Ammonia induces MK-801-sensitive nitration

8/8/2019 Ammonia induces MK-801-sensitive nitration

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The FASEB Journalexpress article 10.1096/fj.01-0862fje. Published online March 26, 2002.

Ammonia induces MK-801-sensitive nitration and

phosphorylation of protein tyrosine residues in rat

astrocytes

Freimut Schliess*, Boris Grg*, Richard Fischer

*, Paul Desjardins

, Hans J. Bidmon

,

Andreas Herrmann, Roger F. Butterworth

, Karl Zilles

,, and Dieter Hussinger

*

*Clinic for Gastroenterology, Hepatology and Infectiology, Heinrich-Heine-University,

Dsseldorf, Germany;

Neuroscience Research Unit, Hpital Saint-Luc, University of Montreal,

Quebec, Canada;C. & O. Vogt Institut for Brain Research, Heinrich-Heine-University

Dsseldorf, Germany;Cardion AG, Erkrath, Germany; and

Institut for Internal Medicine,

Research Center Jlich, Germany

Corresponding author: D. Hussinger, Medizinische Einrichtungen der Heinrich-HeineUniversitt, Klinik fr Gastroenterologie, Hepatologie und Infektiologie, Moorenstrasse 5, D-

40225 Dsseldorf, Germany. E-mail: [email protected]

ABSTRACT

Astrocytes play a key role in the pathogenesis of ammonia-induced neurotoxicity and hepatic

encephalopathy. As shown here, ammonia induces protein tyrosine nitration in cultured rat

astrocytes, which is sensitive to the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801.A similar pattern of nitrated proteins is produced by NMDA. Ammonia-induced tyrosine

nitration depends on a rise in [Ca2+

]i, IB degradation, and NO synthase (iNOS) induction,

which are prevented by MK-801 and the intracellular Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA-AM). Moreover, the increase in

tyrosine nitration is blunted by L-NMMA, 1400W, uric acid, Cu,Zn-superoxidedismutase/catalase treatment, and methionine-sulfoximine, which indicate the involvement ofreactive nitrogen intermediates and intracellular glutamine accumulation. Such reactive nitrogen

intermediates additionally mediate ammonia-induced phosphorylation of the MAP-kinases Erk-

1/Erk-2 and p38MAPK

. Among the proteins, which are tyrosine-nitrated by ammonia,glyceraldehyde-3-phosphate dehydrogenase, the peripheral-type benzodiazepine receptor, Erk-1,

and glutamine synthetase are identified. Ammonia-induced nitration of glutamine synthetase is

associated with a loss of enzymatic activity. Astroglial protein tyrosine nitration is found inbrains from rats after acute ammonia-intoxication or after portacaval anastomosis, indicating the

in vivo relevance of the present findings. The production of reactive nitrogen intermediates andprotein tyrosine nitration may alter astrocyte function and contribute to ammonia neurotoxicity.

Key words: MAP kinases calcium hepatic encephalopathy nitric oxide NMDA receptor

glutamine synthetase liver


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Ammonia is a key factor in the pathogenesis of hepatic encephalopathy (HE), which is a

major complication in acute and chronic liver failure and other hyperammonemic states,such as inborn errors of urea synthesis. HE symptoms are highly variable and range from

mild personality changes to deep coma, but are, at each level of severity, potentially reversible

[for review, see refs 1-4]. The molecular mechanisms underlying ammonia neurotoxicity are

incompletely understood. Cerebral ammonia is eliminated by astrocytes, the only cellularcompartment in the brain expressing glutamine synthetase (5). Intracellular glutamine

accumulation due to increased ammonia detoxification leads to astrocyte swelling, which was

recognized as an early pathogenic event already in subclinical HE in cirrhotic patients (6) andwhich may contribute to the severe rise in intracranial pressure in patients with fulminant hepatic

failure (3). Inhibition of glutamine synthetase by methionine-sulfoximine (MSO) reduces

glutamine accumulation, astrocyte swelling, and brain edema in acutely hyperammonemic rats[e.g., (7)]. It was suggested that HE represents a primary gliopathy and that neuronal dysfunction

is a secondary event due to impaired communication between astrocytes and neurons (4).

Other lines of evidence point to a role of NMDA receptors in ammonia neurotoxicity. NMDA

receptor inhibition prevents the decrease in antioxidant enzyme activities, the generation ofreactive oxygen intermediates (ROIs) (8), and the changes in mitochondrial Ca2+

homeostasis (9)

induced by acute hyperammonemia in rat brain. In addition, high doses of ammonia increasecGMP concentrations in cerebrospinal fluid by a mechanism sensitive to NMDA receptor

antagonists, and the increase in cGMP correlates well with the severity of the neurological

symptoms (10).

Recent hypotheses suggest that an impaired glutamate clearance by the swollen astrocytes

stimulates neuronal NMDA receptors, which triggers an intracellular Ca2+

signal and activatesneuronal nitric oxide (NO) synthase (nNOS) in a Ca

2+/calmodulin-dependent way (3). nNOS-

catalyzed NO synthesis would in turn stimulate formation of cGMP, which may contribute toaltered neuronal function, vasodilation, and brain edema. However, a role of glutamate in

NMDA receptor activation following acute ammonia intoxication of rats was questioned recently

(10), because NMDA receptor inhibition even blocked glutamate release into the extracellularspace. This finding suggested that glutamate release was a consequence but not the cause of

NMDA receptor activation. In addition, selective inhibition of nNOS had no effect on ammonia

toxicity (11), whereas a wide-spectrum NOS inhibitor protected animals from ammonia toxicity

(12).

Whether astrocytes contribute to ammonia-induced NO production is unknown. However,

ammonia stimulates arginine uptake in cultured astrocytes (13) and high ammonia concentrationsinduce the expression of argininosuccinate synthetase and argininosuccinate lyase in astrocytes

but not neurons (14). Moreover, antagonizing astrocyte swelling by methionine-sulfoximine

normalizes brain output of circulating NOx in ammonia-intoxicated rats (7), whereby the sourceof NOx remained unclear.

