K. V. Rama Rao

Ammonia-induced production of free radicals in primary cultures of rat astrocytes

Elevated levels of ammonia in blood and brain result in derangement of cerebral function. Recently, lipid peroxidation and oxidative stress have been implicated in ammonia neurotoxicity. Because ammonia is primarily detoxified in astrocytes, we postulated that pathophysiological concentrations of ammonia might induce free radical formation in these cells. To test this hypothesis, we examined the extent of free radical production in primary cultures of astrocytes that had been preloaded with the fluorescent dye 5‐ (and 6‐)carboxy‐2′,7′‐dichlorodihydrofluorescein diacetate (DCFDA). DCFDA fluoresence was found to be increased in a dose‐dependent manner when astrocytes were exposed to 1, 5, and 10 mM NH4Cl. This phenomenon was transitory; it peaked at 2.5 min after exposure and declined subsequently. By 2 hr after treatment, DCFDA fluorescence was below control level. Addition of catalase or superoxide dismutase to 5 mM NH4Cl‐treated astrocytes reduced free radical formation. Pretreatment with 3 mM methionine sulfoximine, an inhibitor of glutamine synthetase, also suppressed free radical formation by 5 mM NH4Cl. The results of this study suggest that elevated concentrations of ammonia induce the formation of free radicals in astrocytes and that this process is associated with the synthesis of glutamine. We propose that astrocyte‐derived free radicals may be responsible for some of the pathophysiological changes associated with hyperammonemic conditions. J. Neurosci. Res. 66:282–288, 2001.

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.

The Mitochondrial Permeability Transition in Neurologic Disease

Mitochondria, being the principal source of cellular energy, are vital for cell life. Yet, ironically, they are also major mediators of cell death, either by necrosis or apoptosis. One means by which these adverse effects occur is through the mitochondrial permeability transition (mPT) whereby the inner mitochondrial membrane suddenly becomes excessively permeable to ions and other solutes, resulting in a collapse of the inner membrane potential, ultimately leading to energy failure and cell necrosis. The mPT may also bring about the release of various factors known to cause apoptotic cell death. The principal factors leading to the mPT are elevated levels of intracellular Ca2+ and oxidative stress. Characteristically, the mPT is inhibited by cyclosporin A. This article will briefly discuss the concept of the mPT, its molecular composition, its inducers and regulators, agents that influence its activity and describe the consequences of its induction. Lastly, we will review its potential contribution to acute neurological disorders, including ischemia, trauma, and toxic-metabolic conditions, as well as its role in chronic neurodegenerative conditions such as Alzheimers disease, Parkinsons disease, Huntingtons disease and amyotrophic lateral sclerosis.

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.

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.

Induction of the mitochondrial permeability transition in cultured astrocytes by glutamine.

Ammonia is a toxin that has been strongly implicated in the pathogenesis of hepatic encephalopathy (HE), and astrocytes appear to be the principal target of ammonia toxicity. Glutamine, a byproduct of ammonia metabolism, has been implicated in some of the deleterious effects of ammonia on the CNS. We have recently shown that ammonia induces the mitochondrial permeability transition (MPT) in cultured astrocytes, but not in neurons. We therefore determined whether glutamine is also capable of inducing the MPT in cultured astrocytes. Astrocytes were treated with glutamine (4.5 mM) for various time periods and the MPT was assessed by changes in 2-deoxyglucose (2-DG) mitochondrial permeability, calcein fluorescence assay, and by changes in cyclosporin A (CsA)-sensitive inner mitochondrial membrane potential (deltapsi(m)) using the potentiometric dye, JC-1. Astrocytes treated with glutamine significantly increased 2-DG permeability (120%, P<0.01), decreased mitochondrial calcein fluorescence, and concomitantly dissipated the deltapsi(m). All of these effects were blocked by CsA. These data indicate that glutamine induces the MPT in cultured astrocytes. The induction of the MPT by glutamine in astrocytes, and the subsequent development of mitochondrial dysfunction, may partially explain the deleterious affects of glutamine on the CNS in the setting of hyperammonemia.