ATP-synthase inhibitor) or (mitochondrial uncoupler) and Fas-activating antibody (CH11) facilitated rapid cell death that was not associated with decreased ATP production or increased DEVDase activity and cytochrome c release. However, a decrease in cellular ROS production was associated with CH11 treatment, and combinations of CH11 with oligomycin or FCCP further inhibited cellular ROS production. Thus, decreased ROS production is correlated with enhanced cell death. A transition from state 3 to state 4 mitochondrial respiration accounted for the attenuated ROS production and membrane potential. Similar observations were demonstrated in isolated rat liver mitochondria. These data show that ROS production is important in receptor-mediated apoptosis, playing a pivotal role in cell survival.     

Arachidonic acid promotes glutamate-induced cell death associated with necrosis by 12- lipoxygenase activation in glioma cells

Glutamate induced glutathione (GSH) depletion in C6 rat glioma cells, which resulted in cell death. This cell death seemed to be apoptosis through accumulation of reactive oxygen species (ROS) or hydroperoxides representing cytochrome c release from mitochondria and internucleosomal DNA fragmentation. A significant increase of 12-lipoxygenase enzyme activity was observed in the presence of arachidonic acid (AA) under GSH depletion induced by glutamate. AA promoted the glutamate-induced cell death, which reduced caspase-3 activity and diminished internucleosomal DNA fragmentation. Furthermore, AA reduced intracellular NAD, ATP and membrane potentials, which indicated dysfunction of the mitochondrial membrane. Protease inhibitors such as N-alpha-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and 3, 4-dichloroisocumarin (DCI) but no Ac-DEVD, a caspase inhibitor, suppressed the glutamate-induced cell death. AA reduced the inhibitory effect of TPCK and DCI on the glutamate-induced cell death. These results suggest that AA promotes cell death by inducing necrosis from caspase-3-independent apoptosis. This might occur through lipid peroxidation initiated by ROS or lipid hydroperoxides generated during GSH depletion in C6 cells.     

Selenoprotein S silencing triggers mouse hepatoma cells apoptosis and necrosis involving in intracellular calcium imbalance and ROS-mPTP-ATP

Selenoprotein S (SelenoS) is one of the cellular endoplasmic reticulum (ER) and membrane located selenoproteins, and it has the main functions of anti-oxidation, anti-apoptosis and anti-ER stress. To investigate the effect of SelenoS silencing on mouse hepatoma cell death and the intracellular biological function of SelenoS, we knocked down SelenoS in Hepa1-6 cells, and detected ER stress, intracellular calcium homeostasis, mitochondrial dynamics, apoptosis and necrosis. To further explore whether reactive oxygen species (ROS) has an effect on apoptosis and necrosis under SelenoS silencing, we used NAC (2.5?mM) to pretreat cells, and detected ΔΨm, ATP, and apoptosis and necrosis rates. SelenoS silencing broke the intracellular calcium homeostasis, induced mitochondrial dynamic disorder, ROS accumulation, loss of ΔΨm and ATP, and triggered apoptosis and necrosis in mouse hepatoma cells. The clearance of ROS alleviated the loss of ΔΨm and ATP caused by silencing of SelenoS, reduced cell necrosis and increased apoptosis. However, SelenoS silencing did not cause ER stress in Hepa1-6 cells. These results indicate that SelenoS silencing triggers mouse hepatoma cells apoptosis and necrosis through affecting intracellular calcium homeostasis and ROS-mPTP-ATP participates in cell death transformation from apoptosis to necrosis to rise damage.     

Mitochondrial metabolism underlies hyperoxic cell damage

Exposure of mammals to hyperoxia causes pulmonary and ocular pathology. Hyperoxic damage and cell death may derive from enhanced intracellular formation of reactive oxygen species (ROS), probably of mitochondrial origin. There is, however, controversy on this point. When wild-type and respiration-deficient (rho(o)) HeLa cells were cultured in 80% O2, wild-type cells stopped growing after 5 days and died thereafter whereas rho(o) cells survived and grew to confluence. This tolerance of rho(o) cells for hyperoxia was not associated with greater resistance to oxidants such as hydrogen peroxide and t-butyl hydroperoxide. Under both 20% and 80% O2, rho(o) cells exhibited substantially decreased ROS production, and, under 80% O2, rho(o) cells showed no suppression of aconitase activity or mitochondrial protein carbonyl formation. Replacement of normal mitochondria in rho(o) cells restored ROS production and susceptibility to hyperoxia. Two other approaches that diminished mitochondrial ROS generation also increased tolerance for hyperoxia. HeLa cells constantly exposed to the protonophoric uncoupler carbonyl cyanide m-chlorophenylhydrazone, which enhances respiration but decreases ROS production, showed preferential survival under 80% O2, as did HeLa cells treated with chloramphenicol, which suppresses both respiration and mitochondrial ROS production. We conclude that interactions between respiring mitochondria and O2 are primarily responsible for hyperoxic cell damage.     

Rat cerebellar granule cells are protected from glutamate-induced excitotoxicity by S-nitrosoglutathione but not glutathione

In cultured rat cerebellar granule cells, glutamate or N-methyl-D-aspartate (NMDA) activation of the NMDA receptor caused a sustained increase in cytosolic Ca²⁺ levels ([Ca²⁺]_i), reactive oxygen species (ROS) generation, and cell death (respective EC₅₀ values for glutamate were 12, 30, and 38 µM) but no increase in caspase-3 activity. Removal of extracellular Ca²⁺ blocked all three glutamate-induced effects, whereas pretreatment with an ROS scavenger inhibited glutamate-induced cell death but had no effect on the [Ca²⁺]_i increase. This indicates that glutamate-induced cell death is attributable to [Ca²⁺]_i increase and ROS generation, and the [Ca²⁺]_i increase precedes ROS generation. Apoptotic cell death was not seen until 24 h after exposure of cells to glutamate. S-nitrosoglutathione abolished glutamate-induced ROS generation and cell death, and only a transient [Ca²⁺]_i increase was seen; similar results were observed with another nitric oxide (NO) donor, S-nitroso-N-acetylpenicillamine, but not with glutathione, which suggests that the effects were caused by NO. The transient [Ca²⁺]_i increase and the abolishment of ROS generation induced by glutamate and S-nitrosoglutathione were still seen in the presence of an ROS scavenger. Glial cells, which were present in the cultures used, showed no [Ca²⁺]_i increase in the presence of glutamate, and glutamate-induced granule cell death was independent of the percentage of glial cells. In conclusion, NO donors protect cultured cerebellar granule cells from glutamate-induced cell death, which is mediated by ROS generated by a sustained [Ca²⁺]_i increase, and glial cells provide negligible protection against glutamate-induced excitotoxicity. cytosolic calcium concentration; N-methyl-D-aspartate; reactive oxygen species     

Taurine as a Modulator of Excitatory and Inhibitory Neurotransmission

We present data that summarize our findings on the role of taurine in the central nervous system and in particular taurine's interaction with the inhibitory and excitatory systems. In taurine-fed mice, the expression level of glutamic acid decarboxylase (GAD), the enzyme responsible for GABA synthesis, is elevated. Increased expression of GAD was accompanied by increased levels of GABA. We also found in vitro, that taurine regulates neuronal calcium homeostasis and calcium-dependent processes, such as protein kinase C (PKC) activity. This calcium-dependent kinase was regulated by taurine, whereas the activity of protein kinase A (PKA), a cAMP-dependent, calcium-independent kinase, was not affected. Furthermore, as a consequence of calcium regulation, taurine counteracted glutamate-induced mitochondrial damage and cell death.