[Contrast-enhanced ultrasound (CEUS) for characterisation of focal liver lesions]. / Kontrastverstärkte Sonografie (CEUS) zur Diagnostik fokaler Leberläsionen.

Due to the trend towards increased use of imaging and rising awareness among high-risk patients, gastroenterologists and hepatologists are more frequently confronted with patients with focal liver lesions. In the differentiation of these lesions, CT and MRI have increasingly found their way into primary diagnostic steps in everyday clinical practice. Contrast-enhanced sonography, on the other hand, is a very effective and cost-efficient method for assessing focal liver lesions. The success of the method is not only based on the visualisation of microvascularisation in real time. If sonography is performed by the treating physician, he can use the exact knowledge of history and clinical findings to specifically adapt the examination procedure and to interpret the sonographic findings with greater accuracy ("clinical sonography"). At the same time, the method enables the practitioner to combine diagnostics and management decisions in his or her own hands. To achieve excellent results with contrast-enhanced sonography-as with any other imaging method-it is necessary that the examiner is sufficiently qualified.This article systematically presents the sonographic characteristics of the most common liver lesions and clearly shows their contrast patterns using videos (available via QR code). The article illustrates that CEUS could-and from the authors' point of view, should-have an even greater significance in the future.

Non-alcoholic fatty liver disease is associated with an increased risk of type 2 diabetes.

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) has become the most common liver disease worldwide and represents the leading cause of liver-related morbidity and mortality. Its all-cause mortality is often driven by co-existing metabolic diseases such as type 2 diabetes (T2DM), which share many pathophysiological characteristics. The risk of developing T2DM among NAFLD patients in Germany is only poorly described. METHODS: A cohort of 17 245 NAFLD patients and a propensity score-matched cohort of equal size were identified from the Disease Analyzer database (IQVIA) between 2005 and 2020. The incidence of T2DM was evaluated as a function of NAFLD during a 5-year study period using Cox-regression models. RESULTS: Within 5 years of the index date, 18.8% and 11.7% of individuals with and without NAFLD were diagnosed with T2DM ( P  < 0.001). Regression analysis revealed a hazard ratio of 1.77 [95% confidence interval (CI), 1.68-1.88] for the development of T2DM among NAFLD patients. Subgroup analyses confirmed this association for all age groups (18-50, 51-60, 61-70 and >70 years), male and female patients, as well as normal weight (BMI < 25 kg/m 2 ), overweighted (BMI 25-30 kg/m 2 ) and obese (BMI > 30 kg/m 2 ) patients. CONCLUSION: Our data revealed a significantly increased incidence of T2DM among NAFLD patients in Germany. Given the dramatically increasing global relevance of NAFLD, we believe that prevention and regular screening programs for T2DM in NAFLD patients could help to reduce its high mortality and morbidity in the future.

Hyperammonemia-induced changes in the cerebral transcriptome and proteome.

Molecular alterations underlying cerebral impairment in hyperammonemic disorders such as in hepatic encephalopathy (HE) are only poorly understood. Using transcriptomics and proteomics on brains of mice with systemic hyperammonemia resulting from knockout of hepatic glutamine synthetase (LGS-KO) we identified up to 214 genes and 34 proteins whose expressions were altered in brains of LGS-KO mice in a brain region-specific way. Differentially expressed genes were enriched for those related to oxidative stress, cell proliferation, heme metabolism and others. Due to their particularly high expression changes, coactivator associated arginine methyltransferase 1 (CARM1), TROVE2 and Lipocalin-2 (LCN2) were selected for further analyses. All selected candidates were expressed by astrocytes in rodent brain and challenging cultured astrocytes with NH4Cl changed their protein and mRNA levels similar to what was found in brains of LGS-KO mice. Further functional analyses suggested a role of CARM1 for senescence, TROVE2 for RNA quality control and LCN2 for disturbed iron homeostasis in ammonia-exposed astrocytes. LCN2 protein and Trove2 mRNA were also elevated in cerebral cortex of ammonium acetate-challenged rats and in post mortem brain tissue from patients with liver cirrhosis and HE, respectively. This study identified new molecular players potentially relevant for cerebral dysfunction in HE.

