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Diadenosine polyphosphates facilitate the evoked release of acetylcholine from rat hippocampal nerve terminals
Brain Research 879 (2000) 50–54 www.elsevier.com / locate / bres
Research report
Diadenosine polyphosphates facilitate the evoked release of acetylcholine from rat hippocampal nerve terminals c ´ ´ Hernandez ´ ´ Pintor c,d , Maria Teresa Miras-Portugal c , M. Fatima Pereira a , Miguel Diaz , Jesus a,b , a Rodrigo A. Cunha *, J. Alexandre Ribeiro b
a Laboratory of Neurosciences, Faculty of Medicine, University of Lisbon, 1649 -028 Lisbon, Portugal Department of Chemistry and Biochemistry, Faculty of Sciences, University of Lisbon, 1749 -016 Lisbon, Portugal c ´ , Complutense University, 28040 Madrid, Spain Department of Biochemistry, Faculty of Veterinary, E.U. Optica d ´ , Faculty of Veterinary, Complutense University, 28040 Madrid, Spain E.U. Optica
Accepted 18 July 2000
Abstract Diadenosine polyphosphates are present in synaptic vesicles, are released upon nerve stimulation and possess membrane receptors, namely in presynaptic terminals. However, the role of diadenosine polyphosphates to control neurotransmitter release in the CNS is not known. We now show that diadenosine pentaphosphate (Ap 5 A, 3–100 mM) facilitated in a concentration dependent manner the evoked release of acetylcholine from hippocampal nerve terminals, with a maximal facilitatory effect of 116% obtained with 30 mM Ap 5 A. The selective diadenosine polyphosphate receptor antagonist, diinosine pentaphosphate (Ip 5 I, 1 mM), inhibited by 75% the facilitatory effect of Ap 5 A (30 mM), whereas the P2 receptor antagonists, suramin (100 mM) and pyridoxal-phosphate-6-azophenyl-29,49-disulfonic acid (PPADS, 10 mM) only caused a 18–24% inhibition, the adenosine A 1 receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (20 nM), caused a 36% inhibition and the adenosine A 2A receptor antagonist, 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo [2,3-a][1,3,5]triazin-5ylamino]ethyl)phenol (ZM 241385, 20 nM), was devoid of effect. These results show that diadenosine polyphosphates act as neuromodulators in the CNS, facilitating the evoked release of acetylcholine mainly through activation of diadenosine polyphosphate receptors. 2000 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters and receptors Topic: Acetylcholine Keywords: Ap 5 A; Diadenosine polyphosphate; Nerve terminal; Hippocampus; ATP; Adenosine; Receptor
1. Introduction It is now commonly accepted that several transmitters are involved in the chemical transmission of information at most individual synapses (e.g. [24]). Among the signalling molecules stored in synaptic vesicles, adenine nucleotides are a class of molecules with a potentially more generalised importance, since they are co-stored and co-released with several neurotransmitters (reviewed in [27]). The more abundant adenine nucleotide in synaptic vesicles is *Corresponding author. Tel.: 1351-21-7936787; fax: 1351-217936787. E-mail address: [email protected] (R.A. Cunha).
ATP, but another class of adenine nucleotides, diadenosine polyphosphates (Ap n A), has been identified in synaptic vesicles [17,18]. Like ATP [6], Ap n A (Ap 4 A, Ap 5 A and Ap 6 A) are released upon stimulation of nerve terminals [17]. Ap n A can activate some P2 receptor subtypes [20,26] or be extracellularly metabolised into adenosine [11], a neuromodulator on its own [2], but can also activate receptors selective for Ap n A [15,16,19,21]. Ap n A may potentially act as neuromodulator in the CNS, since activation of Ap n A receptors in rat or human midbrain nerve terminals cause an increase in intraterminal calcium [16,22]. However, the effect of Ap n A receptor activation on the release of neurotransmitters is not known. Since clear evidence for the presence of Ap n A in
0006-8993 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )02726-8
M.F. Pereira et al. / Brain Research 879 (2000) 50 – 54
cholinergic synaptic vesicles has been provided [18], we now tested the effect of Ap n A on acetylcholine release from hippocampal nerve terminals. We found that Ap 5 A facilitated the evoked release of acetylcholine and that this effect was mostly due to activation of Ap n A receptors rather than nucleotide P2 receptors or adenosine A 1 or A 2A receptors.
