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Abstract

Ammonia is a regular metabolite in the central nervous system (CNS). However, when it enters the brain from blood in excessive quantities it becomes toxic to CNS cells. Therefore, ammonia is a causative factor in neurological disorders associated with increased blood ammonia, among which hepatic encephalopathy (HE) is a public health problem. Astrocytes are the cells that in the CNS where ammonia is metabolized in a reaction of glutamine synthesis from glutamate and ammonia, and are the primary victim of ammonia toxicity. Bioenergetic failure, oxidative or nitrosative stress and excessive accumulation of glutamine are the interrelated aspects of ammonia-induced astrocytic impairment that contribute to cerebral edema – a major cause of death associated with acute HE. Effects of ammonia on astrocytes radiate to neurons and affect the astrocytic-neuronal interactions. Interference of ammonia with the various steps of the glutamine-glutamate cycle in astrocytes lead to alterations in amino acidergic (glutaminergic and GABAergic) neurotransmission. Ammonia increases GABA-ergic tone by stimulating peripheral benzodiazepine receptors on astrocytes, in this way enhancing the synthesis of neurosteroids that are positive modulators of the GABAA receptors. Direct effects of ammonia on neurons are highlighted by the changes in the NMDA receptor/nitric oxide/cGMP pathway. Overactivation of NMDA receptors in the acute phase of ammonia toxicity is responsible for oxidative and nitrosative stress in neurons (and perhaps in astrocytes), whereas their downregulation upon prolonged exposure to ammonia.leads to impairement of cGMP synthesis held responsible for intellectual and memory deficits of chronic HE patients. Acute hyperammonemia is often associated with increased cerebral blood flow that by a complex mechanism contributes to hyperammonemic brain edema. Ammonia increases the transport across the blood-brain barrier of aromatic amino acids that are precursors of catecholamines: serotonin and dopamine. Ensuing derangements of catecholaminergic transmission are held responsible for sedative effects anfd motor impairment, respectively. Pharmacological interventions to attenuate individual neurotoxic effects of ammonia have in no case reached the stage of clinical trial: slowing the general metabolism with hypothermia is the most recently introduced life-saving paradigm in patients with advanced HE.

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... Hyperammonemia can also trigger severe cerebral edema, brain stem herniation, HE, cognitive impairment, seizures, and cerebral palsy, and, in much more severe cases, may cause neurodevelopmental intellectual disability and even death (Cagnon and Braissant, 2007;Häberle, 2013). Hyperammonemia seems to affect brain function via several mechanisms, with astrocytes the cells primarily affected, due to their topographical position in the blood-brain barrier (Albrecht, 2007), with astrocyte damage, known as astrocytic swelling, playing a key role in brain damage. Astrocytes play an important role in the maintenance of CNS function by means of their interaction with other neural cells, such as neurons and endothelial Frontiers in Neuroscience | www.frontiersin.org ...
... cells, and their modulation of both excitatory and inhibitory neurotransmission. One of the mechanisms known to be produced by hyperammonemia is a bioenergetic failure that can be caused by increased ATP consumption in astrocytes due to increased glutamine synthesis, enhanced Na-K-ATPase activity, and even the inhibition of enzymes within the tricarboxylic acid cycle (TCA cycle) (Farinelli and Nicklas, 1992;Waniewski, 1992;Albrecht, 2007;Albrecht et al., 2009). ...
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Chapter
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: Rates of glutamine formation and of carbon dioxide production (as an indication of oxidative deamination of glutamate) were determined in primary cultures of astrocytes exposed to 50 μM labeled glutamate in the absence or presence of added ammonia (0.1–3 mM). Glutamine formation (1.7 nmol/min/mg protein) was unaffected by all concentrations of added ammonia. This probably reflects the presence of a low content of ammonia (0.1–0.2 mM), originating from degradation of glutamine, in the cells even in the absence of added ammonia, and it shows that pathophysiological concentrations of ammonia do not increase the formation of glutamine from exogenous glutamate. The carbon dioxide production rate was 5.9 nmol/min/mg protein, i.e., three to four times higher than the rate of glutamine formation. It was significantly reduced (to 3.5 nmol/min/mg protein) in the presence of 1 mM or more of ammonia. This is in keeping with suggestions by others that toxic levels of ammonia affect oxidative metabolism.
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The aim of this work was to assess whether ammonia concentrations similar to the increase found in the brain of hyperammonemic rats (100 μm), impair N-methyl-d-aspartate (NMDA) receptor-mediated signal transduction. We first measured glutamate neurotoxicity, which in these neurons is mediated by activation of NMDA receptors, as an initial parameter reflecting activation of NMDA receptor-mediated pathways. Long-term treatment of cultured neurons with ammonia prevents glutamate-induced neuronal death. The EC50 was 20 μm, and at 100 μm the protection was complete. The induction of the protective effect was not immediate, but took several hours.  Treatment with 100 μm ammonia did not prevent a glutamate- or NMDA-induced rise of intracellular calcium. Ammonia impaired the glutamate–nitric oxide–cGMP (3′,5′-cyclic guanosine monophosphate) pathway in a dose- and time-dependent manner. Glutamate-induced formation of cGMP was reduced by 42%, while activation of nitric oxide synthase was not affected. Ammonia reduced by 31% cGMP formation induced by S-nitroso-N-acetyl-penicillamine (SNAP), a NO-generating agent, confirming that the interference occurs at the level of guanylate cyclase activation by nitric oxide.  To assess whether chronic moderate hyperammonemia in vivo also impairs the glutamate–nitric oxide–cGMP pathway, we determined by in vivo brain microdialysis in freely moving rats the formation of cGMP induced by NMDA. In hyperammonemic rats, the formation of cGMP induced by NMDA and SNAP was reduced by ca. 60 and 41%, respectively, indicating that chronic hyperammonemia in the animal in vivo also impairs the glutamate–nitric oxide–cGMP pathway. Impairment of this pathway can contribute to the neurological alterations found in hyperammonemia and hepatic encephalopathy.
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Ammonia is a neurotoxin implicated in the pathogenesis of hepatic encephalopathy, Reye's 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. © 2004 Wiley-Liss, Inc.
Article
Brain edema is a serious complication of hepatic encephalopathy associated with fulminant hepatic failure (FHF). A major component of the edema seems to be cytotoxic, involving astrocyte swelling. Although the mechanism of brain edema in FHF is incompletely understood, it is generally believed that ammonia is involved critically in this process. Recent studies have shown that exposure of cultured astrocytes to ammonia results in the mitochondrial permeability transition (MPT), a phenomenon associated with mitochondrial failure and subsequent cellular dysfunction. The present study examined the potential role of the MPT in the astrocyte swelling associated with ammonia toxicity. Treatment of cultured astrocytes with ammonia (5 mM) caused a time-dependent increase in astrocyte cell volume (swelling), which was completely inhibited by the MPT inhibitor cyclosporin A (CsA). In this study, CsA also inhibited the ammonia-induced aquaporin 4 (AQP4) upregulation, which had been shown previously to be increased in cultured astrocytes by ammonia treatment. These findings suggest that the MPT plays a significant role in the ammonia-induced astrocyte swelling and may contribute to the brain edema associated with FHF. © 2003 Wiley-Liss, Inc.
