Neuroprotective effect of guanosine against glutamate-induced cell death in rat hippocampal slices is mediated by the phosphatidylinositol-3 kinase/Akt/ glycogen synthase kinase 3β pathway activation and inducible nitric oxide synthase inhibition.

Abstract

Excitotoxicity and cell death induced by glutamate are involved in many neurodegenerative disorders. We have previously demonstrated that excitotoxicity induced by millimolar concentrations of glutamate in hippocampal slices involves apoptotic features and glutamate-induced glutamate release. Guanosine, an endogenous guanine nucleoside, prevents excitotoxicity by its ability to modulate glutamate transport. In this study, we have evaluated the neuroprotective effect of guanosine against glutamate-induced toxicity in hippocampal slices and the mechanism involved in such an effect. We have found that guanosine (100 μM) was neuroprotective against 1 mM glutamate-induced cell death through the inhibition of glutamate release induced by glutamate. Guanosine also induced the phosphorylation and, thus, activation of protein kinase B (PKB/Akt), a downstream target of phosphatidylinositol-3 kinase (PI3K), as well as phosphorylation of glycogen synthase kinase 3β, which has been reported to be inactivated by Akt after phosphorylation at Ser9. Glutamate treated hippocampal slices showed increased inducible nitric oxide synthase (iNOS) expression that was prevented by guanosine. Slices preincubated with SNAP (an NO donor), inhibited the protective effect of guanosine. LY294002 (30 μM), a PI3K inhibitor, attenuated guanosine-induced neuroprotection, guanosine prevention of glutamate release, and guanosine-induced GSK3β(Ser9) phosphorylation but not guanosine reduction of glutamate-induced iNOS expression. Taken together, the results of this study show that guanosine protects hippocampal slices by a mechanism that involves the PI3K/Akt/GSK3β(Ser9) pathway and prevention of glutamate-induced glutamate release. Furthermore, guanosine also reduces glutamate-induced iNOS by a PI3K/Akt-independent mechanism.

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Neuroprotective Effect of Guanosine
Against Glutamate-Induced Cell Death in
Rat Hippocampal Slices Is Mediated by the
Phosphatidylinositol-3 Kinase/Akt/
Glycogen Synthase Kinase 3b Pathway
Activation and Inducible Nitric Oxide
Synthase Inhibition
Simone Molz,1,2*Tharine Dal-Cim,1Josiane Budni,1M.D. Martı ´n-de-Saavedra,3
Javier Egea,3Alejandro Romero,3Laura del Barrio,3Ana L.S. Rodrigues,1
Manuela G. Lo ´pez,3and Carla I. Tasca1
1Departamento de Bioquı ´mica, Centro de Cie ˆncias Biolo ´gicas, Universidade Federal de Santa Catarina,
Trindade, Floriano ´polis, SC, Brasil
2Curso de Farma ´cia, Universidade do Contestado, Canoinhas, SC, Brasil
3Instituto Te ´ofilo Hernando, Departamento de Farmacologı ´a y Terape ´utica, Facultad de Medicina,
Universidad Auto ´noma de Madrid, Madrid, Spain
Excitotoxicity and cell death induced by glutamate are
involved in many neurodegenerative disorders. We
have previously demonstrated
induced by millimolar concentrations of glutamate in
hippocampal slices involves apoptotic features and glu-
tamate-induced glutamate release. Guanosine, an en-
dogenous guanine nucleoside, prevents excitotoxicty
by its ability to modulate glutamate transport. In this
study, we have evaluated the neuroprotective effect of
guanosine against glutamate-induced toxicity in hippo-
campal slices and the mechanism involved in such an
effect. We have found that guanosine (100 lM) was
neuroprotective against 1 mM glutamate-induced cell
death through the inhibition
induced by glutamate. Guanosine also induced the
phosphorylation and, thus, activation of protein kinase
B (PKB/Akt), a downstream target of phosphatidylinosi-
tol-3 kinase (PI3K), as well as phosphorylation of glyco-
gen synthase kinase 3b, which has been reported to
be inactivated by Akt after phosphorylation at Ser9.
Glutamate treatedhippocampal
increased inducible nitric oxide synthase (iNOS) expres-
sion that was prevented by guanosine. Slices pre-
incubated with SNAP (an NO donor), inhibited the
protective effect of guanosine. LY294002 (30 lM), a
PI3K inhibitor, attenuated guanosine-induced neuropro-
tection, guanosine prevention of glutamate release, and
guanosine-induced GSK3bSer9phosphorylation but not
guanosinereduction of
expression. Taken together, the results of this study
show that guanosine protects hippocampal slices by a
that excitotoxicity
of glutamate release
slicesshowed
glutamate-inducediNOS
mechanism
pathway and prevention of glutamate-induced gluta-
mate release. Furthermore, guanosine also reduces
glutamate-induced iNOS by a PI3K/Akt-independent
mechanism.
