Cross Talk Between A1 and A2A Adenosine Receptors in the Hippocampus and Cortex of Young Adult and Old Rats

Luísa V. Lopes,1 Rodrigo A. Cunha,1,2 and J. A. Ribeiro1

 1Laboratory of Neurosciences, Faculty of Medicine, University of Lisbon, 1649-028 Lisbon; and  2Department of Chemistry and Biochemistry, Faculty of Sciences, University of Lisbon, 1749-016 Lisbon, Portugal


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Lopes, Luísa V., Rodrigo A. Cunha, and J. A. Ribeiro. Cross Talk Between A1 and A2A Adenosine Receptors in the Hippocampus and Cortex of Young Adult and Old Rats. J. Neurophysiol. 82: 3196-3203, 1999. Adenosine modulates synaptic transmission by acting on inhibitory A1 and facilitatory A2A receptors, the densities of which are modified in aged animals. We investigated how A2A receptor activation influences A1 receptor function and whether this interaction is modified in aged rats. In hippocampal and cortical nerve terminals from young adult (6 wk), but not old rats (24 mo), the A2A receptor agonist, 2-[4-(2-carboxyethyl) phenethylamino]-5'-N-ethylcarboxamidoadenosine (CGS 21680; 30 nM) decreased the binding affinity of a selective A1 receptor agonist, cyclopentyladenosine (CPA), an effect prevented by the A2A antagonist, (4-(2-[7-amino-2-(2-furyl {1,2,4}-triazolo{2,3-a {1,3,5}triazin-5-yl-aminoethyl)phenol (ZM 241385, 20 nM). This effect of CGS 21680 required intact nerve terminals and was also observed in the absence of Ca2+. This A2A-induced "desensitization" of A1 receptors was prevented by the protein kinase C inhibitor, chelerythrine (6 µM), and was not detected in the presence of the protein kinase C activator, phorbol-12,13-didecanoate (250 nM), which itself caused a reduction in binding affinity for CPA. The protein kinase A inhibitor, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (10 µM), and the protein kinase A activator, 8-Br-cAMP (1 mM), had no effects on the A2A-induced A1 receptor desensitization. This A2A-induced A1 receptor desensitization had a functional correlation because CGS 21680 (10 nM) attenuated by 40% the inhibition caused by CPA (10 nM) on CA1 area population spike amplitude in hippocampal slices. This A2A/A1 interaction may explain the attenuation by adenosine deaminase (2 U/ml), which removes tonic A1 inhibition, of the facilitatory effect of CGS 21680 on synaptic transmission. The requirement of tonic A1 receptor activation for CGS 21680 to induce facilitation of synaptic transmission was reinforced by the observation that the A1 receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (20 nM) prevented CGS 21680 (10 nM) facilitation of population spike amplitude. The present results show the ability of A2A receptors to control A1 receptor function in a manner mediated by protein kinase C, but not protein kinase A, in young adult but not in aged rats.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Adenosine is a neuromodulator mainly considered an inhibitor of neurotransmitter release and synaptic transmission via activation of A1 receptors in the neocortex and hippocampus (Ribeiro 1995). Adenosine also can activate A2A receptors, which are less abundant than A1 receptors in these brain areas (Cunha et al. 1996b), causing a discrete facilitation of neurotransmitter release and synaptic transmission in the hippocampus (Cunha et al. 1994b, 1997). In contrast with its modest role in the control of neurotransmitter release, presynaptic A2A receptors effectively modulate the action of several receptors, namely calcitonin gene related peptide receptor (Correia-de-Sá and Ribeiro 1994a), metabotropic glutamate receptors (Winder and Conn 1993), nicotinic autofacilitatory receptors (Correia-de-Sá and Ribeiro 1994b), and D2 dopamine receptors (see Ferré et al. 1997). This lead to the suggestion that adenosine A2A receptors mostly behave as regulators of other modulatory systems.

With aging the relative importance of inhibitory A1 and facilitatory A2A receptors is unbalanced in the limbic cortex, since there is lower density of A1 receptors and higher density of A2A receptors (Cunha et al. 1995). These changes are paralleled by a decreased ability of A1 receptor agonists to inhibit and an enhanced efficiency of A2A receptor agonists to facilitate neurotransmitter release and synaptic transmission in the hippocampus (Lopes et al. 1999; Sebastião et al. 1997).

