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Cultured Rat Astrocytes (cultured + rat_astrocyte)
Selected AbstractsEthanol Increases the Neurotoxic Effect of Tumor Necrosis Factor- , in Cultured Rat AstrocytesALCOHOLISM, Issue 1 2000William J. DeVito Background: The central nervous system is particularly sensitive to the cytotoxic effect of ethanol. In vivo and in vitro studies indicate that ethanol decreases cell proliferation in a number of cells types, including neurons and glial cells in the central nervous system. The cellular mechanisms involved in ethanol-induced cell toxicity, however, are unclear. In this study, we examined the effect of ethanol on tumor necrosis factor- , (TNF,)-induced cell death in a homogeneous population of cultured rat astrocytes. Methods: Flow cytometric and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenytetrazolium bromide (MTT) dye reduction analyses were performed on cultured rat astrocytes to determine the effect of alcohol on TNF, -induced cell death. Results: Flow cytometric analysis revealed that, in quiescent astrocytes, high concentrations of ethanol were required to increase DNA fragmentation and decrease cell viability. Preexposure of astrocytes to low concentrations of ethanol (10 to 50 mM), however, increased the sensitivity of astrocytes to TNF, with low TNF, concentrations (25 to 50 ng/ml) resulting in increased DNA fragmentation. Furthermore, MTT dye reduction analysis revealed that exposure of astrocytes to 5 mM ethanol was sufficient to increase the susceptibility of astrocytes to the cytotoxic effect of ethanol. In a number of cell types, TNF, receptor binding results in the activation of specific signal transduction cascades, including the hydrolysis of sphingomyclin to ceramide. We show that preexposure of astrocytes to a low concentration of ethanol increased the sensitivity of astrocytes to sphingomyelinase, and C2 -ceramide resulting in increased DNA fragmentation and decreased cell viability. More importantly, astrocytes prepared from rats exposed to ethanol prenatally showed increased susceptibility to TNF, -induced cell death. Conclusions: These studies suggest that ethanol increases the susceptibility of astrocytes to TNF, -induced cell death by shifting the balance of sphingolipid metabolism in favor of a pathway that increases the susceptibility of astrocytes to the cytotoxic effect of TNF,. [source] Cycloheximide induces apoptosis of astrocytesPATHOLOGY INTERNATIONAL, Issue 3 2002Takahiro Tsuchida Cultured rat astrocytes were incubated in the presence of cycloheximide (CHX; 20 µg/mL), a potent neuroprotective agent. Then cells were subjected to DNA gel electrophoresis. Electrophoresis showed DNA ladder formation, which is characteristic of apoptosis. Inhibitors of interleukin-1,-converting enzyme (ICE) and caspase 32(CPP32), which play critical roles in certain apoptotic pathways, did not block the cycloheximide-induced apoptosis of cultured astrocytes. This observation indicates that the role of ICE and CPP32 is not significant in the CHX-induced astrocyte apoptosis process. When the blood,brain barrier was disrupted in the rat, the number of brain cells undergoing apoptosis was significantly higher after cycloheximide administration, in contrast to controls. Of the cells that produced glial fibrillary acidic protein, some were observed to undergo apoptosis. Although CHX has been shown to be useful as a neuroprotective agent against ischemic neuronal death, astroglial toxicity may be problematic, depending on CHX concentration. Therefore, a prudent use of this compound is recommended. [source] The bile acid receptor TGR5 (Gpbar-1) acts as a neurosteroid receptor in brainGLIA, Issue 15 2010Verena Keitel Abstract TGR5 (Gpbar-1) is a membrane-bound bile acid receptor in the gastrointestinal tract and immune cells with pleiotropic actions. As shown in the present study, TGR5 is also expressed in astrocytes and neurons. Here, TGR5 may act as a neurosteroid receptor, which is activated by nanomolar concentrations