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Keywords:

  • ammonia;
  • astrocyte;
  • brain glutamate;
  • liver failure

Abstract

  1. Top of page
  2. Abstract
  3. Brain glutamate in liver failure
  4. Synthesis, metabolism and intercellular trafficking of glutamate in the central nervous system
  5. Alterations in regulation of the brain glutamate system in liver failure and hyperammonemia
  6. Oxidative/nitrosative stress and the glutamate system
  7. Conclusions and therapeutic implications
  8. Acknowledgements
  9. References

Liver failure results in significant alterations of the brain glutamate system. Ammonia and the astrocyte play major roles in such alterations, which affect several components of the brain glutamate system, namely its synthesis, intercellular transport (uptake and release), and function. In addition to the neurological symptoms of hepatic encephalopathy, modified glutamatergic regulation may contribute to other cerebral complications of liver failure, such as brain edema, intracranial hypertension and changes in cerebral blood flow. A better understanding of the cause and precise nature of the alterations of the brain glutamate system in liver failure could lead to new therapeutic avenues for the cerebral complications of liver disease.

Abbreviations used
ALF

acute liver failure

CSF

cerebrospinal fluid

EAAT

excitatory amino acid transporter

EAAC

excitatory amino acid carrier

GLAST

glutamate aspartate transporter

GLT-1

glutamate transporter-1

AMPA

α-amino-3-hydro-methyl-4-isoxasole-propionic acid

NMDA

N-methyl-D-aspartate

cGMP

cyclic guanosine monophosphate

NO

nitric oxide

NOS

nitric oxide synthase

Alteration of normal brain function is a characteristic complication of both acute and chronic liver failure. The term ‘hepatic encephalopathy’ encompasses a wide spectrum of neurological alterations, ranging from subtle changes of personality and of the sleep-wake cycle, to confusion and coma. In patients with acute liver failure (ALF), coma is often accompanied by the development of brain edema and intracranial hypertension, a major cause of mortality (Ware et al. 1971). Despite the frequently severe impairment of brain function, most episodes of hepatic encephalopathy in patients with liver failure are reversible and are not associated with structural alterations or neuronal cell loss.

Accumulated evidence points to ammonia and the astrocyte as the two major factors involved in the pathogenesis of hepatic encephalopathy and brain edema in liver failure. High ammonia levels in the brain in liver failure result from the defective detoxification of ammonia, as well as from splanchnic-derived blood (rich in ammonia) by-passing the liver and reaching the systemic circulation through portal-systemic shunts. A correlation between arterial ammonia concentrations and development of brain herniation has been described in patients with ALF (Clemmesen et al. 1999). Increased brain lactate noted in patients and animal models of liver failure/hyperammonemia, together with the known inhibitory effect of ammonia on alpha-ketoglutarate dehydrogenase observed in vitro (Lai and Cooper 1986), suggest that liver failure may lead to ammonia-induced alterations of brain energy metabolism.

The major site of ammonia removal in the brain is the astrocyte, due to the selective expression of glutamine synthetase in these cells (Martinez-HernandeZ et al. 1977). The presence of astrocytic edema – in both acute and in chronic liver failure -, as well as of abnormal astrocytes, so-called Alzheimer type II astrocytosis, supports the concept of astrocytes being the cellular target of ammonia toxicity in the brain (Butterworth 2003). In addition to ammonia, other substances not detoxified by the liver (manganese, mercaptans) and additional factors (inflammation, electrolyte alterations, altered cerebral blood flow) are likely involved in the pathogenesis of hepatic encephalopathy (Vaquero et al. 2003).

Alterations of multiple neurotransmitter systems have been described in hepatic encephalopathy (Butterworth 2000). This review will focus on alterations of the brain glutamate system for several reasons. Being the major excitatory neurotransmitter in the mammalian brain, alterations of glutamatergic pathways are likely to affect multiple brain functions. Secondly, given that the removal of ammonia in the brain is directly linked to the fate of glutamate in astrocytes, astrocytic alterations in liver failure could critically affect glutamate-related processes such as the uptake and clearance of glutamate from the synapse or the coupling of excitatory neuronal activity with cerebral blood flow. Finally, alterations of glutamate homeostasis may also contribute to the development of other cerebral complications of ALF such as brain edema and intracranial hypertension.