V V
thatinvolves thePI3K/Akt/GSK3bSer9
C 2011 Wiley-Liss, Inc.
Key words: glutamate;
Akt; iNOS
guanosine;neuroprotection;
Glutamate excitotoxicity is caused by overstimula-
tion of synaptic glutamate receptors and subsequent neu-
ronal injury (Lipton and Rosemberg, 1994). Glutamate
Contract grant sponsor: Conselho de Aperfeic ¸oamento de Pessoal de
Nı ´vel Superior (CAPES); Contract grant number: 173/2008; Contract
grant sponsor: Conselho Nacional de Desenvolvimento Cientı ´fico e Tec-
nolo ´gico (CNPq; to C.I.T. and A.L.S.R.); Contract grant sponsor:
Financiadora de Estudos e Projetos (FINEP); Contract grant number:
01.06.0842-00 (to C.I.T.); Contract grant sponsor: Spanish Ministry of
Science and Innovation; Contract grant number: SAF2009-12150; Con-
tract grant sponsor: Spanish Ministry of Education; Contract grant num-
ber: PBH2007-0004-PC; Contract grant sponsor: Spanish Ministry of
Health (Instituto de Salud Carlos III); Contract grant number: RETICS-
RD06/0026.
*Correspondence to: Simone Molz, Departamento de Bioquı ´mica, CCB,
UFSC, Trindade, 88040-900 Floriano ´polis, SC, Brasil. E-mail: molz.s
@hotmail.com
Received 11 November 2010; Revised 25 March 2011; Accepted 1
April 2011
Published
(wileyonlinelibrary.com). DOI: 10.1002/jnr.22681
online10June2011 inWiley OnlineLibrary
Journal of Neuroscience Research 89:1400–1408 (2011)
' 2011 Wiley-Liss, Inc.

Page 2

receptor overstimulation is a common event in many
neurodegenerative disorders, including ischemic stroke,
Huntington’s disease, amyotrophic lateral sclerosis, and
Alzheimer’s disease, as well as in aging (Mattson and
Magnus, 2006). We have previously reported that exci-
totoxicity in acute hippocampal slices is induced by
millimolar concentrations of glutamate and promotes a
reduction of cell viability with apoptotic features such as
cytochrome c release, caspase-3 activation, and DNA
fragmentation, via p38MAPKsignalling activation (Molz
et al., 2008a). Glutamate toxicity was also related to re-
versalactivity ofglutamate
extracellular glutamate concentration and excitotoxicty
(Molz et al., 2008b).
The inflammatory response is thought to be impor-
tant for secondary damage following glutamate exposure
(Cardenas et al., 2000; Brown and Bal-Price, 2003;
Moro et al., 2004). The inducible nitric oxide synthase
(iNOS) isoform is a mediator in inflammatory reactions
and may catalyze substantial synthesis of NO in the
injured brain, thus contributing to glutamate release by
neurons (Bal-Price and Brown, 2001).
Given that excitotoxicity is related to the major
CNS disorders but that there is lack of effective treat-
ments for such diseases, the search for alternative phar-
macological strategies against excitotoxic-induced cell
death is of great relevance. The nucleoside guanosine
(GUO) and the guanine nucleotides have been impli-
cated in neuroprotection by counteracting glutamate
excitotoxicity in vitro (Molz et al., 2005, 2008b;
Oleskovicz et al., 2008) and in vivo (Schmidt et al.,
2000, 2005).{FN0} GUO displays extracellular effects as
a cell modulator or in intercellular signaling communica-
tion, exerting trophic effects on neurons and astrocytes
(Decker et al., 2007; Rathbone et al., 2008) and modu-
lation of the glutamatergic system (for review see
Schmidt et al., 2007). Guanine nucleotides and GUO
are released from astrocytic cell cultures under basal or
toxic conditions (Ciccarelli et al., 1999, 2001). Alterna-
tively, when nucleotides such as GTP, GDP, and GMP
are released to the extracellular space and metabolized by
ectonucleotidases, extracellular GUO levels can increase
(Caciagli et al., 2000; Ciccarelli et al., 2001). Workers in
our laboratory have demonstrated that GTP is taken up
and stored into synaptic vesicles (Santos et al., 2006),
and indirect evidence indicates that GUO could be
released from synaptosomes (Fredholm and Vernet,
1979).
Astrocytic glutamate uptake is a crucial process for
the maintenance of extracellular glutamate concentra-
tions below toxic levels, thus preventing excitotoxicity
(Danbolt, 2001; Schousboe and Waagepetersen, 2006).
