Endogenous Benzodiazepine Receptor Ligands in Human and Animal Hepatic Encephalopathy

The role of endogenous benzodiazepine receptor ligands in the pathogenesis of hepatic encephalopathy was studied in humans and in rat models of hepatic encephalopathy. Endogenous benzodiazepine ligands were extracted from rat brain and human CSF by acid treatment and purification by HPLC. Detection and partial characterization of these endogenous benzodiazepine ligands were carried out using both radioreceptor binding assays and radioimmunoassays with anti-benzodiazepine antibodies. Four different benzodiazepine receptor ligands were identified in human and rat tissue, two of which may be diazepam and desmethyldiazepam, based on elution profiles and anti-benzo-diazepine antibody reactivity. Human CSF and serum from patients with hepatic encephalopathy contained approximately 10 times more endogenous benzodiazepine receptor ligand than CSF from controls or nonencephalopathic patients with liver disease. The levels of brain benzodiazepine receptor ligand compounds were also increased approximately 10-fold in rats suffering from fulminant hepatic failure, but not in rats with portacaval shunts, a model of chronic hepatic disease. The increased concentrations of these substances could be behaviorally significant and may contribute to the pathogenesis of hepatic encephalopathy.     

Selective Loss of N-Methyl-d-Aspartate-Sensitive l-[3H]Glutamate Binding Sites in Rat Brain Following Portacaval Anastomosis

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.     

Endogenous GABAergic modulators in the pathogenesis of hepatic encephalopathy

Theories on the neurochemical etiology for hepatic encephalopathy have recently focussed on activation of inhibitory neurotransmitter GABA systems. Modulators of the GABA_A receptor complex, including diazepam binding inhibitor, are significantly and selectively altered in hepatic encephalopathy. In animals and humans, benzodiazepine receptor antagonists rapidly ameliorate this syndrome suggesting the possible existence of an endogenous benzodiazepine-like substance. Endogenous GABAergic modulators may contribute to the neurochemical pathogenesis of hepatic encephalopathy.Special issue dedicated to Dr. Erminio Costa     

Hepatic encephalopathy: An update of pathophysiologic mechanisms

Hepatic encephalopathy (HE) is a neuropsychiatric disorder that occurs in both acute and chronic liver failure. Although the precise pathophysiologic mechanisms responsible for HE are not completely understood, a deficit in neurotransmission rather than a primary deficit in cerebral energy metabolism appears to be involved. The neural cell most vulnerable to liver failure is the astrocyte. In acute liver failure, the astrocyte undergoes swelling resulting in increased intracranial pressure; in chronic liver failure, the astrocyte undergoes characteristic changes known as Alzheimer type II astrocytosis. In portal-systemic encephalopathy resulting from chronic liver failure, astrocytes manifest altered expression of several key proteins and enzymes including monoamine oxidase B, glutamine synthetase, and the so-called peripheral-type benzodiazepine receptors. In addition, expression of some neuronal proteins such as monoamine oxidase A and neuronal nitric oxide synthase are modified. In acute liver failure, expression of the astrocytic glutamate transporter GLT-1 is reduced, leading to increased extracellular concentrations of glutamate. Many of these changes have been attributed to a toxic effect of ammonia and/or manganese, two substances that are normally removed by the hepatobiliary route and that in liver failure accumulate in the brain. Manganese deposition in the globus pallidus in chronic liver failure results in signal hyperintensity on T1-weighted Magnetic Resonance Imaging and may be responsible for the extrapyramidal symptoms characteristic of portal-systemic encephalopathy. Other neurotransmitter systems implicated in the pathogenesis of hepatic encephalopathy include the serotonin system, where a synaptic deficit has been suggested, as well as the catecholaminergic and opioid systems. Further elucidation of the precise nature of these alterations could result in the design of novel pharmacotherapies for the prevention and treatment of hepatic encephalopathy.     