Article
Hepatic encephalopathy (HE) results from acute or chronic liver dysfunction and is associated with hyperammonemia. Ammonium ions penetrate from blood to brain, where they form glutamine (Gln) in the reaction with glutamate catalyzed by an astroglia-specific enzyme, glutamine synthetase (GS). Experimental data suggest that many manifestations of HE can be ascribed to increased Gln synthesis and accumulation in the brain. In HE resulting from acute liver failure (“fulminant hepatic failure”), the osmotic action of Gln appears to be in a large degree responsible for cerebral edema and edema-associated disturbances of cerebral blood flow and ionic homeostasis. In chronic HE not accompanied by cerebral edema, Gln contributes to impairment of cerebral energy metabolism, and its increased transport from brain to the periphery accelerates the blood-to-brain transport of aromatic amino acids, of which tryptophen (Trp) is converted to metabolites directly implicated in HE. Most of the evidence that Gln participates in pathological events has been derived from their disappearance or amelioration in HE rats in which the cerebral Gln content was reduced by treatment with a GS inhibitor, methionine sulfoximine. J. Neurosci. Res. 65:1–5, 2001. © 2001 Wiley-Liss, Inc.
Article
The activity of the blood-brain neutral amino acid transport system is increased in rats infused with ammonium salts or rendered hyperammonemic by a portacaval anastomosis. This effect may be due to a direct action of ammonia or to some metabolic consequence of high ammonia levels, such as increased brain glutamine synthesis. To test these possibilities we evaluated the kinetic parameters of blood-brain transport of leucine and phenylalanine in control rats, in rats after continuous 24 h infusion of ammonium salts (NH4+= 2.5 mmol. kg−1 h−1), and in rats treated with methionine sulfoximine, an inhibitor of glutamine synthetase, before infusion of ammonium salts. In ammonia-infused rats without methionine sulfoximine treatment, the KD and Vmax of phenylalanine transport were increased, respectively, about 170% and 80% compared to controls, whereas the Km and Vmax of leucine transport were increased, respectively, about 100% and 200%. Electron microscopy demonstrated marked swelling of astrocytic processes around brain capillaries of ammonia-infused rats; however, capillary permeability to horseradish peroxidase apparently was not increased by ammonia infusion. Administration of methionine sulfoximine before ammonia infusion inhibited glutamine synthesis and prevented the changes in transport of leucine and phenylalanine, but apparently did not reverse the perivascular swelling. These results suggest that the ammonia-induced increase in the activity of transport of large neutral amino acids across the blood-brain barrier requires glutamine synthesis in brain, and is not a direct effect of ammonia.
Article
Some metabolic effects on primary cultures of neurons or astrocytes were studied following acute or chronic exposure to pathophysiological concentrations (usually 3 mM) of ammonia. Three parameters were investigated: (1)14CO2 production from14C-labeled substrates [glucose, pyruvate, branched-chain amino acids (leucine, valine, isoleucine), and glutamate]; (2) interconversion between glutamate and glutamine; and (3) incorporation of label from labeled branched-chain amino acids into proteins. Neither acute nor chronic exposure to ammonia had any effect on14CO2 production from [U-14C]glucose in astrocytes and neurons, whereas under certain conditions14CO2 production from [1-14C]pyruvate in astrocytes was inhibited by ammonia. Production of14CO2 from [1-14C]branched-chain amino acids was inhibited by acute, but stimulated by chronic, exposure to ammonia (3 mM) in astrocytes, with less effect in neurons. Production of14CO2 from [1-14C]glutamate in both astrocytes and neurons was inhibited by acute exposure to ammonia. In astrocytes, glutamate levels tended to decrease and glutamine levels tended to increase following acute exposure to ammonia; in neurons, both glutamine and glutamate levels decreased. Protein content (per culture dish) increased in astrocytes but not in neurons, after chronic exposure to ammonia, possibly as a result of enhanced protein synthesis and/or by inhibition of protein degradation.
Article
Most of the brain glycogen, a major energy reserve that can be mobilized in response to increased neuronal activity, resides in the astrocyte, the site of the neuropathological abnormality found in hepatic encephalopathy (HE). Ammonia, a neurotoxin implicated in the pathogenesis of HE, has been reported to cause a depletion of glycogen in primary astrocyte cultures. To further investigate the action of ammonia on glycogen levels, cultured astrocytes were exposed to ammonium chloride (1–5 mM) for various times up to 7 d. Treatment with ammonia for 24 h did not alter deoxyglucose uptake, but significantly lowered peak glycogen values (found at 1.5 h following feeding with medium containing 5.5 mM glucose) in a concentration-dependent manner. This inhibitory effect was not observed after longer exposure times to ammonia. Three day treatment of cells did, however, significantly reduce norepinephrine-stimulated glycogenolysis, an effect not seen after 1 d of ammonia treatment. Part of the neurotoxic action of long term ammonia exposure in humans and experimental animals may be to inhibit the breakdown of glycogen. The effect of ammonia on astrocyte glycogen synthesis and/or breakdown may disrupt glial neuronal signaling and thus play a role in the pathogenesis of HE.
Article
We proposed that acute ammonia toxicity is mediated by activation of NMDA receptors. To confirm this hypothesis we have tested whether different NMDA receptor antagonists, acting on different sites of NMDA receptors, prevent death of mice induced by injection of 14 mmol/Kg of ammonium acetate, a dose that induces death of 95% of mice. MK-801, phencyclidine and ketamine, which block the ion channel of NMDA receptors, prevent death of at least 75% of mice. CPP, AP-5, CGS 19755, and CGP 40116, competitive antagonists acting on the binding site for NMDA, also prevent death of at least 75% of mice. Butanol, ethanol and methanol which block NMDA receptors, also prevent death of mice. There is an excellent correlation between the EC50 for preventing ammonia-induced death and the IC50 for inhibiting NMDA-induced currents. Acute ammonia toxicity is not prevented by antagonists of kainate/AMPA receptors, of muscarinic or nicotinic acetylcholine receptors or of GABA receptors. Inhibitors of nitric oxide synthase afford partial protection against ammonia toxicity while inhibitors of calcineurin, of glutamine synthetase or antioxidants did not prevent ammonia-induced death of mice. These results strongly support the idea that acute ammonia toxicity is mediated by activation of NMDA receptors.
