Maike M. Schmidt

Differential effects of iodoacetamide and iodoacetate on glycolysis and glutathione metabolism of cultured astrocytes

Iodoacetamide (IAA) and iodoacetate (IA) have frequently been used to inhibit glycolysis, since these compounds are known for their ability to irreversibly inhibit the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). However, the consequences of a treatment with such thiol reagents on the glutathione (GSH) metabolism of brain cells have not been explored. Exposure of astroglia-rich primary cultures to IAA or IA in concentrations of up to 1 mM deprived the cells of GSH, inhibited cellular GAPDH activity, lowered cellular lactate production and caused a delayed cell death that was detectable after 90 min of incubation. However, the two thiol reagents differed substantially in their potential to deprive cellular GSH and to inhibit astrocytic glycolysis. IAA depleted the cellular GSH content more efficiently than IA as demonstrated by half-maximal effects for IAA and IA that were observed at concentrations of about 10 and 100 μM, respectively. In contrast, IA was highly efficient in inactivating GAPDH and lactate production with half-maximal effects observed already at a concentration below 100 μM, whereas IAA had to be applied in 10 times higher concentration to inhibit lactate production by 50%. These substantial differences of IAA and IA to affect GSH content and glycolysis of cultured astrocytes suggest that in order to inhibit astrocytic glycolysis without substantially compromising the cellular GSH metabolism, IA – and not IAA – should be used in low concentrations and/or for short incubation periods.

Zinc prevents the copper-induced damage of cultured astrocytes

Copper is essential for several cellular processes, but an excess of cellular copper is known to be cell toxic. To study the consequences of a copper treatment of astrocytes, we have used astrocyte-rich primary cultures as model system to investigate cellular functions and cellular integrity of these cells after application of micromolar concentrations of copper chloride. After exposure of the cells to copper, the cell-associated copper content increased strongly in a time and concentration dependent manner. While incubation of cultured astrocytes with 3 microM copper hardly affected the cells during incubation for up to 4h, presence of 10 microM or 30 microM copper severly compromised cellular functions as demonstrated by a loss in total and soluble protein contents, a lowered MTT reduction capacity, lowered activities of the enzymes lactate dehydrogenase, glucose-6-phosphate dehydrogenase and glutathione reductase, a lowered cellular glutathione content, an increased lipid peroxidation, and an elevated membrane permeability for propidium iodide. Presence of an excess of zinc inhibited cellular copper accumulation and prevented most of the detrimental consequences of a copper exposure, suggesting that the beneficial effect of zinc against the copper-induced impairment of cultured astrocytes is mediated by inhibition of the cellular copper accumulation.

Fumaric acid diesters deprive cultured primary astrocytes rapidly of glutathione

Fumaric acid esters (FAE) are used for the systemic therapy of psoriasis and are now considered for the treatment of autoimmune-based neurological disorders such as multiple sclerosis. Currently, the cellular metabolism of FAE as well as the mechanisms of their therapeutic action are poorly understood. Since cellular glutathione (GSH) is involved in the detoxification of xenobiotics, we analysed the consequences of an application of FAE on the content of GSH in brain cells using astroglia-rich primary cultures as model system. Micromolar concentrations of dimethyl fumarate (DMF) or diethyl fumarate (DEF) lowered the cellular GSH content in a time- and concentration-dependent manner. Halfmaximal effects after 60 min of incubation were observed for 10 microM DMF or DEF. In contrast to the diesters, monomethyl fumarate (MMF), monoethyl fumarate (MEF) or fumarate had to be applied in concentrations of 10 mM for 60 min to significantly lower the cellular GSH content. During 60 min exposure, DMF or DEF did not significantly affect the cell viability, increase the cellular content of glutathione disulfide, nor altered the specific activities of glucose-6-phosphate dehydrogenase, glutathione reductase, or lactate dehydrogenase. After removal of DMF or DEF, cultured astrocytes restored their cellular GSH content completely within 4h. These data demonstrate that acute exposure to fumaric acid diesters deprives astrocytes of their GSH, most likely by the reaction of the reactive alpha,beta-unsaturated diesters with GSH.