GUO stimulates glutamate uptake under basal conditions
in cultured astrocytes (Frizzo et al., 2001) and brain
slices (Frizzo et al., 2002). Under excitotoxic situations,
the modulatory effect of guanosine on glutamate up
take has been directly related to a neuroprotective role
(Gottfried et al., 2002; Frizzo et al., 2005; Thomazi
et al., 2008). In vitro GUO also protects rat astrocytes
transporters, increasing
from staurosporine-induced apoptosis (Di Iorio et al.,
2004) and SH-SY5Y cells from b-amyloid-induced apo-
ptosis (Pettifer et al., 2004) and MPP1-induced toxicity
(Pettifer et al., 2007) by stimulation of the phosphatidyl-
inositol-3 kinase (PI3K)/protein kinase B (Akt) cell sur-
vival pathway.
The serine/threonine protein kinase Akt is a signal-
ing kinase downstream of PI3K (Cantley, 2002). The
PI3K/Akt pathway is a critical transducer for several
major survival signals in CNS neurons (Datta et al.,
1997). Glycogen synthase kinase 3b (GSK3b), which is
highly expressed in brain tissue, is one of the key targets
of the antiapoptotic signaling mediated by the PI3K/Akt
pathway (Pap and Cooper, 1998). The activity of
GSK3b is negatively regulated by Akt phosphorylation
at the N-terminal serine 9 (Ser9; Cross et al., 1995).
Reduced PI3K signaling also results in Ser9 dephospho-
rylation (Hetman et al., 2000).
The present study investigates the putative neuro-
protective role of GUO against glutamate-induced hip-
pocampal slice injury and the PI3K/Akt/GSK3bSer9
pathway and iNOS inhibition as the possible mechanism
involved in the neuroprotection afforded by GUO. The
results demonstrate that GUO protects hippocampal sli-
ces by a mechanism involving PI3K/Akt/GSK3bSer9
pathway activation and subsequent reduction of gluta-
mate-induced glutamate release. However, GUO inhibi-
tion of iNOS expression induced by glutamate was not
dependent on the PI3K/Akt pathway in hippocampal
slices of rats.
MATERIALS AND METHODS
Animals
Male Wistar rats (23–25 days of postnatal age) main-
tained on a 12-hr light–12-hr dark schedule at 258C, with
food and water ad libitum, were obtained from our local
breeding colony. Experiments followed the principles of labo-
ratory animal care (NIH publication No. 85-23, revised 1985)
and were approved by the local Ethical Committee of Animal
Research (CEUA/UFSC).
Preparation and Incubation of Hippocampal Slices
Rats were killed by decapitation, and the hippocampi
were rapidly removed and placed in ice-cold Krebs-Ringer
bicarbonate buffer (KRB) of the following composition: 122
mM NaCl, 3 mM KCl, 1.2 mM MgSO4, 1.3 mM CaCl2, 0.4
mM KH2PO4, 25 mM NaHCO3, and 10 mM D-glucose.
The buffer was bubbled with 95% O2-5% CO2up to pH 7.4.
Slices (0.4 mm thick) were rapidly prepared using a McIlwain
tissue chopper, separated in KRB at 48C, and allowed
to recover for 30 min in KRB at 378C to slice stabilization
(Oliveira et al., 2002).
Hippocampal Slice Treatments
After the preincubation period, for slice stabilization, the
slices were incubated with glutamate (Sigma, St. Louis, MO;
1 mM) for 1 hr in KRB buffer. After this period, the medium
was withdrawn and replaced by a nutritive culture medium
Guanosine Protects Via Akt Activation and iNOS Inhibition1401
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composed of 50% KRB, 50% Dulbecco’s modified Eagle’s
medium (DMEM; Gibco, Grand Island, NY), 20 mM
HEPES, and 100 lg/ml gentamicin, and slices were main-
tained for additional 6 hr in a humidified atmosphere of 95%
air and 5% CO2at 378C to evaluate cell viability (Molz et al.,
2008a).
When present, GUO was added to the incubation me-
dium 30 min before glutamate and maintained during the
1 hr of incubation with glutamate. The PI3K inhibitor 2-(4-
morpholinyl)-8-phenyl-4H-1benzo-pyran-4-one
30 lM) or the NO donor S-nitroso-N-acetyl-l,l-penicillamine
(SNAP; 1 mM) was added to the incubation medium 15 min
before GUO and maintained during the GUO preincubation
period.
(LY294002;
Evaluation of Cell Viability
Hippocampal cell viability was evaluated 6 hr after glu-
tamate exposure. Cell viability was determined through the
ability of cells to reduce 3-(4,5-dimethylthiazol-2-yl-diphenyl-
tetrazolium bromide (MTT; Sigma; Mosmann, 1983). Hippo-
campal slices were incubated with MTT (0.5 mg/ml) in KRB
for 30 min at 378C. The tetrazolium ring of MTT can be
cleaved by active dehydrogenases in order to produce a pre-
cipitated formazan. The formazan produced was solubilized by
adding 200 ll dimethylsulfoxide (DMSO), resulting in a col-
ored compound whiose optical density was measured in an
ELISA reader (550 nm).
