Na-K-Cl Cotransporter-1 in the mechanism of ammonia-induced astrocyte swelling

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.     

Glutamine in the mechanism of ammonia-induced astrocyte swelling

Brain edema and the subsequent increase in intracranial pressure are the major neurological complications in fulminant hepatic failure (FHF). Brain edema in FHF is predominantly "cytotoxic" due principally to astrocyte swelling. It is generally believed that ammonia plays a key role in this process, although the mechanism by which ammonia brings about such swelling is yet to be defined. It has been postulated that glutamine accumulation in astrocytes subsequent to ammonia detoxification results in increased osmotic forces leading to cell swelling. While the hypothesis is plausible and has gained support, it has never been critically tested. In this study, we examined whether a correlation exists between cellular glutamine levels and the degree of cell swelling in cultured astrocytes exposed to ammonia. Cultured astrocytes derived from rat brain cortices were exposed to ammonia (5 mM) for different time periods and cell swelling was measured. Cultures treated with ammonia for 1-3 days showed a progressive increase in astrocyte cell volume (59-127%). Parallel treatment of astrocyte cultures with ammonia showed a significant increase in cellular glutamine content (60-80%) only at 1-4 h, a time when swelling was absent, while glutamine levels were normal at 1-3 days, a time when peak cell swelling was observed. Thus no direct correlation between cell swelling and glutamine levels was detected. Additionally, acute increase in intracellular levels of glutamine by treatment with the glutaminase inhibitor 6-diazo-5-oxo-L-norleucine (DON) after ammonia exposure also did not result in swelling. On the contrary, DON treatment significantly blocked (66%) ammonia-induced astrocyte swelling at a later time point (24 h), suggesting that some process resulting from glutamine metabolism is responsible for astrocyte swelling. Additionally, ammonia-induced free radical production and induction of the mitochondrial permeability transition (MPT) were significantly blocked by treatment with DON, suggesting a key role of glutamine in the ammonia-induced free radical generation and the MPT. In summary, our findings indicate a lack of direct correlation between the extent of cell swelling and cellular levels of glutamine. While glutamine may not be acting as an osmolyte, we propose that glutamine-mediated oxidative stress and/or the MPT may be responsible for the astrocyte swelling by ammonia.     

Astrocyte swelling and protein tyrosine nitration in hepatic encephalopathy

Hepatic encephalopathy (HE) is a neuropsychiatric syndrome during the course of acute or chronic liver disease. It is functional in nature, potentially reversible and precipitated by rather heterogeneous factors. Current evidence suggests that HE is the consequence of a low grade chronic glial edema with subsequent alterations of glioneuronal communication. Other lines of evidence support a role of NMDA receptor activation and oxidative/nitrosative stress in the pathogenesis of HE. Different HE-precipitating factors, such as ammonia, benzodiazepines, inflammatory cytokines and hyponatremia induce or aggravate astrocyte swelling and additionally increase oxidative/nitrosative stress and protein tyrosine nitration. Recent findings suggest a close interrelation between astrocyte swelling, NMDA receptor signaling, glutamate, oxidative stress and nitric oxide, which may result in mutual amplification of swelling and oxidative stress. Via such an autoamplificatory loop astrocytes may integrate HE-relevant signals triggered by HE-precipitating factors. This review focuses on the involvement of astrocyte swelling in protein tyrosine nitration and potential consequences in the setting of HE.     

Glutamine in the pathogenesis of acute hepatic encephalopathy

Hepatic encephalopathy (HE) is the major neurological disorder associated with liver disease. It presents in chronic and acute forms, and astrocytes are the major neural cells involved. While the principal etiological factor in the pathogenesis of HE is increased levels of blood and brain ammonia, glutamine, a byproduct of ammonia metabolism, has also been implicated in its pathogenesis. This article reviews the current status of glutamine in the pathogenesis of HE, particularly its involvement in some of the events triggered by ammonia, including mitochondrial dysfunction, generation of oxidative stress, and alterations in signaling mechanisms, including activation of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-κB). Mechanisms by which glutamine contributes to astrocyte swelling/brain edema associated with acute liver failure (ALF) will also be described.     