In the hippocampus, A2A receptors are co-expressed and colocalized with A1 receptors (Cunha et al. 1994a), namely in nerve terminals (Cunha et al. 1996b). Besides the established contribution of both receptor subtypes to the overall effect of adenosine, there is increasing evidence that they do not only have opposite effects but also interact with each other. Activation of A2A receptors attenuates the ability of A1 receptors to inhibit population spike amplitude (Cunha et al. 1994a, O'Kane and Stone 1998), but it is not known if this involves summed opposite responses from both receptors or "desensitization" of A1 receptors, as has been shown in the striatum (Dixon et al. 1997). Thus we now investigated the mechanism of the A2A/A1 receptor interaction in the limbic cortex and if this interaction was modified in aged rats.

We found that, in nerve terminals of young adult rats, activation of A2A receptors decreased the binding of A1 receptor agonists, and this effect required activation of protein kinase C but not protein kinase A. This A2A/A1 interaction may be essential for A2A facilitation of synaptic transmission because the effect of 2-[4-(2-carboxyethyl) phenethylamino]-5'-N-ethylcarboxamidoadenosine (CGS 21680) required tonic A1 receptor activation. Finally, in aged rats, A2A receptors no longer caused A1 desensitization, suggesting that the role of A2A receptors change from mostly a modulator of A1 responses in young rats to a direct facilitatory system in aged rats.


    METHODS
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INTRODUCTION
METHODS
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REFERENCES

Hippocampal and cortical synaptosomes preparation

Male Wistar rats 24 mo old (aged rats) or 6 wk old (young adult rats) were decapitated under halothane anesthesia, the brain removed and the two hippocampi and cortices dissected free. Synaptosomes were prepared as previously described (Cunha et al. 1992). The tissue (cortex or hippocampus) was added to 8 ml of a chilled 0.32 M sucrose solution containing 1 mM EDTA, 1 mg/ml bovine serum albumin, and 5 mM HEPES, pH 7.4, and homogenized in a Potter-Elvehjem homogeneizer with a Teflon piston (4 up-and-down strokes) at 4°C, and the volume was completed to 10 ml with the sucrose solution. The suspension was centrifuged at 3,000 g during 10 min, and the supernatant was collected and centrifuged at 14,000 g for 10 min at 4°C. The pellet was resuspended in 2 ml of a 45% vol/vol Percoll solution made up in a Krebs-Ringer solution [composition was (in mM) 140 NaCl, 5 KCl, 25 HEPES, 1 EDTA, and 10 glucose, pH 7.4]. After centrifugation at 14,000 g for 2 min, the top layer was removed (synaptosomal fraction), washed in 1 ml of Krebs-Ringer solution, and resuspended in Krebs/HEPES solution [containing (in mM) 124 NaCl, 3 KCl, 1 MgCl2, 2 CaCl2, and 10 glucose buffered with 25 mM HEPES, pH 7.4) plus 2 U/ml of adenosine deaminase to remove endogenous adenosine.

Synaptosomal membrane preparation

Synaptosomal membranes were prepared as previously described (Cunha et al. 1996b). The synaptosomal fraction obtained as described in the preceding section was resuspended in 5 ml of a chilled 0.32 M sucrose solution with 50 mM Tris, 2 mM EDTA, buffered to pH 7.6 and homogenized in a Potter-Elvehjem homogeneizer with a Teflon piston (4 up-and-down strokes). The suspension was centrifuged at 1,000 g during 10 min, the supernatant collected and centrifuged at 14,000 g for 20 min. The pellet was resuspended in preincubation solution [containing (in mM) 50 Tris, 1 EDTA, and 2 EGTA, pH 7.4, with adenosine deaminase 2 U/ml] and incubated at 37°C for 30 min to remove endogenous adenosine. The suspension then was centrifuged for 20 min at 14,000 g, and the pellet was resuspended in a Tris/Mg2+ solution (50 mM Tris and 2 mM MgCl2, pH 7.4) with 4 U/ml of adenosine deaminase.

