Cultured Rat Hippocampal Astrocytes Research Articles

Blood-brain barrier dysfunction promotes astrocyte senescence through albumin-induced TGFβ signaling activation.

Blood-brain barrier dysfunction (BBBD) and accumulation of senescent astrocytes occur during brain aging and contribute to neuroinflammation and disease. Here, we explored the relationship between these two age-related events, hypothesizing that chronic hippocampal exposure to the blood-borne protein serum albumin could induce stress-induced premature senescence (SIPS) in astrocytes via transforming growth factor beta 1 (TGFβ) signaling. We found that 1 week of albumin exposure significantly increased TGFβ signaling and senescence marker expression in cultured rat hippocampal astrocytes. These changes were preventable by pharmacological inhibition of the type I TGFβ receptor (TGFβR) ALK5. To study these effects in vivo, we utilized an animal model of BBBD in which albumin was continuously infused into the lateral ventricles of adult mice. Consistent with our in vitro results, 1 week of albumin infusion significantly increased TGFβ signaling activation and the burden of senescent astrocytes in hippocampal tissue. Pharmacological inhibition of ALK5 TGFβR or conditional genetic knockdown of astrocytic TGFβR prior to albumin infusion was sufficient to prevent albumin-induced astrocyte senescence. Together, these results establish a link between TGFβ signaling activation and astrocyte senescence and suggest that prolonged exposure to serum albumin due to BBBD can trigger these phenotypic changes.

PPARβ/δ activation protects against corticosterone-induced ER stress in astrocytes by inhibiting the CpG hypermethylation of microRNA-181a

Increasing evidence indicates that peroxisome proliferator-activated receptors (PPARs) play neuroprotective roles in various neurodegenerative disease models in vivo and in vitro. However, the underlying mechanisms remain unclear. Astrocyte proliferation is a key process in neural development and plays significant roles in the regeneration of neural tissue after a penetrating injury. Corticosterone can significantly reduce the expression of glial fibrillary acid protein (GFAP) in cultured rat hippocampal astrocytes in vitro, and induce astrocytic dysfunction. Our research found that corticosterone treatment resulted in astrocyte damage and reduced the expression of PPARβ/δ. GW0742, a selective and high-affinity PPARβ/δ agonist, attenuated the corticosterone-induced astrocyte damage, but also significantly reversed the increase in the expression of GRP78 and CHOP, the two predominant proteins in endoplasmic reticulum (ER) stress. Moreover, GW0742 decreased the levels of caspase-12 and cleaved caspase-3, thereby protecting astrocytes against corticosterone-induced astrocyte apoptosis. We then confirmed that GRP78 was a target gene of microRNA-181a and found that PPARβ/δ activation increased microRNA-181a levels. Finally, we demonstrated that PPARβ/δ activation by GW0742 noticeably inhibited the activities and expression of DNA methyltransferases, and reduced the corticosterone-induced CpG island hypermethylation of microRNA-181a1 in astrocytes. Therefore, the present study is the first to reveal that PPARβ/δ activation suppresses CpG island hypermethylation-associated silencing of microRNA-181a and thereby protects against ER stress-induced damage in astrocytes. Our findings suggest that PPARβ/δ activation in astrocytes might be a promising target for regulating ER stress-induced astrocytic injury.

D-Serine Is a Substrate for Neutral Amino Acid Transporters ASCT1/SLC1A4 and ASCT2/SLC1A5, and Is Transported by Both Subtypes in Rat Hippocampal Astrocyte Cultures.

N-methyl-D-aspartate (NMDA) receptors play critical roles in synaptic transmission and plasticity. Activation of NMDA receptors by synaptically released L-glutamate also requires occupancy of co-agonist binding sites in the tetrameric receptor by either glycine or D-serine. Although D-serine appears to be the predominant co-agonist at synaptic NMDA receptors, the transport mechanisms involved in D-serine homeostasis in brain are poorly understood. In this work we show that the SLC1 amino acid transporter family members SLC1A4 (ASCT1) and SLC1A5 (ASCT2) mediate homo- and hetero-exchange of D-serine with physiologically relevant kinetic parameters. In addition, the selectivity profile of D-serine uptake in cultured rat hippocampal astrocytes is consistent with uptake mediated by both ASCT1 and ASCT2. Together these data suggest that SLC1A4 (ASCT1) may represent an important route of Na-dependent D-serine flux in the brain that has the ability to regulate extracellular D-serine and thereby NMDA receptor activity.

