hyperammonemia impairs long-term potentiation in hippocampus by altering the modulation of...

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Neurobiology of Disease 15 (2004) 1–10

Hyperammonemia impairs long-term potentiation in hippocampus by

altering the modulation of cGMP-degrading phosphodiesterase by

protein kinase G

Pilar Monfort,a Marıa-Dolores Munoz,b and Vicente Felipoa,*

aLaboratory of Neurobiology, Fundacion Valenciana de Investigaciones Biomedicas, 46010 Valencia, SpainbHospital Ramon y Cajal, Neurologıa Experimental, Madrid, Spain

Received 27 March 2003; revised 5 September 2003; accepted 24 September 2003

Hyperammonemia impairs long-term potentiation (LTP) in hippo-

campus, by an unknown mechanism. LTP in hippocampal slices

requires activation of the soluble guanylate cyclase (sGC) protein

kinase G (PKG)-cGMP-degrading phosphodiesterase pathway. The

aim of this work was to assess whether hyperammonemia impairs

LTP by impairing the tetanus-induced activation of this pathway.

The tetanus induced a rapid cGMP rise, reaching a maximum at 10

s, both in the absence or presence of ammonia. The increase in

cGMP is followed in control slices by a sustained decrease in cGMP

due to PKG-mediated activation of cGMP-degrading phosphodies-

terase, which is required for maintenance of LTP. Hyperammonemia

prevents completely tetanus-induced cGMP decrease by impairing

PKG-mediated activation of cGMP-degrading phosphodiesterase.

Addition of 8Br-cGMP to slices treated with ammonia restores

both phosphodiesterase activation and maintenance of LTP. Impair-

ment of LTP in hyperammonemia may be involved in the

impairment of the cognitive function in patients with hepatic

encephalopathy.

D 2003 Elsevier Inc. All rights reserved.

Keywords: cGMP; Soluble guanylate cyclase; Phosphosdiesterase; cGMP-

dependent protein kinase; Nitric oxide; Long-term potentiation; NMDA

receptors; Hyperammonemia; Hepatic encephalopathy

Introduction

Ammonia is a product of degradation of proteins and other

nitrogenated compounds; however, at high concentrations, am-

monia is toxic, leading to impairment of cerebral function

(Breen and Schenker, 1972; Duffiy and Plum, 1982; Plum,

1971). To prevent the toxic effects of ammonia, it is detoxified;

0969-9961/$ - see front matter D 2003 Elsevier Inc. All rights reserved.

doi:10.1016/j.nbd.2003.09.008

* Corresponding author. Laboratory of Neurobiology, Instituto de

Investigaciones Citologicas, Fundacion Valenciana de Investigaciones

Biomedicas, Amadeo de Saboya, 4, 46010 Valencia, Spain. Fax: +34-96-

3601453.

E-mail address: [email protected] (V. Felipo).

Available online on ScienceDirect (www.sciencedirect.com.)

in ureotelic animals, detoxification occurs in the liver by

incorporating ammonia into urea, a process carried out by the

urea cycle. Ammonia detoxification is impaired in liver failure

and in congenital deficiencies of the urea cycle enzymes

(Batshaw et al., 1975; Bruton et al., 1970). Chronic liver

disease is an important cause of death in Western countries.

When the liver fails or when blood is shunted past the liver

(e.g. in liver cirrhosis), ammonia is not adequately detoxified,

leading to hyperammonemia and hepatic encephalopathy with

altered brain function. The signs of hepatic encephalopathy in

patients with chronic liver disease range from alterations in the

sleep–waking cycle and motor coordination to changes in

personality, gradually developing intellectual impairment. Hepat-

ic encephalopathy may lead to coma and death. Hyperammo-

nemia is considered the main factor responsible for the

alterations in cerebral function associated with hepatic enceph-

alopathy (Clemmesen et al., 1999; Msall et al., 1984; Shih,

1978). However, the molecular mechanisms by which hyper-

ammonemia leads to impaired cerebral function have not been

clarified. Glutamatergic neurotransmission is altered in hyper-

ammonemia and liver disease (Butterworth, 1992; Minana et al.,

1997). Chronic hyperammonemia impairs NMDA receptor-asso-

ciated signal transduction in brain in vivo (Hermenegildo et al.,

1998). Long-term potentiation (LTP) is an activity-dependent

form of increased transmission efficacy at synapses and is

considered to be involved in some forms of learning and

memory (Bliss and Collingridge, 1993). Previous studies

showed that hyperammonemia impairs NMDA receptor-depen-

dent LTP in the CA1 region of rat hippocampus (Munoz et al.,

2000). Moreover, LTP in this area involves sequential activation

of soluble guanylate cyclase, cGMP-dependent protein kinase,

and cGMP-degrading phosphodiesterase, and the activation of

this pathway is necessary for proper induction and maintenance

of LTP (Monfort et al., 2002).

The aim of the present work was to assess whether the

impairment of LTP in hippocampal slices in hyperammonemia

is due to alterations in the sGC-PKG-cGMP-degrading phos-

phodiesterase pathway. It is shown that hyperammonemia

impairs PKG-mediated activation of cGMP-degrading phospho-

diesterase and that this impairment is responsible for the loss of

P. Monfort et al. / Neurobiology of Disease 15 (2004) 1–102

the maintenance of LTP in hyperammonemia, and could also be

responsible for the cognitive impairment in hyperammonemia

and hepatic encephalopathy.