Exploring Uncoupling Proteins and Antioxidant Mechanisms under Acute Cold Exposure in Brains of Fish

Exposure to fluctuating temperatures accelerates the mitochondrial respiration and increases the formation of mitochondrial reactive oxygen species (ROS) in ectothermic vertebrates including fish. To date, little is known on potential oxidative damage and on protective antioxidative defense mechanisms in the brain of fish under cold shock. In this study, the concentration of cellular protein carbonyls in brain was significantly increased by 38% within 1 h after cold exposure (from 28°C to 18°C) of zebrafish (Danio rerio). In addition, the specific activity of superoxide dismutase (SOD) and the mRNA level of catalase (CAT) were increased after cold exposure by about 60% (6 h) and by 60%–90% (1 and 24 h), respectively, while the specific glutathione content as well as the ratio of glutathione disulfide to glutathione remained constant and at a very low level. In addition, cold exposure increased the protein level of hypoxia-inducible factor (HIF) by about 50% and the mRNA level of the glucose transporter zglut3 in brain by 50%–100%. To test for an involvement of uncoupling proteins (UCPs) in the cold adaptation of zebrafish, five UCP members were annotated and identified (zucp1-5). With the exception of zucp1, the mRNA levels of the other four zucps were significantly increased after cold exposure. In addition, the mRNA levels of four of the fish homologs (zppar) of the peroxisome proliferator-activated receptor (PPAR) were increased after cold exposure. These data suggest that PPARs and UCPs are involved in the alterations observed in zebrafish brain after exposure to 18°C. The observed stimulation of the PPAR-UCP axis may help to prevent oxidative damage and to maintain metabolic balance and cellular homeostasis in the brains of ectothermic zebrafish upon cold exposure.

The antiretroviral protease inhibitors indinavir and nelfinavir stimulate Mrp1-mediated GSH export from cultured brain astrocytes

J. Neurochem. (2012) 120, 78–92.

Magnetic field-induced acceleration of the accumulation of magnetic iron oxide nanoparticles by cultured brain astrocytes.

Magnetic iron oxide nanoparticles (Fe-NPs) are considered for various biomedical and neurobiological applications that involve the presence of external magnetic fields. However, little is known on the effects of a magnetic field on the uptake of such particles by brain cells. Cultured brain astrocytes accumulated dimercaptosuccinate-coated Fe-NP in a time-, temperature-, and concentration-dependent manner. This accumulation was strongly enhanced by the presence of the magnetic field generated by a permanent neodymium iron boron magnet that had been positioned below the cells. The magnetic field-induced acceleration of the accumulation of Fe-NP increased almost proportional to the strength of the magnetic field applied, increasing the cellular-specific iron content from an initial 10 nmol/mg protein within 4 h of incubation at 37°C to up to 12,000 nmol/mg protein. However, presence of a magnetic field also increased the amounts of iron that attached to the cells during incubation with Fe-NP at 4°C. These results suggest that the presence of an external magnetic field promotes in cultured astrocytes both the binding of Fe-NP to the cell membrane and the internalization of Fe-NP.

Sustained hydrogen peroxide stress decreases lactate production by cultured astrocytes

Oxidative stress and disrupted energy metabolism are common to many pathological conditions of the brain. Because astrocytes play an important role in the glucose metabolism of the brain, we have investigated whether sustained oxidative stress affects astroglial glucose metabolism with cultured primary rat astrocytes as a model system. Cultured astrocytes were exposed to a sustained concentration of approximately 50 μM H2O2 in the presence of [U‐13C]glucose, and cellular and extracellular contents of lactate and glucose were analysed by enzymatic assays and NMR spectroscopy. Exposure of the cells to sustained H2O2 stress for up to 120 min significantly lowered the rate of lactate accumulation in the media to 61% ± 14% of that in cultures incubated without peroxide. In addition, the ratio of lactate release to glucose consumption was lowered in peroxide‐treated astrocytes to 77% ± 13% of that in control cells, and the specific activity of glyceraldehyde‐3‐phosphate dehydrogenase had declined to about 10% of control cells within 90 min. In addition, the 13C enrichment of intracellular and extracellular [13C]lactate was about 30% and 95%, respectively, and was not affected by the presence of peroxide, demonstrating that two metabolic pools of lactate are present in cultured astrocytes. The decreased rate of lactate production by astrocytes that have been exposed to peroxide stress is a new example of an alteration by oxidative stress of an important metabolic pathway in astrocytes. Such alterations could contribute to the pathological conditions that have been connected with oxidative stress and disrupted energy metabolism in the brain.

