Stimulation by Gangliosides of Viability of Rat Brain Neurons and of Neuronal PC12 Cell Line under Conditions of Oxidative Stress

We studied effect of gangliosides on viability of brain neurons and neuronal PC12 cell line exposed to toxic concentrations of compounds activating free radical reactions. It is found that preincubation of cerebellar granule cells and PC12 cells with micromolar concentrations of ganglioside GM1 increases statistically significantly viability of these cells submitted to inductors of oxidative stress, such as hydrogen peroxide and the Fe²⁺-ascorbate system However, the effect of ganglioside GM1 in the PC12 cells failed to be revealed 1–2 days after treatment of the cells with trypsin, which indicates an importance of interaction of gangliosides with surface proteins for realization of their protective action. GM1, GD1a, and other gangliosides were shown to produce the neuroprotective effect on cerebellar granule cells in the presence of toxic glutamate concentrations. Not only micro-, but also nanomolar concentrations of these gangliosides increased statistically significantly the neuronal viability, although at micromolar concentrations this effect as a rule was more pronounced. The obtained data allow suggesting that the neuroprotective action of gangliosides is determined to a considerable degree by their ability to inhibit free-radical reactions in nerve cells.     

Developmental changes in gangliosides in cultured cerebellar granule neurons

The content and composition of gangliosides in cultures enriched in granule neurones and in astrocytes from rat cerebellum (P6–8) showed marked differences; astrocytes contained less than 10% of the amount of granule neurones and the profile was dominated by simple gangliosides with lactosyl ceramide backbone, while gangliosides of the b series, which constitute about 40% in nerve cells, were virtually undetectable. Granule cell maturation was accompanied by a 16-fold increase in the ganglioside content during the initial 8 days in a serum-supplemented medium (S⁺), reaching a plateau much earlier and at a higher level than observed in the cerebellum in vivo. Developmental changes were characterized, as in vivo, by a pronounced decrease in the GD3 proportion and an increase in the b series of gangliosides. Compared with S⁺, adhesion among cells and fibres is different in a serum-free medium (S^–) in which the rise in cellular ganglioside content was less (30%) but the developmental changes in ganglioside profile were similar. However, in cultures in S^– only, GM3 was not detectable, while the distribution of GM1 and GD3 indicated that maturation is retarded relative to cells in S⁺. Surface exposure of gangliosides (studied by the periodate/[³H]borohydride method) was similar under the two culture conditions. There was an initial delay, especially in S^–, in the insertion of gangliosides into the plasma membrane, while the labelling of GD3 (the dominant ganglioside of immature granule cells) was very low compared with all the other species throughout the whole cultivation time.Special issue dedicated to Dr. Frederick E. Samson.     

Functional relationship between ammonia and gangliosides in brain

The functional significance of ammonia production in brain under physiological or pathological conditions is not clearly known. NH₄ ⁺ stimulates Na⁺, K⁺ activated ATPase causing stabilization of neuronal membranes of which gangliosides are major structural components. Moreover ammonia is known to inhibit lysosomal enzymes which include enzymes degrading gangliosides. Gangliosides have been shown to stimulate neuritogenesis in neuronal cultures and prevent the damage of the neurons from glutamate toxicity particularly in areas of brain ischemia. Hyperammonemia without any behavioural changes was induced in experimental rats by intraperitoneal administration of either a single dose (0.8 mmol/100 g wt.) or by six hourly doses (0.6 mmol/100 g wt.) of ammonium acetate. An increase in the content of gangliosides along with a rise in the content of GD_1A and GD_1B without any change in -galactosidase and N-acetylhexosaminidase was observed in cerebral cortex, cerebellum, and brain stem, following the administration of single dose of ammonium acetate. Gangliosides, after extraction from the different brain regions, were estimated by the thiobarbituric acid method and expressed in terms of sialic acid. Individual gangliosides were separated and estimated by thin layer chromatography using resorcinol as the staining agent. These results suggest that ammonia production in the neuronal pathways in brain either as a result of repeated stimulation under physiological conditions or as a result of focal ischemia or injury, may likewise cause an increase in the content of gangliosides which may help in neuritic growth (physiological conditions facilitating synaptic plasticity) and may exert a protective effect on the neurons in the ischemic area against glutamate toxicity.Former Professor of Biochemistry, OMC, Hyderabad.     

