Directory UMM :Data Elmu:jurnal:B:Brain Research:Vol879.Issue1-2.2000:

www.elsevier.com / locate / bres

Research report

Diadenosine polyphosphates facilitate the evoked release of

acetylcholine from rat hippocampal nerve terminals

a c c,d c

´

´

´

´

M. Fatima Pereira , Miguel Diaz Hernandez , Jesus Pintor

, Maria Teresa Miras-Portugal ,

a,b ,

_*

a

Rodrigo A. Cunha

, J. Alexandre Ribeiro

a

Laboratory of Neurosciences, Faculty of Medicine, University of Lisbon, 1649-028 Lisbon, Portugal

b

Department of Chemistry and Biochemistry, Faculty of Sciences, University of Lisbon, 1749-016 Lisbon, Portugal

c _´

Department of Biochemistry, Faculty of Veterinary, E.U. Optica, Complutense University, 28040 Madrid, Spain

d _´

E.U. Optica, Faculty of Veterinary, Complutense University, 28040 Madrid, Spain 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 A, 3–100₅ 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 30mM Ap A. The5

selective diadenosine polyphosphate receptor antagonist, diinosine pentaphosphate (Ip I, 1₅ mM), inhibited by 75% the facilitatory effect of Ap A (305 mM), whereas the P receptor antagonists, suramin (1002 mM) and pyridoxal-phosphate-6-azophenyl-29,49-disulfonic acid (PPADS, 10mM) only caused a 18–24% inhibition, the adenosine A receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (20 nM),₁ caused a 36% inhibition and the adenosine A2A receptor antagonist, 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo [2,3-a][1,3,5]triazin-5-ylamino]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 A; Diadenosine polyphosphate; Nerve terminal; Hippocampus; ATP; Adenosine; Receptor5

1. Introduction ATP, but another class of adenine nucleotides, diadenosine

polyphosphates (Ap A), has been identified in synapticn

It is now commonly accepted that several transmitters vesicles [17,18]. Like ATP [6], Ap A (Ap A, Ap A andn 4 5

are involved in the chemical transmission of information at Ap A) are released upon stimulation of nerve terminals6

most individual synapses (e.g. [24]). Among the signalling [17]. Ap A can activate some P receptor subtypes [20,26]n 2

molecules stored in synaptic vesicles, adenine nucleotides or be extracellularly metabolised into adenosine [11], a are a class of molecules with a potentially more general- neuromodulator on its own [2], but can also activate ised importance, since they are co-stored and co-released receptors selective for Ap A [15,16,19,21]. Ap A mayn n

with several neurotransmitters (reviewed in [27]). The potentially act as neuromodulator in the CNS, since more abundant adenine nucleotide in synaptic vesicles is activation of Ap A receptors in rat or human midbrainn

nerve terminals cause an increase in intraterminal calcium [16,22]. However, the effect of Ap A receptor activationn *Corresponding author. Tel.: 1351-21-7936787; fax: 1

351-21-on the release of neurotransmitters is not known.

7936787.

E-mail address: racunha@neurociencias.pt (R.A. Cunha). Since clear evidence for the presence of Ap A inn

cholinergic synaptic vesicles has been provided [18], we effluent was collected in 2 min fractions for scintillation now tested the effect of Ap A on acetylcholine releasen counting. The synaptosomes were stimulated with ver-from hippocampal nerve terminals. We found that Ap A5 atridine (10mM) for 2 min at 6 and 26 min after starting facilitated the evoked release of acetylcholine and that this sample collection (S and S ). At the end of the experi-1 2

effect was mostly due to activation of Ap A receptorsn ments, the filters were removed from the chambers to rather than nucleotide P receptors or adenosine A or A2 1 2A determine the amount of tritium retained by the

synapto-receptors. somes.

