Pretreatment Of Astrocytes Research Articles

Spinal injection of TNF‐α‐activated astrocytes produces persistent pain symptom mechanical allodynia by releasing monocyte chemoattractant protein‐1

Accumulating evidence suggests that spinal astrocytes play an important role in the genesis of persistent pain, by increasing the activity of spinal cord nociceptive neurons, i.e., central sensitization. However, direct evidence of whether activation of astrocytes is sufficient to induce chronic pain symptoms is lacking. We investigated whether and how spinal injection of activated astrocytes could produce mechanical allodynia, a cardinal feature of chronic pain, in naïve mice. Spinal (intrathecal) injection of astrocytes, which were prepared from cerebral cortexes of neonatal mice and briefly stimulated by tumor necrosis factor-alpha (TNF-α), induced a substantial decrease in paw withdrawal thresholds, indicating the development of mechanical allodynia. This allodynia was prevented when the astrocyte cultures were pretreated with a peptide inhibitor of c-Jun N-terminal kinase (JNK), D-JNKI-1. Of note a short exposure of astrocytes to TNF-α for 15 min dramatically increased the expression and release of the chemokine monocyte chemoattractant protein-1 (MCP-1), even 3 h after TNF-α withdrawal, in a JNK-dependent manner. In parallel, intrathecal administration of TNF-α induced MCP-1 expression in spinal cord astrocytes. In particular, mechanical allodynia induced by TNF-α-activated astrocytes was reversed by a MCP-1 neutralizing antibody. Finally, pretreatment of astrocytes with MCP-1 siRNA attenuated astrocytes-induced mechanical allodynia. Taken together, our results suggest that activated astrocytes are sufficient to produce persistent pain symptom in naïve mice by releasing MCP-1.

Close association of p38 and JNK with plasminogen-dependent upregulation of PAI-1 in rat astrocytes in vitro

As reported previously, stimulation of astrocytes with plasminogen (PLGn) remarkably enhances their production/release of plasminogen activator inhibitor-1 (PAI-1). In addition, both p38 mitogen-activated protein kinase (p38MAPK) and c-Jun N-terminal kinase (JNK) are activated in these astrocytes. However, it remains to be determined whether the MAPK activation is associated with the PAI-1 induction in PLGn-stimulated astrocytes. In the present study, we investigated the relationship between MAPK activity and PAI-1 induction in PLGn-stimulated astrocytes. PLGn stimulation led to definitive phosphorylation of three MAPKs: external signal regulated kinase (ERK), JNK and p38. These results suggest that all of these MAPKs, either alone or in combination, are involved in PAI-1 induction. To verify this association, an inhibition experiment was carried out by using inhibitors specific for each MAPK. The results of the immunoblotting analysis indicated that 20 μM SB203580 (the p38 inhibitor) or SP600125 (the JNK inhibitor) suppressed approximately 85% or 40% of PLGn-inducible PAI-1, respectively. Only 20% inhibition was achieved by pretreatment of astrocytes with 20 μM PD98059 (the inhibitor of MEK1/2, an upstream kinase of ERK). In conclusion, p38 and JNK were shown to be the major MAPKs involved in the signaling cascade leading to PAI-1 induction in astrocytes stimulated with PLGn.

Characteristic response of astrocytes to plasminogen/plasmin to upregulate transforming growth factor beta 3 (TGFβ3) production/secretion through proteinase-activated receptor-1 (PAR-1) and the downstream phosphatidylinositol 3-kinase (PI3K)-Akt/PKB signaling cascade

The effects of microglia-derived plasminogen (PLGn) on the neurotrophic role of astrocytes were investigated in vitro. The treatment of astrocytes with rat PLGn led to a significant increase in transforming growth factor beta3 (TGFβ3) in the conditioned medium (CM). This response of astrocytes to PLGn was characteristic and different from that to other stimulators, including lipopolysaccharide, phorbol-12-myristate-13-acetate, interferon-gamma, and ATP. In surveying the signaling molecules that respond to PLGn in astrocytes, we found that Akt/PKB phosphorylation is promoted. The pretreatment of astrocytes with an Akt inhibitor prior to PLGn stimulation resulted in a significant decrease in TGFβ3 amounts in the CM, suggesting an association of Akt with TGFβ3 production/secretion. Further survey revealed that phosphatidylinositol 3 kinase (PI3K) is closely associated with TGFβ3 production/secretion in astrocytes. In fact, PI3K inhibitor clearly depressed the phosphorylation of Akt, indicating that PI3K is localized upstream of Akt. Moreover, the effects of PLGn to increase TGFβ3 were depressed by pretreatment with a proteinase-activated receptor-1 (PAR-1) inhibitor. Plasmin could mimic the PLGn effects to upregulate TGFβ3, and the plasmin effects were suppressed by pretreatment with the PAR-1 inhibitor, suggesting the association of PLGn/plasmin effects with PAR-1. In addition, Akt phosphorylation caused by plasmin was inhibited in the presence of PAR-1 inhibitor. We have therefore demonstrated that PLGn/plasmin, probably plasmin, facilitates the production/secretion of TGFβ3 in astrocytes through both PAR-1 and the subsequent signaling cascade including PI3K and Akt.

