Introduction

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ATP and other adenine nucleotides behave as neurotransmitters in the central nervous system (Edwards et al., 1992; Evans et al., 1992). Among these, the diadenosine polyphosphates (Ap_nA), formed by two adenosines joined by a variable numbers of phosphates (for review McLennan, 1992), have been also described as neurotransmitters (for review Pintor et al., 2000). These compounds are costored with ATP and other neurotransmitters in synaptic vesicles and released after synaptic terminal stimulation (Richardson and Brown, 1987, Pintor et al., 1992). Nucleotide signaling on the cell surface is mediated through P2 receptors, classified in two families with a large number of subtypes (for review Fredholm et al., 1994; Ralevic and Burnstock 1998). As the response to dinucleotides is not inhibited by ATP and analogs, a specific receptor for these compounds has been proposed. However, responses mediated through P2 receptor subtypes, which do not respond to ATP, are not excluded for dinucleotides (Pintor and Miras-Portugal 1995; Pivorun and Nordone, 1996).

The presence of specific dinucleotide receptor has been described in rat midbrain and guinea pig and mouse synaptic terminals (Pintor and Miras-Portugal, 1995; Pintor et al., 2000). This dinucleotide receptor is an ionotropic receptor, which can be modulated by the action of protein kinases and phosphatases. In this way, activator agents of protein kinase C and protein kinase A, such as phorbol esters or forskolin, produce a reduction of the dinucleotide response. Protein phosphatase inhibitors, such as okadaic acid and microcystin, also reduced the effect of Ap_nA through the dinucleotide receptor (Pintor et al., 1997b).

The dinucleotide response was also studied in hippocampus, showing that Ap₅A induce Ca²⁺ influx in CA3 hippocampal neurones and synaptosomes with a -conotoxin-sensitive component (Panchenko et al., 1996).

The extracellular action of ATP and Ap_nA is terminated by their extracellular hydrolysis through ecto-nucleotidases, with the final formation of adenosine (Mateo et al., 1997; reviewed by Zimmermann, 2000,). This compound is a neuromodulator on its own (reviewed by Cunha, 2001), finishing its actions by the equilibrative nucleoside transporter present at the synaptic terminals (Fideu et al., 1994).

Adenosine controls the release of neurotransmitters by activating presynaptic A₁ and A_2A receptors (Cunha, 2001) and eventually A₃ receptors, whose role is poorly understood. Adenosine also controls the action of several other presynaptic modulatory systems acting via protein kinase C or protein kinase A pathways (Díaz-Hernández et al., 2000; Cunha, 2001).

Since adenosine receptors modify the activity of protein kinases and the dinucleotide receptor is sensitive to those proteins, the modulation of this receptor by different subtypes of adenosine receptors is studied in this work.

	Experimental Procedures

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Synaptosomal Preparation. Synaptosomes were prepared from rat hippocampal cortices of cervically dislocated and decapitated male Wistar rats (6-7 weeks old). The isolation procedure was different depending on the aim of the preparations. Synaptosomes used in pharmacological studies were obtained according to Pintor and Miras-Portugal (1995). Synaptosomes used in calcium measurement assays in isolated single synaptic terminals and immunochemical analysis were obtained through a Percoll gradient, following the procedure described by Dunkley et al. (1986). All experiments carried out at the Universidad Complutense de Madrid followed the guidelines of the International Council for Laboratory Animal Science. Hippocampus was homogenized in a medium containing 0.32 M sucrose, pH = 7.4. The homogenate was spun for 5 min 900g at 4°C and the supernatant spun again at 9500g for 12 min. The pellets formed were resuspended in 8 ml of 0.32 M sucrose, pH = 7.4. Two milliliters of this synaptosomal suspension were placed onto 3 ml of Percoll discontinuous gradients containing 0.32 M sucrose; 1 mM EDTA; 0.25 mM DL-dithiothreitol; and 3, 10, or 23% Percoll, pH = 7.4. The gradients were centrifuged at 25,000g for 10 min at 4°C. Synaptosomes placed between the 10 and 23% Percoll bands were collected and diluted in 30 ml of HEPES buffer medium (140 mM NaCL, 5 mM KCl, 5 mM NaHCO₃, 1.2 mM NaH₂PO₄, 1 mM MgCl₂, 10 mM glucose, and 10 mM HEPES, pH = 7.4) prior to centrifugation at 22,000g for 10 min.