Materials and methods

Long-term potentiation in hippocampal slices

Experiments were performed using transverse hippocampal

slices (400 Am) from male Wistar rats (180–240 g) as previously

described (Munoz et al., 2000). Briefly, the rat was decapitated, its

brain was rapidly removed and dropped into ice-cold standard

medium (in mM): NaCl 119, KCl 2.5, MgSO4 1.3, KH2PO4 1,

NaHCO3 26.2, glucose 11, and CaCl2 2.5 saturated with 95% O2

and 5% CO2 (pH 7.4). The hippocampi were dissected and

transversal slices were obtained using a manual chopper. The

slices were maintained in an interface holding chamber at room

temperature (21–24jC). After at least 1 h, some slices were

transferred to an open submersion-type recording chamber and

continuously perfused (flow rate 1.5–2 ml/min) with the standard

Fig. 1. Hyperammonemia impairs LTP in hippocampal slices. Restoration by 8B

Materials and methods with rat hippocampal slices exposed or not to 1 mM a

electrical pulses and at the time indicated by the arrow tetanus was applied. Black

circles those in slices exposed to ammonia (mean F SEM, n = 6), and black tria

(meanF SEM, n = 6). The fEPSP slopes after tetanus were significantly lower ( P

ammonia fEPSP, slopes were not significantly different from basal (before applica

treated with 8Br-cGMP were significantly higher ( P < 0.005) than in slices expo

fEPSP (before application of the tetanus), showing that 8Br-cGMP restores the m

shown for: (a) before tetanus application, (b) 40 min after tetanus, and (c) 175 min

time.

medium equilibrated with 95% O2 and 5% CO2. The temperature

was maintained at 31 F 1jC. Schaffer collateral–comissural

fibers were stimulated with electrical square pulses of 50–100

AA, 40 As, 0.05 Hz (Grass S88 stimulator) applied through bipolar

microelectrodes (5 mV) in a set of the fibers in the stratum

radiatum. Once the evoked potentials were stable, basal potentials

were recorded for 15 min as reference-evoked potentials. High-

frequency stimulation (HFS) was given to some slices to induce

long-term potentiation (LTP), and consisted in a tetanus of three

trains (100 Hz, 1 s) at 20-s intervals. Evoked field potentials were

recorded from CA1 stratum radiatum with low-resistance glass

micropipettes filled with Ringer solution. Recording micropipettes

were connected to field effect transistors, the outputs were filtered

between 1 Hz and 3 kHz and amplified by CyberAmp 380 (Axon

Instruments). Evoked responses were on-line digitized at 10 kHz

(Digidata 1200 Interface, Axon Instruments). The synaptic strength

was calculated by measuring the slope of initial phase of the field

EPSP (fEPSP) using a program developed by J. Bustamante. Data

were normalized by referring them to the mean values of responses

during the initial 15-min period, before tetanus application. To

assess the effects of hyperammonemia, 1 mM NH4Cl was added to

r-cGMP. Electrophysiological experiments were carried out as described in

mmonia. Schaffer collateral–commissural pathways were stimulated with

circles show the fEPSP slope in control slices (mean F SEM, n = 6), white

ngles fEPSP slopes in slices exposed to ammonia and 200 AM 8Br-cGMP

< 0.0001) in slices exposed to ammonia than in controls. In slices exposed to

tion of the tetanus) at times larger than 135 min. The fEPSP slopes in slices

sed only to ammonia and also significantly higher ( P < 0.001) than basal

aintenance of LTP. Raw evoked field potentials at the indicated times are

after tetanus. Calibration for evoked potential is 1 mM amplitude and 30-ms

Fig. 2. Paired-pulse facilitation ratio is not modified by exposure of the

slices to ammonia. Paired-pulse facilitation experiments were carried out as

described in Materials and methods. Exposure of hippocampal slices to 1

mM ammonia did not change facilitation ratio (second fEPSP slope/first

fEPSP slope) at any of the interstimulus time interval used. White circles

show fEPSP ratio in slices exposed to ammonia (n = 15), and black circles

those in control slices (n = 15).

P. Monfort et al. / Neurobiology of Disease 15 (2004) 1–10 3

the standard medium from the beginning of the experiment and

was maintained until the end. The concentrations of ammonia in

brain in animal models of hepatic encephalopathy are in the range

of 0.5–5 mM and similar levels are considered to be present in

brain of patients with hepatic encephalopathy (Clemmesen et al.,

1999; Hindfelt et al., 1977; Swain et al., 1992a,b). The concentra-

tion used in this study (1 mM) is therefore within the pathologic

range found in liver disease in humans.

To assess the effects of 8Br-cGMP, this compound was included

in the perfusion medium 30 min before application of the tetanus.

To study the prevention of the effects of 8Br-cGMP by Rp-8Br-

cGMPS, this compound was added to the perfusion medium 20

min before addition of 8Br-cGMP.