Ebselen alters cellular oxidative status and induces endoplasmic reticulum stress in rat hippocampal astrocytes.

Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) is an organoselenium radical scavenger compound, which has strong antioxidant and anti-inflammatory effects. Because of its properties, it may be protective against injury to the nervous tissue. However, evidence suggests that its glutathione peroxidase activity could underlie certain deleterious actions on cell physiology. In this study we have analyzed the effect of ebselen on rat hippocampal astrocytes in culture. Cellular oxidative status, cytosolic free-Ca(2+) concentration ([Ca(2+)]c), setting of endoplasmic reticulum stress and phosphorylation of glial fibrillary acidic protein and major mitogen-activated protein kinases were analyzed. Our results show that ebselen induced a concentration-dependent increase in the generation of reactive oxygen species in the mitochondria. We observed a concentration-dependent increase in global cysteine oxidation and in the level of malondialdehyde in the presence of ebselen. We also detected increases in catalase, glutathione S-transferase and glutathione reductase activity. Ebselen also evoked a concentration-dependent increase in [Ca(2+)]c. Moreover, we observed a concentration-dependent increase in the phosphorylation of the unfolded protein response markers, eukaryotic translation initiation factor 2α and X-box binding protein 1. Finally, ebselen also induced an increase in the phosphorylation of glial fibrillary acidic protein, SAPK/JNK, p38 MAPK and p44/42 MAPK. Our results provide strong evidence that implicate endoplasmic reticulum stress and activation of crucial mitogen-activated protein kinases in an oxidative damage of cells in the presence of ebselen. The compound thus might exert deleterious actions on astrocyte physiology that could compromise their function.

Role of astrocytes in neuronal energetic production and its relationship with neurodegeneration

Event Abstract Back to Event Role of astrocytes in neuronal energetic production and its relationship with neurodegeneration Felipe Vargas-Restrepo1, Angelica M. Sabogal-Guaqueta1 and Gloria Patricia Cardona Gomez1* 1 University of Antioquia, Group of Neuroscience of Antioquia, Colombia Astrocytes are specialized glial cells that make important contributions to neuronal support and the blood-brain barrier. This barrier limits the passage of certain fatty acids and lipids to the Central Nervous System (CNS), which the majority of lipids found in this tissue must be synthesized right there. Astrocytes are involved in the conversion and production of fatty acids in the CNS as well as production of lipid messengers that apparently are involved in the activity of neurons and neuroprotective or neurodegenerative processes. For this reason, we review the role of astrocytes in energy production and its relationship to neuronal lipid changes in neuroprotective and neurodegenerative processes. Figure 1 Acknowledgements We want to thank to the Colciencias project # 111565740581. References Amruthesh S.C., Boerschel M.F., McKinney J.S., Willoughby K.A., Ellis E.F. (1993) Metabolism of arachidonic acid to epoxyeicosatrienoic acids, hydroxyeicosatetraenoic acids, and prostaglandins in cultured rat hippocampal astrocytes. J Neurochem. 61, 150-159. Bezzi P., Carmignoto G., Pasti L., Vesce S., Rossi D., Rizzini B.L., et al. (1998) Prostaglandins stimulate calcium-dependent glutamate release in astrocytes. Nature. 391, 281-285. Moore S.A., Yoder E., Murphy S., Dutton G.R., Spector A.A. (1991) Astrocytes, Not Neurons, Produce Docosahexaenoic Acid (22:6ω-3) and Arachidonic Acid (20:4ω-6). Journal of Neurochemistry. 56, 518-524. Posada-Duque R.A., Barreto G.E., Cardona-Gomez G.P. (2014) Protection after stroke: cellular effectors of neurovascular unit integrity. Front Cell Neurosci. 8, 231. doi: 10.3389/fncel.2014.00231. Staub F., Winkler A., Peters J., Goerke U., Kempski O., Baethmann A. (1995) Clearance and metabolism of arachidonic acid by C6 glioma cells and astrocytes. Neurochem Res. 20, 1449-1456. Keywords: Fatty Acids, Astrocytes, neurodegeneration, Neuroprotection, energy production Conference: Latin-American School on glial cells in the diseased brain (IBRO), Bogotá, Colombia, 13 Jul - 17 Jul, 2015. Presentation Type: Poster Presentation Topic: Traumatic and Cerebrovascular diseases Citation: Vargas-Restrepo F, Sabogal-Guaqueta AM and Cardona Gomez G (2015). Role of astrocytes in neuronal energetic production and its relationship with neurodegeneration. Conference Abstract: Latin-American School on glial cells in the diseased brain (IBRO). doi: 10.3389/conf.fncel.2015.35.00006 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: 19 Mar 2015; Published Online: 16 May 2015. * Correspondence: Prof. Gloria Patricia Cardona Gomez, University of Antioquia, Group of Neuroscience of Antioquia, Medellin, Antioquia, 05001000, Colombia, patricia.cardona@neurociencias.udea.edu.co 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 Felipe Vargas-Restrepo Angelica M Sabogal-Guaqueta Gloria Patricia Cardona Gomez Google Felipe Vargas-Restrepo Angelica M Sabogal-Guaqueta Gloria Patricia Cardona Gomez Google Scholar Felipe Vargas-Restrepo Angelica M Sabogal-Guaqueta Gloria Patricia Cardona Gomez PubMed Felipe Vargas-Restrepo Angelica M Sabogal-Guaqueta Gloria Patricia Cardona Gomez 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.