Ca²⁺ Measurements on Synaptosomal Population. Synaptosomal pellets containing 1 mg of protein were resuspended in 1 ml of Elliot's medium (122 mM NaCl, 3.1 mM KCl, 0.4 mM KH₂PO₄, 5 mM NaHCO₃, 1.2 mM MgSO₄, 10 mM glucose, and 20 mM TES buffer, pH 7.4).

Pharmacological Studies. The concentration-response curve for Ap₅A was obtained by testing concentrations of Ap₅A ranging from 10¹² to 10³ M. To test the ability of the P2 receptor antagonists, suramin (100 µM) and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 50 µM), to modify Ap₅A or ATP responses, the P2 receptor antagonists were added 2 min before addition of Ap₅A or ATP. The same protocol was used to test the ability of the dinucleotide receptor antagonist, Ip₅I (1 µM) (Pintor et al., 1997a), to modify Ap₅A or ATP responses.

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Microfluorimetrical Studies in Single Hippocampal Synaptosomes. As described in synaptosomal preparation, the hippocampal synaptosomal pellets containing 0.5 mg of protein were resuspended in 1 ml of incubation medium and loaded with FURA-2/AM (5 µM) for 1 h at 37°C. Synaptosomes were adhered to coverslips pretreated with poly-L-lysine and maintained for 45 min in solution to allow for the intrasynaptosomal hydrolysis of the FURA-2/AM. The coverslips were washed with phosphate-buffer saline (PBS; 137 mM NaCl, 2.6 mM KCl, 1.5 mM KH₂PO₄, 8.1 mM Na₂HPO₄, pH 7.4) medium and mounted in a small superfusion chamber in the stage of a NIKON TE-200 microscope (Nikon, Tokyo, Japan). Synaptosomes were then superfused at 1.2 ml/min with HEPES buffer medium and Ap₅A and ATP at 100 µM concentration in the same medium. A pulse of 30 mM KCl was applied at the end of each experiment to confirm the viability of the synaptosomes under study. The perfusion system avoids the actions due to the "in situ" generation of ATP or Ap₅A metabolites (Díaz-Hernández et al., 2000).

Immunocytochemical Identification of A₁, A_2A, and A₃ Adenosine Receptors on Hippocampal Synaptosomes. After the image experiments, the synaptosomes were treated with paraformaldehyde at 4%, washed twice with PBS medium, and incubated in PBS containing 3% bovine serum albumin (BSA), 0.1% Triton X-100, and 5% normal rat serum for 1 h. The synaptosomes were then washed twice with PBS and incubated with mouse antisynaptophysin antibody (2 µg/ml) and goat polyclonal antibodies anti-A₁, A_2A, or A₃ receptors (0.2 µg/ml) for 1 h at 37°C. The synaptosomes were then washed three times with PBS/BSA (3%) and were incubated for 1 h at 37°C with a rabbit anti-mouse IgG antibodies labeled with fluorescein isothiocyanate (40 µg/ml) and donkey anti-goat IgG antibodies labeled with tetramethylrhodamine isothiocyanate (40 µg/ml). Then, the synaptosomes were washed three times with PBS/BSA (3%) and mounted following standard procedures. Controls were performed by following the same procedure but substituting synaptophysin by PBS in the presence of 3% BSA. Eight bit images were recorded for immunocytochemical analysis. Therefore, synaptosomes were considered positive for the different antibodies if the mean intensity value was >170 on a 0 to 255 scale, with 0 = white and 255 = black. The cut off value of 170 was determined from visual analysis of immunolabeling and by comparison with control (maximal level obtained with preabsorbed antibodies).

