The FASEB Journal express 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 Görg*, Richard Fischer*, Paul Desjardins†, Hans J. Bidmon‡,
Andreas Herrmann§, Roger F. Butterworth†, Karl Zilles‡,║, and Dieter Häussinger*

*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

Corresponding author: D. Häussinger, Medizinische Einrichtungen der Heinrich-Heine

Universität, Klinik für Gastroenterologie, Hepatologie und Infektiologie, Moorenstrasse 5, D-
40225 Düsseldorf, Germany. E-mail: Haeussin@uni-duesseldorf.de

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, IκB 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-superoxide
dismutase/catalase treatment, and methionine-sulfoximine, which indicate the involvement of
reactive 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 in
brains 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 and
protein 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
A mmonia 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 cellular
compartment 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) and
which 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 of
reactive 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 increase
cGMP 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 activates
neuronal nitric oxide (NO) synthase (nNOS) in a Ca2+/calmodulin-dependent way (3). nNOS-
catalyzed NO synthesis would in turn stimulate formation of cGMP, which may contribute to
altered 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 extracellular
space. 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 concentrations
induce 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 source
of 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
IκB degradation. Reactive nitrogen intermediates act as signal metabolites mediating ammonia-
induced protein tyrosine phosphorylation and dual phosphorylation of the MAP-kinases Erk-
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(β-aminoethyl)-N,N,N´N´tetraacetic acid
(EGTA), ionomycin, 3-morpho-linosydnonimine (SIN-1), BAPTA-AM, NG-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 IκBα were from Santa Cruz Biotechnology (Santa Cruz, CA), and the
monoclonal antibodies raised against glutamine synthetase and iNOS, respectively, were from
Transduction Laboratories (San Diego, CA). The anti-phosho-p38MAPK antibody was from
Promega (Madison, WI). Fluorescein isothiocyanate (FITC)- and Cy3-coupled antibodies were
from 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 Dulbecco’s modified Eagle’s 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 0.9±0.1 U/L in the absence of NH4Cl and 1.0±0.3 U/L in the presence of 1mmol/L and
1.0±0.3 after a 24 h incubation in the presence of 5 mmol/L NH4Cl. Cells were serum-starved 24
h before experimental treatment.

Portacaval shunted (PCA) rats

Adult male Sprague-Dawley rats (150–175 g) were anesthetized with halothane and underwent
an end-to-side portacaval anastomosis (PCA), as described previously (17). Four weeks after
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 stored
at –70°C. On the day of protein extraction, thawed tissues were homogenized in 300 µl 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 µmol/L leupeptin, 2 µmol/L
pepstatin A and 1 µg/ml aprotinin]. These animal procedures conformed to guidelines of the
Animal Ethics Committee of Hôpital Saint-Luc and the University of Montreal.

Acute ammonium acetate intoxication of rats

We used male Wister rats (250–300 g). Ammonium acetate (NH4Ac) or, for control, NaCl (4.5
mmol/kg) was injected intraperitoneally. After NH4Ac administration, the rats experienced a
transient 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 the
middle of each hemisphere were prepared. One of these thick sections was frozen for
immunohistochemistry. The one from the other hemisphere was immersion-fixed in modified
Zambonis 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 Animal
Welfare 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 4°
and 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) were
fixed 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 (4°C, 24 h) section was
submerged again in 25% sucrose in PBS, pH 7.4, for cryoprotection, frozen in isopentane, and
further cryosectioned (50-µm sections). After several rinses in PBS endogenous peroxidases
were 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).
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 4°C 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, 0.2
mmol/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-p38MAPK (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 4°C for 2 h, the blots were
washed again and developed by using enhanced chemiluminescent detection (Amersham,
Braunschweig, Germany).

