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Ammonia induces MK-801-sensitive nitration and phosphorylation of protein tyrosine residues in rat astrocytes¹

FREIMUT SCHLIESS, BORIS GÖRG, RICHARD FISCHER, PAUL DESJARDINS^*, HANS J. BIDMON, ANDREAS HERRMANN, ROGER F. BUTTERWORTH^*, KARL ZILLES^, and DIETER HÄUSSINGER²

Clinic for Gastroenterology, Hepatology and Infectiology, Heinrich-Heine-University, Düsseldorf, Germany;
^* Neuroscience Research Unit, Hôpital Saint-Luc, University of Montreal, Quebec, Canada;
C. & O. Vogt Institut for Brain Research, Heinrich-Heine-University Düsseldorf, Germany;
Cardion AG, Erkrath, Germany; and
Institut for Internal Medicine, Research Center Jülich, Germany

²Correspondence: Medizinische Einrichtungen der Heinrich-Heine Universität, Klinik für Gastroenterologie, Hepatologie und Infektiologie, Moorenstrasse 5, D-40225 Düsseldorf, Germany. E-mail: Haeussin{at}uni-duesseldorf.de

SPECIFIC AIMS

Astrocytes are a major target of ammonia action with pathogenetic relevance for hepatic encephalopathy (HE) in liver disease and other hyperammonemic states. Ammonia effects on astroglial protein tyrosine nitration and signal transduction were investigated in cultured rat astrocytes, brains from acutely ammonia-intoxicated rats, and portacaval-shunted rats with chronic hyperammonemia.

PRINCIPAL FINDINGS

1. Ammonia induces protein tyrosine nitration in cultured rat astrocytes
Exposure of cultured rat astrocytes to NH₄Cl increases 3'-nitrotyrosine immunoreactivity (Fig. 1 A–F). Western blot analysis unraveled a nitration pattern produced by NH₄Cl similar to that induced by the NO donor sodium nitroprusside, the peroxynitrite-producing agent 3-morpholinosydnonimine, and interferon + lipopolysaccharide treatment, which triggers iNOS expression in astrocytes.

View larger version (48K): [in this window] [in a new window]   Figure 1. NH4Cl-induced 3-nitrotyrosine and iNOS immunoreactivity in cultured rat astrocytes. Immunoreactivity of 3-nitrotyrosine (NO2Tyr), iNOS, and GFAP was visualized by confocal laser scanning microscopy. Cultured rat astrocytes were maintained in absence of NH4Cl (control; A, C, E, G) or exposed to 1 mmol/l NH4Cl for 24 h (B, D, F, H). Green: GFAP immunoreactivity (A, B, E–H); red: NO2Tyr immunoreactivity (C–F) or iNOS immunoreactivity (G, H); yellow: colocalization of NO2Tyr and GFAP (F) or iNOS and GFAP (H), respectively. Representatives of 3 independent experiments are shown.

2. Ammonia induces iNOS expression and NO production in cultured astrocytes
NH₄Cl induced iNOS expression in cultured rat astrocytes (Fig. 1G, H ). iNOS expression induced by 1 mM NH₄Cl is accompanied by the generation of 1.9 ± 0.3 µmol NO/mg protein/24 h. iNOS induction by ammonia was related to degradation of IB and was sensitive to pyrrolidine dithiocarbamate, suggesting an involvement of the NF-B system.

3. Features of ammonia-induced protein tyrosine nitration in rat astrocytes
Protein tyrosine nitration in response to ammonia was sensitive to the N-methyl-D-aspartate (NMDA) receptor antagonist receptor MK-801 and was mimicked by NMDA. NMDA receptor-mediated elevation of the intracellular Ca²⁺ concentration and iNOS expression contributed to the ammonia effect on protein tyrosine nitration, which is also sensitive to Cu,Zn superoxide dismutase/catalase treatment and uric acid, suggesting a major contribution of peroxynitrite formation.

Some ammonia effects on astrocytes can be ascribed to ammonia-induced cell swelling, which occurs due to intracellular glutamine accumulation. Hypoosmotic (205 mosmol/l) astrocyte swelling mimicked protein tyrosine nitration induced by ammonia, and the glutamine synthetase inhibitor methionine sulfoximine largely prevented the nitration response to ammonia, suggesting a role for astrocyte swelling and glutamine synthesis in triggering ammonia-induced tyrosine nitration.