The present study identifies ammonia as a trigger of protein tyrosine nitration in cultured rat

astrocytes due to an increased iNOS expression via NMDA receptor-mediated Ca2+

signaling and

IB degradation. Reactive nitrogen intermediates act as signal metabolites mediating ammonia-

induced protein tyrosine phosphorylation and dual phosphorylation of the MAP-kinases Erk-


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1/Erk-2 and p38MAPK

. Erk-1, the peripheral-type benzodiazepine receptor, and the

glyceraldehyde-3-phosphate dehydrogenase are targets of ammonia-induced tyrosine-nitration.An in vivo relevance is suggested by the finding that protein tyrosine nitration of astroglial

proteins occurs in brains of acutely ammonia-intoxicated rats and in rats after portocaval

anastomosis.

MATERIALS AND METHODS

Materials

Pluronic F-127 and the acetoxymethylester of fura-2 were purchased from Molecular Probes Inc

(Eugene, OR). Cell culture media and fetal calf serum were from Gibco Life Technologies(Gaithersburg, MD). Catalase (cat) and superoxide dismutase, methionine-sulfoximine, sodium

nitroprusside, uric acid, and the anti-glial fibrillary acidic protein (GFAP) antibody were

obtained from Sigma (Deisenhofen, Germany). Adenosine triphosphate (ATP) was from

Boehringer, (Mannheim, Germany). Ethyleneglycol-bis(-aminoethyl)-N,N,NNtetraacetic acid

(EGTA), ionomycin, 3-morpho-linosydnonimine (SIN-1), BAPTA-AM, N

G

-mono-methyl- L -arginine (L-NMMA), N-(3-aminomethyl)-benzylacetamidine (1400W), and N-(4-aminobuthyl)-5-chloro-1-naphthalenesulfonamide (W13) were from Calbiochem-Novabiochem GmbH (Bad

Soden, Germany). Monoclonal anti-3-nitrotyrosine and anti-phosphotyrosine antibodies and 3-

nitrotyrosine were purchased from Calbiochem-Novabiochem GmbH and Upstate Biotechnology(Lake Placid, NY). Antibodies recognizing Erk-1/Erk-2 were from Upstate Biotechnology.

Antibodies recognizing phospho-Erk-1/Erk-2 (monoclonal) were from New England Biolabs

GmbH (Frankfurt/Main, Germany). The polyclonal antibodies raised against glutamine

synthetase, p38MAPK

and IB were from Santa Cruz Biotechnology (Santa Cruz, CA), and the

monoclonal antibodies raised against glutamine synthetase and iNOS, respectively, were fromTransduction Laboratories (San Diego, CA). The anti-phosho-p38

MAPKantibody was from

Promega (Madison, WI). Fluorescein isothiocyanate (FITC)- and Cy3-coupled antibodies werefrom Jackson Corp. (West Grove, PA).

Preparation, cultivation, and NH4Cl treatment of rat brain astrocytes

We prepared primary astrocytes from cerebral hemispheres of newborn Wistar rats and cultured

them in Dulbeccos modified Eagles medium (DMEM) as described (15). Purity of the cell

culture as determined by GFAP and S-100 immunohistochemical staining was >95%. The potential toxicity of NH4Cl was addressed by determination of cellular lactate dehydrogenase

(LDH) release into the medium. In line with earlier findings (16), incubation of the astrocytes

with up to 5 mmol/L NH4Cl during 24 h did not increase LDH release by the astrocytes, which

was 0.90.1 U/L in the absence of NH4Cl and 1.00.3 U/L in the presence of 1mmol/L and1.00.3 after a 24 h incubation in the presence of 5 mmol/L NH4Cl. Cells were serum-starved 24h before experimental treatment.

Portacaval shunted (PCA) rats

Adult male Sprague-Dawley rats (150175 g) were anesthetized with halothane and underwentan end-to-side portacaval anastomosis (PCA), as described previously (17). Four weeks after


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surgery, shunted and sham-operated rats were killed by decapitation and the brains were

removed rapidly. The frontal cortex was dissected and frozen immediately on dry ice and storedat 70C. On the day of protein extraction, thawed tissues were homogenized in 300 l of ice-

cold buffer [25 mmol/L Tris-HCl, pH 7.4, 2 mmol/L EGTA, 0.1% sodium dodecyl sulfate

(SDS), 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 2 mol/L leupeptin, 2 mol/L

pepstatin A and 1 g/ml aprotinin]. These animal procedures conformed to guidelines of theAnimal Ethics Committee of Hpital Saint-Luc and the University of Montreal.

Acute ammonium acetate intoxication of rats

We used male Wister rats (250300 g). Ammonium acetate (NH4Ac) or, for control, NaCl (4.5

mmol/kg) was injected intraperitoneally. After NH4Ac administration, the rats experienced atransient coma within 15 to 20 min (7 out of 9 rats) or died (2 out of 9 rats). After 1 or 6 h rats

were anesthetized with pentobarbital (50 mg/kg) and transcardially perfused with 20 ml

physiological saline containing heparin (10,000 iU/L, Liquemin, Hoffmann La Roche, Basel,Switzerland) to remove blood cells, followed by 100 ml physiological saline containing 20%

sucrose as a cryoprotectant. Brains were dissected quickly and 4-mm-thick sections from themiddle of each hemisphere were prepared. One of these thick sections was frozen for

immunohistochemistry. The one from the other hemisphere was immersion-fixed in modifiedZambonis fixative (18). The remaining cerebral tissues were divided further into cerebral cortex,

cerebellar cortex, and caudate-basal forebrain complex, which were frozen immediately over

liquid nitrogen for protein extraction. These experiments were approved by the National AnimalWelfare Legislation.

Immunofluorescence staining of 3-nitrotyrosine residues, iNOS,

glial fibrillary acidic protein, and glutamine synthetase

For immunofluorescence, astrocytes were cultured on glass coverslips with a diameter of 10 mm.

At the end of the experimental treatment cells were fixed with paraformaldehyde for 10 min at 4and washed 3 times with ice-cold phosphate buffered saline (PBS). Subsequently, cells were

incubated with Triton X-100 (0.1% in PBS for 10 min). Cells were washed again and incubated

with 25% goat-Ig in the case of staining with anti-NO2Tyr (mAb, 1:85) and anti-GFAP (pAb,

1:200) for at least 2 h at room temperature. iNOS (mAb, 1:100) and GFAP (pAb, 1:200) werefixed with ice-cold methanol for 10 min, followed by staining for 2 h at room temperature. Cells

were washed three times in PBS and then incubated in PBS with 1% bovine serum albumin for 1

h. The cells were washed extensively and incubated for 2 h with fluorescein isothiocyanate(FITC)-conjugated anti-rabbit IgG or Cy3-conjugated anti-mouse IgG at room temperature. We

gathered images from one or two channels at 488- and 568 nm wavelengths.

