Localization and co-localization
This is the first study on A1R and P2Y1R localization and co-localization in hippocampus by postembedding immunogold electron microscopy analysis. This high resolution technique is able to show the precise subcellular localization of receptors in different subcellular compartments and cell populations (e.g. Bergersen et al., 2003). The purinergic receptors proved to be mainly associated with membrane domains. Single-labelling immunolocalization data showed a significant enrichment of both A1R and P2Y1R mainly in postsynaptic membranes at the PSD, in presynaptic active zones, and in astroglial membranes at glutamatergic synapses and surrounding glia in the rat hippocampus. The same conclusion was arrived at by analysing the data in different ways, in order to partly overcome the limits posed by ‘cross firing’ effects antigen located in closely spaced neighbouring membranes. Because of these, the exact labelling densities of the individual membranes cannot be determined. While the three membrane categories mentioned contain higher levels of both of the purinergic receptors studied, low or moderate labelling may exist in other membrane categories. The data suggest that there may be relatively higher densities of A1R than of P2Y1R at the presynaptic compared to the postsynaptic membrane. Part of this observed difference might be attributable to the fact that the antibodies were to extracellular and intracellular epitopes, respectively, although the inner and outer surfaces of the plasma membrane are only 4–5 nm apart, i.e. an order of magnitude less than the lateral resolution of the immunogold method. However, the main conclusion is that the two receptor types are similarly distributed, compatible with a high degree of co-localization. This was born out by double-labelling experiments that showed A1R and P2Y1R to be closely spaced along synaptic and glial membranes.
In parallel, the specificities of A1R and P2Y1R antibodies used in immunohistochemistry, were assessed by immunoblotting assay using rat whole brain and hippocampus membrane fractions. The obtained results were according to literature data (Hoffmann et al., 1999; Blum et al., 2002; Moore et al., 2002; Waldo & Harden, 2004) and confirmed that antibodies selectively recognized A1 and P2Y1 receptors in hippocampal membranes.
Our high resolution data serve to extend and reconcile previous reports obtained with lower resolution methods. Thus A1R immunoreactivity has been reported in hippocampus both at presynaptic and postsynaptic terminals but not at glial cells (Ochiishi et al., 1999); an immunohistochemical study in the rat hippocampus has concluded that A1Rs were mostly located in axons rather than in nerve terminals (Swanson et al., 1995), whereas work on synaptosomal fractions (Rebola et al., 2003) has suggested that A1Rs are enriched in nerve terminals and are mainly located in synapses, both in the presynaptic active zone and in the PSD membranes. P2Y1R immunoreactivity has been found in astroglia and in different kinds of neurons in hippocampus (Moran-Jimenez & Matute, 2000), especially in ischemic sensitive areas while at the same time another study reported a striking neuronal localization for P2Y1R (human brain, Moore et al., 2000). P2Y1Rs have been reported to be present and active on astrocytes all around the brain (Franke et al., 2001; Volontéet al., 2006). A high degree of co-localization of A1R and P2Y1R has been found in rat hippocampus by immunofluorescence experiments but without cellular and subcellular identification (Yoshioka et al., 2002).
We studied A1Rs and P2Y1Rs in the hippocampal region considering the fact that the hippocampus has been identified as a major target site for numerous disease processes (Bachevalier & Meunier, 1996; Harry & Lefebvre d'Hellencourt, 2003), and considering the generally assumed involvement of purinergic receptors in patho-physiological mechanisms and in the modulation of brain damage (Fredholm, 1997; Franke et al., 2006b). Ischemia, to which hippocampus is particularly vulnerable, produces a marked increase in glutamate within the brain extracellular space (Benvensiste et al., 1984; Hagberg et al., 1985), thereby triggering excitotoxic injuries (Choi & Rothman, 1990). Because of the importance of glutamate in pathological conditions and because its release, in neurons and in astrocytes, is modulated both through A1R (Rudolphi et al., 1992; Masino et al., 2002) and P2Y1R (Rodrigues et al., 2005; Franke et al., 2006a; Jourdain et al., 2007), the present study was focused on A1R–P2Y1R localization and co-localization within and in the vicinity of glutamatergic synapses.
Our results provide direct morphological support for the previous suggestions that both of these receptors contribute to and interact in the modulation of glutamate release (Rudolphi et al., 1992; Mendoza-Fernandez et al., 2000; Masino et al., 2002; Kawamura et al., 2004; Rodrigues et al., 2005).
The functional interaction of A1Rs and P2Y1Rs suggested by the morphological observations was subsequently confirmed through measurement of G protein activation initiated by the A1R agonist, CHA, or the P2Y1R agonist, MeSADP, respectively, and modification of the response through pre-incubation with the other agonist. Because the receptors on study are coupled to different G protein subtypes (Munshi et al., 1991; Yoshioka & Nakata, 2004) and to different intracellular signalling pathways, the [35S]GTPγS binding method was chosen to investigate the A1R–P2Y1R interaction and their reciprocal modulation at the membrane level, allowing any change in their functioning to be determined independently of the second messenger systems activated (Lorenzen et al., 1996).
According to literature data (Gao et al., 2003; Dixon et al., 2004; Niebauer et al., 2005), the selected agonist pre-incubation times and concentrations pre-stimulating A1R and P2Y1R (100 nm CHA and 100 nm MeSADP, respectively) allow a selective and maximal receptor activation.