For immunoprecipitation, cell lysates containing defined protein amounts were incubated with
1.5 µg anti-NO2Tyr, anti-Erk-1/Erk-2, anti-glutamine synthetase, antiperipheral-type
benzodiazepin receptor, or anti-GAPDH antibody, respectively. The immune complexes were
collected 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 passaged
onto round 22 mm3 coverslips 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 µmol/l of the fluorescent Ca2+-chelator fura-
2 acetoxymethylester and 0.02% pluronic F-127, for 30 min at 37°C and 5% CO2. For
fluorescence recording, the coverslips were superfused continuously at a rate of 15 ml/min with
KHB at 37°C, 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 µmol/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 µl) in 96-well-plates and then added 50 µl 2% sulfanilamide in 2.5% H3PO4 and 50 µl 0.2%
naphthalene-ethylenediamine in 2.5 % H3PO4. 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 µl cell homogenate were incubated with 100 µl of reaction mixture at 37°C. The final
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 β-mercaptoethanol, 1 mmol/L
ouabain, 13 mmol/L phosphoenolpyruvate, 100 mmol/L hydroxylamine, and 5 units/ml pyruvate
kinase. We stopped the reaction after 1 h by adding 250 µl of a solution containing 500 mmol/L
FeCl3, 400 mmol/L perchloric acid, and 400 mmol/L HCl. After 30 min incubation on ice, the
protein precipitate was sedimented by centrifugation at 20,000 g and 4°C for 10 min. The
absorbance of the supernatant was measured at 492 nm. Standard curves were established by
using γ-glutamyl hydroxamate. Values were calculated as µmol of γ-glutamyl hydroxamate
formed per hour per milligram of protein at 37°C 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 Student’s t-test: P<0.05 was considered statistically significant.

RESULTS

Ammonia-induced nitration and phosphorylation

of protein tyrosine residues in cultured rat astrocytes

Exposure of cultured rat astrocytes to NH4Cl for 24 h increased 3´-nitration of protein tyrosine
residues as detected by probing Western blots with an antibody raised against 3´-nitrotyrosine
(Fig. 1A). Ammonia-induced protein nitration was detectable already at ammonia concentrations
of 250 µmol/L (lane 2), which is well in the range of brain ammonia concentrations found in
portacaval anastomized rats (21). Protein tyrosine nitration increased with increasing NH4Cl
concentrations up to 5 mmol/L (lanes 2–7); that is, the maximal concentration used. As shown in
Figure 1A, 3-nitrotyrosine (10 mmol/L) competes with binding of the anti-3-nitrotyrosine-
antibody to the blotted proteins, indicating the specificity of detection (lanes 8 and 9).

Figure 2 shows the immunoreactivity of 3´-tyrosine-nitrated proteins in cultured rat astrocytes

exposed to NH4Cl (1 mmol/L) for 24 h as visualized by confocal laser scanning microscopy.
Whereas very little tyrosine nitration (red staining) was detectable under control conditions (Fig.
2C), nitration was increased strongly in response to NH4Cl (Fig. 2D). Nitrotyrosine
immunoreactivity exhibited a punctate staining pattern. Specificity of 3-nitrotyrosine staining
was confirmed by blocking its immunoreactivity with 10 mmol/L 3-nitrotyrosine (not shown).
Astrocytes were counterstained with a GFAP antibody (green color, Fig. 2A, B). Some
heterogeneity of the astrocyte population was observed with respect to ammonia-induced protein
nitration, which became obvious by superposition of GFAP and nitrotyrosine staining (Fig. 2E,
F, yellow: colocalization of 3-nitrotyrosine and GFAP immunoreactivity).

Nitrated protein tyrosine residues are indicative for the generation of reactive nitrogen species,
which may derive from NO as metabolic precursor (22). In line with this, a nitration pattern
similar to that induced by NH4Cl was produced by the NO donors sodium nitroprusside (SNP)
and 3-morpholinosydnonimine (SIN-1), which spontaneously decomposes to yield NO and
superoxide, thereby producing peroxynitrite (Fig. 1A, lanes 15 and 16). Also the combined
treatment with interferon γ (IFN-γ) and lipopolysaccharide (LPS), which prototypically induces
NO production catalyzed by the inducible form of the NOS, led to increased levels of tyrosine-
nitrated proteins (Fig. 1A, lane 17).

Ammonia induces iNOS expression in cultured rat astrocytes

As shown in Figure 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 µmol/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 was
predominantly 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 NH4Cl
for 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 µmol NO/mg protein/24 h in presence of
NH4Cl (n=4).