2. Material and methods Diadenosine pentaphosphate (Ap 5 A), hemicholinium-3, neostigmine, choline kinase (EC 2.7.1.32), veratridine and tetraphenylboron were from Sigma. Suramin, 1,3-dipropyl8-cyclopentylxanthine (DPCPX) and pyridoxal-phosphate6-azophenyl-29,49-disulfonic acid (PPADS) were from RBI and 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl) phenol (ZM 241385) was from Tocris. Methyl-[ 3 H]choline chloride (specific activity 76– 86.3 Ci / mmol) was obtained from Amersham. Diinosine pentaphosphate (Ip 5 I) was synthesised as previously described [21]. All other reagents were of the highest purity available. ZM 241385 was made up into a 5 mM stock in dimethylsulfoxide and DPCPX was made up into a 5 mM stock in 99% dimethylsulfoxide–1% NaOH (1 M) (v / v). Aqueous dilution of these stock solutions was made daily. Dimethylsulfoxide, in the maximum concentration applied during drug testing, was devoid of effects on acetylcholine release. The release of [ 3 H]acetylcholine (ACh) from a rat hippocampal synaptosomal fraction prepared from male Wistar rats, anaesthetised under halothane atmosphere, was performed as previously described [4]. Briefly, the synaptosomes were equilibrated at 378C for 10 min in Krebs solution, gassed with 95% O 2 and 5% CO 2 mixture, of the following composition (mM): NaCl, 124; KCl, 3; NaH 2 PO 4 , 1.25; MgSO 4 , 1; CaCl 2 , 2; NaHCO 3 , 26; glucose, 10; pH 7.4. From this time onwards, all solutions applied to the synaptosomes were kept at 378C and gassed with 95% O 2 and 5% CO 2 . After the equilibration period, the synaptosomes were loaded with [ 3 H]choline (10 mCi / ml, 0.125 mM) for 10 min, centrifuged, and washed twice with 1 ml of Krebs solution containing hemicholinium-3 (10 mM), which was present up to the end of the experiment to prevent the reuptake of choline. The synaptosomes were then resuspended in 10 ml of Krebs solution and layered over Whatman GF / C filters into 8 parallel 90 ml superfusion chambers (adapted from Swinny filter holders, Millipore) with the aid of a roller pump (flow rate: 0.6 ml / min, which was kept constant through the experiment). The chamber volume plus dead volume was approximately 0.6 ml. A series of 8 parallel superfusion chambers was used to enable both control and test conditions to be performed in duplicate from the same batch of synaptosomes. After a 15 min washout period, the
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effluent was collected in 2 min fractions for scintillation counting. The synaptosomes were stimulated with veratridine (10 mM) for 2 min at 6 and 26 min after starting sample collection (S 1 and S 2 ). At the end of the experiments, the filters were removed from the chambers to determine the amount of tritium retained by the synaptosomes. At least 12 h after addition of 5 ml of scintillation cocktail (Scintran T, BDH) to the aliquots of effluent samples and synaptosomes retained in the filters, scintillation counting was performed with a 55–60% efficiency during 2 min. The fractional release of tritium was expressed in terms of percentage of total radioactivity present in the tissue at the time of sample collection. The release of tritium evoked by each veratridine pulse, i.e. the evoked release, was calculated by integration of the area of the peak upon subtraction of the estimated basal tritium outflow from the total outflow of tritium due to stimulation. To measure [ 3 H]ACh in the total tritium outflow, the experiments were performed in the presence of neostigmine (20 mM), [ 3 H]ACh was separated using a cation exchanger, tetraphenylboron, after phosphorylation of [ 3 H]choline, as previously described [5]. When the effect of Ap 5 A on the release of ACh was investigated, Ap 5 A was added to the perfusion medium 6 min before S 2 , i.e. 20 min after starting sample collection, and remained in the bath up to the end of the experiment. The effect of Ap 5 A on the evoked release of ACh was expressed by alterations of the ratio between the evoked release due to second stimulation period and the evoked release due to the first stimulation period (S 2 / S 1 ratio). When we evaluated the modifications of the effect of Ap 5 A by an antagonist, the antagonist was applied 15 min before starting sample collection and hence was present during S 1 and S 2 . When present during S 1 and S 2 , none of the antagonists (Ip 5 I, suramin, PPADS, DPCPX or ZM 241385) significantly altered (P.0.05) the S 2 / S 1 as compared to the S 2 / S 1 ratio obtained in the absence of antagonists (data not shown). The values are presented as mean6S.E.M. To test the significance of the effect of Ap 5 A versus control, a paired Student’s t-test was used. When making comparisons from a different set of experiments with control, one-way analysis of variance (ANOVA) was used, followed by Dunnett’s test. P,0.05 was considered to represent a significant difference.