Effects of oxidative stress inhibitors, neurotoxins, and ganglioside GM1 on Na+,K+-ATPase activity in PC12 Cells and brain synaptosomes

To elucidate mechanism of ganglioside neuroprotection, it is important to study their metabolic effects, specifically of action on Na⁺,K⁺-ATPase. It has been shown that under effect of oxidative stress inductors and neurotoxins an oxidative inactivation of this enzyme takes place in PC12 cells and brain cortex synaptosomes, this inactivation being able to be prevented or decreased by ganglioside GM1. Thus, for instance, 24 h after action of 1 mM H₂O₂, activity of Na⁺,K⁺-ATPase in PC12 cells decreased more than twice. However, in the case of preincubation of the cells with ganglioside GM1 prior to the H₂O₂ action, this enzyme activity did not differ statistically significantly from control. Ganglioside GM1 also was able to increase statistically significantly the enzyme activity decreased by action on the PC12 cells of amyloid β-peptide (Aβ) causing lesion of neurons in Alzheimer’s disease and of low H₂O₂ concentrations. Experiments on brain cortex synaptosomes have established that not only antioxidants—α-tocopherol and superoxide dismutase (SOD)—but also ganglioside GM1 prevent the glutamate-produced Na⁺,K⁺-ATPase oxidative inactivation. The obtained data agree with a suggestion that the ganglioside neuroprotective effect at action on nerve cells of such toxins as Aβ, glutamate or reactive oxygen species is due to their ability to inhibit the free-radical reactions.     

Metabolic effects of ganglioside GM1 on PC12 cells in oxidative stress depend on modulation of activity of tyrosine kinase Trk of receptors

There have been obtained evidences that not only GM1, but also other main brain gangliosides (GD1a, GD1b, and GT1b) increase viability of cells of the neuronal line PC12 under action of H₂O₂. By the example of GM1 and GD1a, gangliosides have been shown to produce a protective effect on PC12 cells under conditions of oxidative stress both at micro- and nanomolar concentrations that are physiological concentrations of gangliosides in cerebrospinal fluid. For the first time, GM1 at nanomolar concentrations was shown to decrease the H₂O₂-induced formation of reactive oxygen species (ROS). It was found that in the presence of inhibitor of tyrosine kinase Trk of receptors K-252a, GM1 at concentrations of 10 μM and 10 nM lost its ability to produce such metabolic effects as a decrease of ROS accumulation and of the degree of oxidative inactivation of Na⁺,K⁺-ATPase in PC12 cells, as well as ceased to increase viability of these cells under conditions of oxidative stress. The dependence of protective and metabolic effects of gangliosides GM1 in PC12 cells treated with H₂O₂ on modulation of activity of activity of tyrosine kinase Trk receptors (i.e., from the same signal system) agrees with concept about the essential role of oxidant effect of GM1 in its increase of cell viability.     

Effect of Glutamate and Inhibitors of Various Pathways of Free Radical Production on Na+,K+-ATPase Activity and Accumulation of Lipids Peroxidation Products in Rat Brain Cortex Synaptosomes

