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Neurobiology of Disease 19 (2005) 150–161
Neurons exposed to ammonia reproduce the differential alteration
in nitric oxide modulation of guanylate cyclase in the cerebellum
and cortex of patients with liver cirrhosis
Regina Rodrigo, Slaven Erceg, and Vicente Felipo*
Laboratory of Neurobiology, Fundacion Valenciana de Investigaciones Biomedicas, Amadeo de Saboya, 4, 46010, Valencia, Spain
Received 3 March 2004; revised 8 November 2004; accepted 2 December 2004
Available online 16 February 2005
The activation of soluble guanylate cyclase by nitric oxide is increased
in the frontal cortex but is reduced in the cerebellum of patients who
died with liver cirrhosis. The aims of this work were to assess whether
hyperammonemia is responsible for the region-selective alterations in
guanylate cyclase modulation in liver cirrhosis and to assess whether
the alteration occurs in neurons or in astrocytes. The activation of
guanylate cyclase by nitric oxide was lower in cerebellar neurons
exposed to ammonia (1.5-fold) than in control neurons (3.3-fold). The
activation of guanylate cyclase by nitric oxide was higher in cortical
neurons exposed to ammonia (8.7-fold) than in control neurons (5.5-
fold). The activation was not affected in cerebellar or cortical
astrocytes. These findings indicate that hyperammonemia is respon-
sible for the differential alterations in the modulation of soluble
guanylate cyclase in cerebellum and cerebral cortex of cirrhotic
patients. Moreover, the alterations occur specifically in neurons and
not in astrocytes.
D 2004 Elsevier Inc. All rights reserved.
Keywords: Hyperammonemia; cGMP; Nitric oxide; Hepatic encephalo-
pathy; Soluble guanylate cyclase
Introduction
Chronic liver disease leads to altered cerebral function resulting
in hepatic encephalopathy (HE), a complex neuropsychiatric
syndrome that can present different degrees. The molecular bases
of the neurological alterations found in hepatic encephalopathy are
not clearly understood. Hyperammonemia is considered one of the
main factors responsible for the neurological alterations found in
hepatic encephalopathy (Felipo and Butterworth, 2002). Both
hyperammonemia and hepatic failure alter glutamatergic neuro-
transmission at different steps (Hermenegildo et al., 1998;
Lombardi et al., 1994; Marcaida et al., 1995; Michalak et al.,
0969-9961/$ - see front matter D 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.nbd.2004.12.001
* Corresponding author. Fax: +34 96 3601453.
E-mail address: [email protected] (V. Felipo).
Available online on ScienceDirect (www.sciencedirect.com).
1997; Minana et al., 1997; Monfort et al., 2002; Moroni et al.,
1983; Rao and Murthy, 1991). Some reviews on the effects of
hyperammonemia and liver failure on glutamatergic neurotrans-
mission have been published (Albrecht, 1998; Butterworth, 1992;
Michalak et al., 1997; Minana et al., 1997; Monfort et al., 2002). It
has been suggested that hepatic encephalopathy may be a
consequence of altered glutamatergic neurotransmission (Butter-
worth, 1992).
The activation of ionotropic (mainly NMDA) glutamate
receptors leads to increased intracellular free calcium which, after
binding to calmodulin, activates nitric oxide synthase, leading to
increased production of nitric oxide (NO), which, in turn, activates
soluble guanylate cyclase, resulting in increased formation of
cGMP. This glutamate–NO–cGMP pathway modulates important
cerebral processes and alterations in the pathway may be
responsible for some of the neurological alterations found in
hepatic encephalopathy.
The function of the glutamate–NO–cGMP pathway is altered in
the brain in vivo in rats with chronic hyperammonemia without
(Hermenegildo et al., 1998) or with liver failure (porta-caval
anastomosis) (Monfort et al., 2001), as determined by in vivo brain
microdialysis in freely moving rats. Moreover, the alteration occurs
at the level of activation of soluble guanylate cyclase by NO
(Corbalan et al., 2002; Hermenegildo et al., 1998).
We have recently shown that the activation of soluble guanylate
cyclase by NO is also altered in brains from patients who died with
liver cirrhosis (Corbalan et al., 2002). Moreover, the alteration is
different in different brain areas, the activation of guanylate cyclase
by NO is increased in the frontal cortex but is reduced in the
cerebellum of patients with liver cirrhosis (Corbalan et al., 2002).
These region-selective alterations in the modulation of soluble
guanylate cyclase by NO are faithfully reproduced in rats with
porta-caval anastomosis (Corbalan et al., 2002).
Chronic hyperammonemia without liver failure also induces a
reduction in the activation of guanylate cyclase by NO in
cerebellum (Hermenegildo et al., 1998), suggesting that hyper-
ammonemia may be responsible for the altered modulation of
guanylate cyclase by NO in brain in cirrhotic patients.
R. Rodrigo et al. / Neurobiology of Disease 19 (2005) 150–161 151
The aim of the present work was to assess whether hyper-
ammonemia is responsible for the region-selective alterations in the
modulation of guanylate cyclase by NO in liver cirrhosis.
Soluble guanylate cyclase is present both in neurons and in
astrocytes (e.g. Baltrons et al., 1995; Teunissen et al., 2000). We
also studied whether the alteration occurs mainly in neurons or in
astrocytes.
To reach these aims, we used primary cultures of neurons or of
astrocytes, from the cerebral cortex or from the cerebellum, and
studied whether exposure to 0.1 mM ammonia (the increase found
in brain of hyperammonemic rats) alters the modulation of soluble
guanylate cyclase by NO.
Materials and methods
Materials
Basal Eagle’s medium, Dulbecco’s modified Eagle’s medium,
fetal bovine serum, horse serum, and gentamycin, penicillin, and
streptomycin were from GIBCO BRL (Spain). DNase I (Deoxi-
ribonuclease I, E.C 3.1.21.1) and Dispase II were from Boehringer
Mannheim (Germany). 3-Isobutyl-1-methylxanthine (IBMX) was
from Sigma Chemical Co. (St. Louis, MO, USA). SNAP (S-
nitroso-N-acetyl-penicillamine) was from Molecular Probes
Europe (Amsterdam, Netherlands).
Primary cultures of cerebellar neurons
Primary cultures of cerebellar neurons were prepared from
cerebellum of 7–8 day-old Wistar rats as previously described
(Minana et al., 1988). The cell suspension was filtered through a
mesh with a pore size of 90 Am and resuspended in basal Eagle’s
medium supplemented with 10% heat-inactivated foetal bovine
serum, 2 mM glutamine, 100 mg/ml gentamycin, 5 mg/ml
fungizone, and 25 mM KCl. Cells were seeded onto polylysine-
coated plates (35-diameter) and, after 15 min at 378C, the medium
containing unattached cells was removed, and fresh medium was
added. To prevent the proliferation of non-neuronal cells, cytosine
arabinoside (10 AM) was added to the culture medium 20 h after
seeding. Cells were incubated at 378C in 5% CO2 atmosphere.