Fig. 3. Hyperammonemia alters the changes in cGMP induced by the tetanus in hip

(white points) or not (black points) to 1 mM ammonia as described in Materials

tetanus: 0, 10, and 30 s and 1, 5, 30, and 60 min. Values are expressed as percentag

the mean F SEM of 10–27 samples per point for controls and of 10–16 samples

0.001) from basal cGMP before the tetanus are indicated by ‘‘a’’ for control slice

different in slices exposed to ammonia than in control slices are indicated by ast

before application of the tetanus was 0.31 F 0.07 pmol/mg protein (n = 61) in con

pmol/mg protein, n = 54).

Paired-pulse facilitation

Paired-pulse facilitation was carried out as described by Zucker

(1989). Stimuli were given at 0.05 Hz with 25–300-ms intervals.

Paired-pulse ratio was calculated as P2/P1 (P1 = slope of the first

fEPSP and P2 = slope of the second fEPSP).

Determination of cGMP in hippocampal slices

Slices were treated as described above and were collected from

the recording chamber under basal conditions and at different times

after application of the tetanus. To assess the effects of hyper-

ammonemia, 1 mM NH4Cl was added to the standard medium,

from the beginning of the experiment and was maintained until the

slices were collected. Samples were immediately homogenized and

sonicated in the kit (see below) assay buffer containing 4 mM

EDTA. Samples were centrifuged (12,000 � g, 5 min) and cGMP

was measured in the supernatant using the BIOTRAK cGMP

enzyme-immunoassay kit from Amersham. Pellets were suspended

in 0.2 N NaOH and protein concentration was measured using the

Lowry’s procedure.

Determination of soluble guanylate cyclase activity in

hippocampal slices

Slices were treated as for determination of cGMP and collect-

ed under basal conditions or 5 or 60 min after application of the

tetanus. Slices were homogenized in ice-cold buffer containing

HEPES 50 mM, pH 7.4, EDTA 4 mM, 0.01% bacitracin, 50%

glycerol, sucrose 250 mM, and dithiothreitol 1 mM. The homo-

genates were centrifuged for 45 min at 430,000 � g (4jC) and the

supernatant was used to measure the activity of sGC. To initiate

the enzymatic reaction, samples were mixed rapidly with an equal

volume of a buffer containing 50 mM HEPES, pH 7.4, 2 mM

isobutylmethylxanthine, 4 mM GTP, 60 mM phosphocreatine, 800

Ag/ml creatine kinase (185 U/mg), 1 mg/ml bovine serum

albumin, and 8 mM MnCl2. Samples were incubated at 37jC

pocampal slices. The tetanus was applied to rat hippocampal slices exposed

and methods. Slices were taken at different times after application of the

e of basal cGMP concentration before application of the tetanus. Values are

per point for slices exposed to ammonia. Values significantly different ( P <

s and by ‘‘b’’ for slices exposed to ammonia. Values that are significantly

erisks as follows: *P < 0.05; **P < 0.0001. Basal concentration of cGMP

trol slices and was not affected in slices exposed to ammonia (0.34 F 0.09

Fig. 4. Effects of hyperammonemia on tetanus-induced increase in the

activity of soluble guanylate cyclase in hippocampal slices. (A) The tetanus

was applied to rat hippocampal slices exposed or not to 1 mM ammonia as

described in Materials and methods. Slices were taken at 0, 5, and 60 min

after application of the tetanus and the activity of soluble guanylate cyclase

was assayed. Values are the mean F SEM of eight experiments. Values

significantly different from basal activity before the tetanus are indicated by

‘‘a’’ for control slices ( P < 0.01) and by ‘‘b’’ for slices exposed to ammonia

( P < 0.005). Values that are significantly different ( P < 0.02) in slices

exposed to ammonia than in control slices are indicated by asterisks. (B)

The initial content of cGMP in the slices used for the assay of soluble

guanylate cyclase shown in A, i.e., the content of cGMP at time 0 of the

guanylate cyclase assay. Values are the mean F SEM of six to eight

experiments and are given as percentage of basal cGMP concentration

before application of the tetanus. Values significantly different from basal

cGMP before the tetanus are indicated by ‘‘a’’ for control slices ( P < 0.005)

and by ‘‘b’’ for slices exposed to ammonia ( P < 0.05). Values that are

significantly different in slices exposed to ammonia than in control slices

are indicated by asterisks as follows: *P < 0.001, **P < 0.0001.


and duplicate 40-Al aliquots were removed from the incubation

mixture at 0, 5, and 10 min, pipetted into 100 Al of 6%

trichloroacetic acid (TCA), and placed on ice. After centrifugation

at 12,000 � g for 10 min at 4jC, the supernatants were

transferred to new tubes and the TCA was extracted with diethyl

ether until pH in the aqueous phase was neutral. Then, this phase

was lyophilized and reconstituted in cGMP assay buffer. cGMP

was measured using the BIOTRAK cGMP enzyme-immunoassay

kit from Amersham.