Epoxyeicosatrienoic acids pretreatment improves amyloid β-induced mitochondrial dysfunction in cultured rat hippocampal astrocytes

Amyloid-β (Aβ) has long been implicated as a causative protein in Alzheimer's disease. Cellular Aβ accumulation is toxic and causes mitochondrial dysfunction, which precedes clinical symptoms of Alzheimer's disease pathology. In the present study, we explored the possible use of epoxyeicosatrienoic acids (EETs), epoxide metabolites of arachidonic acid, as therapeutic target against Aβ-induced mitochondrial impairment using cultured neonatal hippocampal astrocytes. Inhibition of endogenous EET production by a selective epoxygenase inhibitor, MS-PPOH, caused a greater reduction in mitochondrial membrane potential in the presence of Aβ (1, 10 μM) exposure versus absence of Aβ. MS-PPOH preincubation also aggravated Aβ-induced mitochondrial fragmentation. Preincubation of the cells with either 14,15- or 11,12-EET prevented this mitochondrial depolarization and fragmentation. EET pretreatment also further improved the reduction observed in mitochondrial oxygen consumption in the presence of Aβ. Preincubation of the cells with EETs significantly improved cellular respiration under basal condition and in the presence of the protonophore, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP). The uncoupling of ATP synthase from the electron transfer chain that occurred in Aβ-treated cells was also prevented by preincubation with EETs. Lastly, cellular reactive oxygen species production, a hallmark of Aβ toxicity, also showed significant reduction in the presence of EETs. We have previously shown that Aβ reduces EET synthesis in rat brain homogenates and cultured hippocampal astrocytes and neurons (Sarkar P, Narayanan J, Harder DR. Differential effect of amyloid beta on the cytochrome P450 epoxygenase activity in rat brain. Neuroscience 194: 241-249, 2011). We conclude that reduction of endogenous EETs may be one of the mechanisms through which Aβ inflicts toxicity and thus supplementing the cells with exogenous EETs improves mitochondrial dynamics and prevents metabolic impairment.

Ebselen Alters Mitochondrial Physiology and Reduces Viability of Rat Hippocampal Astrocytes

The seleno-organic compound and radical scavenger ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) have been extensively employed as an anti-inflammatory and neuroprotective compound. However, its glutathione peroxidase activity at the expense of cellular thiols groups could underlie certain deleterious actions of the compound on cell physiology. In this study, we have analyzed the effect of ebselen on rat hippocampal astrocytes in culture. Cellular viability, the intracellular free-Ca(2+) concentration ([Ca(2+)]c), the mitochondrial free-Ca(2+) concentration ([Ca(2+)]m), and mitochondrial membrane potential (ψm) were analyzed. The caspase-3 activity was also assayed. Our results show that cell viability was reduced by treatment of cells with ebselen, depending on the concentration employed. In the presence of ebselen, we observed an initial transient increase in [Ca(2+)]c that was then followed by a progressive increase to an elevated plateau. We also observed a transient increase in [Ca(2+)]m in the presence of ebselen that returned toward a value over the prestimulation level. The compound induced depolarization of ψm and altered the permeability of the mitochondrial membrane. Additionally, a disruption of the mitochondrial network was observed. Finally, we did not detect changes in caspase-3 activation in response to ebselen treatment. Collectively, these data support the likelihood of ebselen, depending on the concentration employed, reduces viability of rat hippocampal astrocytes via its action on the mitochondrial activity. These may be early effects that do not involve caspase-3 activation. We conclude that, depending on the concentration used, ebselen might exert deleterious actions on astrocyte physiology that could compromise cell function.