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,

scintilla-2. Material and methods tion counting was performed with a 55–60% efficiency

during 2 min. The fractional release of tritium was Diadenosine pentaphosphate (Ap A), hemicholinium-3,5 expressed in terms of percentage of total radioactivity neostigmine, choline kinase (EC 2.7.1.32), veratridine and present in the tissue at the time of sample collection. The tetraphenylboron were from Sigma. Suramin, 1,3-dipropyl- release of tritium evoked by each veratridine pulse, i.e. the 8-cyclopentylxanthine (DPCPX) and pyridoxal-phosphate- evoked release, was calculated by integration of the area of 6-azophenyl-29,49-disulfonic acid (PPADS) were from RBI the peak upon subtraction of the estimated basal tritium and 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]- outflow from the total outflow of tritium due to

stimula-3

triazin-5-ylamino]ethyl) phenol (ZM 241385) was from tion. To measure [ H]ACh in the total tritium outflow, the

3

Tocris. Methyl-[ H]choline chloride (specific activity 76– experiments were performed in the presence of

neostig-3

86.3 Ci / mmol) was obtained from Amersham. Diinosine mine (20 mM), [ H]ACh was separated using a cation pentaphosphate (Ip I) was synthesised as previously de-5 exchanger, tetraphenylboron, after phosphorylation of

3

scribed [21]. All other reagents were of the highest purity [ H]choline, as previously described [5].

available. ZM 241385 was made up into a 5 mM stock in When the effect of Ap A on the release of ACh was5

dimethylsulfoxide and DPCPX was made up into a 5 mM investigated, Ap A was added to the perfusion medium 65

stock in 99% dimethylsulfoxide–1% NaOH (1 M) (v / v). min before S , i.e. 20 min after starting sample collection,2

Aqueous dilution of these stock solutions was made daily. and remained in the bath up to the end of the experiment. Dimethylsulfoxide, in the maximum concentration applied The effect of Ap A on the evoked release of ACh was5

during drug testing, was devoid of effects on acetylcholine expressed by alterations of the ratio between the evoked

release. release due to second stimulation period and the evoked

3

The release of [ H]acetylcholine (ACh) from a rat release due to the first stimulation period (S / S2 1 ratio). hippocampal synaptosomal fraction prepared from male When we evaluated the modifications of the effect of Wistar rats, anaesthetised under halothane atmosphere, was Ap A by an antagonist, the antagonist was applied 15 min5

performed as previously described [4]. Briefly, the before starting sample collection and hence was present synaptosomes were equilibrated at 378C for 10 min in during S and S . When present during S and S , none of1 2 1 2

Krebs solution, gassed with 95% O and 5% CO mixture,2 2 the antagonists (Ip I, suramin, PPADS, DPCPX or ZM5

of the following composition (mM): NaCl, 124; KCl, 3; 241385) significantly altered (P.0.05) the S / S2 1 as NaH PO , 1.25; MgSO , 1; CaCl , 2; NaHCO , 26;2 4 4 2 3 compared to the S / S2 1 ratio obtained in the absence of glucose, 10; pH 7.4. From this time onwards, all solutions antagonists (data not shown).

applied to the synaptosomes were kept at 378C and gassed The values are presented as mean6S.E.M. To test the with 95% O and 5% CO . After the equilibration period,2 2 significance of the effect of Ap A versus control, a paired5

3

the synaptosomes were loaded with [ H]choline (10mCi / Student’s t-test was used. When making comparisons from ml, 0.125mM) for 10 min, centrifuged, and washed twice a different set of experiments with control, one-way with 1 ml of Krebs solution containing hemicholinium-3 analysis of variance (ANOVA) was used, followed by (10 mM), which was present up to the end of the Dunnett’s test. P,0.05 was considered to represent a experiment to prevent the reuptake of choline. The significant difference.

synaptosomes were then resuspended in 10 ml of Krebs solution and layered over Whatman GF / C filters into 8

parallel 90ml superfusion chambers (adapted from Swinny 3. Results

filter holders, Millipore) with the aid of a roller pump (flow

rate: 0.6 ml / min, which was kept constant through the Two periods of stimulation (S and S ) with veratridine1 2

experiment). The chamber volume plus dead volume was (10mM) caused a similar evoked release of tritium from approximately 0.6 ml. A series of 8 parallel superfusion superfused hippocampal synaptosomes, with an S / S2 1

chambers was used to enable both control and test con- ratio of 0.9860.03 (n514). This evoked release of tritium

21 3

When Ap A (305 mM) was added to superfused hip- absence and presence of Ap A (305 mM), respectively. In