Displacement binding curves

Competition curves of the A1 receptor antagonist, [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX), by the A1 agonist, cyclopentyladenosine (CPA), were performed in the absence and in the presence of the A2A receptor agonist, CGS 21680. The appropriate (hippocampus or cortex) synaptosome or membrane preparation (200 µl containing 165-331 µg protein) was incubated with [3H]DPCPX (2 nM) and 10 different concentrations of the displacer, CPA (ranging from 0.1 nM to 1 µM) in Tris/Mg2+ solution in a final volume of 300 µl. All samples were assayed in triplicate. Nonspecific binding was evaluated in the presence of 2-chloroadenosine (100 µM) and represented nearly 20% of total binding. The test tubes were incubated for 2 h at room temperature (20-25°C), rapidly filtered through GF/C filters using a cell harvester (Whatman), and washed twice with ice-cold Tris/Mg2+ solution. The dried filters were placed in scintillation vials containing 5 ml of scintillation liquid (Optiphase HiSafe Scintillation Cocktail, Wallac, Turku, Finland). Radioactivity was determined after 12 h with an efficiency of 55-60% for 2 min. Membrane protein was determined according to Peterson et al. (1977). The IC50 values were converted into Ki values on nonlinear fitting of the semi-logarithmic curves derived from the competition curves. An F test (P < 0.05) was used to determine whether the competition curves were best fitted by a one or two independent binding site equation. The Ki values are presented as mean with 95% confidence interval (CI).

Electrophysiological recordings in hippocampal slices

Male Wistar rats (5-6 wk old) were decapitated after halothane anesthesia, and the hippocampus dissected free in ice-cold Krebs solution of the following composition (in mM): 124 NaCl, 3 KCl, 26 NaHCO3, 1,25 NaH2PO4, 1 MgSO4, 2 CaCl2, and 10 glucose, gassed with a 95% O2-5% CO2 mixture. Slices were cut (400 µm) with a McIlwain tissue chopper and allowed to recover for >= 1 h in a resting chamber within the same gassed medium at room temperature (20-25°C). Individual slices were transferred to a submersion recording chamber (1-ml capacity) and continuously superfused at a rate of 3 ml/min with the same gassed solution at 30.5°C. Stimulation was delivered to the Schaffer collateral/commissural fibers by a bipolar concentric wire electrode and rectangular pulses of 0.1-ms duration were applied every 15 s. The initial intensity of the stimulus was that eliciting ~50% of maximal population spike amplitude. The population spikes were recorded extracellularly from CA1 stratum pyramidale by use of micropipettes filled with 4 M NaCl and of 2-4 MOmega resistance and displayed on a Tektronix digitizing oscilloscope (Cunha et al. 1996b). The averages of eight consecutive responses were obtained, graphically plotted, and recorded for further analysis with locally developed software. The population spike amplitude was measured as the difference between the spike peak negativity and the following positivity of the potential. The amplitudes were determined for individual responses and then averaged during the predrug control, during drug superfusion and during the postdrug washout period; at least six responses were included in each average. In all of the experiments, the data were analyzed as mean percentage change in response amplitude when compared with responses obtained during the control period. The values are shown as mean ± SE of the mean of n (number of experiments), except where otherwise indicated. The significance of differences was evaluated by the paired Student's t-test. P values <0.05 were considered significant.

Drugs

N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004), CGS 21680, N6-cyclopentyladenosine (CPA), and chelerythrine were purchased from Research Biochemicals International, Natick, MA. Phorbol-12,13-didecanoate, 4alpha -phorbol-12,13-didecanoate, 8-Br-cAMP, and 2-chloroadenosine were from Sigma, Poole Dorset, UK. (4-(2-[7-amino-2-(2-furyl {1,2,4}-triazolo{2,3-a {1,3,5}triazin-5-yl-aminoethyl)phenol (ZM 241385) was from Tocris, Bristol, UK. [3H]DPCPX was from DuPont-NEN, Stevenage, Hertfordshire, UK and adenosine deaminase [from calf intestine, 200 U/mg protein---2 mg/10 ml solution in 50% glycerol (vol/vol), 10 mM potassium phosphate, pH 6.0] was from Boehringer Manheim, Germany. All drugs were diluted daily into the appropriate media from 5 mM stock solutions made up in DMSO stored at -20°C, except adenosine deaminase and [3H]DPCPX, which were prepared directly into the incubation solution each day.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

A2A-induced change in A1 receptor affinity in hippocampal and cortical synaptosomes from young adult rats