A2a Adenosine Receptor Mediates PKA-Dependent Glutamate Release from Synaptic-like Vesicles and Ca2+ Efflux from an IP3- and Ryanodine-Insensitive Intracellular Calcium Store in Astrocytes

Background/Aims: The mechanism underlying transmitter release from astrocytes is not fully understood. The present study examined A_2a adenosine receptor-mediated glutamate release and intracellular Ca²⁺ rise in cultured rat hippocampal astrocytes. Methods: Intracellular amino acids were measured with HPLC. Glutamate release from astrocytes and intracellular Ca²⁺ mobilizations were monitored in the NADH imaging, FM1-43 imaging, and fura-2 imaging. The siRNA to silence the A_2a adenosine receptor-targeted gene was constructed and transfected into cells. Results: Glutamate was condensed in ‘synaptic-like vesicle’ fractions. In the NADH imaging, CGS21680, an agonist of A2a adenosine receptors, increased NADH fluorescent signals, that reflects glutamate release, and the effect was inhibited by DMPX, an inhibitor of A_2a adenosine receptors, H-89, a PKA inhibitor, vesicular transport inhibitors, or botulinum toxin-A, an exocytosis inhibitor. In the FM1-43 imaging to see vesicular recycling, CGS21680 decreased FM1-43 fluorescent signals, that was also prevented by DMPX, H-89, vesicular transport inhibitors, or botulinum toxin-A. CGS21680 increased intracellular Ca²⁺ concentrations both in Ca²⁺-containing and -free extracellular solution. The Ca²⁺ rise was inhibited by DMPX, H-89, or the vesicular transport inhibitor brefeldin A, but it was not affected by inhibitors for phospholipase C, IP₃ receptor, and ryanodine receptor. CGS21680-induced glutamate release and intracellular Ca²⁺ rise were prevented by knocking-down A_2a adenosine receptor. Conclusion: The results of the present study show that A_2a adenosine receptor/PKA promotes glutamate release from synaptic-like vesicles and stimulates Ca²⁺ efflux from an IP₃- and ryanodine-insensitive intracellular calcium store.

Ratiometric high-resolution imaging of JC-1 fluorescence reveals the subcellular heterogeneity of astrocytic mitochondria

Using the mitochondrial potential (ΔΨm) marker JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide) and high-resolution imaging, we functionally analyzed mitochondria in cultured rat hippocampal astrocytes. Ratiometric detection of JC-1 fluorescence identified mitochondria with high and low ΔΨm. Mitochondrial density was highest in the perinuclear region, whereas ΔΨm tended to be higher in peripheral mitochondria. Spontaneous ΔΨm fluctuations, representing episodes of increased energization, appeared in individual mitochondria or synchronized in mitochondrial clusters. They continued upon withdrawal of extracellular Ca2+, but were antagonized by dantrolene or 2-aminoethoxydiphenylborate (2-APB). Fluo-3 imaging revealed local cytosolic Ca2+ transients with similar kinetics that also were depressed by dantrolene and 2-APB. Massive cellular Ca2+ load or metabolic impairment abolished ΔΨm fluctuations, occasionally evoking heterogeneous mitochondrial depolarizations. The detected diversity and ΔΨm heterogeneity of mitochondria confirms that even in less structurally polarized cells, such as astrocytes, specialized mitochondrial subpopulations coexist. We conclude that ΔΨm fluctuations are an indication of mitochondrial viability and are triggered by local Ca2+ release from the endoplasmic reticulum. This spatially confined organelle crosstalk contributes to the functional heterogeneity of mitochondria and may serve to adapt the metabolism of glial cells to the activity and metabolic demand of complex neuronal networks. The established ratiometric JC-1 imaging—especially combined with two-photon microscopy—enables quantitative functional analyses of individual mitochondria as well as the comparison of mitochondrial heterogeneity in different preparations and/or treatment conditions.

