The endogenous guanine-based nucleoside, guanosine (GUO), plays a key role in cells both as a regulatory element and as a structural component. In the central nervous system (CNS) the extracellular guanosine (eGUO) concentration under basal conditions is around 500 nM (Zetterstrom et al. 1982) and this is achieved by the action of 5′-ectonucleotidases that metabolize guanine nucleotides and by neurons and glial cells (i.e. astrocytes and oligodendrocytes), which are able to release it. eGUO displays a large array of biological effects including mitosis stimulation, synthesis and release of trophic factors (e.g. nerve growth factor, fibroblast growth factor, and adenosine), and induction of cellular differentiation, within others (Rathbone et al. 2008). Interestingly, under neuropathological conditions the eGUO concentration can largely increase (Ciccarelli et al. 1999), thus a good correlate between eGUO concentration and excitotoxic conditions could be established. Indeed, it has been demonstrated that GUO is neuroprotective upon several stimuli, for instance GUO protects from seizures induced by several glutamatergic agents, it exerts neuroprotection in ischemia models and also GUO chronic treatment is anxiolytic (for review see (Schmidt et al. 2007). In addition, GUO can induce glutamate uptake after ischemic damage (Schmidt et al. 2007). Therefore, a regulatory role of GUO controlling extracellular levels of glutamate has been demonstrated, thus contributing to the protection of neural cells against glutamate-mediated excitotoxicity.
GUO effects are both dependent and independent of intracellular mechanisms, and thus some of the extracellular effects of this nucleoside have been clearly related to neuroprotection in different neurotoxicity models (Schmidt et al. 2007). Despite this, the precise signalling pathways by which eGUO triggers neuroprotection were not fully explored. In the article by Dal-Cim et al. (2013) in the present issue of the Journal of Neurochemistry, the authors demonstrate that GUO neuroprotects hippocampal slices against oxygen/glucose deprivation (OGD)/reoxygenation-induced neuronal death by synchronizing several intricate signalling pathways. Consequently, the mechanism behind this GUO-mediated neuroprotection implicates various neural functions, thus it involves a reduction in the reactive oxygen species (ROS) production, it prevents mitochondrial transmembrane potential disruption, it mediates the inhibition of nuclear factor kappa B (NF-κB) activation, it reduces the induction of the inducible nitric oxide synthase (iNOS) and also it promotes glutamate uptake (Dal-Cim et al. 2013) (Fig. 1). In a comprehensive sequence of experiments the authors demonstrated the involvement of GUO in hippocampal neuroprotection. Accordingly, they found that the presence of GUO (100 μM) during the reoxygenation period prevented loss of mitochondrial membrane potential and decreased ROS production in hippocampal slices subjected to OGD. Interestingly, this effect was precluded when adenosine A1 receptors (A1Rs) were blocked. In an attempt to elucidate the mechanism behind this phenomenon the authors corroborated that this GUO-mediated neuroprotective effect was mitogen-activated protein kinase (MAPK)/ERK-driven. Further experiments also demonstrated that GUO prevented OGD-induced inflammatory responses involving NF-κB activation. Thus, the presence of GUO during the reoxygenation period subsequent to OGD reduced the translocation of the NF-κB active subunit into the nucleus. In addition, GUO was also able to reduce OGD-mediated iNOS induction. Interestingly, both phenomena were mediated by the A1R-MAPK/ERK signalling pathway. Similarly, the authors explored the involvement of the phosphatidylinositol-3kinase (PI3K)/Akt signalling pathway, which was also shown to play a key role in the GUO-mediated neuroprotection (Oleskovicz et al. 2008). Interestingly, this signalling pathway was neither involved in the GUO control of mitochondrial membrane potential and ROS production nor in the NF-κB activation. However, the PI3K/Akt signalling pathway mediated in the OGD-induction of iNOS expression. Finally, the GUO-mediated decrease in ROS production upon OGD was precluded by blocking the large conductance Ca2+-activated K+ (BK) channels, thus a relationship between GUO and a putative BK/PI3K/Akt signalling pathway was postulated (Fig. 1).
It has been proposed that the neuroprotective effect of GUO may be related to its ability to modulate glutamate uptake (Dal-Cim et al. 2011). Consequently, the authors investigated the relationship between GUO-mediated neuroprotection and glutamate uptake in hippocampal slices. Indeed, in hippocampal slices GUO was able to restore the OGD-mediated glutamate uptake impairment. Interestingly, this effect was blocked by pertussis toxin, thus suggesting the involvement of a Gαi/o-protein coupled signalling pathways. In the light of their previous results Dal-Cim et al. explored the involvement of A1R, an adenosine Gαi/o-protein coupled receptor, in the GUO-induced stimulation of glutamate uptake under OGD conditions. Surprisingly, this GUO effect was MAPK/ERK-driven but A1R independent, thus suggesting a differential signalling fingerprint when compared to the GUO-mediated neuroprotection against OGD-induced oxidative and inflammatory processes. Finally, the authors evaluated the role of A2AR in this GUO-induced stimulation of glutamate uptake upon OGD conditions as this adenosine receptor (AR) has been also described to modulate glutamate uptake (Ciruela et al. 2006). Interestingly, A2AR activation counteracted GUO-induced neuroprotection and stimulation of glutamate uptake upon OGD. Overall, these results suggested a genuine and complex functional interplay between GUO and ARs (Fig. 1).
Dal-Cim's work has aided to decipher some of the signalling pathways by which GUO exerts its neuroprotective effect in hippocampal slices (Fig. 1). Despite this, the precise mechanism of action of eGUO still unknown and the experimental evidences point to a complex relationship between several signalling events. Likewise, the findings presented by Dal-Cim and colleagues (Dal-Cim et al. 2013) raise a number of interesting possibilities, first the existence of specific GUO G protein-coupled receptors (GRs), second the interaction of GUO with BK channels and third the co-existence of eGUO-mediated trophic effects. Thus, at this point we cannot discard any of these possibilities and more work is needed to unravel the precise mechanism of action of the eGUO.
The GRs existence has been postulated for more than a decade. Indeed, the presence of a single high affinity binding site for [3H]-GUO with a KD of 95 nM and Bmax of 0.6 pmol/mg protein has been described in rat brain membranes (Traversa et al. 2002). In addition, more recently it has been demonstrated that GUO is able to activate a putative yet unknown G-protein coupled receptor in rat brain membranes different from the well-characterized ARs (Volpini et al. 2011). However, nowadays the big question still open: why GUO still is an orphan neuromodulator? or in other words, why GRs have not been cloned yet? A number of tantalizing mechanistic implications come to mind in light of Dal-Cim's work. We can speculate that if a new – not yet discovered – GR exists this would share some particular features with ARs, and issue always controversial as some contradictory results has been described. Alternatively, if putative GRs do not exist it could be the case that GUO might signals through existing receptorial complexes containing A1R and/or A2AR (i.e. ARs-containing oligomers). This somehow could explain the divergences observed in the GUO intrinsic activity related to prototypical ARs. Therefore, follow-up experiments should now focus both in the ability of GUO to modulate AR-containing oligomers and in the search of yet uncloned GRs. In the same line of inquiry, further experimentation is needed to uncover the mechanism of GUO-mediated BK channel activation as this might constitute an independent and genuine mechanism behind the GUO-mediated neuroprotection. Taken together, the findings presented here suggest that eGUO may have distinct cellular targets besides the already known prototypical ARs. However, it is important to mention here that until the eGUO mechanism of action (i.e. GUO receptorial proteins) is not found, the formal acceptance of a GUO-based purinergic system in the mammalian CNS is difficult to assume.