Primary cultures of cortical neurons
Neurons from cerebral cortex were prepared from rat fetuses at
embryonic day 14 or 15. Fetuses were decapitated and the
neocortex was dissected out. The neocortices were cut into
1 mm cubes and mechanically dissociated 10–15 times through a
pipette in a tube with 10 mL of Dulbecco’s modified Eagle’s
medium containing 10% heat-inactivated horse serum and anti-
biotics. The cell suspension was filtered through a mesh with a
pore size of 90 Am and was spun down for 5 min. The cellular
pellet was resuspended in Dulbecco’s modified Eagle’s medium
containing 10% heat-inactivated horse serum, 20 U/mL penicillin,
and 20 Ag/mL streptomycin. Cells were seeded (~600.000 cells/
mL) onto polylysine-coated plates (35-diameter) and, after 24 h at
378C, the medium containing unattached cells was removed.
Astrocytes conditioned medium (see below) supplemented with N2
medium (50 Ag/ml bovine insulin, 100 Ag/ml human transferrin
human, 20 nM progesterone, 100 AM putrescine HCl, 30 nM
sodium selenite) was added. To prevent the proliferation of non-
neuronal cells, cytosine arabinoside (5 AM) was added to the
culture medium 72 h after seeding. The cells were incubated at
378C in 5% CO2 atmosphere.
Primary cultures of cortical or cerebellar astrocytes
Primary cultures of astrocytes were prepared from newborn
(cortex) or 7-day-old (cerebellum) Wistar rats. Tissue was
mechanically dissociated by gently shaking for 1 min and
successive passages through nylon cloths having 135- and 90-Ammesh openings. Cells were resuspended in Dulbecco’s modified
Eagle’s medium containing 10% heat-inactivated fetal bovine
serum, 2 mM glutamine, 20 U/mL penicillin, 20 Ag/mL strepto-
mycin, and 5 mg/mL fungizone. Cells were seeded in 35-diameter
plastic Petri dishes at 20 mL/cortex or cerebellum, and maintained
at 378C in 5% CO2 atmosphere. Medium was changed once a
week, and cells were used after 14 days in culture.
Preparation of the medium conditioned by astrocytes
The primary cultures of cortical astrocytes were prepared from
newborn Wistar rats as described above. Cells were seeded in
25 cm2 plastic flask at 30 mL/cortex and maintained at 378C in 5%
CO2 atmosphere. The medium was changed once a week. After 2
weeks, the culture medium was removed, and a serum-free
medium with antibiotics was added and maintained for 48 h. This
conditioned medium was then taken, filtered, and kept at 48C.Fresh serum-free medium was added and maintained for 48 h, then
was filtered and kept at 48C. Cells were allowed to recover for 4 or
5 days in a medium with bovine serum and then two new cycles of
serum-free medium (addition-recovery and filtration) were carried
out. The filtered conditioned serum-free medium was added to
primary culture of cortical neurons as described above.
Exposure of neurons or astrocytes to ammonia
100 AM NH4Cl was added to the culture medium 24 h after
seeding the neurons or astrocytes as previously reported (Herme-
negildo et al., 1998). This concentration was chosen to reproduce
the increase in brain ammonia in hyperammonemic rats (Azorin et
al., 1989). Exposure to this low level of ammonia does not affect
the viability of neurons or astrocytes in culture nor the cell
composition or glial contamination of neuronal cultures.
Activation of soluble guanylate cyclase by nitric oxide in
cultured cells
Primary cultures of astrocytes and of cerebellar neurons were
used 14 days after seeding. Primary cultures of cortical neurons
were used 10 days after seeding.
To activate soluble guanylate cyclase, the nitric oxide-generat-
ing agent S-nitroso-N-acetyl-penicillamine (SNAP) was used and
the content of cGMP in the presence or the absence of SNAP was
measured. To prevent degradation of cGMP by phosphodiesterases,
the general phosphodiesterases inhibitor 3-isobutylmethylxanthine
was added to some samples before the addition of SNAP. Two series
of experiments were carried out, one series with addition of 3-
isobutylmethylxanthine and another series without the addition of
the inhibitor. The results of both series are presented separately.
Neurons or astrocytes in culture were washed three times with
prewarmed Locke’s solution without magnesium (in mM: NaCl,
R. Rodrigo et al. / Neurobiology of Disease 19 (2005) 150–161152
154; KCl, 5.6; NaHCO3, 3.6; CaCl2, 2.3; HEPES, 5; glucose, 5.6;
pH 7.4). The assays of SNAP-induced formation of cGMP were
carried out in the same solution. When added, 300 AM 3-
isobutylmethylxanthine was added 3 min before the addition of
SNAP. Treatment with SNAP 0.1 mM was for 5 min at 378C.Additional experiments were carried out with different SNAP
concentrations (10, 100, and 1000 AM) and different incubation
periods (1, 2, and 5 min) for cerebellar neurons.
cGMP was determined using the BIOTRAK cGMP enzyme
immunoassay kit from Amersham. After treatments, the cells were
resuspended in 250 Al of the kit assay buffer containing 4 mM
EDTA and disrupted by sonication. Samples were centrifuged
(14,000 � g, 5 min) and cGMP was measured in the supernatant.
Pellets were resuspended in 250 Al of 0.25 M NaOH, and protein
was measured by the bichinconic acid procedure. Values are given
as the mean F standard deviations of at least eight different
cultures. For each experiment, samples were measured in triplicate.
Measurement of soluble guanylate cyclase activity in cell extracts
from cerebellar or cortical neurons
Soluble guanylate cyclase activity was measured essentially as
described by Prabhakar et al. (1997). Primary cultures of cerebellar
neurons were used 14 days after seeding and those from cortical
neurons 10 days after seeding. Neurons in culture were washed
three times with prewarmed Locke’s solution without magnesium
(in mM: NaCl, 154; KCl, 5.6; NaHCO3, 3.6; CaCl2, 2.3; HEPES,
5; Glucose, 5.6; pH 7.4). Cells were homogenized in 750 AL of
ice-cold buffer containing 50 mM HEPES, pH 7.4, 1 mM EDTA
(ethylene diamine tetraacetic acid), 50% glycerol, 250 mM
sucrose, 1 mM dithiothreitol, and 0.01% bacitracin and were
sonicated for 20 s. Homogenates were centrifuged for 45 min at
100,000 � g and 48C. The activity of the soluble guanylate cyclasewas assayed in the supernatants. 50 Al of the supernatant was
mixed with an equal volume of buffer containing 50 mM HEPES,
pH 7.4, with 2 mM isobutylmethylxanthine (Germany, Sigma),
4 mM GTP, 60 mM phosphocreatine, 800 Ag/ml creatine kinase
(185 U/mg), 1 mg/ml BSA, and 8 mM MgCl2. When the effect of
SNAP (S-nitroso-N-acetyl-penicillamine, Molecular Probes,
Eugene, OR, USA) was tested, this compound was added (100
AM) to the buffer. Samples were incubated at 378C. 1 mM 3-
isobutylmethylxanthine was added 3 min before the addition of
SNAP. Treatment with SNAP 0.1 mM was for 5 min. Triplicate
50 Al aliquots were taken at 0 and 5 min, boiled for 5 min and kept
on ice for at least 10 min. After centrifugation at 12,000 � g for 10
min at 48C, the supernatant was collected and cGMP was
measured using the BIOTRAK cGMP enzyme immunoassay kit
from Amersham Pharmacia, UK. The activity of sGC is expressed
as picomols of cGMP synthesized per minute per milligram of
protein. Protein was determined by the bicinchonic acid method
(Pierce, Rockford, IL, USA). For each experiment, the samples
were measured in triplicate.
