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. 2002 Jun;136(3):434–440. doi: 10.1038/sj.bjp.0704712

Effects of adenosine A1 and A2A receptor activation on the evoked release of glutamate from rat cerebrocortical synaptosomes

Mario Marchi 1,2,*, Luca Raiteri 1, Francesca Risso 1, Annalisa Vallarino 1, Andrea Bonfanti 1, Angela Monopoli 3, Ennio Ongini 3, Maurizio Raiteri 1,2
PMCID: PMC1573357  PMID: 12023946

Abstract

  1. The effects of adenosine A2A and A1 receptor activation on the release of glutamate were studied in rat cerebral cortex synaptosomes exposed in superfusion to adenosine receptor ligands.

  2. Adenosine (0.1 μM) produced a significant potentiation of the Ca2+-dependent K+(15 mM)-evoked [3H]-D-aspartate overflow (20.4±3.5%), which was blocked by A2A blocker SCH58261 (0.1 μM). At higher concentrations (10 – 1000 μM) adenosine inhibited in a DPCPX-sensitive manner the Ca2+-dependent K+-evoked [3H]-D-aspartate overflow. The inhibitory effect of adenosine at 1000 μM was significantly increased by SCH58261. This inhibition was antagonized by 1 μM DPCPX. Adenosine did not produce any effect on basal release.

  3. The A2A receptor agonist CGS 21680 was ineffective on basal release, but stimulated the Ca2+-dependent K+-evoked overflow of [3H]-D-aspartate (EC50 ≃ 1 pM). The effect of 0.01 nM CGS 21680 was totally sensitive to the A2A receptor antagonist SCH58261 (IC50 ≃5 nM).

  4. The A1 receptor agonist CCPA inhibited the Ca2+-dependent K+-evoked [3H]-D-aspartate overflow (EC50 ≃ 20 nM). The effect of 100 nM CCPA was abolished by 100 nM of the A1 receptor antagonist DPCPX.

  5. The K+(15 mM)-evoked overflow of endogenous glutamate was enhanced by CGS 21680 (0.01 nM) and inhibited by CCPA (0.1 μM). The effect of CGS 21680 was abolished by SCH58261 (0.1 μM) and that of CCPA by DPCPX (0.1 μM).

  6. It is concluded that adenosine and adenosine receptor agonists modulate glutamate release by activating inhibitory A1 and excitatory A2A receptors present on glutamatergic terminals of the rat cerebral cortex.

Keywords: Adenosine, A1 receptors, A2A receptors, glutamate release, cerebral cortex, SCH58261, ischaemia

Introduction

Adenosine is an ubiquitous neuromodulator that acts at four G-protein-coupled receptors termed A1, A2A, A2B, and A3 (Fredholm et al., 2001). One major role of A1 and A2A receptors is to modulate neurotransmitter release (for a review see Sebastião & Ribeiro (1996)). The distribution of these two receptors is discrete: A2A receptors are abundant in the corpus striatum but rather sparse in the cortex and hippocampus, where the density of A1 is higher than that of A2A receptors (Cunha et al., 1996; Fredholm et al., 1998).

There is evidence that adenosine mediates inhibition of neurotransmitter release through receptors of the A1 type, whereas A2A receptor activation is thought to facilitate release (for a review see Sebastião & Ribeiro (1996)). Important exceptions exist, however. For instance, adenosine A2A receptors when activated enhance the evoked release of GABA from rat hippocampus synaptosomes (Cunha & Ribeiro, 2000) whereas, in synaptosomes of rat striatum, they mediate inhibition of the evoked GABA release (Kirk & Richardson, 1994; Kurokawa et al., 1994).

