Regulation of respiration and ATP synthesis in higher organisms: Hypothesis

The present view on the regulation of respiration and ATP synthesis in higher organisms implies only Michaelis-Menten type kinetics and respiratory control as regulatory principles. Recent experimental observations, suggesting further regulatory mechanisms at respiratory chain complexes, are reviewed. A new hypothesis is presented implying regulation of respiration and ATP synthesis in higher organisms mainly via allosteric modification of respiratory chain complexes, in particular of cytochromec oxidase. The allosteric effectors, e.g., metabolites, cofactors, ions, hormones, and the membrane potential are suggested to change the activity and the coupling degree of cytochromec oxidase by binding to specific sites at nuclear coded subunits. Recent results on the structure and activity of cytochromec oxidase, supporting the hypothesis, are reviewed.Dedicated to Professor Dr. Carl Martius on the occasion of his 80th birthday.     

Differential gene expression during apoptosis induced by a serum factor: Role of mitochondrial F<Subscript>0</Subscript>-F<Subscript>1</Subscript> ATP synthase complex

The number of genes that are up regulated or down regulated during apoptosis is large and still increasing. In an attempt to characterize differential gene expression during serum factor induced apoptosis in AK-5 cells (a rat histiocytoma), we found subunit 6 and subunit 8 of the transmembrane proton channel and subunit alpha of the catalytic core of the mitochondrial F₀-F₁ ATP synthase complex to be up regulated during apoptosis. The increase in the expression levels of these subunits was concomitant with a transient increase in the intracellular ATP levels, suggesting that the increase in cellular ATP content is a result of the increase in the expression of ATP synthase subunits' gene and de novo protein synthesis. Depleting the cellular ATP levels with oligomycin inhibited apoptosis significantly, pointing to the requirement of ATP during apoptosis. Caspase 1 and caspase 3 activity and the loss of mitochondrial membrane potential were also inhibited by oligomycin during apoptosis in these cells, suggesting that the oligomycin induced inhibition of apoptosis could be due to inhibition of caspase activity and inhibition of mitochondrial depolarization. However, cytochrome C release during apoptosis was found to be completely independent of intracellular ATP content. Besides the ATP synthase complex genes, other mitochondrial genes like cytochrome C oxidase subunit II and III also showed elevated levels of expression during apoptosis. This kind of a mitochondrial gene expression profile suggests that in AK-5 cells, these genes are upregulated in a time-linked manner to ensure sufficient intracellular ATP levels and an efficient functioning of the mitochondrial respiratory chain for successful completion of the apoptotic pathway.     

Yeast cells depleted in Atp14p fail to assemble Atp6p within the ATP synthase and exhibit altered mitochondrial cristae morphology

Within the yeast mitochondrial ATP synthase, subunit h is a small nuclear encoded protein belonging to the so-called "peripheral stalk" that connects the enzyme catalytic F(1) component to the mitochondrial inner membrane. This study examines the role of subunit h in ATP synthase function and assembly using a regulatable, doxycycline-repressible subunit h gene to overcome the strong instability of the mtDNA previously observed in strains lacking the native subunit h gene. Yeast cells expressing less than 3% of subunit h, but still containing intact mitochondrial genomes, grew poorly on respiratory substrates because of a major impairment of ATP synthesis originating from the ATP synthase, whereas the respiratory chain complexes were not affected. The lack of ATP synthesis in the subunit h-depleted (deltah) mitochondria was attributed to defects in the assembly/stability of the ATP synthase. A main feature of deltah-mitochondria was a very low content (<6%) in the mitochondrially encoded Atp6p subunit, an essential component of the enzyme proton channel, which was in large part because of a slowing down in translation. Interestingly, depletion of subunit h resulted in dramatic changes in mitochondrial cristae morphology, which further supports the existence of a link between the ATP synthase and the folding/biogenesis of the inner mitochondrial membrane.     