3. Results Two periods of stimulation (S 1 and S 2 ) with veratridine (10 mM) caused a similar evoked release of tritium from superfused hippocampal synaptosomes, with an S 2 / S 1 ratio of 0.9860.03 (n514). This evoked release of tritium was mostly Ca 21 -dependent and constituted by [ 3 H]ACh [5,6].
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M.F. Pereira et al. / Brain Research 879 (2000) 50 – 54
When Ap 5 A (30 mM) was added to superfused hippocampal synaptosomes from 6 min before S 2 onwards, there was a significantly larger evoked release of tritium compared to control (see Fig. 1A), and the S 2 / S 1 ratio was increased by 116614% (n56). In contrast, Ap 5 A (30 mM) did not cause any significant modification of basal tritium outflow. When we quantified the amount of [ 3 H]ACh in the effluent, we found that [ 3 H]ACh accounted for 7864% and 7863% (n54) of the evoked tritium release in the
Fig. 1. Facilitation by Ap 5 A of [ 3 H]ACh release from rat hippocampal synaptosomes. The synaptosomes were loaded with [ 3 H]choline, superfused and effluent samples analysed by scintillation counting. In (A) is compared the time course of tritium release in a typical experiment, in the absence (s) and in the presence of Ap 5 A (30 mM) (d). The synaptosomes were challenged with veratridine (10 mM) for 2 min at 6 min (S 1 ) and 26 min (S 2 ) after starting sample collection, as indicated by the bars above the abscissa. Ap 5 A (30 mM) was applied through the superfusate to two of the four parallel superfusion chambers containing the synaptosomes 6 min before S 2 , as indicated by the bar above the abscissa in A. In (B) is shown the concentration-dependent effect of Ap 5 A (3–100 mM) on evoked tritium release. The effect of each concentration of Ap 5 A on evoked release was calculated as the percentage variation of the amount of tritium released in S 2 / amount of tritium released in S 1 in the presence of Ap 5 A during S 2 versus the S 2 / S 1 ratio in control conditions in the same experiment. The results are mean6S.E.M. of 4–6 experiments. *P,0.05 versus 0%.
absence and presence of Ap 5 A (30 mM), respectively. In the absence of stimulation, [ 3 H]ACh accounted for 4264% and 4863% (n54) of basal tritium outflow in the absence and presence of Ap 5 A (30 mM), respectively. As illustrated in Fig. 1B, Ap 5 A caused a concentration dependent facilitation of the evoked tritium release. The estimated EC 50 was 4.6 mM and the maximal facilitatory effect (116614%, n56) was observed at 30 mM. Increasing concentrations of Ap 5 A (100 mM) were unable to cause a larger facilitation of the evoked release of tritium (Fig. 1B). The facilitatory effect of Ap 5 A (30 mM) was inhibited by 75617% (n54) by a supramaximal concentration of the diadenosine polyphosphate antagonist, Ip 5 I (1 mM) [21], and attenuated by 2463% (n54) and 1862% (n53) by supramaximal concentrations of the P2 receptor antagonists, suramin (100 mM) and PPADS (10 mM) [23], respectively (Fig. 2). A supramaximal concentration of the adenosine A 1 receptor antagonist, DPCPX (20 nM) [1], attenuated by 3666% (n54) the facilitatory effect of Ap 5 A (30 mM) on ACh release, whereas a supramaximal concentration of the adenosine A 2A receptor antagonist, ZM 241385 (20 nM, n54) [7], was devoid of effect (Fig. 2).
Fig. 2. Effect of the Ap n A receptor antagonist, Ip 5 I, of the P2 receptor antagonists, suramin and PPADS, of the adenosine A 2A receptor antagonist, ZM 241385, and of the adenosine A 1 receptor antagonist, DPCPX, on the facilitatory effect of Ap 5 A (30 mM) on [ 3 H]ACh release from rat hippocampal synaptosomes. The synaptosomes were loaded with [ 3 H]choline, superfused and effluent samples analysed by scintillation counting. Ap 5 A was added 6 min before S 2 , whereas the antagonists were added 15 min before starting sample collection and thus were present during S 1 and S 2 . The effect of drugs was calculated by modification of the S 2 / S 1 ratio. 0% corresponds to an S 2 / S 1 ratio equal to control (i.e. in the absence of any drug) and 100% corresponds to an S 2 / S 1 ratio twice of control. The absence (2) or presence (1) of each drug during S 2 or during S 1 and S 2 is indicated below each bar. The results are mean6S.E.M. of 4 experiments. *P,0.05 when compared with the effect of Ap 5 A (30 mM).
