Neurochemical Research, Vol. 24, No. 11, 1999, pp. 1399-1401

Evidence for an Astrocytic Glutamate Transporter Deficitin Hepatic Encephalopathy*

Helen Chan1 and Roger F. Butterworth1,2

(Accepted January 8, 1999)

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

KEY WORDS: Glutamate transport; hepatic encephalopathy; glutamate; GLT-1; [3H]D-Aspartate; acuteliver failure.

INTRODUCTION

Hepatic Encephalopathy (HE) is a neuropsychi-atric disorder resulting from acute or chronic liverfailure. HE may present as one of two clinically andpathologically distinct forms. HE in acute liver failureresulting, for example, from viral hepatitis or aceta-minophen overdose, has a rapid fulminating courseand is characterized by brain edema, increased in-tracranial pressure and, if not treated promptly by livertransplantation, a high mortality rate. HE in chronic

1 Neuroscience Research Unit, CHUM (Campus Saint-Luc), Mon-treal, Quebec, Canada.

2 Address reprints requests to: Roger F. Butterworth, Ph.D., D.Sc.,Neuroscience Research Unit, C.H.U.M./Campus Saint-Luc, 1058St-Denis Street, Montreal, Quebec H2X 3J4 Canada. Phone: (514)281-2444 ext. 5759, FAX: (514) 281-2492, Email: butterwr@ere.umontreal.ca

* Special issue dedicated to Dr. Nico M. van Gelder.

liver failure (cirrhosis), generally referred to as portal-systemic encephalopathy (PSE), on the other hand, hasa slower course starting with alterations of personalityand sleep patterns progressing through motor incoor-dination, stupor and coma. Cirrhosis may be alcoholicor non-alcoholic in etiology. PSE is frequently precip-itated by a gastrointestinal bleed associated with por-tal hypertension or ingestion of a high protein meal ora sedative. Neuropathologically, PSE is characterizedby astrocytic (rather than neuronal) changes referred toas Alzheimer type II astrocytosis.

Pathophysiologic mechanisms implicated in HEare incompletely understood. However, deficits in neu-rotransmitter function rather than primary cerebral en-ergy failure are generally considered to play a majorrole. In particular, there is a growing body of evidenceto suggest that the glutamatergic neurotransmitter sys-tem is impaired in HE. Glutamate concentrations arereduced in autopsied brain tissue from patients who

13970364-3190/99/1100-1397$ 16.00/0 C 1999 Plenum Publishing Corporation

1398 Chan and Butterworth

died in hepatic coma resulting from either acute (1) orchronic (2) liver failure. Brain glutamate concentra-tions are also reduced in both acute (ischemic) liverfailure and in thioacetamide-induced liver damage inthe rat (3). In this latter study, reductions in brain glu-tamate were found to parallel the deterioration of neu-rological status and cerebrospinal fluid (CSF) concen-trations of glutamate were concomitantly increased (3).In another recent study of the correlation of alterationsof various brain metabolites with clinical grading ofencephalopathy in rats with acute liver failure, a signif-icant negative correlation between severity of clinicalsymptoms and brain glutamate concentrations was re-ported (4). Furthermore, reductions in brain glutamatewere noted even at early stages of encephalopathy.These reports prompted a series of investigations ofpre- and postsynaptic glutamatergic neurotransmitterfunction in human and experimental HE.

Glutamate Release in Brain in Experimental HE.In acute HE induced in rats by hepatic devasculariza-tion, increased extracellular concentrations of glutamatehave been consistently reported using the technique ofin vivo cerebral microdialysis (5,6). These changes wereselective for glutamate; extracellular concentrationsof other neuroactive amino acids including GABAand aspartate were unchanged during progression ofacute liver failure. Similar findings were reportedin rabbits with experimental acute liver failure (7).However, release of glutamate from synaptosomal pre-parations from rats with acute liver failure resultingfrom thioacetamide-induced hepatotoxicity was un-changed (8).

Several studies have addressed the issue of gluta-mate release from brain in chronic liver failure in therat following end-to-side portacaval anastomosis. Usingthe "cortical cup" approach to study glutamate release,it was demonstrated that extracellular concentrationsof glutamate were increased following anastomosis (9).However, subsequent studies using the technique ofin vivo cerebral microdialysis with dialysis probes inseveral brain structures including frontal cortex, pari-etal cortex and striatum did not reveal any significantalterations of extracellular glutamate concentrations(10-12). In contrast to findings in acute liver failure,extracellular concentrations of aspartate were signifi-cantly increased in the brains of rats following porta-caval anastomosis (10,12). However, in common withfindings in acute liver failure, extracellular concen-trations of GABA were unchanged, suggesting thatthe brain GABA system is not directly implicated inthe pathogenesis of hepatic encephalopathy in eitheracute or chronic live failure.