Increased levels of pregnenolone and its neuroactive metabolite allopregnanolone in autopsied brain tissue from cirrhotic patients who died in hepatic coma

It has been suggested that neurosteroids with agonist properties at the central GABA-A receptor are implicated in the pathogenesis of hepatic encephalopathy (HE) in chronic liver disease. In order to address this issue, gas chromatography/mass spectrometry was used to measure the neurosteroids pregnenolone, allopregnanolone, and tetrahydrodeoxycorticosterone (THDOC) in postmortem brain tissue from controls, cirrhotic patients who died without HE, a patient who died in uremic coma, and cirrhotic patients who died in hepatic coma. Exposure of rat cerebral cortical membranes to brain extracts from hepatic coma patients resulted in a 53% (p < 0.001) increase in binding of [3H]muscimol, a GABA-A receptor ligand. Subsequent GC/MS analysis showed that concentrations of the GABA-A receptor agonist neurosteroid allopregnanolone were significantly increased in brain tissue from hepatic coma patients compared to patients without HE or controls (p < 0.001). Brain allopregnanolone concentrations were significantly correlated with the magnitude of induction of [3H]muscimol binding (r2 = 0.82, p < 0.0001). Concentrations of allopregnanolone comparable to those observed in hepatic coma brains are pathophysiologically relevant. Concentrations of the neurosteroid precursor pregnenolone were also increased in brain tissue from hepatic coma patients, while those of a second neurosteroid THDOC were below the levels of detection in all groups. Brain concentrations of benzodiazepine receptor ligands estimated by radioreceptor assay were not significantly increased in cirrhotic patients with or without hepatic coma. These findings suggest that increased levels of allopregnanolone rather than "endogenous benzodiazepines" offer a cogent explanation for the phenomenon of "increased GABAergic tone" previously proposed in HE.     

GABAA Receptor Complex in an Experimental Model of Hepatic Encephalopathy: Evidence for Elevated Levels of an Endogenous Benzodiazepine Receptor Ligand

The involvement of the gamma-aminobutyric acidA (GABAA) receptor complex in the pathogenesis of hepatic encephalopathy was examined in thioacetamide-treated rats with fulminant hepatic failure. Partially purified extracts from encephalopathic rat brain were approximately three times more potent in inhibiting [3H]Ro 15-1788 binding to benzodiazepine receptors than identically prepared extracts from control rats. High levels of inhibitory activity were also found in extracts of plasma, heart, and liver from thioacetamide-treated rats. The inhibition of [3H]Ro 15-1788 binding by brain extracts appeared to be competitive and reversible and was unaffected by treatment with either proteolytic enzymes or boiling. Further, GABA significantly enhanced the potency of these extracts in inhibiting [3H]flunitrazepam binding. In contrast, no differences were found in radioligand binding to the constituent recognition sites of the GABAA receptor complex in well-washed brain membranes prepared from control and encephalopathic animals. These findings suggest that the recognition-site qualities of the constituent proteins of the GABAA receptor complex are unchanged in an experimental model of hepatic encephalopathy. However, significant elevations in the level of a substance or substances with neurochemical properties characteristic of a benzodiazepine receptor agonist may contribute to the electrophysiological and behavioral manifestations of hepatic encephalopathy.     

Portacaval anastomosis causes selective alterations of peripheral-type benzodiazepine receptor expression in rat brain and peripheral tissues.

There is a growing body of evidence to suggest that peripheral-type benzodiazepine receptors (PTBRs) and their endogenous ligands are implicated in the pathogenesis of end-organ failure in chronic liver disease. Portal-systemic encephalopathy, a major neuropsychiatric complication associated with chronic liver disease, results in activation of brain PTBR and probably in peripheral organs. In order to address these issues, PTBR mRNA was measured using semi-quantitative RT-PCR in extracts of cerebral cortex, kidney and testis of rats four weeks after end-to-side portacaval anastomosis and sham-operation (controls). Densities of PTBR sites were measured concomitantly by in vitro receptor binding using the selective PTBR ligand [3H]PK11195. Portacaval shunting resulted in a 2 to 3-fold increase in expression of PTBR in brain and kidney and a 37% reduction in expression in testis. Densities of [3H]PK11195 sites changed in parallel with the alterations of gene expression. These findings suggest that selective alterations of PTBR expression are implicated in the pathogenesis of peripheral tissue hypertrophy (kidney) and/or atrophy (testis) which accompanies portal-systemic shunting in chronic liver failure. In brain, activation of PTBR could result in an increase in the production of neurosteroids with potent inhibitory action in the CNS, which could contribute to the pathogenesis of portal-systemic encephalopathy.     

