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Guanosine (GUO) is an endogenous modulator of glutamatergic excitotoxicity and has been shown to promote neuroprotection in in vivo and in vitro models of neurotoxicity. This study was designed to understand the neuroprotective mechanism of GUO against oxidative damage promoted by oxygen/glucose deprivation and reoxygenation (OGD). GUO (100 μM) reduced reactive oxygen species production and prevented mitochondrial membrane depolarization induced by OGD. GUO also exhibited anti-inflammatory actions as inhibition of nuclear factor kappa B activation and reduction of inducible nitric oxide synthase induction induced by OGD. These GUO neuroprotective effects were mediated by adenosine A1 receptor, phosphatidylinositol-3 kinase and MAPK/ERK. Furthermore, GUO recovered the impairment of glutamate uptake caused by OGD, an effect that occurred via a Pertussis toxin-sensitive G-protein-coupled signaling, blockade of adenosine A2A receptors (A2AR), but not via A1 receptor. The modulation of glutamate uptake by GUO also involved MAPK/ERK activation. In conclusion, GUO, by modulating adenosine receptor function and activating MAPK/ERK, affords neuroprotection of hippocampal slices subjected to OGD by a mechanism that implicates the following: (i) prevention of mitochondrial membrane depolarization, (ii) reduction of oxidative stress, (iii) regulation of inflammation by inhibition of nuclear factor kappa B and inducible nitric oxide synthase, and (iv) promoting glutamate uptake.
Cerebral ischemic injury remains a leading cause of death and disability in industrialized countries. The lack of oxygen and glucose because of ischemic injury results in a cascade of events such as loss of mitochondrial membrane potential and reduction in ATP production that impairs ATP-dependent processes as Na+/K+ ATPase activity. These events result in disruption of membrane potential triggering a release of excitatory amino acids such as glutamate (Bonde et al. 2003). Excessive extracellular glutamate leads to overstimulation of glutamate receptors and consequent influx of Na+, Cl−, and Ca2+ ions through the channels gated by those receptors. The increase in Ca2+ levels inside the cell results in activation of Ca2+-dependent enzymes which leads to processes such as proteolysis, hydrolysis, lipid peroxidation, and reactive oxygen species (ROS) production (Choi 1988). Glutamate uptake is a crucial process to maintain extracellular glutamate concentrations below toxic levels. This effect is achieved through specific high-affinity sodium-dependent excitatory amino acid transporters that are mainly present in astrocytes. Glutamate transporters are modulated by the cell redox status; thus, increased ROS production may result in glutamate uptake impairment (Trotti et al. 1998).
Excessive ROS production during an ischemic event can trigger an inflammatory response through activation of the transcriptional nuclear factor kappa B (NF-κB) (Gloire et al. 2006). NF-κB dimer is retained in the cytosol by interacting with inhibitory IκB proteins. When active, NF-κB subunit translocates to the nucleus and promotes expression of a large number of genes related with pathology of cerebral ischemia, including those involved in inflammatory response, such as interleukin-1, tumor necrosis factor-alpha and enzymes like inducible nitric oxide synthase (iNOS) and cyclooxigenase-2 (Sethi et al. 2008). The iNOS isoform is a mediator of inflammatory reactions and may catalyze substantial synthesis of nitric oxide (NO) in the injured brain, thus contributing to glutamate excitotoxicity (Bal-Price and Brown 2001).
There are increasing evidences that point to the importance of purines during hypoxic/ischemic events (Thauerer et al. 2012). For example, guanine derivatives can be released from astrocytes when subjected to hypoxia or hypoglycemia (Ciccarelli et al. 1999). Furthermore, guanine nucleotides and nucleoside levels increase during and after hypoxic or hypoglycemic periods. In the particular case of guanosine (GUO), its levels increase progressively under hypoxic or hypoglycemic conditions as a result of extracellular hydrolysis of nucleotides like as GTP, GDP, and GMP (Ciccarelli et al. 2001).
Extracellular effects of GUO have been implicated in neuroprotection by counteracting glutamate excitotoxicity. It has been shown that GUO protects from seizures induced by quinolinic acid in vivo (Schmidt et al. 2000; de Oliveira et al. 2004), and it exerts neuroprotection in in vivo and in vitro models of ischemia (Oleskovicz et al. 2008; Rathbone et al. 2011). Moreover, GUO protects hippocampal slices from glutamate-induced cell death by a mechanism that involves reduction in iNOS expression (Molz et al. 2011).