For immunohistochemistry of brain slices, the thick Zamboni-fixed (4C, 24 h) section was

submerged again in 25% sucrose in PBS, pH 7.4, for cryoprotection, frozen in isopentane, and

further cryosectioned (50-m sections). After several rinses in PBS endogenous peroxidaseswere blocked in PBS containing 3% hydrogen peroxide, and sections were prepared for

immunohistochemistry according to previous protocols by using either the ABC technique or

immunofluorescence and laser scanning microscopy (18). As primary monoclonal antibodies, we

used the antinitrotyrosine antibody (final dilution 1:75).


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Immunoprecipitations and Western blot analysis

At the end of the experimental astrocyte treatment, medium was removed from the culture and

cells were lysed immediately at 4C by using 10 mmol/L Tris/HCl buffer (pH 7.4) containing

1% Triton X-100, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 20 mmol/L NaF, 0.2mmol/L PMSF, and 0.5% NP-40. We performed Western blot analysis by using a semidry

transfer apparatus (Pharmacia, Freiburg, Germany) as described (15). Blots were probed with

antisera against NO2-Tyr (mAb, Calbiochem, 1:5,000), Phospho-Tyr (1:5,000), phospho-Erk-1/Erk-2 (1:5,000), or phospho-p38

MAPK(1:5,000), respectively, for 2 h. Following washing and

incubation with horseradish peroxidase-coupled anti-rabbit-IgG antibody or horseradish

peroxidase-coupled anti-mouse-IgG antibody diluted 1:20,000 at 4C for 2 h, the blots werewashed again and developed by using enhanced chemiluminescent detection (Amersham,

Braunschweig, Germany).

For immunoprecipitation, cell lysates containing defined protein amounts were incubated with

1.5 g anti-NO2Tyr, anti-Erk-1/Erk-2, anti-glutamine synthetase, antiperipheral-type benzodiazepin receptor, or anti-GAPDH antibody, respectively. The immune complexes werecollected by using protein A/G sepharose (Santa Cruz), washed five times, and then subjected to

SDS-polyacrylamide gel electrophoresis.

Calcium imaging at the single cell level

Astrocytes were grown on coverslips in DMEM + 10% FCS. Subconfluent cells were passagedonto round 22 mm

3coverslips and grown for another 4 days in DMEM + 10% FCS. They were

serum-starved for 24 h and incubated with Krebs-Henseleit medium (KHB: 115 mmol/L

NaCl/25 mmol/L NaHCO3/5.9 mmol/L KCl/1.18 mmol/L MgCl2/1.23 mmol/L NaH2PO4/1.2

mmol/L Na2SO4/1.25 mmol/L CaCl2), containing 5 mol/l of the fluorescent Ca

2+

-chelator fura-2 acetoxymethylester and 0.02% pluronic F-127, for 30 min at 37C and 5% CO 2. Forfluorescence recording, the coverslips were superfused continuously at a rate of 15 ml/min with

KHB at 37C, equilibrated with O2/CO2 (95/5; v/v), resulting in pH 7.4. Measurement of

cytosolic calcium concentration [Ca2+

]i was performed with an inverted fluorescence microscope

(Zeiss, Axiovert) as described (19). For control, we added 5 mol/L ATP after each experiment.

Determination of NO2-(Griess reaction)

Astrocytes were treated with 1 mmol/L NH4Cl for 24 h. We sampled culture supernatants (each

100 l) in 96-well-plates and then added 50 l 2% sulfanilamide in 2.5% H3PO4 and 50 l 0.2%

naphthalene-ethylenediamine in 2.5 % H3PO

4. After 10 min, the formation of the azo-dye

product of the Griess reaction was colorimetrically (540 nm) determined. NaNO2 was converted

in the Griess reaction as a standard.

Determination of glutamine synthetase activity

Glutamine synthetase activity was measured according to Ward and Bradford (20). Aliquots of

120 l cell homogenate were incubated with 100 l of reaction mixture at 37C. The final


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concentrations used were as follows: 100 mmol/L imidazole (pH 7.2), 12.5 mmol/L MgCl2, 10

mmol/L ATP, 50 mmol/L potassium glutamate, 20 mmol/L -mercaptoethanol, 1 mmol/Louabain, 13 mmol/L phosphoenolpyruvate, 100 mmol/L hydroxylamine, and 5 units/ml pyruvate

kinase. We stopped the reaction after 1 h by adding 250 l of a solution containing 500 mmol/LFeCl3, 400 mmol/L perchloric acid, and 400 mmol/L HCl. After 30 min incubation on ice, the

protein precipitate was sedimented by centrifugation at 20,000g and 4C for 10 min. Theabsorbance of the supernatant was measured at 492 nm. Standard curves were established by

using -glutamyl hydroxamate. Values were calculated as mol of-glutamyl hydroxamateformed per hour per milligram of protein at 37C and expressed as percentage of control. The

reaction was tested for linearity and dependence on the protein concentration.

Analysis of results

Results from n independent experiments are expressed as means SE. Results were compared by

using the Students t-test:P


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treatment with interferon (IFN-) and lipopolysaccharide (LPS), which prototypically induces

NO production catalyzed by the inducible form of the NOS, led to increased levels oftyrosine-

nitrated proteins (Fig. 1A, lane 17).

Ammonia induces iNOS expression in cultured rat astrocytes

As shown inFigure 3, ammonia induces expression of iNOS in cultured astrocytes. NH4Cl (5

mmol/L)-induced iNOS protein expression in astrocytes was seen already after 6 h, was maximal

after 24 h and declined towards basal levels within 72 h (Fig. 3A). At the 24 h time point,

increased iNOS expression was detectable already in response to 100 mol/L NH4Cl (Fig. 3B).iNOS protein was hardly detectable in the absence of NH4Cl. In line with these Western blot

data, confocal laser scanning images showed an ammonia-induced iNOS immunoreactivity

(iNOS staining, red; GFAP, green; colocalization, yellow) in cultured astrocytes, which waspredominantly perinuclear (Fig. 2G, H).