Effect of propofol upon ammonia-induced neocortical astrocyte swelling and aquaporin-4 expression

To investigate the effect of propofol upon ammonia-induced neocortical astrocyte swelling and aquaporin-4 expression in rat astrocyte. Methods Astrocytes were isolated from newborn Sprague Dawley rats. After a 3-week culture, cell immunofluorescence was employed to label glial fibrillary acidic protein, the specific protein of astrocyte. Astrocytes were cultured with NH4Cl (5 mmol/L) for 6, 12, 24 and 48h. Astrocytes were pretreated by propofol and DMSO respectively for 30 min and then exposed to NH4Cl for 24h. The expression of AQP4 was detected by the Western blot; cell morphology assessed by light microscopy and cell viability measured by MTF reduction assay. The expression of AQP4 was elevated after a 12h exposure to ammonia, lasted to 48h and peaked at 24h. Astrocytes were found significantly swelling in the NH4Cl-24h and DMSO pretreated groups as compared with the propofol pretreated group. The over-expression of aquaporin-4 was attenuated by propofol pretreatment and the cell viability of astrocytes in the propofol pretreated group was higher than that in the NH4Cl-24h group (P < 0.05). Pretreatment of astrocytes with propofol can inhibit the over-expression of AQP4, relieve cellular swelling and reduce the ammonia-induced decline in cell viability of astrocytes.

Reactive oxygen species‐induced cell death of rat primary astrocytes through mitochondria‐mediated mechanism

Astrocytes, the most abundant glial cell population in the central nervous system (CNS), play physiological roles in neuronal activities. Oxidative insult induced by the injury to the CNS causes neural cell death through extrinsic and intrinsic pathways. This study reports that reactive oxygen species (ROS) generated by exposure to the strong oxidizing agent, hexavalent chromium (Cr(VI)) as a chemical-induced oxidative stress model, caused astrocytes to undergo an apoptosis-like cell death through a caspase-3-independent mechanism. Although activating protein-1 (AP-1) and NF-kappaB were activated in Cr(VI)-primed astrocytes, the inhibition of their activity failed to increase astrocytic cell survival. The results further indicated that the reduction in mitochondrial membrane potential (MMP) was accompanied by an increase in the levels of ROS in Cr(VI)-primed astrocytes. Moreover, pretreatment of astrocytes with N-acetylcysteine (NAC), the potent ROS scavenger, attenuated ROS production and MMP loss in Cr(VI)-primed astrocytes, and significantly increased the survival of astrocytes, implying that the elevated ROS disrupted the mitochondrial function to result in the reduction of astrocytic cell viability. In addition, the nuclear expression of apoptosis-inducing factor (AIF) and endonuclease G (EndoG) was observed in Cr(VI)-primed astrocytes. Taken together, evidence shows that astrocytic cell death occurs by ROS-induced oxidative insult through a caspase-3-independent apoptotic mechanism involving the loss of MMP and an increase in the nuclear levels of mitochondrial pro-apoptosis proteins (AIF/EndoG). This mitochondria-mediated but caspase-3-independent apoptotic pathway may be involved in oxidative stress-induced astrocytic cell death in the injured CNS.

Cruciferous Nutraceutical 3H-1,2-dithiole-3-thione Protects Human Primary Astrocytes Against Neurocytotoxicity Elicited by MPTP, MPP+, 6-OHDA, HNE and Acrolein