Ammonia determination
Ammonia concentration in culture medium was measured
essentially as previously described (Hermenegildo et al., 2000).
Medium samples were desproteinized with equal volume of ice-
cold 6% trichloroacetic acid, and kept on ice for 15 min. After
centrifugation at 12,000 � g, for 10 min at 48C, the supernatants
were collected, neutralized with 2% KHCO3 (pH ~7.0), and
centrifuged at 12,000 � g, for 10 min at 48C. Neutralized
supernatants were used to measure ammonia. Samples contained,
in a final volume of 100 AL, 30 AL, or 60 AL of sample, 30 mmol/L
a-ketoglutarate and 0.5 mmol/L nicotinamide adenine dinucleotide
(reduced form) in potassium phosphate buffer (8.0). After record-
ing initial fluorescence, reactions were started by the addition of
5 Ag of glutamate deshydrogenase (Boehringer Mannheim,
Germany) and followed in the Fluoroskan for at least 70 min.
Standards containing up to 20 nmol of ammonia were included in
each assay. The assay was performed using Costar 96-well UV
plates (cat. No. 3635; Corning Costar Corporation, Cambridge,
MA).
Effect of ammonia on NO release and half-life
NO release from SNAP in the presence of different ammonia
concentrations was assayed by measuring the stable end product of
NO, NO2� according to the previously described Griess reaction
adapted from Chao et al. (1996). Briefly, different SNAP
concentrations (10 and 100 AM) mixed with a fixed ammonia
concentration (100 AM) or different ammonia concentrations (0,
10, 100, and 1000 AM) were mixed with a fixed SNAP
concentration (100 AM). These samples were mixed in wells of a
96-well plate with 100 AL of the Griess reagent, made of a 1/1
mixture of 1% (w/v) sulfanilamide in 30% acetic acid and 0.5% (w/
v) of N-1-naphtylethyllenediamine dihydrochloride in 60% acetic
acid. This compound reacts with nitrite to form a chromophore that
was measured spectrophotometrically (580 nm) using a Multiskan
reader. The concentration of nitrite was calculated using calibration
with known concentrations of NaNO2�.
Analysis of soluble guanylate cyclase subunit and glutamine
synthetase content in cultured cells from cerebellum or cerebral
cortex
The primary cultures of astrocytes and of cerebellar neurons
were used 13 days after seeding. Primary cultures of cortical
neurons were used 9 days after seeding. Cells were homogenized
in medium containing 66 mM Tris–HCl (pH 7.4), 1% SDS, 1 mM
EGTA (ethylene glycol-bis (h-aminoethyl ether) tetraacetic acid),
10% glycerol, 1 mM sodium orthovanadate, leupeptin 20 Ag/mL,
4 Ag/mL aprotinin, and 1 mM sodium fluoride. The homogenates
were boiled for 5 min, and protein was determined by the
bicinchonic acid method (Pierce, Rockford, IL, USA). Samples
were subjected to gel electrophoresis and immunoblotting as
previously described (Felipo et al., 1988) using an antibody against
soluble guanylate cyclase (1:1250; Cayman Chemical Co., Ann
Arbor, MI, USA) which recognizes both alpha 1 and beta 1
subunits; an antibody against alpha 2 subunit (Bamberger et al.,
2001) (1:500) and an antibody against glutamine synthetase
(1:1000; Transduction Laboratories). After development with
alkaline phosphatase color development (Sigma, Germany), the
image was captured using the GELPRINTER PLUS System from
TDI (Spain), and the intensities of the spots were measured
using the software Intelligent Quantifierk Version 2.5.0 from
BioImageR.The experimental procedures have been approved by the
Institute and meet the guidelines of the European Union for
treatment and use of experimental animals. All efforts have been
made to minimize the number of animals to be used and their
suffering.
R. Rodrigo et al. / Neurobiology of Disease 19 (2005) 150–161 153
Statistical analysis
Data are expressed as mean F standard deviation. Statistical
analyses were performed using the GraphPad Prism program.
Differences in the activity of soluble guanylate cyclase were
analyzed by ANOVA and Student t test. A confidence level of 95%
was accepted as significant.
Results
The effect of exposure to ammonia on the activation of soluble
guanylate cyclase by NO in cerebellar neurons in culture is shown
in Fig. 1. Fig. 1A shows the results obtained in experiments in
which the neurons were pre-incubated with IBMX before the
addition of SNAP, and Fig. 1B, the results in experiments without
the addition of IBMX.
In the presence of IBMX, the basal content of cGMP was
similar in control neurons (9.8 F 3.8 pmol/mg protein) and in
neurons exposed to ammonia (9.7 F 3.8 pmol/mg protein).
Following the addition of SNAP, cGMP increased to 33 F16 pmol/mg protein in control neurons and the increase was
Fig. 1. Exposure of cerebellar neurons to ammonia reduces the activation of
soluble guanylate cyclase by NO. Primary cultures of cerebellar neurons
were prepared from 7–8 day-old rats. 24 h after seeding, 100 AMammonium chloride was added to the culture medium of one half of the
plates (A); the other half were the controls (C). Cultures were used 14 days
after seeding. Neurons were treated with the NO-generating agent SNAP
(100 AM) for 5 min in the presence (Panel A) or absence (Panel B) of
IBMX (300 AM). The content of cGMP in neurons treated or not (basal)
with SNAP was measured as indicated in Materials and methods. Values are
the mean F SEM of triplicate samples from eight different cultures. Values
significantly different ( P b 0.05) from basal cGMP before the addition of
SNAP are indicated by baQ for control neurons and by bbQ for neurons
exposed to ammonia. Values that are significantly different in neurons
exposed to ammonia from control neurons are indicated by asterisks (*P b
0.05, **P b 0.01 by analysis of variance and Student t test).