Article
Hepatic encephalopathy (HE) and portal-systemic encephalopathy (PSE) are the terms used interchangeably to describe a complex neuropsychiatric syndrome associated with acute or chronic hepatocellular failure, increased portal systemic shunting of blood, or both. Hepatic encephalopathy complicating acute liver failure is referred to as fulminant hepatic failure (FHF). The clinical manifestations of HE or PSE range from minimal changes in personality and motor activity, to overt deterioration of intellectual function, decreased consciousness and coma, and appear to reflect primarily a variable imbalance between excitatory and inhibitory neurotransmission. Pathogenic mechanisms that may be responsible for HE have been extensively investigated using animal models of HE, or cultures of CNS cells treated with neuroactive substances that have been implicated in HE. Of the many compounds that accumulate in the circulation as a consequence of impaired liver function, ammonia is considered to play an important role in the onset of HE. Acute ammonia neurotoxicity, which may be a cause of seizures in FHF, is excitotoxic in nature, being associated with increased synaptic release of glutamate (Glu), the major excitatory neurotransmitter of the brain, and subsequent overactivation of the ionotropic Glu receptors, mainly the N-methyl-D-aspartate (NMDA) receptors. Hepatic encephalopathy complicating chronic liver failure appears to be associated with a shift in the balance between inhibitory and excitatory neurotransmission towards a net increase of inhibitory neurotransmission, as a consequence of at least two factors. The first is down-regulation of Glu receptors resulting in decreased glutamatergic tone. The down-regulation follows excessive extrasynaptic accumulation of Glu resulting from its impaired re-uptake into nerve endings and astrocytes. Liver failure inactivates the Glu transporter GLT-1 in astrocytes. The second factor is an increase in inhibitory neurotransmission by gamma-aminobutyric acid (GABA) due to (a) increased brain levels of natural benzodiazepines; (b) increased availability of GABA at GABA-A receptors, due to enhanced synaptic release of the amino acid; (c) direct interaction of modestly increased levels of ammonia with the GABA-A-benzodiazepine receptor complex; and (d) ammonia-induced up-regulation of astrocytic peripheral benzodiazepine receptors (PBZR). Brain ammonia is metabolised in astrocytes to glutamine (Gln), an osmolyte, and increased Gln accumulation in these cells may contribute to cytotoxic brain edema, which often complicates FHF. Glutamine efflux from the brain is an event that facilitates plasma-to-brain transport of aromatic amino acids. Tryptophan and tyrosine are direct precursors of the aminergic inhibitory neurotransmitters, serotonin and dopamine, respectively. Changes in serotonin and dopamine and their receptors may contribute to some of the motor manifestations of HE. Finally, oxindole, a recently discovered tryptophan metabolite with strong sedative and hypotensive properties, has been shown to accumulate in cirrhotic patients and animal models of HE.
Article
Rats treated with oxindole (10-100 mg/kg i.p.), a putative tryptophan metabolite, showed decreased spontaneous locomotor activity, loss of the righting reflex, hypotension, and reversible coma. Brain oxindole levels were 0.05 +/- 0.01 nmol/g in controls and increased to 8.1 +/- 1.7 or 103 +/- 15 nmol/g after its administration at doses of 10 or 100 mg/kg i.p., respectively. To study the role that oxindole plays in the neurological symptoms associated with acute liver failure, we measured the changes of its concentration in the brain after massive liver damage, and we investigated the possible metabolic pathways leading to its synthesis. Rats treated with either thioacetamide (0.2 and 0.4 g/kg i.p., twice) or galactosamine (1 and 2 g/kg i.p.) showed acute liver failure and a large increase in blood or brain oxindole concentrations (from 0.05 +/- 0.01 nmol/g in brains of controls to 1.8 +/- 0.3 nmol/g in brains of thioacetamide-treated animals). Administration of tryptophan (300-1,000 mg/kg p.o.) caused a twofold increase, whereas administration of indole (10-100 mg/kg p.o.) caused a 200-fold increase, of oxindole content in liver, blood, and brain, thus suggesting that indole formation from tryptophan is a limiting step in oxindole synthesis. Oral administration of neomycin, a broad-spectrum, locally acting antibiotic agent able to reduce intestinal flora, significantly decreased brain oxindole content. Taken together, our data show that oxindole is a neurodepressant tryptophan metabolite and suggest that it may play a significant role in the neurological symptoms associated with acute liver impairment.
Article
The distribution of glutamine synthetase was determined in rat brain by ultrastructural immunocytochemistry. Except for trace amounts in a rare indeterminate glial cell, all the reaction product was located in astrocytes. Specifically, none was observed in neurons, synaptic endings, oligodendrocytes, microglial cells, pericytes, endothelial cells and other mesenchymal vascular elements. The results of this study clearly indicate that the astrocyte forms the compartment in brain concerned with glutamine synthesis, thereby assigning a key role to the astrocyte in the metabolism of ammonia and the putative neurotransmitters, glutamic acid and gamma-aminobutyric acid. The localization of glutamine synthetase in astrocytes additionally provides a valuable marker for a number of neurobiological studies.
Article
Effects of 1 and 5 mM ammonium acetate on glucose metabolism were studied in astrocytes. But for an elevation in the levels of fructose-6-phosphate, phosphoenol pyruvate, and pyruvate, glucose metabolism was unaltered in the presence of 1 mM ammonium acetate. With 5 mM ammonium acetate, but for unaltered lactate, ADP, ATP and decreased aspartate, levels of several intermediates were elevated. Similar results were obtained when astrocytes isolated from hyperammonemic rats were incubated with glucose except for an enhanced production of 14CO2 from [U-14C]glucose. It is suggested that glucose metabolism of astrocytes may not be severely affected in astrocytes of cerebral cortex in acute hyperammonemic states.
Article
Previous experiments in our laboratory suggested that ammonium toxicity could be mediated by the NMDA type of glutamate receptors. To assess this hypothesis we tested if MK-801, a specific antagonist of the NMDA receptor, is able to prevent ammonium toxicity. Mice and rats were injected i.p. with 12 and 7 mmol/kg of ammonium acetate, respectively. 73% of the mice and 70% of the rats died. However, when the animals were injected i.p. with 2 mg/kg of MK-801, 15 min before ammonium injection, only 5% of the mice and 15% of the rats died. The remarkable protection afforded by MK-801 indicates that ammonia toxicity is mediated by the NMDA receptor.