Immunobloting
To evaluate Akt (Ser 473) and GSK3b (Ser 9) phospho-
rylation, slices were incubated for 90 min under control con-
ditions or were treated with GUO (100 lM) for 30, 60, or
90 min. Then, the slices were homogenized in 100 ll ice-
cold lysis buffer (1% Nonidet P-40, 10% glycerol, 137 mM
NaCl, 20 mM Tris-HCl, pH 7.5, 1 lg/ml leupeptin, 1 mM
phenylmethylsulfonyl fluoride, 20 mM NaF, 1 mM sodium
pyrophosphate, and 1 mM Na3VO4). Proteins (30 lg) from
these cell lysates were resolved by SDS-PAGE and transferred
to nitrocellulose membranes (GE Healthcare, Chalfont St.
Giles, United Kingdom). Membranes were incubated with
antitotal-Akt (1:1,000), antiphospho-Akt (1:1,000; Cell Signal-
ing,Izasa,Barcelona, Spain),
(1:1,000), antitotal GSK3b (1:1,000), anti-iNOS (:1,000; Cell
Signaling, Izasa), or anti-b-actin (1:100,000; Sigma). Appro-
priate peroxidase-conjugated secondary antibodies (1:10,000)
were used to detect proteins by enhanced chemiluminescence.
Different band intensities corresponding to immunoblot detec-
tion of protein samples were quantified in Scion Image (Scion
Corporation, Frederick, MD).
antiphospho-GSK3bSer9
Glutamate Release
After the preincubation period to slice recovery (30
min), hippocampal slices were incubated in Hank’s balanced
salt solution (HBSS; composition in mM 1.29 CaCl2, 136.9
NaCl, 5.36 KCl, 0.65 MgSO4, 0.27 Na2HPO4, 1.1 KH2PO4,
2 glucose, and 5 HEPES). When present, GUO was incu-
bated for 30 min and maintained during glutamate exposure.
LY294002 (30 lM) was preincubated for 15 min before
GUO. Glutamate (1 mM) was incubated for 15 min, and glu-
tamate uptake was assessed by adding 0.33 lCi/ml D-
[3H]aspartate with 100 lM unlabeled aspartate for 7 min and
stopped by three ice-cold washes with 1 ml HBSS. D-
[3H]aspartate instead of L-[3H]glutamate was used in order to
avoid glutamate metabolization in intracellular compartments,
although similarresultswere
[3H]aspartate or L-[3H]glutamate. The slices were then further
incubated for 15 min in HBSS, and the supernatant was
collected to measure the amount of released D-[3H]aspartate.
Slices were disrupted by overnight incubation with 0.1%
NaOH/0.01% SDS, and aliquots of lysates were taken for
determination of intracellular D-[3H]aspartate content (Molz
et al., 2008b). Intracellular and extracellular D-[3H]aspartate
content were determined through scintillation counting, cal-
culated as nmol aspartate, and the amount of released aspartate
was expressed as percentage of total D-[3H]aspartate.
obtained byusingD-
Statistical Analysis
Comparisons among groups were performed by one-
way ANOVA, followed by Duncan’s test if necessary, with
P < 0.05 considered to be statistically significant.
RESULTS
GUO Protects Hippocampal Slices Against
Glutamate-Induced Cell Death
Exposure of hippocampal slices to 1 mM glutamate
resulted in a significant decrease in cell viability mea-
sured as reduction of MTT. Treatment of the slices with
GUO (100 lM) significantly prevented the reduction in
cell viability induced by glutamate. Such neuroprotec-
tion was not observed when slices were preincubated
with 30 lM or 300 lM GUO (Fig. 1). None of the
GUO concentrations used was able to alter cellular via-
bility per se.
Involvement of Glutamate Transport and PI3K/
Akt/GSK3bSer9Pathway Activation in the
Protective Effects Afforded by GUO
We have previously demonstrated that glutamate
excitotoxicity in hippocampal slices involves the reversal
of activity of glutamate transporters and consequent glu-
tamate release (Molz et al., 2008b). GUO has been
shown to exert neuroprotection against excitotoxicty by
its ability to modulate glutamate transport (Moretto
et al., 2005). Therefore, glutamate release was evaluated
in the presence of 100 lM GUO. Figure 2 shows that
GUO was able to prevent glutamate-induced D-
[3H]aspartate release (i.e., glutamate release) from hippo-
campal slices.
The presence of the PI3K/Akt inhibitor LY294002
counteracted the release of glutamate as well as partially
preventing the protective effects afforded by GUO in
hippocampal slices exposed to glutamate (Figs. 2, 3).