The effect of A2A receptor activation on the affinity of A1 receptors was evaluated by displacement of the A1 receptor antagonist, [3H]DPCPX (2 nM), by the A1 receptor agonist, CPA, in the absence and in the presence of the A2A receptor agonist, CGS 21680. In hippocampal synaptosomes of young adult rats, in control conditions (i.e., with no CGS 21680), the displacement binding curve of CPA (0.1 nM to 1 µM) was fitted best by a single binding site equation with a Ki of 2.29 nM (95% confidence interval: 1.27-3.31 nM, n = 4; Fig. 1A). The A2A agonist, CGS 21680, in a maximally effective concentration (30 nM) applied to slices of young adult rats (Cunha et al. 1997), caused a shift to the right of the displacement binding curve of CPA, resulting in a Ki of 7.84 nM (95% CI: 7.52-8.15 nM, n = 4; Fig. 1A). This CGS 21680-induced increase in the Ki of CPA in hippocampal synaptosomes was prevented by the A2A receptor antagonist, ZM 241385 (20 nM). The Ki of CPA to displace [3H]DPCPX binding was 2.98 nM (95% CI: 0.84-6.81 nM, n = 3) in the presence of ZM 241385 (20 nM) and 2.49 nM (95% CI: 0.52-4.45 nM, n = 3) in the simultaneous presence of ZM 241385 (20 nM) and CGS 21680 (30 nM; Fig. 1B).



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Fig. 1. Displacement binding curves of [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX; 2 nM) by N6-cyclopentyladenosine (CPA; 0.1 nM to 1 µM) in hippocampal synaptosomes of young adult (6-wk-old) rats (A and B) or aged rats (C) in the absence ( and open circle ) and in the presence ( and ) of the A2A agonist, 2-[4-(2-carboxyethyl) phenethylamino]-5'-N-ethylcarboxamidoadenosine (CGS 21680; 30 nM), in the absence (A) or in the presence (B) of the A2A receptor antagonist, (4-(2-[7-amino-2-(2-furyl {1,2,4}-triazolo {2,3-a {1,3,5}-triazin-5-yl-aminoethyl)phenol (ZM 241385; 20 nM). Ordinates represent the percentage of [3H]DPCPX obtained on subtraction of the nonspecific binding, determined in the presence of 100 µM 2-chloroadenosine, from the total binding. Each point is the mean ± SE of 4 experiments in A, 3 experiments in B, and 2 experiments in C, performed in triplicate. Note that CGS 21680 (30 nM) caused a shift to the right of the displacement binding curve (P < 0.05) in hippocampal synaptosomes of young adult rats, which was prevented by ZM 241385 but was devoid of effect in aged rats.

In synaptosomes prepared from the cerebral cortex of young adult rats, we observed a similar shift caused by CGS 21680 (30 nM) in the displacement binding curve of CPA (Table 1), which was prevented by the A2A-selective antagonist ZM 241385 (20 nM; Table 1), in a similar manner to that observed in the hippocampus. This A2A-induced A1 receptor desensitization is apparently not mediated by a Ca2+-dependent release of any mediator because CGS 21680 (30 nM) was still able to increase the Ki of displacement of [3H]DPCPX by CPA in young rat cortical synaptosomes even in the absence of added extracellular calcium (Table 1).


                              
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Table 1. Ability of CGS 21680 to attenuate the displacement of [3H]DPCPX binding by CPA to rat cortical synaptosomes or from membranes from cortical synaptosomes of young adult rats

A2A-induced change in A1 receptor affinity in membranes from hippocampal and cortical synaptosomes from young adult rats

To ascertain whether this effect of CGS 21680 on the affinity of A1 receptors in hippocampal and cortical synaptosomes might result from a direct receptor-receptor interaction (see Ferré et al. 1997) or it requires recruitment of intracellular transduction systems, we compared the competition curves by CPA of [3H]DPCPX binding in membranes derived from hippocampal or cortical synaptosomes. In membranes from hippocampal synaptosomes, the Ki of displacement of [3H]DPCPX by CPA was 1.57 nM (95% CI: 0.69-2.45 nM, n = 3) in the absence and 1.63 nM (95% CI: 0.92-2.33 nM, n = 3) in the presence of CGS 21680 (30 nM). Similarly, CGS 21680 (30 nM) also failed to cause any modification in the Ki of displacement of [3H]DPCPX by CPA in membranes from hippocampal synaptosomes (Table 1), in contrast to what was observed in intact cortical synaptosomes.