Brain glutamate in liver failure

  1. Top of page
  2. Abstract
  3. Brain glutamate in liver failure
  4. Synthesis, metabolism and intercellular trafficking of glutamate in the central nervous system
  5. Alterations in regulation of the brain glutamate system in liver failure and hyperammonemia
  6. Oxidative/nitrosative stress and the glutamate system
  7. Conclusions and therapeutic implications
  8. Acknowledgements
  9. References

In rats with acute hyperammonemia or ALF induced by thioacetamide or hepatic devascularization, decreased total brain glutamate has consistently been reported (Bosman et al. 1990; Swain et al. 1992b; Peeling et al. 1993; Hilgier and Olson 1994). Decreased concentrations of glutamate were also found in prefrontal cortex, caudate nucleus and cerebellar vermis of patients with cirrhosis who died in hepatic coma (Lavoie et al. 1987).

The concentration of glutamate in brain homogenates (mM range) reflects mainly intracellular pools. Extracellular glutamate is maintained at very low (μM) concentrations to ensure a high signal-to-noise ratio in neurotransmission. In contrast to the decrease of glutamate reported in whole brain homogenates, extracellular brain concentrations of glutamate as well as those of cerebrospinal fluid (CSF) are increased in many different models of ALF and hyperammonemia. Such models include rats or rabbits with liver failure induced by toxins (Hamberger and Nystrom 1984; McArdle et al. 1996) or by hepatic devascularization (de Knegt et al. 1994b), as well as animals exposed to acute hyperammonemia (Suzuki et al. 1992). In rats with hepatic devascularization, cerebral microdialysis studies reveal that extracellular glutamate increases as a function of the deterioration of neurological status (Michalak et al. 1996). Importantly, the prevention of encephalopathy and brain edema by mild hypothermia in these rats was accompanied by attenuation of the increase in extracellular brain glutamate (Rose et al. 2000). Increased extracellular glutamate has also been demonstrated in patients with ALF using similar microdialysis techniques (Tofteng et al. 2002). In these patients, development of intracranial hypertension was positively correlated with high initial values of extracellular brain glutamate. Increased CSF concentrations of glutamate have also been reported in experimental chronic liver failure (Therrien and Butterworth 1991). It is important to note that the increases of extracellular brain glutamate found in liver failure are lower than those reported in experimental ischemia or brain injury associated with glutamate receptor-mediated excitotoxicity. In contrast to these conditions, no evidence of excitotoxic mechanisms has been noted in ALF or acute hyperammonemia, as assessed by 45CaCl2 autoradiography (de Knegt et al. 1994a), and neuronal cell death is generally not considered to be a feature of liver failure.

In addition to glutamate, the brain concentration of other amino acids is also altered in liver failure, and could contribute to the disturbance of other neurotransmitter systems like those of serotonin, dopamine or histamine (Butterworth 2000).

Synthesis, metabolism and intercellular trafficking of glutamate in the central nervous system

  1. Top of page
  2. Abstract
  3. Brain glutamate in liver failure
  4. Synthesis, metabolism and intercellular trafficking of glutamate in the central nervous system
  5. Alterations in regulation of the brain glutamate system in liver failure and hyperammonemia
  6. Oxidative/nitrosative stress and the glutamate system
  7. Conclusions and therapeutic implications
  8. Acknowledgements
  9. References

The blood–brain barrier is virtually impermeable to glutamate. Consequently, all brain glutamate is synthesized locally from glucose, glutamine or from the degradation of proteins (Gruetter et al. 1994). Figure 1 is a schematic representation of some key elements involved in the synthesis, metabolism and intercellular transport of glutamate in the brain. The figure also illustrates the strategic anatomical position of the astrocyte, with functional contacts to both neurons and blood vessels. Evidence for a role of the astrocyte in the coupling of glutamatergic activity to cerebral blood flow has been recently provided in brain slice preparations and involves both astrocytic Ca2+ signalling and transmission of Ca2+-waves along the astrocytic network (Zonta et al. 2003).