M.F. Pereira et al. / Brain Research 879 (2000) 50 – 54
4. Discussion The present results show that Ap 5 A facilitates the evoked release of ACh in the hippocampus. Previous suggestions for a presynaptic role of Ap n A were fostered by the observation that Ap n A increase the influx of calcium in to nerve terminals, causing an increase in basal and K 1 -evoked intracellular calcium concentration ([Ca 21 ] i ) in nerve terminals [16]. Since basal tritium outflow from synaptosomes labelled with [ 3 H]choline does not reflect [ 3 H]ACh release [4,5], it is not possible to conclude on the effect of Ap 5 A on basal ACh release, but only on the evoked release of tritium, which is Ca 21 dependent and mostly constituted by [ 3 H]ACh. Thus, the facilitation by Ap n A of the evoked, i.e. synchronous, release of ACh provide the first direct demonstration for a facilitatory neuromodulatory role of Ap n A in the CNS. One important issue to evaluate the physiological relevance of the presently observed facilitation of ACh release by Ap 5 A is whether the extracellular concentration of Ap 5 A in the synaptic cleft might reach low micromolar levels, compatible with the observed EC 50 of nearly 5 mM. Ap n A are stored in synaptic vesicles with a concentration ratio of 1:25 when compared with ATP [18]. However, the rate of extracellular catabolism of Ap n A in the nervous tissue is 20–50 times lower than that of ATP [15]. Thus, it is likely that the repetitive stimulation of nerve terminals may lead to a localised synaptic extracellular accumulation of Ap n A similar to that estimated for adenine nucleotides (7–25 mM) [3,25]. In different systems, Ap n A may activate Ap n A receptors [15,16,19,21,22], some P2 receptor subtypes [20,26], or be extracellularly metabolised into adenosine [11], and activate adenosine receptors [14]. The facilitation by Ap 5 A of ACh release was strongly inhibited by Ip 5 I, a Ap n A receptor antagonist [21], and only slightly attenuated by the P2 receptor antagonists, suramin and PPADS [23]. Besides potently blocking native Ap n A receptors, Ip 5 I is also an antagonist of some P2x receptor subtypes [13] and some P2 receptors have a low sensitivity to suramin and / or PPADS [23]. But, since ACh release from hippocampal nerve terminals is not under P2 receptor control [4], the inhibition by Ip 5 I of the facilitatory effect of Ap 5 A on ACh release strongly suggests the involvement of an Ap n A receptor. The possibility that activation of adenosine receptors might be responsible for the facilitation of ACh release by Ap 5 A was also not supported by the present results. It is known that the evoked release of ACh in the hippocampus is facilitated by A 2A receptor activation and is inhibited by A 1 receptor activation [5,7]. Also, there is an high and efficient ecto-nucleotidase pathway activity in the hippocampus [4,8] with adenosine being generated within milliseconds after ATP application [10]. However, blockade of A 2A receptors with ZM 241385 failed to modify the facilitatory effect of Ap 5 A on ACh release. Blockade
53
of inhibitory A 1 receptors would be expected to cause no effect or to eventually enhance the facilitation of ACh release by Ap 5 A. In contrast, we found that blockade of A 1 receptors inhibited the facilitatory effect of Ap 5 A on ACh release. It has previously been shown that the efficiency of Ap n A receptor activation is decreased upon removal of endogenous extracellular adenosine [9], indicating the existence of an interaction between A 1 and Ap n A receptors controlling the neuromodulatory effect of Ap n A [15]. In conclusion, the present results provide the first direct demonstration for a neuromodulatory role of Ap n A receptor activation in the CNS. The observation that Ap n A receptor activation facilitated ACh release opens a new avenue in the therapeutical possibilities of enhancing ACh release in situations of cholinergic deficits, which occur in some types of dementia or cognitive deficits [12].
Acknowledgements This work was supported by research grants from the o C.A.M. (n 8012 / 98), the Spanish Ministry of Education, ˜ para a Ciencia ˆ Fundac¸ao e Tecnologia, Culture DGCYT PM 98-0089 and the European Union (BIOMED 2, PL 950676). RAC is in debt to Professor Moniz Pereira (Faculty of Pharmacy, Lisbon) for scintillation counting facilities and to Professor Silva Carvalho (Department Physiology, Faculty Medicine, Lisbon) for animal house facilities.