Article
The effect of ammonia on glutamate accumulation and metabolism was examined in astrocyte cultures prepared from neonatal rat cortices. Intact astrocytes were incubated with 70 microM L-[14C(U)]glutamate and varying amounts of ammonium chloride. The media and cells were analyzed separately by HPLC for amino acids and labelled metabolites. Extracellular glutamate was reduced to 8 microM by 60 min. Removal of glutamate from the extracellular space was not altered by addition of ammonia. The rate of glutamine synthesis was increased from 3.6 to 9.3 nmol/mg of protein/min by addition of 100 microM ammonia, and intracellular glutamate was reduced from 262 to 86 nmol/mg of protein after 30 min. The metabolism of accumulated glutamate was matched nearly perfectly by the synthesis of glutamine, and both processes were proportional to the amount of added ammonia. The transamination and deamination products of glutamate were minor metabolites that either decreased or remained unchanged with increasing ammonia. Thus, ammonia addition stimulates the conversion of glutamate to glutamine in intact astrocyte cultures. At physiological concentrations of ammonia, glutamine synthesis appears to be limited by the rate of glutamate accumulation and the activity of competing reactions and not by the activity of glutamine synthetase.
Article
Glutamate metabolism in rat cortical astrocyte cultures was studied to evaluate the relative rates of flux of glutamate carbon through oxidative pathways and through glutamine synthetase (GS). Rates of 14CO2 production from [1-14C]glutamate were determined, as was the metabolic fate of [14C(U)]glutamate in the presence and absence of the transaminase inhibitor aminooxyacetic acid and of methionine sulfoximine, an irreversible inhibitor of GS. The effects of subculturing and dibutyryl cyclic AMP treatment of astrocytes on these parameters were also examined. The vast majority of exogenously added glutamate was converted to glutamine and exported into the extracellular medium. Inhibition of GS led to a sustained and greatly elevated intracellular glutamate level, thereby demonstrating the predominance of this pathway in the astrocytic metabolism of glutamate. Nevertheless, there was some glutamate oxidation in the astrocyte culture, as evidenced by aspartate production and labeling of intracellular aspartate pools. Inhibition of aspartate aminotransferase caused a greater than 70% decrease in 14CO2 production from [1-14C]glutamate. Inhibition of GS caused an increase in aspartate production. It is concluded that transamination of glutamate rather than oxidative deamination catalyzed by glutamate dehydrogenase is the first step in the entry of glutamate carbon into the citric acid cycle in cultured astrocytes. This scheme of glutamate metabolism was not qualitatively altered by subculturing or by treatment of the cultures with dibutyryl cyclic AMP.
Article
The release of newly loaded L-[14C]glutamine (L-Gln) from rat cerebral cortical capillaries was stimulated by L-transport system substrates: tryptophan (TRY), leucine (leu), and nonlabeled L-Gln, respectively, by 32, 50, and 40% above the basal release resulting from superfusion with standard Krebs–Henseliet buffer. However, no stimulation was observed upon treatment with D-Gln or L-glutamate (L-Glu), which are not the L-system substrates, or with ammonium chloride. The stimulatory effect of TRY was temperature dependent but sodium independent, and was abolished in the presence of a sulfhydryl reagent N-ethylmaleimide (NEM). The results support the view that the L-Gln-stimulated uptake of large neutral amino acids (LNAA) across the blood–brain barrier involves the L-system mediated Gln-LNAA exchange. The TRY-stimulated Gln release was enhanced in vitro by simultaneous addition of ammonium chloride, and in capillaries derived from rats with acute hepatic encephalopathy (HE). These results confirm the role of Gln-LNAA exchange in the excessive accumulation of LNAA in brain observed in a variety of hyperammonemic conditions. Superfusion of L-Gln-loaded capillaries in a buffer containing γ-glutamyl transpeptidase (GGT) inhibitors, serine borate (SB) or 6-diazo-5-oxo-L-norleucine (DON), increased the basal L-Gln release and made it irresponsive to subsequent treatment with TRY. However, the basal release was also increased by superfusion with serine alone or Leu, and this treatment abolished the subsequent effect of TRY as well. Moreover, DON stimulated L-Gln release from capillaries superfused in a standard way, and the effects of DON and TRY were additive. Hence, in the present conditions, SB and DON acted as L-system substrates rather than as GGT inhibitors. Taken together, the results do not support the concept that GGT mediates the Gln-LNAA exchange.
Article
In previous studies we documented an increase in the levels of the serotonin metabolite, 5-hydroxyindoleacetic acid, in the congenitally hyperammonemic sparse fur mouse. To extend these findings, brain serotonin receptors were studied in these animals. Radioligand binding assays were performed using [3H]ketanserin to label serotonin2 sites and 8-[3H]hydroxy(di-n-propylamino)tetralin to label serotonin1A sites in cortical membrane homogenates. The capacity (Bmax) for [3H]ketanserin binding was significantly lower (-21%; p less than 0.05) in sparse fur animals than in control animals; there was no change in affinity (KD). In contrast, the capacity for 8-[3H]hydroxy(di-n-propylamino)tetralin binding was significantly greater (26%; p less than 0.05) in sparse fur compared with control animals. No difference in affinity was observed. Using two behavioral assays, the functional responsiveness of these serotonin receptors was compared in sparse fur and control animals. Head twitch activity elicited by administration of the serotonin agonist quipazine was studied as a behavior mediated by serotonin2 receptors. Compared with controls, sparse fur mice demonstrated a significantly decreased head twitch response (p less than 0.005). Hypothermia elicited by administration of 8-hydroxy(di-n-propylamino)tetralin was studied as a physiologic response mediated by serotonin1A receptors. Although there were not overall group differences in the dose-response data, there was a significant increase in the hypothermia induced by 8-hydroxy(di-n-propylamino)tetralin in sparse fur compared with control mice (p less than 0.02) at the highest dose. These data provide further support for a link between hyperammonemia and alterations in the serotonin system.
Article
Diazepam binding inhibitor (DBI) is a 9-kD polypeptide that was first isolated in 1983 from rat brain by monitoring its ability to displace diazepam from the benzodiazepine (BZD) recognition site located on the extracellular domain of the type A receptor for gamma-aminobutyric acid (GABAA receptor) and from the mitochondrial BZD receptor (MBR) located on the outer mitochondrial membrane. In brain, DBI and its two major processing products [DBI 33-50, or octadecaneuropeptide (ODN) and DBI 17-50, or triakontatetraneuropeptide (TTN)] are unevenly distributed in neurons, with the highest concentrations of DBI (10 to 50 microMs) being present in the hypothalamus, amygdala, cerebellum, and discrete areas of the thalamus, hippocampus, and cortex. DBI is also present in specialized glial cells (astroglia and Bergmann glia) and in peripheral tissues. In the periphery, the highest concentration of DBI occurs in cells of the zona glomerulosa and fasciculata of the adrenal cortex and in Leydig cells of the testis; interestingly, these are the same cell types in which MBRs are highly concentrated. Stimulation of MBRs by appropriate ligands (including DBI and TTN) facilitates cholesterol influx into mitochondria and the subsequent formation of pregnenolone, the parent molecule for endogenous steroid production; this facilitation occurs not only in peripheral steroidogenic tissues, but also in glial cells, the steroidogenic cells of the brain. Some of the steroids (pregnenolone sulfate, dehydroepiandrosterone sulfate, 3 alpha-hydroxy-5 alpha-pregnan-20-one, and 3 alpha, 21-dihydroxy-5 alpha-pregnan-20-one) produced in brain (neurosteroids) function as potent (with effects in the nanomolar concentration range) positive or negative allosteric modulators of GABAA receptor function. Thus, accumulating evidence suggests that the various neurobiological actions of DBI and its processing products may be attributable to the ability of these peptides either to bind to BZD recognition sites associated with GABAA receptors or to bind to glial cell MBRs and modulate the rate and quality of neurosteroidogenesis. The neurobiological effects of DBI and its processing products in physiological and pathological conditions (hepatic encephlopaty, depression, panic) concentrations may therefore be explained by interactions with different types of BZD recognition site. In addition, recent reports that DBI and some of its fragments inhibit (in nanomolar concentrations) glucose-induced insulin release from pancreatic islets and bind acyl-coenzyme A with high affinity support the hypothesis that DBI isa precursor of biologically active peptides with multiple actions in the brain and in peripheral tissues.
