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 Previous Article | Next Article 
The Journal of Neuroscience, March 15, 1998, 18(6):1987-1995
Inhibition by ATP of Hippocampal Synaptic Transmission Requires
Localized Extracellular Catabolism by Ecto-Nucleotidases into Adenosine
and Channeling to Adenosine A1 Receptors
Rodrigo A.
Cunha2,
Ana
M.
Sebastião1, and
J. A.
Ribeiro1
1 Laboratory of Neurosciences, Faculty of Medicine, and
2 Department of Chemistry and Biochemistry, Faculty of
Sciences, University of Lisbon, 1600 Lisbon, Portugal
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ABSTRACT |
ATP analogs substituted in the  -phosphorus (ATPS,
 ,-imido-ATP, and ,-methylene-ATP) were used to probe the
involvement of P2 receptors in the modulation of synaptic
transmission in the hippocampus, because their extracellular catabolism
was virtually not detected in CA1 slices. ATP and -substituted
analogs were equipotent to inhibit synaptic transmission in CA1 pyramid
synapses (IC50 of 17-22 µM). The inhibitory
effect of ATP and -phosphorus-substituted ATP analogs (30 µM) was not modified by the P2 receptor
antagonist suramin (100 µM), was inhibited by 42-49% by
the ecto-5'-nucleotidase inhibitor and  ,-methylene ADP (100 µM), was inhibited by 74-85% by 2 U/ml adenosine
deaminase (which converts adenosine into its inactive
metabolite-inosine), and was nearly prevented by the adenosine
A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (10 nM). Stronger support for the involvement of
extracellular adenosine formation as a main requirement for the
inhibitory effect of ATP and -substituted ATP analogs was the
observation that an inhibitor of adenosine uptake, dipyridamole (20 µM), potentiated by 92-124% the inhibitory effect of
ATP and -substituted ATP analogs (10 µM), a
potentiation similar to that obtained for 10 µM adenosine
(113%). Thus, the present results indicate that inhibition by
extracellular ATP of hippocampal synaptic transmission requires localized extracellular catabolism by ecto-nucleotidases and channeling of the generated adenosine to adenosine A1 receptors.
Key words:
ATP; adenosine; ecto-nucleotidases; hippocampus; ATP
analogs; P2 receptors; A1 receptors
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INTRODUCTION |
ATP is currently recognized as a
neurotransmitter and a neuromodulator in the nervous system (for
review, see Zimmermann, 1994 ). In the hippocampus, ATP is released on
stimulation (Terrian et al., 1989; Cunha et al., 1996a; Wieraszko,
1996) and is a potent inhibitor of hippocampal neuronal excitability
(Lee et al., 1981; Stone and Cusack, 1989; Cunha et al., 1996b). ATP
may directly control neuronal activity either by activating hippocampal
P2 receptors (Kidd et al., 1995; Collo et al., 1996; Inoue
et al., 1996) or by acting as a substrate of ecto-protein kinase during synaptic plasticity phenomena (Wieraszko, 1996). ATP may also indirectly modulate neuronal excitability after its extracellular catabolism by the ecto-nucleotidase cascade (Lee et al., 1981; Cunha et
al., 1992), generating adenosine that modulates synaptic transmission
through inhibitory adenosine A1 receptors or facilitatory A2A receptors (Cunha et al., 1994a).
The proposal that P2 receptors are involved in the
modulation of synaptic transmission mostly relies on the use of
metabolically stable ATP analogs (Fredholm et al., 1994). However, it
has been proposed that metabolically stable ATP analogs may inhibit
neurotransmission either through direct action on inhibitory adenosine
A1 receptors (Hourani et al., 1991; Bailey et al., 1992;
Piper and Hollinsworth, 1996) or indirectly after their localized
catabolism into adenosine (Bruns, 1990; Cascalheira and
Sebastião, 1992).
In the present work we investigated the relationship between the
effects of ATP analogs on synaptic transmission in the hippocampus and
their extracellular catabolism in this preparation. We observed a
mismatch between the absence of extracellular catabolism of -substituted ATP analogs in CA1 slices with the modification of the
inhibitory effect of -substituted ATP analogs by inhibitors of
extracellular purinergic metabolism. This lead us to conclude that
-substituted ATP analogs might undergo minute localized catabolism
into adenosine, and this adenosine is channeled to adenosine
A1 receptors. It is suggested therefore that the
possibility of mechanisms of preferential substrate delivery between
ecto-nucleotidases and adenosine receptors ought to be taken into
account and tested through functional assays using modulators of purine
metabolism whenever effects of apparently stable adenine nucleotides
are observed. This might be of particular relevance when the actions of
adenine nucleotides do not easily fit to P2X or
P2Y receptor-mediated actions.