Characterization of Myelin Sheath F<Subscript>o</Subscript>F<Subscript>1</Subscript>-ATP Synthase and its Regulation by IF<Subscript>1</Subscript>

F_oF₁-ATP synthase is the nanomotor responsible for most of ATP synthesis in the cell. In physiological conditions, it carries out ATP synthesis thanks to a proton gradient generated by the respiratory chain in the inner mitochondrial membrane. We previously reported that isolated myelin vesicles (IMV) contain functional F_oF₁-ATP synthase and respiratory chain complexes and are able to conduct an aerobic metabolism, to support the axonal energy demand. In this study, by biochemical assay, Western Blot (WB) analysis and immunofluorescence microscopy, we characterized the IMV F_oF₁-ATP synthase. ATP synthase activity decreased in the presence of the specific inhibitors (olygomicin, DCCD, FCCP, valynomicin/nigericin) and respiratory chain inhibitors (antimycin A, KCN), suggesting a coupling of oxygen consumption and ATP synthesis. ATPase activity was inhibited in low pH conditions. WB and microscopy analyses of both IMV and optic nerves showed that the Inhibitor of F₁ (IF₁), a small protein that binds the F₁ moiety in low pH when of oxygen supply is impaired, is expressed in myelin sheath. Data are discussed in terms of the role of IF₁ in the prevention of the reversal of ATP synthase in myelin sheath during central nervous system ischemic events. Overall, data are consistent with an energetic role of myelin sheath, and may shed light on the relationship among demyelination and axonal degeneration.     

F1F0-ATP synthases of alkaliphilic bacteria: Lessons from their adaptations

This review focuses on the ATP synthases of alkaliphilic bacteria and, in particular, those that successfully overcome the bioenergetic challenges of achieving robust H⁺-coupled ATP synthesis at external pH values > 10. At such pH values the protonmotive force, which is posited to provide the energetic driving force for ATP synthesis, is too low to account for the ATP synthesis observed. The protonmotive force is lowered at a very high pH by the need to maintain a cytoplasmic pH well below the pH outside, which results in an energetically adverse pH gradient. Several anticipated solutions to this bioenergetic conundrum have been ruled out. Although the transmembrane sodium motive force is high under alkaline conditions, respiratory alkaliphilic bacteria do not use Na⁺- instead of H⁺-coupled ATP synthases. Nor do they offset the adverse pH gradient with a compensatory increase in the transmembrane electrical potential component of the protonmotive force. Moreover, studies of ATP synthase rotors indicate that alkaliphiles cannot fully resolve the energetic problem by using an ATP synthase with a large number of c-subunits in the synthase rotor ring. Increased attention now focuses on delocalized gradients near the membrane surface and H⁺ transfers to ATP synthases via membrane-associated microcircuits between the H⁺ pumping complexes and synthases. Microcircuits likely depend upon proximity of pumps and synthases, specific membrane properties and specific adaptations of the participating enzyme complexes. ATP synthesis in alkaliphiles depends upon alkaliphile-specific adaptations of the ATP synthase and there is also evidence for alkaliphile-specific adaptations of respiratory chain components.     

Heterogeneity of Nucleotide Receptors in NG108-15 Neuroblastoma and C6 Glioma Cells for Mediating Phosphoinositide Turnover

Abstract: We have compared the characteristics of receptors for nucleotide analogues and the involvement of phospholipase C (PLC) in the effector mechanism in NG108-15 neuroblastoma and C6 glioma cells. The relative potency of these analogues to stimulate inositol phosphate (IP) formation is UTP > UDP ? 2-methylthio-ATP (2-MeSATP), GTP > ATP, CTP > ADP > UMP in NG108-15 cells and ATP > UTP > ADP > GTP > UDP ? 2Me-SATP, CTP, UMP in C6 glioma cells. α,β-Methylene-ATP, β,γ-methylene-ATP, AMP, and adenosine had little or no effect in both types of cells. The EC₅₀ values were 3 and 106 µM for UTP in NG108-15 and C6 glioma cells, respectively. The EC₅₀ value for ATP in C6 glioma cells was 43 µM. 2-MeSATP was threefold more potent than ATP in NG108-15 cells but had little effect in C6 glioma cells at 1 mM. In NCB-20 cells, a similar rank order of potency to that found in NG108-15 cells, i.e., UTP ? GTP > ATP > CTP, was observed. In both NG108-15 and C6 glioma cells, preincubation with ATP or UTP caused a pronounced cross-desensitization of subsequent nucleotide-stimulated IP production. ATP and UTP displayed no additivity in terms of IP formation at maximally effective concentrations. In contrast, endothelin-1, bradykinin, and NaF interacted in an additive manner with either nucleotide in stimulating PI hydrolysis. Pretreatment with pertussis toxin did not affect ATP-, UTP-, and GTP-stimulated IP generation in these cells, indicating that nucleotide receptors coupled to PLC by a pertussis toxin-resistant G protein in both cell types. Short-term treatment of the cells with protein kinase C (PKC) activators [phorbol 12-myristate 13-acetate (PMA) and octylindolactam V] produced a dose-dependent inhibition of ATP- and UTP-induced IP formation with a greater extent and higher susceptibility in C6 glioma cells than in NG108-15 cells. Furthermore, a 24-h exposure of the cells to PMA resulted in an obvious attenuation of nucleotide-induced IP formation in C6 glioma cells but failed to change the response in NG108-15 cells. These results suggest that distinct nucleotide receptors that respond to ATP and UTP with different selectivity exist in NG108-15 and C6 glioma cells. These heterogeneous nucleotide receptors coupled to PLC undergo discriminative modulation by PKC. NG108-15 and NCB-20 neuroblastoma are two cell lines that showed the highest specificity to extracellular UTP rather than ATP among the nucleotide receptors so far studied in various cells, suggesting the presence of a pyrimidine receptor in these cells.     