Because LY294002 prevented the neuroprotective effect
of GUO, we decided to measure Akt (Akt phosphoryla-
tion) by Western blot. For these experiments, hippocam-
pal slices were incubated with 100 lM GUO for different
1402Molz et al.
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periods (30, 60, and 90 min), and cell lysates were then
analyzed by immunoblotting. Those GUO incubation
periods were selected because they represent the GUO
preincubation time (30 min), the intermediate period
when slices are in the presence of glutamate plus GUO
(60 min), and the total time for which slices were incu-
bated in the presence of glutamate plus GUO (90 min).
Figure 4A shows a representative immunoblotting of
phospho-Akt (p-Akt). Thirty minutes of exposure to
GUO was sufficient to increase the amount of p-Akt
by almost twofold compared with control slices; this
phosphorylation was maintained at 60 min and was fur-
ther increased after 90 min incubation with GUO
(Fig. 4A,B).
GSK3b is one of the key targets of the signaling
mediated by the PI3K/Akt pathway (Pap and Cooper,
1998). Akt-mediated phosphorylation of GSK3b at
Ser9 inactivates GSK3b, leading to increased cell sur-
vival against glutamate excitotoxicity (Nishimoto et al.,
2008). Incubation of hippocampal slices with GUO
showed a significant increase in phosphorylation of
GSK3b at Ser9 after 30 min of exposure to GUO;
however, its phosphorylation returned to basal levels
after 60 or 90 min of exposure (Fig. 5A,B). LY294002
prevented the GUO-induced up-regulation of GSK3b
at Ser9 (Fig. 6A,B), indicating that the PI3K/Akt
Fig. 2. GUO reduces glutamate-induced glutamate release via PI3K/
Akt activation. Hippocampal slices were incubated for 15 min with
1 mM glutamate (Glu) in the presence or absence of 100 lM guano-
sine (GUO). When present, GUO was preincubated for 30 min
before the addition of glutamate. LY294002 (30 lM) was added to
the incubation medium 15 min before GUO and maintained during
the GUO preincubation period. Control group was considered as
100% and represents glutamate released from slices incubated only in
HBSS. The values represent mean 6 SEM of at least four experi-
ments carried out in triplicate. *Significantly different from control
group (100%) and Glu 1 GUO (P < 0.05).
Fig. 1. Cell viability of hippocampal slices subjected to glutamate in
the presence of GUO. Hippocampal slices were incubated for 1 hr
with 1 mM glutamate (Glu). After this period, incubation medium
was withdrawn and replaced with fresh culture medium without glu-
tamate, and cells were maintained for an additional 6 hr. When pres-
ent, guanosine (GUO 30, 100, and 300 lM) was preincubated for
30 min before the addition of glutamate and maintained during gluta-
mate exposure. When treated with GUO alone, hippocampal slices
were incubated with GUO for 30 min, and then the incubation
medium was withdrawn and replaced with fresh culture medium,
and cells were maintained for an additional 7 hr. The control group
was considered as 100% viable and represents cell viability of slices
incubated only in culture medium. MTT (0.5 mg/ml) was incubated
for 20 min at 378C, and cell viability was accessed at 550 nm. The
values represent mean 6 SEM of at least six experiments carried
out in triplicate. *Significantly different from control group (100%),
#significantly different from Glu (P < 0.05).
Fig. 3. LY294002 prevents the neuroprotective effect of GUO against
glutamate-induced cell death. Hippocampal slices were incubated for
1 hr with 1 mM glutamate (Glu) in the presence or absence of 100
lM guanosine (GUO), preincubated for 30 min before the addition
of glutamate. LY294002 (30 lM) was added to the incubation me-
dium 15 min before GUO and maintained during the GUO preincu-
bation period. After this period, incubation medium was withdrawn
and replaced with fresh culture medium without glutamate, and cells
were maintained for additional 6 hr. The control group was consid-
ered as 100% viable and represents cell viability of slices incubated
only in culture medium. MTT (0.5 mg/ml) was incubated for 20 min
at 378C, and cell viability was accessed at 550 nm. The values repre-
sent mean 6 SEM of at least four experiments carried out in triplicate.
*Significantly different from control group (100%) and Glu 1 GUO,
#significantly different from control group (P < 0.05).
Guanosine Protects Via Akt Activation and iNOS Inhibition 1403
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pathway is responsible for the modulatory effect of
GUO on GSK3b phosphorylation.
GUO Prevents Glutamate-Induced iNOS
Expression
Studieshavedemonstrated
involves the activation of cellular pathways that lead to
iNOS induction (Brown and Bal-Price, 2003) and that
activation of the PI3K/Akt pathway may diminish the
induction of iNOS (Pahan et al., 1999). Therefore, the
involvement of iNOS in glutamate-induced cell damage
to hippocampal slices and the possible prevention of this
effect by GUO were investigated. We have found
increased levels of iNOS after exposure of hippocampal
slices to 1 mM glutamate. This effect was completely
prevented when slices were preincubated with 100 lM
GUO (Fig. 7). Because GUO was able to activate
PI3K/Akt signaling pathway, which was related to pro-
tection, we decided to investigate whether this signaling
pathway could be involved in the protective effect of
GUO against glutamate-induced iNOS up-regulation.