Non-alcoholic fatty liver disease (NAFLD) is associated with an increased incidence of osteoporosis and bone fractures.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) and osteoporosis are common diseases with a rising incidence worldwide. Both diseases occur in similar patient populations, however, data on their mutual influence are conflicting. Here, we aimed to evaluate the impact of NAFLD on the incidence of osteoporosis and fractures by using the Disease Analyzer database featuring data on diagnoses, prescriptions, and demographic aspects of 7.49 million cases followed in general practices in Germany. METHODS: A total of 50,689 patients with NAFLD diagnosed between 2000 and 2015 were matched by age, sex, index year, and 3 comorbidities (obesity, diabetes mellitus, and vitamin D/calcium deficiency) to a cohort of equal size without NAFLD. Incidence of osteoporosis and bone fractures were compared between both groups within 10 years from the index date. RESULTS: Within the observation period, the incidence of osteoporosis was significantly higher in the NAFLD group (6.4%) compared to patients without NAFLD (5.1%; log-rank, p < 0.001). Similar results were observed for bone fractures (12.6 vs. 10.3 %; log-rank p < 0.001). The difference was more pronounced in women compared to men and observed in all age groups >50 years of age. CONCLUSION: Our data show that NAFLD is significantly associated with osteoporosis as well as bone fractures in a large cohort of patients followed in German general practices. This finding suggests that NAFLD patients might benefit from improved monitoring for the occurrence of bone demineralization and osteoporosis, which in turn could trigger preventive therapeutic measures.

Characterization of the scavenger cell proteome in mouse and rat liver.

The structural-functional organization of ammonia and glutamine metabolism in the liver acinus involves highly specialized hepatocyte subpopulations like glutamine synthetase (GS) expressing perivenous hepatocytes (scavenger cells). However, this cell population has not yet been characterized extensively regarding expression of other genes and potential subpopulations. This was investigated in the present study by proteome profiling of periportal GS-negative and perivenous GS-expressing hepatocytes from mouse and rat. Apart from established markers of GS+ hepatocytes such as glutamate/aspartate transporter II (GLT1) or ammonium transporter Rh type B (RhBG), we identified novel scavenger cell-specific proteins like basal transcription factor 3 (BTF3) and heat-shock protein 25 (HSP25). Interestingly, BTF3 and HSP25 were heterogeneously distributed among GS+ hepatocytes in mouse liver slices. Feeding experiments showed that RhBG expression was increased in livers from mice fed with high protein diet compared to standard chow. While spatial distributions of GS and carbamoylphosphate synthetase 1 (CPS1) were unaffected, periportal areas constituted by glutaminase 2 (GLS2)-positive hepatocytes were enlarged or reduced in response to high or low protein diet, respectively. The data suggest that the population of perivenous GS+ scavenger cells is heterogeneous and not uniform as previously suggested which may reflect a functional heterogeneity, possibly relevant for liver regeneration.

Mechanism of Fully Reversible, pH-Sensitive Inhibition of Human Glutamine Synthetase by Tyrosine Nitration.

Glutamine synthetase (GS) catalyzes an ATP-dependent condensation of glutamate and ammonia to form glutamine. This reaction-and therefore GS-are indispensable for the hepatic nitrogen metabolism. Nitration of tyrosine 336 (Y336) inhibits human GS activity. GS nitration and the consequent loss of GS function are associated with a broad range of neurological diseases. The mechanism by which Y336 nitration inhibits GS, however, is not understood. Here, we show by means of unbiased MD simulations, binding, and configurational free energy computations that Y336 nitration hampers ATP binding but only in the deprotonated and negatively charged state of residue 336. By contrast, for the protonated and neutral state, our computations indicate an increased binding affinity for ATP. pKa computations of nitrated Y336 within GS predict a pKa of ∼5.3. Thus, at physiological pH, nitrated Y336 exists almost exclusively in the deprotonated and negatively charged state. In vitro experiments confirm these predictions, in that, the catalytic activity of nitrated GS is decreased at pH 7 and 6 but not at pH 4. These results indicate a novel, fully reversible, pH-sensitive mechanism for the regulation of GS activity by tyrosine nitration.

