Different iron sources to study the physiology and biochemistry of iron metabolism in marine micro-algae.

We compared ferric EDTA, ferric citrate and ferrous ascorbate as iron sources to study iron metabolism in Ostreococcus tauri, Phaeodactlylum tricornutum and Emiliania huxleyi. Ferric EDTA was a better iron source than ferric citrate for growth and chlorophyll levels. Direct and indirect experiments showed that iron was much more available to the cells when provided as ferric citrate as compared to ferric EDTA. As a consequence, growth media with iron concentration in the range 1-100 nM were rapidly iron-depleted when ferric citrate-but not ferric EDTA was the iron source. When cultured together, P. tricornutum cells overgrew the two other species in iron-sufficient conditions, but E. huxleyi was able to compete other species in iron-deficient conditions, and when iron was provided as ferric citrate instead of ferric EDTA, which points out the critical influence of the chemical form of iron on the blooms of some phytoplankton species. The use of ferric citrate and ferrous ascorbate allowed us to unravel a kind of regulation of iron uptake that was dependent on the day/night cycles and to evidence independent uptake systems for ferrous and ferric iron, which can be regulated independently and be copper-dependent or independent. The same iron sources also allowed one to identify molecular components involved in iron uptake and storage in marine micro-algae. Characterizing the mechanisms of iron metabolism in the phytoplankton constitutes a big challenge; we show here that the use of iron sources more readily available to the cells than ferric EDTA is critical for this task.

Copper accelerates glycolytic flux in cultured astrocytes.

Astrocyte-rich primary cultures were used to investigate the consequences of a copper exposure on the glucose metabolism of astrocytes. After application of CuCl(2) (30 µM) the specific cellular copper content increased from initial 1.5 ± 0.2 nmol/mg to a steady state level of 7.9 ± 0.9 nmol/mg within about 12 h. The copper accumulation was accompanied by a significant increase in the extracellular lactate concentration. The stimulating effect of copper on the lactate production remained after removal of extracellular copper. Copper treatment accelerated the rates of both glucose consumption and lactate production by about 60%. The copper induced acceleration of glycolytic flux was prevented by inhibition of protein synthesis, and additive to the stimulation of glycolysis observed for inhibitors of respiration or prolyl hydroxylases. A copper induced stimulation of glycolytic flux in astrocytes could have severe consequences for the glucose metabolism of the brain in conditions of copper overload.

Synergistic accumulation of iron and zinc by cultured astrocytes.

Iron and zinc are essential for normal brain function, yet the mechanisms used by astrocytes to scavenge non-transferrin-bound iron (NTBI) and zinc are not well understood. Ischaemic stroke, traumatic brain injury and Alzheimer's disease are associated with perturbations in the metabolism of NTBI and zinc, suggesting that these two metals may collectively contribute to pathology. The present study has investigated the accumulation of NTBI and zinc by rat primary astrocyte cultures. It was found that astrocytes express mRNA for both divalent metal transporter 1 (DMT1) and Zip14, indicating the potential for these transporters to contribute to the accumulation of NTBI and zinc by these cells. Astrocytes were found to accumulate iron from ferric chloride in a time- and dose-dependent manner, and the rate of accumulation was strongly stimulated by co-incubation with zinc acetate. In addition, cultured astrocytes rapidly accumulated zinc from zinc acetate, and this accumulation was stimulated by co-incubation with ferric chloride. Because a synergistic stimulation of iron and zinc accumulation is inconsistent with the known properties of DMT1 and Zip14, the present results suggest that additional mechanisms assist astrocytes to scavenge iron and zinc when they are present together in the extracellular compartment. These mechanisms may be involved in disorders that involve elevations in the extracellular concentrations of these metal ions.

Manganese Uptake by A549 Cells is Mediated by Both ZIP8 and ZIP14.