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MATERIALS AND METHODS |
ATP, ADP, AMP, adenosine,
adenosine-5'-O-(,-methylene)-diphosphonate sodium salt
(AOPCP), ,-imido ATP tetralithium salt (,-imido-ATP),
,-methylene ATP sodium salt (,-methylene-ATP), ,-methylene ATP dilithium salt (,-methylene-ATP), adenosine 5'-O-(3-thio)-triphosphate tetralithium salt (ATPS),
2'-deoxyadenosine, 2'-deoxyadenosine-5'-triphosphate (2'-dATP),
p-nitrophenylphosphate, hypoxanthine, and inosine were from
Sigma (St. Louis, MO). Adenosine deaminase (type VI, 1803 U/ml, EC
3.5.4.4) was also purchased from Sigma, in a suspension in 50%
(vol/vol) glycerol in potassium phosphate, pH 6.0, and dilutions of
this suspension were used, together with appropriate corrections of
glycerol and KH2PO4 content of all control
solutions. 2-Methylthio-ATP tetrasodium salt (2-methylthio-ATP), 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), suramin, reactive blue 2, and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) were
from RBI (Natick, MA). 2-Methylthio-ADP trilithium salt and
2-methylthio-AMP dilithium salt were from Boehringer Mannheim
(Indianapolis, IN), and dipyridamole was from Boehringer Ingelheim.
KH2PO4 (Aristar) was from BDH Chemicals (Poole,
UK), methanol (Chromosolv) was from Riedel, and tetrabutylammonium (PIC-A) was from Waters Associates (Milford, MA). All other reagents were of the highest purity available.
All nucleotide stock solutions were made up into a 5 mM
stock solution in water. Because most commercially available ATP
analogs are contaminated (up to 5%) by ATP, ADP, and other
unidentified substances, ATP analogs (except 2'-dATP) were separated by
HPLC (as described below) before their use in electrophysiological or
kinetic experiments. It was observed (data not shown) that the HPLC
eluent did not affect synaptic transmission or ATP catabolism in CA1
hippocampal synaptosomes. DPCPX was made up into a 5 mM stock solution in 99% dimethylsulfoxide/1% NaOH (1 M)
(vol/vol). Dipyridamole was made up into a 5 mM
dimethylsulfoxide solution. All stock solutions were stored as frozen
aliquots at  20°C. Aqueous dilutions of these stock solutions were
made daily and appropriate solvent controls were performed. The pH of
the superfusion solution did not change by the addition of the drugs in
the maximum concentrations used.
Electrophysiological recordings of synaptic
transmission. Rat hippocampal slices (400 µm thick) were
prepared (Cunha et al., 1994a) and allowed to recover for 1 hr at room
temperature in artificial cerebrospinal fluid of the following
composition (in mM): NaCl 124, KCl 3, KH2PO4 1.25, MgSO4 1, CaCl2 2, NaHCO3 26, glucose 10, pH 7.4, gassed
with a 95% O2 and 5% CO2 mixture. One slice
was transferred to a 1 ml recording chamber for submerged slices and
superfused continuously with gassed artificial cerebrospinal fluid,
kept at 30.5°C, at a flow rate of 3 ml/min. Drugs were added to this
superfusion solution.
Electrophysiological recordings of field EPSPs (fEPSPs) were obtained
as described previously (Cunha et al., 1994a). Monopolar stimulation
(rectangular pulses of 0.1 msec applied once every 10 sec) was
delivered through an electrode placed on the Schaffer fibers, in the
stratum radiatum near the CA3/CA1 border. Orthodromically evoked fEPSPs
were recorded through an extracellular microelectrode (4 M
NaCl, 2-6 M resistance) placed in the stratum radiatum of the CA1
area. The intensity of the stimulus (130-310 µA) was adjusted to
evoke the largest fEPSP without population spike contamination. The
individual responses were displayed on a Tektronix (2430A) oscilloscope, and the averages of eight consecutive responses were
digitally recorded. fEPSP responses were quantified as the initial
slope of the averaged fEPSPs. Perfusion of a slice with any tested
drugs was started after a stable response was recorded.
When concentration response curves for ATP analogs were performed, each
compound was added in increasing concentrations in a cumulative manner,
followed by washout. When the ability of a given substance to modify
the effect of ATP analogs was tested, ATP analogs were first tested in
the absence of the substance, the substance was then applied to the
preparation for at least 30 min, and the effect of the ATP analogs was
then tested in the presence of the substance. Usually the substance was
washed out, and the effect of ATP analogs was tested again in the
absence of the substance.
Extracellular catabolism of ATP analogs in CA1 slices.
Slices from the CA1 area were manually dissected (Cunha et al., 1994b) and allowed to recover for 1 hr at room temperature in artificial cerebrospinal fluid gassed with a 95% O2 and 5%
CO2 mixture. Groups of three CA1 slices were then
transferred to incubation vials with 500 µl of Krebs' solution of
the following composition (in mM): NaCl 125, KCl 3, KH2PO4 1.25, MgSO4 1, CaCl2 2, HEPES 25, glucose 10, pH 7.4, kept at 30.5°C
under continuous orbital swirling. After 10 min incubation, the initial
substrate, i.e., ATP or an ATP analog, was added at a final
concentration of 30 µM. The kinetic protocols consisted
of a 20 min incubation period with sample collection (50 µl) at 0, 1, 2, 5, 10, 15, and 20 min. The zero time was defined as the sample
collected immediately after (~2-5 sec) addition of the initial
substrate. The samples were stored on ice and analyzed by HPLC. After
the incubation, the remaining bathing solution was removed, and the
slices were homogenized in 200 µl of 2% (vol/vol) Triton X-100 to
determine total lactate dehydrogenase activity and protein content.
