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Neuroscience 183 (2011) 212–220

GUANOSINE IS NEUROPROTECTIVE AGAINST OXYGEN/GLUCOSEDEPRIVATION IN HIPPOCAMPAL SLICES VIA LARGE CONDUCTANCECA2�-ACTIVATED K� CHANNELS, PHOSPHATIDILINOSITOL-3 KINASE/

ROTEIN KINASE B PATHWAY ACTIVATION AND GLUTAMATE

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A. R. S. SANTOSb AND C. I. TASCAa*aDepartamento de Bioquímica, Centro de Ciências Biológicas, Univer-idade Federal de Santa Catarina, Trindade, 88040-900 Florianópolis,C, Brazil

bDepartamento de Ciências Fisiológicas, Centro de Ciências Biológi-as, Universidade Federal de Santa Catarina, Trindade, 88040-900lorianópolis, SC, Brazil

Abstract—Guanine derivatives (GD) have been implicated inmany relevant brain extracellular roles, such as modulationof glutamate transmission and neuronal protection againstexcitotoxic damage. GD are spontaneously released to theextracellular space from cultured astrocytes and during ox-ygen/glucose deprivation (OGD). The aim of this study hasbeen to evaluate the potassium channels and phosphatidili-nositol-3 kinase (PI3K) pathway involvement in the mecha-nisms related to the neuroprotective role of guanosine in rathippocampal slices subjected to OGD. The addition ofguanosine (100 �M) to hippocampal slices subjected to 15min of OGD and followed by 2 h of re-oxygenation is neuro-protective. The presence of K� channel blockers, glibencl-amide (20 �M) or apamin (300 nM), revealed that neuropro-tective effect of guanosine was not dependent on ATP-sen-sitive K� channels or small conductance Ca2�-activated K�

channels. The presence of charybdotoxin (100 nM), a largeconductance Ca2�-activated K� channel (BK) blocker, inhib-ted the neuroprotective effect of guanosine. Hippocampallices subjected to OGD and re-oxygenation showed a sig-ificant reduction of glutamate uptake. Addition of guanosine

n the re-oxygenation period has blocked the reduction oflutamate uptake. This guanosine effect was inhibited whenippocampal slices were pre-incubated with charybdotoxinr wortmanin (a PI3K inhibitor, 1 �M) in the re-oxygenation

period. Guanosine promoted an increase in Akt protein phos-phorylation. However, the presence of charybdotoxin blockedsuch effect. In conclusion, the neuroprotective effect of guanos-ine involves augmentation of glutamate uptake, which is mod-ulated by BK channels and the activation of PI3K pathway.Moreover, neuroprotection caused by guanosine depends on

*Corresponding author. Tel: �55-48-3721-5046; fax: �55-48-3721-9672.-mail address: tasca@ccb.ufsc.br (C. I. Tasca).bbreviations: Akt, protein kinase B; BK, large (big) conductancea2�-activated K� channels; GD, guanine derivatives; GDP, guanos-

ne 5=-diphosphate; GMP, guanosine 5=-monophosphate; GTP,uanosine 5=-triphosphate; GUO, guanosine; HBSS, Hank’s balancedalt solution; KATP, ATP-sensitive K� channels; KCa, Ca2�-activated

K� channels; KRB, Krebs-Ringer bicarbonate buffer; MEK, mitogen-activated protein kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-nyltetrazolium bromide; NMDA, N-methyl-D-aspartate; OGD, oxygen/

dglucose deprivation; PI3K, phosphatidilinositol-3kinase; ROS, reactiveoxygen species; TEA, tetraethylammonium.

0306-4522/11 $ - see front matter © 2011 IBRO. Published by Elsevier Ltd. All righdoi:10.1016/j.neuroscience.2011.03.022

212

the increased expression of phospho-Akt protein. © 2011 IBRO.Published by Elsevier Ltd. All rights reserved.

Key words: guanosine, oxygen/glucose deprivation, gluta-mate uptake, Akt phosphorylation, BK activation, hippocam-pal slices.

Stroke is a major cause of disability and death, worldwide(Mattson, 2000). Ischemic stroke accounts for, approxi-mately, 80% of all strokes and it results from a thromboticor embolic occlusion of a major cerebral artery (Durukanand Tatlisumak, 2007). Experimental in vitro models forischemia comprise advantages for the study of cellularpathophysiological responses. Oxygen and/or glucose de-privation in brain tissue preparations reproduce severalpathological states induced by brain energy failure. Energydeprivation leads to the production of reactive oxygenspecies (ROS), (White et al., 2000) which causes therelease of neurotransmitters such as glutamate (Bonde etal., 2003).