The astrocytic ("peripheral-type") benzodiazepine receptor: role in the pathogenesis of portal-systemic encephalopathy

An increasing body of evidence supports the notion that activation of astrocytic (peripheral-type) benzodiazepine receptors contributes to the pathogenesis of the central nervous system symptoms which are characteristic of portal-systemic encephalopathy (PSE). Binding site densities for the PTBR ligand [3H-PK11195] are increased in autopsied brain tissue from PSE patients as well as in the brains of animals with experimental chronic liver failure. In the case of the animal studies, increased PTBR sites resulted from increased PTBR gene expression. Exposure of cultured astrocytes to ammonia or manganese (two neurotoxic agents which under normal circumstances are removed by the hepatobiliary system and which are found to accumulate in brain in PSE) results in increased densities of [3H-PK11195] binding sites. Activation of PTBR is known to result in increased cholesterol uptake and increased synthesis in brain of neurosteroids some of which have potent positive allosteric modulator properties on the GABA-A receptor system. Accumulation of such substances in the brain in chronic liver failure could explain the neural inhibition characteristics of PSE.     

Involvement of diazepam binding inhibitor and its fragment octadecaneuropeptide in social isolation stress-induced decrease in pentobarbital sleep in mice.

Diazepam binding inhibitor (DBI) and its fragment, octadecaneuropeptide (ODN), are putative endogenous ligands for benzodiazepine (BZD) receptors and have been shown to act as an inverse BZD receptor agonist in the brain. A previous study suggested that the social isolation stress-induced decrease in pentobarbital sleep in mice was partly due to endogenous substances with an inverse BZD receptor agonist-like property. In this study, we examined the effects of DBI and ODN on pentobarbital sleep in group-housed and socially isolated mice to test the possible involvement of DBI and ODN in a social isolation-induced decrease in pentobarbital sleep. The socially isolated mice showed significantly shorter durations of pentobarbital (50 mg/kg, intraperitoneally, i. p.) sleep compared to the group-housed animals. When injected intracerebroventricularly (i.c.v.), DBI and ODN (3 and 10 nmol) dose-dependently shortened the pentobarbital-induced sleeping time in group-housed mice at the same dose range, but these peptides had no effect on the sleeping time in socially isolated animals. In contrast, flumazenil (16.5-33 nmol, i.c.v.), a BZD receptor antagonist, reversed the pentobarbital sleeping time in socially isolated mice to the level of group-housed animals without affecting the sleeping time in group-housed animals. The effects of DBI and ODN in group-housed mice were significantly blocked by flumazenil (33 nmol, i.c.v.). Moreover, the effect of flumazenil in socially isolated mice was significantly attenuated by DBI and ODN (10 nmol, i.c.v.). These results suggest that the changes in the activity of DBI and/or ODN are partly involved in the social isolation-induced decrease in the hypnotic action of pentobarbital in mice.     

New developments on the search for the endogenous ligand(s) of central benzodiazepine receptors

This review describes three new research developments that have occurred since 1983, in relation to the possible identification of endogenous ligand(s) for the benzodiazepine central receptor (BZD-R). The polypeptides diazepam binding inhibitor (DBI) and the ODN of Guidotti and Costa, as well as the endozepines of Shoyab and Todaro are considered in their affinities and pharmacological actions. The work of the De Blas group on the presence of benzodiazepines in brain, confirmed by us and other groups, is commented and the discovery, in our own laboratory, of n-butyl-β-carboline-3-carboxylate as a possible putative ligand, having high affinity for the BZD-R and showing proconvulsant and “anxiogenic” properties, is described. In the concluding remarks, the possibility that two or more endogenous ligands with opposing activity could regulate the BZD-GABA receptor complex is postulated.     