Identification of A₃ Receptor Binding Sites in Hippocampal Nerve Terminals. Membranes from whole rat hippocampus or from hippocampal synaptosomes were prepared as previously described (Cunha et al., 1996) and resuspended in a reaction buffer containing 50 mM Tris and 2 mM MgCl₂, pH 7.4. This reaction buffer also contained the A₁ receptor antagonist, DPCPX, and the A_2A receptor antagonist, ZM 241385 (50 nM). Binding of [¹²⁵I]4-aminobenzyl-5'-N-methylcarboxamideoadenosine ([¹²⁵I]AB-MECA) was for 60 min at 25°C with 620 to 810 µg of membrane protein in a final volume of 250 µl in the reaction buffer, essentially as described previously (Jacobson et al., 1993). Specific binding was determined by subtraction of the nonspecific binding, which was measured in the presence of 100 µM 2-chloroadenosine. The binding reactions were stopped by vacuum filtration through Whatman GF/C glass fiber filters (Whatman, Inc., Clifton, NJ), followed by washing of the filters and reaction tubes with 8 ml of reaction buffer, kept at 4°C. Radioactivity bound to the filters was determined in a gamma counter. Saturation curves were performed in duplicate with six different [¹²⁵I]AB-MECA concentrations ranging from 0.15 to 4.5 nM. Competition curves were performed in duplicate with 0.5 nM [¹²⁵I]AB-MECA and eight different concentrations of competitors ranging from 0.1 nM to 10 µM.

Materials. ATP, Ap₅A, CPA, CGS 21680, and the antibody antisynaptophysin SY-38 were obtained from Roche Applied Science (Indianapolis, IN); suramin was obtained from Bayer AG (Wuppertal, Germany); PPADS, DPCPX, and Cl-IB-MECA were purchased from Sigma/RBI (Natick, MA); ZM 241385 was obtained from Tocris Cookson Ltd. (Bristol, UK); and 2-chloroadenosine and the rabbit anti-mouse IgG were purchased from Sigma-Aldrich (St. Louis, MO). Fura-2/AM and donkey antibody anti-goat IgG were purchased from Molecular Probes, Inc (Leiden, The Netherlands). The antibodies anti-A₁, A_2A, and A₃ adenosine receptors were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). [¹²⁵I]AB-MECA was obtained from PerkinElmer Life Sciences (Boston, MA), and MRS 1191 was a kind gift of Dr. K. A. Jacobson (National Institutes of Health, Bethesda, MD). Other reagents were analytical grade acquired from Merck (Darmstadt, Germany). The Kalcium PC software was purchased from Kinetic Imaging, Ltd. The TE-200 microscope was purchased from Nikon, Tokyo (Japan), and the C-4880-80 multiformat CCD camera was from Hamamatsu.

Statistical Analysis. Data are presented as mean ± S.E.M of three or more determinations in triplicate from different synaptosomal preparations. Comparisons between experimental samples and untreated controls were carried out using nonparametric Mann-Whitney U test (two-tailed) or ANOVA test as indicated in each case. Dose-response curves plotting and fitting was carried out by computer program FigP (Biosoft, Cambridge, UK). When appropriate, single experimental traces are represented in the figures.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Effect of Ap₅A and ATP on Hippocampal Synaptic Terminals. The incubation of hippocampal synaptosomes with Ap₅A (10⁷-10³ M) caused a concentration-dependent increase of [Ca²⁺]_i, with an EC₅₀ value of 33.5 ± 4.5 µM and a maximal effect corresponding to a 24.4 ± 2.1 nM increase of [Ca²⁺]_i (n = 3) (Fig. 1A, Table 1).

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Concentration-response curve for Ap5A on intracellular calcium increase in rat hippocampal synaptosomes, absence of cross-desensitization and pharmacological characterization of Ap5A and ATP responses. A, concentration-response curve of Ap5A on the intrasynaptosomal calcium levels. Data represent mean ± S.E.M. of three experiments in triplicate. B, cross-desensitization study between ATP and Ap5A responses. In upper trace, application of 100 µM ATP followed by 100 µM Ap5A; lower trace application of Ap5A (100 µM) and further application of ATP (100 µM). C, effect of selective P2 antagonists, PPADS (50 µM), suramin (100 µM), and of the selective dinucleotide receptor antagonist, Ip5I (1 µM), on the Ap5A (100 µM)- and ATP (100 µM)-induced raise in intrasynaptosomal calcium. The results represent the mean ± S.E.M. of four experiments in duplicate. ***, p < 0.001 ANOVA test versus control.