Assay of cGMP-degrading phosphodiesterase activity

Phosphodiesterase activity was determined by measuring the

amount of inorganic phosphate released from cGMP. GMP

hydrolyzed from cGMP was converted to guanosine and

phosphate with alkaline phosphatase. Two slices were homog-

enized in 0.3 ml of ice-cold medium, consisting of 20 mM

Tris–HCl pH 7.5, 0.25 M sucrose, 0.1 mM EDTA-K+, 2 mM

MgCl2, 0.4 mM phenyl-methylsulfonyl fluoride, 5 AM leupep-

tin, and 10 mM dithiothreitol. The homogenate (100 Al) was

added to 300 Al of the incubation medium consisting of 40

mM Tris–HCl pH 7.5, 0.1 mM EDTA-K+, 2 mM MgCl2, 0.2

mM cGMP and 7 units of alkaline phosphatase. Samples were

incubated for 20 min at 37jC. The reaction was stopped by

adding 0.7 ml of a solution containing 2% ascorbic acid and

10% trichloroacetic acid in water. Samples were centrifuged for

5 min at 3000 � g. The inorganic phosphate was determined in

the supernatant by measuring the formation of the blue phos-

phomolybdous complex at 700 nm as described by Baginski

et al. (1974).

Assay of the effects of 8Br-cGMP and Rp-8Br-cGMPS on the

impairment by 1 mM ammonia of activation of cGMP-degrading

phosphodiesterase

Slices were obtained as described above, 1 mM ammonia was

present in the medium in all samples. Slices were treated in the

incubation chamber as follows: slices were collected under basal

conditions or 60 min after application of the tetanus to determine

cGMP-degrading phosphodiesterase activity as above. To assess

the effects of 8Br-cGMP (200 AM) or 8Br-cGMP (200 AM) + Rp-

8Br-cGMPS (10 AM; a selective inhibitor of cGMP-dependent

protein kinase), these drugs were included in the perfusion medi-

um. Some slices were incubated with 8Br-cGMP for 30 min before

application of the tetanus and the drug was maintained in the

perfusion medium until removal of the slices for determination of

cGMP-degrading phosphodiesterase activity. Others slices were

preincubated with Rp-8Br-cGMPS for 20 min before addition of

8Br-cGMP; the tetanus was applied 30 min later and both drugs

were maintained in the perfusion medium until removal of the

slices for determination of cGMP-degrading phosphodiesterase

activity.

Determination of GMP in hippocampal slices

Slices were treated as described above and were collected

from the recording chamber under basal conditions or at different

times after application of the tetanus. The slices were immedi-

ately frozen in liquid nitrogen and homogenized in 0.6 N HClO4.

The homogenates were left to stand on ice for 10 min and then

centrifuged for 15 min at 14,000 � g at 4jC. The pellet was usedfor determination of protein according to the method of Lowry.

The supernatant was neutralized with 30% KOH and solid

potassium carbonate. After standing for 10 min on ice, KClO4

crystals were precipitated by centrifugation as above and the

supernatant was used for determination of GMP. GMP was

measured as described by Keppler and Kaiser (1985) with the

modifications described by Montoliu et al. (1999). Fifty micro-

liters of deproteinized samples were incubated with 30 Al of

reaction mixture containing 435 mM triethanolamine pH 7.5, 13

mM Mg(CH3CO2)2, 2.1 mM phosphoenolpyruvate, 0.2 mM

NADH, 0.23 mM ATP, 0.03 mg/ml pyruvate kinase, 0.015 mg/

ml lactate dehydrogenase, and 0.012 mg/ml myokinase. Myoki-

nase was added to the assay mixture to remove AMP in the

sample before initiation of the GMP assay with guanylate kinase.

The reaction (80 Al) was started by addition of 2 milliunits of

guanylate kinase. After incubation for 30–40 min at 25jC, the

Fig. 5. Hyperammonemia prevents tetanus-induced activation of cGMP-degrading phosphodiesterase. The tetanus was applied to rat hippocampal slices

exposed or not to 1 mM ammonia as described in Materials and methods. Some slices were also treated with 8 Br-cGMP (200 AM). Slices were taken at 0, 5,

and 60 min after application of the tetanus and the activity of cGMP-degrading phosphodiesterase was assayed. Values are the mean F SEM of 12–14

experiments. Values significantly different from basal activity before the tetanus are indicated by ‘‘a’’ for control slices ( P < 0.001). In slices exposed to

ammonia, there is no significant difference from basal activity. Values that are significantly different in slices exposed to ammonia than in control slices are

indicated by asterisks as follows: *P < 0.05, **P < 0.0001. ‘‘b’’ indicates that this value is significantly different from its basal value in absence of tetanus

application ( P < 0.005).


amount of NAD+ formed was measured fluorimetrically. NADH

in excess was destroyed by adding 2 Al of 10 M HCl. Then, the

solution was made 6 M in NaOH by addition of 15.6 M NaOH

and incubated for 60 min at 37jC. The samples were diluted 10

times with distilled water and fluorescence was measured, using

an excitation wavelength of 355 nm and an emission wavelength

of 460 nm.

Statistical analysis

The data shown are the mean F SEM of the number of experi-

ments indicated in the legend to each figure. Statistical analysis was

carried out using Student’s t test, except for Fig. 1 in which

statistical analysis was performed using ANOVA and Tukey–

Kramer tests. A value of P < 0.05 was considered significant.

Results

Fig. 6. Hyperammonemia prevents tetanus-induced increase in GMP in

hippocampal slices. The tetanus was applied to rat hippocampal slices

exposed or not to 1 mM ammonia as described in Materials and methods.