Energetic and Morphological Plasticity of C6 Glioma Cells Grown on 3-D Support; Effect of Transient Glutamine Deprivation

The energetic metabolism of rat C6 glioma cells has been investigated as a function of the proliferative and differentiation states under three-dimensional (3-D) growing conditions on microcarrier beads. First, the transient deprivation of glutamine from the culture medium induced a marked decrease in the growth rate and a differentiation of C6 cells through the oligodendrocytic phenotype. Second, the respiratory capacity of the C6 cells during short-term subcultures with or without glutamine continuously declined as a function of the cell density, in part due to the mitochondrial content decrease. During the transition from the early exponential to the plateau growth phase in glutamine-containing medium, the oxygen consumption rate per single cell decreased concomitantly with a decrease in the glucose consumption and lactate production rates. This phenomenon led to a sixfold decrease in the total ATP production flux, without significantly affecting the cellular ATP/ADP ratio, thus indicating that some ATP-consuming processes were simultaneously suppressed during C6 proliferation. In glutamine-free medium, the cellular ATP/ADP ratio transiently increased due to growth arrest and to a reduced ATP turnover. Moreover, the results indicated that glutamine is not an essential respiratory substrate for rat C6 glioma under short-term glutamine deprivation. Worth noting was the high contribution of the mitochondrial oxidative phosphorylation toward the total ATP synthesis (about 80%), regardless of the proliferation or the differentiation status of the C6 cells.     

The F(1)F(0) ATP synthase and mitochondrial respiratory chain complexes are present on the plasma membrane of an osteosarcoma cell line: An immunocytochemical study

F(1)F(0) ATP synthase is ectopically expressed on the surface of several cell types, including endothelium and cancer cells. This study uses immunocytochemical detection methods via highly specific monoclonal antibodies to explore the possibility of plasma membrane localization of other mitochondrial proteins using an osteosarcoma cell line in which the location of the mitochondrial reticulum can be clearly traced by green fluorescent protein tagging of the organelle. We found that subunits of three of the four respiratory chain complexes were present on the surface of these cells. Additionally, we show for the first time that F(0) subunits d and OSCP of the ATP synthase are ectopically expressed. In all cases the OXPHOS proteins show a punctate distribution, consistent with data from proteome analysis of isolated lipid rafts that place the various mitochondrial proteins in plasma membrane microdomains. We also examined the cell surface for marker membrane proteins from several other intracellular organelles including ER, golgi and nuclear envelope. They were not found on the surface of the osteosarcoma cells. We conclude that mitochondrial membrane proteins are ectopically expressed, but not proteins from other cellular organelles. A specific mechanism by which the mitochondrion and plasma membrane fuse to deliver organellar proteins is suggested.     

36° step size of proton‐driven c‐ring rotation in FoF1‐ATP synthase

Synthesis of adenosine triphosphate ATP, the ‘biological energy currency’, is accomplished by F_oF₁‐ATP synthase. In the plasma membrane of Escherichia coli, proton‐driven rotation of a ring of 10 c subunits in the F_o motor powers catalysis in the F₁ motor. Although F₁ uses 120° stepping during ATP synthesis, models of F_o predict either an incremental rotation of c subunits in 36° steps or larger step sizes comprising several fast substeps. Using single‐molecule fluorescence resonance energy transfer, we provide the first experimental determination of a 36° sequential stepping mode of the c‐ring during ATP synthesis.     