Fig. 2. Exposure of cerebellar astrocytes to ammonia does not affect the
activation of soluble guanylate cyclase by NO. Primary cultures of
cerebellar astrocytes were prepared from 7–8 day-old rats. 24 h after
seeding, 100 AM ammonium chloride was added to the culture medium of
one half of the plates (A); the other half were the controls (C). Cultures
were used 14 days after seeding. Astrocytes were treated with the NO-
generating agent SNAP (100 AM) for 5 min in the presence (Panel A) or
absence (Panel B) of IBMX (300 AM). The content of cGMP in astrocytes
treated or not (basal) with SNAP was measured as indicated in Materials
and methods. Values are the mean F SEM of triplicate samples from eight
different cultures. Values significantly different ( P b 0.05) from basal
cGMP before the addition of SNAP are indicated by baQ for control
astrocytes and by bbQ for astrocytes exposed to ammonia.
significantly lower (P = 0.025) in neurons exposed to ammonia,
reaching only 16 F 9 pmol/mg protein (Fig. 1A). This indicates
that exposure to ammonia reduces significantly the activation of
soluble guanylate cyclase by NO in cerebellar neurons in culture.
The activation of the enzyme by NO (NO-induced increase in
cGMP) was 3.4 F 1.3-fold in control neurons but only 1.6 F 0.7-
fold (P = 0.004) in cerebellar neurons exposed to ammonia.
When the experiments were carried out in the absence of
IBMX, the results obtained were different. The basal content of
cGMP was similar in control neurons (3.7 F 1.7 pmol/mg protein)
and in neurons exposed to ammonia (3.3 F 1.7 pmol/mg protein).
Following the addition of SNAP, cGMP increased to 6.8 F 3.8
pmol/mg protein in control neurons and the increase was similar in
neurons exposed to ammonia, reaching 6.1 F 2.2 pmol/mg protein
(Fig. 1B). Under these conditions, in the absence of IBMX, the
increase in cGMP induced by SNAP was similar in control neurons
and in those exposed to ammonia.
In contrast to the effects found in cerebellar neurons in
culture, exposure of cerebellar astrocytes in culture did not affect
the modulation of soluble guanylate cyclase by NO (Fig. 2).
Either in the presence or the absence of added IBMX, the
response was similar in control astrocytes and in those exposed to
ammonia. In the presence of IBMX, the basal content of cGMP
was similar in control astrocytes (1.2 F 0.5 pmol/mg protein) and
R. Rodrigo et al. / Neurobiology of Disease 19 (2005) 150–161154
in astrocytes exposed to ammonia (1.0 F 0.4 pmol/mg protein).
Following the addition of SNAP, cGMP increased to 5.9 F 1.8
pmol/mg protein in control astrocytes and to 4.6 F 1.4 pmol/mg
protein in astrocytes exposed to ammonia. This indicates that
exposure to ammonia does not affect the activation of soluble
guanylate cyclase by NO in cerebellar astrocytes in culture. The
activation of the enzyme by NO was 4.9 F 1.6-fold in control
astrocytes and 4.6 F 1.9-fold in astrocytes exposed to ammonia
(Fig. 2A).
In the absence of IBMX, the basal content of cGMP was also
similar in control astrocytes (0.9 F 0.3 pmol/mg protein) and in
astrocytes exposed to ammonia (1.0 F 0.4 pmol/mg protein).
Following the addition of SNAP, cGMP increased to 2.2 F 1.0
pmol/mg protein in control astrocytes and to 2.0 F 0.9 pmol/mg
protein in astrocytes exposed to ammonia (Fig. 2B). This indicates
that exposure to ammonia does not affect the increase in cGMP
induced by SNAP in cerebellar astrocytes, either in the presence or
the absence of IBMX.
The effect of exposure to ammonia on the activation of soluble
guanylate cyclase by NO in cortical neurons in culture is shown in
Fig. 3. Fig. 3A shows the results obtained in experiments in which
the neurons were pre-incubated with IBMX before the addition of
Fig. 3. Exposure of cortical neurons to ammonia increases the activation of
soluble guanylate cyclase by NO. Primary cultures of cortical neurons were
prepared from rat fetuses at embryonic day 15. 24 h after seeding, 100 AMammonium chloride was added to the culture medium of one half of the
plates (A); the other half were the controls (C). Cultures were used 10 days
after seeding. Neurons were treated with the NO-generating agent SNAP
(100 AM) for 5 min in the presence (Panel A) or absence (Panel B) of IBMX
(300 AM). The content of cGMP in neurons treated or not (basal) with SNAP
was measured as indicated in Materials and methods. Values are the meanFSEM of triplicate samples from five different cultures. Values significantly
different ( P b 0.05) from basal cGMP before the addition of SNAP are
indicated by baQ for control neurons and by bbQ for neurons exposed to
ammonia. Values that are significantly different in neurons exposed to
ammonia from control neurons are indicated by asterisks (*P b 0.05, **P b
0.01 by analysis of variance and Student t test) and values.
SNAP, and Fig. 3B, the results in experiments without the addition
of IBMX.
In the presence of IBMX, the basal content of cGMP was
similar in control neurons (3.4 F 0.7 pmol/mg protein) and in
neurons exposed to ammonia (3.0 F 0.6 pmol/mg protein).
Exposure to ammonia significantly increased the activation of
soluble guanylate cyclase by NO in cortical neurons. Following the
addition of SNAP, cGMP increased to 19 F 4 pmol/mg protein in
control neurons and to 26 F 7 pmol/mg protein (P = 0.03) in
neurons exposed to ammonia. This indicates that exposure to
ammonia increases significantly the activation of soluble guanylate
cyclase by NO in cortical neurons in culture. The activation of the
enzyme by NO was 5.5 F 1.7-fold in control neurons and
increased significantly (P = 0.01) to 8.7 F 2.5-fold in cortical
neurons exposed to ammonia (Fig. 3A).
When the experiments were carried out in the absence of
IBMX, the results obtained were different. The basal content of
cGMP was similar in control neurons (3.9 F 1.2 pmol/mg protein)
and in neurons exposed to ammonia (3.2 F 1.0 pmol/mg protein).
Following the addition of SNAP, cGMP was not increased in
cortical neurons in culture, remaining at 3.2F 1.0 pmol/mg protein
in control neurons and at 3.3 F 0.82 pmol/mg protein in neurons
exposed to ammonia (Fig. 3B).