The alveolar epithelia of the lungs require manganese (Mn) as an essential nutrient, but also provide an entry route for airborne Mn that can cause neurotoxicity. Transporters involved in Mn uptake by alveolar epithelial cells are unknown. Recently, two members of the Zrt- and Irt-like protein (ZIP) family of metal transporters, ZIP8 and ZIP14, have been identified as crucial Mn importers in vivo. ZIP8 is by far most abundantly expressed in the lungs, whereas ZIP14 expression in the lungs is low compared to other tissues. We hypothesized that Mn uptake by alveolar epithelial cells is primarily mediated by ZIP8. To test our hypothesis, we used A549 cells, a type II alveolar cell line. Mirroring the in vivo situation, A549 cells expressed higher levels of ZIP8 than cell models for the liver, intestines, and kidney. Quantification of ZIP8 and ZIP14 revealed a strong enrichment of ZIP8 over ZIP14 in A549 cells. Using siRNA technology, we identified ZIP8 and ZIP14 as the major transporters mediating Mn uptake by A549 cells. To our surprise, knockdown of either ZIP8 or ZIP14 impaired Mn accumulation to a similar extent, which we traced back to similar amounts of ZIP8 and ZIP14 at the plasma membrane. Our study highlights the importance of both ZIP8 and ZIP14 in Mn metabolism of alveolar epithelial cells.

Metabolism and functions of copper in brain.

Copper is an important trace element that is required for essential enzymes. However, due to its redox activity, copper can also lead to the generation of toxic reactive oxygen species. Therefore, cellular uptake, storage as well as export of copper have to be tightly regulated in order to guarantee sufficient copper supply for the synthesis of copper-containing enzymes but also to prevent copper-induced oxidative stress. In brain, copper is of importance for normal development. In addition, both copper deficiency as well as excess of copper can seriously affect brain functions. Therefore, this organ possesses ample mechanisms to regulate its copper metabolism. In brain, astrocytes are considered as important regulators of copper homeostasis. Impairments of homeostatic mechanisms in brain copper metabolism have been associated with neurodegeneration in human disorders such as Menkes disease, Wilson's disease and Alzheimer's disease. This review article will summarize the biological functions of copper in the brain and will describe the current knowledge on the mechanisms involved in copper transport, storage and export of brain cells. The role of copper in diseases that have been connected with disturbances in brain copper homeostasis will also be discussed.

Astrocyte functions in the copper homeostasis of the brain.

Copper is an essential element that is required for a variety of important cellular functions. Since not only copper deficiency but also excess of copper can seriously affect cellular functions, the cellular copper metabolism is tightly regulated. In brain, astrocytes appear to play a pivotal role in the copper metabolism. With their strategically important localization between capillary endothelial cells and neuronal structures they are ideally positioned to transport copper from the blood-brain barrier to parenchymal brain cells. Accordingly, astrocytes have the capacity to efficiently take up, store and to export copper. Cultured astrocytes appear to be remarkably resistant against copper-induced toxicity. However, copper exposure can lead to profound alterations in the metabolism of these cells. This article will summarize the current knowledge on the copper metabolism of astrocytes, will describe copper-induced alterations in the glucose and glutathione metabolism of astrocytes and will address the potential role of astrocytes in the copper metabolism of the brain in diseases that have been connected with disturbances in brain copper homeostasis.

Copper metabolism of astrocytes.

This short review will summarize the current knowledge on the uptake, storage, and export of copper ions by astrocytes and will address the potential roles of astrocytes in copper homeostasis in the normal and diseased brain. Astrocytes in culture efficiently accumulate copper by processes that include both the copper transporter Ctr1 and Ctr1-independent mechanisms. Exposure of astrocytes to copper induces an increase in cellular glutathione (GSH) content as well as synthesis of metallothioneins, suggesting that excess of copper is stored as complex with GSH and in metallothioneins. Furthermore, exposure of astrocytes to copper accelerates the release of GSH and glycolytically generated lactate. Astrocytes are able to export copper and express the Menkes protein ATP7A. This protein undergoes reversible, copper-dependent trafficking between the trans-Golgi network and vesicular structures. The ability of astrocytes to efficiently take up, store and export copper suggests that astrocytes play a key role in the supply of neurons with copper and that astrocytes should be considered as target for therapeutic interventions that aim to correct disturbances in brain copper homeostasis.