Size- and age-dependent changes in adductor muscle swimming physiology of the scallop Aequipecten opercularis.

SUMMARY The decline of cellular and especially mitochondrial functions with age is, among other causes, held responsible for a decrease in physiological fitness and exercise capacity during lifetime. We investigated size- and age-related changes in the physiology of exercising specimens of the short lived swimming scallop Aequipecten opercularis (maximum life span 8 to 10 years) from the Isle of Man, UK. A. opercularis swim mainly to avoid predators, and a decrease in swimming abilities would increase the risk of capture and lower the rates of survival. Bigger (older) individuals were found to have lower mitochondrial volume density and aerobic capacities (citrate synthase activity and adenylates) as well as less anaerobic capacity deduced from the amount of glycogen stored in muscle tissue. Changes in redox potential, tissue pH and the loss of glutathione in the swimming muscle during the exercise were more pronounced in young compared to older individuals. This indicates that older individuals can more effectively stabilize cellular homeostasis during repeated exercise than younger animals but with a possible fitness cost as the change in physiology with age and size might result in a changed escape response behaviour towards predators.

Glutamate induces release of glutathione from cultured rat astrocytes--a possible neuroprotective mechanism?

Glutamate is the major excitatory amino acid of the mammalian brain but can be toxic to neurones if its extracellular levels are not tightly controlled. Astrocytes have a key role in the protection of neurones from glutamate toxicity, through regulation of extracellular glutamate levels via glutamate transporters and metabolic and antioxidant support. In this study, we report that cultures of rat astrocytes incubated with high extracellular glutamate (5 mM) exhibit a twofold increase in the extracellular concentration of the tripeptide antioxidant glutathione (GSH) over 4 h. Incubation with glutamate did not result in an increased release of lactate dehydrogenase, indicating that the rise in GSH was not because of membrane damage and leakage of intracellular pools. Glutamate‐induced increase in extracellular GSH was also independent of de novo GSH synthesis, activation of NMDA and non‐NMDA glutamate receptors or inhibition of extracellular GSH breakdown. Dose–response curves indicate that GSH release from rat astrocytes is significantly stimulated even at 0.1 mM glutamate. The ability of astrocytes to increase GSH release in the presence of extracellular glutamate could be an important neuroprotective mechanism enabling neurones to maintain levels of the key antioxidant, GSH, under conditions of glutamate toxicity.

Metabolic and physiological responses in tissues of the long-lived bivalve Arctica islandica to oxygen deficiency

In Arctica islandica, a long lifespan is associated with low metabolic activity, and with a pronounced tolerance to low environmental oxygen. In order to study metabolic and physiological responses to low oxygen conditions vs. no oxygen in mantle, gill, adductor muscle and hemocytes of the ocean quahog, specimens from the German Bight were maintained for 3.5 days under normoxia (21 kPa=controls), hypoxia (2 kPa) or anoxia (0 kPa). Tissue levels of anaerobic metabolites octopine, lactate and succinate as well as specific activities of octopine dehydrogenase (ODH) and lactate dehydrogenase (LDH) were unaffected by hypoxic incubation, suggesting that the metabolism of A. islandica remains fully aerobic down to environmental oxygen levels of 2 kPa. PO(2)-dependent respiration rates of isolated gills indicated the onset of metabolic rate depression (MRD) below 5 kPa in A. islandica, while anaerobiosis was switched on in bivalve tissues only at anoxia. Tissue-specific levels of glutathione (GSH), a scavenger of reactive oxygen species (ROS), indicate no anticipatory antioxidant response takes place under experimental hypoxia and anoxia exposure. Highest specific ODH activity and a mean ODH/LDH ratio of 95 in the adductor muscle contrasted with maximal specific LDH activity and a mean ODH/LDH ratio of 0.3 in hemocytes. These differences in anaerobic enzyme activity patterns indicate that LDH and ODH play specific roles in different tissues of A. islandica which are likely to economize metabolism during anoxia and reoxygenation.