beta-Adrenergic receptor desensitization stimulates glucose uptake in C6 rat glioma cells

When C₆ glioma cells were stimulated by β -adrenergic ligands, [³H]-deoxyglucose uptake by the cells decreased in the first 30 min, followed by its acceleration. The stimulation of deoxyglucose uptake was attributable to desensitization of β-adrenergic receptor-adenylate cyclase system. When the cells were treated with quinacrine or tetracaine, phospholipase inhibitors, the stimulation of deoxyglucose uptake by isoproterenol was diminished without changing the basal rate. On the other hand, when C₆ glioma cells were treated with melittin or phorbol ester, phospholipase A₂ activators, the deoxyglucose uptake increases even in the absence of isoproterenol. Since these compounds inhibit or enhance phospholipase A₂ as well as the desensitization of β -adrenergic receptors (Proc. Natl. Acad. Sci. USA 77, 1341–1345, 1980), these results suggest that turnovers of phospholipids in the vicinity of β -adrenergic receptors modify the glucose uptake of C₆ glioma cells.     

Resveratrol increases glutamate uptake and glutamine synthetase activity in C6 glioma cells

Resveratrol, a phytoalexin found mainly in grapes, is a promising natural product with anti-cancer and cardio-protective activities. Here, we investigated, in C6 glioma cells, the effect of resveratrol on some specific parameters of astrocyte activity (glutamate uptake, glutamine synthetase and secretion of S100B, a neurotrophic cytokine) commonly associated with the protective role of these cells. Cell proliferation was significantly decreased by 8% and 26%, following 24h of treatment with 100 and 250 microM resveratrol. Extracellular S100B increased after 48 h of resveratrol exposure. Short-term resveratrol exposure (from 1 to 100 microM) induced a linear increase in glutamate uptake (up to 50% at 100 microM resveratrol) and in glutamine synthetase activity. Changes in these glial activities can contribute to the protective role of astrocytes in brain injury conditions, reinforcing the putative use of this compound in the therapeutic arsenal against neurodegenerative diseases and ischemic disorders.     

Ca regulation of connexin 43 hemichannels in C6 glioma and glial cells

Connexin hemichannels have a low open probability under normal conditions but open in response to various stimuli, forming a release pathway for small paracrine messengers. We investigated hemichannel-mediated ATP responses triggered by changes of intracellular Ca²⁺ ([Ca²⁺]_i) in Cx43 expressing glioma cells and primary glial cells. The involvement of hemichannels was confirmed with gja1 gene-silencing and exclusion of other release mechanisms. Hemichannel responses were triggered when [Ca²⁺]_i was in the 500 nM range but the responses disappeared with larger [Ca²⁺]_i transients. Ca²⁺-triggered responses induced by A23187 and glutamate activated a signaling cascade that involved calmodulin (CaM), CaM-dependent kinase II, p38 mitogen activated kinase, phospholipase A2, arachidonic acid (AA), lipoxygenases, cyclo-oxygenases, reactive oxygen species, nitric oxide and depolarization. Hemichannel responses were also triggered by activation of CaM with a Ca²⁺-like peptide or exogenous application of AA, and the cascade was furthermore operational in primary glial cells isolated from rat cortex. In addition, several positive feed-back loops contributed to amplify the responses. We conclude that an elevation of [Ca²⁺]_i triggers hemichannel opening, not by a direct action of Ca²⁺ on hemichannels but via multiple intermediate signaling steps that are adjoined by distinct signaling mechanisms activated by high [Ca²⁺]_i and acting to restrain cellular ATP loss.     

Zinc homeostasis in C6 glioma cells

Zinc homeostasis in mammalian cells is precisely regulated by cellular signal transduction mechanisms. The main result of this study is the finding that modulators of phospholipase C (PLC) activity affect cellular zinc export. Two different PLC inhibitors caused an increase of the total cellular zinc level whereas two different PLC activators caused a decrease. Furthermore, both the inhibition of cyclic nucleotide phosphodiesterases as well as the administration of 8-bromo-cAMP evoked a drop in the intracellular zinc level, indicating the involvement of cAMP in the control of cellular zinc export. It is concluded that the activity of PLC controls cellular zinc transport and that the effect of elevated zinc concentrations on PLC activity might be mediated by cAMP. However, modulation of other major signaling enzymes did not affect the cellular zinc homeostasis. These include activation and inhibition of guanylate cyclase, activation of protein kinase G, activation of protein kinase A, and activation or inhibition of protein kinase C. Furthermore there was no evidence for the existence of a zinc-sensing receptor in C6 glioma cells, which would stimulate PLC activity and evoke a mobilization of intracellular free-calcium levels.     