Effect of Ap₅A and ATP on Single Hippocampal Nerve Terminals. To understand if the responses to Ap₅A and ATP occurred in the same hippocampal nerve terminals or in different nerve terminals, we recorded the effect of Ap₅A and ATP on [Ca²⁺]_i in single synaptosomes. Four different populations of synaptosomes were identified according to their responses to both nucleotides. The first group of synaptosomes responded to Ap₅A (100 µM) but not to ATP (100 µM), as it is the case of the synaptosome labeled as 1 (Fig. 2B, upper trace). This type of response occurred in 20% of the total functional synaptic terminals analyzed. The second group of synaptosomes (labeled 2) displayed the opposite behavior, responding to 100 µM ATP but not to 100 µM Ap₅A (17% of total functional synaptic terminals) (Fig. 2B). The third group of synaptosomes responded to both Ap₅A and ATP (100 µM). An example of this third group is the synaptosome labeled as 3, which accounted for 11% of total functional synaptic terminals. Finally, the fourth group of synaptosomes, nearly half of the functional synaptosomes, failed to respond to either Ap₅A or ATP.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Single hippocampal synaptic terminals responses to Ap5A and ATP. A, an image of isolated nerve terminals (synaptosomes) glued to coverslip with poly-L-lysine and loaded with FURA-2 dye (bar 1 µm). B, the time course of fluorescence changes recorded for the terminals labeled with numbers 1, 2, and 3 in panel A. Agonist stimulation was for 30 s and are indicated by the horizontal solid bars.

Effect of Adenosine A₁ Receptor Activation on Ap₅A Responses. Binding studies have previously shown that A₁ receptor binding sites are enriched in hippocampal nerve terminals (Cunha et al., 1996). Using an immunocytochemical approach, we confirmed that about 25% of the hippocampal synaptic terminals, identified with anti-synaptophysin antibodies, also displayed binding of anti-adenosine A₁ receptor antibodies (Fig. 3A).

View larger version (31K): [in this window] [in a new window]   Fig. 3.   Identification of adenosine receptors A1 and A2A in hippocampal synaptic terminals by immunocytochemistry. The fluorescent images showed the fluorescein-tagged anti-synaptophysin antibodies and rhodamin-tagged anti-A1 (A), and A2A (B) adenosine receptors antibody binding to hippocampal nerve terminals. The yellow color represents the coexpression of both proteins.

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View larger version (13K): [in this window] [in a new window]   Fig. 4.   Effect of A1 adenosine receptor activation on Ap5A response. A, concentration-response curve of Ap5A on [Ca2+]i in the absence (dashed line) and in the presence of the A1 receptor agonist, CPA (25 nM), and ADA 0.2 U/ml. B, effect of adenosine receptor antagonists (DPCPX, ZM 241385, and MRS 1191) in the two components of the Ap5A concentration-response curve in the presence of 25 nM CPA. *, p < 0.01, **, p < 0.005 and ***, p < 0.001 ANOVA test versus control. The result are mean ± S.E.M. of three experiments in triplicate. One hundred percent corresponds to the effect of 100 µM Ap5A in absence of any drug.

Effect of Adenosine A_2A Receptor Activation on Ap₅A Response. Beside A₁ receptors, previous binding studies have also shown an enrichment in the number of adenosine A_2A receptor-like binding sites in hippocampal nerve terminals (Cunha et al., 1996). Using an immunocytochemical approach, it was now confirmed that about 30% of the hippocampal synaptic terminals, identified with anti-synaptophysin antibodies, indeed displayed binding of anti-adenosine A_2A receptor antibodies (Fig. 3B).

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Effect of A2A adenosine receptor activation on Ap5A response. A, concentration-response curve of Ap5A on [Ca2+]i in the absence (dashed line) and in the presence of the A2A receptor agonist, CGS 21680 (10 nM), and ADA 0.2 U/ml. B, effect of adenosine receptor antagonists (DPCPX, ZM 241385, and MRS 1191) in the two components of the Ap5A concentration-response curve in the presence of 10 nM CGS 21680. **, p < 0.005 and ***, p < 0.001 ANOVA test versus control. The results are mean ± S.E.M. of four experiments in duplicate. One hundred percent corresponds to the effect of 100 µM Ap5A in absence of any drug.