Oxidative and nitrosative stress in ammonia neurotoxicity

Increased ammonia accumulation in the brain due to liver dysfunction is a major contributor to the pathogenesis of hepatic encephalopathy (HE). Fatal outcome of rapidly progressing (acute) HE is mainly related to cytotoxic brain edema associated with astrocytic swelling. An increase of brain ammonia in experimental animals or treatment of cultured astrocytes with ammonia generates reactive oxygen and nitrogen species in the target tissues, leading to oxidative/nitrosative stress (ONS). In cultured astrocytes, ammonia-induced ONS is invariably associated with the increase of the astrocytic cell volume. Interrelated mechanisms underlying this response include increased nitric oxide (NO) synthesis which is partly coupled to the activation of NMDA receptors and increased generation of reactive oxygen species by NADPH oxidase. ONS and astrocytic swelling are further augmented by excessive synthesis of glutamine (Gln) which impairs mitochondrial function following its accumulation in there and degradation back to ammonia (“the Trojan horse” hypothesis). Ammonia also induces ONS in other cell types of the CNS: neurons, microglia and the brain capillary endothelial cells (BCEC). ONS in microglia contributes to the central inflammatory response, while its metabolic and pathophysiological consequences in the BCEC evolve to the vasogenic brain edema associated with HE. Ammonia-induced ONS results in the oxidation of mRNA and nitration/nitrosylation of proteins which impact intracellular metabolism and potentiate the neurotoxic effects. Simultaneously, ammonia facilitates the antioxidant response of the brain, by activating astrocytic transport and export of glutathione, in this way increasing the availability of precursors of neuronal glutathione synthesis.     

Suppression of NF-κB and NF-κB regulated oxidative stress and neuroinflammation by BAY 11-7082 (IκB phosphorylation inhibitor) in experimental diabetic neuropathy

Inflammation is an emerging patho-mechanism of diabetes and its complications. NF-κB pathway is one of the central machinery initiating and propagating inflammatory responses. The present study envisaged the involvement of NF-κB inflammatory cascade in the pathophysiology of diabetic neuropathy using BAY 11-7082, an IκB phosphorylation inhibitor. Streptozotocin was used to induce diabetes in Sprauge Dawley rats. BAY 11-7082 (1 &; 3 mg/kg) was administered to diabetic rats for 14 days starting from the end of six weeks post diabetic induction. Diabetic rats developed deficits in nerve functions and altered nociceptive parameters and also showed elevated expression of NF-κB (p65), IκB and p-IκB along with increased levels of IL-6 &; TNF-α and inducible enzymes (COX-2 and iNOS). Furthermore, there was an increase in oxidative stress and decrease in Nrf2/HO-1 expression. We observed that BAY 11-7082 alleviated abnormal sensory responses and deficits in nerve functions. BAY 11-7082 also ameliorated the increase in expression of NF-κB, IκB and p-IκB. BAY 11-7082 curbed down the levels of IL-6, TNF-α, COX-2 and iNOS in the sciatic nerve. Lowering of lipid peroxidation and improvement in GSH levels was also seen along with increased expression of Nrf2/HO-1. Thus it can be concluded that NF-κB expression and downstream expression of proinflammatory mediators are prominent features of nerve damage leading to inflammation and oxidative stress and BAY 11-7082 was able to ameliorate experimental diabetic neuropathy by modulating neuroinflammation and improving antioxidant defence.     

Tetrandrine suppresses LPS-induced astrocyte activation via modulating IKKs-IκBα-NF-κB signaling pathway

Astrocyte activation has been implicated in the pathogenesis of many neurological diseases. These reactive astrocytes are capable of producing a variety of proinflammatory mediators and potentially neurotoxic compounds, such as nitric oxide (NO), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and interleukin-1beta (IL-1beta). In this study, we examined the suppressive effects of Tetrandrine (TET) on astrocyte activation induced by lipopolysaccharide (LPS) in vitro. We found that TET decreased the release of NO, TNF-alpha, IL-6 and IL-1beta in LPS-activated astrocytes. Also mRNA expression levels of inducible nitric oxide synthase (iNOS), macrophage inflammatory protein-1alpha (MIP-1alpha) and vascular cell adhesion molecule-1 (VCAM-1) were inhibited in TET pretreated astrocytes. Such suppressive effects might be resulted from the inhibition of nuclear factor kappa B (NF-kappaB) activation through downregulating IkappaB kinases (IKKs) phosphoration, which decreased inhibitor of nuclear factor-kappaB-alpha (IkappaBalpha) phosphoration and degradation. Our results suggest that TET acted to regulate astrocyte activation through inhibiting IKKs-IkappaBalpha-NF-kappaB signaling pathway.     