Evidence for functional selectivity in TUDC- and norUDCA-induced signal transduction via α5ß1 integrin towards choleresis.

Functional selectivity is the ligand-specific activation of certain signal transduction pathways at a receptor and has been described for G protein-coupled receptors. However, it has not yet been described for ligands interacting with integrins without αI domain. Here, we show by molecular dynamics simulations that four side chain-modified derivatives of tauroursodeoxycholic acid (TUDC), an agonist of α5ß1 integrin, differentially shift the conformational equilibrium of α5ß1 integrin towards the active state, in line with the extent of ß1 integrin activation from immunostaining. Unlike TUDC, 24-nor-ursodeoxycholic acid (norUDCA)-induced ß1 integrin activation triggered only transient activation of extracellular signal-regulated kinases and p38 mitogen-activated protein kinase and, consequently, only transient insertion of the bile acid transporter Bsep into the canalicular membrane, and did not involve activation of epidermal growth factor receptor. These results provide evidence that TUDC and norUDCA exert a functional selectivity at α5ß1 integrin and may provide a rationale for differential therapeutic use of UDCA and norUDCA.

Biallelic mutation of human SLC6A6 encoding the taurine transporter TAUT is linked to early retinal degeneration.

We previously reported that inactivation of the transmembrane taurine transporter (TauT or solute carrier 6a6) causes early retinal degeneration in mice. Compatible with taurine's indispensability for cell volume homeostasis, protein stabilization, cytoprotection, antioxidation, and immuno- and neuromodulation, mice develop multisystemic dysfunctions (hearing loss; liver fibrosis; and behavioral, heart, and skeletal muscle abnormalities) later on. Here, by genetic, cell biologic, in vivo1H-magnetic resonance spectroscopy and molecular dynamics simulation studies, we conducted in-depth characterization of a novel disorder: human TAUT deficiency. Loss of TAUT function due to a homozygous missense mutation caused panretinal degeneration in 2 brothers. TAUTp.A78E still localized in the plasma membrane but is predicted to impact structural stabilization. 3H-taurine uptake by peripheral blood mononuclear cells was reduced by 95%, and taurine levels were severely reduced in plasma, skeletal muscle, and brain. Extraocular dysfunctions were not yet detected, but significantly increased urinary excretion of 8-oxo-7,8-dihydroguanosine indicated generally enhanced (yet clinically unapparent) oxidative stress and RNA oxidation, warranting continuous broad surveillance.-Preising, M. N., Görg, B., Friedburg, C., Qvartskhava, N., Budde, B. S., Bonus, M., Toliat, M. R., Pfleger, C., Altmüller, J., Herebian, D., Beyer, M., Zöllner, H. J., Wittsack, H.-J., Schaper, J., Klee, D., Zechner, U., Nürnberg, P., Schipper, J., Schnitzler, A., Gohlke, H., Lorenz, B., Häussinger, D., Bolz, H. J. Biallelic mutation of human SLC6A6 encoding the taurine transporter TAUT is linked to early retinal degeneration.

Regulation of Plasma Membrane Localization of the Na⁺-Taurocholate Co-Transporting Polypeptide by Glycochenodeoxycholate and Tauroursodeoxycholate.