Effect of Adenosine A₃ Receptor Activation on Dinucleotide Response. Beside A₁ and A_2A receptors, there are two other adenosine receptors, A_2B and A₃ receptors. No role or evidence for a presynaptic location of A_2B receptors has yet been forwarded, but it was reported that A₃ receptor activation decreased presynaptic A₁ receptor-mediated responses (Dunwiddie et al., 1997) as well as metabotropic glutamate receptor responses in the rat hippocampus (Macek et al., 1998). We now tried to confirm using both binding studies and immunocytochemical studies that A₃ receptors were indeed located in hippocampal nerve terminals. We found that the A₃ receptor agonist [¹²⁵I]AB-MECA, in the presence of the A₁ and A_2A receptors antagonists, DPCPX (50 nM) and ZM 241385 (50 nM), bound to rat hippocampal membranes with a K_D of 0.91 to 1.23 nM and a B_max of 30.5 to 36.9 fmol/mg of protein (n = 2). The binding of [¹²⁵I]AB-MECA was nearly 3-fold greater in membranes from rat hippocampal nerve terminals (B_max of 91.8-102.6 fmol/mg of protein, n = 2), with no change in K_D (1.09-1.11 nM, n = 2) (Fig. 6A). This [¹²⁵I]AB-MECA binding might correspond to adenosine A₃ receptor binding since the selective A₃ receptor antagonist, MRS 1191, completely displaced [¹²⁵I]AB-MECA binding with a K_i of 37.2 to 46.7 nM (n = 2) (Fig. 6B). The location of A₃ receptors in rat hippocampal nerve terminals was further confirmed by the labeling of synaptic terminals with an anti-A₃ receptor antibody, showing that the A₃ receptor is present in 29% of the terminals (Fig. 6C).

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Localization of A3 adenosine receptors on hippocampal nerve terminals. A, the binding of the A3 receptor agents, [125I]AB-MECA, to rat hippocampal membranes (squares) and to hippocampal synaptosomal membranes (circles). B, displacement curve of [125I]AB-MECA (0.5 nM) by the A3 receptor antagonist, MRS 1191 from rat hippocampal synaptosomal membranes. C, fluorescent image showing the fluorescein-tagged anti-synaptophysin antibodies and rhodamin-tagged anti-A3 adenosine receptor antibody binding to hippocampal nerve terminals. The yellow color represents the coexpression of both proteins.

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View larger version (12K): [in this window] [in a new window]   Fig. 7.   Effect of A3 adenosine receptor activation on Ap5A response. A, concentration-response curve of Ap5A on [Ca2+]i in the absence (dashed line) and in the presence of the A3 receptor agonist, Cl-IB-MECA (100 nM), and ADA 0.2 U/ml. B, effect of adenosine receptor antagonists (DPCPX, ZM 241385, and MRS 1191) on the Ap5A response in the presence of 100 nM Cl-IB-MECA. **, p < 0.005 and ***, p < 0.01 ANOVA test versus control. The results are mean ± S.E.M of four experiments in duplicate. One hundred percent corresponds to the effect of 100 µM Ap5A in absence of any drug.

	Discussion

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The results obtained in the present study indicate the presence of pharmacologically different presynaptic receptors for dinucleotides and for ATP, both increasing [Ca²⁺]_i in rat hippocampal nerve terminals. Furthermore, it was shown that adenosine receptors modulate the dinucleotide receptor-mediated responses, so that A₁ and A_2A receptors potentiated Ap₅A responses and A₃ receptors depressed Ap₅A responses.