NH4Cl-induced NO formation in astrocytes was confirmed by measuring nitrite release into the

medium, which results from NO oxidation. Incubation of the astrocytes with 1 mmol/L NH4Clfor 24 h increased NO2

-concentration in the supernatant to 223 21.4 % of control (NH4Cl

absent), corresponding to the generation of 1.9 0.3 mol NO/mg protein/24 h in presence ofNH4Cl (n=4).

NH4Cl decreased the IB expression level in astrocytes (Fig. 3C), which is indicative of an

activation of the NFB system by ammonia. MG-132, which inhibits proteasomal IB

degradation (23), and pyrrolidine dithiocarbamate, which inhibits NFB activation, inhibited

both the decrease of IB and the increase of iNOS expression in response to ammonia (Fig. 3C).

These findings suggest the involvement of NFB in mediating iNOS induction in response toammonia.

Features of ammonia-induced protein tyrosine nitration in rat astrocytes

The ammonia-induced protein tyrosine nitration in astrocytes was characterized furtherpharmacologically. Astrocytes were exposed to 1 mmol/L NH4Cl for 6 h, which was sufficient to

induce significant protein tyrosine nitration. L-NMMA, a widely used NOS inhibitor of broad

specificity (24), reduced protein tyrosine nitration in presence of NH4Cl almost to basal levels(Fig. 1B, lane 7). Likewise, 1400W, a specific iNOS inhibitor (25) largely blocked the ammonia

effect on protein tyrosine nitration (Fig. 1B, lane 8). W13, a calmodulin antagonist (26), had no

significant effect on ammonia-induced Tyr-nitration (Fig. 1B, lane 3). These findings suggestthat ammonia-induced protein nitration in cultured astrocytes depends on the ammonia-induced

up-regulation of iNOS expression (Fig. 3), whereas the Ca2+/calmodulin-regulated eNOS andnNOS do probably not contribute significantly.

Tyrosine nitration occurs secondarily to the reaction of NO with ROIs. Peroxynitrite is formed in

a diffusion-limited reaction between NO and superoxide and represents a very potent nitrating

species (22) (see also the effect of SIN-1 in Fig. 1A, lane 16). Previous studies have shown that peroxynitrite formation can be blocked effectively by Cu,Zn-superoxide dismutase (SOD) in

combination with cat (27). Indeed, SOD/cat treatment of the astrocytes completely abolished the


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ammonia-induced protein tyrosine nitration (Fig. 1B, lane 9). Likewise, uric acid, a potent

scavenger of peroxynitrite (28), blocked the ammonia-induced nitration of proteins (Fig. 1B, lane14). These findings suggest a major contribution of peroxynitrite to protein tyrosine nitration

induced by ammonia.

As demonstrated in Figure 4A, NH4Cl induced a rapid increase in the cytosolic calciumconcentration [Ca2+

]i, which was small compared with the [Ca2+

]i response triggered by

extracellular ATP. The [Ca2+

]i response to NH4Cl was abolished completely in the presence of

BAPTA-AM (Fig. 4B). BAPTA-AM also antagonizes the ammonia-induced IB degradation,iNOS expression (Fig. 3C) and protein tyrosine nitration (Fig. 1B, lane 4), consistent with an

involvement of the ammonia-induced [Ca2+

]i -signal at the level of NH4Cl-induced NFBactivation.

MK-801 affects glutamate signaling by inhibiting NMDA receptor-gated Ca2+

channels (29). At

a concentration of 100 mol/L, MK-801 largely abolished the NH4Cl-induced [Ca2+

]i elevation

(Fig. 4C), IB degradation, iNOS expression (Fig. 3C), and protein tyrosine nitration (Fig. 1B,

line 12). Ammonia-induced protein tyrosine nitration was already sensitive to 50 and 100 nmol/LMK-801 and was fully blunted at a concentration of 100 mol/L (Fig. 1B, lanes 1012).Consistent with an involvement of NMDA receptor activation in ammonia-induced tyrosine

nitration, NMDA produced a pattern of tyrosine-nitrated proteins similar to that found after

NH4Cl treatment of the astrocytes (Fig. 1A, lane 14).

These findings suggest that ammonia in astrocytes generates an NMDA-receptor-dependent Ca2+

signal, which is apparently required for NFB-mediated iNOS expression. iNOS-dependentgeneration of reactive nitrogen intermediates, such as peroxynitrite, results in protein tyrosine

nitration.

Ammonia is known to induce astrocyte swelling, which is largely due to an intracellularglutamine accumulation (3, 6, 16), and several ammonia effects on astrocytes can be ascribed to

ammonia-induced cell swelling (6, 30). Like ammonia (Fig. 4A), hypoosmotic astrocyte swelling produces a [Ca

2+]i signal (15, 19, 31). As shown in Fig. 1A (lanes 1013) hypoosmotic (205

mosmol/L) astrocyte swelling induced a pattern of protein nitration, which resembled that

induced by ammonia (1 mmol/L). Further, inhibition of ammonia-induced glutamine synthesis

by methionine sulfoximine (MSO) largely prevented protein nitration in response to ammonia(Fig. 1B, lane 13). These data suggest that ammonia-induced protein nitration may at least in partbe ascribed to ammonia-induced glutamine synthesis and astrocyte swelling.

Ammonia induces protein tyrosine phosphorylation in astrocytes

Besides protein tyrosine nitration, NH4Cl induced a dose-dependent phosphorylation of protein

tyrosine residues in cultured astrocytes as shown in Fig. 5A (lanes 17).Similar to protein

tyrosine nitration, the ammonia-induced tyrosine phosphorylation was sensitive to treatment ofthe cells with L-NMMA, MK-801, uric acid, and SOD/cat (Fig. 5B, lanes 813). It is suggested

that reactive nitrogen intermediates may act as signal metabolites, which trigger not only protein

tyrosine nitration but also protein tyrosine phosphorylation in response to ammonia.


