Arumugam R. Jayakumar

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

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 NH4Cl for different time periods (1–72 h) significantly increased phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), p38MAPK, 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.

Ammonia induces the mitochondrial permeability transition in primary cultures of rat astrocytes

Ammonia is a toxin that has been strongly implicated in the pathogenesis of hepatic encephalopathy (HE), and the astrocyte appears to be the principal target of ammonia toxicity. The specific neurochemical mechanisms underlying HE, however, remain elusive. One of the suggested mechanisms for ammonia toxicity is impaired cellular bioenergetics. Because there is evidence that the mitochondrial permeability transition (MPT) is associated with mitochondrial dysfunction, we determined whether the MPT might be involved in the bioenergetic alterations related to ammonia toxicity. Accordingly, we examined the mitochondrial membrane potential (Δψm) in cultured astrocytes and neurons using laser‐scanning confocal microscopy after loading the cells with the voltage‐sensitive dye JC‐1. We found that ammonia induced a dissipation of the Δψm in a time‐ and concentration‐dependent manner. These findings were supported by flow cytometry using the voltage‐sensitive dye tetramethylrhodamine ethyl ester (TMRE). Cyclosporin A, a specific inhibitor of the MPT, completely blocked the ammonia‐induced dissipation of the Δψm. We also found an increase in the mitochondrial permeability to 2‐deoxyglucose in astrocytes that had been exposed to 5 mM NH4Cl, further supporting the concept that ammonia induces the MPT in these cells. Pretreatment with methionine sulfoximine, an inhibitor of glutamine synthetase, blocked the ammonia‐induced collapse of Δψm, suggesting a role of glutamine in this process. Over a 24‐hr period, ammonia had no effect on the Δψm in cultured neurons. Collectively, our data indicate that ammonia induces the MPT in cultured astrocytes, which may be a factor in the mitochondrial dysfunction associated with HE and other hyperammonemic states.

Mechanisms of ammonia-induced astrocyte swelling.

Astrocyte swelling represents the major factor responsible for the brain edema associated with fulminant hepatic failure (FHF). The edema may be of such magnitude as to increase intracranial pressure leading to brain herniation and death. Of the various agents implicated in the generation of astrocyte swelling, ammonia has had the greatest amount of experimental support. This article reviews mechanisms of ammonia neurotoxicity that contribute to astrocyte swelling. These include oxidative stress and the mitochondrial permeability transition (MPT). The involvement of glutamine in the production of cell swelling will be highlighted. Evidence will be provided that glutamine induces oxidative stress as well as the MPT, and that these events are critical in the development of astrocyte swelling in hyperammonemia.

New concepts in the mechanism of ammonia-induced astrocyte swelling

It is generally accepted that astrocyte swelling forms the major anatomic substrate of the edema associated with acute liver failure (ALF) and that ammonia represents a major etiological factor in its causation. The mechanisms leading to such swelling, however, remain elusive. Recent studies have invoked the role of oxidative stress in the mechanism of hepatic encephalopathy (HE), as well as in the brain edema related to ALF. This article summarizes the evidence for oxidative stress as a major pathogenetic factor in HE/ALF and discusses mechanisms that are triggered by oxidative stress, including the induction of the mitochondrial permeability transition (MPT) and activation of signaling kinases. We propose that a cascade of events initiated by ammonia-induced oxidative stress results in cell volume dysregulation leading to cell swelling/brain edema. Blockade of this cascade may provide novel therapies for the brain edema associated with ALF.

Signaling factors in the mechanism of ammonia neurotoxicity

Mechanisms involved in hepatic encephalopathy (HE) still remain poorly understood. It is generally accepted that ammonia plays a major role in this disorder, and that astrocytes represent the principal target of ammonia neurotoxicity. In recent years, studies from several laboratories have uncovered a number of factors and pathways that appear to be critically involved in the pathogenesis of this disorder. Foremost is oxidative and nitrosative stress (ONS), which is largely initiated by an ammonia-induced increase in intracellular Ca2+. Such increase in Ca2+ activates a number of enzymes that promote the synthesis of reactive oxygen-nitrogen species, including constitutive nitric oxide synthase, NADPH oxidase and phospholipase A2. ONS subsequently induces the mitochondrial permeability transition, and activates mitogen-activated protein kinases and the transcription factor, nuclear factor-kappaB (NF-κB). These factors act to generate additional reactive oxygen-nitrogen species, to phosphorylate various proteins and transcription factors, and to cause mitochondrial dysfunction. This article reviews the role of these factors in the mechanism of HE and ammonia toxicity with a focus on astrocyte swelling and glutamate uptake, which are important consequences of ammonia neurotoxicity. These pathways and factors provide attractive targets for identifying agents potentially useful in the therapy of HE and other hyperammonemic disorders.

Oxidative Stress in the Pathogenesis of Hepatic Encephalopathy

The pathogenesis of hepatic encephalopathy (HE) remains elusive. While it is clear that ammonia is the likely toxin and that astrocytes are the main target of its neurotoxicity, precisely how ammonia brings about cellular injury is poorly understood. Studies over the past decade have invoked the concept of oxidative stress as a pathogenetic mechanism for ammonia neurotoxicity. This review sets out the arguments in support of this concept based on evidence derived from human observations, animal studies, and cell culture investigations. The consequences and potential therapeutic implications of oxidative stress in HE are also discussed.