Lactate dehydrogenase activity, an index of cellular disruption (Cunha
et al., 1992), was quantified in the bathing solution and was always
lower than 2% of total lactate dehydrogenase activity in the CA1
slices.
Extracellular catabolism of adenine nucleotides in CA1
synaptosomes. Synaptosomes from the CA1 area were obtained from
homogenized, manually dissected CA1 slices by a sucrose-Percoll method
(Cunha et al., 1992). Aliquots of 100 µl of the synaptosomes
(resuspended in 800 µl of Krebs' solution) were then added to
incubation vials with 400 µl of Krebs' solution kept at 30.5°C.
After a 10 min incubation, the initial substrate, i.e., ATP, or an ATP
analog, or AMP in the presence of ATP analogs, was added at a final
concentration of 30 µM. The kinetic protocols were
identical to these described for CA1 slices. Each collected sample was
centrifuged (14,000 × g for 15 sec) in a refrigerated
centrifuge (4°C), and the supernatant (50 µl) was stored on ice for
HPLC analysis. After the 20 min incubation, the synaptosomes were
pelleted by centrifugation (14,000 × g for 15 sec) in
a refrigerated centrifuge (4°C). The pelleted synaptosomes were
homogenized in 200 µl of 2% (vol/vol) Triton X-100 to determine
total lactate dehydrogenase activity and protein content. The remaining
bathing solution was used to quantify lactate dehydrogenase activity,
which was always lower than 3% of total lactate dehydrogenase activity
in the CA1 synaptosomes.
HPLC analysis. Separation of nucleotides and their
degradation products was performed by ion-pair reverse-phase HPLC
analysis of nonextracted samples (20 µl) as described previously
(Cascalheira and Sebastião, 1992), with minor modifications,
using a Beckman 126 solvent delivery module equipped with a 210A sample
injection valve with a 20 µl loop coupled with a Beckman 166 UV
detector set at 254 nm, both connected to a Compaq 286e computer using the Gold software package. Separations were performed at room temperature with LiChrospher 100 RP-18 (5 µm) cartridges (Merck, Darmstadt, Germany) fitted into a Manu-cart holder (Merck). The columns
were protected by LiChrospher 60 RP-select B (5 µm) pre-columns (Merck). The eluent, pH 6.0, was composed of
KH2PO4 60 mM, tetrabutylammonium 5 mM, and 5-35% (vol/vol) methanol. A 10 min linear
gradient from 5 to 35% (vol/vol) methanol was performed, starting
after sample injection, with a constant flow rate of 1.5 ml/min. Under
these conditions, the retention times of the substances used as
standards were as follows: hypoxanthine (1.2 min), inosine (1.7 min),
adenosine (3.6 min), AMP (4.7 min), AOPCP (5.8 min), ADP (6.8 min),
,-methylene-ATP (7.8 min), ,-imido-ATP (7.9 min),
,-methylene-ATP (8.0 min), ATP (8.3 min), 2-methylthio-AMP (9.8 min), ATPS (10.7 min), 2-methylthio-ADP (10.9 min), and
2-methylthio-ATP (11.6 min). Linear calibration curves were obtained
for each substance after electronic integration of the peak area for
each substance (1-1000 pmol in 20 µl injected). Identification of
the peaks was performed by comparison of relative retention times with
standards.
Kinetic analysis. The concentrations of the products at the
different times of sample collection were corrected by subtracting the
concentrations of products eventually present at zero time. The
concentration of products, in samples collected from the same batch of
slices or synaptosomes, incubated without adding substrate, were also
subtracted for correction of spontaneous release. Plots of
concentration of the substrate and products as a function of time
(progress curves) were constructed. The relative extent of catabolism
of ATP analogs was compared by calculating the amount of substrate
catabolized after 20 min. The specific activity of ecto-ATPase activity
in hippocampal preparations was calculated by linear regression of the
decrease of the amount of extracellular ATP during the first 5 min of
catabolism, normalized per milligram of protein. To test the effects of
p-nitrophenylphosphate on the catabolism of ATP analogs,
progress curves of ATP analogs were performed in parallel batches of
slices or synaptosomes from the same group of animals, one in the
absence and the other in the presence of
p-nitrophenylphosphate (1 mM).
The extracellular catabolism of AMP in the presence of ATP analogs was
analyzed as described previously (Cunha et al., 1992).
Other determinations. Lactate dehydrogenase (EC 1.1.1.27)
activity was assayed by the method of Keiding et al. (1974). Protein concentration of the slices and of the synaptosomes was determined by
the Bradford method, modified according to Spector (1978).