It has been shown that treatment of the rat brain cortex synaptosomes with glutamate produced both a significant reduction in Na⁺,K⁺-ATPase activity and accumulation of products of lipid peroxidation (LPO) like malone dialdehyde, dienoic conjugates, and Schiff bases. A suppression of different routes of free radical production in cytosol by quinacrine, indomethacin, and allopurinol (blockers of phospholipase A₂, cyclooxygenases, and xanthine oxidases, respectively) as well as by MK-801 (a antagonist of MDA-receptors) prevented or lowered significantly the effect of glutamate on Na⁺,K⁺-ATPase activity. No significant effect of glutamate on the Na⁺,K⁺-ATPase activity was also observed in the presence of L-NAME (inhibitor of NO-synthase). Inhibitors of the arachidonate and NO-synthase pathway of free radical production also prevented accumulation of LPO end products in the rat brain cortex under the effect of glutamate. In the presence of rotenone and olygomycin (blockers of mitochondrial electron transport and ATP synthase, respectively), glutamate led to even a greater inactivation of Na⁺,K⁺-ATPase and accumulation of malone dialdehyde. The data obtained suggest that at early stages of ischemia the neurotoxic effect of glutamate is due to an inflow of calcium ions through NMDA receptors and activation of different pathways of free radical production in cytosol of nerve cells. At these stages, protective functions of mitochondria appear to predominate due to their ability to accumulate calcium ions and to prevent an excessive increase of the cytosol calcium concentration under the effect of excitatory amino acids.     

Gangliosides Attenuate Ethanol-Induced Apoptosis in Rat Cerebellar Granule Neurons

Ethanol significantly enhances cell death of differentiated rat cerebellar granule neurons on culture in a serum-free medium containing a depolarizing concentration of KCl (25 mM), 5 M MK-801 (an NMDA receptor antagonist), and 20–200 mM ethanol for 1–4 days. Cell death augmented by ethanol was concentration- and time-dependent with neurons displaying hallmark apoptotic morphology and DNA fragmentation that correlated with the activation of cytosolic caspase-3. Inclusion of 5 M MK-801 or 100 M glycine in culture media did not alter rates of cell death indicating ethanol toxicity is mediated via an NMDA receptor-independent pathway. Preincubation with 50 M gangliosides GM1, GD1a, GD1b or GT1b for 2 h, or preincubation with 10 M LIGA₂₀ (a semisynthetic GM1 with N-dichloroacetylsphingosine) for 10 min, attenuated caspase-3 activity and ethanol-induced cell death. Data show native gangliosides and a synthetic derivative are potently neuroprotective in this model of ethanol toxicity, and potentially serve as useful probes to further unravel the mechanisms relevant to neuronal apoptosis.     

Mechanistic study of modulation of SR Ca2+-ATPase activity by gangliosides GM1 and GM3 through some biophysical measurements

On the basis of confirming the antagonistic effects of GM1 and GM3 on the activity of Ca²⁺-ATPase, we further demonstrated that some of the components of these two gangliosides, including sialic acid (NeuNAc), asialo-GM1, asialo-GM3 and ceramide, failed to show any effects on the activity of Ca²⁺-ATPase. Thus it is apparent that the intact molecules of these two gangliosides with their specific conformations were needed to perform their effects on Ca²⁺-ATPase. From the fluorescence resonance energy transfer measurements, the energy transfer between Cys 670/674 and Lys 515 was decreased by GM1 and increased by GM3, indicating GM1 induced the conformation of the hydrophilic region of Ca²⁺-ATPase to be less compact, while GM3 induced it to be more compact. From the CD spectra measurements, GM1 and GM3 both reduced the content of -helical structures of Ca²⁺-ATPase, but GM1 caused a stronger decrease than that of GM3. Using DPH as the probe, we found that the membrane lipid fluidity of the proteoliposomes containing Ca²⁺-ATPase was decreased by GM1 and tend to increase by GM3.     

Interaction of the B subunit of cholera toxin with endogenous ganglioside GM1 causes changes in membrane potential of rat thymocytes

