Ch.R.K. Murthy

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.

Fulminant hepatic failure induced oxidative stress in nonsynaptic mitochondria of cerebral cortex in rats.

Fulminant hepatic failure (FHF) is a condition with sudden onset of necrosis of hepatocytes and degeneration of liver tissue without any established liver disease. FHF is associated with increased ammonia levels in blood and brain, which is supposed to be neurotoxic, ultimately leading to neuronal death. Evidences from previous studies suggest for mitochondrial dysfunctions under hyperammonemic conditions. In the present investigation, on thioacetamide-induced FHF rat models, studies were undertaken on cerebral nonsynaptic mitochondrial oxidative stress. The results of the present study reveal elevated lipid peroxidation along with reduced total thiol levels in the cerebral cortex mitochondria of experimental animals compared to saline treated control rats. In addition, the enzymatic activities of glutathione peroxidase and glutathione reductase were decreased, with an elevation in Mn-SOD activity. Overall, thioacetamide-induced FHF in rats enhanced the levels of lipid peroxidation coupled with impaired antioxidant defenses in the cerebral nonsynaptic mitochondria.

Activities of pyruvate dehydrogenase, enzymes of citric acid cycle, and aminotransferases in the subcellular fractions of cerebral cortex in normal and hyperammonemic rats.

Activity levels of pyruvate dehydrogenase, enzymes of citric acid cycle, aspartate and alanine aminotransferases were estimated in mitochondria, synaptosomes and cytosol isolated from brains of normal rats and those injected with acute and subacute doses of ammonium acetate. In mitochondria isolated from animals treated with acute dose of ammonium acetate, there was an elevation in the activities of pyruvate, isocitrate and succinate dehydrogenases while the activities of malate dehydrogenase (malate→oxaloacetate), aspartate and alanine aminotransferases were suppressed. In subacute conditions a similar profile of change was noticed excepting that there was an elevation in the activity of α-ketoglutarate dehydrogenase in mitochondria. In the synaptosomes isolated from animals administered with acute dose of ammonium acetate, there was an increase in the activities of pyruvate, isocitrate, α-ketoglutarate and succinate dehydrogenases while the changes in the activities of malate dehydrogenase, asparatate and alanine amino transferases were suppressed. In the subacute toxicity similar changes were observed in this fraction except that the activity of malate dehydrogenase (oxaloacetate→malate) was enhanced. In the cytosol, pyruvate dehydrogenase and other enzymes of citric acid cycle except malate dehydrogenase were enhanced in both acute and subacute ammonia toxicity though their activities are lesser than that of mitochondria. In this fraction malate dehydrogenase (oxaloacetate→malate), was enhanced while activities of malate dehydrogenase (malate→oxaloacetate), aspartate, and alanine aminotransferases were suppressed in both the conditions. Based on these results it is concluded that the decreased activities of malate dehydrogenase (malate→oxaloacetate) in mitochondria and of aspartate, aminotransferase in mitochondria and cytosol may be responsible for the disruption of malate-aspartate, shuttle in hyperammonemic state. Possible existence of a small vulnerable population of mitochondria in brain which might degenerate and liberate their contents into cytosol in hyperammonemic states is also suggested.

Glutamatergic synaptic dysfunction in hyperammonemic syndromes

Ammonia is a normal biochemical component of brain produced and utilized in several reactions of nitrogen metabolism. Blood and brain ammonia levels are in dynamic equilibrium. As a result, any increase in blood ammonia levels are rapidly reflected in brain. This becomes important in certain pathological states wherein blood ammonia levels are elevated. It has been conclusively shown that such circumstances are associated with derangement of cerebral function (Bruton et al., 1970; Cooper and Plum, 1987; Butterworth et al., 1987). Elevation of blood ammonia levels (hyperammonemia) is encountered chiefly in diseases associated with hepatic dysfunction. These may be of the acute type such as fulminant hepatic failure (acute necrosis of liver cells due to drugs, toxins or viral infections) or the chronic type such as liver cirrhosis. The term hepatic encephalopathy was coined to describe the spectrum of neurological changes associated with liver dysfunction. Initial stages of hepatic encephalopathy (HE) are associated with personality, emotional and neuropsychological disturbances (Gilberstadt et al . , 1980; Levy et al . , 1987; Pomier-Layrargues et al., 1991). As encephalopathy progresses, hyperventilation, confusion, drowsiness, hyperthermia and, ultimately coma are observed. In the later stages of encephalopathy, decerebrate rigidity, and convulsions may occur. In liE, ammonia is considered the chief culprit causing neurological dysfunction (Butterworth et al., 1987; Lockwood et al., 1991). However, involvement of other substances along with ammonia could also contribute. Such substances include mercaptans, phenols (Zieve and Brunner, 1985) and short chain fatty acids (Zieve, 1985).