Statistical calculations. The values are presented as
mean ± SEM, with n being the number of animals or
different groups of animals used. The significance of the differences
between the means was calculated by a one-way ANOVA followed by a
paired Student's t test or a Dunnett's test. p
values of <0.05 were considered to represent significant
differences.
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RESULTS |
Effects of ATP analogs on hippocampal synaptic transmission
ATP, in the micromolar range, inhibited in a
concentration-dependent manner the fEPSP slope in stimulated CA1
pyramid synapses, with an IC50 of 20 ± 3 µM (n = 4) and was slightly less potent than adenosine (IC50 of 12 ± 2 µM;
n = 4). ATP analogs substituted in the -phosphorus
also inhibited synaptic transmission in a concentration-dependent
manner (Fig. 1); the IC50
values were 17 ± 3 µM (n = 4) for
ATPS, 22 ± 5 µM (n = 4) for
,-imido-ATP, and 20 ± 2 µM (n = 4) for ,-methylene-ATP. ,-methylene-ATP, an ATP analog
substituted in the -phosphorus, was less potent than ATP (Fig. 1),
with a maximal inhibition of 19 ± 2% at 60 µM
(n = 4; p < 0.05). ATP analogs
substituted in the purine moiety, namely in the ribose ring, such as
2-methylthio-ATP (Fig. 1), were also less potent than ATP, with a
maximum inhibition of 25 ± 5% at 60 µM
(n = 4; p < 0.05), or even devoid of
effects, such as 2'-dATP (10-100 µM; n = 2).
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Figure 1.
Inhibition of the fEPSP slope, recorded
extracellularly in hippocampal CA1 pyramids, by exogenously added
adenosine ( ), ATP ( ), ATPS ( ), ,-imido-ATP ( ),
,-methylene-ATP ( ), 2-methylthio-ATP ( ), and
,-methylene-ATP ( ). The ordinates represent the percentage of
inhibition of fEPSP slope produced by adenosine, ATP, or ATP analogs in
relation to the fEPSP slope in control conditions (i.e., in the absence
of any added drug to the perfusion solution). 0% corresponds to the
fEPSP slope in control conditions (i.e., without any added drug), and
100% corresponds to blockade of fEPSP. The results are mean ± SEM of two to five experiments. The SEMs are shown when they exceed the
symbols in size.
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As shown in Figure 2, the inhibitory
effect of ATP and of ATP analogs (30 µM) was not modified
(n = 2-3) by the P2 receptor antagonist
suramin (100 µM). The inhibitory effect of ATP and of
-phosphorus-substituted ATP analogs was also virtually unaffected by
the P2 receptor antagonists, reactive blue 2 (30 µM; n = 2), or PPADS (30 µM; n = 1). By itself, suramin (100 µM; n = 3), reactive blue 2 (30 µM; n = 2), and PPADS (30 µM; n = 1) caused a 16 ± 1%,
21 ± 4%, and 15% inhibition of fEPSP slope, respectively.
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Figure 2.
Modification of the inhibition by adenosine,
ATP, and ATP analogs of fEPSPs by 100 µM suramin, a
P2 receptor antagonist (A), by 100 µM ,-methylene ADP (AOPCP), an inhibitor of
ecto-5'-nucleotidase (B), by 2 U/ml adenosine
deaminase (ADA), the enzyme that converts adenosine into its inactive
metabolite, inosine (C), by 10 nM 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), an adenosine A1
receptor antagonist (D), and by 20 µM dipyridamole, an inhibitor of adenosine uptake
(E). The ordinates represent the percentage
inhibition of fEPSP slope by adenosine or ATP, ATPS (S),
,-imido-ATP (,-Im), ,-methylene-ATP (,-Me),
, -methylene-ATP (Me), and 2-methylthio-ATP (2MeS) in the
absence (open bars) and presence (filled
bars) of the drugs indicated in each panel. 0% corresponds to
the fEPSP slope in control conditions (i.e., without any added drug or
after addition of suramin, AOPCP, adenosine deaminase, DPCPX, or
dipyridamole), and 100% corresponds to blockade of fEPSPs. In each
experiment, suramin, AOPCP, adenosine deaminase, DPCPX, or dipyridamole
were applied to the preparations 30-45 min before the effect of
adenosine, ATP, or ATP analogs was tested in their presence. The effect
of adenosine, ATP, or ATP analogs in the absence and presence of
suramin, AOPCP, adenosine deaminase, DPCPX, or dipyridamole was always
compared in the same experiment. * p < 0.05 (paired Student's t test) when comparing with the
effect of adenosine, ATP, or ATP analogs alone. The results are
mean ± SEM of three to four experiments. The SEMs are shown when
they exceed the symbols in size.
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In contrast, the inhibitor of ecto-5'-nucleotidase, AOPCP (100 µM), inhibited by 42-49% (n = 3) the
inhibitory effect of ATP and -substituted ATP analogs (Fig. 2). This
effect was not attributable to any effect of AOPCP per se on purine
receptors because the inhibitory effect of adenosine (30 µM) was not modified by 100 µM AOPCP
(n = 3). As described previously (Cunha et al., 1996b), AOPCP (100 µM) by itself caused a 18 ± 2%
inhibition of fEPSP slope (n = 3).