Exposure of cortical astrocytes to ammonia did not affect the
modulation of soluble guanylate cyclase by NO. Either in the
presence or the absence of added IBMX, the response was similar in
control astrocytes and in those exposed to ammonia. In the presence
of IBMX, the basal content of cGMP in astrocytes exposed to
ammonia (1.5F 0.9 pmol/mg protein) was not different from that in
control astrocytes (0.9 F 0.4 pmol/mg protein). Following the
addition of SNAP, cGMP increased to 4.4F 2.1 pmol/mg protein in
control astrocytes and to 5.7 F 2.8 pmol/mg protein in astrocytes
exposed to ammonia. This indicates that exposure to ammonia does
not affect the activation of soluble guanylate cyclase by NO in
cortical astrocytes in culture. The activation of the enzyme by NO
was 4.9 F 2.7-fold in control astrocytes and 3.8 F 2.0-fold in
astrocytes exposed to ammonia (Fig. 4A).
In the absence of IBMX, the basal content of cGMP was also
similar in control astrocytes (2.2 F 1.7 pmol/mg protein) and in
astrocytes exposed to ammonia (2.1 F 1.1 pmol/mg protein).
Following the addition of SNAP, cGMP was not increased in
cortical astrocytes in culture, remaining at 1.9 F 0.40 pmol/mg
protein in control astrocytes and at 2.3 F 0.9 pmol/mg protein in
astrocytes exposed to ammonia (Fig. 4B).
This indicates that exposure to ammonia does not affect the
increase in cGMP induced by SNAP in cortical astrocytes, either in
the presence or the absence of IBMX.
The above results show that the increase in cGMP induced by
SNAP is reduced in intact cerebellar neurons in culture exposed to
ammonia. To further confirm that this is due to the reduced
activation of soluble guanylate cyclase by NO, we measured the
enzymatic activity in extracts of cultured cerebellar or cortical
neurons. Neurons were treated with ammonia for 13 or 10 days,
respectively, and then were lysed, and the in vitro activation of
soluble guanylate cyclase was assayed. As shown in Fig. 5, the
activation of soluble guanylate cyclase by NO was reduced in
extracts from cerebellar neurons and increased in extracts of cortical
neurons exposed to ammonia, thus confirming that hyperammone-
mia actually alters the sensitivity of the enzyme to NO.
As cGMP responses to SNAP in cultured neurons may depend
on the concentration of SNAP and on the incubation time, we
Fig. 4. Exposure of cortical astrocytes to ammonia does not affect the
activation of soluble guanylate cyclase by NO. Primary cultures of cortical
astrocytes were prepared from 1-day-old rats. 24 h after seeding, 100 AMammonium chloride was added to the culture medium of one half of the
plates (A); the other half were the controls (C). Cultures were used 14 days
after seeding. Astrocytes were treated with the NO-generating agent SNAP
(100 AM) for 5 min in the presence (Panel A) or absence (Panel B) of
IBMX (300 AM). The content of cGMP in astrocytes treated or not (basal)
with SNAP was measured as indicated in Materials and methods. Values are
the mean F SEM of triplicate samples from four different cultures. Values
significantly different ( P b 0.05) from basal cGMP before the addition of
SNAP are indicated by baQ for control astrocytes and by bbQ for astrocytesexposed to ammonia.
Fig. 5. Exposure of cultured neurons to ammonia also alters the modulation
of soluble guanylate cyclase by NO in neuronal extracts. Primary cultures
of cerebellar (Panel A) or cortical (Panel B) neurons were prepared and,
24 h after seeding, 100 AM ammonium chloride was added to the culture
medium of one half of the plates (A); the other half were the controls (C).
Cerebellar neurons were used 14 days after seeding and cortical neurons, 10
days after seeding. Neuronal extracts were prepared as described in
Materials and methods and treated with the NO-generating agent SNAP
(100 AM) for 5 min. The content of cGMP in neuronal extracts treated or
not (basal) with SNAP was measured as indicated in Materials and
methods. Values are the mean F SEM of triplicate samples from six
different cultures. Values that are significantly different in neurons exposed
to ammonia from the control neurons are indicated by asterisks (*P b 0.05,
by analysis of variance and Student t test).
R. Rodrigo et al. / Neurobiology of Disease 19 (2005) 150–161 155
assessed in primary cultures of cerebellar neurons whether
exposure to ammonia also impairs the activation of soluble
guanylate cyclase when the experiments are performed at lower
or higher concentrations of SNAP or at shorter incubation times.
The effects of different concentrations of SNAP on the activation
of soluble guanylate cyclase in control neurons and in those
exposed to ammonia are shown in Fig. 6A. For 10, 100, and 1000
AM SNAP, the activity of the enzyme increased to 175 F 62%,
340 F 130%, and 569 F 184% of basal, respectively, in control
neurons. The increase was significantly lower in neurons exposed
to ammonia at all SNAP concentrations, reaching 117 F 17%,
160 F 68%, and 245 F 133% of basal for 10, 100, and 1000 AMSNAP, respectively.
Fig. 6B shows the activation of soluble guanylate cyclase in
control neurons and in those exposed to ammonia incubated with
100 AM SNAP for different times. For 1, 2, and 5 min, the activity
of the enzyme increased to 357 F 33%, 410 F 170%, and 330 F75% of basal, respectively, in control neurons. The increase was
significantly lower in neurons exposed to ammonia, reaching
219 F 55%, 322 F 135%, and 150 F 40% of basal for 1, 2, and 5
min of incubation, respectively.
The above results show that the activation of soluble guanylate
cyclase by NO is reduced in cerebellar neurons exposed to
ammonia at all the incubation times and concentrations of SNAP
tested. This indicates that the effect does not depend on the
experimental conditions and reflects a real alteration of soluble
guanylate cyclase modulation by NO.
Astrocytes in primary culture express glutamine synthetase, that
can metabolize ammonia by incorporating it into glutamine, while
neurons in culture do not express this enzyme. Moreover, the
culture medium contains glutamine that is metabolized by the cells
to form glutamate and ammonia, thus increasing the ammonia
concentration in the culture medium. This may lead to different
ammonia levels in cultures of astrocytes and neurons and at
different periods of culture. The concentrations of ammonia in the
culture medium of cerebellar and cortical neurons at different days
of culture are shown in Fig. 7. The concentration of ammonia in
the medium of cerebellar neurons was significantly higher (c100
AM) in neurons treated with 100 AM ammonia than in control
neurons at all the culture times used (Fig. 7A). For cortical
neurons, the concentration of ammonia was higher in neurons
treated with ammonia at 2 days in culture (310 F 44 AM for
control neurons and 416 F 30 AM for neurons treated with
ammonia) but was not significantly different at longer culture times
(Fig. 7B). Cultured astrocytes, both from cerebellum or cerebral
cortex, are able to metabolize the ammonia added and no
difference in ammonia concentration in the medium was found
Fig. 6. Effect of different SNAP concentrations and incubation times on the
activation of soluble guanylate cyclase in cerebellar neurons in culture
exposed or not to ammonia. Primary cultures of cerebellar neurons were
prepared from 7–8 day-old rats. 24 h after seeding, 100 AM ammonium
chloride was added to the culture medium of one half of the plates; the other
half were the controls. Cultures were used 14 days after seeding. Neurons
were treated with different SNAP concentrations (10, 100, and 1000 AM)
for 5 min (Panel A) or with 100 AM SNAP for 1, 2, or 5 min (Panel B). The
content of cGMP in neurons treated or not (basal) with SNAP was
measured as indicated in Materials and methods. The activation of soluble
guanylate cyclase (sGC) was calculated dividing the content of cGMP in
the presence of SNAP by its content in the absence of SNAP. Values are the
mean F SEM of triplicate samples from five different cultures. Values that
are significantly different from control neurons are indicated by asterisks
(*P b 0.01, **P b 0.001 by analysis of variance with post-hoc Newman–
Keuls test).
