The benzodiazepine receptor in cultured astrocytes from genetically epilepsy-prone rats

Ducis, Ilze; Norenberg, Luz Oliva B.; Norenberg, Michael D.

doi:10.1016/0006-8993(90)90793-b

Cited by 68 publications

(41 citation statements)

References 24 publications

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“…Primary cultures of cortical astrocytes were prepared as described previously (Ducis et al, 1990). Briefly, brains of 1-to 2-d-old rat pups were seeded on 35 mm culture dishes in DMEM containing penicillin, streptomycin, and 15% fetal bovine serum and incubated at 37°C with 5% CO 2 and 95% air.…”

Section: Methodsmentioning

confidence: 99%

Oxidative Stress and Mitogen-Activated Protein Kinase Phosphorylation Mediate Ammonia-Induced Cell Swelling and Glutamate Uptake Inhibition in Cultured Astrocytes

Jayakumar¹,

Panickar

Murthy³

et al. 2006

J. Neurosci.

192

158

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Hepatic encephalopathy (HE) is a major neurological complication in patients with severe liver failure. Elevated levels of ammonia have been strongly implicated as a factor in HE, and astrocytes appear to be the primary target of its neurotoxicity. Mechanisms mediating key aspects of ammonia-induced astrocyte dysfunction such as cell swelling and inhibition of glutamate uptake are not clear. We demonstrated previously that cultured astrocytes exposed to ammonia increase free radical production. We now show that treatment with antioxidants significantly prevents ammonia-induced astrocyte swelling as well as glutamate uptake inhibition. Because one consequence of oxidative stress is the phosphorylation of mitogen-activated protein kinases (MAPKs), we investigated whether phosphorylation of MAPKs may mediate astrocyte dysfunction. Primary cultured astrocytes exposed to 5 mM NH 4 Cl for different time periods (1-72 h) significantly increased phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), p38 MAPK , and c-Jun N-terminal kinase (JNK) 1/2/3, which was inhibited by appropriate MAPK inhibitors 1, 4-diamino-2, 3-dicyano-1, 4-bis (2-aminophenylthio) butadiene (UO126; for ERK1/2), trans-1-(4-hydroxyclyclohexyl)-4-(4-fluorophenyl)-5-(2-methoxypyrimidin-4-yl)imidazole (SB 239063; for p38MAPK ), and anthra[1,9-cd]pyrazol-6(2H)-one (SP600125; for JNK1/2/3), as well as by antioxidants. Kinase inhibitors partially or completely prevented astrocyte swelling. Although SB239063 and SP600125 significantly reversed glutamate uptake inhibition and ammonia-induced decline in glutamate-aspartate transporter protein levels, UO126 did not, indicating a differential effect of these kinases in ammonia-induced astrocyte swelling and glutamate transport impairment. These studies strongly suggest the involvement of oxidative stress and phosphorylation of MAPKs in the mechanism of ammonia-induced astrocyte dysfunction associated with ammonia neurotoxicity.

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Section: Methodsmentioning

confidence: 99%

Oxidative Stress and Mitogen-Activated Protein Kinase Phosphorylation Mediate Ammonia-Induced Cell Swelling and Glutamate Uptake Inhibition in Cultured Astrocytes

Jayakumar¹,

Panickar

Murthy³

et al. 2006

J. Neurosci.

192

158

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“…Primary cultured astrocytes were prepared from cerebral cortices of 1 day old rat pups by the method of Ducis et al 26 Briefly, cortices were freed of meninges, minced and dissociated by trituration, passed through sterile nylon sieves, and then placed in Dulbecco's modified Eagle medium (DMEM) containing penicillin, streptomycin, and 15% fetal bovine serum. Approximately 0.5 Â 10 6 cells were seeded in 35 mm culture plates and maintained at 371C in an incubator equilibrated with 5% CO 2 and 95% air.…”

Section: Materials and Methods Astrocyte Culturesmentioning

confidence: 99%

The mitochondrial permeability transition, and oxidative and nitrosative stress in the mechanism of copper toxicity in cultured neurons and astrocytes