Supramolecular organization of the yeast F1Fo-ATP synthase

Background information. The yeast mitochondrial F₁F_o‐ATP synthase is a large complex of 600 kDa that uses the proton electrochemical gradient generated by the respiratory chain to catalyse ATP synthesis from ADP and P_i. For a large range of organisms, it has been shown that mitochondrial ATP synthase adopts oligomeric structures. Moreover, several studies have suggested that a link exists between ATP synthase and mitochondrial morphology. Results and discussion. In order to understand the link between ATP synthase oligomerization and mitochondrial morphology, more information is needed on the supramolecular organization of this enzyme within the inner mitochondrial membrane. We have conducted an electron microscopy study on wild‐type yeast mitochondria at different levels of organization from spheroplast to isolated ATP synthase complex. Using electron tomography, freeze‐fracture, negative staining and image processing, we show that cristae form a network of lamellae, on which ATP synthase dimers assemble in linear and regular arrays of oligomers. Conclusions. Our results shed new light on the supramolecular organization of the F₁F_o‐ATP synthase and its potential role in mitochondrial morphology.     

Supramolecular organization of protein complexes in the mitochondrial inner membrane

The liquid state model that envisions respiratory chain complexes diffusing freely in the membrane is increasingly challenged by reports of supramolecular organization of the complexes in the mitochondrial inner membrane. Supercomplexes of complex III with complex I and/or IV can be isolated after solubilisation with mild detergents like digitonin. Electron microscopic studies have shown that these have a distinct architecture and are not random aggregates. A 3D reconstruction of a I1III2IV1 supercomplex shows that the ubiquinone and cytochrome c binding sites of the individual complexes are facing each other, suggesting a role in substrate channelling. Formation of supercomplexes plays a role in the assembly and stability of the complexes, suggesting that the supercomplexes are the functional state of the respiratory chain. Furthermore, a supramolecular organisation of ATP synthases has been observed in mitochondria, where ATP synthase is organised in dimer rows. Dimers can be isolated by mild detergent extraction and recent electron microscopic studies have shown that the membrane domains of the two partners in the dimer are at an angle to each other, indicating that in vivo the dimers would cause the membrane to bend. The suggested role in crista formation is supported by the observation of rows of ATP synthase dimers in the most curved parts of the cristae. Together these observations show that the mitochondrial inner membrane is highly organised and that the molecular events leading to ATP synthesis are carefully coordinated.     

Oxidative phosphorylation supported by an alternative respiratory pathway in mitochondria from Euglena

The effect of antimycin, myxothiazol, 2-heptyl-4-hydroxyquinoline-N-oxide, stigmatellin and cyanide on respiration, ATP synthesis, cytochrome c reductase, and membrane potential in mitochondria isolated from dark-grown Euglena cells was determined. With L-lactate as substrate, ATP synthesis was partially inhibited by antimycin, but the other four inhibitors completely abolished the process. Cyanide also inhibited the antimycin-resistant ATP synthesis. Membrane potential was collapsed (<60 mV) by cyanide and stigmatellin. However, in the presence of antimycin, a H(+)60 mV) that sufficed to drive ATP synthesis remained. Cytochrome c reductase, with L-lactate as donor, was diminished by antimycin and myxothiazol. Cytochrome bc(1) complex activity was fully inhibited by antimycin, but it was resistant to myxothiazol. Stigmatellin inhibited both L-lactate-dependent cytochrome c reductase and cytochrome bc(1) complex activities. Respiration was partially inhibited by the five inhibitors. The cyanide-resistant respiration was strongly inhibited by diphenylamine, n-propyl-gallate, salicylhydroxamic acid and disulfiram. Based on these results, a model of the respiratory chain of Euglena mitochondria is proposed, in which a quinol-cytochrome c oxidoreductase resistant to antimycin, and a quinol oxidase resistant to antimycin and cyanide are included.     