Astrocytes possess important roles in maintaining normal brain function and providing trophic support to the neurons. They also suffer a range of toxic insults, being a chief target of prooxidants such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 1-methyl-4-phenylpyridinium (MPP(+)), 6-hydroxydopamine (6-OHDA), 4-hydroxy-2-nonenal (HNE), and acrolein. Recently, we have observed that the cellular antioxidants and phase 2 enzymes can be upregulated by 3H-1,2-dithiole-3-thione (D3T), a nutraceutical found in cruciferous vegetables, against many prooxidants in human neuroblastoma cell lines (SH-SY5Y). However, the regulation of the above cellular factors by D3T in astrocytes and their role in ameliorating the neurotoxic effects of the above neurotoxins have not been investigated. In this study, we show that incubation of human primary astrocytes with micromolar concentrations (5-100 microM) of D3T for 24 h resulted in significant increases in the levels of reduced glutathione (GSH), glutathione reductase (GR), and the phase 2 enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1). D3T treatment also caused time-dependent increases in mRNA expression of the gamma-glutamylcysteine ligase catalytic subunit (GCLC), GR, and of NQO1 in these cells. Pretreatment of astrocytes with D3T was found to afford remarkable protection against the neurocytotoxicity elicited by MPTP, MPP(+), 6-OHDA, HNE and acrolein. Taken together, this study demonstrates for the first time that in human astrocytes, the cruciferous nutraceutical D3T potently induces the cellular GSH system and the phase 2 enzyme NQO1, which is accompanied by dramatically increased resistance of these cells to the damage induced by various neurotoxicants. The results of this study may have important implications for the development of novel neuroprotective strategies.

A new perspective for the role of octadecaneuropeptide ODN in neurodegenerative diseases

Event Abstract Back to Event A new perspective for the role of octadecaneuropeptide ODN in neurodegenerative diseases Olfa Masmoudi-Kouki1*, Yosra Hamdi1, Hadhemi Kaddour1, David Vaudry2, 3, Faouzi Jenhani4, Jerome Leprince2, 3, Marie-Christine Tonon2, 3 and Mohamed Amri1 1 Faculty of Science of Tunis, University Tunis El Manar, Laboratory of Functional Neurophysiology and Pathology, 00/UR/08-01, Department of Biological Sciences, Faculty of Science of Tunis, Tunisia 2 Inserm U413-EA4310, Inserm U413-EA4310, France 3 Laboratory of Neuronal and Neuroendocrine Communication and differentiation, European Institute for Peptide Research (IFRMP 23), Regional Platform for Cell Imaging of Haute-Normandie (PRIMACEN) , France 4 National Centre of Blood Transfusion, El Manar II 2092, Laboratory of Cellular Immunology and Cytometry, Tunisia Oxidative stress, resulting from accumulation of reactive oxygen species, plays a critical role on astroglial cell death associated with neurodegenerative diseases and stroke. Astroglial cells synthesize and release endozepines, a family of biologically active peptides that have been implicated in cellular protection. Thus, the purpose of the present study was to investigate the potential protective effect of one of the endozepines, the octadecaneuropeptide ODN, on H2O2-induced oxidative stress and cell death in rat astrocytes. Incubation of cultured astrocytes with graded concentrations of H2O2 for 1 h provoked a dose-dependent reduction of the number of living cells as evaluated by flow cytometric analysis and lactate dehydrogenase assay. The cytotoxic effect of H2O2 was associated with morphological modifications that are characteristic of apoptotic cell death. H2O2-treated cells exhibited high level of reactive oxygen species associated with a reduction of both superoxide dismutases (SOD) and catalase antioxidant enzymatic activities. Pre-treatment of astrocytes with very low concentrations of ODN (0.01 pM – 0.1 nM), dose-dependently prevented cell death induced by H2O2, and the effect of ODN was accompanied by a marked attenuation of ROS accumulation. ODN stimulated SOD and catalase activities in a concentration-dependent manner, and blocked H2O2-evoked inhibition of SOD and catalase activities. In addition, ODN totally suppressed H2O2-induced reduction of mitochondrial membrane potential. Taken together, these data demonstrate for the first time that the endozepine ODN is a potent protective agent that prevents oxidative stress-induced apoptotic cell death. Endozepines are thus potential candidates for development into therapeutically valuable agents for the treatment of cerebral injuries. Keywords: Apoptosis, Astrocytes, Catalase, Superoxide Dismutase Conference: 3rd Mediterranean Conference of Neuroscience , Alexandria, Egypt, 13 Dec - 16 Dec, 2009. Presentation Type: Poster Presentation Topic: CNS Diseases Citation: Masmoudi-Kouki O, Hamdi Y, Kaddour H, Vaudry D, Jenhani F, Leprince J, Tonon M and Amri M (2009). A new perspective for the role of octadecaneuropeptide ODN in neurodegenerative diseases. Front. Neurosci. Conference Abstract: 3rd Mediterranean Conference of Neuroscience . doi: 10.3389/conf.neuro.01.2009.16.156 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 25 Nov 2009; Published Online: 25 Nov 2009. * Correspondence: Olfa Masmoudi-Kouki, Faculty of Science of Tunis, University Tunis El Manar, Laboratory of Functional Neurophysiology and Pathology, 00/UR/08-01, Department of Biological Sciences, Faculty of Science of Tunis, 2092 Tunis, Tunisia, olfa.masmoudi@fst.utm.tn Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Olfa Masmoudi-Kouki Yosra Hamdi Hadhemi Kaddour David Vaudry Faouzi Jenhani Jerome Leprince Marie-Christine Tonon Mohamed Amri Google Olfa Masmoudi-Kouki Yosra Hamdi Hadhemi Kaddour David Vaudry Faouzi Jenhani Jerome Leprince Marie-Christine Tonon Mohamed Amri Google Scholar Olfa Masmoudi-Kouki Yosra Hamdi Hadhemi Kaddour David Vaudry Faouzi Jenhani Jerome Leprince Marie-Christine Tonon Mohamed Amri PubMed Olfa Masmoudi-Kouki Yosra Hamdi Hadhemi Kaddour David Vaudry Faouzi Jenhani Jerome Leprince Marie-Christine Tonon Mohamed Amri Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Astrocyte-Derived CO Is a Diffusible Messenger That Mediates Glutamate-Induced Cerebral Arteriolar Dilation by Activating Smooth Muscle Cell K Ca Channels