Transducing system involved in the A2A-induced A1 receptor desensitization in synaptosomes from young adult rats

To investigate the transduction mechanism involved in this A2A/A1 interaction in young adult rats, we tested the action of a protein kinase C inhibitor, chelerythrine, and of a protein kinase A inhibitor, HA1004, on the decrease in affinity of A1 receptors induced by CGS 21680 in cortical synaptosomes. By themselves, neither chelerythrine (6 µM) nor HA1004 (10 µM) caused any significant modification of the Ki of CPA to displace [3H]DPCPX (Table 2). But the presence of chelerythrine (6 µM, n = 3) prevented, while HA 1004 (10 µM, n = 3) did not affect the effect of CGS 21680 on A1 receptor binding (Table 2), suggesting the involvement of protein kinase C, but not protein kinase A, on the A2A-induced A1 receptor desensitization. This idea was reinforced by the observation that the protein kinase C activator, phorbol-12,13-didecanoate (250 nM, n = 5), mimicked the effect of CGS 21680 to increase the Ki of CPA to displace [3H]DPCPX (Table 2). Furthermore the addition of CGS 21680 (30 nM) on top of phorbol-12,13-didecanoate (250 nM, n = 3) failed to cause further modifications of the Ki of [3H]DPCPX by CPA when compared with phorbol-12,13-didecanoate (250 nM) alone (Table 2). In contrast, the inactive analogue of phorbol-12,13-didecanoate (250 nM), 4-alpha phorbol-12,13-didecanoate (250 nM) was devoid of effects (n = 2) and failed to prevent the increase induced by CGS 21680 (30 nM) in the Ki of CPA to displace [3H]DPCPX (n = 3; Table 2). Consistent with the lack of involvement of protein kinase A in this phenomenon is the observation that the protein kinase A activator, 8-Br-cAMP (1 mM), was devoid of effect (n = 4) on the ability of CPA to displace [3H]DPCPX in cortical synaptosomes of young adult rats.


                              
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Table 2. Ability of protein kinase C modifiers, and lack of effect of cAMP/protein kinase A modifiers, to mimick and to prevent the CGS-21680-induced increase of the Ki of CPA to displace [3H]DPCPX binding to rat cortical nerve terminals of young adult rats

Effect of A2A receptor activation on A1 receptor binding in hippocampal and cortical synaptosomes from aged rats

In contrast to what was observed in hippocampal or cortical synaptosomes from young adult rats, CGS 21680 (30 nM) failed to significantly modify the ability of CPA to displace [3H]DPCPX in synaptosomes from aged rats. In hippocampal synaptosomes from aged rats, the Ki of displacement of [3H]DPCPX by CPA was 0.996-1.228 nM (n = 2) in the absence and 1.164-1.396 nM (n = 2) in the presence of CGS 21680 (30 nM; Fig. 1C). Likewise, in cortical synaptosomes from aged rats, the Ki of displacement of [3H]DPCPX by CPA was 1.07 nM (95% CI: 0.48-1.67 nM, n = 3) in the absence and 1.28 nM (95% CI: 0.48-2.08 nM, n = 3) in the presence of CGS 21680 (30 nM).

This disruption of A2A/A1 interaction in aged rats was accompanied by the inability of protein kinase C activation to affect A1 receptor binding. In contrast with its effect in young adult rats, phorbol-12,13-didecanoate (250 nM, n = 3) did not affect A1 receptor binding in cortical synaptosomes from aged rats, the same occurring with 8-Br-cAMP (1 mM, n = 2). In cortical synaptosomes from aged rats, the Ki of displacement of [3H]DPCPX by CPA was 1.78 nM (95%CI: 1.03-1.40 nM, n = 3) in the absence of any drug, 1.22 nM (95%CI: 0.51-3.01 nM, n = 3) in the presence of phorbol-12,13-didecanoate (250 nM) and 0.79-2.01 nM (n = 2) in the presence of 8-Br-cAMP (1 mM).

Attenuation by A2A receptors of A1-receptor-mediated responses in hippocampal slices from young adult rats

We tested this A2A/A1 interaction at a functional level, through extracellular electrophysiological recordings from CA1 area of hippocampal slices of young adult rats. By itself CGS 21680 (10 nM) elicited a moderate facilitatory effect of 10.3 ± 4.6% on population spike amplitude. This effect of CGS 21680 (10 nM) was observed in four of seven experiments. The lack of response to CGS 21680 on hippocampal slices in some animals has been noted previously (Cunha et al. 1997; Li and Henry 1998), and the reason for this individual variation is not known. In these four experiments in which CGS 21680 caused a facilitation of population spike amplitude, we compared the effect of the selective A1 receptor agonist, CPA, in the absence and in the presence of CGS 21680. CPA (10 nM) alone caused a 63.4 ± 10.3% (n = 4) inhibition of population spike amplitude. In the presence of CGS 21680 (10 nM), CPA (10 nM) only reduced by 40.5 ± 13.1% the population spike amplitude (Fig. 2) compared with the effect caused by the first application of CPA (10 nM) alone in the same slice. Two successive applications of CPA (10 nM) alone elicited a similar inhibition of population spike amplitude (the ratio between CPA inhibitory effect elicited by the 2nd and 1st application was 1.05 ± 0.05, n = 2).