Summary The fluorescent anionic dye, bisoxonol, and flow cytometry have been used to monitor changes in the membrane potential of rat thymocytes exposed to the B subunit of cholera toxin. The B subunit induced a rapid hyperpolarization, which was due to activation of a Ca²⁺-sensitive K⁺ channel. Reduction of extracellular Ca²⁺ to <1 m by the addition of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid immediately abolished the hyperpolarization caused by the B subunit. Cells treated with quinine and tetraethylammonium lost their ability to respond to the B subunit, whereas 4-aminopyridine did not have any effect. Thus, calcium-sensitive and not voltage-gated K⁺ channels appeared to be responsible for the hyperpolarization. The results of ion substitution experiments indicated that extracellular Na⁺ was not essential for changes in membrane potential. Further studies with ouabain, amiloride and furosemide demonstrated that electrogenic Na⁺/K⁺ ATPase, Na⁺/H⁺ antiporter and Na⁺/K⁺/Cl^– cotransporter, respectively, were not involved in the hyperpolarization process induced by the B subunit. Thus, crosslinking of several molecules of ganglioside GM1 on the cell surface of rat thymocytes by the pentavalent B subunit of cholera toxin modulated plasma membrane permeability to K⁺ by triggering the opening of Ca²⁺-sensitive K⁺ channels. A role for gangliosides in regulating ion permeability would have important implications for the function of gangliosides in various cellular phenomena.     

Glutamate Neurotoxicity Is Independent of Calpain I Inhibition in Primary Cultures of Cerebellar Granule Cells

Glutamate-induced neurotoxicity and calpain activity were studied in primary cultures of rat cerebellar granule neurons and glial cells. Calpain activation, as monitored by quantitative immunoblotting of spectrin, required micromolar concentrations of Ca2+ in neuronal homogenates (calpain I) and millimolar Ca2+ concentrations in glial homogenates (calpain II). Glutamate-induced toxicity and calpain activation were observed in neuronal, but not in glial, cultures. In neurons, calpain I activation by glutamate was dose-dependent and persisted after withdrawal of neurotoxic doses of glutamate. Natural (GM1) and semisynthetic (LIGA4) gangliosides or the glutamate receptor blocker MK-801 prevented calpain I activation and delayed neuronal death elicited by glutamate. GM1 and LIGA4 had no effect on calpain I activity in neuronal homogenates, however. Furthermore, two calpain I inhibitors (leupeptin and N-acetyl-Leu-Leu-norleucinal) prevented glutamate-induced spectrin degradation, but failed to affect glutamate neurotoxicity. These results thus suggest that glutamate-induced neurotoxicity is independent of calpain I activation.     

Developmental Changes in Ganglioside Composition and Synthesis in Embryonic Rat Brain

Developmental changes in ganglioside composition and biosynthesis was studied in rat brain between embryonic day (E) 14 and birth. In E14 brains, GM3 and GD3 were predominant. At E16, "b" series gangliosides, such as GD1b, GT1b, and GQ1b, increased in content. After E18, "a" series gangliosides such as GM1, GD1a, and GT1a increased in content, and the content of GM3 and GD3 markedly decreased. Because of these changes in composition, we determined the activities, in homogenates of embryonic brains, of two key enzymes of ganglioside synthesis: sialyltransferase for the synthesis of GD3 from GM3 and N-acetylgalactosaminyltransferase for GM2 synthesis from GM3. The sialyltransferase activity (GM3----GD3) was constant between E14 and E18 but decreased rapidly from E18 to birth. In contrast, the N-acetylgalactosaminyltransferase activity (GM3----GM2) increased between E14 and E18 but was constant from E18 to birth. These changes in ganglioside composition and enzymatic activities indicate that during development there is a shift from synthesis of the simplest gangliosides of the "a" and "b" pathways to synthesis of the more complex gangliosides.     

Inhibition of Na+, K+-ATPase activity by δ-aminolevulinic acid

-Aminolaevulinic acid (ALA) has been shown to be toxic to cultured neurons and glia at concentrations as low as 10 M. In an attempt to elucidate the mechanism of toxicity, the effects of ALA on membrane ATPase activity were investigated. Exposure of neuron cultures to 1 mM ALA for 7 days caused a substantial decrease in both Na⁺, K⁺-ATPase and Mg²⁺-ATPase activities. At lower concentrations, ALA affected only the Na⁺, K⁺-component. ALA appeared to act directly, inhibiting Na⁺, K⁺-ATPase activity in rat brain cortex membrane preparations at 10 M Although this effect was slight, it may well represent the mechanism of action of ALA, since ouabain, a potent inhibitor of Na⁺, K⁺-ATPase activity, proved to be more toxic to cultured neurons than ALA. Furthermore, cardiac glycoside overdosage causes neurological disturbances which are very similar to those observed in the acute attack of porphyria.     