image

Figure 1.  Schematic representation of the synthesis, metabolism and intercellular transport of glutamate in the brain. The synthesis of glutamate from glucose involves the partial oxidation of glucose via the glycolytic pathway and the tricarboxylic acid cycle, followed by the amination of alpha-ketoglutarate by glutamate dehydrogenase or transaminases. In addition, glutamate is synthetized in neurons from glutamine by glutaminase. Upon depolarisation, glutamate concentrated in pre-synaptic vesicles is released into the synaptic cleft, where it can bind to diverse ionotropic (NMDA, AMPA, Kainate) and metabotropic glutamate receptors localized on pre- and post-synaptic neuronal and astrocytic membranes, resulting in depolarisation of the post-synaptic neuron and modulation of excitatory neurotransmission. High-affinity glutamate transporters localized on astrocytes (EAAT-1/GLAST, EAAT-2/GLT-1) and on the post-synaptic neuron (EAAT-3/EAAC-1) mediate the rapid and efficient clearance of glutamate from the extracellular space. In astrocytes, glutamate is amidated by glutamine synthetase, an ATP-dependent reaction, and the resulting – electrochemically ‘inactive’– glutamine can be transported back to the neuron as a precursor for glutamate, the so-called ‘glutamate-glutamine cycle’. Ammonia exposure leads to decreased expression of the astrocytic glutamate transporters EAAT-1 and EAAT-2, and stimulates Ca2+-dependent release from both astrocytes and neurons. Ammonia also inhibits glutaminase, a principally neuronal enzyme. Extracellular brain glutamate is increased in a wide range of hyperammonemic disorders including liver failure.

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Alterations in regulation of the brain glutamate system in liver failure and hyperammonemia

  1. Top of page
  2. Abstract
  3. Brain glutamate in liver failure
  4. Synthesis, metabolism and intercellular trafficking of glutamate in the central nervous system
  5. Alterations in regulation of the brain glutamate system in liver failure and hyperammonemia
  6. Oxidative/nitrosative stress and the glutamate system
  7. Conclusions and therapeutic implications
  8. Acknowledgements
  9. References

Alterations at all levels of regulation of the brain glutamate system depicted in Fig. 1 have been described in liver failure and hyperammonemic conditions.

Glutamate synthesis and metabolism

The brain has an extraordinary ability to concentrate ammonia under hyperammonemic conditions as reflected by the increased brain concentrations of ammonia (up to 5 mm; normal range 0.05–0.1 mm) in animals with ALF (Swain et al. 1992a). Potential mechanisms explaining this phenomenon have been recently reviewed (Ott and Larsen 2004), and include an increased permeability of the blood–brain barrier to ammonia (Lockwood et al. 1991). Increased glutamine synthesis in astrocytes is one of the earliest consequences of this increased availability of ammonia, and could contribute to the decrease of total brain concentrations of glutamate in hyperammonemia. Studies using in vivo and ex vivo NMR spectroscopy reveal that the increased glutamine synthesis in liver failure or hyperammonemia is due, at least to a large extent, to the stimulation of the anaplerotic activity of astrocytes (Sibson et al. 1997; Zwingmann et al. 2003). Reductions of brain glutamate in hyperammonemic syndromes including liver failure could also be due to loss from pools other than the astrocyte. In accordance with this notion, the glutamate content of cultured cortical neurons is decreased when exposed to ammonia (Hertz et al. 1987), and ammonia can inhibit phosphate-activated glutaminase (Hogstad et al. 1988), an enzyme expressed principally by neurons. 1H,13C-NMR spectroscopic studies in rats with ALF confirm that the reduction of brain glutamate results at least in part from impaired de novo synthesis from glucose in neurons (Zwingmann et al. 2003). Interestingly, mild hypothermia normalizes the de novo synthesis of glutamate via pyruvate dehydrogenase (expressed both by astrocytes and neurons) and prevents the reduction of brain glutamate in ALF (Chatauret et al. 2003).

Ammonia-induced depletion of brain glutamate could impair the malate-aspartate shuttle, which mediates the transfer of reducing equivalents between cytosol and mitochondria. Supporting this hypothesis, addition of glutamate to the incubation medium significantly ameliorated the reduction of the pyruvate/lactate ratio, but not the increased production of lactate, induced by ammonia exposure of cultured cortical astrocytes (Kala and Hertz 2005). Ammonia inhibits the tricarboxylic acid cycle enzyme alpha-ketoglutarate dehydrogenase in brain preparations in vitro (Lai and Cooper 1986) and stimulates phospho-fructokinase and other glycolytic enzymes (Ratnakumari and Murthy 1993).