Slices were taken at 0, 5, and 60 min after application of the tetanus and the

content of GMP was measured. Values are the mean F SEM of 8–11

experiments. Values significantly different from basal GMP before the

tetanus are indicated by ‘‘a’’ ( P < 0.001) or ‘‘b’’ ( P < 0.0001) for control

slices. In slices exposed to ammonia, there is no significant difference from

basal GMP. Values that are significantly different in slices exposed to

ammonia than in control slices are indicated by asterisks as follows: *P <

0.001, **P < 0.0001.

Tetanic stimulation of Schaffer collateral–commissural path-

way induced LTP in the CA1 in hippocampal slices of control rats.

The excitatory postsynaptic potential (EPSP) slope increased

significantly (P < 0.01, n = 6) to around 250% of basal and was

maintained at this level during more than 180 min (Fig. 1).

In hippocampal slices exposed to 1 mM ammonia, the appli-

cation of the tetanus also induced a potentiation of the synaptic

response, but the magnitude of the EPSP increased only by 80%

(Fig. 1). This increase was significantly lower than that in control

slices (P < 0.01, n = 6). This indicates that hyperammonemia

reduces the magnitude of the potentiation induced by the tetanus.

Moreover, exposure to ammonia also impairs the maintenance

of the potentiation. While in control slices the potentiation is

maintained for more than 3 h, in slices exposed to ammonia it is

maintained for ca. 45 min, after which it began to decrease. The

potentiation is completely lost 160 min after tetanus, when the

Fig. 7. 8Br-cGMP restores tetanus-induced activation of phosphodiesterase in slices exposed to ammonia. Prevention by inhibition of PKG. The tetanus was

applied to rat hippocampal slices exposed to 1 mM ammonia. Some slices were also treated with 8Br-cGMP (200 AM, n = 12) or with 8Br-cGMP (200 AM) +

Rp-8Br-cGMPS (10 AM, n = 11) as described in Materials and methods. Slices were taken at 0 and 60 min after application of the tetanus and the activity of

cGMP-degrading phosphodiesterase was assayed. The activities at 60 min are given as percentage of basal values. Values are the mean F SEM of 20 samples

for slices treated only with ammonia and of the number of samples indicated above for each treatment. Values that are significantly different ( P < 0.0001) from

slices treated only with ammonia are indicated by ‘‘a’’. Values that are significantly different ( P < 0.002) from samples treated with ammonia + 8Br-cGMP are

indicated as ‘‘b’’.


EPSP is not significantly different from the basal EPSP before

application of the tetanus (Fig. 1).

These results show that hyperammonemia induces two

effects: reduces the magnitude of the potentiation and impairs

its maintenance.

Fig. 8. Effects of 8Br-cGMP on fEPSP in control hippocampal slices. Schaffer col

the time indicated by the arrow 8Br-cGMP (200 AM) was added to Ringer so

significantly different between with or without 8Br cGMP. Values are the meanF S

basal value (a), and for exposure to 8Br cGMP (b). Calibrations: 1 mV and 20 m

To assess whether the effects of ammonia on LTP may be

due to an alteration in the presynaptic release machinery, we

studied the effects of exposure to ammonia on paired-pulse

facilitation. As shown in Fig. 2, paired-pulse facilitation was

not affected by ammonia at any time interval, indicating that

lateral–commissural pathways were stimulated with electrical pulses and at

lution and maintained along the experiment. The fEPSP slopes were not

EM, n = 4. Raw evoked field potentials at the indicated times are shown for

s.


the impairment of LTP by ammonia is due to postsynaptic

mechanisms.

The reduced magnitude of the LTP in slices exposed to

ammonia is not due to a modification of the basal synaptic

transmission. The magnitudes of the EPSP evoked by the control

low-frequency stimulus are the same in slices exposed to

ammonia and in control slices. EPSP slopes at 50–10 AAstimulus intensity were 0.62 F 0.01 (n = 15) and 0.61 F 0.11

(n = 15) in control slices and in slices exposed to ammonia,

respectively.

We studied the mechanism by which hyperammonemia affects

LTP. As it has been reported that LTP requires activation of

nitric-oxide-sensitive soluble guanylate cyclase and a transient

increase in cGMP (Monfort et al., 2002) and that hyperammo-

nemia reduces nitric-oxide-induced increase in cGMP in rat brain

(Hermenegildo et al., 1998), one possibility would be that the

reduced formation of cGMP would be responsible for the

impairment of LTP by ammonia. We assessed whether addition

of 8Br-cGMP (200 AM), a membrane-permeable analog of

cGMP, to slices treated with ammonia restores the potentiation

induced by the tetanus. As shown in Fig. 1, 8Br-cGMP did not

increase the magnitude of the potentiation, but completely re-

stored its maintenance.

This suggests that the impairment of LTP by hyperammonemia

would be due to an altered response in cGMP metabolism

following application of the tetanus.

Fig. 9. Rp 8Br cGMPS impairs LTP in hippocampal slices exposed to ammonia.

basal recordings, 10 AM Rp 8Br cGMPS was added to the perfusion fluid (arro

remained along the experiment. Thirty minutes later, the tetanus was applied (arrow

(mean F SEM, n = 5). Raw evoked field potentials at the indicated times are show

(b), for slices in the presence of Rp 8Br cGMPS plus 8Br cGMP (c), at 20 min af

To confirm this possibility, we studied the changes in cGMP

concentration in slices at different times after application of the

tetanus in the absence or the presence of ammonia.