Our proposal for the existence of different receptors mediating the responses to dinucleotides and ATP is based on the following three concurring observations: 1) cross-desensitization studies showing that one of the nucleotides did not change the response to the other, 2) single nerve terminal microfluorimetric studies revealing the existence of nerve terminals sensitive only to Ap₅A but not to ATP and others sensitive only to ATP but not to Ap₅A, and 3) pharmacological studies showing that the dinucleotide receptor antagonist, Ip₅I (Pintor et al., 1997a), abolished Ap₅A-induced [Ca²⁺]_i transients without affecting ATP responses, whereas the P2 receptor antagonist, suramin (Ralevic and Burnstock, 1998), blocked ATP but not Ap₅A responses. An interesting point was the lack of inhibitory effect by PPADS on ATP responses. These results could suggest that the ionotropic ATP receptor present in hippocampal synaptic terminals is formed by P2X₄ and/or P2X₆ subunits (Ralevic and Burnstock, 1998). However, the pharmacological properties of the different heteromeric combination of P2X subunits is not yet well understood, and the abundant presence of P2X₃ subunits has been recently described in rat midbrain synaptic terminals (Díaz-Hernández et al., 2001a). On the other hand, the lack of antagonism by suramin and PPADS on Ap₅A response confirms and extends previous findings that Ap₅A- or Ap₄A-induced Ca²⁺ transients were mediated by different receptors from those operated by ATP in hippocampal nerve terminals (Panchenko et al., 1996), as well as in other central nervous system areas (Pivorun and Nordone, 1996; Miras-Portugal et al., 1998). Therefore, it is reasonable to think that the synaptic terminals that only responded to diadenosine polyphosphates or ATP express dinucleotide receptors or P2X receptors, respectively, but it is more difficult to know what receptor or receptors are present in the synaptosomes that responded to both agonists. Although the possibility of the existence of a third receptor type, which would be sensitive to ATP and Ap₅A, can not be totally discarded (Miras-Portugal et al., 1999; Diaz-Hernandez et al., 2001b), the results obtained with the selective P2 and dinucleotide receptor antagonists favor the idea that the dinucleotide and P2X receptors may be coexpressed on some hippocampal synaptic terminals. It is important to stress that the present finding demonstrates that the presynaptic response to Ap₅A can be pharmacologically dissociated from that triggered by ATP. These data do not exclude the possibility that the dinucleotide receptor might be a different P2 receptor with a particular arrangement of subunits that renders the receptor insensitive to ATP and to P2 receptor antagonists and particularly sensitive to Ap₅A and other dinucleotides, as shown in previous studies (Pintor and Miras-Portugal, 1995; Pintor et al., 2001). Only the cloning of a different molecular entity will exclude this hypothesis.

The major finding of the present work is the marked modification of dinucleotide responses by adenosine receptor activation. Activation of A₁ and A_2A receptors produced a dramatic transformation of the original sigmoid concentration-response curve for Ap₅A into a biphasic one with two clearly separated components. The new component of the Ap₅A concentration-response curve displayed an EC₅₀ value in the picomolar/low nanomolar range. The second component showed an EC₅₀ value in the low micromolar range, similar to that of the Ap₅A concentration-response curve in the absence of A₁ or A_2A adenosine receptor agonists. This ability of high-affinity adenosine receptors to induce the appearance of a high-affinity state of dinucleotide receptors had already been anticipated in rat midbrain synaptosomes where addition of alkaline phosphatase (which increases the levels of extracellular adenosine) also produces the appearance of a high-affinity component in the Ap₅A concentration-response curve (Díaz-Hernández et al., 2000). From a physiological point of view, it is relevant to mention that the Ap₅A extracellular concentration measured in rat brain sample perfusates of conscious rats is in the low nanomolar range (Pintor et al., 1995). In that situation, the adenosine modulation via A₁ and A_2A receptors appears necessary for the dinucleotide receptor response.

Notably, A₁ and A_2A receptors produced a qualitatively similar effect on Ap₅A responses, whereas in most situations A₁ and A_2A receptors cause opposite modulatory effects (reviewed by Lopes et al., 1999a). However, there are situations where A₁ and A_2A receptors not only produce qualitatively similar effects but even cooperate in potentiating their respective responses (Ogata et al., 1996). The pharmacological characterization of the effect of adenosine receptor agonists is compatible with the involvement of both A₁ and A_2A receptors. Thus, low nanomolar concentrations of CPA selectively activate hippocampal presynaptic A₁ receptors (Cunha, 2001), an effect prevented by DPCPX. Also, low nanomolar concentrations of CGS 21680 selectively activate hippocampal presynaptic A_2A receptors (Cunha, 2001), an effect prevented by ZM 241385 but not by DPCPX, indicating the involvement of A_2A receptors (Cunha, 2001). The mechanism by which A₁ and A_2A receptors modulate Ap₅A responses was not directly investigated in the present study. But since activation of protein kinase A or protein kinase C cause an inhibition of Ap₅A-induced raise in [Ca²⁺]_i, the effect of both A₁ and A_2A receptors might be related to their dual ability to trigger G_i/G_o proteins (Freissmuth et al., 1991; Cunha et al., 1999) potentially decreasing protein kinase A activity. Indeed, activation of high-affinity adenosine receptors decreases protein kinase A- and protein kinase C-mediated effects in hippocampal synaptic terminals (Bouron, 1999).