Co-administration of C-Phycocyanin ameliorates thioacetamide-induced hepatic encephalopathy in Wistar rats

Fulminant hepatic failure (FHF) is a condition with a sudden onset of necrosis followed by degeneration of hepatocytes, without any previously established liver disease, generally occurring within hours or days. FHF is associated with a wide spectrum of neuropsychiatric alterations ranging from stupor to coma, culminating in death. In the present study FHF was induced in rats by the administration of thioacetamide (TAA). Oxidative stress is thought to play a prominent role in the pathophysiology of cerebral changes during FHF leading to the assumption that antioxidants might offer protection. Hence, in the present study the protective effect of C-Phycocyanin (C-PC), a natural antioxidant, was evaluated on TAA-induced tissue damage. C-Phycocyanin was administered intraperitoneally twice at 24 h interval (50 mg/kg body weight) along with the hepatotoxin TAA (300 mg/kg body weight). The animals were sacrificed 18 h after the second injection of TAA treatment and various biochemical parameters were analysed in liver, serum and brain tissues. These studies revealed significant prevention of TAA-induced liver damage by C-PC, as evidenced by a) increase in survival rate; b) the prevention of leakage of liver enzymes (AAT and AST) and ammonia into serum; c) increase in prothrombin time and d) liver histopathology. Ultrastructural studies of astrocytes of different regions of brain clearly showed a decrease in edema after C-PC treatment. TAA-induced histopathological lesions in different regions of the brain namely cerebral cortex, cerebellum and pons medulla were significantly reduced by the co-administration of C-PC with TAA. Further C-PC treatment resulted in a) decrease in the levels of tryptophan and markers of lipid peroxidation and b) elevation in the activity levels of catalase, glutathione peroxidase in different regions of brain. These studies reveal the potential of C-PC in ameliorating TAA-induced hepatic encephalopathy by improving antioxidant defenses.

Hyperammonemic alterations in the uptake and release of glutamate and aspartate by rat cerebellar preparations.

Release and uptake of neurotransmitter amino acids, glutamate and aspartate, were studied in the synaptosomes, astrocytes and in the perikarya of granule neurons isolated from the cerebella of normal and hyperammonemic rats. During acute hyperammonemia, depolarization-induced release of both the amino acids from the synaptosomes was elevated. The Vmax values of high-affinity uptake systems were elevated without alterations in the Km values for these two amino acids during acute hyperammonemic states. In the case of the low-affinity uptake system of these two amino acids, there was a decrease in the Km values without alterations in the Vmax values. These results are discussed in relation to the mechanism of ammonia toxicity.

Ammonia-induced alterations in glutamate and muscimol binding to cerebellar synaptic membranes

Binding of glutamate and muscimol (an agonist for GABAA receptors) to their respective receptors has been studied in the cerebellum of normal and hyperammonemic rats. There was a decrease in both high- and low-affinity binding of glutamate in the cerebellum during hyperammonemia. Kinetic studies revealed that the decrease is due to a reduction in the number of binding sites, but not due to changes in the binding affinities. Further studies also revealed that the decrease was only in the N-methyl-D-aspartate (NMDA)-specific binding sites without any alterations in the binding to non-NMDA sites represented by kianic acid (KA)- and quisqualic acid (QQ)-sensitive receptor sites. These effects were also mimicked when the membrane preparations from the cerebellum of normal animals were incubated with ammonium acetate. Enhancement of muscimol binding was observed in animals injected with ammonium acetate. It is concluded that hyperammonemic states, even in the presence of a functional liver, are capable of altering amino acid neurotransmission and this might play an important role in cerebral dysfunction under these conditions.

Isolation of astrocytes, neurons, and synaptosomes of rat brain cortex: distribution of enzymes of glutamate metabolism

A simplified method was developed for the bulk separation of neuronal perikarya and astroglial celis from adult rat brain without the involvement of density gradients. Activities of various enzymes involved in glutamate metabolism were estimated and compared with those of synaptosomes. The activities of glutamate dehydrogenase and aspartate aminotransferase were higher in synaptosomes than in neuronal perikarya or glia. Glutamine synthetase was distributed in all the three fractions while glutaminase activity was higher in astrocytes than in synaptosomes and was not detectable in neuronal perikarya. The significance of these results in relation to metabolic compartmentation was discussed.

Acute metabolic effects of ammonia on the enzymes of glutamate metabolism in isolated astroglial cells

Enzymes of glutamate metabolism were studied in the astrocytes isolated from rats injected with a large dose of ammonium acetate and compared with those isolated from controls. The activities of glutamate dehydrogenase (GDH) and glutaminase decreased while those of glutamine synthetase (GS) and aspartate aminotransferase (AAT) increased both in convulsive and comatose states. The activity of alanine aminotransferase (A1AT) increased only in convulsive state. The results suggested that glutamate required for the formation of glutamine in astrocytes might have its origin in nerve endings and the depletion of citric acid cycle intermediates might occur in nerve endings at least in acute ammonia toxicity.

Acute effects of ammonia on the enzymes of citric acid cycle in rat brain

Activities of the enzymes of citric acid cycle were determined along with aspartate and alanine aminotransferases and NADP(+)-isocitrate dehydrogenase in the brains of rats treated with an acute dose of ammonium acetate and compared with those of normal animals. Elevation in the activities of pyruvate, ?-ketoglutarate and succinate dehydrogenases and citrate synthase was observed in hyperammonemic animals. The activities of malate, NADP(+)-isocitrate dehydrogenases and aminotransferases decreased under these conditions. The results suggest that ammonia toxicity might not be due to the depletion of ?-ketoglutarate from citric acid cycle.