Astrocyte signals can modulate arteriolar tone, contributing to regulation of cerebral blood flow, but specific intercellular communication mechanisms are unclear. Here we used isolated cerebral arteriole myocytes, astrocytes, and brain slices to investigate whether carbon monoxide (CO) generated by the enzyme heme oxygenase (HO) acts as an astrocyte-to-myocyte gasotransmitter in the brain. Glutamate stimulated CO production by astrocytes with intact HO-2, but not those genetically deficient in HO-2. Glutamate activated transient K(Ca) currents and single K(Ca) channels in myocytes that were in contact with astrocytes, but did not affect K(Ca) channel activity in myocytes that were alone. Pretreatment of astrocytes with chromium mesoporphyrin (CrMP), a HO inhibitor, or genetic ablation of HO-2 prevented glutamate-induced activation of myocyte transient K(Ca) currents and K(Ca) channels. Glutamate decreased arteriole myocyte intracellular Ca2+ concentration and dilated brain slice arterioles and this decrease and dilation were blocked by CrMP. Brain slice arteriole dilation to glutamate was also blocked by L-2-alpha aminoadipic acid, a selective astrocyte toxin, and paxilline, a K(Ca) channel blocker. These data indicate that an astrocytic signal, notably HO-2-derived CO, is used by glutamate to stimulate arteriole myocyte K(Ca) channels and dilate cerebral arterioles. Our study explains the astrocyte and HO dependence of glutamatergic functional hyperemia observed in the newborn cerebrovascular circulation in vivo.

Glutamate transporter blockade affects Ca2+ responses in astrocytes

Brief pretreatment of astrocytes in culture with glutamate (500 μM for 20 min), was earlier shown to significantly enhance the Ca2+ responses to a depolarizing pulse. It is known that malfunction of glutamate transporters increases extracellular glutamate concentration. We hypothesized that pretreatment of astrocytes with glutamate in conditions where the glutamate transporter activity is blocked should cause further elevation of the Ca2+ responses to a depolarizing pulse. To test the hypothesis we pretreated astrocytes in culture (primary rat astrocyte cultures) with glutamate (500 μM) and glutamate transport inhibitor, threo-β-hydroxy-aspartate (200 μM, TBHA) or glutamate (500 μM) in Na+ free extracellular solution for 20 min. The Ca2+ responses were elicited by depolarization of the astrocyte to evoke voltage-gated Ca2+ currents. Paradoxical attenuation of the Ca2+ transients was observed when the glutamate pretreatment was done in conditions that blocked glutamate transport, accompanied by faster rise and decay times. When the experiments were done on astrocyte pairs that were pretreated with glutamate and TBHA, we observed attenuated Ca2+ responses in the adjoining cell when compared with the depolarized cell. The results were contrary to our earlier observation of heightened responses in the adjoining cell of the astrocyte pair, in cells pretreated with glutamate alone. The attenuated Ca2+ responses in astrocytes would imply decrease in the vesicular release of glutamate and ATP. Extracellular glutamate concentration dependent regulation of the Ca2+ signaling mechanism thus seems to operate in astrocytes, which may be important in regulating the neurotoxic accumulation of glutamate in the extracellular space and the synapse.