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Effect of ammonia on MAP-kinases Erk-1/Erk-2 and p38MAPK

in astrocytes

The Erk-1/Erk-2-type and the p38-type MAP-kinases are key signaling elements, which are

involved in the differential regulation of cell function in response to stress, growth, and

differentiation factors [reviewed in (32)]. MAP-kinases are activated by MAP-kinase-kinases of

dual specificity, which phosphorylate Thr/Tyr-residues within a characteristic three amino acidmotif (Erk-1/Erk-2, Thr-Glu-Tyr; p38MAPK

,: Thr-Gly-Tyr). As shown in Figure 6A exposure of

astrocytes to NH4Cl for 24 h induced the presence of dual phosphorylated Erk-1/Erk-2 (ppErk-1

and ppErk-2) and p38MAPK

(ppP38). NH4Cl (1 mmol/L)-induced dual phosphorylation ofMAP-kinases was already seen after 6 h (Fig. 6B). The inhibitor studies shown in Figure 6B revealed,

that NH4Cl-induced dual phosphorylation of Erk-1/Erk-2 and p38MAPK

was sensitive to MK-801

(50 nM100 M), L-NMMA, uric acid, and SOD/cat treatment. Thus, reactive nitrogenintermediates apparently are generated in a MK-801-sensitive manner and mediate the dual

phosphorylation of Erk-1/Erk-2 and p38MAPK

in response to ammonia.

Ammonia-induced tyrosine nitration of Erk-1/Erk-2 and p38MAPK

was examined by

immunoprecipitation studies. Astrocytes were exposed to NH4Cl (1 and 5 mmol/L) for 24 h.Immunoprecipitated Erk-1/Erk-2 or p38MAPK

were analyzed by Western blot with the anti-3-

nitrotyrosine antibody. As a countercheck, we analyzed immunoprecipitated 3-tyrosine-nitratedproteins in Western blot for the presence of Erk-1/Erk-2 and p38

MAPK. As shown in Figure 6C,

NH4Cl induced tyrosine nitration of Erk-1 (NO2Tyr-Erk-1), but not of Erk-2 (NO2Tyr-Erk-2) or

p38MAPK

(NO2Tyr-P38). At least a fraction of tyrosine-nitrated Erk-1 seems to be dualphosphorylated (Figure 6C). These findings suggest a differential sensitivity of the protein

kinases with respect to ammonia-induced tyrosine nitration.

Ammonia induces nitration of glutamine synthetase, the peripheral-type benzodiazepine

receptor, and the glyceraldehyde-3-phosphate dehydrogenase in cultured astrocytes

The effect of NH4Cl on tyrosine nitration of glutamine synthetase was studied in cultured

astrocytes. 3-tyrosine-nitrated proteins were immunoprecipitated and analyzed by Western blotfor the presence of glutamine synthetase (GS). Conversely, glutamine synthetase was

precipitated and tested by Western blot analysis for the presence of nitrotyrosine residues

(NO2Tyr-GS). As shown in Figure 7A, exposure ofrat astrocytes to NH4Cl for 24 h induced a

dose-dependent tyrosine nitration of the glutamine synthetase. In addition, NH4Cl induced asignificant decrease of glutamine synthetase activity by about 30% in the astrocytes (Fig. 7A).

Removal of tyrosine-nitrated proteins from the cell lysate by immunoprecipitation did not reduce

glutamine synthetase activity further (Fig. 7A). This suggests that tyrosine-nitrated glutaminesynthetase does not contribute to enzyme activity and that nitration of tyrosine residues is

associated with an inactivation of glutamine synthetase in NH4Cl-treated astrocytes.

Apart from Erk-1 and glutamine synthetase, ammonia also induced tyrosine nitration of the

peripheral-type benzodiazepine receptor (NO2Tyr-PBR) and the glycolytic enzyme

glyceraldehyde-3-phosphate dehydrogenase (NO2Tyr-GAPDH), as shown by their presence in

the anti-nitrotyrosine immunoprecipitates (Fig. 7B). However, ammonia did not induce tyrosinenitration of glial fibrillary acidic protein (not shown).


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In vivo relevance

To test the in vivo relevance of ammonia-induced protein nitration in astrocytes, we studied

animal models for chronic and acute hyperammonemia. Portacaval anastomized rats (PCA rats)

are frequently used for studies on HE pathophysiology and represent a model for chronic

hyperammonemia (33, 34). Four weeks after PCA surgery, these animals developed liver atrophyand chronic hyperammonemia with blood ammonia concentrations of about 300 mol/L (sham-

operated rats: 170 mol/L) (21). Ammonia concentrations about 500 mol/L and a glutamine

content of about 15 mol/g were found in the cerebral cortex (21). These values compare to 160mol/L ammonia and 4.7 mol/g glutamine, in the cortex of sham-operated rats (21),

respectively. As shown in Figure 8, elevated levels of protein tyrosine nitration were found in the

cerebral cortex of PCA rats when compared with sham-operated controls. Immunoprecipitated3-tyrosine-nitrated proteins were about sixfold enriched with glutamine synthetase (NO2Tyr-

GS) in PCA rats, com pared with sham-operated control rats (Fig. 8). Because glutamine

synthetase is found almost exclusively in astrocytes (5), these findings indicate that proteintyrosine nitration develops in astrocytes in response to portosystemic blood shunting.

As a model of acute hyperammonemia, rats were injected with NH4Ac (4.5 mmol/kg) and killed

1 or 6 h later. Also the brains of these animals contained increased levels of protein tyrosinenitration as shown by Westernblot analysis (Fig. 8) and elevated NO2Tyr immunoreactivity in

brain slices (Fig. 9A-D). Again, immunoprecipitated 3-tyrosine-nitrated proteins were markedly

enriched with glutamine synthetase (Fig. 8). In addition, a hyperammonemia-induced increase intyrosine nitration of astroglial proteins was visualized by confocal laser scanning microscopy

(Fig. 9 EK): 3-nitrotyrosine immunoreactivity was enriched along the blood vessels and

colocalized with GFAP. The findings suggest, that tyrosine nitration of astroglial proteins isenhanced markedly in vivo under conditions of chronic and acute hyperammonemia.

DISCUSSION

Astrocytes represent the major target of ammonia action with pathogenetic relevance for HE in

liver disease and other hyperammonemic states (14). The ammonia-induced protein tyrosinenitration of astroglial cells in culture and in vivo shown in this study is a new aspect of cerebral

ammonia action.

