Freimut Schliess

Hepatic encephalopathy in chronic liver disease: a clinical manifestation of astrocyte swelling and low-grade cerebral edema?

EPATIC ENCEPHALOPATHY (HE) iS a frequent COmH plication of chronic liver disease. Its pathogenesis is not understood, although there is agreement on the important role of neurotoxins, especially ammonia (1). In the brain of HE patients, neurons appear morphologically normal, but astrocytes exhibit signs of Alzheimer type II degeneration with nuclear enlargement, peripheral margination of chromatin and prominent nucleoli. Functional alterations in HE include selective alterations of blood-brain barrier permeability, changes in cerebral energy metabolism, an increased GABA-ergic tone and changes in several other neurotransmitter systems and their receptors (for reviews see (2-8)). However, previous hypotheses about the pathogenesis of HE were unable to explain all the facets of this clinical syndrome. Clearly, pathogenetic models have to explain the functional nature and reversibility of HE symptoms, and their precipitation by heterogeneous factors, such as infections, diuretics, sedatives, trauma, bleeding or high protein intake.

Pathogenetic mechanisms of hepatic encephalopathy

Hepatic encephalopathy (HE) in liver cirrhosis is a clinical manifestation of a low-grade cerebral oedema, which is exacerbated in response to ammonia and other precipitating factors. This low-grade cerebral oedema is accompanied by an increased production of reactive oxygen and nitrogen oxide species (ROS/RNOS), which trigger multiple protein and RNA modifications, thereby affecting brain function. The action of ammonia, inflammatory cytokines, benzodiazepines and hyponatraemia integrates at the level of astrocyte swelling and oxidative stress. This explains why heterogenous clinical conditions can precipitate HE episodes. Oxidised RNA species, which are formed in response to oxidative stress, also participate in local postsynaptic protein synthesis in neurons, which is required for memory formation. Although the functional consequences of RNA oxidation in this context remain to be established, these findings bear a potential biochemical explanation for the multiple alterations of neurotransmitter receptor systems and of synaptic plasticity. Such changes may in part also underlie the pathologically altered oscillatory networks in the brain of HE patients in vivo, as detected by magnetencephalography. These disturbances of oscillatory networks, which in part are triggered by hypothalamic structures, can explain the motor and cognitive deficits in patients with HE. Current therapeutic strategies aim at the elimination of precipitating factors. The potential of therapies targeting downstream pathophysiological events in HE has not yet been explored, but offers novel potential sites of therapeutic intervention.

Hypoosmotic swelling and ammonia increase oxidative stress by NADPH oxidase in cultured astrocytes and vital brain slices.

The role of NADPH oxidase (NOX) and the regulatory subunit p47phox for hypoosmotic ROS generation was studied in cultured rat astrocytes and brain slices of wilde type and p47phox knock‐out mice. Cultured rat astrocytes express mRNAs encoding for the regulatory subunit p47phox, NOX1, 2, and 4, and the dual oxidases (DUOX)1 and 2, but not NOX3. Hypoosmotic (205 mosmol/L) swelling of cultured astrocytes induced a rapid generation of ROS that was accompanied by serine phosphorylation of p47phox and prevented by the NADPH oxidase inhibitor apocynin. Apocynin also impaired the hypoosmotic tyrosine phosphorylation of Src. Both, hypoosmotic ROS generation and p47phox serine phosphorylation were sensitive to the acidic sphingomyelinase inhibitors AY9944 and desipramine, the protein kinase C (PKC)ζ‐inhibitory pseudosubstrate peptide, the NMDA receptor antagonist MK‐801 and the intracellular Ca2+ chelator BAPTA‐AM. Also hypoosmotic exposure of wilde type mouse cortical brain slices increased ROS generation, which was allocated in part to the astrocytes and which was absent in presence of apocynin and in cortical brain slices from p47phox knock‐out mice. Also ammonia induced a rapid ROS production in cultured astrocytes and brain slices, which was sensitive to apocynin. The data suggest that astrocyte swelling triggers a p47phox‐dependent NADPH oxidase‐catalyzed ROS production. The findings further support a close interrelation between osmotic and oxidative stress in astrocytes, which may be relevant to different brain pathologies including hepatic encephalopathy.