3

pocampal synaptosomes from 6 min before S2 onwards, the absence of stimulation, [ H]ACh accounted for 4264% there was a significantly larger evoked release of tritium and 4863% (n54) of basal tritium outflow in the absence compared to control (see Fig. 1A), and the S / S ratio was2 1 and presence of Ap A (305 mM), respectively.

increased by 116614% (n56). In contrast, Ap A (305 mM) As illustrated in Fig. 1B, Ap A caused a concentration5

did not cause any significant modification of basal tritium dependent facilitation of the evoked tritium release. The

3

outflow. When we quantified the amount of [ H]ACh in the estimated EC50 was 4.6 mM and the maximal facilitatory

3

effluent, we found that [ H]ACh accounted for 7864% effect (116614%, n56) was observed at 30 mM. Increas-and 7863% (n54) of the evoked tritium release in the ing concentrations of Ap A (1005 mM) were unable to cause a larger facilitation of the evoked release of tritium (Fig. 1B).

The facilitatory effect of Ap A (305 mM) was inhibited by 75617% (n54) by a supramaximal concentration of the diadenosine polyphosphate antagonist, Ip I (15 mM) [21], and attenuated by 2463% (n54) and 1862% (n53) by supramaximal concentrations of the P receptor antago-2

nists, suramin (100 mM) and PPADS (10 mM) [23], respectively (Fig. 2). A supramaximal concentration of the adenosine A1 receptor antagonist, DPCPX (20 nM) [1], attenuated by 3666% (n54) the facilitatory effect of Ap A (305 mM) on ACh release, whereas a supramaximal concentration of the adenosine A2A receptor antagonist, ZM 241385 (20 nM, n54) [7], was devoid of effect (Fig. 2).

3

Fig. 1. Facilitation by Ap A of [ H]ACh release from rat hippocampal5 3

synaptosomes. The synaptosomes were loaded with [ H]choline,

super-fused and effluent samples analysed by scintillation counting. In (A) is Fig. 2. Effect of the Ap A receptor antagonist, Ip I, of the P receptorn 5 2

compared the time course of tritium release in a typical experiment, in the antagonists, suramin and PPADS, of the adenosine A2Areceptor antago-absence (s_{) and in the presence of Ap A (30} mM) (d_{). The synapto- nist, ZM 241385, and of the adenosine A receptor antagonist, DPCPX,}

5 1

3

somes were challenged with veratridine (10mM) for 2 min at 6 min (S )1 on the facilitatory effect of Ap A (305 mM) on [ H]ACh release from rat and 26 min (S ) after starting sample collection, as indicated by the bars2 hippocampal synaptosomes. The synaptosomes were loaded with

3

above the abscissa. Ap A (305 mM) was applied through the superfusate to [ H]choline, superfused and effluent samples analysed by scintillation two of the four parallel superfusion chambers containing the synapto- counting. Ap A was added 6 min before S , whereas the antagonists were5 2

somes 6 min before S , as indicated by the bar above the abscissa in A. In2 added 15 min before starting sample collection and thus were present (B) is shown the concentration-dependent effect of Ap A (3–1005 mM) on during S and S . The effect of drugs was calculated by modification of1 2

evoked tritium release. The effect of each concentration of Ap A on5 the S / S ratio. 0% corresponds to an S / S ratio equal to control (i.e. in2 1 2 1

evoked release was calculated as the percentage variation of the amount the absence of any drug) and 100% corresponds to an S / S ratio twice2 1

of tritium released in S / amount of tritium released in S in the presence2 1 of control. The absence (2) or presence (1) of each drug during S or2

of Ap A during S versus the S / S ratio in control conditions in the same5 2 2 1 during S1 and S2 is indicated below each bar. The results are experiment. The results are mean6S.E.M. of 4–6 experiments. *P,0.05 mean6S.E.M. of 4 experiments. *P,0.05 when compared with the effect