Despite the evidence showing that GUO displays a relevant neuroprotective effect in different neurotoxicity models (for review, see Schmidt et al. 2007), its extracellular site of interaction and its intracellular signaling pathway have not yet been fully characterized. It is suggested that GUO modulates cell proliferation, neurite outgrowth and cellular protection by a mechanism that involves adenosine receptors activation (Ciccarelli et al. 2000; Thauerer et al. 2010; Dal-Cim et al. 2012). However, GUO and GTP bind to adenosine receptors with a very low affinity (Muller and Scior 1993). Interestingly, in a recent study, we have showed that GUO is able to afford protection to hippocampal slices against oxygen/glucose deprivation and reoxygenation (OGD) by stimulating glutamate uptake, which is dependent on large conductance Ca2+-activated K+ (BK) channels and phosphatidylinositol-3 kinase (PI3K) pathway activation, (Dal-Cim et al. 2011). Taken together, these findings suggest that GUO may have distinct cellular targets besides the already known purinergic receptors.
The results of this study show that GUO, by modulating adenosine receptors and activating the MAPK extracellular-signal regulated kinase (ERK) kinase (MEK), promotes neuroprotection of hippocampal slices subjected to OGD. The neuroprotective mechanism of action of GUO involves reduction ROS production, prevention of mitochondrial membrane potential disruption, inhibition of NF-κB and iNOS induction, and promotion of glutamate uptake. Some of the data present here have appeared in the form of an abstract (Tasca et al. 2011).
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- Materials and methods
This study shows for the first time that guanosine protects hippocampal slices against oxidative and inflammatory processes induced by OGD via a mechanism that involves MAPK/ERK and A1R activation. Moreover, guanosine attenuates OGD-induced glutamate uptake impairment by activating MAPK/ERK, Gi/Go-proteins-coupled signaling and blockade of A2A adenosine receptors.
Disruption of mitochondrial membrane potential is a marker of mitochondrial dysfunction that contributes to cell death by reducing ATP production, increasing ROS production and releasing signaling molecules that regulate apoptotic cell death (Christophe and Nicolas 2006). The regulation of mitochondrial function via therapeutic approaches may facilitate neuroprotective cell responses. Recently, we demonstrated that GUO protects SH-SY5Y neuroblastoma cells from oxidative damage induced by mitochondrial activity impairment (Dal-Cim et al. 2012). Here, we show that GUO prevents disruption of the mitochondria potential induced by OGD in hippocampal slices. These effects point to the fact that GUO plays an important role in mitochondrial homeostasis in events that cause energetic unbalance. Reinforcing this notion, we show that GUO reduced ROS production induced by OGD. The precise mechanism by which GUO exerts these effects is not yet completely understood, but evidence suggests that it involves heme oxygenase-1 expression, via PI3K pathway, A1R and A2AR modulation and also BK channel activation (Dal-Cim et al. 2011, 2012).
In this study, we have found that the PI3K pathway does not directly mediate GUO′s protective effect on oxidative damage induced in an in vitro model of brain ischemia. We have also observed that BK channels mediated GUO-induced protective effect only against ROS production in the CA1 region of hippocampus, which usually is the most susceptible hippocampal region in an ischemic insult (Kirino and Sano 1984; Stanika et al. 2010). However, GUO maintained the mitochondrial potential and decreased ROS levels via activation of MAPK/ERK and A1R in the CA1 region of the hippocampus, indicating that GUO may have distinct cellular targets and mechanisms of action depending on the intensity of the insult and the seriousness of the cellular consequence observed.
The A1R is traditionally coupled to members of Pertussis toxin-sensitive family of G-proteins (Gi/Go) and their activation inhibits the adenylyl cyclase activity or modulates calcium channels activity. Activation of A1R at nerve endings inhibits transient calcium channels, whereas the same receptor in nerve cell bodies and dendrites may preferentially regulate potassium channels conductance (Boison et al. 2010). Thus, it is feasible that GUO could modulate A1R activity promoting a restoration of ionic gradient that was disrupted after OGD in ischemic events. Furthermore, intracellular signaling activated by A1R could contribute to GUO-induced neuroprotection.