Article
Alpha-ketoglutarate together with an amino group donor (alanine) was shown to be able to serve as a precursor for the glutamate pool which is released by potassium-induced depolarization (i.e., transmitter glutamate) in cerebellar granule cells. However, these compounds could not be utilized as precursors for intracellular glutamate or for release of transmitter aspartate. The formation of transmitter glutamate was inhibited by the transamination inhibitor aminooxyacetic acid but not by phenylsuccinate, an inhibitor of the dicarboxylate carrier in the mitochondrial membrane. Both of these inhibitors have previously been found to inhibit synthesis of transmitter glutamate from glutamine. The results support the hypothesis that alpha-ketoglutarate and alanine undergo transmination in the cytosol to form pyruvate and glutamate, and that this glutamate pool is available for transmitter release of glutamate but does not constitute the major intracellular pool of glutamate.
Article
The mechanism of brain swelling during hyperammonemia is not understood, but glutamine accumulation is consistently observed. We tested the hypothesis that brain swelling associated with hyperammonemia is a consequence of the osmotic effect of intracellular glutamine accumulation in brain. Increases in plasma ammonium levels from 31 +/- 3 to 601 +/- 38 mumol/l (+/- SE) were produced by 6 h of infusion of ammonium acetate in anesthetized rats. Hyperammonemia resulted in increased brain water content accompanied by more than a tripling of brain glutamine concentration compared with control rats receiving sodium acetate (5.6 +/- 0.4 vs. 18.8 +/- 0.4 mmol/kg). Inhibition of glutamine synthetase activity by pretreatment with L-methionine sulfoximine prevented both the increase in brain glutamine levels and the increase in brain water content despite elevated plasma ammonium levels (908 +/- 196 mumol/l). Thus cerebral edema during hyperammonemia is associated with glutamine accumulation. We suggest that accumulated glutamine may serve as an idiogenic osmole causing swelling. Because brain swelling eventually leads to increased intracranial pressure and tissue hypoxia, these data suggest a unifying mechanism to account for the many pathophysiological abnormalities found during coma associated with various forms of liver disease, inborn errors of metabolism, and Reye's syndrome.
Article
The effects of in vitro treatment with ammonium chloride, hepatic encephalopathy (HE) due to thioacetamide (TAA) induced liver failure and chronic hyperammonemia produced by i.p. administration of ammonium acetate on the activity of the two malate-aspartate shuttle enzymes: aspartate aminotransferase (AAT), malate dehydrogenase (MDH), and on the pyruvate carboxylase (PC) activity were examined in synaptic and nonsynaptic mitochondria from rat brain. With regard to the shuttle enzymes the response to ammonium ions in vitro (3mM NH4Cl) was observed in nonsynaptic mitochondria only, and was manifested by a 27% decrease of AAT activity and a 16% decrease in MDH activity. By contrast, both in vivo conditions primarily affected the synaptic mitochondrial enzymes: TAA-induced HE produced a 26% decrease of synaptic mitochondrial AAT and a 50% decrease of synaptic mitochondrial MDH. Hyperammonemia inhibited synaptic mitochondrial AAT by 30% and synaptic mitochondrial MDH by 45%. HE produced no effect at all in nonsynaptic mitochondria while hyperammonemia produced a 30% increase in the AAT activity, but no changes in MDH. All the experimental conditions affected the nonsynaptic mitochondria PC: ammonium chloride in vitro produced a 20% decrease, TAA-induced HE--a 30% decrease, whereas hyperammonemia inhibited the enzyme by 53%. The PC activity in synaptic mitochondria was very low (about 2% of that measured in nonsynaptic mitochondria), which is consistent with the primarily astrocytic localization of the enzyme.
Article
Excitatory amino acids have been implicated in the pathogenesis of hepatic encephalopathy. In the present study, kainate, quisqualate and N-methyl-D-aspartate (NMDA) subclasses of L-glutamate receptors were measured in adult rat brain by quantitative receptor autoradiography following surgical construction of an end-to-side portacaval anastomosis (PCA). PCA resulted in sustained hyperammonemia and decreased binding of L-glutamate to the NMDA receptor when compared to sham-operated controls. Decreases in binding ranged from 17 to 39% in several regions of cerebral cortex, hippocampus, striatum, and thalamus. Binding to quisqualate and kainate receptor subtypes was not altered. PCA leads to astrocytic changes in brain but does not result in any measurable loss of neuronal integrity. It is therefore proposed that decreased glutamate binding to the NMDA receptor following PCA results from increased extracellular glutamate caused by decreased reuptake into perineuronal astrocytes and a compensatory down-regulation of these receptors. Such changes could be of pathophysiological significance in hepatic encephalopathy.
Article
Peripheral-type benzodiazepine receptors were evaluated using the specific ligand [3H]-PK 11195 in brain homogenates from nine cirrhotic patients who died in hepatic coma and from an equal number of age-matched control subjects. Histopathological studies showed evidence of severe Alzheimer type II astrocytosis in the brains of all cirrhotic patients. Saturation-binding assays revealed a single saturable binding site for [3H]-PK 11195 in brain, with affinities in the 2- to 3-nmol/L range. Diazepam was found to be a relatively potent inhibitor of 3H-PK 11195 binding (IC50 = 253 nmol/L), whereas the central benzodiazepine antagonist Ro 15-1788 displaced 3H-PK 11195 binding with low affinity (IC50 greater than 40 mumols/L). Densities of [3H]-PK 11195 binding sites were found to be increased by 48% (p less than 0.01) and 25% (p less than 0.05) in frontal cortex and caudate nuclei, respectively, from cirrhotic patients. Densities of [3H]-PK 11195 binding sites in frontal cortex from two nonencephalopathic cirrhotic patients were not significantly different from control values. No concomitant changes of affinities of these binding sites were observed. Because it has been suggested that peripheral-type benzodiazepine receptors may be localized on mitochondrial membranes and may therefore be involved in cerebral oxidative metabolism, the alterations observed in this study could be of pathophysiological significance in hepatic encephalopathy.