Hyperosmolarity and CD95L trigger CD95/EGF receptor association and tyrosine phosphorylation of CD95 as prerequisites for CD95 membrane trafficking and DISC formation

The mechanisms underlying CD95 ligand (CD95L)‐ and hyperosmolarity‐induced activation of the CD95 system [Reinehr, R., Graf, D., Fischer, R., Schliess, F., and Haussinger, D. (2002) Hepatology 36, 602‐614] as initial steps of apoptosis were studied. Hyperosmotic exposure (405 mosmol/l) of rat hepatocytes induced within 1 min oxidative stress and antioxidant‐sensitive activation of the epidermal growth factor receptor (EGFR) and c‐Jun‐N‐terminal‐kinase (JNK). After 30 min of hyperosmotic exposure EGFR associated with CD95 and CD95 became tyrosine phosphorylated. Inhibition of JNK or protein kinase C (PKC) had no effect on EGFR phosphorylation but abolished CD95/EGFR association, CD95‐tyrosine phosphorylation, membrane targeting, and Fas‐associated death domain/caspase 8 recruitment to CD95 [death‐inducing signaling complex (DISC) formation]. Inhibition of EGFR tyrosine kinase activity prevented CD95 tyrosine phosphorylation and DISC formation but not hyperosmolarity‐induced EGFR phosphorylation and EGFR association with CD95. Tyrosine‐phosphorylated CD95 was enriched in the plasma membrane. All maneuvers preventing CD95 tyrosine phosphorylation inhibited CD95 membrane trafficking and DISC formation. Stimulation of EGFR by EGF induced EGFR phosphorylation but no association with CD95 or CD95 phosphorylation. Addition of CD95L also induced EGFR and JNK activation, EGFR/CD95 association, CD95 tyrosine phosphorylation, DISC formation, and CD95 membrane targeting with an inhibitor sensitivity profile similar to that of hyperosmotic CD95 activation, except that inhibition of PKC was ineffective. The data suggest that moderate hyperosmolarity or CD95L trigger oxidative stress and EGFR activation followed by a JNK‐dependent EGFR/CD95association and CD95 tyrosine phosphorylation, probably through EGFR tyrosine kinase activity. This provides a signal for CD95 membrane trafficking and DISC formation.

Ammonia Induces RNA Oxidation in Cultured Astrocytes and Brain In Vivo

Oxidative stress plays a major role in cerebral ammonia toxicity and the pathogenesis of hepatic encephalopathy (HE). As shown in this study, ammonia induces a rapid RNA oxidation in cultured rat astrocytes, vital mouse brain slices, and rat brain in vivo. Ammonia‐induced RNA oxidation in cultured astrocytes is reversible and sensitive to MK‐801, 1,2‐Bis(o‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid, apocynin, epigallocatechin gallate, and polyphenon 60, suggesting the involvement of N−methyl−D‐aspartic acid (NMDA) receptor activation, Ca2+, nicotinamide adenine dinucleotide phosphate, and reduced form (NADPH) oxidase‐dependent oxidative stress. Also, hypo‐osmolarity, tumor necrosis factor alpha (TNF‐α), and diazepam increase RNA oxidation in cultured astrocytes, suggesting that the action of different HE‐precipitating factors converges at the level of RNA oxidation. Among the oxidized RNA species, 18S‐rRNA and the messenger RNA (mRNA) coding for the glutamate/aspartate transporter (GLAST) were identified. Cerebral RNA oxidation in acutely ammonia‐loaded rats in vivo is reversible and predominates in neuronal soma and perivascular astrocyte processes. In neuronal dendrites, oxidized RNA colocalizes with the RNA‐binding splicing protein neurooncological ventral antigen (NOVA)‐2 within putative RNA transport granules, which are also found in close vicinity to postsynaptic spines. This indicates that oxidized RNA species may participate in postsynaptic protein synthesis, which is a biochemical substrate for learning and memory consolidation. Neuronal and astroglial RNA oxidation increases also in vital mouse brain slices treated with ammonia and TNF‐α, respectively. Conclusion: Cerebral RNA oxidation is identified as a not yet recognized consequence of acute ammonia intoxication. RNA oxidation may affect gene expression and local protein synthesis and thereby provide another link between reactive oxygen species (ROS)/reactive nitrogen oxide species (RNOS) production and ammonia toxicity. (HEPATOLOGY 2008.)

Reversible inhibition of mammalian glutamine synthetase by tyrosine nitration

The effect of tyrosine nitration on mammalian GS activity and stability was studied in vitro. Peroxynitrite at a concentration of 5 μmol/l produced tyrosine nitration and inactivation of GS, whereas 50 μmol/l peroxynitrite additionally increased S‐nitrosylation and carbonylation and degradation of GS by the 20S proteasome. (−)Epicatechin completely prevented both, tyrosine nitration and inactivation of GS by peroxynitrite (5 μmol/l). Further, a putative “denitrase” activity restored the activity of peroxynitrite (5 μmol/l)‐treated GS. The data point to a potential regulation of GS activity by a reversible tyrosine nitration. High levels of oxidative stress may irreversibly damage and predispose the enzyme to proteasomal degradation.