To achieve this, hipocampal slices were exposed to
that excitotoxicity
LY294002, before the preicubation period with GUO.
LY294002 did not modify the effect of GUO on gluta-
mate-induced iNOS, which indicates that the PI3K/Akt
pathway is not involved in the inhibition of iNOS
induction caused by GUO (Fig. 8).
The combination of SNAP (an NO donor) plus
glutamate did not further reduce cell viability of hippo-
campal slices in comparison with glutamate alone. Addi-
tionally, preincubation of hippocampal slices with SNAP
(1 mM), prior to GUO treatment significantly abolished
the protective effect of GUO against glutamate-induced
cell damage (Fig. 9). These data point out to a neuro-
protective mechanism of action of GUO against gluta-
mate excitotoxicity, likely involving either decrease
of iNOs expression or NO production in hippocampal
slices.
DISCUSSION
The present study demonstrates that GUO prevents
glutamate-induced cell death of hippocampal slices by a
mechanism involving inhibition of glutamate-induced
glutamate release through activation of the PI3K/Akt
Fig. 4. Immunodetection of phosphorylated and total Akt in hippo-
campal slices treated with GUO. Protein extracts from hippocampal
slice lysates were subjected to Western blotting analysis to determine
phosphorylated (p-Akt) and total (t-Akt) Akt as described in Materials
and Methods. A: Representative Western blot of phosphorylated Akt
(p-Akt) and total Akt (t-Akt) from hippocampal slices exposed
to GUO (100 lM) for 30, 60, or 90 min. B: Quantitative analysis of
p-Akt/t-Akt in optical density (O.D.). The control values were nor-
malized to 100%, and other treatments were expressed in relation to
this value. The values represent mean 6 SEM of four independent
experiments. *Significantly different from control group (P < 0.05).
**Significantly different from control group (P < 0.01).
Fig. 5. Immunodetection of phosphorylated GSK3bSer9and total
GSK3b in hippocampal slices treated with GUO. Protein extracts from
hippocampal slice lysates were subjected to Western blotting analysis
for phosphorylated (p-GSK3bSer9) and total (t-GSK3b) measurement
as described in Materials and Methods. A: Representative Western blot
of phosphorylated GSK3b at Ser9 (p-GSK3bSer9) and total GSK3b (t-
GSK3b) obtained from hippocampal slices exposed to GUO (100 lM)
for 30, 60, or 90 min. B: Quantitative analysis of p-GSK3bSer9/t-
GSK3b in optical density (O.D.). The control values were normalized
to 100%, and other treatments were expressed in relation to this value.
The values represent mean 6 SEM of six independent experiments.
*Significantly different from control group (P < 0.05).
1404 Molz et al.
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pathway and a consequent inhibition of GSK3b as well as
the reduction of glutamate-induced iNOS expression in
rat hippocampal slices. The regulation of excitotoxicity
relies on the function of glutamate transporters present on
astrocytes and, to a lesser extent, in neurons (Anderson
and Swanson, 2000). These transporters are coupled to
Na1/K1-ATPase activity in order to remove excessive
glutamate from the brain’s extracellular space, preventing
excitotoxicity (Danbolt, 2001). In situations in which the
membrane Na1gradient is disrupted, such as after respira-
tory chain inhibition resulting from Ca21overload and
oxidative stress, glutamate can be released to the synaptic
cleft by reversal of glutamate transporters (Rossi et al.,
2000; Camacho and Massieu, 2006). In a previous study,
we demonstrated that glutamate-induced toxicity in hip-
pocampal slices was due to its ability to trigger glutamate
releaseby reversed glutamate
DL-TBOA, a glutamate transport inhibitor, prevented the
loss of cell viability induced by glutamate and glutamate-
induced glutamate release (Molz et al., 2008b).
transport, insofaras
Hippocampal slice preincubation with GUO (100
lM) prevented glutamate-induced cell damage as well as
glutamate-induced glutamate release from hippocampal
slices (Figs. 1, 2), indicating that the neuroprotective
effect of GUO is due to its ability to prevent glutamate-
induced reversal of uptake, leading to reduced glutamate
release and thus preventing excitotoxicity.