Glutamine transport in C6 glioma cells

Glutamine transport across the cell membranes of a variety of mammalian tissues is mediated by at least four transport systems: a sodium-independent system L, and sodium-dependent systems A, ASC and N, the latter occurring in different tissue-specific variants. In this study we assessed the contribution of these systems to the uptake of [(3)H]glutamine in C6 rat glioma cells. The sodium-dependent uptake, which accounted for more than 80% of the total uptake, was not inhibited by 2-methylaminoisobutyric acid (MeAIB), indicating that system A was inactive, possibly being depressed by glutamine present in the culture medium. About 80% of the sodium-dependent uptake was mediated by system ASC, which differed from system ASC common to other CNS- and non-CNS tissues by its pH-dependence and partial lithium tolerance. The residual 20% of sodium-dependent uptake appeared to be mediated by system N, which was identified as a component resistant to inhibition by MeAIB+threonine. The system N in C6 cells appeared to be neither fully compatible with the neuronal system Nb, nor with the N system described in astrocytes: it differed from the former in being strongly inhibited by histidine and showing fair tolerance for lithium, and from the latter in its pH-insensitivity and strong inhibition by glutamate. The sodium-independent glutamine uptake differed from the astrocytic or neuronal uptake in its relatively weak inhibition by system L substrates and a strong inhibition by system ASC substrates, indicating a possible contribution of a variant of the ASC system.     

ANG II increases 2-deoxyglucose uptake in mouse embryonic stem cells

It is now suggested that all components of the renin-angiotensin system are present in many tissues, including the embryo and may play a major role in embryo development and differentiation. However, little is known regarding whether ANG II regulates glucose transport in mouse embryonic stem (ES) cells. Thus, the effects of ANG II on [3H]-2-deoxyglucose (2-DG) uptake and its related signal pathways were examined in mouse ES cells. ANG II significantly increased cell proliferation and 2-DG uptake in concentration- and time-dependent manner (>18 h, >10(-8) M) and increased mRNA and protein level of GLUT1 by 31+/-7% and 22+/-5% compared to control, respectively. Actinomycin D and cycloheximide completely blocked the effect of ANG II on 2-DG uptake. ANG II-induced increase of 2-DG uptake was blocked by losartan, an ANG II type 1 (AT1) receptor blocker, but not by PD 123319, an ANG II type 2 (AT2) receptor blocker. In addition, ANG II-induced stimulation of 2-DG uptake was attenuated by phospholipase C (PLC) inhibitors, neomycin and U 73122 and ANG II increased inositol phosphates (IPs) formation by 37+/-8% of control. Protein kinase C (PKC) inhibitors, staurosporine, bisindolylmaleimide I, and H-7 also blocked ANG II-induced stimulation of 2-DG uptake. Indeed, ANG II activated a PKC translocation from the cytosolic to membrane fraction, suggesting a role of PKC. A 23187 (Ca2+ ionophore) increased 2-DG uptake and nifedifine (L-type Ca2+ channel blocker) blocked it. In conclusion, ANG II increased 2-DG uptake by PKC activation via AT1 receptor in mouse ES cells.     

Methylene blue stimulates 2-deoxyglucose uptake in L929 fibroblast cells

Methylene blue (MB), a common cell stain, has been shown to inhibit nitric oxide synthase and guanylate cyclase, which has led to the recent use of MB in nitric oxide signaling studies. This study documents the effects of MB on 2-deoxyglucose (2DG) uptake in L929 fibroblast cells where uptake is controlled by a single glucose transporter, GLUT 1. MB significantly activates cytochalasin B-inhibitable glucose transport in a dose dependent fashion within 10 min. A maximal stimulation of up to 800% was achieved by 50 microM MB after a 45-min exposure. The Vmax of transport increased without a change in the Km, which was accomplished without a significant change in the GLUT 1 content. The reduced form of MB, did not stimulate 2DG uptake and potassium ferricyanide, an extracellular redox agent, prevented both the staining and stimulatory effects of MB suggesting MB is reduced at the cell surface before it enters L929 cells. Phenylarsine oxide did not block cell staining as noted in other cells lines, but it did inhibit both basal and MB-stimulated 2DG uptake. Likewise, methyl-beta-cyclodextrin, an agent used to remove membrane cholesterol, blocked both the staining and stimulatory effects of MB. The AMP analog, AICAR, inhibited rather than activated basal 2DG uptake, and it did not alter MB-stimulated uptake suggesting that AMP kinase activation is not critical to the MB effect. Wortmannin, an inhibitor of PI kinase, had no effect on MB-stimulated 2DG uptake. These data provide additional insight into the acute regulation of GLUT 1 transport activity in L929 cells.     