It is generally believed that excessive release of the excitatory amino acids glutamate/aspartate is involved in a number of neuropathological conditions. There is evidence that adenosine can play protective roles against excitotoxicity and that extracellular levels of adenosine are elevated following brain oxidative stress and metabolic dysfunction that occur after interruption of the cerebral flow (Rudolphi et al., 1992; von Lubitz, 1999 and references therein; Moreau & Huber, 1999 and references therein). A reasonable hypothesis implies that endogenous adenosine inhibits glutamate release by activating A1 receptors. On the other hand, activation of A2A receptors by the compound CGS 21680 was reported to enhance excitatory transmitter release from ischaemic rat cerebral cortex (Simpson et al., 1992) suggesting that, under some conditions, endogenous adenosine can activate excitatory A2A receptors. Accordingly, A2A receptor antagonists limited cerebral damage caused by global ischaemia (Gao & Phillis, 1994; von Lubitz, 1999) and reduced cortical infarct volume following focal ischaemia (Monopoli et al., 1998). Furthermore, brain injury induced by transient focal ischaemia was attenuated in A2A receptor-deficient mice (Chen et al., 1999). These results appear compatible with the hypothesis that release-inhibiting A1 receptors and release-enhancing A2A receptors exist on glutamatergic terminals of the cerebral cortex. However, as discussed in the review by Sebastião & Ribeiro (1996), whether elevation and decrease of glutamate release can be produced by adenosine acting directly on glutamatergic nerve terminals or indirectly through more or less complex circuits, has not been established.

In order to ascertain if cerebrocortex glutamatergic nerve endings possess release-regulating adenosine heteroreceptors, we studied the effects of some adenosine receptor ligands on glutamate release using thin layers of synaptosomes in superfusion, a preparation that permits one to establish the localization of a given receptor on a particular family of nerve endings (see Raiteri & Raiteri, 2000). Modulation of glutamate release by selective A1 and A2A receptor agonists as well as by adenosine was investigated.

Methods

Animals

Adult male Sprague – Dawley rats (200 – 250 g) were used. Animals were housed at constant temperature (22±1°C) and relative humidity (50%) under a regular light-dark schedule (light 0700 – 1900 h). Food and water were freely available.

Preparation of synaptosomes

Crude synaptosomes were prepared from rat cerebral cortex as previously described (Raiteri et al., 1984). Briefly, rats were killed by decapitation and the cerebral cortex was rapidly dissected. The tissue was homogenized in 40 volumes of 0.32 M sucrose buffered at pH 7.4 with phosphate. The homogenate was centrifuged (5 min, 1000×g) to remove nuclei and debris; synaptosomes were then isolated from the supernatant by centrifugation at 12,000×g for 20 min. The synaptosomal pellet was finally resuspended in a physiological medium having the following composition (mM): NaCl 125, KCl 3, MgSO4 1.2, CaCl2 1.2, NaH2PO4 1.0, NaHCO3 22 and glucose 10 (gassed with 95% O2 – 5% CO2 at 37°C), pH 7.2 – 7.4.

Release experiments

Synaptosomes were incubated 15 min at 37°C with [3H]-D-aspartate ([3H]-D-ASP; spec. act. 16.3 Ci mmol−1; final concentration 0.08 μM) in an atmosphere of 95% O2 and 5% CO2. [3H]-D-aspartate is almost universally used (the ratio of papers is about 100 : 1) instead of [3H]-glutamate because it is non-metabolizable; but it is a substrate for the glutamate transporters (Drejer et al., 1985). In experiments of endogenous glutamate release, preincubation was performed in the same conditions and no labelling substance was added. Thereafter synaptosomes were distributed in parallel superfusion chambers. (Raiteri et al., 1974; Raiteri & Raiteri, 2000). Superfusion was started at a rate of 0.6 ml min−1 with aerated standard medium for 46 min in order to remove excess of tritium and/or to equilibrate the system. When Ca2+-independent release was to be studied, Ca2+-deprived medium (containing MgCl2 8.8 mM and EGTA 0.1 mM) was used throughout the superfusion. In K+-evoked release experiments, after 46 min of superfusion the following consecutive samples were collected: basal release (B1; 3 min); K+ evoked release (S; 6 min); after-depolarization basal release (B2; 3 min). Synaptosomes were exposed to the depolarizing stimulus (15 mM KCl containing medium) for 90 s, starting at the end of the first fraction collected (B1). Agonists were added concomitantly with the stimulus, whereas antagonists were added 8 min in advance. Antagonists, when used alone to see their effects on release, were added at the end of fraction B1.