Brain glutamate transporters in liver failure

The removal of glutamate from the synaptic cleft is a high affinity, high capacity process mediated by a family of glutamate transporters (Danbolt 2001). Altered transporter function is suggested by the decreased uptake of glutamate reported in a wide range of preparations, including rat hippocampal slices exposed to serum and CSF from patients with chronic liver disease (Schmidt et al. 1990), hippocampal tissue from patients who died in hepatic coma, and synaptosomal preparations from rats with thioacetamide-induced ALF (Oppong et al. 1995).

Studies of the expression of glutamate transporters in brain in liver failure have also been performed. Decreased mRNA and protein expression of the astrocytic glutamate transporter EAAT-2 (the major glutamate transporter in rat forebrain) have been reported in rats with ALF (Knecht et al. 1997; Desjardins et al. 2001) (Fig. 2a–c), or following acute ammonia intoxication (Norenberg et al. 1997). Decreases in the expression of the glutamate transporters EAAT-1 (GLAST), EAAT-2 (GLT-1) and EAAT-3 (EAAC-1) have also been reported in cerebellum of the rat several weeks after portacaval anastomosis (Suarez et al. 2000). Exposure of primary cultures of astrocytes to ammonia for 3–7 days resulted in significant decreases in the high affinity uptake of l-glutamate (Bender and Norenberg 1996) and of d-aspartate (a non-metabolizable glutamate analogue) (Chan et al. 2000), which were associated with a decreased expression of EAAT-1 both at the mRNA and protein levels (Zhou and Norenberg 1999; Chan et al. 2000). Ammonia exposure led also to a time- and dose- dependent decrease of d-aspartate uptake in cultures of rat cerebellar granule neurons, but this was not associated with a down-regulation of the neuronal glutamate transporter EAAT-3 (EAAC-1) (Chan et al. 2003). Taken together, these studies demonstrate that hyperammonemia leads to limitations in the capacity of glutamate uptake in both astrocytes and neurons through distinct mechanisms. The accumulation of extracellular brain glutamate in liver failure is probably the consequence of these deficits in cellular uptake (Fig. 2d).

image

Figure 2.  Acute liver failure (ALF) is associated with altered astrocytic glutamate transporter EAAT-2 expression and with extracellular accumulation of glutamate. (a) Reverse transcriptase-polymerase chain reaction analysis of EAAT-2 mRNA expression in frontal cortex of control (sham-operated) rats and of rats with ALF induced by hepatic devascularization. GAPDH is shown as control. (b) Western blot analysis of EAAT-2 protein expression in frontal cortex from control and ALF rats. (c) Quantification of binding sites for 3H-D-Aspartate, a non-metabolizable glutamate analogue, in frontal cortex from control and ALF rats. (d) Extracellular concentration of glutamate in frontal cortex from control rats, ALF rats 1 h before the appearance of encephalopathy (loss of righting reflex) and ALF rats at coma stages (loss of corneal reflex). Samples were obtained by cerebral microdialysis and analysed by HPLC with fluorescence detection. a, b and c were adapted from (Desjardins et al. 2001), and d was adapted from (Michalak et al. 1996) with permission.

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Additional factors with the potential to contribute to decreased brain glutamate transport in liver failure include lactic acidosis, oxidative stress and energy failure. Increased brain lactate synthesis is a feature of both ALF and hyperammonemia, and lactic acid has been shown to decrease astrocytic glutamate transport in experimental preparations through a direct effect on pH (Swanson et al. 1995; Bender et al. 1997). Oxidative stress in the brain has been demonstrated in liver failure and hyperammonemia (Murthy et al. 2001; Song et al. 2002), and it also has the potential to modify glutamate transporter activity even in the absence of changes in transporter expression (Blanc et al. 1998). Finally, energy failure could impair the maintenance of the transmembrane potassium gradient that drives the transport of glutamate. However, despite reports of decreased brain glucose oxidation in liver failure, there is no clear evidence for a loss of high energy phosphates in brain in either acute or chronic liver failure (Hindfelt and Siesjo 1971; Deutz et al. 1988; Bates et al. 1989).

Astrocyte swelling is a characteristic feature of acute and, to a lesser extent, chronic liver failure. Even modest degrees of astrocyte swelling have the potential to lead to changes in transmembrane ion transport, in the conformation of the plasma membrane and in the diffusion distance of glutamate from the synaptic cleft to the transporter proteins, all of which could alter normal glutamate transport processes.

