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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 M
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, 4-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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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).
|
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).
|
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-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.
|
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).
|
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
metaboliteinosine). 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.
|
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.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|