Revisiting Kadenbach: Electron flux rate through cytochrome c‐oxidase determines the ATP‐inhibitory effect and subsequent production of ROS

Mitochondrial respiration is the predominant source of ATP. Excessive rates of electron transport cause a higher production of harmful reactive oxygen species (ROS). There are two regulatory mechanisms known. The first, according to Mitchel, is dependent on the mitochondrial membrane potential that drives ATP synthase for ATP production, and the second, the Kadenbach mechanism, is focussed on the binding of ATP to Cytochrome c Oxidase (CytOx) at high ATP/ADP ratios, which results in an allosteric conformational change to CytOx, causing inhibition. In times of stress, ATP‐dependent inhibition is switched off and the activity of CytOx is exclusively determined by the membrane potential, leading to an increase in ROS production. The second mechanism for respiratory control depends on the quantity of electron transfer to the Heme aa3 of CytOx. When ATP is bound to CytOx the enzyme is inhibited, and ROS formation is decreased, although the mitochondrial membrane potential is increased.     

Nitric oxide synthase induction in glial cells: Effect on neuronal survival

In primary rat cortical glial cell cultures lipopolysaccharide (LPS) induced a dose- and time-dependent increase of intracellular cyclic GMP concentration associated with a release of nitrite. The LPS-induced cyclic GMP and nitrite increase was enhanced by interferon-γ and was prevented by L-N^G- nitroarginine, dexamethasone and cycloheximide. Thus indicates that LPS effect occured via the production of nitric oxide (NO) and involved new protein synthesis suggesting the induction of NO syntahse in these cells. Furthermore this induction was Ca²⁺-independent and was blocked by an inhibitor of the synthesis of tetrahydrobiopterin. The inducible NO synthase was also expressed by C6 glioma cells. In primary mixed cultures containing both neuronal and glial cells, the effects of LPS were less important than in primary glial cell cultures suggesting that glial cells rather than neurons expressed the inducible form of NO synthase. On the other hand no change on neuronal viability was observed after NO synthase induction by LPS in this culture type. This study indicates that glial cells are able to induce NO synthase without affecting neuronal survival.     

The Sur7 protein regulates plasma membrane organization and prevents intracellular cell wall growth in Candida albicans

The Candida albicans plasma membrane plays important roles in cell growth and as a target for antifungal drugs. Analysis of Ca-Sur7 showed that this four transmembrane domain protein localized to stable punctate patches, similar to the plasma membrane subdomains known as eisosomes or MCC that were discovered in S. cerevisiae. The localization of Ca-Sur7 depended on sphingolipid synthesis. In contrast to S. cerevisiae, a C. albicans sur7Δ mutant displayed defects in endocytosis and morphogenesis. Septins and actin were mislocalized, and cell wall synthesis was very abnormal, including long projections of cell wall into the cytoplasm. Several phenotypes of the sur7Δ mutant are similar to the effects of inhibiting β-glucan synthase, suggesting that the abnormal cell wall synthesis is related to activation of chitin synthase activity seen under stress conditions. These results expand the roles of eisosomes by demonstrating that Sur7 is needed for proper plasma membrane organization and cell wall synthesis. A conserved Cys motif in the first extracellular loop of fungal Sur7 proteins is similar to a characteristic motif of the claudin proteins that form tight junctions in animal cells, suggesting a common role for these tetraspanning membrane proteins in forming specialized plasma membrane domains.     

Ectoadenylate kinase and plasma membrane ATP synthase activities of human vascular endothelial cells

Formation of ATP from ADP on the external surface of vascular endothelial cells has been attributed to plasma membrane ATP synthase, ectoadenylate kinase (ecto-AK), and/or ectonucleoside diphosphokinase. These enzymes or their catalytic products have been causatively linked to the elaboration of vascular networks and the regulation of capillary function. The amount of ATP generated extracellularly is small, requiring sensitive analytical methods for quantification. Human umbilical vein endothelial cells were used to revisit extracellular ATP synthesis using a reliable tetrazolium reduction assay and multiwell plate cultures. Test conditions compatible with AK stability were established. Extracellular AK activity was found to be <1% of the total (intracellular and extracellular), raising the possibility that the external enzyme could have leaked from living cells and/or a few dying cells. To determine whether AK inadvertently leaked from the cells, the activity of another cytoplasmic enzyme, glucose-6-phosphate dehydrogenase (G6PD), was also measured. G6PD is present in the cytoplasm in similar abundance to AK. The activity ratio of G6PD (extracellular/total) was found to be similar to that of AK. Because G6PD in the medium was probably due to leakage, other cytoplasmic macromolecules, including AK, should be released proportionately from the cells. The role of plasma membrane ATP synthase in extracellular ATP formation was examined using Hanks' balanced salt solution with and without selective inhibitors of AK and ATP synthase activities. With P(1),P(5)-di(adenosine 5')-pentaphosphate (inhibitor of AK activity), no extracellular ATP synthesis was detected, whereas with oligomycin, piceatannol, and aurovertin (inhibitors of F(1)F(0)-ATP synthase and F(1)-ATPase activities), no inhibition of extracellular ATP synthesis was observed. AK activity alone could account for the observed extracellular ATP synthesis. The possible impact of ADP impurity in the assays is discussed.     