Angiotensin II induces proliferation of cultured rat astrocytes through c-Jun N-terminal kinase

Previously we showed that tyrosine kinases and ERK1/2 mediate angiotensin II (Ang II)-induced cellular proliferation in astrocytes. In the current study, we investigated whether Ang II activates c-Jun N-terminal kinase (JNK) and determined if JNK mediates Ang II-induced astrocyte growth in cultured brainstem astrocytes. Ang II activated JNK in a time- and concentration-dependent manner. Maximal stimulation of 14-fold over basal occurred with 100 nM Ang II and was significantly apparent 5 min after treatment. Ang II-induced JNK phosphorylation was abolished by co-treatment with the AT 1 receptor antagonist, losartan, but not by PD123319 (an AT 2 receptor antagonist). The JNK inhibitor, SP600125 (10 μM), markedly inhibited Ang II-induced JNK phosphorylation (by 84%) and astrocyte proliferation (by over 90%). Pretreatment of astrocytes with 10 μM PP2 (Src inhibitor) inhibited Ang II stimulation of JNK (by 90%) whereas, the protein kinase C (PKC) inhibitor, Go6976, failed to inhibit Ang II-mediated JNK phosphorylation. In conclusion, we showed for the first time that JNK mediates Ang II-specific astrocyte proliferation. In addition, Ang II activation of the JNK pathway is mediated by Src and not through PKC activation. This study is the first to show that JNK mediates Ang II effects in astrocytes and may provide a better understanding of the functions of Ang II in astrocytes from brainstem in particular and of glial cells in general.

Activation of P2X7 Receptors Stimulates the Expression of P2Y2 Receptor mRNA in Astrocytes Cultured from Rat Brain

Under pathological conditions brain cells release ATP at concentrations reported to activate P2X(7) ionotropic receptor subtypes expressed in both neuronal and glial cells. In the present study we report that the most potent P2X(7) receptor agonist BzATP stimulates the expression of the metabotropic ATP receptor P2Y(2) in cultured rat brain astrocytes. In other cell types several kinds of stimulation, including stress or injury, induce P2Y(2) expression that, in turn, is involved in different cell reactions. Similarly, it has recently been found that in astrocytes and astrocytoma cells P2Y(2) sites can trigger neuroprotective pathways through the activation of several mechanisms, including the induction of genes for antiapoptotic factors, neurotrophins, growth factors and neuropeptides. Here we present evidence that P2Y(2) mRNA expression in cultured astrocytes peaks 6 h after BzATP exposure and returns to basal levels after 24 h. This effect was mimicked by high ATP concentrations (1 mM) and was abolished by P2X(7)-antagonists oATP and BBG. The BzATP-evoked P2Y(2) receptor up-regulation in cultured astrocytes was coupled to an increased UTP-mediated intracellular calcium response. This effect was inhibited by oATP and BBG and by P2Y(2)siRNA, thus supporting evidence of increased P2Y(2) activity. To further investigate the mechanisms by which P2X(7) receptors mediated the P2Y(2) mRNA up-regulation, the cells were pre-treated with the chelating agent EGTA, or with inhibitors of mitogen-activated kinase (MAPK) (PD98059) or protein kinase C, (GF109203X). Each inhibitor significantly reduced the extent to which BzATP induced P2Y(2) mRNA. Both BzATP and ATP (1 mM) increased ERK1/2 activation. P2X(7)-induced ERK1/2 phosphorylation was unaffected by pre-treatment of astrocytes with EGTA whereas it was inhibited by GF109203X. Phorbol-12-myristate-13-acetate (PMA), an activator of PKCs, rapidly increased ERK1/2 activation. We conclude that activation of P2X(7) receptors in astrocytes enhances P2Y(2) mRNA expression by a mechanism involving both calcium influx and PKC/MAPK signalling pathways.

Manganese suppresses ATP-dependent intercellular calcium waves in astrocyte networks through alteration of mitochondrial and endoplasmic reticulum calcium dynamics