Article
Exposure of primary astrocyte cultures to ammonia caused a dose- and time-dependent reduction of isoproterenol-stimulated cyclic AMP (cAMP) production. This treatment did not affect basal cAMP levels. This defect in receptor-linked cAMP production may contribute to the pathogenesis of hepatic and ammonia encephalopathies.
Article
The effects of ammonium chloride (3 mM) and beta-methylene-DL-aspartate (BMA; 5 mM) (an inhibitor of aspartate aminotransferase, a key enzyme of the malate-aspartate shuttle (MAS] on the metabolism of glutamate and related amino acids were studied in primary cultures of astrocytes and neurons. Both ammonia and BMA inhibited 14CO2 production from [U-14C]- and [1-14C]glutamate by astrocytes and neurons and their effects were partially additive. Acute treatment of astrocytes with ammonia (but not BMA) increased astrocytic glutamine. Acute treatment of astrocytes with ammonia or BMA decreased astrocytic glutamate and aspartate (both are key components of the MAS). Acute treatment of neurons with ammonia decreased neuronal aspartate and glutamine and did not apparently affect the efflux of aspartate from neurons. However, acute BMA treatment of neurons led to decreased neuronal glutamate and glutamine and apparently reduced the efflux of aspartate and glutamine from neurons. The data are consistent with the notion that both ammonia and BMA may inhibit the MAS although BMA may also directly inhibit cellular glutamate uptake. Additionally, these results also suggest that ammonia and BMA exert differential effects on astroglial and neuronal glutamate metabolism.
Article
Evoked release of glutamate and aspartate from cultured cerebellar granule cells was studied after preincubation of the cells in tissue culture medium with glucose (6.5 mM), glutamine (1.0 mM), D[3H] aspartate and in some cases aminooxyacetate (5.0 mM) or phenylsuccinate (5.0 mM). The release of endogenous amino acids and of D-[3H] aspartate was measured under physiological and depolarizing (56 mM KCl) conditions both in the presence and absence of calcium (1.0 mM), glutamine (1.0 mM), aminooxyacetate (5.0 mM) and phenylsuccinate (5.0 mM). The cellular content of glutamate and aspartate was also determined. Of the endogenous amino acids only glutamate was released in a transmitter fashion and newly synthesized glutamate was released preferentially to exogenously supplied D-[3H] aspartate, a marker for exogenous glutamate. Evoked release of endogenous glutamate was reduced or completely abolished by respectively, aminooxyacetate and phenylsuccinate. In contrast, the release of D-[3H] aspartate was increased reflecting an unaffected release of exogenous glutamate and an increased "psuedospecific radioactivity" of the glutamate transmitter pool. Since aminooxyacetate and phenylsuccinate inhibit respectively aspartate aminotransferase and mitochondrial keto-dicarboxylic acid transport it is concluded that replenishment of the glutamate transmitter pool from glutamine, formed in the mitochondrial compartment by the action of glutaminase requires the simultaneous operation of mitochondrial keto-dicarboxylic acid transport and aspartate aminotransferase which is localized both intra- and extra-mitochondrially. The purpose of the latter enzyme apparently is to catalyze both intra- and extra-mitochondrial transamination of alpha-ketoglutarate which is formed intramitochondrially from the glutamate carbon skeleton and transferred across the mitochondrial membrane to the cytosol where transmitter glutamate is formed.(ABSTRACT TRUNCATED AT 250 WORDS)
Article
Prolonged thioacetamide treatment increased gamma-glutamyl transpeptidase (GGT) activity in the rat liver and induced neurological symptoms of hepatic encephalopathy (HE). The enzyme activity measured without an amino acid or peptide acceptor was increased in cortical capillaries and synaptosomes, but remained unchanged in astroglia isolated from the brains of hyperammonemic rats. In the presence of L-glutamine the activity of GGT was stimulated by about 60% in astroglial cells while in the capillaries and synaptosomes the amino acid stimulation was less pronounced. Glycylglycine also stimulated the GGT activity in the astroglia more (4-fold) than in cortical capillaries or synaptosomes (3-fold). Similar stimulatory effects of these gamma-glutamyl moiety acceptors on the GGT activity were observed in capillaries, glial cells and synaptosomes derived from the brains of rats with HE. These results indicate that GGT may be involved in the excessive accumulation of large neutral amino acids (and some peptides) in the brain of rats with HE.
Article
Na+/K+-ATPase activity and GABA uptake were measured in the bulk isolated astrocytes and synaptosomes from rats in which an early, metabolic phase of hepatogenic encephalopathy (HE) was induced by the treatment with thioacetamide (TAA). Both the enzyme activity and the amino acid neurotransmitter uptake were increased above control in the astroglial fraction but remained unaffected in synaptosomes. The results lend support to the earlier observations that the astrocytes are the primary target cells in HE. Furthermore, they may be interpreted as indicating that the early astroglial reaction to HE comprises stimulation of the astrocytes' function, especially concerning clearance of K+ ions and neurotransmitters from the extracellular space of CNS.
Article
1. Previously we have shown that sera from patients with fulminant hepatic failure (FHF) will inhibit partially purified rat brain Na+,K+-ATPase and sodium efflux from human leucocytes in vitro. Similar inhibition may be involved in the pathogenesis of encephalopathy and cerebral oedema in these patients. 2. In the present study we have attempted to establish whether the activity of brain Na+,K+-ATPase is decreased in vivo in rats with d-galactosamine induced hepatic failure using homogenates of snap-frozen brains. 3. Na+,K+-ATPase activity was significantly reduced in the forebrain region at the stage of mild encephalopathy (43 h after injection), while at the deeper stage of coma (43–53 h after injection) enzyme activity was further reduced in the forebrain region and was also significantly reduced in the hindbrain region. Ouabain insensitive ATPase activity was not significantly altered at any time. 4. While a significant increase in the water content (0.5%) of the hindbrain region was found 43 h after galactosamine, there was no clear correlation between the development of cerebral oedema and the reduction of Na+,K+-ATPase activity. 5. The activity of partially purified normal rat brain Na+,K+-ATPase was 15% lower when incubated with sera from rats in the deep stage of coma compared with control sera. 6. These data support other evidence that the reduction in brain Na+,K+-ATPase is likely to be due to toxic substance circulating in serum which have been shown to inhibit this enzyme in vitro and to cause coma when administered to normal animals.