Membrane-related processes and overall energy metabolism inTrypanosoma brucei and other kinetoplastid species

An electrochemical proton gradient exists across the plasma membrane and the mitochondrial membrane of the bloodstream form ofTrypanosoma brucei. The membrane potential across the plasma membrane and the regulation of the internal pH depend on the temperature.Leishmania donovani regulates its internal pH and maintains a constant electrochemical proton gradient across its plasma membrane under all conditions examined. The mitochondrion of theT. brucei bloodstream form is energized, even though the reactions taking place in it do not result in net ATP synthesis and the Kreb's cycle and the respiratory chain are absent. Glucose is transported across the plasma membrane ofT. brucei by a facilitated diffusion carrier, that can transport a wider range of substrates than its mammalian counterparts. Pyruvate exits the cell via a facilitated diffusion transporter as well. Conflicting evidence exists for the mechanism of glucose transport inL. donovani; biochemical evidence suggests proton/glucose symport, while facilitated diffusion is indicated by physiological data.     

Inhibition of Iron-oxidizing Activity by Bisulfite Ion in Thiobacillus ferrooxidans

Growth of Thiobacillus ferrooxidans on iron- and sulfur-salts media and iron oxidizing activity of this bacterium were strongly inhibited by bisulfite ion. The mechanism of inhibition by bisulfite ion of iron-oxidizing activity was studied with the plasma membrane of T. ferrooxidans AP19-3. The c-type cytochrome in the plasma membrane was reduced by ferrous ion and the cytochrome reduced by Fe²⁺ was oxidized by cytochrome c oxidase in the plasma membrane. In contrast, c-type cytochrome was reduced by bisulfite ion, but it was not oxidized by cytochrome c oxidase in the membrane. Cytochrome c-oxidizing activity was also inhibited by the ion when mammalian cytochrome c was used as an electron donor, suggesting that cytochrome c oxidase, one of the component of iron oxidase, is the site of inhibition by bisulfite ion.     

A comparative study of ectonucleotidase and P2 receptor mRNA profiles in C6 cell line cultures and C6 <Emphasis Type="Italic">ex vivo</Emphasis> glioma model

Glioblastoma multiforme is the most common type of primary brain tumour and has the worst clinical outcome. Nucleotides represent an important class of extracellular molecules involved in cell proliferation, differentiation and apoptosis. Alterations in purinergic signalling have been implicated in pathological processes, such as cancer, and glioma cell lines are widely employed as a model to study the biology of brain tumours. Increasing evidence, however, suggests that glioma cell lines may not present all the phenotypic and genetic characteristics of the primary tumours. We have compared the biological characteristics of C6 rat glioma cells in culture and the same cells after their implantation in the rat brain and growth in culture (denominated as the C6 ex vivo culture model). Parameters evaluated included cell morphology, differentiation, angiogenic markers, purinergic receptors and ecto-nucleotidase mRNA profile/enzymatic activity. Analysis of the C6 glioma cell line and C6 ex vivo glioma cultures revealed distinct cell morphologies, although cell differentiation and angiogenic marker expressions were similar. Both glioma models co-expressed multiple P2X and P2Y receptor subtypes with some differences. In addition, the C6 glioma cell line and C6 ex vivo glioma cultures exhibited similar extracellular ATP metabolism and cell proliferation behaviour when exposed to cytotoxic ATP concentrations. Thus, the disruption of purinergic signalling is a feature shown not only by glioma cell lineages, but also by primary glioma cultures. Our results therefore suggest the participation of the purinergic system in glioma malignancy. This study was supported by grants from the Brazilian agencies CNPq, FAPERGS and Fundo de Incentivo à Pesquisa e Eventos (HCPA). E. Braganhol and D. Huppes were recipients of Brazilian CNPq fellowships; A. Bernardi was the recipient of a CAPES fellowship.