In basal release experiments samples were collected in five consecutive fractions of 3 min each (B1 – B5). Agonists were added at the end of fraction B1, while antagonists were present in the superfusion medium 8 min before the sample collection started.

In both experimental procedures fraction B1 was not influenced by the presence of agonist drugs, while antagonists could not produce any effect, since the neurotransmitters were immediately removed from the superfusion chambers after being released and did not act on presynaptic receptors.

Calculation

In experiments with synaptosomes prelabelled with [3H]-D-ASP (used as a marker for glutamate release; see, for instance, Drejer et al., 1985) the fractions collected and the superfused synaptosomes were counted for radioactivity. The amount of radioactivity released into each fraction was expressed as a percentage of the total synaptosomal tritium present at the start of the respective collection period.

The K+-evoked overflow was estimated subtracting from the K+-evoked release (S) the basal release (B1+B2). The Ca2+-dependent K+-evoked overflow was calculated, in each experiment, by subtracting the Ca2+-independent K+-evoked overflow from the K+-evoked overflow. In our experiments the Ca2+-independent K+-evoked release amounted to 40 – 50% of the K+-evoked overflow. All the drugs studied did not modify the Ca2+-independent K+-evoked overflow. Drug effects were evaluated as the per cent ratio of the overflow in presence of the drug vs the overflow in control conditions (run in parallel).

In basal release experiments neurotransmitter efflux was calculated as fractional rate by performing the ratio between the radioactivity present in the second and following fractions and that collected in the first (Bx/B1). Drug effects were evaluated by comparing each ratio in presence of the drug with the corresponding ratio in control conditions (run in parallel) and expressing variations in per cent. Final data are means±s.e.mean of the given number of experiments (n).

Data were analysed by one-way ANOVA followed by Dunnett's multiple comparison test and considered significant for P<0.05 at least.

Determination of endogenous glutamate

Endogenous glutamate was determined by high performance liquid chromatography analysis following precolumn derivatization with o-phthalaldehyde and separation on a C18 reverse-phase chromatographic column (Chrompack, Middleburg, The Netherlands; 10×4.6 mm, 3 μm; 30°C) coupled with fluorometric detection (excitation wavelength 350 nm; emission wavelength 450 nm). Buffers and the gradient program were as follows: solvent A, 0.1 M sodium acetate (pH 5.8)/methanol 80 : 20; solvent B, 0.1 M sodium acetate (pH 5.8)/methanol, 20 : 80; solvent C, sodium acetate (pH 6.0)/methanol, 80 : 20; gradient program, 15% B and 85% C for 1 min from the initiation of the program; 50% B and 50% C in 1 min; 30% B and 70% A in 1 min; 100% B in 0.5 min; isocratic flow 2 min; flow rate 0.9 ml min−1. Homoserine was used as internal standard.

The amount of endogenous glutamate released was expressed as pmol mg protein−1. The KCl-evoked overflow was estimated by subtracting the amount of neurotransmitter in the two 3-min fractions representing the basal release from the neurotransmitter present in the 6-min fraction collected during and after the depolarization pulse. Protein content was determined according to Bradford (1976).

Drugs

[3H]-D-aspartate (specific activity, 16.3 Ci mmol−1) was purchased from Amersham Radiochemical Centre (Buckinghamshire, U.K.). Adenosine, 2-chloro-N6-cyclopentyladenosine (CCPA) and 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) were obtained from Sigma Chemical Co. (St Louis, MO, U.S.A.). CGS 21680 hydrochloride ¼ H2O was produced by Tocris Cookson Ltd. (Bristol, U.K.). SCH58261 was synthesized at the Schering-Plough Research Institute (Kenilworth, NJ, U.S.A.).