That the effect of ATP and -phosphorus-substituted ATP analogs
depended mainly on the formation of extracellular adenosine was also
supported by the observation that adenosine deaminase (2 U/ml), which
converts adenosine into its inactive metabolite inosine, inhibited by
74-85% (n = 3-4) the inhibitory effect of ATP and
-substituted ATP analogs (30 µM) (Fig. 2). By itself, adenosine deaminase (2 U/ml) caused a 23 ± 2% increase of fEPSP slope (n = 4), which is compatible with an inhibitory
"tonus" by endogenous adenosine (Cunha et al., 1996b).
The requirement for activation of inhibitory adenosine A1
receptors to observe the inhibitory effect of ATP and
-phosphorus-substituted ATP analogs was suggested from the
observation that 10 nM DPCPX, an adenosine A1
receptor antagonist that blocks the inhibitory effect of maximally
effective concentrations of 2-chloroadenosine in the hippocampus
(Sebastião et al., 1990), inhibited by 80-93% (n = 3-4) the inhibitory effect of ATP and
-substituted ATP analogs (30 µM) (Fig. 2). By itself,
DPCPX (10 nM) caused a 19 ± 3% increase of fEPSP
slope (n = 4).
Stronger support for the involvement of extracellular adenosine
formation as a main requirement for the inhibitory effect of ATP and
-substituted ATP analogs was derived from the observation that
dipyridamole (20 µM), which supramaximally inhibits
adenosine uptake (Morgan and Marangos, 1987), potentiated the
inhibitory effect of ATP and -phosphorus-substituted ATP analogs (10 µM) (Fig. 3). Thus,
dipyridamole (20 µM) potentiated by 92-124%
(n = 3-4) the inhibitory effect of ATP and
-substituted ATP analogs (10 µM), and it potentiated
the inhibitory effect of adenosine (10 µM) by a similar
amount (113 ± 26%; n = 4). By itself,
dipyridamole (20 µM) caused a 30 ± 5% inhibition
of fEPSP slope (n = 4), probably by the ability of
dipyridamole to increase the endogenous levels of extracellular
adenosine (Mitchell et al., 1993).
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Figure 3.
Effect of the inhibitor of adenosine uptake,
dipyridamole to potentiate the inhibitory effect of ATPS
(1), ,-imido-ATP (2),
,-methylene-ATP (3), 2-methylthio-ATP
(4), ATP (5), adenosine (6), and ,-methylene-ATP
(7) on fEPSP slope. In A is
shown the time course of the slope of averages of eight consecutive
fEPSPs recorded from the CA1 area of the hippocampus. The
hippocampal slice was perfused with adenosine, ATP, or ATP analogs (10 µM) either in the absence or in the presence of
dipyridamole (20 µM), as shown in the top
bars. In B, C, and
D are shown recordings of fEPSPs, corresponding to the
absence of any added drug in the perfusion medium (i in
B, C, and D), the presence
of ATP (B, ii), ATPS
(C, ii), and adenosine (D,
ii), the presence of dipyridamole (iii in
B, C, and D), the
simultaneous presence of dipyridamole and ATP (B,
iv), the simultaneous presence of dipyridamole and ATPS (C, iv), and the simultaneous
presence of dipyridamole and adenosine (D,
iv). Each recording is composed of a stimulus artifact followed by the presynaptic volley and the fEPSP and corresponds to the
average of eight consecutive responses. Calibration bars (shown in
B for B-D): 500 µV, 5 msec. Note that
dipyridamole (20 µM) potentiated to a similar extent the
inhibitory effect of -phosphorus-substituted ATP analogs
(1, 2, and 3), ATP
(5), and adenosine (6),
whereas the inhibitory effect of 2-methylthio-ATP
(4) and ,-methylene-ATP (7) was not modified appreciably.
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The small inhibitory effect of ,-methylene-ATP (30 µM) was more potently inhibited (65 ± 5%;
n = 3) by 100 µM AOPCP than the
inhibitory effect of -phosphorus-substituted ATP analogs, less
inhibited by 10 nM DPCPX (60 ± 5%; n = 3), and completely prevented (n = 2) by 2 U/ml
adenosine deaminase (Fig. 2). The potentiation of the inhibitory effect
of ,-methylene-ATP (10 µM) by 20 µM
dipyridamole (32 ± 15%; n = 3) was also lower
than that observed for -phosphorus-substituted ATP analogs (Fig.
2).
The small inhibitory effect of 2-methylthio-ATP (30 µM)
was also less inhibited by 2 U/ml adenosine deaminase (44 ± 5%;
n = 3) and by 10 nM DPCPX (67 ± 6%;
n = 2) than the inhibitory effect of
-phosphorus-substituted ATP analogs and was not consistently modified by 100 µM AOPCP (n = 3) or by 20 µM dipyridamole (n = 3) (Fig. 2).