4. Discussion of inhibitory A receptors would be expected to cause no1

effect or to eventually enhance the facilitation of ACh The present results show that Ap A facilitates the5 release by Ap A. In contrast, we found that blockade of A5 1

evoked release of ACh in the hippocampus. Previous receptors inhibited the facilitatory effect of Ap A on ACh5

suggestions for a presynaptic role of Ap A were fosteredn release. It has previously been shown that the efficiency of by the observation that Ap A increase the influx ofn Ap A receptor activation is decreased upon removal ofn

calcium in to nerve terminals, causing an increase in basal endogenous extracellular adenosine [9], indicating the 1

and K -evoked intracellular calcium concentration existence of an interaction between A and Ap A receptors1 n 21

([Ca ] ) in nerve terminals [16]. Since basal tritiumi controlling the neuromodulatory effect of Ap A [15].n 3

outflow from synaptosomes labelled with [ H]choline does In conclusion, the present results provide the first direct

3

not reflect [ H]ACh release [4,5], it is not possible to demonstration for a neuromodulatory role of Ap A re-n

conclude on the effect of Ap A on basal ACh release, but5 ceptor activation in the CNS. The observation that Ap An 21

only on the evoked release of tritium, which is Ca - receptor activation facilitated ACh release opens a new

3

dependent and mostly constituted by [ H]ACh. Thus, the avenue in the therapeutical possibilities of enhancing ACh facilitation by Ap A of the evoked, i.e. synchronous,n release in situations of cholinergic deficits, which occur in release of ACh provide the first direct demonstration for a some types of dementia or cognitive deficits [12]. facilitatory neuromodulatory role of Ap A in the CNS.n

One important issue to evaluate the physiological

rele-vance of the presently observed facilitation of ACh release _{Acknowledgements} by Ap A is whether the extracellular concentration of5

Ap A in the synaptic cleft might reach low micromolar5 _{This work was supported by research grants from the} o

levels, compatible with the observed EC50 of nearly 5mM. _{C.A.M. (n 8012 / 98), the Spanish Ministry of Education,} Ap A are stored in synaptic vesicles with a concentrationn Fundac¸ao para a Ciencia e Tecnologia, Culture DGCYT˜ ˆ ratio of 1:25 when compared with ATP [18]. However, the _{PM 98-0089 and the European Union (BIOMED 2, PL} rate of extracellular catabolism of Ap A in the nervousn _{950676). RAC is in debt to Professor Moniz Pereira}

tissue is 20–50 times lower than that of ATP [15]. Thus, it _{(Faculty of Pharmacy, Lisbon) for scintillation counting} is likely that the repetitive stimulation of nerve terminals _{facilities and to Professor Silva Carvalho (Department} may lead to a localised synaptic extracellular accumulation _{Physiology, Faculty Medicine, Lisbon) for animal house} of Ap A similar to that estimated for adenine nucleotidesn _facilities.

(7–25mM) [3,25].

In different systems, Ap A may activate Ap A receptorsn n

[15,16,19,21,22], some P receptor subtypes [20,26], or be2 _References

extracellularly metabolised into adenosine [11], and

acti-vate adenosine receptors [14]. The facilitation by Ap A of5 _{[1] R.F. Bruns, J.H. Fergus, E.W. Badger, J.A. Bristol, L.A. Santay, J.D.}

ACh release was strongly inhibited by Ip I, a Ap A5 n Hartman, S.J. Hays, C.C. Huang, Binding of the A -selective1

receptor antagonist [21], and only slightly attenuated by adenosine antagonist 8-cyclopentyl-1,3-dipropylxanthine to rat brain membranes, Naunyn-Schmiedeberg’s Arch. Pharmacol. 335 (1987)

the P2 receptor antagonists, suramin and PPADS [23].

59–63.

Besides potently blocking native Ap A receptors, Ip I isn 5 _{[2] R.A. Cunha, Adenosine as a neuromodulator and as a homeostatic}

also an antagonist of some P2x receptor subtypes [13] and _{regulator in the nervous system: different roles, different sources and} some P receptors have a low sensitivity to suramin and / or2 different receptors, Neurochem. Int. (2000) in press.