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Fig. 2. Attenuation by CGS 21680 (10 nM) of the CPA (10 nM)-mediated inhibition of population spike amplitude in the CA1 area of hippocampal slices from young adult rats (6 wk old). A: raw data obtained in an individual assay as the mean of 8 population spike. A, top: control population spike, followed by a population spike obtained 30 min after superfusion of the slice with CPA (10 nM), and finally a population spike obtained on washout of CPA. Bottom: population spike in the presence of CGS 21680 (10 nM) obtained in the same slice, followed by a population spike obtained 30 min after superfusion of the slice with CPA (10 nM) in the presence of CGS 21680 (10 nM) and finally a population spike obtained on washout of CPA but still in the presence of CGS 21680 (10 nM). B: ability of CPA (10 nM) to inhibit population spike amplitude in the absence and in the presence of CGS 21680 (10 nM) is compared, as indicated by the symbols under each column. Results are means ± SE of 4 experiments. * P < 0.05 vs. 0%; ** P < 0.05 between the 2 situations.

Attenuation of A2A responses by removing tonic A1 inhibition in hippocampal slices from young adult rats

The observation that the facilitatory effect on neuronal excitability elicited by CGS 21680 was larger in magnitude over CPA responses than alone raised the question whether its facilitatory effect mainly results from an attenuation of tonic inhibition by endogenous adenosine through A1 receptors. Thus we compared the effect of CGS 21680 on population spike amplitude in hippocampal slices in the absence and in the presence of adenosine deaminase (which converts adenosine to its inactive metabolite---inosine). Two successive applications of CGS 21680 (10 nM) caused a similar facilitation of population spike amplitude (the ratio between CGS 21680 facilitatory effect elicited by the 2nd and 1st application was 0.94 ± 0.12, n = 3). Adenosine deaminase (2 U/ml, n = 3) facilitated by 24 ± 3% population spike amplitude, by removing the tonic A1-receptor-mediated inhibition caused by endogenous adenosine (Cunha et al. 1996a). As shown in Fig. 3A, when adenosine deaminase (2 U/ml) was present, CGS 21680 (10 nM) failed to modify population spike amplitude, whereas on washout of adenosine deaminase, CGS 21680 (10 nM) now enhanced the amplitude of population spikes. This attenuation by adenosine deaminase of CGS 21680-facilitation of population spike amplitude was observed either without or on adjusting the intensity of stimulation to yield a population spike amplitude in the presence of adenosine deaminase identical to that of control (data not shown). As illustrated in Fig. 3B, this dependency on the presence of endogenous adenosine of the facilitatory effect of A2A receptor activation on synaptic transmission was observed in three of five experiments. In the other two experiments, CGS 21680 (10 nM) alone was devoid of effect.



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Fig. 3. Ability of adenosine deaminase (which converts adenosine into its inactive metabolite---inosine) to attenuate the facilitation caused by the A2A receptor agonist, CGS 21680 (10 nM), on neuronal excitability in the CA1 area of hippocampal slices from young adult rats (6 wk old). A: time course of the averaged amplitude of 8 consecutive population spikes in a representative experiment. Hippocampal slice was first superfused with CGS 21680 (10 nM) first in the presence then in the absence of adenosine deaminase (ADA, 2U/ml), as indicated by the upper horizontal bars. B: ability of CGS 21680 (10 nM) to enhance population spike amplitude in the absence and in the presence of adenosine deaminase (2 U/ml) is compared, as indicated by the symbols under each column. Results are means ± SE of 3 experiments. * P < 0.05 vs. 0%; ** P < 0.05 between the 2 situations.