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Research report
Diadenosine polyphosphates facilitate the evoked release of acetylcholine from rat hippocampal nerve terminals c ´ ´ Hernandez ´ ´ Pintor c,d , Maria Teresa Miras-Portugal c , M. Fatima Pereira a , Miguel Diaz , Jesus a,b , a Rodrigo A. Cunha *, J. Alexandre Ribeiro b
a Laboratory of Neurosciences, Faculty of Medicine, University of Lisbon, 1649 -028 Lisbon, Portugal Department of Chemistry and Biochemistry, Faculty of Sciences, University of Lisbon, 1749 -016 Lisbon, Portugal c ´ , Complutense University, 28040 Madrid, Spain Department of Biochemistry, Faculty of Veterinary, E.U. Optica d ´ , Faculty of Veterinary, Complutense University, 28040 Madrid, Spain E.U. Optica
Accepted 18 July 2000
Abstract Diadenosine polyphosphates are present in synaptic vesicles, are released upon nerve stimulation and possess membrane receptors, namely in presynaptic terminals. However, the role of diadenosine polyphosphates to control neurotransmitter release in the CNS is not known. We now show that diadenosine pentaphosphate (Ap 5 A, 3–100 mM) facilitated in a concentration dependent manner the evoked release of acetylcholine from hippocampal nerve terminals, with a maximal facilitatory effect of 116% obtained with 30 mM Ap 5 A. The selective diadenosine polyphosphate receptor antagonist, diinosine pentaphosphate (Ip 5 I, 1 mM), inhibited by 75% the facilitatory effect of Ap 5 A (30 mM), whereas the P2 receptor antagonists, suramin (100 mM) and pyridoxal-phosphate-6-azophenyl-29,49-disulfonic acid (PPADS, 10 mM) only caused a 18–24% inhibition, the adenosine A 1 receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (20 nM), caused a 36% inhibition and the adenosine A 2A receptor antagonist, 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo [2,3-a][1,3,5]triazin-5ylamino]ethyl)phenol (ZM 241385, 20 nM), was devoid of effect. These results show that diadenosine polyphosphates act as neuromodulators in the CNS, facilitating the evoked release of acetylcholine mainly through activation of diadenosine polyphosphate receptors. 2000 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters and receptors Topic: Acetylcholine Keywords: Ap 5 A; Diadenosine polyphosphate; Nerve terminal; Hippocampus; ATP; Adenosine; Receptor
1. Introduction It is now commonly accepted that several transmitters are involved in the chemical transmission of information at most individual synapses (e.g. [24]). Among the signalling molecules stored in synaptic vesicles, adenine nucleotides are a class of molecules with a potentially more generalised importance, since they are co-stored and co-released with several neurotransmitters (reviewed in [27]). The more abundant adenine nucleotide in synaptic vesicles is *Corresponding author. Tel.: 1351-21-7936787; fax: 1351-217936787. E-mail address: [email protected] (R.A. Cunha).
ATP, but another class of adenine nucleotides, diadenosine polyphosphates (Ap n A), has been identified in synaptic vesicles [17,18]. Like ATP [6], Ap n A (Ap 4 A, Ap 5 A and Ap 6 A) are released upon stimulation of nerve terminals [17]. Ap n A can activate some P2 receptor subtypes [20,26] or be extracellularly metabolised into adenosine [11], a neuromodulator on its own [2], but can also activate receptors selective for Ap n A [15,16,19,21]. Ap n A may potentially act as neuromodulator in the CNS, since activation of Ap n A receptors in rat or human midbrain nerve terminals cause an increase in intraterminal calcium [16,22]. However, the effect of Ap n A receptor activation on the release of neurotransmitters is not known. Since clear evidence for the presence of Ap n A in
0006-8993 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )02726-8
M.F. Pereira et al. / Brain Research 879 (2000) 50 – 54
cholinergic synaptic vesicles has been provided [18], we now tested the effect of Ap n A on acetylcholine release from hippocampal nerve terminals. We found that Ap 5 A facilitated the evoked release of acetylcholine and that this effect was mostly due to activation of Ap n A receptors rather than nucleotide P2 receptors or adenosine A 1 or A 2A receptors.