Excessive extracellular glutamate leads to overstimu-lation of glutamate receptors and consequent influx ofNa�, Cl� and Ca2� ions, through the channels gated byhose receptors. The increase of Ca2� levels inside the cellresults in the activation of Ca2�-dependent enzymes

hich lead to processes such as proteolysis, hydrolysis,ipid peroxidation and ROS production (Choi, 1988). Glu-amate uptake is a crucial process to maintain extracellularlutamate concentrations below toxic levels. This effect ischieved through specific high-affinity sodium-dependentxcitatory amino acid transporters that are mainly present

n astrocytes. Glutamate transporters are modulated by theell redox status (Anderson and Swanson, 2000), thus

ncreased ROS production may result in impairment oflutamate uptake (Trotti et al., 1998).

Cell injuries, like hypoxia and hypoglycemia, arehought to cause remarkable outflow of purines (Ciccarellit al., 1999; Jurányi et al., 1999). Nucleotides are rapidlyatabolized extracellularly and monophosphate nucleo-ides and nucleosides accumulate within the traumatic tis-ue, reaching high concentrations in the extracellularpace (Zimmermann et al., 1998; Ciccarelli et al., 2001;aciagli et al., 2000). Guanine derivatives (GD) are re-

eased in amounts three-fold greater than their adenine

erivatives counterparts (Ciccarelli et al., 1999).

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The nucleoside guanosine (GUO) and guanine nucle-tides have been implicated in neuroprotection by coun-eracting glutamate excitotoxicity, which was observed iniverse models of glutamate toxicity. It has been shownD protect from seizures induced by quinolinic acid in vivo

Schmidt et al., 2000, 2005; Oliveira et al., 2004). Quino-inic acid is a known N-methyl-D-aspartate (NMDA) recep-tor agonist and it has also been revealed as a modulator ofastrocytic and neuronal glutamate transport, causing anexcessive stimulation of the glutamatergic system (Tava-res et al., 2000, 2002). Thus, the neuroprotective effects ofGD on quinolinic acid-induced damage may involve themodulation of glutamate receptors or transporters.

Studies from our laboratory have demonstrated thatguanosine-5=-monophosphate (GMP) is able to reduceneuronal damage in hippocampal slices submitted to an invitro model of ischemia (Oliveira et al., 2002), or to ahypoglycemia-like insult induced by a glucose-deprivedincubation medium (Molz et al., 2005). GMP has alsoprevented the reduction in cell viability and apoptotic DNAfragmentation induced by NMDA (Molz et al., 2008a), al-though it might be toxic for neurons at elevated concen-trations (Molz et al., 2009). In cultured hippocampal neu-rons, guanine nucleotides, namely guanosine 5=-triphos-phate (GTP), guanosine 5=-diphosphate (GDP) and GMP,inhibited NMDA- or kainate-mediated toxicity, whereasguanosine did not shown any protective effect (Morciano etal., 2004). However, it was also previously shown thatguanosine was able to increase glutamate uptake in cor-tical slices maintained at basal conditions or submitted tooxygen/glucose deprivation (OGD) (Frizzo et al., 2002).Moreover, we have shown that guanosine promotes neu-roprotection depending on extracellular Ca2� levels andmodulation of protein kinase A, protein kinase C, mitogen-activated protein kinase (MEK) and phosphatidilinositol-3kinase (PI3K) pathways in slices subjected to OGD (Oles-kovicz et al., 2008).

Increasing evidence show that guanosine displays arelevant neuroprotective effect within in vivo and in vitroneurotoxicity models (for review, see Schmidt et al., 2007).However, the exact extracellular site of interaction (puta-tive receptor) and mechanisms of action for this nucleosidehave not yet been fully characterized. Nevertheless, weshowed that guanosine neuroprotection depends on K�

channels activation in hippocampal slices subjected toOGD (Oleskovicz et al., 2008), and Benfenati et al. (2006)showed that guanosine promotes an increased expressionof functional K� rectifying channels in astrocytes culture.