The protective effect of GUO against the gluta-
mate-induced cellviability
prevented by LY294002, a PI3K inhibitor (Fig. 3). Fur-
thermore, the ability of GUO to reduce glutamate
release was inhibited by LY294002 (Fig. 2), Also, treat-
ment of hippocampal slices with GUO showed increased
levels of p-Akt (Fig. 4). Taken together, these results
indicate that GUO prevents glutamate release and pro-
tects hippocampal slices through a mechanism that
involves activation of PI3K/Akt pathway. Data from the
literature show that activation of the PI3K/Akt pathway
can stimulate glutamate uptake as well as trafficking of
glutamate transporters and their expression in the cellular
decreasewas partially
Fig. 6. LY294002 prevents GUO-induced up-regulation of GSK3b
at Ser9. Hippocampal slices were exposed to GUO (100 lM) for
30 min. When present, LY294002 (30 lM) was added to the incuba-
tion medium 15 min before GUO and maintained during the GUO
preincubation period. Protein extracts from hippocampal slice lysates
were subjected to Western blotting analysis to determine phosphoryl-
ated (p-GSK3bSer9) and total (t-GSK3b) as described in Materials
and Methods. A: Representative Western blot of phosphorylated
GSK3b at Ser9 (p-GSK3bSer9) and total GSK3b (t-GSK3b). B:
Quantitative analysis of p-GSK3bSer9/t-GSK3b in optical density
(O.D.). The control values were normalized to 100%, and other
treatments were expressed in relation to this value. The values repre-
sent mean 6 SEM of five independent experiments. *Significantly
different from control group (P < 0.05).
Fig. 7. Immunodetection of iNOS in hippocampal slices treated with
glutamate in the presence of GUO. Untreated (control, C) or 1 mM
glutamate (Glu)-treated hippocampal slices were incubated for 1 hr in
culture medium. Slices were maintained for an additional 6 hr in
fresh culture medium without glutamate. When present, guanosine
(GUO 30, 100, or 300 lM) was preincubated for 30 min. Protein
extracts from hippocampal slice lysates were subjected to Western
blot analysis for iNOS detection as described in Materials and Meth-
ods. A: Representative Western blot of iNOS in hippocampal slices
exposed to Glu or Glu 1 GUO. B: Quantitative analysis by optical
density of iNOS expression related to b-actin. The control values
were normalized to 100%, and other treatments were expressed in
relation to this value. The values represent mean 6 SEM of four in-
dependent experiments. *Significantly different from control group,
and#significantly different from Glu (P < 0.05).
Guanosine Protects Via Akt Activation and iNOS Inhibition1405
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Page 7

membrane (Sims et al., 2000; Krizman-Genda et al.,
2005; Guillet et al., 2005). In agreement with our find-
ings that GUO modulates glutamate transport, other
studies have demonstrated that GUO stimulates gluta-
mate uptake under physiological (Frizzo et al., 2001) as
well as under excitotoxic (Oliveira et al., 2004; Moretto
et al., 2005; Thomazi et al., 2008) conditions. Our
results contribute to characterizing better the modulatory
effect of GUO on glutamate transport.
PI3K/Akt signaling pathway has been shown to act
as an upstream mechanism of GSK3b activity regulation,
insofar as PI3K/Akt might directly phosphorylate Ser9
of GSK3b, leading to GSK3b inactivation. It has been
reported that GSK3b activation may be involved in sev-
eral apoptotic signaling pathways that lead to activation
of caspase-3 (Grimes and Jope, 2001; Bhat et al., 2004).
Glutamate toxicity in hippocampal slices is also mediated
by caspase-3 activation (Molz et al., 2008a). In astrocyte
cultures, GUO prevented the apoptotic effects of stauro-
sporine by inhibiting caspase-3 and activating PI3K/Akt
pathway, with subsequent inhibition of GSK3b (Di Iorio
et al., 2004). Here we have also observed that preincu-
bation of hippocampal slices with GUO (30 min) is suf-
ficient to protect hippocampal slices against subsequent
glutamate-induced cell death by a mechanism that
involves inhibition of GSK3b activity through activation
of the PI3K/Akt pathway, in that LY294002, an PI3K
inhibitor, was able to prevent GUO-induced GSK3bSer9
phosphorylation (Figs. 5, 6). GUO-induced Akt phos-
phorylation was observed at 30 min and sustained until
90 min (Fig. 4). Otherwise, increased GSK3b phospho-
rylation at Ser9 was observed at 30 min incubation with
GUO and then returned to basal levels (Fig. 5). GSK3b
phosphorylation at Ser9 is very dynamic, because a num-
ber of pathways and kinases converge at GSK3b, and its
dephosphorylation is regulated by protein phosphatase 1
(Bennecib et al., 2000; Zhang et al., 2003).
One of the hallmarks of the inflammatory process
in the CNS is the expression of iNOS by activated glia.