The data presented here suggest astroglial NMDA receptor activation to be an early event inammonia-induced signal transduction. The NMDA receptor antagonist MK-801 blocked the

ammonia-induced rise in [Ca2+

]i (Fig. 4), degradation of IB, and i NOS expression (Fig. 3),overall protein tyrosine nitration and phosphorylation (Fig. 1 and 5) and dual phosphorylation of

the MAP-kinases Erk-1/Erk-2 and p38

MAPK

(Fig. 6). In addition, the ammonia effect on proteintyrosine nitration was mimicked largely byNMDA (Fig. 1). The mechanism of NMDA receptoractivation by ammonia remains speculative. On the one hand, ammonia depolarizes astrocytes

(35), and membrane depolarization may remove the Mg2+

blockade from the NMDA receptor

(36). On the other hand [Ca2+

]i elevation in astrocytes appears sufficient to induce astroglialglutamate release (37), which could account for NMDA receptor activation. However, the

ammonia-induced [Ca2+

]i signal was downstream to NMDA receptor activation (Fig. 4C). Thus,we speculate that a NMDA receptor-mediated rise in [Ca

2+]i induced by ammonia may stimulate


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glutamate release, which in turn promotes further NMDA receptor signaling. Consistent with this

idea, glutamate release into the extracellular space in brains from acutely ammonia-intoxicatedrats was shown to be sensitive to the NMDA receptor antagonist MK-801 (10).

Ammonia-induced NO production and protein tyrosine nitration in cultured astrocytes is

attributable largely to iNOS induction, which is downstream of a Ca

2+

-dependent IBdegradation (Figs. 1 and 3). Besides NO production, the ammonia-induced Ca

2+signal may

trigger the mitochondrial generation of further ROIs. NMDA receptor-mediated [Ca2+

]i changesare tightly coupled to mitochondrial Ca

2+uptake (38). Consistently, in brains of acutely

ammonia-intoxicated rats, MK-801 blocked both the elevation of Ca2+

levels in nonsynaptic

mitochondria and the increase of superoxide formation (8, 9). It is feasible that ammoniaactivates astroglial NMDA receptors to generate NO and superoxide simultaneously, which may

combine to the tyrosine-nitrating agent peroxynitrite.

The data presented in Figures 5and 6 suggest an involvement of ROIs, including those derived

from NO in ammonia-induced protein tyrosine phosphorylation and dual phosphorylation of the

MAP-kinases Erk-1/Erk-2 and p38

MAPK

. The role of ROIs as signal metabolites is wellestablished, and peroxynitrite was shown recently to trigger tyrosine phosphorylation-dependentsignal transduction (39). Further studies are required to identify the primary signaling elements

upstream of the MAP-kinases Erk-1/Erk-2 and p38MAPK

targeted by peroxynitrite and other ROIs

in astrocytes.

NH4Cl induces cell swelling in cultured astrocytes, which is abrogated by the glutamine

synthetase inhibitor MSO (16, 40). Protein tyrosine nitrationby ammonia was also sensitive toMSO (Fig. 1B) and the pattern of protein tyrosine nitration induced by ammonia was roughly

mimicked by hypoosmotic astrocyte swelling (Fig. 1A), which suggests some contribution of

glutamine synthesis and cell swelling. Ammonia treatment ofastrocytes for 3 h is sufficient for

glutamine accumulation and volume regulatory osmolyte depletion, which are both counteracted by MSO (16). The release of excitatory amino acids contributes to volume regulation ofhypoosmotically swollen astrocytes (31), and it seems conceivable that an MSO-sensitive

glutamate release counteracts ammonia-induced swelling, thereby promoting NMDA receptor

signaling.

In astrocytes, tyrosine nitration may result in a modification of enzyme activities and a

predisposition of proteins for proteasomal degradation (22). Tyrosine nitration of glutamine

synthetase is associated with its inactivation (Fig. 7). It seems well conceivable that the decreasein glutamine synthetase activity found in the cortex of PCA rats (41) is caused by tyrosine

nitration and/or oxidative modifications ofthe enzyme. Inactivation of glutamine synthetase may

reduce glutamine accumulation and thereby attenuate astrocyte swelling and possibly ammoniatoxicity. Inactivation of glutamine synthetase by tyrosine nitration might reduce glutamine

accumulation, astrocyte swelling, Ca2+

influx, NO, and ROI production, and thereby might

attenuate ammonia toxicity.

Tyrosine nitration of GAPDH was shown recently to inactivate the enzyme, when treated with

authentic peroxynitrite (42), which suggests that ammonia-induced GAPDH nitration (Fig. 7B)

could contribute to compromised energy metabolism found in HE (2). Ammonia is known to


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increase ligand binding to the PBR and to augment PBR-mediated neurosteroid synthesis in

cultured astrocytes and hepatic encephalopathy (2). It remains to be established to what extentprotein tyrosine nitration (Fig. 6B) contributes to altered PBR function.

SOD/cat added to cultured cells are not expected to permeate the plasmamembrane freely, which

leads to the suggestion that their effectiveness to block ammonia-induced tyrosine nitration (Fig.1B) is attributable to interception of reactive intermediates released by the astrocytes. The

astroglial release of ROIs, NO, and peroxynitrite might alter the function of astrocytes and

neurons, which are in close vicinity to the astrocytes. Coculture of iNOS expressing astrocyteswith neurons led to increased sensitivity of the neurons to glutamate, impairment of the

respiratory chain, and ATP depletion in neurons in a reversible fashion (43). Therefore, it seems

possible that astroglial release of reactive nitrogen intermediates leads to the ammonia-inducedreversible changes in neurotransmission, which are a feature of HE in acute and chronic liver

disease.

The data in this study show that ammonia-induced protein tyrosine nitration occurs not only in

cultured astrocytes in vitro but that it is also found in in vivo models of acute and chronichyperammonemia (Figs. 8 and 9). The involvement of astrocytes in these in vivo models is

demonstrated by the strong tyrosine nitration of glutamine synthetase (Figs. 7and 8), which isalmost specific for astrocytes (5). Following acute ammonia intoxication, tyrosine nitration was

most pronounced in the perivascular area and was colocalized with GFAP-positive cells (Fig. 9

EK). Astrocytes are known to be important constituents of the blood-brain barrier, andpermeability changes of the latter are a hallmark of HE in acute and chronic liver disease (2).