Previous reports have shown that A1R activation is linked to modulation of PI3K and MAPK/ERK pathways (Jacobson and Gao 2006; Thauerer et al. 2012). Notwithstanding, activation of MEK in ischemic events is controversial. Studies have reported the involvement of MEK activation with pro-inflamatory processes and cell death following ischemia (Alessandrini et al. 1999; Maddahi et al. 2011). However, it was also demonstrated that MAPK/ERK pathway was involved in purine nucleoside-mediated protection of hippocampal slices and astrocytic or neuronal cells following hypoxic insults (Ciccarelli et al. 2007; Oleskovicz et al. 2008; Tomaselli et al. 2008). The results obtained in this study reinforce the importance of MAPK/ERK intracellular signaling activation by purine nucleosides, specifically GUO, since we demonstrate that blockade of MEK prevented GUO-induced neuroprotection against oxidative damage, impairment of glutamate uptake and inflammatory process via NF-κB and iNOS, which were induced by ischemia.
The transcriptional factor NF-κB is considered the major inflammatory mediator in neuronal tissue. Its activation during brain ischemia has been related to excessive ROS production. Activation of NF-κB leads to nuclear translocation of the p65 and/or p50 subunits to modulate the transcription of NF-κB responsive genes such as IL-6 and iNOS (Madrigal et al. 2006). The participation of GUO in inflammatory processes was previously reported by D′Alimonte that showed that GUO treatment (300 μM) inhibits TNF-α and amyloid-β peptide-induced p65 phosphorylation in mouse microglial cells (D'Alimonte et al. 2007). In this study, we show that lower concentrations of GUO (100 μM) are also capable of decreasing nuclear p65 expression induced by an oxidative insult because of OGD in hippocampal cells. Furthermore, analysis of the expression of iNOS, a NF-κB responsive gene, showed that GUO treatment could reduce iNOS levels under OGD conditions. Induction of iNOS results in excessive amounts of NO that changes its physiological neuromodulatory actions to neurotoxic effects such as inhibition of the complex-I of the electron transport chain. In addition, NO may react with superoxide anions to form nitrite peroxide, a strong pro-oxidant, that ultimately leads to irreversible cellular damage (Brown 2010). Thus, inhibition of iNOS has been proposed as an important therapeutic strategy against ischemic insults (Iadecola and Ross 1997; Moro et al. 2004). The mechanism elicited by GUO to counteract NF-κB activation and iNOS induction was mediated by the transduction signaling A1R and MAPK/ERK.
Several evidence support the notion that the neuroprotective effect of GUO against excitotoxicity is related to the modulation of glutamate uptake (Oliveira et al. 2004; Moretto et al. 2005; Schmidt et al. 2007), thus facilitating the clearance of glutamate from the synaptic cleft. Recently, we demonstrated that GUO treatment prevents glutamate release induced by glutamate excitotoxicity and it also increases glutamate uptake into hippocampal slices subject to OGD, effects that are mediated by activation of PI3K pathway (Dal-Cim et al. 2011; Molz et al. 2011). Here, we observed that GUO-induced stimulation of glutamate uptake was totally prevented by MEK inhibition. Thus, it seems that the effect of GUO on glutamate uptake may be related to its ability to reduce ROS production, since MAPK/ERK pathway also mediates GUO′s action on oxidative damage induced by OGD. In addition, it has been reported that signaling pathways such as PI3K and MAPK/ERK are involved in regulation of glutamate transporters activity, expression and insertion in the cell membrane (Li et al. 2006; Frizzo et al. 2007; Dal-Cim et al. 2011) thus, MEK activation by GUO could result in modulation of glutamate transporters activity, contributing in this way to its neuroprotective profile.