Results

Adenosine has rarely been used in neurotransmitter release studies. Since the characteristics of our superfusion technique (see Raiteri & Raiteri, 2000) permit the agents added to the medium to hit their targets directly before being metabolized or taken up by transporters, we studied the effects of adenosine on the release of [3H]-D-ASP (used as a tracer for endogenous glutamate/aspartate). Rat cortical synaptosomes were prelabelled with [3H]-D-ASP and then exposed during superfusion to adenosine. The drug added at 0.1, 10 μM or 1 mM did not produce any effect on the basal release of [3H]-D-ASP (values of B2/B1 ratio: controls=0.99±0.03, n=5; in presence of 0.1 μM adenosine=1.030±0.04, n=5; in presence of 10 μM adenosine=0.99±0.05; n=5; in presence of 1 mM adenosine=1.05±0.06, n=5).

The effect of adenosine was therefore studied in synaptosomes depolarized with 15 mM KCl. When synaptosomes were exposed to a large range of adenosine concentrations (from 0.01 μM to 1 mM), a significant potentiation of the Ca2+-dependent K+-evoked [3H]-D-ASP overflow could be observed only at 0.1 μM concentration (20.4±3.5%) (Figure 1A). This effect was totally counteracted by the A2A blocker SCH58261 (0.1 μM; Figure 1B). Adenosine significantly inhibited the Ca2+-dependent K+-evoked [3H]-D-ASP overflow when added at concentrations higher than 1 μM with a maximal inhibitory effect of 37.44±2.6% (Figure 1A). The inhibition produced by 1 mM adenosine was significantly increased in presence of A2A antagonist SCH58261 (0.1 μM), but completely abolished by 1 μM of the A1 receptor antagonist DPCPX (Figure 1B).

Figure 1.

Figure 1

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(A) Effects of adenosine in presence or in absence of 10−6 DPCPX, on the 15 mM Ca2+-dependent KCl-evoked [3H]-D-ASP overflow from rat cortical synaptosomes. Data are means±s.e.mean of at least six experiments run in triplicate. *P<0.05 at least versus controls. (B) Effects of adenosine in presence of the A1 receptor antagonist DPCPX or the A2A receptor antagonist SCH58261 on the 15 mM KCl-evoked [3H]-D-ASP overflow from rat cortical synaptosomes. Data are means±s.e.mean values of at least five experiments run in triplicate. *P<0.05 at least versus controls; #P<0.05 at least versus 10−3 M adenosine; §P<0.05 at least versus 10−7 M adenosine.

The modest stimulation brought about by 0.1 μM adenosine acting at A2A receptors might have been due to concomitant activation of inhibitory A1 receptors. However, the presence of 1 μM DPCPX was unable to potentiate the releasing effect of 0.1 μM adenosine (Figure 1A). The Figure also shows that the effect of 1 μM adenosine (slightly inhibitory) was reverted into a stimulatory one by 1 μM DPCPX. Also the maximal inhibitory effect obtained with 10 μM adenosine became a stimulatory effect in presence of 1 μM DPCPX. However, the stimulations of [3H]-D-ASP release observed with 1 or 10 μM adenosine (having blocked A1 receptors) did not differ significantly from that produced by 0.1 μM alone. Added at 1 mM with 1 μM DPCPX, adenosine was unable to cause any effect on [3H]-D-ASP release (Figure 1A). Panel B of Figure 1 shows that 1 μM DPCPX or 0.1 μM SCH58261 did not modify, on its own, the K+-evoked release of [3H]-D-ASP, as expected considering the characteristics of the superfusion technique used in this work.

As illustrated in Figure 2A, CGS 21680 increased, in a concentration-dependent manner, the Ca2+-dependent K+-evoked overflow of [3H]-D-ASP from cortical synaptosomes. The EC50 value amounted to about 1 pM, the maximal effect (47.69±6.2%) being reached at ∼ 0.01 nM. The basal release remained unaffected by CGS 21680 (not shown).

Figure 2.