NH4Cl decreased the IκB expression level in astrocytes (Fig. 3C), which is indicative of an
activation of the NFκB system by ammonia. MG-132, which inhibits proteasomal IκB
degradation (23), and pyrrolidine dithiocarbamate, which inhibits NFκB activation, inhibited
both the decrease of IκB and the increase of iNOS expression in response to ammonia (Fig. 3C).
These findings suggest the involvement of NFκB in mediating iNOS induction in response to
ammonia.

Features of ammonia-induced protein tyrosine nitration in rat astrocytes

The ammonia-induced protein tyrosine nitration in astrocytes was characterized further

pharmacologically. 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 suggest
that 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 and
nNOS 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
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, lane
14). 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 calcium
concentration [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 IκB 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 NFκB
activation.

MK-801 affects glutamate signaling by inhibiting NMDA receptor-gated Ca2+ channels (29). At
a concentration of 100 µmol/L, MK-801 largely abolished the NH4Cl-induced [Ca2+]i elevation
(Fig. 4C), IκB 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/L
MK-801 and was fully blunted at a concentration of 100 µmol/L (Fig. 1B, lanes 10–12).
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 NFκB-mediated iNOS expression. iNOS-dependent
generation 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 intracellular

glutamine 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 [Ca2+]i signal (15, 19, 31). As shown in Fig. 1A (lanes 10–13) 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 part
be 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 1–7).Similar to protein
tyrosine nitration, the ammonia-induced tyrosine phosphorylation was sensitive to treatment of
the cells with L-NMMA, MK-801, uric acid, and SOD/cat (Fig. 5B, lanes 8–13). 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.
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 acid
motif (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 of MAP-
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 nM–100 µM), L-NMMA, uric acid, and SOD/cat treatment. Thus, reactive nitrogen
intermediates 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-nitrated
proteins in Western blot for the presence of Erk-1/Erk-2 and p38MAPK. 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 dual
phosphorylated (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 blot
for 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 of rat astrocytes to NH4Cl for 24 h induced a
dose-dependent tyrosine nitration of the glutamine synthetase. In addition, NH4Cl induced a
significant 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 glutamine
synthetase 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 tyrosine
nitration of glial fibrillary acidic protein (not shown).
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 atrophy
and chronic hyperammonemia with blood ammonia concentrations of about 300 µmol/L (sham-
operated rats: 170 µmol/L) (21). Ammonia concentrations about 500 µmol/L and a glutamine
content of about 15 µmol/g were found in the cerebral cortex (21). These values compare to 160
µmol/L ammonia and 4.7 µmol/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. Immunoprecipitated
3´-tyrosine-nitrated proteins were about sixfold enriched with glutamine synthetase (NO2Tyr-
GS) in PCA rats, compared with sham-operated control rats (Fig. 8). Because glutamine
synthetase is found almost exclusively in astrocytes (5), these findings indicate that protein
tyrosine 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 tyrosine
nitration as shown by Western blot 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 in
tyrosine nitration of astroglial proteins was visualized by confocal laser scanning microscopy
(Fig. 9 E–K): 3´-nitrotyrosine immunoreactivity was enriched along the blood vessels and
colocalized with GFAP. The findings suggest, that tyrosine nitration of astroglial proteins is
enhanced 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 (1–4). The ammonia-induced protein tyrosine
nitration 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 in
ammonia-induced signal transduction. The NMDA receptor antagonist MK-801 blocked the
ammonia-induced rise in [Ca2+]i (Fig. 4), degradation of IκB, and iNOS 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 p38MAPK (Fig. 6). In addition, the ammonia effect on protein
tyrosine nitration was mimicked largely by NMDA (Fig. 1). The mechanism of NMDA receptor
activation 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 astroglial
glutamate 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 [Ca2+]i induced by ammonia may stimulate
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-intoxicated
rats 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 Ca2+-dependent IκB
degradation (Figs. 1 and 3). Besides NO production, the ammonia-induced Ca2+ signal may
trigger the mitochondrial generation of further ROIs. NMDA receptor-mediated [Ca2+]i changes
are tightly coupled to mitochondrial Ca2+ 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 ammonia
activates astroglial NMDA receptors to generate NO and superoxide simultaneously, which may
combine to the tyrosine-nitrating agent peroxynitrite.