Catabolism of ATP analogs in hippocampal CA1 slices
and synaptosomes
As shown in Figure
4A, extracellular ATP
(30 µM) was catabolized by hippocampal CA1 slices
(0.75 ± 0.02 mg protein), with a half-degradation of 8 ± 2 min (n = 5). The ATP metabolites detected in the bath
were ADP, the concentration of which reached a maximum of 7 ± 2 µM at 10 min; AMP, the concentration of which reached a
maximum of 9 ± 1 µM at 15 min; and adenosine, the
maximum concentration of which (12.5 ± 0.9 µM) was
reached at 20 min.
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Figure 4.
Progress curves of ATP and ATP analog catabolism
in CA1 hippocampal slices and synaptosomes. ATP or ATP analogs (30 µM) were incubated at zero time with CA1 slices or
synaptosomes. Samples (50 µl) were collected from the bath (600 µl)
at the times indicated in the abscissa and analyzed by HPLC. In
A is shown the catabolism of ATP () into ADP (),
AMP (), and adenosine () in CA1 slices. In B is
shown the catabolism of ATP () into ADP (), AMP (), and
adenosine ( ) in CA1 synaptosomes. In C is shown the
catabolism of ATPS (, ) into AMP (,) in CA1 slices
(filled symbols, filled lines) and CA1
synaptosomes (open symbols, broken lines). In
D is shown the catabolism of ,-imido-ATP (,
) into AMP (,) in CA1 slices (filled symbols,
filled lines) and CA1 synaptosomes (open symbols, broken
lines). In E is shown the catabolism of ,-methylene-ATP (, ) into AMP (,) in CA1 slices
(filled symbols, filled lines) and CA1
synaptosomes (open symbols, broken lines). In
F is shown the catabolism of ,-methylene ATP (, ) into ,-methylene ADP ( ,  ) in CA1 slices
(filled symbols, filled lines) and CA1
synaptosomes (open symbols, broken lines). In
G is shown the catabolism of 2-methylthio-ATP () into
2-methylthio-ADP (), 2-methylthio-AMP () and into an unidentified
compound () in CA1 slices. In H is shown the
catabolism of 2-methylthio-ATP () into 2-methylthio-ADP (),
2-methylthio-AMP () and into an unidentified compound () in CA1
synaptosomes. Each point is the average of four to five experiments.
The vertical bars represent the SEM and are shown when
they exceed the symbols in size. The concentrations of inosine and
adenosine detected under control conditions, without addition of
initial substrate, were subtracted. The concentration of any purine
metabolite present at time 0 was also subtracted.
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The catabolism of ATP (30 µM) showed a similar pattern in
hippocampal CA1 synaptosomes (2.6 ± 0.2 mg protein;
n = 4), with a half-degradation time of 0.8 ± 0.1 min. The ATP metabolites detected in the bath were ADP, the
concentration of which reached a maximum of 10 ± 1 µM at 1 min; AMP, the concentration of which reached a
maximum of 11 ± 2 µM at 10 min; and adenosine, the
concentration of which reached a maximum of 17 ± 3 µM at 15 min (Fig. 4B).
Surprisingly, the extent of catabolism of -phosphorus-substituted
ATP analogs (30 µM) was not significant in hippocampal CA1 slices (n = 5). The concentration of these ATP
analogs did not change significantly during the 20 min incubation
period, nor did any purine metabolite appear in the bath in measurable amounts (Fig. 4C-E).
In contrast, -phosphorus-substituted ATP analogs (30 µM) were catabolized in hippocampal CA1 synaptosomes
(Fig. 4C-E). After 20 min, 5.1 ± 0.5 µM
ATPS had been catabolized (n = 4), and 1.0 ± 0.1 µM AMP appeared in the bath; 3 ± 1 µM ,-imido-ATP had been catabolized
(n = 4), and 0.8 ± 0.1 µM AMP
appeared in the bath after 20 min; and 4.0 ± 0.2 µM
,-methylene-ATP had been catabolized (n = 4), and
1.0 ± 0.2 µM AMP appeared in the bath after 20 min.
In hippocampal CA1 synaptosomes, when ATPS, ,-imido-ATP, or
,-methylene-ATP were assayed as initial substrates, ADP never appeared in the bath in measurable amounts. The presence of
p-nitrophenylphosphate (1 mM) did not
appreciably change the amounts of ATPS, ,-imido-ATP or
,-methylene-ATP catabolized after 20 min, nor did
p-nitrophenylphosphate appreciably change the amounts of AMP
formed as a result of -phosphorus-substituted ATP analogs (30 µM) catabolism.
When ,-methylene-ATP (30 µM) was used as
initial substrate, the only metabolite appearing in the bath was AOPCP,
which reached a maximum concentration of 10.4 ± 0.7 µM in CA1 slices (n = 5) and 13 ± 1 µM in CA1 synaptosomes (n = 4) (Fig.
4F).
2-Methylthio-ATP (30 µM) was catabolized in both CA1
slices (Fig. 4G) and CA1 synaptosomes (Fig.