In addition to ammonia, manganese that accumulates in basal ganglia structures in both experimental (Rose et al. 1999) and human liver failure (Layrargues et al. 1995) could contribute to altered glutamate transport in brain. Indeed, exposure of cultured astrocytes to manganese leads to decreased uptake of d-aspartate, an effect that is additive to the decreased uptake induced by exposure to ammonia (Hazell and Norenberg 1997).

Glutamate release in liver failure

An inhibitory effect of ammonia was reported on electrical- or KCl-induced release of endogenous glutamate from hippocampal slices of normal rats (Cummins et al. 1981). On the other hand, studies in cultured astrocytes suggest that ammonia exposure leads to increased astrocytic glutamate release (Rose 2002). Swelling of astrocytes (a feature of liver failure) by exposure to hypo-osmotic media results in release of endogenous glutamate, an effect that does not involve the reversal of Na+-dependent glutamate transporters (Kimelberg et al. 1990). Increased astrocytic release of glutamate may also occur through a Ca2+-dependent mechanism (Hertz and Zielke 2004). A recent study systematically re-evaluated this issue, and found that the release of glutamate from cultured astrocytes exposed to ammonia involved a pH-mediated, Ca2+-dependent mechanism (Rose et al. 2005). Together, these studies confirm that ammonia influences the mechanisms regulating glutamate release from astrocytes and neurons in a cell-selective manner. Alterations of glutamate and calcium dynamics in astrocytes could interfere with the normal coupling of glutamatergic activity to cerebral blood flow (Zonta et al. 2003), and, in this way, contribute to the altered cerebrovascular haemodynamics characteristic of ALF (Vaquero et al. 2004). In contrast to glutamate, the release of GABA is inhibited in cultured astrocytes after an acute exposure to ammonia (Albrecht et al. 1994).

Ammonia and glutamatergic synaptic transmission

In rat hippocampal slices, ammonia exhibits a direct inhibitory effect on glutamatergic neurotransmission, which is also evident after the iontophoretic application of glutamate, suggesting that such inhibition is mediated via a post-synaptic action (Fan et al. 1990). Ammonia profoundly depresses AMPA receptor-mediated currents, but potentiates those mediated by NMDA receptors. On the other hand, exposure of mouse cortical preparations to ammonia leads to decreased depolarisation induced by NMDA and AMPA receptor ligands, as well as to decreased formation of inositol-3-phosphate induced by agonists of metabotropic glutamate receptors (Lombardi et al. 1994). These observations suggest that ammonia has direct but distinct effects on glutamatergic synaptic transmission, and that such effects vary depending on the receptor subtype and brain region under study.

Decreased densities of binding sites for ligands of AMPA/kainate receptors were reported in the brains of rats with ALF induced by hepatic devascularization (Michalak and Butterworth 1997). In contrast, densities of NMDA receptor ligands were shown to be unchanged in this model (Michalak et al. 1996) as well as in brain preparations from rabbits with ALF or acute hyperammonemia (de Knegt et al. 1993). Subsequent evidence that both neurons and astrocytes may express similar subclasses of glutamate receptors (Carmignoto 2000), however, hampers the interpretation of these findings.

Although NMDA receptor expression is unchanged, excessive activation of NMDA receptors has been implicated in the pathogenesis of the cerebral manifestations of acute ammonia neurotoxicity, as suggested by the protective effects of a wide variety of NMDA receptor antagonists against seizures and death induced by the administration of a lethal dose of ammonium acetate to mice (Hermenegildo et al. 1996). In rats with ALF, the administration of memantine, an NMDA receptor antagonist, also led to an improvement of encephalopathy (Vogels et al. 1997). Activation of NMDA receptors leads to an increase of intracellular calcium, which, in conjunction with calmodulin, activates nitric oxide synthase resulting in increased synthesis of nitric oxide from l-arginine (Fig. 3). Nitric oxide subsequently activates soluble guanylate cyclase, leading to increased synthesis of cyclic guanosine monophosphate (cGMP). In rats monitored by in vivo microdialysis, acute ammonia intoxication led to an increase of cerebellar cGMP that was attenuated by the NMDA receptor antagonist MK-801, confirming ammonia-induced over-activation of this signal-transduction pathway (Hermenegildo et al. 2000). Analysis of the time course of the increase of ammonia, glutamate and cGMP in ammonia-intoxicated animals revealed that NMDA receptor activation occurred before the increase of extracellular glutamate, leading the authors to suggest that NMDA receptor activation was a direct effect of ammonia. Such signal transduction mechanisms do not appear to hold in the case of chronic liver failure, where a down-regulation of NMDA receptor sites (Peterson et al. 1990) and a decrease in the activity of NMDA receptor-induced production of cGMP (Erceg et al. 2005) have been reported. Activation or attenuation of the NMDA receptor-mediated NO-cGMP signal transduction pathway may be pathogenetically relevant, as this pathway has been implicated in processes that are frequently altered in patients with hepatic encephalopathy, such as the circadian rhythmicity (sleep-waking cycle), learning ability and memory function.