The activation of K� channels in nerve cells is linked tohe control of membrane potential. Activation of some K�

channels types, such as Ca�2-activated K� channels, oc-cur in response to increased Ca�2 ions concentrationsnside the cells and cell membrane depolarization (Burg etl., 2006). The ATP-sensitive K� channels (KATP) are reg-

ulated by the intracellular ATP concentration. The modu-lation of large conductance Ca2�-activated K� channels(BK) and KATP channels activity has been also described in

he literature as a mechanism of neuroprotection after an

schemic damage (Gribkoff et al., 2001; Yamada and Ina-aki, 2005).

Accumulating evidences suggest PI3K/protein kinase(Akt) pathway plays a crucial role in neuronal survival

fter cerebral ischemia. Immediately after global ischemia,drastic decrease in the levels of phosphorylated-Akt

p-Akt) occurs, and this event precedes the release ofytochrome c and caspases activation (Ouyang et al.,999). However, the activation of PI3K/Akt signaling path-ay may mediate survival of vulnerable hippocampal CA1eurons after transient global cerebral ischemia (Endo etl., 2006). The mechanism of neuroprotection promoted byuanosine against OGD was shown to involve the activa-ion of PI3K/Akt pathway (Oleskovicz et al., 2008).

Therefore, the aim of this study has been to evaluatehe involvement of different subtypes of potassium chan-els and PI3K/Akt pathway in the mechanisms of neuro-rotection promoted by guanosine. Herein, we show thathe addition of guanosine to hippocampal slices subjectedo 15 min of OGD and followed by 2 h of re-oxygenationas been neuroprotective. The mechanism of neuropro-ection promoted by guanosine involves the activation ofK channels and augmentation of glutamate uptake, which

s modulated by BK channels and PI3K pathway activation.uanosine promoted increased phosphorylated levels ofkt that resulted in neuroprotection against the damage

nduced by the OGD. Some of the data present here haveppeared in the form of an abstract (Dal-Cim et al., 2010).

EXPERIMENTAL PROCEDURES

Animals

Male adult Wistar rats (60–90 days postnatal) maintained on a12 h light–12 h dark schedule at 25 °C, with food and water adlibitum, have been obtained from our local breeding colony. Ex-periments have followed the “Principles of laboratory animal care”(NIH publication No. 85-23, revised in 1985) and they have beenapproved by the local Ethical Committee for Animal Research.

Preparation and incubation of hippocampal slices

Rats were killed by decapitation and hippocampi were rapidlyremoved and placed in an ice-cold Krebs-Ringer bicarbonatebuffer (KRB) of the following composition (in mM): 122 NaCl, 3KCl, 1.2 MgSO4, 1.3 CaCl2, 0.4 KH2PO4, 25 NaHCO3 and 10D-glucose. The buffer was bubbled with 95% O2–5% CO2 up to pH7.4. Slices (0.4 mm) were prepared using a McIlwain TissueChopper, separated in KRB at 4 °C, and one slice per tube wasallowed to recover for 30 min in KRB at 37 °C.

In the OGD model, hippocampal slices were incubated in anOGD buffer with a similar salt composition as described above.The OGD buffer consists of a Hank’s balanced salt solution(HBSS), composition in mM: 1.3 CaCl2, 137 NaCl, 5 KCl, 0.65MgSO4, 0.3 Na2HPO4, 1.1 KH2PO4, 5 HEPES and 10 2-deoxy-glucose (Pocock and Nicholls, 1998), and the buffer was bubbledwith nitrogen throughout the incubation period (Strasser and Fi-scher, 1995). During the re-oxygenation period, the OGD bufferwas replaced by physiological KRB and bubbled with 95% O2–5%

O2.The hippocampal slices were subjected to three different

conditions: control—hippocampal slices were maintained in KrebsRinger buffer for 15 min; OGD—hippocampal slices were only

subjected to OGD buffer for 15 min; OGD and re-oxygenation

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group—hippocampal slices were subjected to 15 min for OGDand followed by 2 h of re-oxygenation (Brongholi et al., 2006).

Slices treatment

Guanosine (100 �M, Sigma, St. Louis, MO, USA) treatment wasperformed as follows: guanosine was added to the control group;or guanosine was added during the OGD period only; or in thegroup subjected to OGD followed by re-oxygenation, whenguanosine was added during re-oxygenation period only (Olesk-ovicz et al., 2008). Potassium channels blockers: Apamin (300nM, Sigma, St. Louis, MO, USA), Charybdotoxin (100 nM, Sigma,St. Louis, MO, USA), Glibenclamide (20 �M, Sigma, St. Louis,MO, USA), Tetraethylammonium (TEA, 1 mM, Sigma, St. Louis,MO, USA) and PI3K pathway inhibitor, Wortmanin (1 �M, Alo-mone, Jerusalem, Israel) were used to assess the mechanism ofneuroprotection promoted by guanosine. In the re-oxygenationperiod, the inhibitors were preincubated for 15 min before addingguanosine and they were kept together for 2 h.