Figure 2

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(A) Effects of CGS 21680 on the Ca2+-dependent 15 mM KCl-evoked [3H]-D-aspartate ([3H]-D-ASP) overflow from rat cortical synaptosomes. Data are means±s.e.mean of at least six experiments run in triplicate. Where s.e.mean is not shown, data are mean values of single experiments run in triplicate. *P<0.05 at least versus controls. (B) Effect of the A2A receptor antagonist SCH58261 on the 10−11 M CGS 21680-evoked potentiation of the [3H]-D-ASP overflow from rat cortical synaptosomes. Data are means±s.e.mean of six experiments run in triplicate. Where s.e.mean is not shown, data are mean values of single experiments run in triplicate.

The potentiating effect of 0.01 nM CGS 21680 on the Ca2+-dependent K+-evoked [3H]-D-ASP overflow was counteracted by the selective A2A receptor antagonist SCH58261 (Zocchi et al., 1996) in a concentration-dependent manner (Figure 2B). The calculated IC50 value amounted to 5 nM.

The A1 receptor agonist CCPA produced a concentration-dependent inhibition of the Ca2+-dependent K+-evoked overflow of [3H]-D-ASP from superfused rat cortical synaptosomes (Figure 3). The calculated EC50 of the compound was ∼ 20 nM; the maximal effect (50.18±4.8% inhibition) was reached when CCPA was added to the superfusion medium at 100 nM. This effect was completely antagonized by 100 nM DPCPX.

Figure 3.

Figure 3

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Effects of the A1 receptor agonist CCPA in presence or in absence of 10−6 M DPCPX on the Ca2+-dependent 15 mM KCl-evoked [3H]-D-ASP overflow from rat cortical synaptosomes. Data are means±s.e.mean values of at least five experiments run in triplicate. Where s.e.mean is not shown, data are mean values of single experiments run in triplicate. *P<0.05 at least versus controls.

A set of experiments was finally performed to investigate the effects of A1 or A2A receptor ligands on the depolarization-evoked overflow of endogenous glutamate from rat cortical synaptosomes. As illustrated in Figure 4, the K+(15 mM)-evoked overflow of endogenous glutamate was significantly potentiated by 0.01 nM CGS 21680; the potentiation was counteracted by 0.1 μM SCH58261. Conversely, the A1 receptor agonist CCPA, added at 0.1 μM, inhibited the depolarization-evoked overflow of endogenous glutamate, an effect antagonized by 0.1 μM DPCPX.

Figure 4.

Figure 4

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Effect of CGS 21680, alone or in presence of SCH58261, and of CCPA, alone or in presence of DPCPX, on the 15 mM KCl-evoked glutamate overflow from rat cortical synaptosomes. Data are mean±s.e.mean of n experiments run in triplicate. *P<0.05 at least versus controls; #P<0.05 at least versus 10−11 M CGS 21680; °P<0.05 at least versus 10−7 M CCPA. The basal release of endogenous glutamate, calculated on an average of six experiments, was 300±20 pmoles mg protein−1.

Discussion

The technique here used to monitor transmitter release (a very thin layer of synaptosomes up-down superfused) is best suited to identify and characterize targets that are located on a given family of nerve endings. As described in detail in a recent article (Raiteri & Raiteri, 2000), all the substances released by the preparation are quickly removed by the superfusion fluid before they can feedback on the releasing terminal or act on neighbouring particles. The targets present on the terminals remain therefore virtually ligand-free but can be activated by agents added to the superfusion medium. These agents reach their targets at the desired concentration which is maintained by the continuous flow in spite of possible enzymatic degradation and uptake. In such a system, indirect effects are prevented or minimized and modulations of release of a given transmitter by the compounds added to the superfusion medium can be assumed to be consequent to direct actions on the nerve terminals releasing that transmitter. This is very important in studies of adenosine as modulator of transmitter release because the contribution of endogenously released adenosine, of adenosine originating from released ATP or of ATP itself are minimal, if any, because of the quick removal of these agents.