CAT2 arginine transporter deficiency significantly reduces iNOS-mediated NO production in astrocytes

We have previously demonstrated that genetic ablation of cationic amino acid transporter 2 (Cat2) significantly inhibits nitric oxide (NO) production by inducible nitric oxide synthase (iNOS) in activated macrophages. Here we report that iNOS activity is impaired by 84% in activated Cat2-deficient astrocytes. Cat2 ablation appears to reduce astrocyte NO synthesis by decreasing the uptake of the sole precursor, arginine, as well as by reducing the expression of iNOS following activation. Excessive or dysregulated NO production by activated astrocytes and other CNS cell types has been implicated in the pathogenesis of neurological disorders. Our results support the idea that manipulation of CAT2 transporter function might be useful for the therapeutic modulation of iNOS activity.     

Ammonia Neurotoxicity and the Mitochondrial Permeability Transition

Ammonia is a neurotoxin that predominantly affects astrocytes. Disturbed mitochondrial function and oxidative stress, factors implicated in the induction of the mitochondrial permeability transition (MPT), appear to be involved in the mechanism of ammonia neurotoxicity. We have recently shown that ammonia induces the MPT in cultured astrocytes. To elucidate the mechanisms of the MPT, we examined the role of oxidative stress and glutamine, a byproduct of ammonia metabolism. The ammonia-induced MPT was blocked by antioxidants, suggesting a causal role of oxidative stress. Direct application of glutamine (4.5-7.0 mM) to cultured astrocytes increased free radical production and induced the MPT. Treatment of astrocytes with the mitochondrial glutaminase inhibitor, 6-diazo-5-oxo-L-norleucine, completely blocked free radical formation and the MPT, suggesting that high ammonia concentrations in mitochondria resulting from glutamine hydrolysis may be responsible for the effects of glutamine. These studies suggest that oxidative stress and glutamine play major roles in the induction of the MPT associated with ammonia neurotoxicity.     

The regulation of inducible nitric oxide synthase gene expression induced by lipopolysaccharide and tumor necrosis factor-alpha in C6 cells: involvement of AP-1 and NFkappaB

The roles of AP-1 and NFkappaB in the regulation of inducible nitric oxide synthase (iNOS) mRNA expression induced by the combination of lipopolysaccharide and tumor necrosis factor-alpha (LT) in C6 cells were examined in the present study. The iNOS mRNA level and NO release were increased by several cytokines alone or combination treatments at 24 hr. LT-induced iNOS mRNA level was maximally increased at 6 hr and maintained at higher level at least up to 24 hr. At 6 hr, iNOS protein level and NO release were also increased by LT. By western blot analysis, AP-1, such as Fra-1, Jun B, and phospho-CREB protein levels were increased by LT and translocation of NFkappaB p52 from the cytoplasm to the nucleus was increased. In addition, phosphorylations of MAPKs (ERK 1/2, p38, JNK 1/2) were increased by LT. LT-induced iNOS mRNA level was inhibited by PD98059 (MEK 1/2 inhibitor), SB203580 (p38 inhibitor), and cycloheximide (a protein synthesis blocker), indicating that the phosphorylation of ERK 1/2 and p38, and on-going protein synthesis are necessary for LT-induced iNOS expression. Electrophoretic mobility shift assay (EMSA) showed that AP-1 and NFkappaB DNA binding activities were increased at 6 hr and these AP-1 and NFkappaB DNA bands increased by LT were super-shifted when Fra-1, Jun B, or NFkappaB p50 antibody was coincubated. These findings strongly suggest that, in C6 cells, Fra-1, Jun B, NFkappaB p50, and NFkappaB p52 appear to be involved in the regulation of iNOS mRNA induced by LT.     