Analysis of cell viability

Cell viability was determined through MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction assay (Mos-mann, 1983; Liu et al., 1997). The tetrazolium ring of MTT iscleaved by various dehydrogenases in active mitochondria, andthen precipitated as a blue formazan product. After the treatmentof hippocampal slices, they were incubated with MTT (0.5 mg/ml)in KRB, for 20 min at 37 °C. The medium was withdrawn, theprecipitated formazan was solubilized with dimethyl sulfoxide andviable cells were quantified spectrophotometrically at a wave-length of 550 nm. Variability due to slice size differences wasminimal.

L-[3H]glutamate uptake

L-[3H]glutamate uptake was evaluated as previously described(Molz et al., 2009). After OGD and re-oxygenation protocol, hip-pocampal slices were washed for 15 min at 37 °C in a HBSS,composition in mM: 1.3 CaCl2, 137 NaCl, 5 KCl, 0.65 MgSO4, 0.3Na2HPO4, 1.1 KH2PO4, 2 glucose and 5 HEPES. Uptake wasssessed by adding 0.33 �Ci/ml L-[3H]glutamate with 100 �Mnlabeled glutamate in a final volume of 300 �l. Incubation wastopped immediately after 7 min by discarding the incubationedium, and slices were subjected to two ice-cold washes with 1l HBSS. Slices were solubilized by adding a solution with 0.1%aOH/0.01% SDS and incubated overnight. Aliquots of slice ly-ates were taken for determination of the intracellular content of

L-[3H]glutamate by scintillation counting. Sodium-independent up-take was determined by using choline chloride, instead of sodiumchloride in the HBSS buffer. Unspecific sodium-independent up-take (approximately 30% of total glutamate uptake) was sub-tracted from total uptake to obtain the specific sodium-dependentglutamate uptake. Results were expressed in nmol of L-[3H]gluta-

ate and taken up per mg of protein per min.

Immunoblotting analysis

Expression of Akt phosphorylation and immunocontent was de-termined by Western blot analysis as described in Molz et al.(2008b), with some modifications. Slices were solubilized withSDS-stopping solution (4% SDS, 2 mM EDTA, 8% �-mercapto-ethanol, and 50 mM Tris, pH 6.8). Samples (60 �g of total protein/track) were separated by 12% SDS-PAGE and transferred tonitrocellulose membranes. Membranes were blocked with 5%skim milk (1 h) in TBS (Tris 10 mM, NaCl 150 mM, pH 7.5),followed by three times washing with TBS-T (Tris 10 mM, NaCl150 mM, Tween-20 0.05%, pH 7.5). Membranes were incubated

with primary antibodies anti-phospho Akt (Sigma, dilution 1:2000) v

or anti Akt (Cell signalling, dilution 1:1000) overnight, at 4 °C, andthen, they were exposed to a secondary antibody (anti-rabbit IgG,dilution 1:8000, Cell Signaling) for 1 h at room temperature. Im-munocomplexes were visualized using the enhancing chemilumi-nescence (ECL) detection system (GE Healthcare). Densitometricanalyses were performed for the quantification of the immunoblotsusing the Scion Image Software (Scion Corporation).

Protein measurement

Protein content was evaluated by the method of Lowry et al.(1951) for glutamate uptake, and by the method of Peterson1977) for Western blot analysis. Bovine serum albumin (Sigma)as used as standard.

Statistical analysis

Comparisons among groups were performed by one-way analysisof variance (ANOVA) followed by Duncan’s test if necessary, withP�0.05 considered to be statistically significant.

RESULTS

Guanosine prevented the reduction in glutamateuptake induced by OGD and re-oxygenation

We have previously shown that guanosine is neuroprotec-tive in hippocampal slices subjected to OGD followed byre-oxygenation, and that guanosine-induced neuroprotec-tion involves K� channels activation (Oleskovicz et al.,008). In this study, we have evaluated the subtypes ofotassium channels involved in guanosine-induced neuro-rotective effect and assessed glutamate uptake and Akthosphorylation, on this in vitro model of ischemia.