BACKGROUND/AIMS: Hydrophobic bile salts, such as glycochenodeoxycholate (GCDC) can trigger hepatocyte apoptosis, which is prevented by tauroursodesoxycholate (TUDC), but the effects of GCDC and TUDC on sinusoidal bile salt uptake via the Na⁺-taurocholate transporting polypeptide (Ntcp) are unclear. METHODS: The effects of GCDC and TUDC on the plasma membrane localization of Ntcp were studied in perfused rat liver by means of immunofluorescence analysis and super-resolution microscopy. The underlying signaling events were investigated by Western blotting and inhibitor studies. RESULTS: GCDC (20 µmol/l) induced within 60 min a retrieval of Ntcp from the basolateral membrane into the cytosol, which was accompanied by an activating phosphorylation of the Src kinases Fyn and Yes. Both, Fyn activation and the GCDC-induced Ntcp retrieval from the plasma membrane were sensitive to the NADPH oxidase inhibitor apocynin, the antioxidant N-acetylcysteine and the Src family kinase inhibitors SU6656 and PP-2, whereas PP-2 did not inhibit GCDC-induced Yes activation. Internalization of Ntcp by GCDC was also prevented by the protein kinase C (PKC) inhibitor Gö6850. TUDC (20 µmol/l) reversed the GCDC-induced retrieval of Ntcp from the plasma membrane and prevented the activation of Fyn and Yes in GCDC-perfused rat livers. Reinsertion of Ntcp into the basolateral membrane in GCDC-perfused livers by TUDC was sensitive to the protein kinase A (PKA) inhibitor H89 and the integrin-inhibitory peptide GRGDSP, whereas the control peptide GRADSP was ineffective. Ex posure of cultured rat hepatocytes to GCDC (50 µmol/l, 15min) increased the fluorescence intensity of the reactive oxygen fluorescent indicator DCF to about 1.6-fold of untreated controls in a TUDC (50 µmol/l)-sensitive way. GCDC caused a TUDC-sensitive canalicular dilatation without evidence for Bsep retrieval from the canalicular membrane. CONCLUSION: The present study suggests that GCDC triggers the retrieval of Ntcp from the basolateral membrane into the cytosol through an oxidative stress-dependent activation of Fyn. TUDC prevents the GCDC-induced Fyn activation and Ntcp retrieval through integrin-dependent activation of PKA.

Taurine transporter (TauT) deficiency impairs ammonia detoxification in mouse liver.

Hepatic ammonia handling was analyzed in taurine transporter (TauT) KO mice. Surprisingly, hyperammonemia was present at an age of 3 and 12 months despite normal tissue integrity. This was accompanied by cerebral RNA oxidation. As shown in liver perfusion experiments, glutamine production from ammonia was diminished in TauT KO mice, whereas urea production was not affected. In livers from 3-month-old TauT KO mice protein expression and activity of glutamine synthetase (GS) were unaffected, whereas the ammonia-transporting RhBG protein was down-regulated by about 50%. Double reciprocal plot analysis of glutamine synthesis versus perivenous ammonia concentration revealed that TauT KO had no effect on the capacity of glutamine formation in 3-month-old mice, but doubled the ammonia concentration required for half-maximal glutamine synthesis. Since hepatic RhBG expression is restricted to GS-expressing hepatocytes, the findings suggest that an impaired ammonia transport into these cells impairs glutamine synthesis. In livers from 12-, but not 3-month-old TauT KO mice, RhBG expression was not affected, surrogate markers for oxidative stress were strongly up-regulated, and GS activity was decreased by 40% due to an inactivating tyrosine nitration. This was also reflected by kinetic analyses in perfused liver, which showed a decreased glutamine synthesizing capacity by 43% and a largely unaffected ammonia concentration dependence. It is concluded that TauT deficiency triggers hyperammonemia through impaired hepatic glutamine synthesis due to an impaired ammonia transport via RhBG at 3 months and a tyrosine nitration-dependent inactivation of GS in 12-month-old TauT KO mice.

Inhibiting Glutamine-Dependent mTORC1 Activation Ameliorates Liver Cancers Driven by ß-Catenin Mutations.