Glutamate can induce proinflamatory cytokines that in
turn can trigger iNOS expression (Cardenas et al., 2000;
Moro et al., 2004). In this study, we show that 100 lM
GUO prevents glutamate-induced iNOS expression in
hippocampal slices (Fig. 7). However, GUO at concen-
trations of 30 or 300 lM was unable to counteract
Fig. 8. Guanosine reduction of glutamate-induced iNOS expression
does not involve the PI3K/Akt pathway. Hippocampal slices were
incubated for 1 hr with 1 mM glutamate (Glu) in the presence or ab-
sence of 100 lM guanosine (GUO), preincubated for 30 min before
the addition of glutamate. LY294002 (30 lM) was added to the
incubation medium 15 min before GUO and maintained during
the GUO preincubation period. Protein extracts from hippocampal
slice lysates were subjected to Western blot analysis to determine
iNOS expression as described in Materials and Methods. A: Repre-
sentative Western blot of iNOS in hippocampal slices. B: Quantita-
tive analysis by optical density of iNOS expression related to b-actin.
The values represent mean 6 SEM of four independent experiments.
*Significantly different from all other groups (P < 0.05).
Fig. 9. Guanosine neuroprotection against glutamate-induced cell
death is prevented by an NO donor. Hippocampal slices were incu-
bated for 1 hr with 1 mM glutamate (Glu) in the presence or absence
of 100 lM guanosine (GUO). When present, GUO was preincu-
bated for 30 min before the addition of glutamate, and SNAP
(1 mM) was added to incubation medium 15 min before GUO and/
or Glu and maintained during the 1-hr incubation period. After that,
incubation medium was withdrawn and replaced by fresh culture me-
dium without glutamate, and cells were maintained for an additional
6 hr. Control group was considered as 100% and represents cell via-
bility of slices incubated only in culture medium. MTT (0.5 mg/ml)
was incubated for 20 min at 378C, and cell viability was measured at
550 nm. The values represent mean 6 SEM of at least four experi-
ments carried out in triplicate. *Significantly different from control
group (100%) and Glu 1 GUO (P < 0.05).
1406 Molz et al.
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Page 8

glutamate-induced iNOS; this can be correlated with the
previous observation that these same GUO concentra-
tions were also unable to prevent loss of cell viability
induced by glutamate (Fig. 1). Thus, if iNOS suppres-
sion is important to counteract cell death induced by
glutamate, a concentration that cannot elicit such an
event cannot be neuroprotective.
The possible involvement of PI3K/Akt pathway as
the upstream mediator of GUO suppression of gluta-
mate-induced iNOS up-regulation was also investigated.
Our results showed that PI3K/Akt is not the upstream
signalling pathway involved in this GUO effect, because
LY294002 did not abolish the ability of GUO to reduce
the amount of iNOS (Fig. 8). This is in agreement with
the viability results shown in figure 3; the PI3K inhibitor
LY294002 only partially prevented the neuroprotective
effect of GUO against glutamate-induced cell death, indi-
catingthat another signalling
involved. Astrocytes or C6 glioma cells treated with a
PI3K inhibitor increased iNOS expression in response to
bacterial lipopolysaccharide or cytokines, demonstrating
that the activity of PI3K pathway is important to coun-
teract iNOS expression (Pahan et al., 1999). Further-
more, the aggravation of brain injury by Wortmannin
(another PI3K inhibitor) was not associated with iNOS
expression (Kilic et al., 2006). Our results clearly indicate
that the neuroprotective effect of GUO involves iNOS
modulation. To understand better the mechanism by
which GUO could be reducing iNOS, we used SNAP
(an NO donor) at a concentration that it does not reduce
cellular viability. The combination of SNAP plus gluta-
mate did not further reduce cell viability of hippocampal
slices in comparison with glutamate alone (Fig. 9). These
results suggest that the NO produced by SNAP is not
able to induce cell death per se or to increase glutamate-
induced cell death, indicating that NO produced via
iNOS expression is sufficient to induce cell death and
cannot be further increased by NO donated by SNAP.
However, the NO that is produced by SNAP is able to
modulate the protective effect of GUO against gluta-
mate-induced cell death, indicating that the protective
mechanism of GUO involves its ability to modulate the
amount of NO in order to prevent cell death by a mech-
anism that involves regulation of iNOS expression and
not sequestration of NO production.
Taken together, these observations support the hy-
pothesis that GUO protects against neurodegeneration by
a mechanism involving PI3K/Akt activation and inacti-
vation of GSK3b, with subsequent inhibition of gluta-
mate-induced glutamate release in hippocampal slices.
The blockade of iNOS expression is also important for
the neuroprotective effect of GUO against glutamate-
induced toxicity.
Inflammation, impairment of glutamate transport,
and excitotoxiciy are all processes involved in pathoge-
nesis of neurodegenerative diseases in the CNS (Brown
and Bal-Price, 2003; Maragakis and Rothstein, 2004).
Therefore, the protective effects of GUO, which nor-
malize the activity of glutamate transporters, counteract-
pathway should be
ing excitotoxicity, may be considered an important
neuroprotective strategy for degeneration of the CNS.
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