Ethanol reduces kainate-evoked glutamate secretion in rat hippocampal astrocytes

In this study we have used rat hippocampal astrocytes in culture to investigate the effect of ethanol on kainate-induced glutamate secretion. Our results show that kainate (10 μM to 500 μM) stimulated glutamate release from astrocytes. Preincubation of astrocytes in the presence of ethanol induced a concentration-dependent (1 mM–50 mM) inhibition of glutamate release caused by stimulation of cells with 100 μM kainate. Inhibition of alcohol-dehydrogenase, by preincubation of astrocytes in the presence of 4-methylpyrazole (1 mM), abolished ethanol-induced inhibition of glutamate release in response to kainate. On the other hand, preincubation of astrocytes in the presence of the antioxidant cinnamtannin B-1 (10 μM) also blocked ethanol inhibitory action on glutamate release in response to kainate. Ethanol (50 mM) reduced Ca 2+ mobilization in response to kainate, whereas cinnamtannin B-1 reversed the inhibitory action of ethanol on Ca 2+ mobilization by kainate. Our results are consistent with an inhibitory action of ethanol on glutamate secretion from hippocampal astrocytes. The inhibitory effects of ethanol are probably due to its oxidative metabolization, involves reactive oxygen species production, and a lower Ca 2+ mobilization by kainate. Taking into account the pivotal role that astrocytes play within the central nervous system, especially in relation to neurons, the negative effects of ethanol on the release of glutamate might affect neuron-glia communication in the hippocampus, which might lead to functional defects in the brain.

Transglutaminase 2 expression induced by lipopolysaccharide stimulation together with NO synthase induction in cultured astrocytes

Activation of glia has been observed in neurodegenerative diseases such as Parkinson's disease (PD), Alzheimer's disease (AD), multiple sclerosis and brain ischemia. Excessive production of nitric oxide (NO), as a consequence of increased inducible NO synthase (iNOS) in glia, contributes to neurodegeneration. Transglutaminase 2 (TG2) is a cross-linking enzyme, which is activated in neurodegenerative diseases such as PD, AD and Huntington's diseases. However, mechanisms contributing to the increased TG activity in neurodegenerative diseases remain to be clarified. In the present study, we examined the expression of TG2 in cultured rat hippocampal astrocytes activated with lipopolysaccharide (LPS), which is generally used for a stimulant of iNOS induction. The expressions of TG2 mRNA and protein were increased by stimulation with LPS in a dose-dependent manner. The LPS-induced TG2 expression was diminished by ammonium pyrrolidine-1-carbodithioate; an inhibitor for nuclear factor (NF)-κB activation, suggesting the factors involved. Both expressions of TG2 and iNOS induced by LPS stimulation were suppressed by an antioxidant, ethyl pyruvate, in a dose-dependent manner. Furthermore, they were also suppressed by cystamine, an inhibitor of TG activity. These results suggest that the level of TG2 expression is regulated by oxidative stress and the activity of TG itself, and that the induction of iNOS and NO production are closely associated with TG2 expression in LPS-stimulated activation of astrocytes.

Extracellular high K+ stimulates vesicular glutamate release from astrocytes by activating voltage‐dependent calcium channels

Extracellular high K(+) (75 mM) increased intracellular Ca(2+) concentrations in cultured rat hippocampal astrocytes, and the Ca(2+) rise was abolished by deleting extracellular Ca(2+) or cadmium, a non-selective inhibitor of voltage-dependent calcium channels (VDCCs). In the reverse transcription-polymerase chain reaction analysis, cultured astrocytes expressed mRNAs for L type-VDCC subunits such as alpha1B, alpha1C, alpha1D, and alpha1E. Extracellular high K(+) (75 mM) stimulated glutamate release from astrocytes. The glutamate release was not prevented by the glutamate transporter inhibitor, L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC), or deleting extracellular Na(+), but otherwise it was clearly inhibited by deleting extracellular Ca(2+), cadmium, vesicular transport inhibitors such as brefeldin A, bafilomycin A1, and latrunculin B, or botulinum toxin-A, an exocytosis inhibitor. Extracellular high K(+) (75 mM) bleached fluorescent signals of FM1-43, taken up into the vesicular membrane in astrocytes, that was also inhibited by deleting extracellular Ca(2+), cadmium, brefeldin A, bafilomycin A1, latrunculin B, or botulinum toxin-A, but not by PDC. Taken together, the results of the present study indicate that extracellular high K(+)-evoked depolarization activates VDCCs expressed in astrocytes, causing an increase in intracellular Ca(2+) concentrations through VDCCs, which triggers vesicular glutamate release from astrocytes, independently of reverse transport through glutamate transporters.