Hippocampal slices subjected to 15 min of OGDhowed a significant decrease in glutamate uptake whenompared to the control group. The addition of guanosine100 �M) during the OGD period did not alter the decreasen glutamate uptake. When slices were subjected to OGDnd followed by 2 h of re-oxygenation, a significant de-rease in glutamate uptake was also observed. However,he presence of guanosine during the re-oxygenation pe-iod blocked the reduction in glutamate uptake induced byGD followed by re-oxygenation (Fig. 1).

europrotective effect of guanosine involves BKhannels activation

n order to evaluate the K� channel subtype involved inguanosine-induced neuroprotection, hippocampal sliceswere incubated with selective K� channel blockers. Duringhe ischemic episode a drastic reduction in the amount ofntracellular ATP occurs (White et al., 2000), which mayctivate KATP channels, allowing the opening of these

channels and subsequent hyperpolarization of cell mem-brane (Burg et al., 2006). In order to evaluate the involve-ment of these KATP channels in guanosine-induced neu-oprotection, KATP channel blocker glibenclamide (20 �M)

was used. The presence of glibenclamide did not alter thedecreased cell viability induced by OGD. Glibenclamidewas not able to block the neuroprotective effect of guanos-ine in the re-oxygenation period (Fig. 2A).

In order to investigate the involvement of Ca2�-acti-

ated K� channels (KCa) in the protective role of guanos-

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T. Dal-Cim et al. / Neuroscience 183 (2011) 212–220 215

ine, a non-selective KCa blocker, TEA, was used. WhenTEA (1 mM) was incubated in the control group and duringthe OGD, it did not alter cell viability. However, the pres-ence of TEA during re-oxygenation period, inhibits theneuroprotection promoted by guanosine (Fig. 2B). By us-ing selective KCa blockers, we have discriminated a puta-ive guanosine effect among subtypes of KCa. When slicesere incubated with apamin (300 nM), a small conduc-

ance KCa channel blocker, the neuroprotective effect ofguanosine was not altered (Fig. 2C). However, the pres-ence of charybdotoxin (100 nM), a BK blocker, preventedthe guanosine-induced neuroprotection during re-oxygen-ation period (Fig. 2D). These results showed the involve-ment of BK channels in the mechanism of neuroprotectionafforded by guanosine.

Since the blockade of BK channels was able to inhibitthe protection promoted by guanosine, we also looked forthe participation of this K� channel on glutamate transport-ers activity. When present, charybdotoxin per se, did notalter glutamate uptake into hippocampal slices (Fig. 3).Guanosine was able to recover decreased glutamate up-take promoted by OGD followed by the re-oxygenation.This guanosine effect was abolished when hippocampalslices were incubated with charybdotoxin before addingguanosine in the group subjected to OGD and followed bythe re-oxygenation (Fig. 3).

Guanosine is neuroprotective via PI3K activation

A recent report from our laboratory showed that neuropro-tection promoted by guanosine depends on PI3K pathwayactivation (Oleskovicz et al., 2008). In order to evaluate theputative involvement of the PI3K pathway in the mecha-nism of guanosine-induced recovery of glutamate uptake,

Fig. 1. Effect of guanosine (GUO) on glutamate uptake into hip-pocampal slices subjected to oxygen/glucose deprivation (OGD) andre-oxygenation (R). Slices were incubated for 15 min in the control (C)or in OGD buffer and subjected or not to 2 h of re-oxygenation inpresence or absence of GUO (100 �M). The values are expressed asnmol L-[3H]glutamate/mg protein/min and represent mean�standardrror of five experiments carried out in triplicates. � represents meanignificantly different from control groups, P�0.05; # represents meanignificantly different from OGD R groups, P�0.05 (ANOVA followedy Duncan’s test).

slices were incubated with wortmanin, a PI3K inhibitor. The

preincubation with wortmanin in the re-oxygenation period,blocked the increase of glutamate uptake promoted byguanosine (Fig. 4).

Guanosine has been shown to induce Akt phosphory-lation (Pettifer et al., 2004; D’Alimonte et al., 2007). Thus,considering PI3K is involved in the augmentation of gluta-mate uptake promoted by guanosine, we evaluated theguanosine effect on Akt phosphorylation. Slices subjectedto OGD and OGD followed by re-oxygenation showed adecreased Akt phosphorylation when compared to control.However, when guanosine was present during re-oxygen-ation, a recovery on Akt phosphorylation was observed.The presence of charybdotoxin, a BK channel inhibitor,blocked the increase on Akt phosphorylation promoted byguanosine (Fig. 5), showing that guanosine-induced neu-roprotection occurs via BK channels and PI3K/Akt pathwayinvolvement.