Regional Differences in the Capacity for Ammonia Removal by Brain Following Portocaval Anastomosis

Portocaval anastomosis (PCA) in the rat leads, within 4 weeks, to severe liver atrophy, sustained hyperammonemia, and increased brain ammonia. Because brain is not equipped with an effective urea cycle, removal of ammonia involves glutamine synthesis and PCA results in significantly increased brain glutamine. Glutamine synthetase activities, however, are decreased by 15% in cerebral cortex and are unchanged in brainstem of shunted rats. Administration of ammonium acetate to rats following PCA results in severe encephalopathy (loss of righting reflex and, ultimately, coma). Glutamine concentrations in brainstem of comatose rats are increased a further two-fold, whereas those of cerebral cortex are unchanged. Consequently, ammonia levels in cerebral cortex reach disproportionately high levels (of the order of 5 mM). These findings suggest a limitation in the capacity of cerebral cortex to remove additional blood-borne ammonia by glutamine formation following PCA. Such mechanisms may explain the hypersensitivity of rats with PCA and of patients with portal-systemic shunting to small increases of blood ammonia. Disproportionately high levels of brain ammonia in certain regions, such as cerebral cortex, may then result in alterations of inhibitory neurotransmission and, ultimately, loss of cellular (astrocytic) integrity.     

Regional alterations of dopamine and its metabolites in rat brain following portacaval anastomosis

Hyperammonemia and changes in brain monoamine metabolism have been proposed to contribute to the pathogenesis of the neuropsychiatric symptoms characteristic of human portal-systemic encephalopathy (PSE) resulting from chronic liver disease. Portacaval anastomosis (PCA) in the rat leads to sustained hyperammonemia and mild encephalopathy. In order to evaluate the role of dopamine (DA) metabolism in PSE, levels of DA and its metabolites were measured by HPLC with electrochemical detection in brain regions of rats with PCA at various stages of encephalopathy precipitated by ammonium acetate administration. Following ammonium acetate administration, rats with PCA rapidly develop severe neurological signs of encephalopathy progressing through loss of righting reflex to coma; sham-operated control animals administered ammonium acetate showed no such neurological deterioration. Concentrations of the DA metabolites DOPAC and HVA as well as [DA metabolites]/[DA] ratios, an indirect measure of DA turnover in brain, were increased in caudate-putamen, in cingulate and pyriform entorhinal cortices as well as in raphe nucleus and locus coeruleus. Increased DA metabolites, however, did not worsen at coma states of PSE. Increased DA turnover thus appears to relate to early neuropsychiatric and extrapyramidal symptoms of PSE.     

The "peripheral-type" benzodiazepine (omega 3) receptor in hyperammonemic disorders

Increased levels of brain ammonia occur in both congenital and acquired hyperammonemic syndromes including hepatic encephalopathy, fulminant hepatic failure, Reye's syndrome and congenital urea cycle disorders. In addition to its effect on neurotransmission and energy metabolism, ammonia modulates the expression of various genes including the astrocytic "peripheral-type" benzodiazepine (or omega 3) receptor (PTBR). Increased expression of the isoquinoline carboxamide binding protein (IBP), one of the components of the PTBR complex, is observed in brain and peripheral tissues following chronic liver failure as well as in cultured astrocytes exposed to ammonia. Increased densities of binding sites for the PTBR ligand [3H]-PK11195 are also observed in these conditions as well as in brains of animals with acute liver failure, congenital urea cycle disorders and in patients who died in hepatic coma. The precise role of PTBR in brain function has not yet fully elucidated, but among other functions, PTBR mediates the transport of cholesterol across the mitochondrial membrane and thus plays a key role in the biosynthesis of neurosteroids some of which modulate major neurotransmitter systems such as the gamma-aminobutyric acid (GABA(A)) and glutamate (N-methyl-D-aspartate (NMDA)) receptors. Activation of PTBR in chronic and acute hyperammonemia results in increased synthesis of neurosteroids which could lead to an imbalance between excitatory and inhibitory neurotransmission in the CNS. Preliminary reports suggest that positron emission tomography (PET) studies using [11C]-PK11195 may be useful for the assessment of the neurological consequences of chronic liver failure.     