In fact, the natural ligand has rarely been employed as an exogenous release modulator in biochemical studies of transmitter release. In a very recent report (Bennett & Boarder, 2000) adenosine (300 μM) was found to attenuate the depolarization-evoked release of glutamate from rat striatal slices. Previously, adenosine had been reported to inhibit glutamate and aspartate release evoked by electrical stimuli from rat hippocampal slices (Corradetti et al., 1984). The veratridine-evoked release of [3H]-acetylcholine from rat cortical synaptosomes was inhibited by exogenous adenosine apparently with no potentiation at low concentrations (Cunha et al., 1994).

The findings illustrated in Figure 1 are in keeping with the affinity reported for adenosine on A1 and A2A receptors. The A2A receptor is thought to have high affinity (0.1 – 1 μM) for adenosine (Daly et al., 1983; Ribeiro, 1999); according to Nishimura et al. (1990), adenosine is excitatory on neurotransmission between 0.01 and 1 μM, but becomes inhibitory between 10 μM and 1 mM, reflecting a lower affinity for A1 receptors. Based on the characteristics of the superfusion technique employed, our results show that glutamatergic axon terminals in the rat cerebral cortex are endowed with A2A and A1 receptors mediating respectively potentiation and inhibition of the evoked glutamate release.

CGS 21680 is currently used as a selective A2A receptor agonist (see Ongini & Fredholm, 1996). However, in brain regions where the A2A receptors are sparse, such as the cerebral cortex, CGS 21680 binds to additional non-specific undefined sites (Johansson & Fredholm, 1995; Cunha et al., 1996). On the other hand, these sites are insensitive to the selective A2A receptor antagonist SCH58261 indicating that they are unrelated to A2A receptors (Lindström et al., 1996). As shown in Figure 2B, the elevation of the K+-evoked [3H]-D-ASP overflow produced by the maximally effective concentration (0.01 nM) of CGS 21680 was abolished by SCH58261. The IC50 for SCH58261 is 5 nM, which is close to the reported affinity from binding studies (Ki values around 2 nM) and the IC50 values obtained in functional tests (see Ongini & Fredholm, 1996). Our results show that the CGS 21680 effect on the release of [3H]-D-ASP or of endogenous glutamate from cortical nerve endings is exclusively mediated by A2A receptors.

Comparing the results obtained with low concentrations of adenosine and those obtained with CGS 21680 it can be seen that the release potentiation produced by the natural modulator acting at A2A receptor (∼ 20%) is significantly lower than that caused by the A2A agonist (∼ 50%). The possibility that 0.1 μM adenosine concomitantly activates release-inhibiting A1 receptors, thus counteracting the A2A-mediated release stimulation, seems unlikely, however, because the effect of adenosine did not increase when the A1 receptors were blocked by DPCPX. No further potentiation of release could be observed when, keeping blocked A1 receptors, the concentrations of adenosine were increased to 1 or 10 μM, suggesting that, under our experimental conditions, adenosine can produce only a relatively modest potentiation of glutamate release. Why release potentiation by adenosine is lower than that provoked by CGS 21680 is at present unclear.

Adenosine and CGS 21680 were ineffective on the basal release from superfused synaptosomes. In this system the compounds seem to enhance the Ca2+-dependent exocytotic component of the release of glutamate evoked by depolarization, but are ineffective on the Ca2+-independent component of the K+-evoked overflow. The latter (40 – 50% of the total overflow) occurs by transporter reversal, being almost totally blocked (85 – 90%) by the glutamate EAAT1-EAAT3 transporter inhibitor DL-TBOA added at 10 μM. Similar results had been obtained when the release of [3H]-GABA or [3H]-acetylcholine was studied in superfused hippocampal or striatal synaptosomes. CGS 21680 facilitated the evoked [3H]-GABA or [3H]-acetylcholine release (30 or 15%, respectively) but had no effect on the basal release (Cunha et al., 1995; Cunha & Ribeiro, 2000).