DISCUSSION

This study showed that guanosine protects hippocampalslices subjected to 15 min of OGD followed by 2 h ofre-oxygenation and this effect involves BK channels acti-vation. Moreover, OGD and OGD followed by re-oxygen-ation promoted a reduction in glutamate uptake. Guanos-ine prevented such reduction via BK channels and PI3Kpathway activation, as it was demonstrated by pharmaco-logical blockade with selective inhibitors. Additionally,guanosine increases Akt phosphorylation in slices sub-jected to OGD and re-oxygenation, an effect also pre-vented by pharmacological blockade of BK channels.

The respiratory substrates, oxygen and glucose, areimportant factors in cellular death and deprivation of thesesubstrates leads to different cell death pathways, whichare dependent on concentration of these substrates indifferent ischemic zones (Lobysheva et al., 2009). Be-cause brain tissue depends, almost exclusively, on oxida-tive phosphorylation for energy production, oxygen andglucose deprivation results in energy failure, disruption ofthe ionic gradient and deregulation of glutamate uptake(Camacho and Massieu, 2006). In this study, we haveconfirmed that slices subjected to OGD followed by re-oxygenation presented a decreased glutamate uptake, aspreviously demonstrated by us (Brongholi et al., 2006) andother groups (Thomazi et al., 2008). Therefore, the use ofagents that modulate glutamate uptake or minimize theeffects of glutamate toxicity (Kajta et al., 2009) appears tobe an interesting strategy of neuroprotection against theischemic damage. Evidences support the hypothesis thatneuroprotective effect of guanosine against excitotoxicityis related to the modulation of glutamate uptake (Frizzo etal., 2002; Oliveira et al., 2004; Moretto et al., 2005).Herein, we showed that the presence of guanosine duringthe OGD period only, was not able to block the decreasedglutamate uptake. On the other hand, when present in there-oxygenation period, guanosine prevents the decreasedglutamate uptake and promotes neuroprotection. The de-creased energetic charge during the ischemic episode

(OGD) might be responsible for the inability of guanosine

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T. Dal-Cim et al. / Neuroscience 183 (2011) 212–220216

to recover an ATP-dependent process as glutamate up-take (which relies on Na�–K�–ATPase activity). However,during the re-oxygenation when energy substrates areprovided to cells, guanosine showed an increased glu-tamate reuptake and induced cell survival. Therefore,our results corroborate with previous evidence showingguanosine has a specific modulatory effect on glutamateuptake.

The neuroprotective effect afforded by guanosine hasbeen demonstrated in the literature. Guanosine has beenshown to protect human neuroblastoma SH-SY5Y cellsagainst amyloid-beta and 1-methyl-4-phenylpyridinium ion(MPP�)-induced apoptosis (Pettifer et al., 2004, 2007) andt prevented seizures induced by several glutamatergic

Fig. 2. Evaluation of the involvement of potassium channels in theoxygen/glucose deprivation (OGD) and re-oxygenation(R). Slices wechannels blockers and subjected or not to 2 h of reperfusion. In the rebsence of K� channel blockers. Slices were pre-incubated with: (A) AT

K� channels blocker, TEA (1 mM). (C) Small conductance Ca2�-activaCa2�-activated K� channels blocker, charybdotoxin (Cha, 100 nM). Theassay and represent mean�SD of four experiments carried out in trip# represents mean significantly different from OGD and all the other O

gents in rodents (Lara et al., 2001; Schmidt et al., 2000).

onsidering we have recently demonstrated that neuro-rotection induced by guanosine during OGD depends onxtracellular calcium and occurs via K�-channels activa-

tion (Oleskovicz et al., 2008), here we extended the studyof guanosine-induced neuroprotection against OGD in hip-pocampal slices, revealing the putative interaction site ofguanosine and its mechanism of action.