4H). During the 20 min incubation period, 9.3 ± 0.6 µM and 13.9 ± 0.6 µM
2-methylthio-ATP were catabolized in both CA1 slices (n = 5) and synaptosomes (n = 4), respectively. 2-Methylthio-ATP was converted into 2-methylthio-ADP and
2-methylthio-AMP and in another nonidentified product with a retention
time of 7.9 min, which might correspond to 2-methylthio-adenosine,
although no standard is commercially available to identify this peak
(Fig. 4G,H).
The catabolism of AMP (30 µM) resulted in the formation
of adenosine, inosine, and hypoxanthine, as described previously (Cunha et al., 1992). The rate of AMP catabolism during the first 5 min of
incubation was decreased by 12-21% by ,-methylene-ATP (30 µM; n = 2), and was not modified by
ATPS, ,-imido-ATP, or ,-methylene-ATP (30 µM; n = 2).
| |
DISCUSSION |
The present results show that the inhibition of
synaptic transmission in Schaffer fibers/CA1 pyramid synapses of rat
hippocampal slices by ATP and -phosphorus-substituted ATP analogs is
mediated by activation of inhibitory A1 adenosine receptors
after extracellular catabolism of these nucleotides
into adenosine by ecto-nucleotidases. Thus, the inhibitory effect on
synaptic transmission of ATP and -substituted ATP analogs was
inhibited (1) by AOPCP, an inhibitor of ecto-5'-nucleotidase, the
enzyme that forms adenosine from extracellular adenine nucleotides, (2)
by adenosine deaminase, which converts adenosine into its inactive
metabolite inosine, and (3) by DPCPX, an adenosine A1
receptor antagonist, whereas (4) it was potentiated by the
adenosine uptake inhibitor dipyridamole.
The inability of the nucleotide P2 receptor
antagonists suramin reactive blue 2 and PPADS to modify the inhibitory
effect of ATP and -substituted ATP analogs further supports the idea that extracellular catabolism of ATP and -substituted ATP analogs into adenosine is mainly responsible for this inhibitory effect. We
tested three chemically distinct P2 antagonists to exclude the possibility that diffusion barriers might exist to access P2 receptors. It should be stressed, however, that the
possibility of P2 receptors having a role in the
hippocampus cannot be excluded. Indeed, P2 receptors are
expressed in the hippocampus (Kidd et al., 1995; Buell et al., 1996;
Collo et al., 1996), the effects of ATP on synaptic transmission in the
hippocampus are not completely prevented by pharmacological tools
interfering with adenosine neuromodulation (Lee et al., 1981; Cunha et
al., 1996b), and antagonists of P2 receptors produce
effects in hippocampal preparations (Motin and Bennett, 1995), although
they also directly affect ionotropic GABA and glutamate receptors
(Nakazawa et al., 1995) and modify ecto-nucleotidase activity
(Ziganshin et al., 1996). Also, P2 receptors insensitive to
the commonly used P2 receptor antagonists have been
described (Buell et al., 1996). Thus, it is possible that at higher
frequencies of stimulation, for instance, P2 receptors may
have a physiological role in modulating synaptic transmission in the
hippocampus, as has been shown to occur with ecto-protein kinases
(Wieraszko, 1996); although at lower frequencies of stimulation, as
used in the present work, the effect of ATP is mostly mediated by
A1 receptors after extracellular catabolism into adenosine by ecto-nucleotidases. Previous work also concluded that the absence of
P2 receptor-mediated modulation of transmission in Schaffer fibers/CA1 pyramid synapses of rat hippocampal slices based on the use
of ATP analogs derived from the inactive enantiomer of adenosine
L-adenosine (Stone and Cusack, 1989).