Thus, one is tempted to speculate that protein nitration of perivascular astrocytes might affect

transastrocytic substrate transport, which would correspond to altered blood-brain barrierpermeability. In clinical settings, HE is known to be precipitated by a variety of factors, such as

bleeding, high protein intake, electrolyte disturbances, sepsis, and infections (2, 3, 30). In viewof this, it is important to note that not only ammonia, but also hyponatremia and inflammatory

cytokines were shown here to increase protein tyrosine nitration. The question arises to what

extent clinical symptoms of HE can be attributed to ammonia-induced protein nitration. Clearly,this cannot be answered. However, the findings that MSO and NMDA receptor antagonists

abolish ammonia-induced protein nitration (this paper), on the one hand, and were reported, on

the other, to improve clinical signs of ammonia toxicity in experimental animals (e.g., refs 7, 10,

44)] argue for a role of protein nitration in the pathogenesis of HE symptoms. Further studies arerequired to settle this issue.

ACKNOWLEDGMENTS

We gratefully acknowledge fruitful discussions with Ralf Kubitz. Also the technical assistance of

Nicole Beyen is greatly acknowledged. This study was supported by DeutscheForschungsgemeinschaft through Sonderforschungsbereich 575 Experimentelle Hepatologie

(Dsseldorf).

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Received November 28, 2001; revised January 30, 2002.


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Fig. 1

Figure 1: Ammonia-induced protein tyrosine nitration in cultured rat astrocytes. Protein tyrosine nitration wasdetected by Western Blot analysis with an antibody raised against 3-nitrotyrosine. Representatives of at least three

independent experiments are shown. Apparent differences in tyrosine nitration patterns between the groups shown result

from Western blot analysis of different gel electrophoretic separations. A) Protein tyrosine nitration. Astrocytes were

exposed to NH4Cl (0.15 mmol/L as indicated, lines 27) for 24 h. Alternatively, astrocytes were exposed to

hypoosmolarity (205 mosmol/L, 6 h, lane 12), NH4Cl (1 mmol/L, 6 h, lane 13), NMDA (1 mmol/L, 6 h, lane 14), SNP

(100 mol/L, 1 h, lane 15), SIN-1 (1 mmol/L, 1 h, lane 16), or LPS (1 g/ml) + IFN (100U/ml,

24 h, lane 17). Controls:astrocytes remained untreated for 6 (lanes 10 and 18) or 24 h (lane 1). ypoosmolarity was adjusted by dilution of the

medium with the appropriate volume of NaCl-free medium. The normoosmotic control condition (305 mosmol/L) was

achieved by addition of an identical volume of normoosmotic medium (lane 11). Specificity of 3-nitrotyrosine detection

was demonstrated by a reduced immunoreactivity when blots were incubated with the anti-3-nitrotyrosine antibody in

presence of 3-nitrotyrosine (lanes 8 and 9). B) Pharmacological characterization of ammonia-induced protein tyrosine

nitration. Astrocytes remained untreated (controls, lines 1and 5) or were exposed to NH4Cl (1 mmol/L, 6 h) without

further pretreatment (lines 2 and 6) or after a 30-min preincubation with L-NMMA (100 mol/l, lane 7), 1400 W

(2 mol/L, lane 8), BAPTA-AM (10 mol/L, lane 4), W13 (50 mol/L, lane 3), SOD/cat (6000 and 8000 units,

respecticvely, lane 9), uric acid (200 mol/L, lane 14.), MSO (3 mmol/L,lane 13), MK-801 (100 mol/L, 100 nmol/L or

50 nmol/L as indicated, lanes 10, 11, 12 ).


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Fig. 2

Figure 2. NH4Cl-induced 3-nitrotyrosine and iNOS immunoreactivity in cultured rat astrocytes.Immunoreactivities of 3-nitrotyrosine (NO2Tyr), iNOS and GFAP were visualized by confocal laser scanning microscopy

Cultured rat astrocytes were maintained in absence of NH4Cl (control; A,C,E,G) or were exposed to 1 mmol/L NH4Cl for

24 h (B,D,F,H). Green: GFAP immunoreactivity (A,B,E,F,G,H), red: NO2Tyr immunoreactivity (C,D,E,F) or iNOS

immunoreactivity (G, H), yellow: colocalization of NO2Tyr and GFAP (F, Q) or iNOS and GFAP (H), respectively.

Representatives of 3 independent experiments are shown. NO2Tyr immunoreactivity was blocked in presence of 10

mmol/L nitrotyrosine (not shown).


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Fig. 3

Figure 3. NH4Cl-induced iNOS expression and IB degradation in cultured rat astrocytes. iNOS and IBexpression levels were determined by Western blot analysis. Representatives of at least three independent experiments are

shown. A) Time course of NH4Cl-induced iNOS expression. Astrocytes were exposed to 5 mmol/L NH4Cl for 6, 24, or 72

h as indicated. Alternatively, cells were treated with LPS (1 g/ml) + IFN-(100U/ml)for 24 h. Controls: cells remained

without further treatment. B) Dose dependence of NH4Cl-induced iNOS expression. Astroytes were exposed to the

indicated concentrations of NH4Cl for 24 h. C) Pharmacological characterization of NH4Cl-induced iNOS expression and

I B degradation. Astrocytes were exposed to 5 mmol/L NH4Cl for 6 h without further treatment or in presence of PDTC

(100 mol/L), MG-132 (30 mol/L), MK-801 (100 mol/L), or BAPTA-AM (50 mol/L), respectively.


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Fig. 4

Figure 4. NH4Cl-induced increase of cytosolic Ca2+ concentration [Ca2+]i in cultured rat astrocytes. Representative

tracings from at least 10 experiments are shown. Astrocytes were exposed to 5 mmol/L NH4Cl for the indicated time

period without further treatment (A) or in presence of BAPTA-AM (50 mol/L, 30 min pretreatment) (B) or MK-801

(100 mol/L, 30 min pretreatment) (C).