The neurotoxicity of manganese [Mn] is due in part to glutamate excitotoxicity. Release of ATP by astrocytes is a critical modulator of glutamatergic neurotransmission, which is regulated by calcium (Ca 2+) waves that propagate through astrocytic networks in response to synaptic activity. It was postulated that Mn alters ATP-dependent intracellular Ca 2+ dynamics in astrocytes, thereby suppressing Ca 2+ wave activity. Confluent primary cultures of cortical astrocytes were loaded with the Ca 2+-sensitive dye fluo-4 and examined by fluorescence microscopy for Ca 2+ wave activity following micropipet mechanical stimulation of a single cell. Mitochondrial Ca 2+ was evaluated by fluorescence microscopy following addition of ATP using the mitochondrial-specific Ca 2+ dye rhod-2-AM. Imaging studies revealed that pretreatment of astrocytes with 1–10 μM Mn significantly reduced the rate, area, and amplitude of mechanically induced Ca 2+ waves. This attenuation was not a result of inhibited mitochondrial calcium uptake because robust calcium waves were still observed following pretreatment of astrocytes with Ru360, an inhibitor of mitochondrial Ca 2+ uptake, either in coupling or uncoupling conditions. However, determination of endoplasmic reticulum (ER) Ca 2+ levels in cells using the sarco/endoplasmic reticulum Ca 2+-ATPase inhibitor thapsigargin indicated that Mn reduced the available pool of releasable ER Ca 2+ at concentrations as low as 1 μM. Examination of ATP-stimulated changes in mitochondrial Ca 2+ indicated that, in cells pretreated with Mn, mitochondria retained high levels of Ca 2+. It is concluded that exposure of astrocytes to low concentrations of Mn 2+ results in sequestration of Ca 2+ within the mitochondria that reduces the available pool of releasable Ca 2+ within the ER, thereby inhibiting calcium wave activity.

Functional demonstration of surface carbonic anhydrase IV activity on rat astrocytes

Buffering of the brain extracellular fluid is catalyzed by carbonic anhydrase (CA) activity. Whereas the extracellular isoform CA XIV has been localized exclusively to neurons in the brain, and to glial cells in the retina, there has been uncertainty regarding the form or forms of CA on the surface of brain astrocytes. We addressed this issue using physiological methods on cultured and acutely dissociated rat astrocytes. Prior work showed that the intracellular lactate-induced acidification (LIA) of astrocytes is diminished by benzolamide, a poorly permeant, nonspecific CA inhibitor. We demonstrate that pretreatment of astrocytes with phosphatidylinositol-specific phospholipase C (PI-PLC) results in a similar inhibition of the mean LIA (by 66 +/- 3%), suggesting that the glycosylphosphatidylinositol-anchored CA IV was responsible. Pretreatment of astrocytes with CA IV inhibitory antisera also markedly reduced the mean LIA in both cultured cortical (by 46 +/- 4%) and acutely dissociated hippocampal astrocytes (by 54 +/- 8%). Pre-immune sera had no effect. The inhibition produced by PIPLC or CA IV antisera was not significantly less than that by benzolamide, suggesting that the majority of detectable surface CA activity was attributable to CA IV. Thus, our data collectively document the presence of CAIV on the surface of brain astrocytes, and suggest that this is the predominant CA isoform on these cells.

Sequential induction of heme oxygenase-1 and manganese superoxide dismutase protects cultured astrocytes against nitric oxide

Nitric oxide (NO) is a widely recognized mediator of physiological and pathophysiological signal transmission. In an attempt to better understand the molecular actions of NO in astrocytes, stress protein expression in response to NO donor sodium nitroprusside was investigated. Heme oxygenase-1 (HO-1) has been identified as an inducer of manganese superoxide dismutase (MnSOD), playing a cytoprotective role under the condition of nitrosative stress. We present evidence that the sequential induction of HO-1 and MnSOD protects astrocytes from NO toxicity: (1) both HO-1 and MnSOD expression were induced by NO; (2) NO-mediated increase in MnSOD activity was partly abolished by HO-1 inhibitor Zn(II) protoporphyrin IX (ZnPP); (3) pretreatment of astrocytes with a nontoxic dose of NO protected the cells against the later treatment with a toxic dose of NO; (4) inhibition of HO-1 by ZnPP sensitized astrocytes to the nontoxic dose of NO resulting in a marked cytotoxicity; and (5) adenovirus-mediated overexpression of MnSOD protected astrocytes from the NO toxicity. The molecular action of NO in astrocytes appears to be dose-dependent. While a high dose of NO exerts cytotoxicity leading to the tissue damage in the central nervous system, a low dose of NO may act as an important signaling molecule in astrocytes with concurrent induction of cytoprotective proteins such as HO-1 and MnSOD.