As shown in Fig. 3, application of the tetanus to control slices

induced a rapid rise in cGMP that reached 132 F 4% of basal (n =

20) at 10 s. In slices exposed to ammonia, cGMP also increased to

142 F 4% of basal (n = 15) 10 s after the tetanus. These results

show that ammonia does not affect the initial transient increase in

cGMP induced by the tetanus.

In control slices, this initial transient increase in cGMP is

followed by a sustained decrease in cGMP that is significantly

lower than the basal concentration (before tetanus) at 5 min (88 F3%, n = 28, P < 0.001), 30 min (88 F 5%, n = 13, P < 0.001), and

60 min (78 F 3%, n = 15, P < 0.001) after tetanus.

In contrast (Fig. 3), in slices exposed to ammonia, the content of

cGMP is significantly higher than basal at 5 min after the tetanus

(115 F 3%, n = 10, P < 0.001) and is not different from basal at

30 min (95 F 4%, n = 11, p = 0.24) or 60 min (91 F 7, n = 16,

P = 0.21).

The concentration of cGMP in slices exposed to ammonia is

significantly higher than in control slices at any time from 5 to 60

min after tetanus. These results clearly show that hyperammonemia

alters the tetanus-induced changes in cGMP.

Tetanus-induced changes in cGMP are due to sequential

activation of soluble guanylate cyclase, cGMP-dependent protein

kinase, and cGMP-degrading phosphodiesterase (Monfort et al.,

Electrophysiological experiments were carried as in Fig. 1. After 20 min of

w); 20 min later, 200 AM of 8Br-cGMP was also added (arrow) and both

). The fEPSP slopes were not significantly different from basal at any time

n for basal ammonia slices (a), for slices in the presence of Rp 8Br cGMPS

ter tetanus (d), and at 160 min after tetanus (e). Calibrations: 1 mV, 10 ms.


2002). We studied which step of this pathway is altered in

hyperammonemia.

Application of the tetanus increased guanylate cyclase activity

by 34 F 4% in control slices and by 101 F 17% in slices exposed

to ammonia (Fig. 4A). Guanylate cyclase activity returned to basal

levels 60 min after tetanus both in control slices and in those

exposed to ammonia.

The content of cGMP in the same slices in which the activity of

guanylate cyclase was measured is shown in Fig. 4B. In control

slices, application of the tetanus leads to a significant decrease in

cGMP both at 5 min (71 F 4%, n = 7, P < 0.005) and at 60 min

(74 F 5%, n = 5, P < 0.005). In contrast, in slices exposed to

ammonia, cGMP increased significantly at 5 min (128 F 10%,

n = 8, P < 0.05) and returned to basal levels (102 F 9%, n = 6,

p = 0.7) 60 min after tetanus.

We then studied the effects of hyperammonemia on tetanus-

induced activation of the phosphodiesterase that degrades cGMP

(Fig. 5). The basal activity of phosphodiesterase is similar in

control slices [10.8 F 0.5 nmol/(mg protein min), n = 16] and in

slices exposed to ammonia [11.2 F 0.6 nmol/(mg protein min), n =

11]. In control slices, application of the tetanus increased signif-

icantly phosphodiesterase activity at 5 min (by 42F 7%, n = 9, P <

0.0001) and 60 min (by 36 F 3%, n = 15, P < 0.0001). In contrast,

in slices exposed to ammonia, the tetanus did not induce any

activation of the phosphodiesterase (Fig. 5), which remained at

basal levels both at 5 min (111F 7%, n = 11, P = 0.14) and 60 min

(90 F 3%, n = 12, P = 0.17).

The increase in the activity of cGMP-degrading phosphodi-

esterase must lead to an increase in the product of degradation

of cGMP: GMP. To further confirm the effects of the tetanus on

phosphodiesterase activity, we measured the levels of GMP

(Fig. 6). As expected, in control slices, in which phosphodies-

terase activity is increased, the content of GMP was also

significantly increased over basal levels both at 5 min (140 F3%, n = 11, P < 0.005) and 60 min (188 F 13%, n = 8, P <

0.005) after tetanus. In contrast, in slices exposed to ammonia,

GMP content remained at basal values both at 5 min (108 F13%, n = 8, P = 0.45) and 60 min (93 F 6%, n = 8, P = 0.32)

after tetanus (Fig. 6). The lack of changes in GMP in hyper-

ammonemia is in agreement with the lack of activation of

phosphodiesterase shown in Fig. 5.

The above results indicate that in hyperammonemia the tetanus

does not induce the sustained decrease in cGMP found in control

slices because hyperammonemia interferes with the tetanus-in-

duced activation of cGMP-degrading phosphodiesterase.

As shown in Fig. 1, addition of 8Br-cGMP to slices exposed

to ammonia restores LTP. We studied whether this restoration may

be due to a recovery of the tetanus-induced activation of cGMP-

degrading phosphodiesterase. As shown in Fig. 7, application of

the tetanus to slices exposed to ammonia and treated with 200 AM8Br-cGMP leads to a significant activation of the phosphodies-

terase (125 F 4%, n = 12, P = 0.0001). Treatment with 8Br-

cGMP alone (without tetanus) did not increase phosphodiesterase

activity in slices exposed to ammonia (87 F 12% of slices

without 8Br-cGMP), but increased significantly the phosphodies-

terase activity (141 F 10% of basal, n = 14, P = 0.009) (Fig. 5)

and the formation of GMP (122 F 9% of basal, n = 19, P =

0.045) in control slices. Treatment of control slices with 8Br-

cGMP also increases slightly, but not significantly, the magnitude

of the EPSP (Fig. 8).