Reddy

Rao

Norenberg

2008

Laboratory Investigation

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Copper is an essential element and an integral component of various enzymes. However, excess copper is neurotoxic and has been implicated in the pathogenesis of Wilson's disease, Alzheimer's disease, prion conditions, and other disorders. Although mechanisms of copper neurotoxicity are not fully understood, copper is known to cause oxidative stress and mitochondrial dysfunction. As oxidative stress is an important factor in the induction of the mitochondrial permeability transition (mPT), we determined whether mPT plays a role in copper-induced neural cell injury. Cultured astrocytes and neurons were treated with 20 mM copper and mPT was measured by changes in the cyclosporin A (CsA)-sensitive inner mitochondrial membrane potential (DCm), employing the potentiometric dye TMRE. In astrocytes, copper caused a 36% decrease in the DCm at 12 h, which decreased further to 48% by 24 h and remained at that level for at least 72 h. Cobalt quenching of calcein fluorescence as a measure of mPT similarly displayed a 45% decrease at 24 h. Pretreatment with antioxidants significantly blocked the copper-induced mPT by 48-75%. Copper (24 h) also caused a 30% reduction in ATP in astrocytes, which was completely blocked by CsA. Copper caused death (42%) in astrocytes by 48 h, which was reduced by antioxidants (35-60%) and CsA (41%). In contrast to astrocytes, copper did not induce mPT in neurons. Instead, it caused early and extensive death with a concomitant reduction (63%) in ATP by 14 h. Neuronal death was prevented by antioxidants and nitric oxide synthase inhibitors but not by CsA. Copper increased protein tyrosine nitration in both astrocytes and neurons. These studies indicate that mPT, and oxidative and nitrosative stress represent major factors in copper-induced toxicity in astrocytes, whereas oxidative and nitrosative stress appears to play a major role in neuronal injury.

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“…Astrocyte Cultures-Astrocyte cultures were prepared from brains of 1-2-day-old rat pups by the method of Ducis et al (31). Briefly, cerebral cortices were freed of meninges, minced, dissociated by trituration and vortexing, passed through sterile nylon sieves, placed in Dulbecco's modified Eagle's medium containing penicillin, streptomycin, and fetal bovine serum, and incubated at 37°C in a humidified chamber provided with 5% CO 2 and 95% air.…”

Section: Methodsmentioning

confidence: 99%

Na-K-Cl Cotransporter-1 in the Mechanism of Ammonia-induced Astrocyte Swelling

Jayakumar

Liu

Moriyama

et al. 2008

Journal of Biological Chemistry

Self Cite

114

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Brain edema and the consequent increase in intracranial pressure and brain herniation are major complications of acute liver failure (fulminant hepatic failure) and a major cause of death in this condition. Ammonia has been strongly implicated as an important factor, and astrocyte swelling appears to be primarily responsible for the edema. Ammonia is known to cause cell swelling in cultured astrocytes, although the means by which this occurs has not been fully elucidated. A disturbance in one or more of these systems may result in loss of ion homeostasis and cell swelling. In particular, activation of the Na-K-Cl cotransporter (NKCC1) has been shown to be involved in cell swelling in several neurological disorders. We therefore examined the effect of ammonia on NKCC activity and its potential role in the swelling of astrocytes. Cultured astrocytes were exposed to ammonia (NH 4 Cl; 5 mM), and NKCC activity was measured. Ammonia increased NKCC activity at 24 h. Inhibition of this activity by bumetanide diminished ammonia-induced astrocyte swelling. Ammonia also increased total as well as phosphorylated NKCC1. Treatment with cyclohexamide, a potent inhibitor of protein synthesis, diminished NKCC1 protein expression and NKCC activity. Since ammonia is known to induce oxidative/ nitrosative stress, and antioxidants and nitric-oxide synthase inhibition diminish astrocyte swelling, we also examined whether ammonia caused oxidation and/or nitration of NKCC1. Cultures exposed to ammonia increased the state of oxidation and nitration of NKCC1, whereas the antioxidants N-nitro-L-arginine methyl ester and uric acid all significantly diminished NKCC activity. These agents also reduced phosphorylated NKCC1 expression. These results suggest that activation of NKCC1 is an important factor in the mediation of astrocyte swelling by ammonia and that such activation appears to be mediated by NKCC1 abundance as well as by its oxidation/nitration and phosphorylation.

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The benzodiazepine receptor in cultured astrocytes from genetically epilepsy-prone rats

Cited by 68 publications

References 24 publications

Oxidative Stress and Mitogen-Activated Protein Kinase Phosphorylation Mediate Ammonia-Induced Cell Swelling and Glutamate Uptake Inhibition in Cultured Astrocytes

Oxidative Stress and Mitogen-Activated Protein Kinase Phosphorylation Mediate Ammonia-Induced Cell Swelling and Glutamate Uptake Inhibition in Cultured Astrocytes

The mitochondrial permeability transition, and oxidative and nitrosative stress in the mechanism of copper toxicity in cultured neurons and astrocytes

Na-K-Cl Cotransporter-1 in the Mechanism of Ammonia-induced Astrocyte Swelling

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