The finding that glutamate release is enhanced during depolarization with relatively low [K+] excludes involvement of gliasomes contaminating synaptosomal preparations. In fact, glial preparations release glutamate when exposed to very high concentrations of K+ (⩾90 mM; Longuemare & Swanson, 1997).

Of interest, the A2A receptors present on glutamatergic terminals exhibit an apparent affinity for CGS 21680 (EC50 ≃ 1 pM) about three orders of magnitude higher than that of the A2A receptors present on hippocampal GABAergic terminals (EC50 ≃ 1 nM; Cunha & Ribeiro, 2000). On the other hand, CGS 21680 inhibited the K+(15 mM)-evoked release of [3H]-GABA from superfused striatal synaptosomes with an EC50 value of 1 pM (Kirk & Richardson, 1994).

The transducing mechanisms by which adenosine and CGS 21680 enhance the evoked release of glutamate remain to be investigated. As discussed recently (Moreau & Huber, 1999; Cunha & Ribeiro, 2000), the facilitation of the K+-evoked release of GABA from hippocampal synaptosomes appears to involve activation of adenylate cyclase and protein kinase A, protein kinase C and P-type Ca2+ channels.

The Ca2+-dependent K+-evoked [3H]-D-ASP overflow was inhibited by adenosine and by the A1 receptor agonist CCPA with similar maximal effects. Both the inhibitions produced by adenosine and by CCPA were counteracted by the selective A1 receptor antagonist DPCPX. Similar results were obtained by monitoring the K+-evoked release of endogenous glutamate.

Activation of A1 receptors is thought to inhibit the release of various transmitters. In particular, the A1 agonist N6-cyclohexyladenosine depressed the K+- or the 4-aminopyridine-evoked release of endogenous glutamate from guinea-pig cerebral cortex or rat striatal synaptosomes and inhibition of voltage-sensitive Ca2+ channels was associated with the A1 receptor-mediated reduction of release (Barrie & Nicholls, 1993; Ambrósio et al., 1996). The results obtained by concomitantly adding adenosine at high concentrations and the A2A receptor antagonist indicate that the potentiating effect of adenosine is still present and do not desensitize. This data is in agreement with previous findings showing that A2A receptors remain unaltered upon repeated administration of receptor agonists (Casati et al., 1994; Adami et al., 1995).

The present demonstration that adenosine receptors of the A2A receptor type able to enhance glutamate release exist on glutamatergic axon terminals in the rat cerebral cortex may help to explain, at least in part, the reduction of the cortical infarct volume observed in rats administered the A2A antagonist SCH58261 after focal cerebral ischaemia (Monopoli et al., 1998) and, more generally, the neuroprotective effects of adenosine A2A receptor antagonists. It is worth recalling that extracellular K+ concentrations increase to depolarizing levels during ischaemia and this would facilitate activation of A2A receptors on glutamate terminals. The A2A agonist CGS 21680 was reported to enhance the ischaemia-evoked release of aspartate/glutamate from the rat cerebral cortex in vivo, while the basal release was not affected (O'regan et al., 1992). Finally, the above-mentioned higher apparent affinity of CGS 21680 for the A2A receptors present on glutamatergic terminals versus those present on GABAergic terminals, implying a preferential release of glutamate versus that of GABA, may deserve consideration.

Acknowledgments

This work was supported by MURST network grants (1998 and 2000 to M. Marchi and M. Raiteri), by Progetto Ricerca Ateneo (2001 to M. Marchi and M. Raiteri). The financial support of Telethon Italy (grant no. 1164) is gratefully acknowledged. We are grateful to Mrs Maura Agate for her valuable aid in preparing the manuscript.

Abbreviations

CCPA

2-chloro-N6-cyclopentyladenosine

CGS 21680

2-4-(-2-carboxyethyl)phenethylamino-5′-N-ethylcarboxamidoadenosine

DPCPX

3-cyclopentyl-1,3-dipropylxanthine

SCH58261

7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazole-[4,3-e]-1,2,4 triazolo [1,5-c]pyrimidine

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