Tetanus-induced activation of the phosphodiesterase is mediat-

ed by PKG (Monfort et al., 2002). To assess whether the restora-

tion of activation of the phosphodiesterase by 8Br-cGMP in slices

exposed to ammonia is mediated by activation of PKG, we

assessed whether an inhibitor of this kinase, Rp-8Br-cGMPS,

prevents this restoration. As shown in Fig. 7, Rp-8Br-cGMPS

completely prevented (P = 0.0017) the increase in the activity of

the phosphodiesterase that remained at 87 F 6% (n = 10). Neither

8Br-cGMP nor Rp-8Br-cGMPS plus 8Br-cGMP affected the basal

activity of phosphodiesterase. Rp-8Br-cGMPS also completely

prevented the induction of LTP (Fig. 9). This is in agreement with

previous reports showing that inhibition of PKG inhibits LTP

induction in control slices (Zhuo et al., 1994).

Discussion

Hyperammonemia impairs LTP in hippocampus (Munoz et al.,

2000), but the mechanism responsible for this impairment has not

been studied. Moreover, LTP requires sequential activation of

soluble guanylate cyclase, protein kinase G, and cGMP-degrading

phosphodiesterase, resulting in transient increase followed by

sustained decrease in cGMP that is required for proper induction

and maintenance of LTP (Monfort et al., 2002). It has been shown

that hyperammonemia impairs the modulation of soluble guanylate

cyclase by nitric oxide in cerebellum of rats in vivo and in cultured

neurons (Hermenegildo et al., 1998). It therefore seemed likely that

the impairment of LTP by hyperammonemia are due to alterations in

the modulation of cGMP levels following application of the tetanus.

As a first step to assess whether changes in cGMP response are

responsible for the impairment of LTP in hyperammonemia, we

assessed whether LTP may be restored in slices exposed to

ammonia by addition of the membrane-permeable analog of cGMP

8Br-cGMP. As shown in Fig. 1, exposure to ammonia induces two

different effects on LTP:

(1) Reduces the magnitude of the potential, which in control slices

increases up to around 250% of basal, while in slices exposed

to ammonia it increases only to around 185% of basal.

(2) Impairs the maintenance of LTP, which begins to be lost after

about 45 min after tetanus and is completely lost after about 2 h.

Treatment of the slices exposed to ammonia with 8Br-cGMP

completely restored the maintenance of LTP (Fig. 1). The magni-

tude of the potentiation remained at ca. 185% of basal for more

than 3 h. However, 8Br-cGMP did not increase the magnitude of

the potentiation in slices exposed to ammonia. These results

indicate that hyperammonemia induces two effects on LTP: de-

crease in magnitude and impairment of maintenance that are

mediated by different molecular mechanisms. The impairment in

the maintenance would be due to altered responses in cGMP

modulation following the tetanus and is completely restored by

8Br-cGMP; in contrast, the reduced magnitude of the potentiation

would be due to other mechanisms. One possibility is that the

degree of activation of NMDA receptors could be reduced in slices

exposed to 1 mM ammonia. It has been reported that exposure of

cerebellar neurons in culture to this concentration of ammonia

reduces activation of NMDA receptors (Marcaida et al., 1995). If a

similar reduction in NMDA receptors’ activation following appli-


cation of the tetanus occurs in the slices, this may lead to a

reduction in the magnitude of the potentiation.

Both in control slices and in those exposed to ammonia,

there is a rapid and transient increase in cGMP after the tetanus

that is not affected by hyperammonemia. However, in control

slices, the transient increase is followed by a significant sus-

tained decrease in cGMP, which remains below basal levels for

at least 60 min, while in slices exposed to ammonia this

decrease in cGMP is completely prevented. The impairment of

this decrease may be responsible for the loss of the maintenance

of LTP in hyperammonemia.

The initial increase is due to activation of soluble guanylate

cyclase while the subsequent decrease is due to activation of

cGMP-degrading phosphodiesterase. As shown in Fig. 4A, hyper-

ammonemia did not prevent tetanus-induced activation of soluble

guanylate cyclase. In fact, the activation of the cyclase was

significantly higher in slices exposed to ammonia (200% of basal)

than in control slices (135% of basal). The increase in the activity

of soluble guanylate cyclase induced by the tetanus involves two

different mechanisms. The tetanus induces activation of NMDA

receptors, leading to increased formation of nitric oxide, which

activates soluble guanylate cyclase. However, this activation of

soluble guanylate cyclase would not be detected in the in vitro

assay we used to measure soluble guanylate cyclase activity in

homogenates from hippocampal slices, since nitric oxide is very

reactive and is no longer present when we carried out the in vitro

determinations of activity. The increase in soluble guanylate

cyclase detected in the in vitro assay after application of the tetanus

must be due to a covalent modification of the enzyme that remains

in the in vitro assay. This modification may be a change in

phosphorylation (Ferrero et al., 2000; Louis et al., 1993; Monfort

et al., 2002; Zwiller et al., 1981, 1985).