Article
Oxygen consumption was measured polarographically in fractions enriched in astrocytes or neurons, and in synaptosomes derived from rats in which two subsequent stages of acute hepatic encephalopathy were induced by thioacetamide treatment. A 30% decrease of oxygen consumption was noted in astrocytes from animals with coma, well in agreement with the decrease of whole cerebral oxygen consumption and the increase of whole brain ammonia. In contrast, at the same stage the oxygen consumption in neurons was increased by some 35%, whereas synaptosomes remained unaffected. The results are in keeping with the view that astrocytes are the cells whose metabolism is primarily affected during hepatic encephalopathy. On the other hand, they support recent pathophysiologic evidence that ammonia-induced neuronal dysfunction is not a consequence of impaired energy metabolism in the nerve cells.
Article
Light microscopic studies of primary astrocyte cultures following exposure to ammonia have shown several alterations. To determine the nature and significance of these changes, electron microscopic studies were performed. Ultrastructural changes consisted of proliferation, pleomorphism and swelling of mitochondria, condensation of the mitochondrial matrix, cytoplasmic lucency and vacuolization, disaggregation of polyribosomal clusters, an initial increase followed by degranulation of rough endoplasmic reticulum, proliferation of smooth endoplasmic reticulum, an accumulation of dense bodies and a loss of intermediate glial filaments. The early alterations appeared reactive and perhaps reflected ammonia detoxification. Some changes were degenerative and support the view that ammonia exerts a direct toxic effect on astrocytes. It is postulated that these changes may interfere with critical astroglial functions and thereby play a key role in the neurologic dysfunction seen in hyperammonemia.
Article
Morphological features of three models of portalsystemic encephalopathy in the rat were studied and compared with plasma ammonia levels and clinical observations. Carbon tetrachloride-induced cirrhosis with terminal coma produced a wide variety of structural changes in the brain whose severity was related to plasma ammonia levels at the time of death. These changes included diffuse gliosis, Alzheimer cells and focal neuronal necrosis but did not include spongiform changes in cerebral or cerebellar cortex. Porta-caval anastomosis (PCA) did not appear to produce any significant neurological symptoms. Rats with PCA of durations 1–30 weeks were studied and over this time the structural changes included astrocytic nuclear swelling, swelling of perivascular astrocytic foot-processes and spongiform change in the molecular layer of the cerebellum. No evidence of Alzheimer cells or gliosis was seen and plasma ammonia levels at no stage exceed twice the normal levels. Porta-caval anastomosis followed by gavage feeding with ammoniated cationic exchange resin produced severe neurological symptoms and marked hyperammonaemia. In these animals not only astrocytes but oligodendrocytes and neurons showed nuclear and cytoplasmic swelling and numerous Alzheimer type II cells were seen, together with a diffuse gliosis, but no evidence of spongiform change in the cerebral or cerebellar cortex was seen. It is concluded that ammonium ions are important in the genesis of morphological changes in the brain in rat models of portal-systemic encephalopathy, but the relevance of these changes to neurological dysfunction is uncertain.
Article
Affinities and densities of binding sites for the 5HT1A receptor ligand [3H]8-hydroxy(di-n-propylamino)tetralin ([3H]8-OH-DPAT) and the 5HT2 receptor ligand [3H]ketanserin were measured using a rapid filtration assay in crude membrane preparations from frontal cortex and hippocampus of nine cirrhotic patients who died in hepatic encephalopathy and from an equal number of age-matched subjects free from hepatic, neurological or psychiatric disorders. Binding site densities (Bmax) obtained by Scatchard analysis of saturation binding isotherms for [3H]8-OH-DPAT were decreased in frontal cortex (by 56%, P < 0.05) and hippocampus (by 30%, P < 0.05). [3H]ketanserin binding sites were concomitantly increased (by 55%, P < 0.05) in hippocampus of cirrhotic patients. Ligand binding affinities were within normal ranges in all cases. Previous reports have described the accumulation of the 5HT metabolite 5-hydroxyindoleacetic acid and increased activities of the 5HT-metabolizing enzyme MAOA in this same material from patients with hepatic encephalopathy. Taken together, these findings suggest that alterations of serotoninergic function in brain could be responsible for some of the neuropsychiatric symptoms of hepatic encephalopathy observed in humans with chronic liver disease.
Article
The binding parameters of [3H]SCH 23390 and [3H]spiperone (radioligands for dopamine D1 and D2 receptors, respectively) were investigated in autopsied frontal cortex, caudate nucleus and globus pallidus/putamen of cirrhotic patients who died in hepatic coma as well as in age- and sex-matched controls. Specific [3H]SCH 23390 binding site densities were unchanged in all regions; in contrast, specific [3H]spiperone binding site density was decreased (by 44%, P < 0.001) in the globus pallidus/putamen of patients with HE. Decreased densities of pallidal D2 binding sites could relate to the motor dysfunctions commonly encountered in human HE.
Article
We measured the brain uptake index (BUI) for radiolabelled L-ornithine (ORN) in rats with acute hepatic encephalopathy (HE) induced by two (onset stage) or three (comatous stage) administrations of a hepatotoxin-thioacetamide (TAA). In the comatose group, an increase of the BUI to 275% of control was measured at 24 h post-treatment. In the onset group, the BUI for ORN increased gradually with time: it reached 220% of control at 7 days post-treatment and 442% of control at 21 days post-treatment. HE did not raise the BUI for a blood-brain barrier (BBB) non-penetrable amino acid L-aspartate (ASP), indicating that HE activates ORN transport but does not produce BBB leakage. ORN transport through BBB was not increased in rats with hyperammonemia comparable to that accompanying HE, but was induced without liver damage. Considering recent evidence that ORN acting intracerebrally ameliorates pathophysiological symptoms of HE, increased transport ORN across BBB should facilitate HE therapy based on systemic administration of this amino acid.
Article
Using radioenzymatic assays, activities of MAOA and MAOB were measured in autopsied brain tissue from cirrhotic patients who died in hepatic coma and in material from an equal number of age-matched subjects who were free from hepatic, neurological or psychiatric disorders. Activities of both MAOA and MAOB were significantly increased in frontal cortex and caudate nucleus, two brain regions shown previously to be the site of functional and morphological alterations of astrocytes and increased concentrations of the acid metabolites of dopamine and serotonin. These findings suggest that increased monoamine metabolism and subsequent modifications of monoaminergic synaptic function could contribute to the pathogenesis of hepatic encephalopathy.