Effects of Ammonia on Glutamate release in Brain.In both acute and chronic liver failure, brain ammoniaconcentrations are increased and may attain millimolarlevels (14). Recent studies using Positron Emission To-mography suggest that brain ammonia concentrationsin the brains of humans with liver failure are also in-creased (15). It is well established that glutamate syn-thesis and release in brain in vitro is regulated by theavailability of ammonia (13). Exposure of hippocampalslices to millimolar concentrations of ammonia (ie:concentrations similar to those reported in liver failure)results in a decrease in K+-evoked release of glutamatewithout any significant alteration of spontaneous re-lease (16). A similar lack of effect of ammonia on K+-evoked release of glutamate by synaptosomal prepara-tions has also been reported (17). These findings alongwith the negative findings in thioacetamide-inducedliver failure (8) suggest that the increased extracellu-lar glutamate concentrations in the brains of animalswith acute or chronic liver failure (above) are not theresult of a direct action of ammonia on the release ofglutamate from nerve terminals. Rather, they may re-sult from an inhibitory effect of ammonia on glutamateremoval from the synaptic cleft.

Glutamate Uptake by Brain Preparations in Ex-perimental HE. The synaptic action of glutamate israpidly and efficiently terminated by high affinity trans-port systems located on nerve terminals and on per-ineuronal astrocytes. Uptake of [3H]-glutamate has beenassessed using brain slices and synaptosomes from ratswith chronic liver failure resulting from portal veinand bile duct ligation (18); no significant alterationswere noted. On the other hand, a marked decrease incapacity for synaptosomal glutamate uptake was re-ported in rats with acute liver failure resulting fromthioacetamide-induced hepatotoxicity (18). Further-more, using an autoradiographic procedure and [3H]D-aspartate, a non-metabolizeable glutamate analogue, arecent study revealed a significant loss of glutamateuptake sites in the frontal cortex of rats with acuteliver failure due to hepatic devascularization (19).These findings again demonstrate important differ-ences between the effects of acute versus chronic liverfailure on the brain glutamate system.

Effects of Ammonia on Glutamate Uptake by BrainPreparations. Exposure of rat hippocampal slices toblood extracts from patients with varying severity of HEresulted in inhibition of D-aspartate uptake and the rel-ative potency of inhibition was found to correlate withammonia concentrations of the extracts (20).

Furthermore, exposure of primary cortical astro-cytes to millimolar concentrations of ammonia also re-

Glutamate Transport in Hepatic Encephalopathy 1399

suited in a significant reduction in uptake of [3H]D-aspartate (21). On the other hand, the effects of am-monia on glutamate uptake by synaptosomal prepara-tions are equivocal with both significant reductions(22) and lack of changes (18) being reported.

Glutamate Transporter Expression in Brain inHE. Recently, three major high affinity, Na+-dependentrat brain glutamate transporters have been cloned andsequenced (23,24). Immunocytochemical and in situhybridization studies reveal that these transporters dif-fer with respect to their cellular and regional localiza-tion. GLT-1 and GLAST are preferentially expressedon astrocytic membranes whereas EAAC-1 appears tobe neuronal in localization (Fig. 1). It has been sug-gested that GLT-1 is the glutamate transporter whichplays the major role in clearance of extracellular glu-tamate in the rat forebrain (25).

Interest in GLT-1 in relation to HE in acute liverfailure was kindled by the findings of increased ex-tracellular glutamate in the brains of rats with acuteliver failure (5,6) as well as the report of increasedextracellular glutamate concentrations in the brainsof GLT-1 knockout mice (26) coupled with the ob-servation that these animals manifest convulsions andbrain edema (26), two important features of acute

liver failure. GLT-1 protein and gene expressionwere therefore studied in extracts from cerebral cor-tex of rats at coma stages of encephalopathy follow-ing hepatic devascularization (27) and a significantloss of GLT-1 expression was noted. A similar loss ofGLT-1 expression was also described in the brains ofmice with thioacetamide-induced liver failure (28).GLT-1 is regulated both transcriptionally and post-transcriptionally (25,30). Transcriptional regulationof GLT-1 is mediated by cyclic AMP analogues aswell as by glutamate, glutamate receptor agonists andby 8-opioid receptor stimulation (25,31). Rapid regu-lation of GLT-1 independent of protein synthesis mayalso occur by diverse signaling molecules includingprotein kinase C, arachidomic acid and cytokines (25).That the loss of GLT-1 expression in brain in acuteliver failure is the consequence of ammonia toxicity issupported by the observations that 1) hyperammone-mic mice manifest a significant loss of GLT-1 expres-sion in brain (28); 2) exposure of cultured astrocytesto millimolar concentrations of ammonia results in aloss of GLT-1 gene expression (29) and 3) reducedGLT-1 expression results in increased extracellularglutamate concentrations which are positively corre-lated with arterial ammonia levels (6).

Fig. 1. Glutamate transporter localization.

1400 Chan and Butterworth

In summary, there is convincing evidence to sug-gest that acute liver failure results in decreased expres-sion of the astrocytic glutamate transporter GLT-1 andthat this decreased expression results in increased ex-tracellular glutamate in forebrain. Studies in culturedastrocytes exposed to pathophysiologically relevantconcentrations of ammonia suggest that the loss ofGLT-1 expression in brain in acute liver failure is theconsequence of ammonia toxicity. The effect of ammo-nia on GLT-1 expression in these cells appears to in-volve a transcriptional mechanism. Increased extra-cellular glutamate resulting from decreased astrocyticremoval could explain the hyperexcitability and brainedema which occurs in acute liver failure.

ACKNOWLEDGMENTS

Studies from the author's research unit were funded by grantsfrom The Medical Research Council of Canada. The authors thankDominique Roy for her assistance with the preparation of the man-uscript.

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