R. Rodrigo et al. / Neurobiology of Disease 19 (2005) 150–161156
between astrocytes treated or not with ammonia at any time of
culture (not shown).
The difference in ammonia concentration is high at the moment
of addition of 100 AM ammonia, both in cerebellar and cortical
neurons. At this time, the concentration of ammonia is negligible in
control neurons and reaches suddenly 100 AM in neurons exposed
to ammonia. As shown in Fig. 7, the relative difference in ammonia
concentration decreases with the incubation time as ammonia
raises progressively, both in control neurons and in those exposed
to ammonia. In cerebellar neurons, those treated with ammonia are
always exposed to c100 AM more ammonia than the control
neurons are, while in cortical neurons, this difference is only
present at short incubation periods. This suggests that ammonia
could exert its effects on the modulation of soluble guanylate
cyclase due to the sudden initial rise up to 100 AM that leads to a
long-lasting alteration that remains for several days.
To shed some light on this matter, we studied the time-course of
the alterations in basal and NO-stimulated soluble guanylate
cyclase activity in cerebellar neurons. As shown in Fig. 8, NO-
stimulated sGC activity is not altered 2 h after the addition of
ammonia but is significantly reduced (42%) 4 h after the addition
of ammonia. This inhibition is maintained thereafter and remains
similar at 12 and 24 h (Fig. 8) and at 13 days (Fig. 1) after the
addition of ammonia. This indicates that ammonia needs some time
to induce the effect (more than 2 h), which is then long-lasting.
An alternative explanation for the altered modulation of soluble
guanylate cyclase by NO could be that ammonia might affect the
release of NO from SNAP or the half-life of NO. To assess this
possibility, we incubated SNAP (100 AM) with different concen-
trations of ammonia (0, 10, 100, and 1000 AM). As shown in
Fig. 9A, ammonia does not affect the release or the half-life of NO
formation from SNAP. Similar experiments were carried out with
10 or 100 AM SNAP in the presence or the absence of 100 AMammonia. As shown in Fig. 9B, ammonia did not affect NO release
nor half-life for any of the conditions tested.
This again supports that exposure to ammonia alters the
modulation of soluble guanylate cyclase by NO. To assess whether
this alteration could be associated with an alteration in the content
of soluble guanylate cyclase subunits, we determined by immuno-
blotting the effects of exposure to ammonia on the content of the
alpha 1, alpha 2, and beta 1 subunits of the enzyme in the different
cell types. The results are shown in Fig. 10 and Table 1. Exposure
to ammonia increased slightly (ca. 20%) the content of the alpha
subunits of guanylate cyclase in cerebellar neurons but not in the
other cell types analyzed (Table 1). This suggests that the altered
content of soluble guanylate cyclase subunits does not play a
significant role in the altered modulation of the enzyme by NO.
Fig. 10 and Table 1 also show the effects of exposure to
ammonia on the content of glutamine synthetase. The content of
this enzyme in cultures of cerebellar neurons was very slight; in
cortical neurons, it was present at low levels and it was abundant in
cultured astrocytes. Exposure to ammonia did not affect its content
in neuronal cultures but increased it slightly (25–31%) in cultures
of both cerebellar and cortical astrocytes.
Discussion
To discern the possible role of soluble guanylate cyclase and of
cGMP-degrading phosphodiesterases to the effects of ammonia on
the modulation by NO of cGMP levels, we carried out two series of
experiments. One series was performed in the presence of IBMX,
that inhibits most cGMP-degrading phosphodiesterases, and
another in the absence of phosphodiesterase inhibitors.
Both for cerebellar and cortical astrocytes in culture, the levels
of cGMP in the presence or the absence of IBMX and/or SNAP
were the same in control astrocytes and in those exposed to
ammonia. This indicates that hyperammonemia does not affect
cGMP levels nor its modulation by NO in astrocytes.
The results reported show that, in the presence of IBMX, the
addition of SNAP results in increased cGMP both in cerebellar and
cortical astrocytes and the increase is of the same range, ca. 5-fold
(Figs. 2A and 4A).
In contrast, in the absence of IBMX, the addition of SNAP
results in increased cGMP in cerebellar astrocytes (Fig. 2B) but not
in cortical astrocytes. This suggests that the activity of cGMP-
degrading phosphodiesterase in cortical astrocytes is very high and
is enough to keep the content of cGMP at a constant level either in
the presence of the nitric oxide generating agent SNAP at 100 AM.
Moreover, this confirms that, under the conditions used, IBMX is
inhibiting most of the phosphodiesterase activity, allowing the
Fig. 7. Time-course of ammonia concentration in the culture medium of cerebellar and cortical neurons. Primary cultures of cerebellar (Panel A) or cortical
(Panel B) neurons were prepared from 7–8 day-old rats. 24 h after seeding, 100 AM ammonium chloride was added to the culture medium of one half of the
plates; the other half were the controls. Ammonia concentration was measured in the culture medium at the indicated days after seeding as described in
Materials and methods. Values are the mean F SEM of triplicate samples from eight different cultures. Values that are significantly different from control
neurons are indicated by asterisks (*P b 0.01 by analysis of variance with post-hoc Newman–Keuls test).
R. Rodrigo et al. / Neurobiology of Disease 19 (2005) 150–161 157
accumulation of cGMP, in spite of the high phosphodiesterase
activity.
In contrast with the results in cortical astrocytes, in cerebellar
astrocytes, the activity of the phosphodiesterase is not so high, and
Fig. 8. Time-course of the ammonia-induced alterations NO-stimulated
soluble guanylate cyclase activity in cerebellar neurons. Primary cultures of
cerebellar neurons were prepared from 7–8 day-old rats. 24 h after seeding,
100 AM ammonium chloride was added to the culture medium of one half
of the plates (A); the other half were the controls (C). Cultures were used at
the indicated times after the addition of ammonia. Neurons were treated
with the NO-generating agent SNAP (100 AM) for 5 min. The content of
cGMP in neurons treated or not (basal) with SNAP was measured as
indicated in Materials and methods. Values are the mean F SEM of
triplicate samples from five different cultures. Values that are significantly
different in neurons exposed to ammonia from control neurons are indicated
by asterisks (*P b 0.05, **P b 0.01 by analysis of variance and Student t
test).
cGMP content increases in the presence of SNAP because the
formation by soluble guanylate cyclase exceeds its degradation by
the phosphodiesterase. The highest activity of phosphodiesterase in
the cerebral cortex than in the cerebellum is in agreement with
previous reports (Aoki et al., 1985; Baltrons et al., 1999; Wykes
et al., 2002).