of 5,-pregnan-3,-ol-20-one and micromolar concentrations of 5,-pregnan-3,-17,-21-triol-20-one and 5,-pregnan-3,-ol-20-one (allopregnanolone). TGR5 stimulation in astrocytes and neurons is coupled to adenylate cyclase activation, elevation of intracellular Ca2+ and the generation of reactive oxygen species. In cultured rat astrocytes, TGR5 mRNA is downregulated in the presence of neurosteroids and ammonia already at concentrations of 0.5 mmol L,1. Furthermore, TGR5 protein levels are significantly reduced in isolated rat astrocytes after incubation with ammonia. A marked downregulation of TGR5 mRNA is also found in cerebral cortex from cirrhotic patients dying with hepatic encephalopathy (HE) when compared with brains from noncirrhotic control subjects. It is concluded that TGR5 is a novel neurosteroid receptor in brain with implications for the pathogenesis of HE. © 2010 Wiley-Liss, Inc. [source] Existence and distinction of acid-evoked currents in rat astrocytesGLIA, Issue 12 2010Chao Huang Abstract Astrocytes are vital structures that support and/or protect neighboring neurons from pathology. Although it is generally accepted that glutamate receptors mediate most astrocyte effects, acid-evoked currents have recently attracted attention for their role in this regard. Here, we identified the existence and characteristics of acid-sensing ion channels (ASICs) and the transient receptor potential vanilloid type 1 (TRPV1) in astrocytes. There were two types of currents recorded under the application of acidic solution (pH 6.0) in cultured rat astrocytes. Transient currents were exhibited by 10% of the astrocytes, and sustained currents were exhibited by the other 90%, consistent with the features of ASIC and TRPV1 currents, respectively. Western blotting and immunofluorescence confirmed the expression of ASIC1, ASIC2a, ASIC3, and TRPV1 in cultured and in situ astrocytes. Unlike the ASICs expressed in neurons, which were mainly distributed in the cell membrane/cytoplasm, most of the ASICs in astrocytes were expressed in the nucleus. TRPV1 was more permeable to Na+ in cultured astrocytes, which differed from the typical neuronal TRPV1 that was mainly permeable to Ca2+. This study demonstrates that there are two kinds of acid-evoked currents in rat astrocytes, which may provide a new understanding about the functions of ligand-gated ion channels in astrocytes. © 2010 Wiley-Liss, Inc. [source] Beta-amyloid peptide stimulates endozepine release in cultured rat astrocytes through activation of N -formyl peptide receptorsGLIA, Issue 13 2008Tursonjan Tokay Abstract Astroglial cells synthesize and release endozepines, a family of neuropeptides derived from diazepam-binding inhibitor (DBI). The authors have recently shown that ,-amyloid peptide (A,) stimulates DBI gene expression and endozepine release. The purpose of this study was to determine the mechanism of action of A, in cultured rat astrocytes. A,25,35 and the N -formyl peptide receptor (FPR) agonist N -formyl-Met-Leu-Phe (fMLF) increased the secretion of endozepines in a dose-dependent manner with EC50 value of ,2 ,M. The stimulatory effects of A,25,35 and the FPR agonists fMLF and N -formyl-Met-Met-Met (fMMM) on endozepine release were abrogated by the FPR antagonist N - t -Boc-Phe-Leu-Phe-Leu-Phe. In contrast, A,25,35 increased DBI mRNA expression through a FPR-independent mechanism. A,25,35 induced a transient stimulation of cAMP formation and a sustained activation of polyphosphoinositide turnover. The stimulatory effect of A,25,35 on endozepine release was blocked by the adenylyl cyclase inhibitor somatostatin, the protein kinase A (PKA) inhibitor H89, the phospholipase C inhibitor U73122, the protein kinase C (PKC) inhibitor chelerythrine and the ATP binding cassette transporter blocker glyburide. Taken together, these data demonstrate for the first time that A,25,35 stimulates endozepine release from rat astrocytes through a FPR receptor positively coupled to PKA and PKC. © 2008 Wiley-Liss, Inc. [source] Activation of CysLT receptors