Based on their lobule location, hepatocytes display differential gene expression, including pericentral hepatocytes that surround the central vein, which are marked by Wnt-ß-catenin signaling. Activating ß-catenin mutations occur in a variety of liver tumors, including hepatocellular carcinoma (HCC), but no specific therapies are available to treat these tumor subsets. Here, we identify a positive relationship between ß-catenin activation, its transcriptional target glutamine synthetase (GS), and p-mTOR-S2448, an indicator of mTORC1 activation. In normal livers of mice and humans, pericentral hepatocytes were simultaneously GS and p-mTOR-S2448 positive, as were ß-catenin-mutated liver tumors. Genetic disruption of ß-catenin signaling or GS prevented p-mTOR-S2448 expression, while its forced expression in ß-catenin-deficient livers led to ectopic p-mTOR-S2448 expression. Further, we found notable therapeutic benefit of mTORC1 inhibition in mutant-ß-catenin-driven HCC through suppression of cell proliferation and survival. Thus, mTORC1 inhibitors could be highly relevant in the treatment of liver tumors that are ß-catenin mutated and GS positive.

Hyperammonemia in gene-targeted mice lacking functional hepatic glutamine synthetase.

Urea cycle defects and acute or chronic liver failure are linked to systemic hyperammonemia and often result in cerebral dysfunction and encephalopathy. Although an important role of the liver in ammonia metabolism is widely accepted, the role of ammonia metabolizing pathways in the liver for maintenance of whole-body ammonia homeostasis in vivo remains ill-defined. Here, we show by generation of liver-specific Gln synthetase (GS)-deficient mice that GS in the liver is critically involved in systemic ammonia homeostasis in vivo. Hepatic deletion of GS triggered systemic hyperammonemia, which was associated with cerebral oxidative stress as indicated by increased levels of oxidized RNA and enhanced protein Tyr nitration. Liver-specific GS-deficient mice showed increased locomotion, impaired fear memory, and a slightly reduced life span. In conclusion, the present observations highlight the importance of hepatic GS for maintenance of ammonia homeostasis and establish the liver-specific GS KO mouse as a model with which to study effects of chronic hyperammonemia.

Locomotor impairment and cerebrocortical oxidative stress in portal vein ligated rats in vivo.

BACKGROUND & AIMS: Oxidative/nitrosative stress plays an important role in the pathogenesis of hepatic encephalopathy and ammonia toxicity. The present study was undertaken in order to investigate the impact of portal vein ligation on cerebrocortical oxidative stress and its relation to locomotor activity. METHODS: Cerebral protein tyrosine nitration, RNA oxidation, locomotor activity, and microglia activation were studied in rats that underwent portal vein ligation (PVL). RESULTS: Two weeks after PVL, increased levels of protein tyrosine nitration and RNA oxidation were found in the brain. PVL rats exhibited hyperammonemia and reduced locomotor behaviour, but displayed no signs of microglia activation or upregulation of the mRNAs for interleukin-1ß and tumor necrosis factor-α. PVL also had no effect on astrocytic glutamate transporter or inducible nitric-oxide synthase expression. Only cerebral Il-6 mRNA levels were increased. Daily administration of indomethacin prevented PVL-induced protein tyrosine nitration, RNA oxidation, Il-6 mRNA increase, and the impairment of locomotor activity, but did not prevent PVL-induced hyperammonemia. CONCLUSIONS: The data suggest that PVL triggers oxidative/nitrosative stress in the brain without activation of microglia and neuroinflammation. Prevention of protein tyrosine nitration and RNA oxidation by indomethacin also prevents the disturbances in locomotor activity pointing to a relevance of oxidative stress in the pathophysiology of HE.

Oxidative stress markers in the brain of patients with cirrhosis and hepatic encephalopathy.