image

Figure 3.  Schematic representation of the Glutamate-NMDA-NO-cGMP signal transduction pathway. Experimental evidence suggests that ammonia stimulates the NMDA receptor-mediated NO-cGMP signal transduction pathway. The NO produced may result in glutamine synthetase tyrosine nitration leading to decreased glutamine synthetase activity and decreased ammonia removal (glutamine production) capacity, resulting in disproportionate increases of brain ammonia. Increased NO synthesis could also result from ammonia-induced increase uptake of the NOS substrate L-Arginine. The precise cellular location (neurons or astrocytes) of the ammonia-induced increases of the NMDA receptor-mediated NO-cGMP signal transduction pathway remains to be established.

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Oxidative/nitrosative stress and the glutamate system

  1. Top of page
  2. Abstract
  3. Brain glutamate in liver failure
  4. Synthesis, metabolism and intercellular trafficking of glutamate in the central nervous system
  5. Alterations in regulation of the brain glutamate system in liver failure and hyperammonemia
  6. Oxidative/nitrosative stress and the glutamate system
  7. Conclusions and therapeutic implications
  8. Acknowledgements
  9. References

Exposure of cultured astrocytes to ammonia results in a dose-dependent increase in the generation of free radicals (Murthy et al. 2001), and the expression of heme oxygenase-1, an oxidative stress-related gene, is increased in the brains of rats with acute (Sawara et al. 2005) or chronic (Song et al. 2002) liver failure. In brain homogenates from rats with acute ammonia intoxication, an increased formation of superoxide and a decreased activity of antioxidant enzymes were also reported (Kosenko et al. 1997). Such changes were prevented by administration of MK-801 (Kosenko et al. 1999), suggesting that the ammonia-induced oxidative stress was mediated by activation of NMDA receptors.

Chronic liver failure resulting from end-to-side portacaval anastomosis in the rat is associated with increased mRNA and protein expression of neuronal nitric oxide synthase (NOS-1), and with increased nitric oxide synthase activity (Rao et al. 1995, 1997a) (Fig. 4a–c). In addition, increased production of nitric oxide was demonstrated in the brains of portacaval-shunted rats following intravenous infusion of ammonia (Master et al. 1999). Increased uptake of l-arginine, the substrate for nitric oxide synthase, was also reported in synaptosomes from portacaval-shunted rats (Rao et al. 1995, 1997b). Exposure of cultured astrocytes to ammonia or manganese also increases the uptake of l-arginine (Hazell and Norenberg 1998), suggesting a further mechanism to explain the increased nitric oxide production in chronic liver failure. Activation of NMDA receptors is one potential explanation for these findings (Fig. 3).

image

Figure 4.  Chronic liver failure resulting from portacaval anastomosis (PCA) results in increased neuronal nitric oxide synthase (NOS-1) expression, NOS activity and glutamine synthetase nitration in the rat brain. (a) Nitric oxide synthase reaction. (b) Reverse transcriptase-polymerase chain reaction analysis of NOS-1 mRNA expression in cerebellum of sham-operated rats (Sham) and of portacaval-shunted rats (PCA). Adapted from (Rao et al. 1997a). (c) NOS activity in cytosolic extracts of cerebral cortex from sham-operated and PCA rats. Adapted from (Rao et al. 1995). (d) Upper lane is a western blot of glutamine synthetase performed on tyrosine-nitrated proteins immunoprecipitated from brain extracts of sham-operated and PCA rats. The lower lane is a western blot of glutamine synthetase on the initial brain extracts (before immunoprecipitation) to correct for potential changes in protein expression associated with the PCA procedure. The graph below represents the densitometric quantification of relative glutamine synthetase nitration in each group. IP: NTyr, Immunoprecipitation with anti-3′-nitrotyrosine antibody; WB, western blot; GS, glutamine synthetase. Adapted from (Schliess et al. 2002) with permission.