Alpha-melanocyte-stimulating hormone increases the expression of vascular endothelial growth factor in rat hippocampal astrocytes in vitro

Alpha-melanocyte-stimulating hormone (α-MSH) is a regulator of many physiological processes that has neuroprotective characteristics. In this work, we showed the expression of the melanocortin receptor MC4R in cultured rat hippocampal astrocytes. We found a twofold increase in the expression of vascular endothelial growth factor in cultured rat hippocampal astrocytes in one hour after application of α-MSH at concentrations of 0.1 to 10 nM. The selective MC4R antagonist HS028 prevented stimulation of VEGF expression. These data suggest that the neuroprotective effects of α-MSH may be mediated by the stimulation of production of growth/neurotrophic factors in hippocampal astrocytes.

Ebselen increases cytosolic free Ca 2+ concentration, stimulates glutamate release and increases GFAP content in rat hippocampal astrocytes

We have investigated the effect of the seleno-organic compound and radical scavenger ebselen on rat hippocampal astrocytes in culture. Throughout our study we carried out determinations of [Ca 2+] c in fura-2-loaded cells by single cell imaging, glutamate secretion employing an enzymatic-based assay and GFAP expression, which was monitorized by immunocytochemistry and confocal microscopy. Our results show that ebselen (1–20 μM) dose dependently increases [Ca 2+] c, stimulates glutamate release and increases GFAP content, a hallmark of astrocyte reactivity. Ebselen did not alter significantly cell viability as assayed by determination of LDH release into the extracellular medium. Ebselen-evoked glutamate release and increase in GFAP content were Ca 2+-dependent, because incubation of astrocytes in the absence of extracellular Ca 2+ (medium containing 0.5 mM EGTA) and in the presence of the intracellular Ca 2+ chelator BAPTA (10 μM) significantly reduced ebselen-evoked changes in these parameters. The effects of ebselen we have observed may underline various signalling pathways which are important for cell proliferation, differentiation and function. However, aberrations in astroglial physiology could significantly compromise brain function, due to their role as modulators of neuron activity. Therefore, we consider that careful attention should be paid when employing ebselen as a prophylactic agent against brain damage.

Ethanol induces glutamate secretion by Ca 2+ mobilization and ROS generation in rat hippocampal astrocytes

In this study we have investigated the effect of ethanol on [Ca 2+] c by microfluorimetry and glutamate secretion using an enzyme-linked system, in rat hippocampal astrocytes in culture. Our results show that ethanol (1–200 mM) evoked a dose-dependent increase in glutamate secretion. 50 mM ethanol, a concentration within the range of blood alcohol levels in intoxicated humans, induced a release of Ca 2+ from intracellular stores in the form of oscillations. Ca 2+-mobilizing effect of ethanol was not prevented by preincubation of cells in the presence of 2 mM of the antioxidant dithiothreitol. Ethanol-evoked glutamate secretion was reduced when extracellular Ca 2+ was omitted (medium containing 0.5 mM EGTA) and following preincubation of astrocytes in the presence of the intracellular Ca 2+ chelator 1,2-bis-( o-aminophenoxy)-ethane- N, N, N′, N′-tetraacetic acid tetraacetoxy-methyl ester (10 μM). Preincubation of astrocytes in the presence of 2 mM of the antioxidant dithiothreitol significantly reduced ethanol-evoked glutamate secretion. Finally, preincubation of astrocytes in the presence of bafilomycin (50 nM) significantly reduced ethanol-induced neurotransmitter release, indicating that exocytosis is involved in glutamate secretion. In conclusion, our results suggest that ethanol mobilizes Ca 2+ from intracellular stores, and stimulates a Ca 2+-dependent glutamate secretion, probably involving reactive oxygen species production, and therefore creating a situation potentially leading to neurotoxicity in the hippocampus.