Alteration in morphology and induction of glutamine synthetase in rat glioma C6BU-1 cells cultured with prostaglandin D2-supplemented media

We evaluated the effects of prostaglandins (PGs) on rat glioma C6BU-1 cells by supplementing the culture media with PGs. In the medium containing PGD₂ (15 or 20 μM), the glial cells showed altered morphology from an elongated fibroblastic form to a spreading multipolar one within 24 h, and their growth rate was suppressed to half of that of control cultures. In these cultures, the specific activity of glutamine synthetase (GS) increased approximately twofold within 48 h in comparison to the value for vehicle-treated controls. Simultaneous treatment with actinomycin D or cycloheximide completely blocked the PGD₂-elicited increase in GS specific activity, suggesting that the increase was due to de novo synthesis of the enzyme. PGD₂-like prostanoids such as PGD₁ and 9-deoxy-Δ⁹, Δ¹²-13,14-dihydro-PGD₂ (Δ¹²-PGJ₂), when added to the culture medium, mimicked the actions of PGD₂ on the C6BU-I cells, though their effective concentrations were not necessarily identical. PGs of the E- and F-series had almost no discernible effect on the glioma. These results might imply a possibility that PGD₂ plays a regulatory effect in growth and/or differentiation of rat glioma C6BU-1 cells.     

Measurement of 2-deoxyglucose and 2-deoxyglucose 6-phosphate in tissues

The enzymatic methods previously described for 2-deoxyglucose (DG) and 2-deoxyglucose 6-phosphate have been refined and adapted to measurements of brain samples ranging from 50 mg wet weight to less than a microgram dry weight. Procedures for preparing such samples for assay are described. Analytical properties of the enzymes employed are given together with means for overcoming their possible short comings. Emphasis is placed on information useful for employing DG to assess rapid changes in glucose metabolism.     

Glutamine transport in C6 glioma cells shows ASCT2 system characteristics

Previous studies from this laboratory have shown that glutamine (Gln) uptake in a rat astrocytoma-derived C6 cell line shows characteristics similar with the uptake of a model ASC system substrate, threonine, whose pH-dependence and partial tolerance of Li(+) substitution for Na(+) resemble the ASCT2 variant of the system. In support of the previous findings, RT-PCR analysis revealed that C6 cells strongly express ASCT2 mRNA, but not at all GlnT mRNA or NAT2 mRNA, the A and N system variants specifically engaged in Gln transport in normal CNS. Other features of Gln transport in C6 cells indicating the involvement of ASCT2 system included its resistance to ouabain and stimulation of Gln efflux from the cells in the presence of excess Gln or cysteine (Cys), demonstrating that the system operates in the exchange mode. Replacement of NaCl in the incubation medium with isoosmotic sucrose did neither significantly affect the kinetics, nor any other major characteristics of Gln or Thr transport, including its pH-dependence, inhibition by ASCT system substrates or resistance to the model system A substrate-N-methylamino-isobutyric acid (MeAiB).     

Beta adrenergic receptor repopulation of C6 glioma cells after irreversible blockade and down regulation

C6 glioma cells possess beta adrenergic receptors coupled with adenylate cyclase which can be irreversibly blocked by bromoacetylaminomethylpindolol (Br-AAM-pindolol), a beta adrenergic antagonist. With 1 microM Br-AAM-pindolol, more than 80% of beta adrenergic receptors, labeled by (3H)-dihydroalprenolol [3H)-DHA), were blocked. After this blockade, new beta adrenergic receptors were synthesized only during cell division. However, at cell confluency when the cell number was constant, turnover of beta adrenergic receptors was barely detectable. Cycloheximide (1 microgram/ml) inhibited cell growth as well as reappearance of beta adrenergic receptors. A 90% loss of beta adrenergic receptors in C6 glioma cells was obtained after down-regulation for 15 h with 10 microM isoproterenol, a beta adrenergic agonist. After removal of the agonist, recovery of beta-adrenergic-sensitive adenylate cyclase was complete within 2 to 3 days, whereas beta adrenergic receptors reached 90% of control value within 6 days. The half-life of the receptor recovery was 2 to 3 days. Pretreatment of C6 glioma cells by Br-AAM-pindolol and subsequent cell exposure to isoproterenol indicated that down regulation and recovery of unblocked beta adrenergic receptors did occur; however isoproterenol did not accelerate the biosynthesis of beta adrenergic receptors. The recovery of both biological response and beta adrenergic receptor occupancy was restored both in the presence or absence of cycloheximide (1 microgram/ml), a concentration which blocked 90% of protein synthesis. Our results suggest that reappearance of beta adrenergic receptors in C6 glioma cells, following isoproterenol-induced down regulation, was not due to synthesis of new receptors but to recycling of the beta adrenergic receptors.     