Cytodifferentiation Enhances Erk Activation Induced by Endothelin-1 in Primary Cultured Astrocytes

We have previously demonstrated that endothelin-1 (ET-1)-induced extracellular signal-regulated kinase (Erk) activity via the ETB receptor (EDNRB) is mediated through two independent pathways, a protein kinase C-dependent pathway and a pertussis toxin (PTX)-sensitive pathway, in astrocytes. In this study, we showed that the molar potency of ET-1 to induce Erk activation was two orders of magnitude higher in dibutyryl cAMP (DBcAMP)-treated astrocytes than in quiescent astrocytes. This DBcAMP-enhanced molar potency of ET-1 in Erk activation was selectively inhibited by pretreatment of astrocytes with PTX. The expression level of EDNRB in astrocytes was markedly upregulated by DBcAMP-induced cytodifferentiation. However, this up-regulation was simply attributed to the high expression of low-affinity sites. The molar potency of ET-1 to induce both stimulation of inositol trisphosphate production and activation of protein kinase C in DBcAMP-treated astrocytes was similar to that in quiescent astrocytes. On the contrary, the molar potency of ET-1 to induce accumulation of Ras-GTP was two orders of magnitude higher in DBcAMP-treated astrocytes than in quiescent astrocytes, which was consistent with the case of ET-1-induced Erk activation. Moreover, the ET-1-induced Ras activation was PTX sensitive. These results suggest that cytodifferentiation selectively enhances the PTXsensitive Ras/Erk pathway induced by ET-1 in astrocytes, and that cytodifferentiation-induced EDNRB up-regulation might not contribute to this selective potentiation of ET-1 signaling.

Properties of Ca(2+)-dependent exocytosis in cultured astrocytes.

Astrocytes, a subtype of glial cells, have numerous characteristics that were previously considered exclusive for neurons. One of these characteristics is a cytosolic [Ca2+] oscillation that controls the release of the chemical transmitter glutamate and atrial natriuretic peptide. These chemical messengers appear to be released from astrocytes via Ca(2+)-dependent exocytosis. In the present study, patch-clamp membrane capacitance measurements were used to monitor changes in the membrane area of a single astrocyte, while the photolysis of caged calcium compounds by a UV flash was used to elicit steps in [Ca2+]i to determine the exocytotic properties of astrocytes. Experiments show that astrocytes exhibit Ca(2+)-dependent increases in membrane capacitance, with an apparent Kd value of approximately 20 microM [Ca2+]i. The delay between the flash delivery and the peak rate in membrane capacitance increase is in the range of tens to hundreds of milliseconds. The pretreatment of astrocytes by the tetanus neurotoxin, which specifically cleaves the neuronal/neuroendocrine type of SNARE protein synaptobrevin, abolished flash-induced membrane capacitance increases, suggesting that Ca(2+)-dependent membrane capacitance changes involve tetanus neurotoxin-sensitive SNARE-mediated vesicular exocytosis. Immunocytochemical experiments show distinct populations of vesicles containing glutamate and atrial natriuretic peptide in astrocytes. We conclude that the recorded Ca(2+)-dependent changes in membrane capacitance represent regulated exocytosis from multiple types of vesicles, about 100 times slower than the exocytotic response in neurons.

The antiapoptotic effect of guanosine is mediated by the activation of the PI 3-kinase/AKT/PKB pathway in cultured rat astrocytes.

Guanosine has many trophic effects in the CNS, including the stimulation of neurotrophic factor synthesis and release by astrocytes, which protect neurons against excitotoxic death. Therefore, we questioned whether guanosine protected astrocytes against apoptosis induced by staurosporine. We evaluated apoptosis in cultured rat brain astrocytes, following exposure (3 h) to 100 nM staurosporine by acridine orange staining or by oligonucleosome, or caspase-3 ELISA assays. Staurosporine promoted apoptosis rapidly, reaching its maximal effect (approximately 10-fold over basal apoptotic values) in 18-24 h after its administration to astrocytes. Guanosine, added to the culture medium for 4 h, starting from 1 h prior to staurosporine, reduced the proportion of apoptotic cells in a concentration-dependent manner. The IC50 value for the inhibitory effect of guanosine is 7.5 x 10(-5) M. The protective effect of guanosine was not affected by inhibiting the nucleoside transporters by propentophylline, or by the selective antagonists of the adenosine A1 or A2 receptors (DPCPX or DMPX), or by an antagonist of the P2X and P2Y purine receptors (suramin). In contrast, pretreatment of astrocytes with pertussis toxin, which uncouples Gi-proteins from their receptors, abolished the antiapoptotic effect of guanosine. The protective effect of guanosine was also reduced by pretreatment of astrocytes with inhibitors of the phosphoinositide 3-kinase (PI3K; LY294002, 30 microM) or the MAPK pathway (PD98059, 10 microM). Addition of guanosine caused a rapid phosphorylation of Akt/PKB, and glycogen synthase kinase-3beta (GSK-3beta) and induced an upregulation of Bcl-2 mRNA and protein expression. These data demonstrate that guanosine protects astrocytes against staurosporine-induced apoptosis by activating multiple pathways, and these are mediated by a Gi-protein-coupled putative guanosine receptor.