Ethanol stimulates ROS generation by mitochondria through Ca 2+ mobilization and increases GFAP content in rat hippocampal astrocytes

We have employed rat hippocampal astrocytes in culture to investigate the effect of ethanol on reactive oxygen species (ROS) production as well as its effect on [Ca 2+] c and GFAP expression. Cells were loaded with the fluorescent probes fura-2 and H 2DCFDA for the determination of changes in [Ca 2+] c and ROS production respectively, employing spectrofluorimetry. GFAP content was determined by immunocytochemistry and confocal scanning microscopy. Our results show ROS production in response to 50 mM ethanol, that was reduced in Ca 2+-free medium (containing 0.5 mM EGTA) and in the presence of the intracellular Ca 2+ chelator BAPTA (10 μM). The effect of ethanol on ROS production was significantly reduced in the presence of the alcohol dehydrogenase inhibitor 4-methylpyrazole (1 mM), and the antioxidants resveratrol (100 μM) or catalase (300 U/ml). Preincubation of astrocytes in the presence of 10 μM antimycin plus 10 μM oligomycin to inhibit mitochondria completely blocked ethanol-evoked ROS production. In addition, ethanol led to a sustained increase in [Ca 2+] c that reached a constant level over the prestimulation values. Finally, incubation of astrocytes in the presence of ethanol increased the content of GFAP that was significantly reduced in the absence of extracellular Ca 2+ and by resveratrol and catalase pretreatment. The data obtained in the present study suggest that astrocytes are able to metabolize ethanol, which induces two effects on intracellular homeostasis: an immediate response (Ca 2+ release and ROS generation) and later changes involving GFAP expression. Both effects may underline various signaling pathways which are important for cell proliferation, differentiation and function.

Contribution of epoxyeicosatrienoic acids to the hypoxia-induced activation of Ca 2+-activated K + channel current in cultured rat hippocampal astrocytes

Brief hypoxia differentially regulates the activities of Ca 2+-activated K + channels (K Ca) in a variety of cell types. We investigated the effects of hypoxia (<2% O 2) on K Ca channel currents and on the activities of cytochrome P450 2C11 epoxygenase (CYP epoxygenase) in cultured rat hippocampal astrocytes. Exposure of astrocytes to hypoxia enhanced macroscopic outward K Ca current, increased the open state probability (NPo) of 71 pS and 161 pS single-channel K Ca currents in cell-attached patches, but failed to increase the NPo of both the 71 pS and 161 pS K Ca channel currents recorded from excised inside-out patches. The hypoxia-induced enhancement of macroscopic K Ca current was attenuated by pretreatment with tetraethylammonium (TEA, 1 mM) or during recording using low-Ca 2+ external bath solution. Exposure of astrocytes to hypoxia was associated with generation of superoxide as detected by staining of cells with the intracellular superoxide detection probe hydroethidine (HE), attenuation of the hypoxia-induced activation of unitary K Ca channel currents by superoxide dismutation with tempol, and as quantitated by high-pressure liquid chromatography/fluorescence assay using HE as a probe. In cultured astrocytes in which endogenous CYP epoxygenase activity has been inhibited with either miconazole or N-methylsulfonyl-6-(2-propargyloxyphenyl) hexanamide (MSPPOH) hypoxia failed to increase the NPo of both the 71 pS and 161 pS K Ca currents and generation of superoxide. Hypoxia increased the level of P450 epoxygenase protein and production of epoxyeicosatrienoic acids (EETs) from cultured astrocytes, as determined by immunohistochemical staining and LC/MS analysis, respectively. Exogenous 11,12-EET increased the NPo of both the 71 pS and 161 pS K Ca single-channel currents only in cell-attached but not in excised inside-out patches of cultured astrocytes. These findings indicate that hypoxia enhances the activities of two types of unitary K Ca currents in astrocytes by a mechanism that appears to involve CYP epoxygenase-dependent generation of superoxide and increased production or release of EETs.