4. Ammonia induces protein phosphorylation in cultured astrocytes
NH₄Cl increased protein tyrosine phosphorylation and induced dual Thr/Tyr phosphorylation of the MAP kinases Erk-1/Erk-2 and p38^MAPK in cultured astrocytes. This was sensitive to NOS inhibition, MK-801, Cu,Zn superoxide dismutase/catalase, and uric acid. These findings suggest that reactive nitrogen intermediates are involved in ammonia-induced signaling via protein phosphorylation.

5. Ammonia induces nitration of glutamine synthetase, the peripheral-type benzodiazepine receptor, the glyceraldehyde-3-phosphate dehydrogenase, and Erk-1 in cultured astrocytes
Ammonia induced tyrosine nitration of glutamine synthetase in cultured astrocytes. This was accompanied by an 30% decrease of total glutamine synthetase activity (Fig. 2 ). Ammonia induced tyrosine nitration of the peripheral-type benzodiazepine receptor, the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase, and the MAP kinase Erk-1 but not of the glial fibrillary acidic protein, Erk-2, and p38^MAPK.

View larger version (37K): [in this window] [in a new window]   Figure 2. Tyrosine nitration of glutamine synthetase, glyceraldehyde-3-phosphate dehydrogenase, and the peripheral-type benzodiazepine receptor in cultured rat astrocytes. GS, glutamine synthetase; PBR, peripheral-type benzodiazepine receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; tyrosine-nitrated GS, PBR, or GAPDH: NO2Tyr-GS, NO2Tyr-PBR, and NO2Tyr-GAPDH, respectively. IP, immunoprecipitation; WB, Western blot. A) Ammonia-induced Tyr nitration of GS is associated with reduced activity. Astrocytes were exposed to NH4Cl (0.1–5 mmol/l) or remained untreated. Proteins precipitated with the anti-3'-nitrotyrosine antibody were analyzed in Western blot for the presence of NO2-Tyr-GS. For control, immunoprecipitated GS was analyzed in Western blot for the presence of 3-nitrotyrosine residues. The anti-GS immunoprecipitation was counterchecked in Western blot with the anti-GS antibody. GS activity was determined in astrocytes treated with 5 mM NH4Cl for 24 h or in untreated cells. To check the contribution of nitrated GS to enzymatic activity, lysates were depleted of tyrosine-nitrated proteins by immunoprecipitation. Nitrotyrosine-containing lysates were clarified with protein A Sepharose. B) Ammonia-induced nitration of PBR and GAPDH. PBR and GAPDH were detected by Western blot in the anti-3-nitrotyrosine immunoprecipitate from ammonia-treated astrocytes.

6. In vivo relevance
Portacaval anastomosed (PCA) rats are often used to study HE pathophysiology and represent a model for chronic hyperammonemia, whereas NH₄Ac intoxication of rats is used to study acute ammonia effects. Elevated levels of protein tyrosine nitration were found in the cerebral cortex of PCA rats and acutely ammonia-intoxicated rats vs. control animals. Tyrosine nitration of glutamine synthetase, which is expressed almost exclusively in astrocytes, was increased by sixfold in the cerebral cortex of both animal models. An increase in tyrosine nitration of astroglial proteins in acutely ammonia-intoxicated rats was visualized by confocal laser scanning microscopy. Nitrotyrosine immunoreactivity was enriched along the blood vessels and colocalized with glial fibrillary acidic protein. The findings suggest that tyrosine nitration of astroglial proteins is markedly enhanced in vivo under conditions of chronic and acute hyperammonemia.