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Fig. 5

Figure 5. Ammonia-induced protein tyrosine nitration and phosphorylation in cultured rat astrocytes. Proteintyrosine phosphorylation was detected by Western blot analysis with antibodies raised against phosphotyrosine,

respectively. Representatives of at least three independent experiments are shown. A) Dose dependence. Astrocytes were

exposed to NH4Cl (0.15 mmol/L as indicated, lines 17) for 24 h. Control: astrocytes remained untreated for 24 h

(lane 1). B) Pharmacological characterization of ammonia-induced protein tyrosine phosphorylation. Astrocytes remained

untreated (control, lines 1) or were exposed to NH4Cl (1 mmol/L, 6 h) without further pretreatment (lines 2) or after a 30-

min preincubation with SOD/cat (6000 and 8000 units, respecticvely, lane 3), uric acid (2 00 mol/L, lane 4), MK-801

(100 mol/L) and L-NMMA (100 mol/l, lane 6).


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Fig. 6

Figure 6. Ammonia-induced dual phosphorylation of the MAP-kinases Erk-1 and Erk-2 and p38MAPK and tyrosinenitration of Erk-1/Erk-2. AbbreviationsppErk-1, ppErk-1, ppP38: dual phosporylated Erk-1, Erk-2, and p38,

respectively. NO2Tyr-Erk-1, NO2-Tyr-Erk-2, NO2Tyr-P38: Erk-1, Erk-2, or P38 containing 3-nitrotyrosine residues.

NO2Tyr-ppErk-1, NO2Tyr-ppErk-2: dual phosphorylated Erk-1 or Erk-2 containing 3-nitrotyrosine residues. IP:

immunoprecipitation. WB: Western blot. A) NH4Cl-induced dual phosphorylation of Erk-1/Erk-2 and p38MAPK

: dose

dependence. Astrocytes were exposed to NH4Cl (0-5 mmol/L as indicated) for 24 h. Dual phosphorylation of the MAP-

kinases was detected by Western blot analysis with antibodies specifically recognizing the dual phosphorylated Erk-

1/Erk-2 or p38MAPK

, respectively. Expression levels of Erk-1/Erk-2 and p38MAPK

were monitored by using antibodies

recognizing the MAP kinases independent from their phosphorylation state. B) Pharmacological characterization of theNH4Cl-induced dual phosphorylation of Erk-1/Erk-2 and p38

MAPK. Astrocytes were exposed to 1 mmol/L NH4Cl for 6 h

without further treatment or after 30 min pretreatment with MK-801 (50 nmol/L, 100 nmol/L, or 100 mol/L,

respectively), L-NMMA (100 mol/L), uric acid (200 mol/L), or SOD/cat (6000 and 8000 units, respectively). Dual

MAP-kinase phosphorylation was monitored as described in (A). C) Ammonia-induced tyrosine nitration of Erk-1.

Astrocytes were exposed to 1 or 5 mmol/L NH4Cl for 24 h or remained without NH4Cl treatment. Proteins precipitated

with the anti-3-nitrotyrosine antibody were analyzed in Western blot for the presence of Erk-1/Erk-2 and p38MAPK.

Immunoprecipitated Erk-1/Erk-2 and p38MAPK were analyzed in Western blot for the presence of 3-nitrotyrosine residues.

For control, immunoprecipitated Erk-1/Erk-2 and p38MAPK were checked in Western blot with anti-Erk-1/Erk-2 and anti-

p38MAPK

antibody, respectively.


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Fig. 7

Figure 7. Tyrosine nitration of glutamine synthetase, glyceraldehyde-3-phosphate dehydrogenase, and theperipheral-type benzodiazepine receptor in cultured rat astrocytes. AbbreviationsGS: glutamine synthetase. PBR:

peripheral-type benzodiazepin receptor. GAPDH: glyceraldehyde-3-phosphate dehydrogenase. NO2Tyr-GS, NO2Tyr-

PBR, and NO2Tyr-GAPDH: tyrosine-nitrated GS, PBR, or GAPDH, respectively. IP: immunoprecipitation. WB: Western

blot. A) Ammonia-induced Tyr nitration of GS is associated with reduced activity. Astrocytes were exposed to NH4Cl(0.15 mmol/L as indicated) 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 from 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 within the anti-3-nitrotyrosine

imunoprecipitate from ammonia-treated astrocytes.


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Fig. 8

Figure 8. Protein tyrosine nitration in brains of portocaval anastomized and acutely ammonia-intoxicated rats.The institution of a portocaval anastomosis and acute ammonia intoxication of rats was performed as described inMaterials and Methods. The PCA rats were killed 4 weeks after PCA institution, and the acutely ammonia-intoxicated rats

were killed 1 h after NH4Ac injection. Controls: rats sham-operated and treated with NaCl, respectively. Brain protein

extracts were analyzed in Western blot for the presence of tyr-nitrated proteins. In addition, brain proteins precipitated

with the anti-3-nitrotyrosine antibody were analyzed in Western blot for the presence of tyrosine-nitrated glutamine

synthetase (NO2Tyr-GS). Neither ammonia intoxication nor the PCA institution changed significantly expression levels of

the GS. Western blot analysis of two representatives from each animal model and densitometric quantification of tyrosine

nitration of GS are given. Tyrosine nitration of GS in PCA rats was increased 6.3 0.3 fold in PCA rats and 6.0 0.2-fold

in acutely NH4Cl-intoxicated rats compared to the respective control animals (P


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Fig. 9

Figure 9. Immunohistochemical analysis of protein tyrosine nitration in brains of acutely ammonia-intoxicatedrats. NH4Cl intoxication was performed as described in Material and Methods. The animals were killed 6 h after ammonia

injection. Controls: NaCl-treated rats. DAB staining shows a NH4Ac-induced increase in NO2Tyr immunoreactivity (A

D). Confocal laserscanning microscopy (EK) unravelled that many cells in the parenchyma show dot-like NO2Tyrinnunoreactivity (red staining). GFAP (green staining)-positive astrocytic fibrils are seen closely associated with NO2Tyr

immunoreactivity along blood vessels, the latter of which contain relatively high levels of NO2Tyr within their wall.

Colocalization (yellow) of NO2Tyr and GFAP immunoreactvity may underrepresent astroglial tyrosine nitration because

only 15% of the astrocytes express GFAP constitutively and GFAP immunoreactivity due to pathological stimuli usually

increases after 4872 h.

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Ammonia induces MK-801-sensitive nitration

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