Notably, we show that the action of GUO on glutamate uptake is not related to its putative interaction with A1R. On the other hand, we previously demonstrated that increased glutamate uptake induced by GUO depends on BK channel activation (Dal-Cim et al. 2011), suggesting that GUO may display distinct cellular effects regarding its extracellular interaction site. Modulation of BK channels by GUO can result in a decline of intracellular Ca2+ levels, thus avoiding the loss of cell membrane potential, a critical event for maintaining the activity of glutamate transporters (Danbolt 2001). Moreover, A1R activation is associated with decreased vesicular glutamate release in pre-synaptic terminals (Sperlagh and Vizi 2011), an effect that does not depend on modulation of cell membrane glutamate transporters. It has been recently demonstrated that A2AR activation in cultured astrocytes, decreases glutamate uptake while activation of A1R has no effect on the modulation of glutamate transport (Matos et al. 2012). These evidences reinforce the idea that activation of A1R does not modulate the activity of glutamate transporters. Surprisingly, we have observed that A2AR stimulation blocks the effect of GUO on glutamate uptake in hippocampal slices subjected to OGD, suggesting that cellular signaling triggered by the activation of A2AR combined with the energy imbalance and oxidative damage caused by OGD dramatically affect the ability of GUO to recover the impaired glutamate transporters activity.
A1R and A2AR may closely interact through the formation of oligomers and in this heteromeric organization, they can modulate glutamate release from pre-synaptic terminals in the striatum (Ciruela et al. 2006), or GABA uptake into cultured cortical astrocytes (Cristóvão-Ferreira et al. 2011). It has been proposed that when A1R and A2AR are forming an oligomer, a hierarchical activation of A2AR over A1R will result in the functional tuning of purinergic transmission (Ciruela et al. 2012). In this scenario, GUO may act as an antagonic A2AR modulator, although it is surprising that GUO effect on glutamate uptake was dependent on Gi-protein coupled signaling, whereas A1R blockade did not alter GUO effect on glutamate uptake. On the other hand, it seems plausible that the blockade of A2AR activation would be involved on GUO effect, since it has been shown that A2AR antagonists may exert neuroprotective actions (Cunha 2005; Gomes et al. 2011).
Although our data strongly indicate an interaction among GUO and adenosine receptors (and in earlier studies, with the BK potassium channel), we cannot exclude the possibility that GUO acts through a selective receptor and secondarily modulates the activity of adenosine receptors and BK channels. Studies supporting this hypothesis have demonstrated specific putative binding sites for GUO (Tasca et al. 1999; Gysbers et al. 2000; Traversa et al. 2002; Volpini et al. 2011). Supporting this idea, we have seen that inhibition of Gi/Go-protein by Pertussis toxin reverses the effect of GUO on cell viability and glutamate uptake in hippocampal slices subject to OGD, indicating that the interaction site of GUO couples to a Gi/Go-protein. Still, the exact mechanism of GUO action on BK channels and/or adenosine receptors remains to be determined.
Concluding, GUO is neuroprotective against oxidative and inflammatory processes induced by ischemia via activation of A1R and MAPK/ERK pathway. GUO also prevented glutamate uptake impairment by modulation of A2A adenosine receptors and a pathway involving Gi/o-proteins-coupling and MAPK/ERK signaling (Fig. 8). The molecular targets and signaling pathways recruited by GUO to display its neuroprotective effects are still under evaluation in our laboratory.
Figure 8. Proposed mechanism of cellular protection afforded by GUO against oxygen/glucose deprivation (OGD) in hippocampal slices. GUO reduces reactive oxygen species levels, maintains mitochondrial membrane potential and inhibits nuclear factor kappa B activation through adenosine A1 receptor (A1R) and MAPK/ERK activation (thick blue arrows). GUO also counteracts inducible nitric oxide synthase induction via A1R, MAPK/ERK (thick blue arrows), and PI3K pathways activation (dotted black arrow). Previous study from our laboratory (Dal-Cim et al. 2011) demonstrated that GUO-induced recovery of decreased glutamate uptake because of OGD involves large conductance Ca2+-activated K+ channels (BK) and PI3K/Akt pathway activation (thin black arrows). In this study, the stimulation of glutamate uptake evoked by GUO involves Gi/o-proteins-coupling and MAPK/ERK signaling (dashed red arrow). Curiously, A1R blockade does not interfere with and A2A receptor (A2AR) activation abolishes GUO effect on glutamate uptake. A putative oligomeric interaction between adenosine receptors may be involved in this GUO effect (small blue arrow between A1R and adenosine A2AR). In this scenario, GUO may act as an antagonic A2AR modulator. Our data strongly indicate an interaction among GUO and adenosine receptors; however, the possibility that GUO acts through a selective receptor and secondarily modulates the activity of adenosine receptors and BK channels cannot be completely excluded. The exact mechanism of GUO action on BK channels and/or adenosine receptors modulation is under investigation.
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