The A1 receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 20 nM, n = 5) facilitated by 17 ± 1% population spike amplitude, by removing the tonic A1-receptor- mediated inhibition caused by endogenous adenosine (Cunha et al. 1996a). As shown in Fig. 4A, CGS 21680 (30 nM) alone enhanced the amplitude of population spikes, whereas in the presence of DPCPX (20 nM), CGS 21680 (10 nM) failed to modify population spike amplitude. This almost complete blockade by DPCPX of the CGS-21680-induced facilitation of population spike amplitude was observed either without or on adjusting the intensity of stimulation to yield a population spike amplitude, in the presence of DPCPX, identical to that of control (as shown in Fig. 4A). As illustrated in Fig. 4B, this dependency on the presence of tonic A1 receptor activation of the facilitatory effect of A2A receptor activation on synaptic transmission, was observed in four of five experiments. In one experiment, CGS 21680 (10 nM) alone was devoid of effect.



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Fig. 4. Ability of the A1 receptor antagonist, DPCPX, to attenuate the facilitation caused by the A2A receptor agonist, CGS 21680 (10 nM), on neuronal excitability in the CA1 area of hippocampal slices from young adult rats (6 wk old). A: time course of the averaged amplitude of 8 consecutive population spikes in a representative experiment. Hippocampal slice first was superfused with CGS 21680 (10 nM) first in the absence then in the presence of DPCPX (20 nM), as indicated by the upper horizontal bars. Arrow indicates a change in the intensity of stimulation to obtain a population spike amplitude similar to that of control conditions. B: ability of CGS 21680 (10 nM) to enhance population spike amplitude in the absence and in the presence of DPCPX (20 nM) is compared, as indicated by the symbols under each column. Results are means ± SE of 3 experiments. * P < 0.05 vs. 0%; ** P < 0.05 between the 2 situations.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present data show that activation of adenosine A2A receptors decreases presynaptic adenosine A1 receptor binding as well as A1 functional responses in young adult but not aged rats. The selective activation of A2A receptors with CGS 21680 (Jarvis et al. 1989) elicited an attenuation of the A1 receptor binding evaluated with CPA and DPCPX, chosen for their high selectivity (Bruns et al. 1987; Williams et al. 1986) in hippocampal and cerebral cortical synaptosomes of young adult rats, as previously shown to occur in the striatum (Dixon et al. 1997). This A2A/A1 molecular interaction correlates functionally with the CGS-21680-induced decrease in the efficiency of CPA to inhibit neuronal excitability (see Cunha et al. 1994a; O'Kane and Stone 1998). A2A receptor attenuation of A1 responses also has been described at the rat neuromuscular junction (Correia-de-Sá and Ribeiro 1994b), although in this study it was not clear whether this A2A attenuation of A1 responses was due to an A2A/A1 receptor cross talk or to a sum of opposite effects. The present demonstration of a decrease in affinity of A1 receptors induced by A2A receptor activation, together with the observation that the attenuation of A1 responses caused by A2A receptor activation was larger in amplitude than the direct facilitatory effect of CGS 21680 on population spike amplitude, suggests than the main role of A2A receptors in young adult rats is to modulate A1 responses rather than to directly facilitate neuronal excitability. This idea was further supported by the fact that adenosine deaminase and DPCPX were able to inhibit CGS-21680-induced facilitation of neuronal excitability, indicating that tonic A1 receptor inhibition by endogenous adenosine (see Cunha et al. 1996a) is required to reveal a clear facilitation induced by A2A receptor activation.

A2A receptors are pleiotropic receptors, mostly coupled to Gs proteins (Cunha et al. 1999; Marala and Mustafa 1993; Olah 1997) but also to other G-protein subtypes (Cunha et al. 1999; Sexl et al. 1997), activating both the adenylate cyclase/cAMP pathway and the protein kinase C pathway (Gubitz et al. 1996; Kirk and Richardson 1995). We now observed that the ability of A2A receptors to decrease A1 receptor affinity depends on the activation of the protein kinase C but not of the adenylate cyclase/cAMP/protein kinase A pathway. This is consistent with previous studies showing that activation of protein kinase C by phorbol esters attenuate A1 adenosine receptors inhibition of neuromuscular transmission (Sebastião and Ribeiro 1990), glutamate release in cortical synaptosomes (Barrie and Nicholls 1993) and in retinotectal synapses (Zhang and Schmidt 1998). In addition, activation of other protein-kinase-C-coupled receptors such as metabotropic glutamate receptors (Budd and Nicholls 1995; de Mendonça and Ribeiro 1997; Di Iorio et al. 1996), adenosine A3 receptors (Dunwiddie et al. 1997), and muscarinic receptors (Worley et al. 1987) also attenuates A1 adenosine receptor inhibitory presynaptic effects. The target of protein kinase C phosphorylation is still unclear. Likely candidates are A1 receptors, which become clustered and desensitize by phosphorylation (Ciruela et al. 1997); Gi/Go proteins which are under protein kinase C control (Katada et al. 1985); and N-type calcium channels, the likely final target of A1 receptors (Ribeiro 1995), which activity increases on phosphorylation (Swartz 1993). The observations that A3 receptor activation desensitizes A1, but not GABAB responses (Dunwiddie et al. 1997), which also are coupled to the same type of G proteins (Thompson et al. 1993), suggest that the target of PKC activation might be A1 receptors rather than Gi/Go proteins or N-type calcium channels.