Effects of gangliosides on the activity of the plasma membrane Ca-ATPase

Control of intracellular calcium concentrations ([Ca^2 +]_i) is essential for neuronal function, and the plasma membrane Ca^2 +-ATPase (PMCA) is crucial for the maintenance of low [Ca^2 +]_i. We previously reported on loss of PMCA activity in brain synaptic membranes during aging. Gangliosides are known to modulate Ca^2 + homeostasis and signal transduction in neurons. In the present study, we observed age-related changes in the ganglioside composition of synaptic plasma membranes. This led us to hypothesize that alterations in ganglioside species might contribute to the age-associated loss of PMCA activity. To probe the relationship between changes in endogenous ganglioside content or composition and PMCA activity in membranes of cortical neurons, we induced depletion of gangliosides by treating neurons with d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (d-PDMP). This caused a marked decrease in the activity of PMCA, which suggested a direct correlation between ganglioside content and PMCA activity. Neurons treated with neuraminidase exhibited an increase in GM1 content, a loss in poly-sialoganglioside content, and a decrease in PMCA activity that was greater than that produced by d-PDMP treatment. Thus, it appeared that poly-sialogangliosides had a stimulatory effect whereas mono-sialogangliosides had the opposite effect. Our observations add support to previous reports of PMCA regulation by gangliosides by demonstrating that manipulations of endogenous ganglioside content and species affect the activity of PMCA in neuronal membranes. Furthermore, our studies suggest that age-associated loss in PMCA activity may result in part from changes in the lipid environment of this Ca^2 + transporter.     

Analysis of glutamate receptors in primary cultured neurons from fetal rat forebrain

In order to further analyze the development of glutamatergic pathways in neuronal cells, the expression of excitatory amino acid receptors was studied in a model of neurons in primary culture by measuring the specific binding of L-[³H]glutamate under various incubation conditions in 8-day-old intact living neurons isolated from the embryonic rat forebrain, as well as in membrane preparations from these cultures and from newborn rat forebrain. In addition, the receptor responsiveness to glutamate was assessed by studying the uptake of tetraphenylphosphonium (TPP⁺) which reflects membrane polarization. In the presence of a potent inhibitor of glutamate uptake, the radioligand bound to a total number of sites of 36.7 pmol/mg protein in intact cells incubated in a Tris buffer containing Na⁺, Ca²⁺, and Cl^–, with a Kd around 2 M. In the absence of the above ions, [³H]glutamate specific binding diminished to 14.2 pmol/mg protein with a Kd-value of 550 nM. Under both of the above conditions, similar Kd were obtained in membranes isolated from cultures and from the newborn brain. However, Bmax-values were significantly lower in culture membranes than in intact cells or newborn membranes. Displacement studies showed that NMDA was the most potent compound to inhibit [³H]glutamate binding in membranes obtained from cultured neurons as well as from the newborn brain, whereas quisqualate, AMPA, kainate andtrans-ACPD were equally effective. According to these data and to the ionic dependence of glutamate binding, it was concluded that cultured neurons from the rat embryo forebrain express various glutamate receptor subtypes, mainly L-AP4 and NMDA receptors, with characteristics close to those in the newborn brain, and which display functional properties since a transient cell exposure to glutamate led to a 70% inhibition of [³H]TPP⁺ uptake.     