2. Material and methods Diadenosine pentaphosphate (Ap 5 A), hemicholinium-3, neostigmine, choline kinase (EC 2.7.1.32), veratridine and tetraphenylboron were from Sigma. Suramin, 1,3-dipropyl8-cyclopentylxanthine (DPCPX) and pyridoxal-phosphate6-azophenyl-29,49-disulfonic acid (PPADS) were from RBI and 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl) phenol (ZM 241385) was from Tocris. Methyl-[ 3 H]choline chloride (specific activity 76– 86.3 Ci / mmol) was obtained from Amersham. Diinosine pentaphosphate (Ip 5 I) was synthesised as previously described [21]. All other reagents were of the highest purity available. ZM 241385 was made up into a 5 mM stock in dimethylsulfoxide and DPCPX was made up into a 5 mM stock in 99% dimethylsulfoxide–1% NaOH (1 M) (v / v). Aqueous dilution of these stock solutions was made daily. Dimethylsulfoxide, in the maximum concentration applied during drug testing, was devoid of effects on acetylcholine release. The release of [ 3 H]acetylcholine (ACh) from a rat hippocampal synaptosomal fraction prepared from male Wistar rats, anaesthetised under halothane atmosphere, was performed as previously described [4]. Briefly, the synaptosomes were equilibrated at 378C for 10 min in Krebs solution, gassed with 95% O 2 and 5% CO 2 mixture, of the following composition (mM): NaCl, 124; KCl, 3; NaH 2 PO 4 , 1.25; MgSO 4 , 1; CaCl 2 , 2; NaHCO 3 , 26; glucose, 10; pH 7.4. From this time onwards, all solutions applied to the synaptosomes were kept at 378C and gassed with 95% O 2 and 5% CO 2 . After the equilibration period, the synaptosomes were loaded with [ 3 H]choline (10 mCi / ml, 0.125 mM) for 10 min, centrifuged, and washed twice with 1 ml of Krebs solution containing hemicholinium-3 (10 mM), which was present up to the end of the experiment to prevent the reuptake of choline. The synaptosomes were then resuspended in 10 ml of Krebs solution and layered over Whatman GF / C filters into 8 parallel 90 ml superfusion chambers (adapted from Swinny filter holders, Millipore) with the aid of a roller pump (flow rate: 0.6 ml / min, which was kept constant through the experiment). The chamber volume plus dead volume was approximately 0.6 ml. A series of 8 parallel superfusion chambers was used to enable both control and test conditions to be performed in duplicate from the same batch of synaptosomes. After a 15 min washout period, the
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effluent was collected in 2 min fractions for scintillation counting. The synaptosomes were stimulated with veratridine (10 mM) for 2 min at 6 and 26 min after starting sample collection (S 1 and S 2 ). At the end of the experiments, the filters were removed from the chambers to determine the amount of tritium retained by the synaptosomes. At least 12 h after addition of 5 ml of scintillation cocktail (Scintran T, BDH) to the aliquots of effluent samples and synaptosomes retained in the filters, scintillation counting was performed with a 55–60% efficiency during 2 min. The fractional release of tritium was expressed in terms of percentage of total radioactivity present in the tissue at the time of sample collection. The release of tritium evoked by each veratridine pulse, i.e. the evoked release, was calculated by integration of the area of the peak upon subtraction of the estimated basal tritium outflow from the total outflow of tritium due to stimulation. To measure [ 3 H]ACh in the total tritium outflow, the experiments were performed in the presence of neostigmine (20 mM), [ 3 H]ACh was separated using a cation exchanger, tetraphenylboron, after phosphorylation of [ 3 H]choline, as previously described [5]. When the effect of Ap 5 A on the release of ACh was investigated, Ap 5 A was added to the perfusion medium 6 min before S 2 , i.e. 20 min after starting sample collection, and remained in the bath up to the end of the experiment. The effect of Ap 5 A on the evoked release of ACh was expressed by alterations of the ratio between the evoked release due to second stimulation period and the evoked release due to the first stimulation period (S 2 / S 1 ratio). When we evaluated the modifications of the effect of Ap 5 A by an antagonist, the antagonist was applied 15 min before starting sample collection and hence was present during S 1 and S 2 . When present during S 1 and S 2 , none of the antagonists (Ip 5 I, suramin, PPADS, DPCPX or ZM 241385) significantly altered (P.0.05) the S 2 / S 1 as compared to the S 2 / S 1 ratio obtained in the absence of antagonists (data not shown). The values are presented as mean6S.E.M. To test the significance of the effect of Ap 5 A versus control, a paired Student’s t-test was used. When making comparisons from a different set of experiments with control, one-way analysis of variance (ANOVA) was used, followed by Dunnett’s test. P,0.05 was considered to represent a significant difference.