Copper export from cultured astrocytes.

Copper is an essential trace metal that is required as a catalytic co-factor or a structural component of several important enzymes. However, since excess of copper can also harm cells due to its potential to catalyse the generation of toxic reactive oxygen species, transport of copper and the cellular copper content are tightly regulated. Astrocytes are known to efficiently take up copper ions, but it was not known whether these cells are also able to export copper. Treatment of astrocyte-rich primary cultures for 24 h with copper chloride caused a concentration-dependent increase in the specific cellular copper content. During further 24 h incubation in the absence of copper chloride, the copper-loaded astrocytes remained viable and released up to 45% of the accumulated copper. The rate of copper export was proportional to the amount of cellular copper, was almost completely prevented by lowering the incubation temperature to 4 °C and was partly prevented by the endocytosis inhibitor amiloride. Copper export is most likely mediated by the copper ATPase ATP7A, since this transporter is expressed in astrocyte cultures and its cellular location is strongly affected by the absence or the presence of extracellular copper. The potential of cultured astrocytes to export copper suggests that astrocytes provide neighbouring cells in brain with this essential trace element.

Copper-treatment increases the cellular GSH content and accelerates GSH export from cultured rat astrocytes.

To test whether copper exposure affects astroglial glutathione (GSH) metabolism, we have exposed astrocyte-rich primary cultures with copper chloride in concentrations of up to 30 µM and investigated cellular and extracellular GSH contents. Cultured astrocytes accumulated copper in a concentration-dependent manner thereby increasing the specific cellular copper content within 24h up to sevenfold. The increase in the cellular copper content was accompanied by a proportional increase in the specific cellular GSH content that reached up to 165% of the values of cells that had been incubated without copper, while the low cellular content of GSH disulfide (GSSG) remained unaltered in copper-treated cells. Also the rate of GSH export was significantly increased after copper exposure reaching up to 177% of control values. The export of GSH from control and copper-treated astrocytes was lowered by more than 70%, if cells were incubated in presence of the multidrug-resistance protein (Mrp) 1 inhibitor MK571 or at a low incubation temperature of 4°C. These data demonstrate that copper accumulation stimulates GSH synthesis and accelerates Mrp1-mediated GSH export from cultured astrocytes. These processes are likely to contribute to the resistance of astrocytes against copper toxicity and could improve the supply of GSH precursors from astrocytes to neurons.

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.

Copper accumulation by cultured astrocytes.

To study copper transport in brain astrocytes, we have used astrocyte-rich primary cultures as model system. Cells in these cultures contained a basal copper content of 1.1+/-0.4 nmol per mg protein. The cellular copper content increased strongly after application of copper chloride in a time and concentration-dependent manner. Analysis of the linear copper accumulation during the first 5 min of copper exposure revealed that cultured astrocytes accumulated copper with saturable kinetics with apparent K(M)- and V(max)-values of 9.4+/-1.8 microM and 0.76+/-0.13 nmol/(min x mg protein), respectively. In contrast, incubation of astrocytes with copper in the presence of ascorbate caused a linear increase of the copper accumulation rates for copper concentrations of up to 30 microM. In addition, copper accumulation was strongly inhibited by the presence of an excess of zinc or of various other divalent metal ions. The presence of mRNA and of immunoreactivity of the copper transport protein Ctr1 in astrocyte cultures suggests that Ctr1 contributes to the observed copper accumulation. However, since some characteristics of the observed copper accumulation are not consistent with Ctr1-mediated copper transport, additional Ctr1-independent mechanism(s) are likely to be involved in astrocytic copper accumulation.