Berberine Inhibits the Release of Glutamate in Nerve Terminals from Rat Cerebral Cortex

Berberine, an isoquinoline plant alkaloid, protects neurons against neurotoxicity. An excessive release of glutamate is considered to be one of the molecular mechanisms of neuronal damage in several neurological diseases. In this study, we investigated whether berberine could affect endogenous glutamate release in nerve terminals of rat cerebral cortex (synaptosomes) and explored the possible mechanism. Berberine inhibited the release of glutamate evoked by the K⁺ channel blocker 4-aminopyridine (4-AP), and this phenomenon was prevented by the chelating extracellular Ca²⁺ ions and the vesicular transporter inhibitor bafilomycin A1, but was insensitive to the glutamate transporter inhibitor DL-threo-beta-benzyl-oxyaspartate. Inhibition of glutamate release by berberine was not due to it decreasing synaptosomal excitability, because berberine did not alter 4-AP-mediated depolarization. The inhibitory effect of berberine on glutamate release was associated with a reduction in the depolarization-induced increase in cytosolic free Ca²⁺ concentration. Involvement of the Ca_v2.1 (P/Q-type) channels in the berberine action was confirmed by blockade of the berberine-mediated inhibition of glutamate release by the Ca_v2.1 (P/Q-type) channel blocker ω-agatoxin IVA. In addition, the inhibitory effect of berberine on evoked glutamate release was prevented by the mitogen-activated/extracellular signal-regulated kinase kinase (MEK) inhibitors. Berberine decreased the 4-AP-induced phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and synapsin I, the main presynaptic target of ERK; this decrease was also blocked by the MEK inhibition. Moreover, the inhibitory effect of berberine on evoked glutamate release was prevented in nerve terminals from mice lacking synapsin I. Together, these results indicated that berberine inhibits glutamate release from rats cortical synaptosomes, through the suppression of presynaptic Cav2.1 channels and ERK/synapsin I signaling cascade. This finding may provide further understanding of the mode of berberine action in the brain and highlights the therapeutic potential of this compound in the treatment of a wide range of neurological disorders.     

Changes in developmental profiles of brain gangliosides during ontogeny of a teleost fish (Sarotherodon mossambicus,Cichlidae)

Summary Ontogenetic changes of brain ganglioside concentration and composition have been followed in the teleost fishSarotherodon mossambicus Cichlidae) from the 1st day post hatching to the adult stage, in order to correlate these with findings in higher vertebrates. During the developmental period from hatching to the transition to free swimming, which comes along with maximum rate of synaptogenesis, a sharp rise in the brain ganglioside content occurs, which is mainly due to the trisialoganglioside GT1b. In the following phase of myelination (characterized in birds and mammals by an increase in GM1, GM1 and GM4) accretion of the novel and so far unidetified fraction (GM2) occurs, which is highly enriched in the brain stem. The results obtained are discussed with respect to gangliosides as useful biochemical markers for brain development and maturation in all vertebrates.     

Environmental Estrogen-Like Chemicals and Hydroxyl Radicals Induced by MPTP in the Striatum: A Review

Oxygen free radical formation has been implicated in lesions caused by the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and iron. Although MPTP produces a parkinsonian syndrome after its conversion to 1-methyl-4-phenylpyridine (MPP⁺) by type B monoamine oxidase (MAO) in the brain, the etiology of this disease remains obscure. This review focuses on the role of an environmental neurotoxin chemically related to MPP⁺-induced free radical generation in the pathogenesis of Parkinson's disease. Environmental-like chemicals, such as para-nonylphenol or bisphenol A, significantly stimulated hydroxyl radical (OH) formation in the striatum. Allopurinol, a xanthine oxidase inhibitor, prevents para-nonylphenol and MPP⁺-induced OH generation. Tamoxifen, a synthetic nonsteroidal antiestrogen, suppressed the OH generation via dopamine efflux induced by MPP⁺. These results confirm that free radical production might make a major contribution at certain stages in the progression of the injury. Such findings may be useful in elucidating the actual mechanism of free radical formation in the pathogenesis of neurodegenerative brain disorders, including Parkinson's disease and traumatic brain injuries.     