The Relationship Between Plasma and Brain Quinolinic Acid Levels and the Severity of Hepatic Encephalopathy in Animal Models of Fulminant Hepatic Failure

Abstract: Quinolinic acid is an excitatory, neurotoxic tryptophan metabolite proposed to play a role in the pathogenesis of hepatic encephalopathy. This involvement was investigated in rat and rabbit models of fulminant hepatic failure at different stages of hepatic encephalopathy. Although plasma and brain tryptophan levels were significantly increased in all stages of hepatic encephalopathy, quinolinic acid levels increased three- to sevenfold only in the plasma, CSF, and brain regions of animals in stage IV hepatic encephalopathy. Plasma-CSF and plasma-brain quinolinic acid levels in rats and rabbits with fulminant hepatic failure were strongly correlated, with CSF and brain concentrations ∼10% those of plasma levels. Moreover, there was no significant regional difference in brain quinolinic acid concentrations in either model. Extrahepatic indoleamine-2,3-dioxygenase activity was not altered in rats in stage IV hepatic encephalopathy, but hepatic l -tryptophan-2,3-dioxygenase activity was increased. These results suggest that quinolinic acid synthesized in the liver enters the plasma and then accumulates in the CNS after crossing a permeabilized blood-brain barrier in the end stages of liver failure. Furthermore, the observation of low brain concentrations of quinolinic acid only in stage IV encephalopathy suggests that the contribution of quinolinic acid to the pathogenesis of hepatic encephalopathy in these animal models is minor.     

State of GABA-benzodiazepine receptor complex in diabetic neuropathy: effect of nicotinamide and nicotinoyl-GABA

An increase in GABA uptake by isolated rat brain synaptic endings as well as a decrease of pharmacologically active GABA analogue muscimol specific binding have indicated a physiologically drastic failure in realization of GABA-mediated inhibitory effects in CNS induced by diabetic encephalopathy. In spite of the impairment of inhibitory function of GABAergic transmission in diabetes a crucial activation of benzodiazepine receptors was determined, as it is tested by the increase in specific binding of flunitrazepam by synaptic membranes. This increase may play an important role in endogenous control of neural activity associated with the factors undefined so far. Using the approach that GABA, and several synthetic GABA agonists, appear to increase the affinity of the benzodiazepine recognition sites for such ligands, presumably by some allosteric mechanism, the findings concerning the in vitro binding assay technique confirm at least some of the functional characteristics observed between GABA and benzodiazepine receptors in vivo under pathological conditions. Indeed, the absence of activating effect on the affinity of flunitrazepam specific binding in the presence of micromolar concentrations of exogenous GABA implicate diabetes-induced alterations in coupling GABA- and benzodiazepine receptors that might be linked to changes in conformantial state of this membrane-bound complex and could partially explain diabetes-induced impairments of GABAergic transmission evaluated in the present study. Our study suggests that nicotinamide and especially GABA play an important role in improving the functioning of brain GABA-benzodiazepine complex impaired in diabetes through specific ligand-mediated mechanism and can be useful in the management of diabetes-associated brain failures.     

Amino acid changes in regions of the CNS in relation to function in experimental portal-systemic encephalopathy

Sustained hyperammonemia resulting from portocaval anastomosis (PCA) in the rat, is accompanied by neurological symptoms and reversible morphological changes in brain, the nature and distribution of which suggest selective vulnerability of certain brain structures. the present study was initiated to investigate the effects of increasing CNS ammonia on the distribution of amino acids in regions of the rat brain in relation to the degree of neurological impairment in PCA rats. Four weeks following PCA, rats were administered ammonium acetate (5.2 mmol/kg, i.p.) to precipitate neurological symptoms of encephalopathy which included diminished locomotor activity, loss of hindlimb extension and righting reflexes and ultimately coma. At various stages during the development of encephalopathy, rats were sacrificed and the amino acids glutamine, glutamate and aspartate measured simultaneously, using a sensitive double-isotope dansyl microassay. Homogenates of the following regions of the CNS were assayed: cerebral cortex, hippocampus, striatum, midbrain, hypothalamus, cerebellum, medulla-pons, spinal cord (gray matter) and spinal cord (white matter). Sustained hyperammonemia associated with PCA alone resulted in a non-uniform 2–4 fold increase of glutamine in all regions of the CNS. Glutamate, on the other hand, was selectively increased in striatum and cerebellum, two regions of brain shown to exhibit early morphologically-characterised astrocytic abnormalities in rats with PCA. Onset of severe neurological dysfunction was accompanied by significantly decreased glutamine and glutamate in striatum and cerebellum. Thus, sustained hyperammonemia in association with portocaval shunting results in region-selective effects with respect to glutamine-glutamate metabolism in the CNS.     