The major conclusion of the present work is the absence of this A2A/A1 receptor cross talk in aged rats. In contrast to young adult rats, activation of A2A receptors failed to modify A1 receptor binding in both hippocampal and cerebral cortical nerve terminals. Previous work in the hippocampus had shown that A2A receptors increase in number and are more tightly coupled to G proteins (Lopes et al. 1999), causing a larger facilitation of neurotransmitter release and synaptic transmission in aged compared with young adult rats (Lopes et al. 1999; Sebastião et al. 1997). Also these A2A-receptor-mediated facilitatory effects do not depend on a tonic A1-receptor-mediated inhibition which is also more enhanced in aged than in young adult rat hippocampal slices (Sebastião et al. 1997). In addition, we have shown previously that CPA is able to inhibit synaptic transmission in the hippocampus of aged rats with a similar maximal effect, although with a lower potency (Sebastião et al. 1997). Therefore this age-related change in A2A/A1 cross talk appears not to be due to a lower efficiency of A2A receptors nor to an absence of A1 receptor responses in aged rats but might be due to modifications of the organization and relative densities of G-protein-coupled receptors and intracellular pathways. Interestingly, direct activation of protein kinase with phorbol esters also failed to modify A1 receptor binding in aged rats, although it decreased A1 receptor binding in young adult cortical nerve terminals. Aging has been reported to decrease (Martini et al. 1994) and to increase (Battaini et al. 1990; Colombo et al. 1997) protein kinase C activity in the limbic cortex. These discrepancies possibly are related to the existence of several isoforms of protein kinase C with different dynamics of subcellular distribution (reviewed by Jaken 1996). It is not known how aging affects the subcellular distribution and enzymatic density of each protein kinase C isoform in the limbic cortex. Also awareness of the importance of docking in promoting interactions between receptors is growing (Tsunoda et al. 1997), but most of these essential ancillary elements have not yet been identified to envisage possible age-related changes. In this respect, it is interesting to note that A2A receptor activation is virtually devoid of effects on cAMP levels in the limbic cortex of young adult rats but causes a marked cAMP accumulation in cortical slices from aged rats (Lopes et. 1999), suggesting a modified coupling of A2A receptors to their transduction systems in aged animals.

We previously have observed that the facilitation of neurotransmitter release and of synaptic transmission mediated by A2A receptors in aged animals is enhanced when compared with young adults (Lopes et al. 1999; Sebastião et al. 1997). Therefore the present results, showing an A2A receptor attenuation of A1 responses in young adult but not in aged rats, suggest an age-related change in the neuromodulatory function of A2A receptors in the limbic cortex: from counteracting A1 responses in young adult rats, with moderate direct effects on neuronal excitability, to a direct facilitatory role, independent of tonic A1 receptor activation, in aged animals. Further exploration remains on the mechanism by which A2A receptors are directly responsible for a facilitation of neurotransmitter release in the hippocampus of aged rats.


    ACKNOWLEDGMENTS

The authors are indebted to A. de Mendonça and A. Alves-Rodrigues for critically reviewing the manuscript. We also are grateful to J. E. Coelho and A. R. Costenla for technical support. R. A. Cunha thanks Prof. Moniz Pereira for scintillation counting facilities.

This work was supported by Fundação para a Ciência e Tecnologia (Praxis/P/SAU/14012/98).


    FOOTNOTES

Address for reprint requests: R. A. Cunha, Laboratory of Neurosciences, Faculty of Medicine, University of Lisbon, Av. Prof. Egas Moniz, 1649-028 Lisbon, Portugal.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 21 June 1999; accepted in final form 4 August 1999.


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society