UNLABELLED: Cell culture studies and animal models point to an important role of oxidative/nitrosative stress in the pathogenesis of cerebral ammonia toxicity. However, it is unknown whether oxidative/nitrosative stress in the brain is also characteristic of hepatic encephalopathy (HE) in humans. We therefore analyzed post mortem cortical brain tissue samples from patients with cirrhosis dying with or without HE in comparison with brains from patients without liver disease. Significantly elevated levels of protein tyrosine-nitrated proteins, heat shock protein-27, and 8-hydroxyguanosine as a marker for RNA oxidation were found in the cerebral cortex of HE patients, but not of patients with cirrhosis but without HE. Glutamine synthetase (GS) activity was significantly decreased, whereas GS protein expression was not significantly affected. Protein expression of the glutamate/aspartate cotransporter was up-regulated in HE, whereas protein expression of neuronal and inducible nitric oxide synthases, manganese-dependent and copper/zinc-dependent superoxide dismutase, and glial glutamate transporter-1 were not significantly increased. CONCLUSION: These data indicate that HE in patients with cirrhosis is associated with oxidative/nitrosative stress, protein tyrosine nitration, and RNA oxidation, suggesting a role of oxidative stress in the pathogenesis of HE in patients with cirrhosis.

Ammonia triggers exocytotic release of L-glutamate from cultured rat astrocytes.

Ammonia toxicity to the brain involves NMDA receptor overactivation and glutamate excitotoxicity. The mechanisms underlying glutamate release from astrocytes in response to ammonia were addressed in this study. In cultured rat astrocytes, glutamate immunoreactivity (IR) was punctate and partly colocalized with transfected VAMP2-YFP. NH(4)Cl (5 mmol/L) and hypoosmotic exposure (205 mosmol/L) induced a rapid colchicine-sensitive loss of cellular glutamate and glutamate appearance in the extracellular space. The NH(4)Cl-induced glutamate loss from astrocytes was strongly blunted after transfection of the cells with VAMP2 siRNA. Ammonia-induced exocytosis of VAMP2-YFP expressing vesicles was shown by total internal reflection fluorescence microscopy (TIRF-M). Glutamate exocytosis in response to ammonia was sensitive to chelation of Ca(2+), cyclooxygenase inhibition by indomethacin and colchicine. Ammonia triggered the rapid formation of prostanoids, which were identified as upstream events in ammonia-induced glutamate exocytosis. Also, addition of prostaglandin E(2) or of tumor necrosis factor (TNF)-alpha triggered glutamate exocytosis. Inhibition of ammonia-induced glutamate exocytosis after transfection of VAMP2 siRNA inhibited ammonia-induced RNA oxidation. It is concluded that ammonia triggers a prostanoid- and Ca(2+)-dependent glutamate exocytosis, which is essential for induction of ammonia-induced RNA oxidation.

Ammonia induces RNA oxidation in cultured astrocytes and brain in vivo.

UNLABELLED: 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, Ca(2+), nicotinamide adenine dinucleotide phosphate, and reduced form (NADPH) oxidase-dependent oxidative stress. Also, hypo-osmolarity, tumor necrosis factor alpha (TNF-alpha), 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-alpha, 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.

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 p47(phox) for hypoosmotic ROS generation was studied in cultured rat astrocytes and brain slices of wilde type and p47(phox) knock-out mice. Cultured rat astrocytes express mRNAs encoding for the regulatory subunit p47(phox), 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 p47(phox) and prevented by the NADPH oxidase inhibitor apocynin. Apocynin also impaired the hypoosmotic tyrosine phosphorylation of Src. Both, hypoosmotic ROS generation and p47(phox) serine phosphorylation were sensitive to the acidic sphingomyelinase inhibitors AY9944 and desipramine, the protein kinase C (PKC)zeta-inhibitory pseudosubstrate peptide, the NMDA receptor antagonist MK-801 and the intracellular Ca(2+) 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 p47(phox) 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 p47(phox)-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.

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 micro mol/l produced tyrosine nitration and inactivation of GS, whereas 50 micro mol/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 micro mol/l). Further, a putative "denitrase" activity restored the activity of peroxynitrite (5 micro mol/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.