The EC50 for CHA in stimulating A1R-G protein coupling was around 30 nm. Although CHA has been reported to block [3H]DPCPX binding at A1R with a Ki of approximately 5 nm in rat hippocampus (Maemoto et al., 1997), according to our data, higher EC50 values for different A1R agonists in the [35S]GTPγS binding assay have been found (Lorenzen et al., 1996; Cordeaux et al., 2004). In order to obtain a selective stimulation of A1R, without interference of other receptor subtypes, we have chosen treatment of hippocampal membranes with 100 nm agonist CHA. In fact in hippocampus, although A1Rs are the mainly expressed adenosine receptors, the presence of low amounts of A2A and A3 receptors have been described (Duarte et al., 2006; Lopes et al., 2003). Nevertheless, these receptors have a different agonist pharmacological profile; CHA, in fact, shows a high affinity towards A1Rs (Murphy & Snyder, 1982; Maemoto et al., 1997) and a low affinity towards A2A ones (Cunha et al., 1996; Gao et al., 2003). Even if, in our experimental conditions, the EC50 for CHA in stimulating A1R-G-protein coupling was around 30 nm, the real binding potency of CHA to A1R sites is around a few nanomolar units or indeed in the subnanomolar range in rat hippocampus, so the agonist at 100 nm is able to selectively saturate A1R binding sites. This was confirmed by an antagonist dose–response curve, DPCPX was able to completely abrogate the effects mediated by CHA from 1 to 100 nm. At higher agonist concentrations the antagonistic effect of DPCPX was reduced suggesting that the agonist binds to a different receptor population, probably identifiable with A2A receptor sites.
On the other hand, in our results, MeSADP showed subnanomolar potency in stimulating P2Y1R. Because of the absolute potency of nucleoside tri- and diphosphates for P2Y receptors is dependent on the levels of receptor protein expression, typical EC50 values are not easily defined for specific agonists at particular P2Y receptor subtypes in different tissue preparations and cell lines (Volontèet al., 2006). In accordance with our results, low nanomolar and subnanomolar EC50 values have been reported for MeSADP towards human P2Y1R, expressed in astrocytoma 1321 N1 cells (Palmer et al., 1998; Niebauer et al., 2005), and rat P2Y1R, expressed in HEK 293 cells (Vohringer et al., 2000).
MeSADP is the principal agonist not only at P2Y1R but also at P2Y12−13 receptors, that are coupled to GI proteins and are expressed (mRNA) in the brain (Hollopeter et al., 2001; Zhang et al., 2002; Sasaki et al., 2003), even if not at high level in the hippocampus (Fumagalli et al., 2004; Amadio et al., 2006). To confirm that in our model the MeSADP-mediated G-protein activation was mainly driven by P2Y1R, we also stimulated hippocampal membranes in the presence of the selective P2Y1R antagonist, MRS2179. MRS2179 was able to block the MeSADP-mediated response, confirming the P2Y1R involvement as no antagonistic effects have been demonstrated on P2Y12−13 receptors at the MRS2179 concentrations used (Moro et al., 1998; Von Kugelgen, 2006).
The results obtained on A1R–P2Y1R cross-talk in hippocampus showed that, stimulating one receptor, the functioning of the other was changed. In particular, P2Y1R pre-activation caused an impairment in A1R–G protein coupling with a reduction in A1R agonist potency; on the other hand, A1R pre-activation induced an increase in P2Y1R functional coupling to G proteins. Our results are in agreement with the previously reported reduction in the A1R ligand affinities in cells co-expressing both A1R and P2Y1R (Yoshioka et al., 2001). Various studies have reported that ATP, massively released after brain damage, acts to modulate not only its own P2Y1R but also A1R (Hourani et al., 1991; Piper & Hollingsworth, 1996; Masino et al., 2002; Fredholm et al., 2003; Yoshioka & Nakata, 2004).
The functional consequence of this A1–P2Y1 receptor cross-talk is complicated by the availability time and the balance of their endogenous ligands. Extracellular ATP, rapidly available due to direct release into the extracellular space, and adenosine, available after ATP breakdown, are tightly regulated by rapid metabolism and re-uptake (Zimmermann, 2000) and can be differently regulated in physiological or pathological conditions; in fact the ecto-nucleotidase chain has proved to be up-regulated in ischemically damaged tissues (Braun et al., 1998).
Data, at present, have shown the A1R–P2Y1R interaction mechanism may be used to fine-tuning the purinergic signalling, including the inhibition of neurotransmission (Nakata et al., 2004). Considering the new information available and the A1R and P2Y1R involvement in glutamatergic transmission modulation (Mendoza-Fernandez et al., 2000; Masino et al., 2002; Kawamura et al., 2004; Rodrigues et al., 2005), we can speculate that there is an A1R–P2Y1R cross-talk in rat hippocampal glutamatergic synapses and surroundings glia, where these receptors are co-localized. This might therefore be one of the mechanisms for the adenine nucleotide-mediated inhibition of glutamatergic neurotransmitter release. Therefore, as suggested for adenosine A1 and A2A receptors in striatal (Ciruela et al., 2006) and hippocampal (Rebola et al., 2005) glutamatergic nerve terminals, a cross-talk/heteromerization of A1R–P2Y1R could exert a fine-tuning modulation of glutamatergic neurotransmission, providing a switch mechanism by which low and high concentrations of adenosine or purines could regulate glutamate release.
Because of the high level of complexity of purinergic receptor signalling (Volontèet al., 2006) and the regulation of glia–neuron and glia–glia communications by extracellular purines (Franke et al., 2006b; Jourdain et al., 2007), the present work opens the way to further investigation of the A1R–P2Y1R system interaction on astrocyte cell populations, which communicate bi-directionally with neurons (Newman, 2003; Bezzi et al., 2004; Jourdain et al., 2007) and contribute to damage or to regeneration after CNS injury (Franke et al., 2001; Anderson et al., 2003).