However, the impairment of LTP in slices exposed to

ammonia is not due to the slight increase in soluble guanylate

cyclase activation but to the reduced activation of cGMP-

degrading phosphodiesterase, as reflected by the fact that cGMP

content is the same in slices exposed to ammonia and in controls

10 s after tetanus; however, 5 min after tetanus, the content of

cGMP is decreased in control slices but not in slices exposed to

ammonia.

Hyperammonemia completely prevents tetanus-induced activa-

tion of the phosphodiesterase (Fig. 5) as well as the increase in

GMP, the product of its reaction (Fig. 6), indicating that the

difference in the time course changes in cGMP (and the impair-

ment of LTP) in slices exposed to ammonia is because hyper-

ammonemia prevents tetanus-induced activation of cGMP-

degrading phosphodiesterase.

As shown in Fig. 1, 8Br-cGMP restores the maintenance of LTP

in slices exposed to ammonia. We therefore assessed whether 8Br-

cGMP is also able to restore tetanus-induced activation of the

phosphodiesterase. As shown in Fig. 7, this is the case: In slices

exposed only to ammonia, phosphodiesterase activity 60 min after

tetanus was 93% of control. However, in slices treated both with

ammonia and 8Br-cGMP, the tetanus significantly increased (P <

0.0001) phosphodiesterase activity to 125 F 4% of basal. This

shows that 8Br-cGMP restores phosphodiesterase activation by the

tetanus in hyperammonemia.

It has been shown that tetanus-induced activation of the

phosphodiesterase is mediated by protein kinase G (Monfort et

al., 2002). It is therefore likely that the lack of activation of the

phosphodiesterase could be due to impaired activation of this

enzyme by PKG and that 8Br-cGMP restores this activation. To

assess this possibility, we tested whether inhibition of PKG by Rp-

8Br-cGMPS prevents the restoration of the activation of the

phosphodiesterase by 8Br-cGMP. As shown in Fig. 7, this was

the case, indicating that 8Br-cGMP restores phosphodiesterase

activation by activating PKG.

The reason by which PKG-mediated activation of the phospho-

diesterase is impaired in hyperammonemia remains unclear. As

shown in Figs. 1 and 4, in slices exposed to ammonia, the

concentration of cGMP does not decrease below basal values

along LTP and remains higher than in control slices between 5

and 60 min after tetanus. The lack of activation is not due therefore

to reduced cGMP concentration. However, activation of the

phosphodiesterase is restored by 8Br-cGMP, a membrane-perme-

able analog of cGMP. This suggests that, in hyperammonemia,

more cGMP is needed to activate PKG and the phosphodiesterase.

One mechanism by which hyperammonemia may affect the mod-

ulation of PKG by cGMP is an alteration in the phosphorylation of

PKG. It has been shown that phosphorylation of PKG increases its

cGMP-binding affinity (Kotera et al., 2003). Hyperammonemia

may reduce the phosphorylation of PKG and its cGMP-binding

affinity, resulting in a PKG that requires more cGMP to be

activated.

PKG is phosphorylated by protein kinase C and this phosphor-

ylation increases the activity of PKG (Hou et al., 2003). It has been

shown that hyperammonemia reduces phosphorylation of protein

kinase C substrates in brain (Felipo et al., 1993; Grau et al., 1996;

Kosenko et al., 1994; Marcaida et al., 1995). A decrease in protein

kinase C-mediated phosphorylation of PKG would also result in

decreased PKG activity and requirement of higher cGMP levels to

reach enough activation of PKG to phosphorylate and activate the

phosphodiesterase.

This differential sensitivity of PKG to activation by cGMP in

slices exposed to ammonia is further supported by the fact that in

control slices, either application of the tetanus or addition of 200

AM 8Br-cGMP is enough to activate PKG and the phosphodies-

terase, while in slices exposed to ammonia neither the tetanus nor

8Br-cGMP alone are able to activate PKG. Moreover, phosphodi-

esterase and simultaneous application of both stimuli are required

to reach activation (Fig. 5).

In summary, the above results show that LTP in hyperammo-

nemia is impaired because tetanus-induced changes in cGMP are

altered. In hyperammonemia, the tetanus does not induce the

activation of cGMP-degrading phosphodiesterase nor the subse-

quent decrease in cGMP that occur in control hippocampal slices.

This would be due to impairment of PKG-medicated phosphory-

lation of the phosphodiesterase. Addition of 8Br-cGMP to slices

exposed to ammonia restores both activation of the phosphodies-

terase by the tetanus and LTP.

These results clearly point out to an alteration in signal

transduction that is responsible for the impairment of LTP in

hyperammonemia. LTP is considered the molecular and cellular

basis for some forms of learning and memory (Bliss and Colling-

ridge, 1993). LTP impairment may be responsible for the reduced

learning ability in hyperammonemic rats (Aguilar et al., 2000) and

in patients with liver disease who show cognitive impairment as

one of the manifestations of hepatic encephalopathy.


Acknowledgments

This work was supported by grants (PM99-0018, SAF2002-

00851) of the Ministerio de Ciencia y Tecnologıa and grant from

Ministerio de Sanidad (Red G03-155) of Spain and by grant from

Escuela Valenciana de Estudios para la Salud (EVES) Consellerıa

de Sanidad, Generalitat Valenciana (BM-028/2002).

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