induces astrocyte proliferation and death after oxygen,glucose deprivationGLIA, Issue 1 2008Xiao-Jia Huang Abstract We recently found that 5-lipoxygenase (5-LOX) is activated to produce cysteinyl leukotrienes (CysLTs), and CysLTs may cause neuronal injury and astrocytosis through activation of CysLT1 and CysLT2 receptors in the brain after focal cerebral ischemia. However, the property of astrocyte responses to in vitro ischemic injury is not clear; whether 5-LOX, CysLTs, and their receptors are also involved in the responses of ischemic astrocytes remains unknown. In the present study, we performed oxygen-glucose deprivation (OGD) followed by recovery to induce ischemic-like injury in the cultured rat astrocytes. We found that 1-h OGD did not injure astrocytes (sub-lethal OGD) but induced astrocyte proliferation 48 and 72 h after recovery; whereas 4-h OGD moderately injured the cells (moderate OGD) and led to death 24,72 h after recovery. Inhibition of phospholipase A2 and 5-LOX attenuated both the proliferation and death. Sub-lethal and moderate OGD enhanced the production of CysLTs that was inhibited by 5-LOX inhibitors. Sub-lethal OGD increased the expressions of CysLT1 receptor mRNA and protein, while moderate OGD induced the expression of CysLT2 receptor mRNA. Exogenously applied leukotriene D4 (LTD4) induced astrocyte proliferation at 1,10 nM and astrocyte death at 100,1,000 nM. The CysLT1 receptor antagonist montelukast attenuated astrocyte proliferation, the CysLT2 receptor antagonist BAY cysLT2 reversed astrocyte death, and the dual CysLT receptor antagonist BAY u9773 exhibited both effects. In addition, LTD4 (100 nM) increased the expression of CysLT2 receptor mRNA. Thus, in vitro ischemia activates astrocyte 5-LOX to produce CysLTs, and CysLTs result in CysLT1 receptor-mediated proliferation and CysLT2 receptor-mediated death. © 2007 Wiley-Liss, Inc. [source] The triakontatetraneuropeptide TTN increases [Ca2+]i in rat astrocytes through activation of peripheral-type benzodiazepine receptorsGLIA, Issue 2 2001Pierrick Gandolfo Abstract Astrocytes synthesize a series of regulatory peptides called endozepines, which act as endogenous ligands of benzodiazepine receptors. We have recently shown that one of these endozepines, the triakontatetraneuropeptide TTN, stimulates DNA synthesis in astroglial cells. The purpose of the present study was to determine the mechanism of action of TTN on cultured rat astrocytes. Binding of the peripheral-type benzodiazepine receptor ligand [3H]Ro5-4864 to intact astrocytes was displaced by TTN, whereas its C-terminal fragment (TTN[17,34], the octadecaneuropeptide ODN) did not compete for [3H]Ro5-4864 binding. Microfluorimetric measurement of cytosolic calcium concentrations ([Ca2+]i) with the fluorescent probe indo-1 showed that TTN (10,10 to 10,6 M) provokes a concentration-dependent increase in [Ca2+]i in cultured astrocytes. Simultaneous administration of TTN (10,8 M) and Ro5-4864 (10,5 M) induced an increase in [Ca2+]i similar to that obtained with Ro5-4864 alone. In contrast, the effects of TTN (10,8 M) and ODN (10,8 M) on [Ca2+]i were strictly additive. Chelation of extracellular Ca2+ by EGTA (6 mM) or blockage of Ca2+ channels with Ni2+ (2 mM) abrogated the stimulatory effect of TTN. The calcium influx evoked by TTN (10,7 M) or by Ro5-4864 (10,5 M) was not affected by the N- and T-type calcium channel blockers ,-conotoxin (10,6 M) and mibefradil (10,6 M), but was significantly reduced by the L-type calcium channel blocker nifedipine (10,7 M). Patch-clamp studies showed that, at negative potentials, TTN (10,7 M) induced a sustained depolarization. Reduction of the chloride concentration in the extracellular solution shifted the reversal potential from 0 mV to a positive potential. These data show that TTN, acting through peripheral-type benzodiazepine receptors, provokes chloride efflux, which in turn induces calcium influx via L-type calcium channels in rat astrocytes. GLIA 35:90,100, 2001. © 2001 Wiley-Liss, Inc. [source] | ||||||||||