Docosahexaenoic acid supplementation induces dose and time dependent oxidative changes in C6 glioma cells

In view of the promising use of n-3 polyunsaturated fatty acids (PUFAs) in the prevention and treatment of neurological diseases, it is necessary to ascertain the lack of detrimental oxidative effects. We evaluated short- and long-term effects of 25, 50 and 75 μM docosahexaenoic acid (DHA) supplementation on the oxidative status of C6 glial cells. DHA was incorporated into cells dose and time dependently without any cytotoxic effect. Reactive oxygen species (ROS) level was related to DHA dose and supplementation time. At the lowest dose no significant increase in ROS values was observed at hour 24. Low doses of DHA strengthened the cellular antioxidant defence system as highlighted by a raise in both GPX and catalase activity, and the decreased levels of lipid peroxidation. This effect was pronounced at 24 h of supplementation, almost disappeared at hour 48, while after 72 h an opposite effect was observed: lipid peroxidation increased concomitantly with DHA doses. Therefore, the final effect of DHA on cellular redox status is dependent on dose and time supplementation.     

Docosahexaenoic acid supplementation induces dose and time dependent oxidative changes in C6 glioma cells

In view of the promising use of n-3 polyunsaturated fatty acids (PUFAs) in the prevention and treatment of neurological diseases, it is necessary to ascertain the lack of detrimental oxidative effects. We evaluated short- and long-term effects of 25, 50 and 75 μM docosahexaenoic acid (DHA) supplementation on the oxidative status of C6 glial cells. DHA was incorporated into cells dose and time dependently without any cytotoxic effect. Reactive oxygen species (ROS) level was related to DHA dose and supplementation time. At the lowest dose no significant increase in ROS values was observed at hour 24. Low doses of DHA strengthened the cellular antioxidant defence system as highlighted by a raise in both GPX and catalase activity, and the decreased levels of lipid peroxidation. This effect was pronounced at 24 h of supplementation, almost disappeared at hour 48, while after 72 h an opposite effect was observed: lipid peroxidation increased concomitantly with DHA doses. Therefore, the final effect of DHA on cellular redox status is dependent on dose and time supplementation.     

Acyl-based anandamide uptake inhibitors cause rapid toxicity to C6 glioma cells at pharmacologically relevant concentrations

Compounds blocking the uptake of the endogenous cannabinoid anandamide (AEA) have been used to explore the functions of the endogenous cannabinoid system in the CNS both in vivo and in vitro. In this study, the effects of four commonly used acyl-based uptake inhibitors [N-(4-hydroxyphenyl)arachidonylamide (AM404), N-(4-hydroxy-2-methylphenyl) arachidonoyl amide (VDM11), (5Z,8Z,11Z,14Z)-N-(3-furanylmethyl)-5,8,11,14-eicosatetraenamide (UCM707) and (9Z)-N-[1-((R)-4-hydroxybenzyl)-2-hydroxyethyl]-9-octadecen-amide (OMDM2)] and the related compound arvanil on C6 glioma cell viability were investigated. All five compounds reduced the ability of the cells to accumulate calcein, reduced the total nucleic acid content and increased the activity of lactate dehydrogenase recovered in the cell medium. AM404 (10 microm) and VDM11 (10 microm) acted rapidly, reducing cell viability after 3 h of exposure when cell densities of 5,000 per well were used. In contrast, UCM707 (30 microm), OMDM2 (10 microm) and the related compound arvanil (10 microm) produced a more slowly developing effect on cell viability, although robust effects were seen after 6-9 h of exposure. At higher cell densities, the toxicities of AM404 and UCM707 were reduced. Comparison of the compounds with arachidonic acid, arachidonic acid methyl ester, AEA, arachidonoyl glycine and oleic acid suggested that the toxicity of the arachidonoyl-based compounds was related primarily to the acyl side-chain rather than the head group. A variety of pre-treatments blocking possible metabolic pathways and receptor targets were tested, but the only consistent protective treatment against the effects of these compounds was the antioxidant N-acetyl-L-cysteine. It is concluded that AM404, VDM11, UCM707 and OMDM2 produce a rapid loss of C6 glioma cell viability over the same concentration range as is required for the inhibition of AEA uptake in vitro, albeit with a longer latency. Such effects should be kept in mind when acyl-derived compounds are used to probe the function of the endocannabinoid system in the CNS, particularly in chronic administration protocols.     