Involvement of p38MAPK in the regulation of proteolysis by liver cell hydration

BACKGROUND & AIMS Liver cell hydration is a major determinant of proteolysis control; however, the underlying mechanisms are unknown. METHODS The role of mitogen-activated protein kinases for proteolysis control was studied in perfused rat liver. RESULTS Hyposmolarity led to a rapid activation of Erk-2 and p38(MAPK), but not of c-Jun-N-terminal kinase 1. Likewise, isosmotic cell swelling induced by insulin, ethanol, or glutamine/glycine activated p38(MAPK). Inhibition of hyposmotic Erk activation by pertussis or cholera toxin, erbstatin, or genistein had no effect on the swelling-induced inhibition of proteolysis. Likewise, wortmannin, rapamycin, and okadaic acid were ineffective, but proteolysis recovery from hyposmotic inhibition was okadaic acid sensitive. SB203580, an inhibitor of p38(MAPK), abolished both the antiproteolytic effect of hyposmotic cell swelling and the hyposmolarity-induced inhibition of autophagic vacuole formation. Also, the antiproteolytic effect of isotonic cell swelling induced by ethanol, glutamine/glycine, or insulin was abolished by SB203580, but not the swelling potency of these agents. SB203580 had no effect on the cell hydration-independent control of proteolysis exerted by NH4Cl, asparagine, or phenylalanine. CONCLUSIONS The data suggest an important role of p38(MAPK) in the regulation of autophagic proteolysis by cell volume in liver.

Involvement of integrins and Src in tauroursodeoxycholate-induced and swelling-induced choleresis

BACKGROUND & AIMS Stimulation of canalicular secretion by tauroursodeoxycholate (TUDC) involves dual activation of p38 mitogen-activated protein kinase (p38(MAPK)) and extracellular signal-regulated kinase (ERK). This study investigates the sensing and upstream signaling events of TUDC-induced choleresis. METHODS TUDC and hypo-osmolarity effects on protein kinase activities and taurocholate excretion were studied in perfused rat liver. RESULTS TUDC induced a rapid activation of focal adhesion kinase (FAK) and Src, as shown by an increase in Y418 phosphorylation and a decrease in Y529 phosphorylation of Src. Inhibition of Src by PP-2 abolished the TUDC-induced activation of p38(MAPK) but not of FAK and ERKs. An integrin-inhibitory peptide with an RGD motif blocked TUDC-induced FAK, Src, ERK, and p38(MAPK) activation, suggesting that integrin signaling toward FAK/Src is required for TUDC-induced MAPK activation. The RGD peptide and PP-2 also abolished the stimulation of taurocholate excretion in perfused rat liver in response to TUDC. Integrin-dependent Src activation was also identified as an upstream event in hypo-osmotic signaling toward MAPKs and choleresis. CONCLUSIONS TUDC-induced stimulation of canalicular taurocholate excretion involves integrin sensing, FAK, and Src activation as upstream events for dual MAPK activation. Integrins may also represent one long-searched sensor for cell hydration changes in response to hypo-osmolarity.

Activation of the Janus Kinase/Signal Transducer and Activator of Transcription Pathway by Osmotic Shock

Numerous cytokines, growth, and differentiation factors elicit their intracellular responses via Janus tyrosine kinases (Jaks) and transcription factors of the STAT (signal transducer and activator of transcription) family. Additionally, environmental stress (UV light, heat, aniso-osmolarity, and radicals) has recently been shown to activate intracellular signaling cascades such as the stress-activated protein kinases and nuclear factor-κB. In this study, we demonstrate that in different cell lines a particular stress, namely hyperosmolarity, results in tyrosine phosphorylation of the Janus kinases Jak1, Jak2, and Tyk2 and in the activation of STAT1 and/or STAT3. Both transcription factors are phosphorylated at a specific tyrosine residue and translocation to the nucleus was demonstrated by the use of a STAT3/green fluorescent protein fusion protein. A prominent role for Jak1 in the activation of STATs by hypertonicity was demonstrated by the use of Jak-deficient cell lines. Stress-activated STAT1 and STAT3 transactivate a reporter gene containing the acute-phase response element of the rat α2-macroglobulin promoter. Experiments using a diffusible solute suggest that not the increase in intracellular osmolarity but the resultant cell shrinkage is the trigger for Jak/STAT activation.

The cellular hydration state: a critical determinant for cell death and survival.

Abstract Alterations in cellular hydration not only contribute to metabolic regulation, but also critically determine the cellular response to different kinds of stress. Whereas cell swelling triggers anabolic pathways and protects cells from heat and oxidative challenge, cellular dehydration contributes to insulin resistance and catabolism and increases the cellular susceptibility to stressinduced damage. Intracellular accumulation of organic osmolytes, cell cycle delay and the expression of heat shock proteins provide cellular tolerance to hyperosmolarity and protect against stressors under dehydrating conditions. This article discusses some mechanisms by which alterations in cell hydration contribute to cytoprotection or cell damage. In addition, the close relationship between osmotic and oxidative stress and the contribution of isoosmotic shrinkage to apoptotic cell death are considered.