Article
Most of the brain glycogen, a major energy reserve that can be mobilized in response to increased neuronal activity, resides in the astrocyte, the site of the neuropathological abnormality found in hepatic encephalopathy (HE). Ammonia, a neurotoxin implicated in the pathogenesis of HE, has been reported to cause a depletion of glycogen in primary astrocyte cultures. To further investigate the action of ammonia on glycogen levels, cultured astrocytes were exposed to ammonium chloride (1-5 mM) for various times up to 7 d. Treatment with ammonia for 24 h did not alter deoxyglucose uptake, but significantly lowered peak glycogen values (found at 1.5 h following feeding with medium containing 5.5 mM glucose) in a concentration-dependent manner. This inhibitory effect was not observed after longer exposure times to ammonia. Three day treatment of cells did, however, significantly reduce norepinephrine-stimulated glycogenolysis, an effect not seen after 1 d of ammonia treatment. Part of the neurotoxic action of long term ammonia exposure in humans and experimental animals may be to inhibit the breakdown of glycogen. The effect of ammonia on astrocyte glycogen synthesis and/or breakdown may disrupt glial neuronal signaling and thus play a role in the pathogenesis of HE.
Article
Liver failure, or shunting of intestinal blood around the liver, results in hyperammonemia and cerebral dysfunction. Recently it was shown that ammonia caused some of the metabolic signs of hepatic encephalopathy only after it was metabolized by glutamine synthetase in the brain. In the present study, small doses of methionine sulfoximine, an inhibitor of cerebral glutamine synthetase, were given to rats either at the time of portacaval shunting or 3-4 weeks later. The effects on several characteristic cerebral metabolic abnormalities produced by portacaval shunting were measured 1-3 days after injection of the inhibitor. All untreated portacaval-shunted rats had elevated plasma and brain ammonia concentrations, increased brain glutamine and tryptophan content, decreased brain glucose consumption, and increased permeability of the blood-brain barrier to tryptophan. All treated rats had high ammonia concentrations, but the brain glutamine content was normal, indicating inhibition of glutamine synthesis. One day after shunting and methionine sulfoximine administration, glucose consumption, tryptophan transport, and tryptophan brain content remained near control values. In the 3-4-week-shunted rats, which were studied 1-3 days after methionine sulfoximine administration, the effect was less pronounced. Brain glucose consumption and tryptophan content were partially normalized, but tryptophan transport was unaffected. The results agree with our earlier conclusion that glutamine synthesis is an essential step in the development of cerebral metabolic abnormalities in hyperammonemic states.
Article
The effects of in vitro treatment with ammonium chloride, hepatic encephalopathy (HE) due to thioacetamide (TAA) induced liver failure and chronic hyperammonemia produced by i.p. administration of ammonium acetate on the two components of the multienzyme 2-oxoglutarate dehydrogenase complex (OGDH): 2-oxoglutarate decarboxylase (E1) and lipoamide dehydrogenase (E3), were examined in synaptic and nonsynaptic mitochondria from rat brain. With regard to E1 the response to ammonium ions in vitro (3 mM NH4Cl) was observed in nonsynaptic mitochondria only and was manifested by a 21% decrease of Vmax and a 35% decrease of Km for 2-oxoglutarate (2-OG). By contrast, both in vivo conditions primarily affected the synaptic mitochondrial E1: TAA-induced HE produced an 84% increase of Vmax and a 38% increase of Km for 2-OG. Hyperammonemia elevated Vmax of E1 by 110% and Km for 2-OG by 30%. HE produced no effect at all in nonsynaptic mitochondria while hyperammonemia produced a 35% increase of Vmax and a 30% increase of Km for 2-OG of E1. Both in vivo conditions produced a 20% increase of E3 activity in synaptic mitochondria, but no effect at all in nonsynaptic mitochondria. The preferential sensitivity of E1 to ammonium chloride in vitro in nonsynaptic mitochondria and hyperammonemic conditions in vivo in synaptic mitochondria may play a crucial role in the compartmentation of OGDH responses under analogous conditions. These results confirm the intrinsic differences between the OGDH properties in the synaptic and nonsynaptic brain compartments.
Article
The effects of 5-2500 microM concentrations of neutral ammonium salts on the binding of ligands to components of the GABAA receptor complex were investigated. [3H]Flunitrazepam binding to the benzodiazepine receptor was enhanced by ammonium (10-500 microM), but not sodium tartrate with EC50 = 98 microM and Emax = 31%. Further increasing ammonium tartrate concentrations (500-2500 microM) decreased [3H]flunitrazepam binding to control levels. The ammonium tartrate-induced increase in [3H]flunitrazepam binding was manifested as a 50% decrease in Kd. Furthermore, GABA increased the potency of ammonium tartrate in enhancing [3H]flunitrazepam binding by 63%. [3H]Ro 15-1788 and [3H]Ro 15-4513 binding to the benzodiazepine receptor was not significantly enhanced by ammonium tartrate (Emax approximately 13%). Ammonium tartrate also increased, then decreased the binding of 500 nM [3H]muscimol to the GABAA receptor (EC50 = 52 microM, Emax = 30%) in a concentration-dependent manner, but had no effect on [3H]SR 95-531 binding (Emax < 16%). The ammonium tartrate-induced alterations in [3H]muscimol binding were demonstrated in saturation assays as the loss of the high affinity binding site and a 27% increase in the Bmax of the low affinity binding site. These results indicate that ammonia biphasically enhances, then returns ligand binding to both the GABA and benzodiazepine receptor components of the GABAA receptor complex to control levels in a barbiturate-like fashion. This suggests that ammonia may enhance GABAergic neurotransmission at concentrations commonly encountered in hepatic failure, an event preceding the suppression of inhibitory neuronal function observed at higher (> 1 mM) ammonia concentrations. This increase in GABAergic neurotransmission is consistent with the clinical picture of lethargy, ataxia and cognitive deficits associated with liver failure and congenital hyperammonemia.
Article
We investigated the role of brain peripheral-type benzodiazepine receptors (PBRs) and pregnenolone (a product of PBRs activation) in hepatic encephalopathy (HE)/hyperammonemia. Administration of the hepatotoxin, thioacetamide, or ammonium acetate to mice for 3 days significantly increased the number of brain PBRs (138-146% of control) and the affinity of the ligands for these receptors (2-fold). The total content of pregnenolone and its rate of synthesis in brain of the experimental animals were significantly increased. Our results suggest a novel integrated mechanism by which ammonia-induced activation of PBRs leads to elevated levels of pregnenolone-derived neurosteroids which are known to enhance GABA-ergic neurotransmission. This mechanism may play a pivotal role in pathogenesis of HE.
Article
The uptake of [3H]glutamine (GLN) to non-synaptic mitochondria isolated from rat cerebral hemispheres was measured in the absence or presence of 3 mM ammonium ion (ammonium chloride; ammonia). Ammonia increased Vmax of the saturable component of GLN uptake by > 20%, without affecting K(m), but did not change a non-saturable component of GLN transport representing diffusion or uptake mediated by a very low affinity carrier. Since GLN is an idiogenic osmole, its increased uptake may contribute to the swelling of astrocytic mitochondria and, subsequently, to a decrease in cerebral energy metabolism usually associated with acute hyperammonemic states. The result is consistent with the recent view that GLN accumulating in the brain in hyperammonemic conditions contributes to ammonia neurotoxicity.