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Immunohistochemical studies of brains from portacaval-shunted rats receiving an ammonia infusion or from rats with acute ammonia intoxication reveal evidence of increased nitrotyrosine formation in astrocytic end-feet surrounding blood vessels (Schliess et al. 2002; Vaquero et al. 2003). Protein tyrosine nitration was also demonstrated in primary cultures of rat cortical astrocytes exposed to ammonia, where it was shown to be dependent on an increase of intracellular Ca2+, on the degradation of IkB and on the induction of iNOS (Schliess et al. 2002). Addition of MK-801 led to attenuation of these changes and to the prevention of protein tyrosine nitration, again suggesting that activation of NMDA receptors is a major initiating event. Consistent with this possibility, exposure of astrocytes to NMDA produced a pattern of protein tyrosine nitration similar to that produced by ammonia.

Glutamine synthetase is among the proteins that have been identified so far as targets of ammonia-induced protein tyrosine nitration (Schliess et al. 2002) (Fig. 4d). Nitration of glutamine synthetase led to decreased activity of the enzyme (Schliess et al. 2002; Gorg et al. 2005). Whether glutamine synthetase protein nitration in liver failure occurs as a consequence of activation of NMDA receptor-mediated NOS-1 induction in neurons or in astrocytes (or both cell types) remains to be established, but loss of glutamine synthetase activity and limited capacity for de novo glutamine synthesis have been consistently reported in brain in liver failure (Girard et al. 1993; Kanamori et al. 1996; Desjardins et al. 1999). Protein tyrosine nitration offers a plausible explanation for this finding and for the precipitously high brain concentrations of ammonia that are encountered in liver failure.

Conclusions and therapeutic implications

  1. Top of page
  2. Abstract
  3. Brain glutamate in liver failure
  4. Synthesis, metabolism and intercellular trafficking of glutamate in the central nervous system
  5. Alterations in regulation of the brain glutamate system in liver failure and hyperammonemia
  6. Oxidative/nitrosative stress and the glutamate system
  7. Conclusions and therapeutic implications
  8. Acknowledgements
  9. References

Multiple alterations of the brain glutamate system are evident in both chronic and ALF. It is likely that increased brain ammonia is the principal neurotoxin responsible for these alterations, although other agents, particularly manganese, may also contribute. Virtually all elements of the glutamate system are altered, including its synthesis and metabolism, its intercellular trafficking, as well as the function and expression of glutamate transporters and receptors. Many alterations responsible for the dysfunction of the glutamatergic system in liver failure reside in astrocytes, reflecting the current view of astrocytes as active players in the modulation of synaptic transmission and blood-flow in normal physiology.

Changes of the brain glutamate system in liver failure offer potential targets for future therapies. Among these therapies, mild hypothermia has been shown to prevent the increase of extracellular brain glutamate and, concomitantly, brain edema in rats with ALF (Rose et al. 2000). Other interventions with more specific actions on the elements of the brain glutamate system, such as diverse NMDA receptor antagonists or sildenafil, have also shown some beneficial effects in experimental models of liver failure (Vogels et al. 1997; Erceg et al. 2005). Measures to increase the expression and function of astrocytic glutamate transporters could also be beneficial. In this regard, a recent report has described up-regulation of brain EAAT-2 (GLT-1) protein by ceftriaxone and other beta-lactams in rodents (Rothstein et al. 2005), an unexpected effect of antibiotics that are frequently used in the clinic. In addition, increasing evidence linking glutamatergic dysfunction with oxidative/nitrosative stress and with the cerebral complications of liver failure open a new area to explore other potential interventions, such as NOS inhibition or antioxidants.

References

  1. Top of page
  2. Abstract
  3. Brain glutamate in liver failure
  4. Synthesis, metabolism and intercellular trafficking of glutamate in the central nervous system
  5. Alterations in regulation of the brain glutamate system in liver failure and hyperammonemia
  6. Oxidative/nitrosative stress and the glutamate system
  7. Conclusions and therapeutic implications
  8. Acknowledgements
  9. References
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