Functional cross-talk of Ca2+-mobilizing endothelin receptors in C6 glioma cells

There are conflicting results concerning the receptor subtype(s) involved in calcium-mediated endothelin signaling in the glial cells. In order to elucidate the role of endothelin A and B receptors in these processes, we have studied the effect of a complex spectrum of endothelin receptor ligands on intracellular calcium concentration changes in proliferating and differentiated C6 rat glioma cells. Cell differentiation was induced by dibutyryl-cAMP and assessed by the glial fibrillar acidic protein content. Intracellular calcium changes were measured in cell suspensions using fluorescent probe Fura-2. The specific endothelin B receptor agonists sarafotoxin S6c and IRL-1620 did not influence the intracellular calcium concentration. However, calcium changes induced by endothelin-1 and especially by endothelin-3 after the pretreatment of cells with one of these endothelin B receptor specific agonists were significantly enhanced even above the values attained by the highest effective endothelin concentrations alone. Such endothelin B-receptor ligand-induced sensitization of calcium signaling was not observed in differentiated C6 cells. Moreover, endothelin-induced calcium oscillations in differentiated C6 cells were less inhibited by BQ-123 and BQ-788 than in their proliferating counterparts. In conclusion, the specific activation of endothelin B receptor in C6 rat glioma cells does not affect intracellular calcium per se, but probably does so through interaction with the endothelin A receptor. The pattern and/or functional parameters of endothelin receptors in C6 rat glioma cells are modified by cell differentiation.     

SIRT2 activity is required for the survival of C6 glioma cells

SIRT2 is a tubulin deacetylase, which can play either detrimental or beneficial roles in cell survival under different conditions. While it has been suggested that reduced SIRT2 expression in human gliomas may contribute to development of gliomas, there has been no study that directly determines the effects of decreased SIRT2 activity on the survival of glioma cells. In this study we applied both pharmacological and molecular approaches to determine the roles of SIRT2 in the survival of glioma cells. Our studies, by conducting such assays as flow cytometry-based Annexin V assay and caspase-3 immunostaining, have indicated that decreased SIRT2 activity leads to apoptosis of C6 glioma cells by caspase-3-dependent pathway. Our experiments have further shown that reduced SIRT2 activity produces necrosis of C6 glioma cells. Moreover, our study applying SIRT2 siRNA has also shown that decreased SIRT2 leads to both necrosis and apoptotic changes of C6 glioma cells. Collectively, our study has provided novel evidence indicating that SIRT2 activity plays a key role in maintaining the survival of glioma cells, and that reduced SIRT2 activity can induce both necrosis and caspase-3-dependent apoptosis of C6 glioma cells. These results have also suggested that inhibition of SIRT2 might become a novel therapeutic strategy for gliomas.     

Study ofO-sialylation of glycoproteins in C6 glioma cells treated with retinoic acid

When treated with retinoic acidin vivo, C6 glioma cells show an enhancement of CMP-Neu5Ac:Gal 1–3 GalNAc-R -2,3 sialyltransferase activity. A 300kDa glycoprotein was detected by lectin affinoblotting in retinoic acid-treated C6 cells which stained weakly or not at all in control cells. Comparative studies with different lectins demonstrated that this glycoprotein contains 2,3 Neu5Ac Gal-GalNAc O-glycan moieties. Cultures in the presence of an inhibitor of O-glycan synthesis (N-acetylgalactosaminide -O-benzyl) demonstrated that enhancement of staining of the 300 kDa glycoprotein was not due to the increase of the 2,3 sialyltransferase but to thede novo synthesis of the polypeptide chain of this glycoprotein.Abbreviations RA retinoic acid - Neu5Ac N-acetylneuraminic acid - CMP-Neu5Ac cytidine 5 monophosphosialate - 2,3 ST CMP-Neu5Ac:Gal 1–3 GalNAc-R -2,3 sialyltransferase - GalNAc-O-benzyl N-acetylgalactosaminide -O-benzyl - Gal1-3GalNAc-O-benzyl Galactosyl 1-3N-acetylgalactosaminide -O-benzyl - TBS Tris-HCl buffer 50mm pH 7.5 containing NaCl 0.15m and Tween 20 0.05% - B1 buffer TBS containing MgCl₂ 1mm, MnCl₂ 1mm and CaCl₂ 1mm