Normal coupling of brain benzodiazepine and neurosteroid modulatory sites on the GABA-A receptor complex in human hepatic encephalopathy

To assess the possible implication of the allosteric coupling of different modulatory sites at the GABA-A receptor complex in hepatic encephalopathy (HE), we investigated in autopsied frontal cortex of six cirrhotic patients and six appropriately-matched controls, the modulatory effects of the benzodiazepine site agonist flunitrazepam on the binding of [3H]muscimol and the effect of the neurosteroid site agonist allopregnanolone (5alpha-pregnan-3alpha-ol-20-one) on the binding of [3H]muscimol and [3H]flunitrazepam. There were no significant differences in either the magnitude E(max): 11.5+/-1.1% (controls) versus 10.2+/-2.2% (HE patients) or the efficacy EC(50): 20.2+/-5.5 nM (controls) versus 17.7+/-6.2 nM (HE patients) of flunitrazepam modulation of [3H]muscimol binding. Allopregnanolone also showed modulation of both sites to a comparable extent in brain tissue from cirrhotic patients and controls E(max): [3H]muscimol, 15.1+/-2.8% (controls) versus 13.8+/-1.9% (HE patients); [3H]flunitrazepam, 17.9+/-2.3% (controls) versus 19.1+/-2.3% (HE patients), EC(50): [3H]muscimol, 386.5+/-25.8 nM (controls) versus 373.8+/-13.1 nM (HE patients); [3H]flunitrazepam, 49.8+/-22.9 nM (controls) versus 55.5+/-14.0 nM (HE patients). These findings demonstrate unequivocally that the GABA-A sites and their benzodiazepine and neurosteroid modulatory sites manifest normal allosteric coupling in brain in human HE. Therefore, if increased "GABAergic tone" is implicated in the pathophysiology of HE, this must be the consequence of increased brain concentrations of endogenous benzodiazepine and/or neurosteroid ligands for components of the GABA-A receptor complex rather than alterations of the receptor proteins themselves.     

Evidence for an Astrocytic Glutamate Transporter Deficit in Hepatic Encephalopathy

There is increasing evidence to suggest that hepatic encephalopathy in acute liver failure is the result of altered glutamatergic function. In particular, the high affinity uptake of glutamate is decreased in brain slices and synaptosomes from rats with acute liver failure as well as by exposure of cultured astrocytes to concentrations of ammonia equivalent to those reported in brain in acute liver failure. Both protein and gene expression of the recently cloned and sequenced astrocytic glutamate transporter GLT-1 are significantly reduced in the brains of rats with acute liver failure. Decreased expression of GLT-1 in brain in acute liver failure results in increased extracellular brain glutamate concentrations which correlates with arterial ammonia concentrations and with the appearance of severe encephalopathy and brain edema in these animals. Ammonia-induced reductions in expression of GLT-1 resulting in increased extracellular glutamate concentrations could explain some of the symptoms (hyperexcitability, cerebral edema) characteristic of hepatic encephalopathy in acute liver failure.     

Loss of noradrenaline transporter sites in frontal cortex of rats with acute (ischemic) liver failure

There is increasing evidence that central noradrenaline (NA) transport mechanisms are implicated in the central nervous system complications of acute liver failure. In order to assess this possibility, binding sites for the high affinity NA transporter ligand [3H]-nisoxetine were measured by quantitative receptor autoradiography in the brains of rats with acute liver failure resulting from hepatic devascularization and in appropriate controls. In vivo microdialysis was used to measure extracellular brain concentrations of NA. Severe encephalopathy resulted in a significant loss of [3H]-nisoxetine sites in frontal cortex and a concomitant increase in extracellular brain concentrations of NA in rats with acute liver failure. A loss of transporter sites was also observed in thalamus of rats with acute liver failure. This loss of NA transporter sites could result from depletion of central NA stores due to a reserpine-like effect of ammonia which is known to accumulate to millimolar concentrations in brain in ischemic liver failure. Impaired NA transport and the consequent increase in synaptic concentrations and increased stimulation of neuronal and astrocytic noradrenergic receptors could be implicated in the pathogenesis of the encephalopathy and brain edema characteristic of acute liver failure.