In any case, the modulation of the levels of cGMP by NO are
not affected by hyperammonemia in cerebellar or cortical
astrocytes. In contrast, it is significantly affected, in opposite
ways, in cerebellar and cortical neurons in culture.
For the control neurons, in the presence of IBMX, SNAP
increased the content of cGMP 3.4-fold in cerebellar neurons and
5.5-fold in cortical neurons. However, in the absence of IBMX,
SNAP increased the content of cGMP only by 84% in cerebellar
neurons, and no increase in cGMPwas observed in cortical neurons.
These data suggest that the activity of cGMP-degrading
phosphodiesterase in cortical neurons is very high and is enough
to keep the content of cGMP at a constant level in the presence of
100 AM SNAP, while in cerebellar neurons, the phosphodiesterase
activity is not so high. Moreover, this confirms that, under the
conditions used, IBMX inhibits most of the phosphodiesterase
activity also in neurons, allowing the accumulation of cGMP, in
spite of the high phosphodiesterase activity.
The results reported also show that SNAP-induced accumu-
lation of cGMP is altered in cultured neurons exposed to ammonia
when assayed in the presence of IBMX but not when assayed in its
absence.
For cortical neurons, in the presence of IBMX, SNAP
induces an accumulation of cGMP in control neurons, reaching
Fig. 9. Effect of ammonia on NO release from SNAP. NO release from SNAP was assayed in presence of different ammonia concentrations (10, 100, and 1000
AM) by measuring the formation of the stable end product of NO, NO2� (Panel A). Values are the mean F SEM of triplicate samples from 5 different
experiments. NO release from different SNAP concentrations (10 and 100 AM) in the presence of 100 AM ammonia was also assessed (Panel B). Values are the
mean F SEM of triplicate samples from 5 different experiments.
R. Rodrigo et al. / Neurobiology of Disease 19 (2005) 150–161158
19 F 4 pmol/mg protein, and a significantly higher accumu-
lation in neurons exposed to ammonia, reaching 26 F 7 pmol/
mg protein. In contrast, in the absence of IBMX, the activity of
the phosphodiesterases is high enough, both in control neurons
and in those exposed to ammonia, to degrade all the cGMP
formed by soluble guanylate cyclase in response to the NO
generated by the SNAP added. This indicates that the increased
accumulation of cGMP in neurons exposed to ammonia would
be due to the increased activation of soluble guanylate cyclase
by NO.
For cerebellar neurons, in the presence of IBMX, the
accumulation of cGMP induced by SNAP was significantly lower
in neurons exposed to ammonia than in control neurons. In the
absence of IBMX, the slight accumulation of cGMP remaining was
similar in control neurons and in those exposed to ammonia. This
indicates that the reduced accumulation of cGMP in neurons
exposed to ammonia would be due to reduced activation of soluble
guanylate cyclase by NO.
Therefore, the above results indicate that exposure to ammonia
increases the activation of soluble guanylate cyclase by NO in
cortical neurons and reduces it in cerebellar neurons. Hyper-
ammonemia does not affect the activation of soluble guanylate
cyclase by NO in cerebellar or cortical astrocytes.
Modulation of soluble guanylate cyclase by NO is strongly
altered in the brain of patients who died with liver cirrhosis
(Corbalan et al., 2002). One outstanding feature of this alteration is
that the effect of liver cirrhosis on the modulation of guanylate
cyclase by NO is the opposite in cerebral cortex and cerebellum.
The activation of the enzyme by NO is increased in cerebral cortex
but is reduced in the cerebellum (Corbalan et al., 2002). The
mechanism(s) by which liver cirrhosis leads to these alterations is
not clear. The liver is the main organ responsible for the
detoxification of both endogenous and exogenous toxic com-
pounds. When the liver fails, these toxic compounds are not
properly detoxified and may reach the brain and affect cerebral
function. One of these toxic compounds is ammonia, and hyper-
ammonemia is considered one of the main factors responsible for
the neurological alterations found in hepatic encephalopathy
(Felipo and Butterworth, 2002).
The main aim of the present work was to assess whether
hyperammonemia is responsible for the alterations in the modu-
lation of soluble guanylate cyclase by NO in brain from patients
who died with liver cirrhosis. To assess this possibility, we have
tested whether ammonia per se is able to induce similar alterations
in the modulation of soluble guanylate cyclase by NO in cerebellar
and cortical cells in culture. Soluble guanylate cyclase is present
Fig. 10. Effect of exposure to ammonia on the content of alpha-1 and alpha-2 isoforms of soluble guanylate cyclase and of glutamine synthetase in cultured
cerebellar and cortical neurons and astrocytes. Cell lysates from neurons and astrocytes of cerebellum or cerebral cortex were prepared and the content of the a-
1, a-2, and h1 subunits of sGC and glutamine synthetase were analyzed by immunoblotting as indicated in Materials and methods. Some representative
immunoblottings of a-1 and a-2 isoforms using samples from different cultures are shown in Panel A. h1 content was not altered and is not presented. Some
representative immunoblottings of glutamine synthetase are shown in Panel B. The numbers above the lines indicate the amount of protein applied. The codes
under the bands indicate different control (C-n) or exposed to ammonia (A-n) samples.
R. Rodrigo et al. / Neurobiology of Disease 19 (2005) 150–161 159
both in neurons and in astrocytes. We have therefore assessed the
effects of exposure to ammonia of cerebellar or cortical neurons or
astrocytes in culture on the modulation of soluble guanylate
cyclase by NO.
The results reported show that exposure to ammonia increases
the activation of guanylate cyclase by NO in cortical neurons and
reduces its activation in cerebellar neurons. These effects repro-
Table 1
Effect of exposure to ammonia on the content of soluble guanylate cyclase
subunits and glutamine synthetase in primary cultures of neurons or
astrocytes exposed to ammonia
Protein content (percent of control)
Alpha 1
sGC
Alpha 2
sGC
Beta 1
sGC
Glutamine
synthetase
Cerebellar neurons 125 F 14* 119 F 18* 94 F 17 94 F 34
Cerebellar astrocytes 100 F 24 111 F 21 112 F 15 125 F 4*
Cortical neurons 98 F 11 107 F 14 89 F 4 83 F 22
Cortical astrocytes 85 F 21 97 F 18 86 F 11 131 F 13*
Cell lysates from neurons and astrocytes of cerebellum or cerebral cortex
treated or not with 0.1 mM ammonia were prepared at 9 days of culture for
cortical neurons and at 13 days of culture for the other cell types. The
content of the a-1, a-2, and h1 subunits of soluble guanylate cyclase and ofglutamine synthetase were analyzed by immunoblotting as indicated in
Materials and methods. The content of each protein in cells exposed to
ammonia is given and is expressed as percentage of the content in the
corresponding control cells. Values that are significantly different from
controls are indicated by asterisks.