Isolation of three novel cholinergic neuron-specific gangliosides from bovine brain and theirin vitro syntheses

In the present study, three extremely minor but novel Chol-1 antigens, termed X1, X2, and X3 have been isolated from bovine brain gangliosides. Based on the results of sialidase degradation, TLC-immunostaining with anti-Chol-1 antibody and fast atom bombardment mass spectrometry, their chemical structures were identified as: $$\begin{gathered} III^6 NeuAc--GgOse4Cer (X1:GM1\alpha ) \hfill \\ III^6 NeuAc,II^3 NeuAc--GgOse4Cer (X2:GT1a\alpha ) \hfill \\ III^6 NeuAc,II^3 NeuAc--NeuGc--GgOse4Cer (X3:GT1b\alpha ) \hfill \\ \end{gathered} $$ The yields of GM1α, GD1aα, and GT1bα, were approximately 150, 20, and 10 µg, respectively, from 10 g of the bovine brain ganglioside mixture. In conjunction with our previous observations, all gangliosides with anti-Chol-1 reactivity were found to contain a common sialyl α2–6N-acetylgalactosamine residue, indicating that this unique sialyl linkage is the specific antigenic determinant. We subsequently examined the biosyntheses of the three novel Chol-1 gangliosides using rat liver Golgi fraction as an enzyme source. The results showed that GM1α, GD1aα, and GT1bα were synthesized from asialo-GM1, GM1a, and GD1b, respectively, by the action of a GalNAc α2-6sialyltransferase.     

Gangliosides GM1 and GD1a normalize respiratory rates of rat brain mitochondria reduced by <Emphasis Type="Italic">tert</Emphasis>-butyl hydroperoxide

The rate of oxygen consumption by glutamate- and malate-energized rat brain mitochondria, which was stimulated by an uncoupler 2,4-dinitrophenol (DNP), declined in the presence of a prooxidant tert-butyl hydroperoxide. Preincubation with gangliosides GM1 or GD1a at micromolar (but not nanomolar) concentrations significantly slowed down this decline in the mitochondrial respiration, as shown by measuring absolute respiratory rates and ratios of the mitochondrial respiratory rate in the presence of DNP to the basal respiratory rate (V _DNP/V ₀). Gangliosides GM1 and GD1a also slowed down a decline in the DNP-stimulated mitochondrial respiration induced by long-term incubation (“aging”) of mitochondria on ice. The data obtained are likely to reflect a prooxidant-induced reduction in the activity of enzymes of the mitochondrial respiratory chain as well as a GM1- and GD1a-induced decrease in the degree of their inactivation. Interestingly, in the presence of the Trk receptor tyrosine kinase inhibitor (K252a) this effect of gangliosides was not manifested in any way. Our data suggest that the direct impact of gangliosides on mitochondrial signaling pathways, specifically on the Trk receptor tyrosine kinase, plays a certain role in the mechanism of their protective effect on cerebral neurons and, probably, neuroglia.     

Taurine Release Is Enhanced in Cell-Damaging Conditions in Cultured Cerebral Cortical Astrocytes

The release of preloaded [³H]taurine from cultured cerebral cortical astrocytes was studied under various cell-damaging conditions, including hypoxia, ischemia, aglycemia and oxidative stress, and in the presence of free radicals. Astrocytic taurine release was enhanced by K⁺ (50 mM), veratridine (0.1 mM) and the ionotropic glutamate receptor agonist kainate (1.0 mM). Metabotropic glutamate receptor agonists had only weak effects on taurine release. Similarly to the swelling-induced taurine release the efflux in normoxia seems to be mediated mainly by DIDS-(diisothiocyanostilbene-2,2-disulphonate) and SITS-(4-acetamido-4-isothiocyanostilbene-2,2-disulphonate) sensitive CI^– channels, since these blockers were able to reduce both basal and K⁺ -stimulated release. The basal release of taurine was moderately enhanced in hypoxia and ischemia, whereas the potentiation in the presence of free radicals was marked. The small basal release from astrocytes signifies that taurine release from brain tissue in ischemia may originate from neurons rather than glial cells. On the other hand, the release evoked by K⁺ in hypoxia and ischemia was greater than in normoxia, with a very slow time-course. The enhanced release of the inhibitory amino acid taurine from astrocytes in ischemia may be beneficial to surrounding neurons, outlasting the initial stimulus and counteracting overexcitation.