Metabotropic glutamate receptors activate phospholipase D in astrocytes through a protein kinase C-dependent and Rho-independent pathway

Metabotropic glutamate receptors (mGluRs) are G protein-coupled receptors that mediate phospholipase D (PLD) activation in brain, but the mechanism underlying this response remains unclear. Here we used primary cultures of astrocytes as a cell model to explore the mechanism that links mGluRs to PLD. Glutamate activated both phospholipase C (PLC) and PLD with equal potency and this effect was mimicked by l-cysteinesulfinic acid, a putative neurotransmitter previously shown to activate mGluRs coupled to PLD, but not PLC, in adult brain. PLD activation by glutamate was dependent on Ca 2+ mobilization and fully blocked by both protein kinase C (PKC) inhibitors and PKC down-regulation, suggesting that PLD activation is secondary to PLC stimulation. Furthermore, brefeldin A, an inhibitor of ADP-ribosylation factor (ARF) activation, partially inhibited the activation of PLD by glutamate. By contrast, pretreatment of astrocytes with Clostridium difficile toxin B, which inactivates small G proteins of the Rho family (Rho, Rac, and Cdc42), had no effect on PLD stimulation by glutamate. Taken together, these results indicate that PLD activation by mGluRs in astrocytes is dependent on PKC and small G proteins of the ARF family, but does not require Rho proteins.

Interleukin-4 and interleukin-10 modulate nuclear factor kappaB activity and nitric oxide synthase-2 expression in Theiler's virus-infected brain astrocytes.

In brain astrocytes, nuclear factor kappaB (NF-kappaB) is activated by stimuli that produce cellular stress causing the expression of genes involved in defence, including the inducible nitric oxide synthase (NOS-2). Theiler's murine encephalomyelitis virus (TMEV) induces a persistent CNS infection and chronic immune-mediated demyelination, similar to human multiple sclerosis. The cytokines interleukin (IL)-4 and IL-10 inhibit the expression of proinflammatory cytokines, counteracting the inflammatory process. Our study reports that infection of cultured astrocytes with TMEV resulted in a time-dependent phosphorylation of IkappaBalpha, degradation of IkappaBalpha and IkappaBbeta, activation of NF-kappaB and expression of NOS-2. The proteasome inhibitor MG-132 blocked TMEV-induced nitrite accumulation, NOS-2 mRNA expression and phospho-IkappaBalpha degradation, suggesting NF-kappaB-dependent NOS-2 expression. Pretreatment of astrocytes with IL-4 or IL-10 decreased p65 nuclear translocation, NF-kappaB binding activity and NOS-2 transcription. IL-4 and IL-10 caused an accumulation of IkappaBalpha in TMEV-infected astrocytes without affecting IkappaBbeta levels. The IkappaB kinase activity and the degradation rate of both IkappaBs were not modified by either cytokine, suggesting de novo synthesis of IkappaBalpha. Indeed, IL-4 or IL-10 up-regulated IkappaBalpha mRNA levels after TMEV infection. Therefore, the accumulation of IkappaBalpha might impair the translocation of the NF-kappaB to the nucleus, mediating the inhibition of NF-kappaB activity. Overall, these data suggest a novel mechanism of action of IL-4 and IL-10, which abrogates NOS-2 expression in viral-infected glial cells.

Peroxynitrite decomposition catalyst prevents apoptotic cell death in a human astrocytoma cell line incubated with supernatants of HIV-infected macrophages

BackgroundOxidative stress has shown to contribute in the mechanisms underlying apoptotic cell death occuring in AIDS-dementia complex. Here we investigated the role of peroxynitrite in apoptosis occurring in astroglial cells incubated with supernatants of HIV-infected human primary macrophages (M/M).ResultsFlow cytometric analysis (FACS) of human cultured astrocytes shortly incubated with HIV-1-infected M/M supernatants showed apoptotic cell death, an effect accompanied by pronounced staining for nitrotyrosine (footprint of peroxynitrite) and by abnormal formation of malondialdehyde (MDA). Pretreatment of astrocytes with the peroxynitrite decomposition catalyst FeTMPS antagonized HIV-related astrocytic apoptosis, MDA formation and nitrotyrosine staining.ConclusionsTaken together, our results suggest that inibition of peroxynitrite leads to protection against peroxidative stress accompanying HIV-related apoptosis of astrocytes. Overall results support the role of peroxynitrite in HIV-related programmed death of astrocytes and suggest the use of peroxynitrite decomposition catalyst to counteract HIV-1-related neurological disorders.