* P b 0.05 by Student’s t test.
duce faithfully the effects found in the cerebral cortex (increased
activation) and the cerebellum (decreased activation) from patients
who died with liver cirrhosis (Corbalan et al., 2002). This supports
that hyperammonemia is responsible for the effects found in the
brain of patients who died with liver cirrhosis, both for the increase
in the activation in cerebral cortex and for the decrease in
cerebellum.
Although it has been suggested that in cerebellar neurons in
culture, a significant fraction of NO is generated by astrocytic
admixture (Malcolm et al., 1996), in our cultures of cerebellar
neurons, astrocytes are less than 3% of the cells present, and
therefore, it is unlikely that they contribute to the effects found in
cerebellar neurons in culture. Moreover, in pure cultures of
astrocytes, ammonia did not affect the modulation of guanylate
cyclase by NO. These results support therefore that exposure to
ammonia alters the modulation of soluble guanylate cyclase in
cultured neurons but not in astrocytes.
This suggests that in the brain of patients who died with liver
cirrhosis, the effects also occur mainly in neurons and not in
astrocytes.
The mechanism by which the same agent (ammonia) can lead to
opposite effects in cerebellar and cortical neurons is not clear by
now. The results obtained suggest that there may be some intrinsic
factor in the neurons, different in each cerebral area, that leads to
opposite responses to ammonia of the modulation of guanylate
cyclase by NO.
Differential modulation of other processes in the cerebellum
and cortex has been previously reported. For example, the
activation of NMDA receptor induces choline release in cortical
R. Rodrigo et al. / Neurobiology of Disease 19 (2005) 150–161160
neurons but not in cerebellar neurons in culture, and this release
has been related with excitotoxic cell death (Gasull et al., 2000).
Also, the regulatory subunits of cAMP-dependent protein kinases
are differentially expressed in cortical and cerebellar neurons,
leading to a differential ability of these neurons to transmit cAMP
signals to the nucleus (Paolillo et al., 1999). Also, the role of
neuronal NO in the coupling between cerebral blood flow and local
neuronal activity is different in these cerebral areas. NO is
important in neurovascular coupling in the cerebellum, but its role
in the neocortex is less important and it seems that in this area,
other neurotransmitters are more relevant in neurovascular
coupling (Hayashi et al., 2002). It is not surprising therefore that
ammonia may affect differentially the modulation of soluble
guanylate cyclase in the cerebellum and cortex.
A possible factor that can be involved in the differential
response of the modulation of the NO-cGMP pathway in cortical
and cerebellar neurons in culture is that the former are GABAergic
and the latter glutamatergic (Westergaard et al., 1991 and
references therein). This difference may reflect upon the regulation
of the NO–cGMP pathway.
Another remarkable feature of the effects reported in the
present work is that ammonia alters the modulation of soluble
guanylate cyclase by NO in neurons but not in astrocytes. A
possible explanation for the selective effect of ammonia on
neurons but not on astrocytes is that ammonia in the brain is
mainly eliminated by glutamine synthetase, which incorporates
ammonia in glutamate to form glutamine. Glutamine synthetase is
specifically expressed in astrocytes and is not present in neurons
(Norenberg and Martinez-Hernandez, 1979). Astrocytes may
therefore eliminate ammonia and neurons cannot. This happens
both in cell cultures and in brain in vivo. It is therefore likely that
both in cultures and in brain in vivo, the neurons are actually
exposed to high ammonia levels while in astrocytes ammonia
levels may be to low to affect the modulation of soluble
guanylate cyclase by NO. In fact, ammonia levels in the culture
medium are not increased in astrocytes exposed to ammonia
compared to control astrocytes. For cerebellar neurons, ammonia
levels in the medium are higher in neurons exposed to ammonia
than in control neurons at any time of culture (Fig. 7A), while for
cortical neurons, ammonia is only increased on day 2 of culture
but not later. These data, together with those of Fig. 8 showing
that ammonia needs more than 2 h but less than 4 h to induce
alterations in modulation of guanylate cyclase by NO, indicate
that a brelatively longQ (more than 2 h), but not necessarily a
bchronicQ, exposure to high ammonia levels is enough to induce
long-lasting alterations in the modulation of guanylate cyclase.
The mechanism by which hyperammonemia leads to altered
modulation of soluble guanylate cyclase by NO remains unclear.
The existence of endogenous allosteric modulators of soluble
guanylate cyclase with the ability to alter its sensitivity to NO has
been proposed (Koesling, 1998). One of these modulators has
been partially purified (Kim and Burstyn, 1994). The activity of
soluble guanylate cyclase is also regulated by phosphorylation by
either protein kinase C (Zwiller et al., 1985), cAMP-dependent
protein kinase (Zwiller et al., 1981), or cGMP-dependent protein
kinase (Murthy, 2001), all of which result in modifications of
enzyme activity (Louis et al., 1993). It is possible that hyper-
ammonemia alters the modulation of soluble guanylate cyclase by
NO by altering the content or function of some of these
endogenous modulators or the phosphorylation of soluble
guanylate cyclase.
The results reported here show that exposure to ammonia of
primary cultures of neurons reproduces faithfully the alterations in
the modulation of soluble guanylate cyclase found in the
cerebellum and cerebral cortex from cirrhotic patients. These
neuronal cultures are therefore a good model system to study
molecular mechanisms responsible for these alterations.
The glutamate–nitric oxide–cGMP pathway modulates impor-
tant cerebral processes, including memory and learning processes
and circadian rhythms. The alteration of one of the steps of this
pathway, the activation of soluble guanylate cyclase by NO, in
hyperammonemia and liver disease may therefore contribute to
the cognitive and intellectual impairment and to the alterations
in the sleep–waking cycle found in patients with hepatic
encephalopathy.
In summary, the present work shows that hyperammonemia is
responsible for the altered modulation of soluble guanylate cyclase
by NO found in the brain of patients who died with liver cirrhosis,
both for the increase in the activation in cerebral cortex and for the
decrease in cerebellum. Moreover, these alterations occur in
neurons but not in astrocytes.
Acknowledgments
This work was supported by grants from the Ministerio de
Ciencia y Tecnologıa (SAF2002-00851) and from Ministerio de
Sanidad (Red G03-155) of Spain and by a grant from Agencia
Valenciana de Ciencia y Tecnologia. Generalitat Valenciana
(Grupos03/001) and by grant GV04B-055 of Generalitat
Valenciana.
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