CONCLUSIONS AND SIGNIFICANCE

Ammonia is a key factor in the pathogenesis of HE, a major complication in acute and chronic liver failure and other hyperammonemic states. Cerebral ammonia is detoxified by astrocytes, which play a primary role in impairment of neuronal function under hyperammonemic conditions. Ammonia-induced tyrosine nitration of astroglial proteins shown in this study represents a new molecular aspect of ammonia toxicity. The relevance of this finding is suggested by the fact that MK-801, NOS inhibitors of broad specificity, and methionine sulfoximine, which block ammonia-induced protein tyrosine nitration (this study), protected animals from ammonia toxicity. However, specific nNOS inhibition was ineffective. Our study suggests that astroglial NMDA receptor activation represents a trigger of cerebral ammonia action (Fig. 3 ): ammonia-induced NMDA receptor activation leads to [Ca²⁺]_i elevation, which triggers iNOS expression and production of NO. The ammonia-induced Ca²⁺ signal may be coupled with elevation of mitochondrial Ca²⁺ concentration and thereby trigger the mitochondrial generation of superoxide, which may combine with NO to the tyrosine-nitrating agent peroxynitrite. Reactive nitrogen intermediates account for protein tyrosine nitration and act as signal metabolites for augmentation of protein phosphorylation in ammonia-exposed astrocytes. The mechanisms of NMDA receptor activation by ammonia in astrocytes are unknown. Ammonia-induced depolarization could remove the Mg²⁺ blockade from the NMDA receptor. The NMDA receptor-mediated [Ca²⁺]_i signal and astrocyte swelling could stimulate glutamate release, in turn promoting further NMDA receptor signaling. Consistent with this idea, glutamate release into the extracellular space in brains of acutely ammonia-intoxicated rats has been shown to be sensitive to the NMDA receptor antagonist MK-801; as shown here, inhibition of glutamine synthetase abolished ammonia-induced tyrosine nitration in astrocytes.

View larger version (17K): [in this window] [in a new window]   Figure 3. Ammonia-induced astrocyte signaling. The tentative scheme summarizes our current view of ammonia-induced signaling leading to tyrosine nitration and phosphorylation in astrocytes. Ammonia induces an NMDA receptor-dependent increase in [Ca2+]i, which mediates iNOS-catalyzed NO production. Ammonia is detoxified by the glutamine synthetase, leading to intracellular glutamine accumulation and astrocyte swelling. NO was also shown to induce astrocyte swelling. [Ca2+]i elevation and astrocyte swelling may trigger glutamate release into the extracellular space, further promoting NMDA receptor signaling. The formation of reactive oxygen and nitrogen intermediates such as peroxynitrite may account for protein tyrosine nitration and stimulation of signaling cascades, resulting in protein tyrosine phosphorylation.

In astrocytes, tyrosine nitration may result in the modification of enzyme activities and a predisposition of proteins for proteasomal degradation. Tyrosine nitration of glutamine synthetase is associated with its inactivation (Fig. 2) . It is conceivable that the decrease in glutamine synthetase activity in the cortex of PCA rats is caused by tyrosine nitration and/or oxidative modifications of the enzyme. GAPDH nitration and/or oxidation (Fig. 2) may contribute to compromised energy metabolism in HE. Ammonia is known to increase ligand binding to the PBR and augment PBR-mediated neurosteroid synthesis in cultured astrocytes and hepatic encephalopathy. It remains to be established to what extent protein tyrosine nitration (Fig. 2) contributes to altered PBR function.

This study shows that ammonia-induced protein tyrosine nitration occurs not only in cultured astrocytes in vitro, but also in in vivo models of acute and chronic hyperammonemia. The involvement of astrocytes in these in vivo models is demonstrated by the strong tyrosine nitration of glutamine synthetase, which is expressed almost exclusively in astrocytes. After acute ammonia intoxication, tyrosine nitration was most pronounced in the perivascular area and colocalized with GFAP-positive cells. Astrocytes are known to be important constituents of the blood–brain barrier. Thus, one is tempted to speculate that protein nitration of perivascular astrocytes may affect transastrocytic substrate transport, which would correspond to the well-known altered blood–brain barrier permeability in HE. In clinical settings, HE is precipitated by bleeding, high protein intake, electrolyte disturbances, sepsis, and infections. Not only ammonia, but also hyponatremia and inflammatory cytokines, were shown here to increase protein tyrosine nitration. Other studies are required to clarify to what extent clinical symptoms of HE can be attributed to ammonia-induced protein nitration.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0862fje; to cite this article, use FASEB J. (March 26, 2002) 10.1096/fj.01-0862fje

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