3. Results Two periods of stimulation (S 1 and S 2 ) with veratridine (10 mM) caused a similar evoked release of tritium from superfused hippocampal synaptosomes, with an S 2 / S 1 ratio of 0.9860.03 (n514). This evoked release of tritium was mostly Ca 21 -dependent and constituted by [ 3 H]ACh [5,6].
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M.F. Pereira et al. / Brain Research 879 (2000) 50 – 54
When Ap 5 A (30 mM) was added to superfused hippocampal synaptosomes from 6 min before S 2 onwards, there was a significantly larger evoked release of tritium compared to control (see Fig. 1A), and the S 2 / S 1 ratio was increased by 116614% (n56). In contrast, Ap 5 A (30 mM) did not cause any significant modification of basal tritium outflow. When we quantified the amount of [ 3 H]ACh in the effluent, we found that [ 3 H]ACh accounted for 7864% and 7863% (n54) of the evoked tritium release in the
Fig. 1. Facilitation by Ap 5 A of [ 3 H]ACh release from rat hippocampal synaptosomes. The synaptosomes were loaded with [ 3 H]choline, superfused and effluent samples analysed by scintillation counting. In (A) is compared the time course of tritium release in a typical experiment, in the absence (s) and in the presence of Ap 5 A (30 mM) (d). The synaptosomes were challenged with veratridine (10 mM) for 2 min at 6 min (S 1 ) and 26 min (S 2 ) after starting sample collection, as indicated by the bars above the abscissa. Ap 5 A (30 mM) was applied through the superfusate to two of the four parallel superfusion chambers containing the synaptosomes 6 min before S 2 , as indicated by the bar above the abscissa in A. In (B) is shown the concentration-dependent effect of Ap 5 A (3–100 mM) on evoked tritium release. The effect of each concentration of Ap 5 A on evoked release was calculated as the percentage variation of the amount of tritium released in S 2 / amount of tritium released in S 1 in the presence of Ap 5 A during S 2 versus the S 2 / S 1 ratio in control conditions in the same experiment. The results are mean6S.E.M. of 4–6 experiments. *P,0.05 versus 0%.
absence and presence of Ap 5 A (30 mM), respectively. In the absence of stimulation, [ 3 H]ACh accounted for 4264% and 4863% (n54) of basal tritium outflow in the absence and presence of Ap 5 A (30 mM), respectively. As illustrated in Fig. 1B, Ap 5 A caused a concentration dependent facilitation of the evoked tritium release. The estimated EC 50 was 4.6 mM and the maximal facilitatory effect (116614%, n56) was observed at 30 mM. Increasing concentrations of Ap 5 A (100 mM) were unable to cause a larger facilitation of the evoked release of tritium (Fig. 1B). The facilitatory effect of Ap 5 A (30 mM) was inhibited by 75617% (n54) by a supramaximal concentration of the diadenosine polyphosphate antagonist, Ip 5 I (1 mM) [21], and attenuated by 2463% (n54) and 1862% (n53) by supramaximal concentrations of the P2 receptor antagonists, suramin (100 mM) and PPADS (10 mM) [23], respectively (Fig. 2). A supramaximal concentration of the adenosine A 1 receptor antagonist, DPCPX (20 nM) [1], attenuated by 3666% (n54) the facilitatory effect of Ap 5 A (30 mM) on ACh release, whereas a supramaximal concentration of the adenosine A 2A receptor antagonist, ZM 241385 (20 nM, n54) [7], was devoid of effect (Fig. 2).
Fig. 2. Effect of the Ap n A receptor antagonist, Ip 5 I, of the P2 receptor antagonists, suramin and PPADS, of the adenosine A 2A receptor antagonist, ZM 241385, and of the adenosine A 1 receptor antagonist, DPCPX, on the facilitatory effect of Ap 5 A (30 mM) on [ 3 H]ACh release from rat hippocampal synaptosomes. The synaptosomes were loaded with [ 3 H]choline, superfused and effluent samples analysed by scintillation counting. Ap 5 A was added 6 min before S 2 , whereas the antagonists were added 15 min before starting sample collection and thus were present during S 1 and S 2 . The effect of drugs was calculated by modification of the S 2 / S 1 ratio. 0% corresponds to an S 2 / S 1 ratio equal to control (i.e. in the absence of any drug) and 100% corresponds to an S 2 / S 1 ratio twice of control. The absence (2) or presence (1) of each drug during S 2 or during S 1 and S 2 is indicated below each bar. The results are mean6S.E.M. of 4 experiments. *P,0.05 when compared with the effect of Ap 5 A (30 mM).