Theophylline Potentiates Lipopolysaccharide-Induced NO Production in Cultured Astrocytes

Elucidation of the functions of astrocytes is important for understanding of the pathogenic mechanism of various neurodegenerative diseases. Theophylline is a common drug for bronchial asthma and occasionally develops side-effects, such as acute encephalopathy; although the pathogenic mechanism of the side-effects is unknown. The lipopolysaccharide (LPS)-induced nitric oxide (NO) production is generally used for an index of the activation of astrocyte in vitro. In this study, in order to elucidate the effect of theophylline on the astrocytic functions, we examined the LPS-induced NO production and the expression of iNOS in cultured rat cortex astrocytes. Theophylline alone could not induce the NO production; however, NO production induced by LPS was enhanced by theophylline in a dose-dependent manner; and by isobutylmethylxanthine, a phosphodiesterase inhibitor. The theophylline enhancement of LPS-induced NO production was further increased by dibutyryl cyclic AMP, a membrane-permeable cAMP analog; and by forskolin, an adenylate cyclase activator. When the cells were preincubated with Rp-8-Br-cAMP, an inhibitor of protein kinase A, the theophylline enhancement of LPS-induced NO production was decreased. The extent of iNOS protein expression induced by LPS was also enhanced by theophylline. It is likely that phosphodiesterase inhibition is a major action mechanism for the theophylline enhancement of LPS-induced NO production in astrocytes. Theophylline-induced acute encephalopathy might be due to the hyper-activation of astrocytes via cAMP signaling to produce excess amount of NO.     

Suppressive effects of genistein on oxidative stress and NFkappaB activation in RAW 264.7 macrophages

This study was designed to investigate whether genistein may ameliorate oxidative stress and nuclear factor kappaB (NFkappaB) activation in the lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophage cell line. Treatment of RAW 264.7 cells with genistein significantly reduced lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production in a dose-dependent manner with an IC50 of 69.4 microM. Genistein at 50 microM and 100 microM concentrations reduced thiobarbituric acid-reactive substances (TBARS) accumulation, increasing the GSH level and antioxidant enzyme activities, such as superoxide dismutase (SOD) and catalase. The specific DNA-binding activities of nuclear factor kappaB (NFkappaB) on nuclear extracts from 50 microM and 100 microM genistein treatments were significantly suppressed. These results suggest that genistein has mild antioxidant activity to suppress intracellular oxidative stress and NFkappaB activation.     

Ammonia-induced Na,K-ATPase/ouabain-mediated EGF receptor transactivation,MAPK/ERK and PI3K/AKT signaling and ROS formation cause astrocyte swelling

Ammonia toxicity is clinically important and biologically poorly understood. We reported previously that 3 mM ammonia chloride (ammonia), a relevant concentration for hepatic encephalopathy studies, increases production of endogenous ouabain and activity of Na,K-ATPase in astrocytes. In addition, ammonia-induced upregulation of gene expression of α₂ isoform of Na,K-ATPase in astrocytes could be inhibited by AG1478, an inhibitor of the EGF receptor (EGFR), and by PP1, an inhibitor of Src, but not by GM6001, an inhibitor of metalloproteinase and shedding of growth factor, suggesting the involvement of endogenous ouabain-induced EGF receptor transactivation. In the present cell culture study, we investigated ammonia effects on phosphorylation of EGF receptor and its intracellular signal pathway towards MAPK/ERK_1/2 and PI3K/AKT; interaction between EGF receptor, α₁, and α₂ isoforms of Na,K-ATPase, Src, ERK_1/2, AKT and caveolin-1; and relevance of these signal pathways for ammonia-induced cell swelling, leading to brain edema, an often fatal complication of ammonia toxicity. We found that (i) ammonia increases EGF receptor phosphorylation at EGFR⁸⁴⁵ and EGFR¹⁰⁶⁸; (ii) ammonia-induced ERK_1/2 and AKT phosphorylation depends on the activity of EGF receptor and Src, but not on metalloproteinase; (iii) AKT phosphorylation occurs upstream of ERK_1/2 phosphorylation; (iv) ammonia stimulates association between the α₁ Na,K-ATPase isoform, Src, EGF receptor, ERK_1/2, AKT and caveolin-1; (v) ammonia-induced ROS production might occur later than EGFR transactivation; (vi) both ammonia induced ERK phosphorylation and ROS production can be abolished by canrenone, an inhibitor of ouabain, and (vii) ammonia-induced cell swelling depends on signaling via the Na,K-ATPase/ouabain/Src/EGF receptor/PI3K-AKT/ERK_1/2, but in response to 3 mM ammonia it does not appear until after 12 h. Based on literature data it is suggested that the delayed appearance of the ammonia-induced swelling at this concentration reflects required ouabain-induced oxidative damage of the ion and water cotransporter NKCC1. This information may provide new therapeutic targets for treatment of hyperammonic brain disorders.