Glutamate Release Through Glial Gap-Junction Hemichannels

Ye et al. investigated the role of gap-junction hemichannels in astrocytes and discovered that they mediated glutamate release; regulation of astrocyte hemichannel activity could thus modulate levels of extracellular glutamate in the central nervous system. Abnormal increases in extracellular glutamate, the principal excitatory neurotransmitter in brain, have been implicated in excitotoxic cell death. Under normal conditions, astrocytes accumulate glutamate, which helps to maintain low extracellular concentrations. Gap junctions, formed by aligned hemichannels on adjacent cells, can mediate the passage of molecules between some cell types, including glia. Unopposed hemichannels have been identified in astrocytes; their functions, however, have been unclear. Ye at al. used immunocytochemistry to detect hemichannels in cultured rat hippocampal astrocytes and confirmed their functionality by measuring increased uptake of a fluorescent dye after exposure to nominally divalent cation-free saline (DFCS). Dye permeability was sensitive to gap-junction blockers. The authors monitored changes in extracellular and intracellular glutamate concentration, under different ionic conditions and in the presence or absence of various pharmacological agents, and determined that glutamate efflux in response to DFCS occurred through hemichannels. Increased glutamate efflux was accompanied by a decrease in glutamate uptake, likely secondary to a disruption of ionic gradients associated with hemichannel opening. Removal of divalent cations also elicited gap-junction-blocker-sensitive release of glutamate and other amino acids from mouse optic nerve (a white matter preparation). These data suggest that astrocytes could release glutamate through hemichannels in response to low extracellular divalent cation concentration in vivo and thereby contribute to various neuropathologies.Z.-C. Ye, M. S. Wyeth, S. Baltan-Tekkok, B. R. Ransom, Functional hemichannels in astrocytes: A novel mechanism of glutamate release. J. Neurosci. 23, 3588-3596 (2003). [Abstract] [Full Text]

Glutamate Release Through Glial Gap-Junction Hemichannels

Ye et al. investigated the role of gap-junction hemichannels in astrocytes and discovered that they mediated glutamate release; regulation of astrocyte hemichannel activity could thus modulate levels of extracellular glutamate in the central nervous system. Abnormal increases in extracellular glutamate, the principal excitatory neurotransmitter in brain, have been implicated in excitotoxic cell death. Under normal conditions, astrocytes accumulate glutamate, which helps to maintain low extracellular concentrations. Gap junctions, formed by aligned hemichannels on adjacent cells, can mediate the passage of molecules between some cell types, including glia. Unopposed hemichannels have been identified in astrocytes; their functions, however, have been unclear. Ye at al . used immunocytochemistry to detect hemichannels in cultured rat hippocampal astrocytes and confirmed their functionality by measuring increased uptake of a fluorescent dye after exposure to nominally divalent cation-free saline (DFCS). Dye permeability was sensitive to gap-junction blockers. The authors monitored changes in extracellular and intracellular glutamate concentration, under different ionic conditions and in the presence or absence of various pharmacological agents, and determined that glutamate efflux in response to DFCS occurred through hemichannels. Increased glutamate efflux was accompanied by a decrease in glutamate uptake, likely secondary to a disruption of ionic gradients associated with hemichannel opening. Removal of divalent cations also elicited gap-junction-blocker-sensitive release of glutamate and other amino acids from mouse optic nerve (a white matter preparation). These data suggest that astrocytes could release glutamate through hemichannels in response to low extracellular divalent cation concentration in vivo and thereby contribute to various neuropathologies. Z.-C. Ye, M. S. Wyeth, S. Baltan-Tekkok, B. R. Ransom, Functional hemichannels in astrocytes: A novel mechanism of glutamate release. J. Neurosci. 23 , 3588-3596 (2003). [Abstract] [Full Text]

The anti-dementia drug FK960 stimulates glial glutamate release via a PKA pathway

The present study was conducted to understand the mechanism underlying the facilitatory action of FK960, an anti-dementia drug, on hippocampal neurotransmission. FK960 facilitated hippocampal neurotransmission in normal mice, and also in mice lacking the glial glutamate transporter, GLT-1 ( glut-1 −/− ), but to a lesser extent. FK960 enhanced glutamate release from cultured hippocampal astrocytes from normal rats and mice, while the drug had no effect on the release from cultured rat hippocampal neurons. The glutamate release was still obtained with cultured hippocampal astrocytes from glut-1 −/− mice, suggesting that the release is not due to GLT-1-mediated counter transport of glutamate. The FK960 action was inhibited by H-89, a selective inhibitor of cAMP-dependent protein kinase (PKA), bafilomycin A1, an inhibitor of vesicular transport, or BAPTA-AM, a chelator of intracellular Ca 2+. FK960 caused an increase in intracellular Ca 2+ concentrations by stored Ca 2+ release in cultured rat hippocampal astrocytes, and H-89 abolished the increase. Forskolin, a PKA activator, mimicked the effect of FK960 on intracellular Ca 2+ mobilizations. Taken together, it appears that FK960 stimulates glutamate release from astrocytes, likely as a result of raising intracellular Ca 2+ concentrations via a PKA pathway. The FK960 action would increase synaptic glutamate concentrations, in part responsible for the facilitation of hippocampal neurotransmission. The results of the present study may provide a new idea that agents targeting astrocytes could serve as anti-dementia drugs.