The discrepancy between the stability of -substituted ATP
analogs in an innervated skeletal muscle preparation and the
modification of their action on neuromuscular transmission by modifiers
of adenosine metabolism lead to the proposal of localized catabolism of
adenine nucleotides coupled to activation of inhibitory adenosine A1 receptors (Bruns, 1990; Cascalheira and Sebastião,
1992). This discrepancy was also observed in the present work, i.e., no
measurable extracellular metabolism of -substituted ATP analogs seems to occur in hippocampal slices, whereas the functional assays revealed an adenosine-mediated action of these nucleotides. The channeling of the product of ecto-nucleotidases activity, adenosine, to
A1 receptors allows us to understand how a very low rate of catabolism of -substituted ATP analogs might allow effective activation of adenosine A1 receptors. Although very low
amounts of adenosine are formed, substrate channeling allows a high
local concentration of adenosine to be reached in the surroundings of A1 receptors. This possibility, which was not tested or
considered in several studies on the physiological effects mediated by
extracellular ATP, has lead to the proposal that -substituted ATP
analogs might directly activate adenosine A1 receptors
(Hourani et al., 1991; Bailey et al., 1992; Piper and Hollingsworth,
1996), although binding studies show that -substituted ATP analogs
are not effective displacers of adenosine A1 receptor
binding (Williams and Braunwalder, 1986). The existence of substrate
channeling properties in the delivery of adenosine formed from
ecto-nucleotidases and the existence of a strong gradient for
extracellular adenosine in a synapse (Cunha, 1997) could also explain
the greater potency of ATP and ATP analogs compared with that of
adenosine to inhibit neurotransmitter release or synaptic transmission
(Silinsky and Ginsborg, 1983; Shinozuka et al., 1990; Cunha et al.,
1994c), which was the basis for proposing the existence of a
P3 receptor (Shinozuka et al., 1990; Cunha et al., 1994c)
(but see Saitoh and Nakata, 1996). Thus, if adenosine is effectively
removed by the nucleoside transport system before reaching adenosine
A1 receptors and adenosine formed from extracellular
adenine nucleotides catabolism is preferentially delivered to adenosine
A1 receptor, it is conceivable that ATP derivatives may
have a greater potency than adenosine itself, although both act via
adenosine A1 receptor activation. Awareness is growing
(Harden et al., 1997) that to establish an effect of ATP as such it is
essential to show that removal of extracellular adenosine, inhibition
of the ecto-nucleotidase cascade, and blockade of
A1-mediated responses does not occlude ATP effect. Only
after the possibility is excluded that the effect of ATP is not
mediated by localized extracellular catabolism into adenosine by the
ecto-nucleotidase cascade will it be possible to consider an effect
mediated by ATP as such.
The extracellular catabolism of ATP by ecto-nucleotidases was
qualitatively similar in hippocampal CA1 slices and synaptosomes, with
the apparent sequential formation of ADP, AMP, and adenosine. Quantitatively, the specific activity of ATP catabolism was higher in
synaptosomes than in slices, as had been observed previously in a
particular synaptosomal fraction (Cunha and Sebastião, 1992). ATP
analogs with substitutions in -phosphorus were also catabolized in
CA1 synaptosomes, but not in slices, although at a lower rate than ATP
catabolism. However, the extracellular catabolism of -phosphorus-substituted ATP analogs was different from that of ATP,
because the only metabolite detected in the bath was AMP, whereas ADP
was never detected. This pattern would be expected if an ecto-ATP
pyrophosphatase (EC 3.6.1.8) instead of an ecto-ATP diphosphohydrolase
(Plesner, 1995) would be catabolizing extracellular ATP, because the
involvement of nonspecific phosphatases was excluded on the basis of
the absence of effect of p-nitrophenylphosphate. The lack of
commercially available inhibitors of ecto-ATP pyrophosphatase precludes
direct testing of this hypothesis. The ability to detect catabolism of
ATP analogs in synaptosomes but not in slices probably reflects a
higher specific activity of catabolism of -substituted ATP analogs
in synaptosomes. It is expected that localized catabolism of
-substituted ATP analogs might occur in the slices, because the
synaptosomes were obtained from the slices, but the rate of catabolism
may be below detection limit.
It is interesting to note that ATP analogs substituted in the
-phosphorus or in the purine ring displayed a functional behavior distinct from that of -substituted ATP analogs. Thus, the modulators of adenosine metabolism/effects (adenosine deaminase, DPCPX) affected the action of ,-methylene-ATP the same as they modify the action of AOPCP in the hippocampus (Cunha et al., 1996b). The observations that a long (30 min) preincubation with ,-methylene-ATP (100 µM) reduces the inhibitory effect of ATP (30 µM) on synaptic transmission by 16-21%
(n = 2; data not shown) and that ,-methylene-ATP
(30 µM) inhibited extracellular AMP catabolism further
supports the possibility that the effect of this -substituted ATP
analog is caused by direct or indirect (after conversion into AOPCP)
(compare Fig. 4F) inhibition of ecto-5'-nucleotidase.
Although presynaptic inhibition of neurotransmitter by activation of
P2Y receptors has been proposed (von Kügelgen, 1996),
the effect of 2-methylthio-ATP, which was smaller than that of the
other ATP analogs, is currently difficult to interpret with the
biochemical and pharmacological approaches used.
In conclusion, the present results demonstrate that ATP and
-phosphorus-substituted ATP analogs have to be extracellularly converted into adenosine to exert their inhibitory effects on synaptic
transmission in the hippocampus, and they highlight the importance of
channeling adenosine to adenosine A1 receptors as a means
for adenine nucleotides to inhibit synaptic transmission.
| |
FOOTNOTES |
Received Sept. 10, 1997; revised Jan. 5, 1998; accepted Jan. 6, 1998.
This work was supported by Junta Nacional de Investigaçao
Cientifica e Tecnologica, Praxis XXI, Gulbenkian Foundation, and European Union (BIOMED 2 programme). We thank Dr. H. Zimmermann for
critically reviewing this manuscript.
Correspondence should be addressed to R. A.Cunha, Laboratory of
Neurosciences, Faculty of Medicine, University of Lisbon, Avenida
Professor Egas Moniz, 1600 Lisboa, Portugal.
| |
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Abstract
Introduction