The data presented in Figures 5 and 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 p38MAPK. The role of ROIs as signal metabolites is well
established, and peroxynitrite was shown recently to trigger tyrosine phosphorylation-dependent
signal 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 nitration by ammonia was also sensitive to
MSO (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 of astrocytes 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 of
hypoosmotically 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 decrease
in glutamine synthetase activity found in the cortex of PCA rats (41) is caused by tyrosine
nitration and/or oxidative modifications of the enzyme. Inactivation of glutamine synthetase may
reduce glutamine accumulation and thereby attenuate astrocyte swelling and possibly ammonia
toxicity. 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
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 extent
protein 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 astrocytes
with 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-induced
reversible 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 chronic
hyperammonemia (Figs. 8 and 9). The involvement of astrocytes in these in vivo models is
demonstrated by the strong tyrosine nitration of glutamine synthetase (Figs. 7 and 8), which is
almost 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
E–K). Astrocytes are known to be important constituents of the blood-brain barrier, and
permeability 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 barrier
permeability. 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 view
of 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 are
required 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 Deutsche
Forschungsgemeinschaft through Sonderforschungsbereich 575 “Experimentelle Hepatologie”
(Düsseldorf).

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

Fig. 1

Figure 1: Ammonia-induced protein tyrosine nitration in cultured rat astrocytes. Protein tyrosine nitration was
detected 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 (0.1–5 mmol/L as indicated, lines 2–7) 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 µmol/L, 1 h, lane 15), SIN-1 (1 mmol/L, 1 h, lane 16), or LPS (1 µg/ml) + IFN (100
U/ml, 24 h, lane 17). Controls:
astrocytes remained untreated for 6 (lanes 10 and 18) or 24 h (lane 1). Ηypoosmolarity 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 µmol/l, lane 7), 1400 W
(2 µmol/L, lane 8), BAPTA-AM (10 µmol/L, lane 4), W13 (50 µmol/L, lane 3), SOD/cat (6000 and 8000 units,
respecticvely, lane 9), uric acid (200 µmol/L, lane 14.), MSO (3 mmol/L,lane 13), MK-801 (100 µmol/L, 100 nmol/L or
50 nmol/L as indicated, lanes 10, 11, 12 ).
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).
Fig. 3

Figure 3. NH4Cl-induced iNOS expression and IκB degradation in cultured rat astrocytes. iNOS and IκB
expression 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 µg/ml) + IFN-γ (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 κB degradation. Astrocytes were exposed to 5 mmol/L NH4Cl for 6 h without further treatment or in presence of PDTC
(100 µmol/L), MG-132 (30 µmol/L), MK-801 (100 µmol/L), or BAPTA-AM (50 µmol/L), respectively.
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 µmol/L, 30 min pretreatment) (B) or MK-801
(100 µmol/L, 30 min pretreatment) (C).
Fig. 5

Figure 5. Ammonia-induced protein tyrosine nitration and phosphorylation in cultured rat astrocytes. Protein
tyrosine 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 (0.1–5 mmol/L as indicated, lines 1–7) 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 (200 µmol/L, lane 4), MK-801
(100 µmol/L) and L-NMMA (100 µmol/l, lane 6).
Fig. 6

Figure 6. Ammonia-induced dual phosphorylation of the MAP-kinases Erk-1 and Erk-2 and p38MAPK and tyrosine
nitration of Erk-1/Erk-2. Abbreviations—ppErk-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 the
NH4Cl-induced dual phosphorylation of Erk-1/Erk-2 and p38MAPK. 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 µmol/L,
respectively), L-NMMA (100 µmol/L), uric acid (200 µmol/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.
Fig. 7

Figure 7. Tyrosine nitration of glutamine synthetase, glyceraldehyde-3-phosphate dehydrogenase, and the

peripheral-type benzodiazepine receptor in cultured rat astrocytes. Abbreviations—GS: 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
(0.1–5 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.
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 in
Materials 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<0.05).
Fig. 9

Figure 9. Immunohistochemical analysis of protein tyrosine nitration in brains of acutely ammonia-intoxicated

rats. 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 (E–K) unravelled that many cells in the parenchyma show dot-like NO2Tyr
innunoreactivity (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 48–72 h.