M.F. Pereira et al. / Brain Research 879 (2000) 50 – 54
4. Discussion The present results show that Ap 5 A facilitates the evoked release of ACh in the hippocampus. Previous suggestions for a presynaptic role of Ap n A were fostered by the observation that Ap n A increase the influx of calcium in to nerve terminals, causing an increase in basal and K 1 -evoked intracellular calcium concentration ([Ca 21 ] i ) in nerve terminals [16]. Since basal tritium outflow from synaptosomes labelled with [ 3 H]choline does not reflect [ 3 H]ACh release [4,5], it is not possible to conclude on the effect of Ap 5 A on basal ACh release, but only on the evoked release of tritium, which is Ca 21 dependent and mostly constituted by [ 3 H]ACh. Thus, the facilitation by Ap n A of the evoked, i.e. synchronous, release of ACh provide the first direct demonstration for a facilitatory neuromodulatory role of Ap n A in the CNS. One important issue to evaluate the physiological relevance of the presently observed facilitation of ACh release by Ap 5 A is whether the extracellular concentration of Ap 5 A in the synaptic cleft might reach low micromolar levels, compatible with the observed EC 50 of nearly 5 mM. Ap n A are stored in synaptic vesicles with a concentration ratio of 1:25 when compared with ATP [18]. However, the rate of extracellular catabolism of Ap n A in the nervous tissue is 20–50 times lower than that of ATP [15]. Thus, it is likely that the repetitive stimulation of nerve terminals may lead to a localised synaptic extracellular accumulation of Ap n A similar to that estimated for adenine nucleotides (7–25 mM) [3,25]. In different systems, Ap n A may activate Ap n A receptors [15,16,19,21,22], some P2 receptor subtypes [20,26], or be extracellularly metabolised into adenosine [11], and activate adenosine receptors [14]. The facilitation by Ap 5 A of ACh release was strongly inhibited by Ip 5 I, a Ap n A receptor antagonist [21], and only slightly attenuated by the P2 receptor antagonists, suramin and PPADS [23]. Besides potently blocking native Ap n A receptors, Ip 5 I is also an antagonist of some P2x receptor subtypes [13] and some P2 receptors have a low sensitivity to suramin and / or PPADS [23]. But, since ACh release from hippocampal nerve terminals is not under P2 receptor control [4], the inhibition by Ip 5 I of the facilitatory effect of Ap 5 A on ACh release strongly suggests the involvement of an Ap n A receptor. The possibility that activation of adenosine receptors might be responsible for the facilitation of ACh release by Ap 5 A was also not supported by the present results. It is known that the evoked release of ACh in the hippocampus is facilitated by A 2A receptor activation and is inhibited by A 1 receptor activation [5,7]. Also, there is an high and efficient ecto-nucleotidase pathway activity in the hippocampus [4,8] with adenosine being generated within milliseconds after ATP application [10]. However, blockade of A 2A receptors with ZM 241385 failed to modify the facilitatory effect of Ap 5 A on ACh release. Blockade
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of inhibitory A 1 receptors would be expected to cause no effect or to eventually enhance the facilitation of ACh release by Ap 5 A. In contrast, we found that blockade of A 1 receptors inhibited the facilitatory effect of Ap 5 A on ACh release. It has previously been shown that the efficiency of Ap n A receptor activation is decreased upon removal of endogenous extracellular adenosine [9], indicating the existence of an interaction between A 1 and Ap n A receptors controlling the neuromodulatory effect of Ap n A [15]. In conclusion, the present results provide the first direct demonstration for a neuromodulatory role of Ap n A receptor activation in the CNS. The observation that Ap n A receptor activation facilitated ACh release opens a new avenue in the therapeutical possibilities of enhancing ACh release in situations of cholinergic deficits, which occur in some types of dementia or cognitive deficits [12].
Acknowledgements This work was supported by research grants from the o C.A.M. (n 8012 / 98), the Spanish Ministry of Education, ˜ para a Ciencia ˆ Fundac¸ao e Tecnologia, Culture DGCYT PM 98-0089 and the European Union (BIOMED 2, PL 950676). RAC is in debt to Professor Moniz Pereira (Faculty of Pharmacy, Lisbon) for scintillation counting facilities and to Professor Silva Carvalho (Department Physiology, Faculty Medicine, Lisbon) for animal house facilities.
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