Glutamine-induced free radical production in cultured astrocytes

Ammonia is a neurotoxin implicated in the pathogenesis of hepatic encephalopathy, Reyes syndrome, inborn errors of the urea cycle, glutaric aciduria, and other metabolic encephalopathies. Brain ammonia is predominantly metabolized to glutamine in astrocytes by glutamine synthetase. While the synthesis of glutamine has generally been viewed as the principal means of ammonia detoxification, this presumed beneficial effect has been questioned as growing evidence suggest that some of the deleterious effects of ammonia may be mediated by glutamine rather than ammonia per se. Since ammonia is known to induce the production of free radicals in cultured astrocytes, we investigated whether such production might be mediated by glutamine. Treatment of astrocytes with glutamine (4.5 mM) increased free radical production at 2–3 min (95%; P < 0.05), as well as at 1 and 3 h (42% and 49%, respectively; P < 0.05). Similarly treated cultured neurons failed to generate free radicals. Free radical production by glutamine was blocked by the antioxidants deferoxamine (40 μM) and α‐phenyl‐N‐tert‐butyl‐nitrone (250 μM), as well as by the nitric oxide synthase inhibitor Nω‐nitro‐L‐arginine methyl ester (500 μM). Free radical production was also blocked by 6‐diazo‐5‐oxo‐L‐norleucine (1 mM), an inhibitor of glutaminase, suggesting that ammonia released by glutamine hydrolysis may be responsible for the generation of free radicals. Additionally, the mitochondrial permeability transition inhibitor, cyclosporin A, blocked free radical production by glutamine. The results indicate that astrocytes, but not neurons, generate free radicals following glutamine exposure. Glutamine‐induced oxidative and/or nitrosative stress may represent a key mechanism in ammonia neurotoxicity.

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 (NH4Cl; 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.

NFκB in the mechanism of ammonia-induced astrocyte swelling in culture

Astrocyte swelling and brain edema are major neuropathological findings in the acute form of hepatic encephalopathy (fulminant hepatic failure), and substantial evidence supports the view that elevated brain ammonia level is an important etiological factor in this condition. Although the mechanism by which ammonia brings about astrocyte swelling remains to be determined, oxidative/nitrosative stress and mitogen‐activated protein kinases (MAPKs) have been considered as important elements in this process. One factor known to be activated by both oxidative stress and MAPKs is nuclear factor κB (NFκB), a transcription factor that activates many genes, including inducible nitric oxide synthase (iNOS). As the product of iNOS, nitric oxide (NO), is known to cause astrocyte swelling, we examined the potential involvement of NFκB in ammonia‐induced astrocyte swelling. Western blot analysis of cultured astrocytes showed a significant increase in NFκB nuclear translocation (a measure of NFκB activation) from 12 h to 2 days after treatment with NH4Cl (5 mM). Cultures treated with anti‐oxidants, including superoxide dismutase, catalase, and vitamin E as well as the MAPKs inhibitors, SB239063 (an inhibitor of p38‐MAPK) and SP600125 (an inhibitor of c‐Jun N‐terminal kinase), significantly diminished NFκB activation by ammonia, supporting a role of oxidative stress and MAPKs in NFκB activation. The activation of NFκB was associated with increased iNOS protein expression and NO generation, and these changes were blocked by BAY 11–7082, an inhibitor of NFκB. Additionally, ammonia‐induced astrocyte swelling was inhibited by the NFκB inhibitors, BAY 11–7082 and SN‐50, thereby implicating NFκB in the mechanism of astrocyte swelling. Our studies indicate that cultured astrocytes exposed to ammonia display NFκB activation, which is likely to be a consequence of oxidative stress and activation of MAPKs. NFκB activation appears to contribute to the mechanism of ammonia‐induced astrocyte swelling, apparently through its up‐regulation of iNOS protein expression and the subsequent generation of NO.

Ammonia Neurotoxicity: Role of the Mitochondrial Permeability Transition

Hepatic encephalopathy (HE) is an important cause of morbidity and mortality in patients with severe liver disease. Although the mechanisms responsible for HE remain elusive, ammonia is generally considered to be involved in its pathogenesis, and astrocytes are thought to be the principal target of ammonia neurotoxicity. Altered bioenergetics and oxidative stress are also thought to play a major role in this disorder. In this paper, we present data invoking the mitochondrial permeability transition (MPT) as a factor in the pathogenesis of HE/hyperammonemia. The MPT is a Ca2+-dependent, cyclosporin A (CsA) sensitive process due to the opening of a pore in the inner mitochondrial membrane that leads to a collapse of ionic gradients and ultimately to mitochondrial dysfunction. Many of the factors that facilitate the induction of the MPT are also known to be implicated in the mechanism of HE, including free radicals, Ca2+, nitric oxide, alkaline pH, and glutamine. We have recently shown that treatment of cultured astrocytes with 5 mM NH4Cl resulted in a dissipation of the mitochondrial membrane potential (ΔΨm), which was sensitive to CsA. Similarly treated cultured neurons failed to show a loss of the ΔΨm. Further support for the ammonia induction of the MPT was obtained by observing an increase in mitochondrial permeability to 2-deoxyglucose-6-phosphate, and a decrease in calcein fluorescence in astrocytes after ammonia treatment, both of which were also blocked by CsA. CsA was likewise capable of exerting a protective effect against hyperammonemia in mice. Taken together, our data suggest that the MPT represents an important component of the pathogenesis of HE and other hyperammonemic states.