The evaluation of putative K�-channels involved in themechanism of action of guanosine showed that a BKblocker (charybdotoxin) was able to prevent guanosine-induced neuroprotection against OGD followed by re-oxy-genation. Modulation of this type of K� channel may be animportant target on cellular protection, since BK channelsactivation depends on depolarization and intracellular cal-

otective effect of guanosine in the hippocampal slices subjected toted for 15 min in the control (C) or OGD buffer with or without K�

ation group guanosine (GUO 100 �M) was added in the presence orive K� channel blocker, glibenclamide (Gli, 20 �M). (B) Ca2�-activatedhannels blocker, apamin (Apa, 300 nM). (D) Large (big) conductancere expressed in % cellular viability, as measured by the MTT reductionrepresents mean significantly different from control groups, P�0.05;

roups, P�0.05 (ANOVA followed by Duncan’s test).

neuroprre incuba-oxygenP-sensitted K� cvalues a

cium concentration increase. BK channels can promote a

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negative feedback regulation of Ca2� influx via voltage-gated-Ca2� channels, regulation of cellular excitability andeurotransmitter release (Hu et al., 2001; Ghatta et al.,006). Data in the literature indicate that BK channel ac-ivity was modulated by PKC phosphorylation at a specificite present in this channel (Kim and Park, 2008; Zhu et al.,009). Accordingly, we previously demonstrated thatuanosine-induced neuroprotection in hippocampal slicesubjected to OGD also depends on PKC activation (Oles-ovicz et al., 2008). Thus, it is possible that guanosineould modulate BK channels activity by PKC activation,nd thereby promote neuroprotection.

Considering that guanosine modulates glutamate up-ake, we have evaluated the involvement of BK channelsn this putative mechanism related to guanosine-inducedeuroprotection. We observed that BK channels inhibitionlocked guanosine-induced increase in glutamate uptakefter OGD followed by re-oxygenation. Modulation of BKhannels activity contributes to the regulation of Ca2�

levels in the cell (Ghatta et al., 2006), thus it is feasible thatguanosine effect on BK channels may contribute to ionichomeostasis and reduction of ROS production, preventingthe impairment of glutamate uptake observed after OGDfollowed by re-oxygenation.

Guanosine may display short and long-term effects inthe brain, acting as a neuromodulator or a neuroprotectiveagent. For instance, neuroprotective effects against OGD

Fig. 3. Evaluation of the involvement of large conductance Ca2�-ctivated K� (BK) channels on guanosine-induced modulation of glu-

amate uptake in hippocampal slices subjected to oxygen/glucoseeprivation (OGD) and re-oxygenation(R). Slices were incubated for5 min in the control (C) or OGD buffer with or without charybdotoxinCha, 100 nM, a BK channels blocker) and subjected or not to 2 h ofeperfusion. In the re-oxygenation group, guanosine (GUO 100 �M)

was added in the presence or absence of charybdotoxin. Control sliceswere maintained in a KRB buffer throughout the experiments. Thevalues are expressed as nmol L-[3H]glutamate/mg protein/min andepresent means�standard error of five experiments carried out inriplicates. * represents mean significantly different from controlroups, P�0.05; # represents mean significantly different from OGD

and all the other OGD R groups, P�0.05 (ANOVA followed by Dun-can’s test).

and chemically-induced seizures have been shown to be f

related to guanosine effect on glutamate uptake, whenguanosine action may limit the toxic glutamate extracellu-lar rise under pathophysiological conditions (Oliveira et al.,2004; Moretto et al., 2005; Vinadé et al., 2005; Thomazi etal., 2008). Glutamate uptake is an electrogenic Na�-de-endent mechanism favoured by membrane hyperpolar-

zation (Anderson and Swanson, 2000). It is conceivablehat activation of K� currents by guanosine could increasethe efficiency of glutamate uptake. Activation of K� chan-els by guanosine has been previously shown (Benfenatit al., 2006). However, in that study, a chronic exposition touanosine (500 �M) was necessary in order to induce an

ncrease in activity/expression of an inward rectifier K�

channel in cultured rat cortical astrocytes, whereas 1–12 hof exposition was not sufficient to induce K� channel func-tional appearance. Therefore, K� channels activation mightbe involved in short- and long-term guanosine effects. In thepresent study, we provided pharmacological evidence thatthe observed short-term guanosine effects, as glutamatetransport modulation and neuroprotection, occurs via activa-tion of BK channels.

Identification of a molecular target to guanosine is offundamental relevance, considering specific receptors toguanosine or GD are poorly characterized (Tasca et al.,1999; Traversa et al., 2002). Whether short- and long-termeffects of guanosine rely on the activation of differentsubtypes of K� channels deserves further investigation.Moreover, considering our neuroprotective observationswere made in brain slices, we cannot affirm in which typeof cells (neurons or astrocytes) K� channels are activated.