One idiosyncratic pharmacological characteristic of A₁ receptor-mediated responses in this study is their functional antagonism by ZM 241385, as reported in Fig. 4. This is in agreement with previous observations reporting the ability of ZM 241385 to antagonize functional responses mediated by pharmacologically defined A₁ receptors in hippocampal preparations without direct A₁ receptor blockade (Lopes et al., 1999b). This atypical behavior is still not understood but may be due to a tight A₁/A_2A receptor cross talk (Dixon et al., 1997; Lopes et al., 1999a), which is probably reflected by the reported existence of atypical adenosine receptors with mixed A_2A/A₁ pharmacological characteristics (Cunha et al., 1996, 1999; Lindström et al., 1996).

The present study also establishes the presence of presynaptic A₃ receptors in the hippocampus based on both binding and immunocytochemical studies. Furthermore, we also observed functional effects resulting from presynaptic A₃ receptor in the hippocampus, as previously observed by others (Dunwiddie et al., 1997; Macek et al., 1998). The activation of A₃ receptors affects Ap₅A responses in a manner opposite to that of A₁ and A_2A receptors, decreasing the potency of Ap₅A to raise [Ca²⁺]_i. The mechanism by which A₃ receptors decrease Ap₅A responses is not known but might be related to the ability of A₃ receptors to activate protein kinase C, namely in hippocampal nerve terminals (Dunwiddie et al., 1997; Macek et al., 1998). One mechanism that can be excluded to explain A₃ receptor action is their ability to decrease A₁ receptor-mediated responses (Dunwiddie et al., 1997), since we found no evidence for a tonic modulation of Ap₅A responses by endogenous extracellular adenosine, and the experiments were performed in the presence of adenosine deaminase.

Despite the mechanism operated by adenosine A₁, A_2A, or A₃ receptors to modulate dinucleotide responses, the different modulatory role of each adenosine receptor subtype reveals a pattern of modulation of presynaptic Ap₅A responses according to the levels of endogenous extracellular adenosine. Thus, at low intensities of functioning of neuronal circuits, the levels of endogenous extracellular adenosine are low and mainly activate A₁ receptors (Díaz-Hernández et al., 2000; reviewed in Cunha, 2001). Increased functioning of neuronal circuits leads to a recruitment of A_2A receptor-mediated responses (Correia-de-Sá et al., 1996; reviewed in Cunha, 2001). The K_D for adenosine of rat A₃ receptors, estimated to be 10⁶ M (Jacobson, 1998) suggests that A₃ receptors will mostly be activated upon abnormal neuronal activation. Thus, the adenosine modulatory system is likely designed to increase Ap₅A responses at lower (via A₁ receptors) and particularly at higher intensities of neuronal activity (via A_2A receptors). The inhibitory effect of A₃ receptor activation may be a safety mechanism to decrease potentially facilitatory Ap₅A responses under abnormal conditions of neuronal firing.

In conclusion, the present results show a clear ability of adenosine receptors to control the potency of Ap₅A to modulate presynaptic responses in the hippocampus at more physiological levels of this dinucleotide (Pintor et al., 1995). Since adenosine is the final product of Ap₅A extracellular catabolism through ecto-nucleotidases (Mateo et al., 1997; reviewed by Zimmermann, 2000), the activation of adenosine A₁ and A_2A receptors may be conceived as a feedback amplification loop to increase sensitivity to Ap₅A. In contrast, activation of A₃ receptors may constitute a safety shut down mechanism to avoid excessive and uncontrolled functioning of this self-activating loop. These results also emphasize the need to activate rather than block adenosine responses when probing the neuromodulatory role of dinucleotides.