Fig. 4. Evaluation of the involvement of the PI3K pathway on guanos-ine-induced modulation of glutamate uptake in hippocampal slicessubjected to oxygen/glucose deprivation (OGD) and re-oxygen-ation(R). Slices were incubated for 15 min in the control (C) or OGDbuffer with or without wortmanin (wort, 1 �M, a PI3K inhibitor) andsubjected or not to 2 h of reperfusion. In the re-oxygenation group,guanosine (GUO 100 �M) was added in the presence or absence of

ortmanin. Control slices were maintained in a KRB buffer throughouthe experiments. The values are expressed as nmol L-[3H]gluta-mate/mg protein/min and represent mean�standard error of four ex-periments carried out in triplicates. * represents mean significantlydifferent from control groups, P�0.05; # represents mean significantlyifferent from OGD and all the other OGD R groups, P�0.05 (ANOVA

ollowed by Duncan’s test).

amtin2

mtPpra(pmetP

seas(brmb

awagP

B

B

absem*

O

T. Dal-Cim et al. / Neuroscience 183 (2011) 212–220218

However, it must be considered that astroglial syncytium ismainly responsible for K� homeostasis (Leis et al., 2005)nd astrocytic glutamate transporters have a crucial role inaintenance of the neurotransmitter extracellular concen-

ration at physiological levels (Danbolt, 2001). Additionally,t is possible that guanosine displays different effects oneurons (Tasca et al., 2010) and astrocytes (Decker et al.,007).

The evaluation of signaling pathways related to theechanisms of guanosine-induced neuroprotection showed

hat modulation of glutamate uptake involves the activation ofI3K/Akt pathway. It has been shown that modulation of PI3Kathway is related to the increase of glutamate uptake and itegulates the expression of glutamate transporters excitatorymino acid carrier 1 (EAAC1) (Sims et al., 2000) and GLT-1Li et al., 2006). Activation of Akt increased the surface ex-ression of EAAC1 in transfected cells and, further, gluta-ate transport (Krizman-Genda et al., 2005). Moreover, sev-ral studies have shown that guanosine promotes neuropro-

ection by activation of PI3K/Akt pathway (Di Iorio et al., 2004;

Fig. 5. Evaluation of Akt phosphorylation (p-Akt) in hippocampalslices subjected to oxygen/glucose deprivation (OGD) and re-oxygen-ation(R). Slices were incubated for 15 min in the control (C) or OGDbuffer with or without charybdotoxin (Cha, 100 nM, a BK channelsblocker) and subjected or not to 2 h of reperfusion. In the re-oxygen-ation group, guanosine (GUO 100 �M) was added in the presence orbsence of charybdotoxin. Control slices were maintained in a KRBuffer throughout the experiments. (A) Representative immunoblottinghowing phospho-Akt and total Akt levels. (B) Quantitative analysisxpressed as the ratio of pAkt/Akt total. The values representeans�standard error of five experiments carried out in triplicates.represents mean significantly different from control group, P�0.05;

# represents mean significantly different from OGD and all the otherGD R groups (ANOVA followed by Duncan’s test).

ettifer et al., 2004; Oleskovicz et al., 2008). Here we ob-

erved that guanosine augments phospho-Akt levels and thisffect involves BK channels activation. In cultured neurons,ctivation of BK channels by a specific agonist has beenhown to result in increased levels of Akt phosphorylationGaspar et al., 2008). Thus, the modulation of BK channelsy GUO could result in Akt protein activation during thee-oxygenation period, which leads to an increased gluta-ate uptake due to the modulation of glutamate transportersy Akt activity.

In conclusion, hippocampal slices exposed to OGDnd re-oxygenation showed a reduced cell viability whichas prevented by guanosine. The neuroprotective mech-nism promoted by guanosine involves an increase oflutamate uptake, which is mediated by BK channels andI3K/Akt activation, thus resulting in neuroprotection.

Acknowledgments—Research supported by grants from the Bra-zilian funding agencies: CNPq (Conselho Nacional de Desenvol-vimento Científico e Tecnológico), CAPES (Conselho de Aper-feiçoamento de Pessoal de Nível Superior), FINEP (Financiadorade Estudos e Projetos—IBN-Net # 01.06.0842-00) and INCT (In-stituto Nacional de Ciência e Tecnologia) for Excitotoxicity andNeuroprotection. C.I.T. and A.R.S.S. are recipients of CNPq pro-ductivity fellowship. T.D.-C. is recipient of CNPq pre-doctoralscholarship and W.C.M. is recipient of CAPES Master scholar-ship. The authors are grateful to F.K. Ludka for English revision.

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(Accepted 9 March 2011)(Available online 22 March 2011)