˜

PPADS [23]. But, since ACh release from hippocampal [3] R.A. Cunha, A.M. Sebastiao, Adenosine and adenine nucleotides are independently released from both the nerve terminals and the muscle

nerve terminals is not under P receptor control [4], the2

fibres upon electrical stimulation of the innervated skeletal muscle

inhibition by Ip I of the facilitatory effect of Ap A on5 5 _{of the frog, Pflugers Arch. 424 (1993) 503–510.¨}

ACh release strongly suggests the involvement of an Ap An [4] R.A. Cunha, J.A. Ribeiro, A.M. Sebastiao, Purinergic modulation of˜

3

receptor. the evoked release of [ H]acetylcholine from the hippocampus and

The possibility that activation of adenosine receptors cerebral cortex of the rat: role of the ectonucleotidases, Eur. J. Neurosci. 6 (1994) 33–42.

might be responsible for the facilitation of ACh release by

˜ [5] R.A. Cunha, E. Milusheva, E.S. Vizi, J.A. Ribeiro, A.M. Sebastiao,

Ap A was also not supported by the present results. It is5 _{Excitatory and inhibitory effects of A and A adenosine receptor}

1 2A

known that the evoked release of ACh in the hippocampus 3

activation on the electrically evoked [ H]acetylcholine release from

is facilitated by A2A receptor activation and is inhibited by different areas of the rat hippocampus, J. Neurochem. 63 (1994)

A₁ receptor activation [5,7]. Also, there is an high and 207–214.

˜

[6] R.A. Cunha, E.S. Vizi, A.M. Sebastiao, J.A. Ribeiro, Preferential

efficient ecto-nucleotidase pathway activity in the

hip-release of ATP and its extracellular catabolism as a source of

pocampus [4,8] with adenosine being generated within

adenosine upon high- but not low-frequency stimulation of rat

milliseconds after ATP application [10]. However, bloc- _{hippocampal slices, J. Neurochem. 67 (1996) 2180–2187.} kade of A2A receptors with ZM 241385 failed to modify [7] R.A. Cunha, M.D. Constantino, J.A. Ribeiro, ZM 241385 is an

adenosine receptor agonists CGS 21680 and HENECA in the rat of diadenosine polyphosphates — Ap A and Ap A — in rat4 5 21

hippocampus, Br. J. Pharmacol. 122 (1997) 1279–1284. synaptic terminals: Ca -dependent release evoked by 4-amino-˜

[8] R.A. Cunha, A.M. Sebastiao, J.A. Ribeiro, Inhibition by ATP of pyridine and veratridine, Neurosci. Lett. 136 (1992) 141–144. hippocampal synaptic transmission requires localized extracellular [18] J. Pintor, H.J. Kowalewski, M. Torres, M.T. Miras-Portugal, H. catabolism by ecto-nucleotidases into adenosine and channeling to Zimmermann, Synaptic vesicle storage of diadenosine polyphos-adenosine A receptors, J. Neurosci. 18 (1998) 1987–1995.1 phates in the Torpedo electric organ, Neurosci. Res. Comm. 10

´ ´

[9] M. Dıaz-Hernandez, J. Pintor, M.T. Miras-Portugal, Modulation of (1992) 9–15. ´

the dinucleotide receptor present in rat midbrain synaptosomes by [19] J. Pintor, M.A. Dıaz-Rey, M.T. Miras-Portugal, Ap A and ADP-4 b-S adenosine and ATP, Br. J. Pharmacol. 130 (2000) 434–440. bindind to P purinoceptors present on rat brain synaptic terminals,2

[10] T.D. Dunwiddie, L. Diao, K. Lee, W.R. Proctor, Adenine nucleotides Br. J. Pharmacol. 108 (1993) 1094–1099.

undergo rapid, quantitative conversion to adenosine in the extracel- [20] J. Pintor, B.F. King, M.T. Miras-Portugal, G. Burnstock, Selectivity lular space in the rat hippocampus, J. Neurosci. 17 (1997) 7673– and activity of adenine dinucleotides at recobinant P2X2 and P2Y1

7682. purinoceptors, Br. J. Pharmacol. 119 (1996) 1006–1012.

[11] T. Emanuelli, C.D. Bonan, J.J. Sarkis, A.M. Battastini, Catabolism [21] J. Pintor, J. Gualix, M.T. Miras-Portugal, Diinosine polyphosphates, of Ap A and Ap A by rat brain synaptosomes, Braz. J. Med. Biol.4 5 a group of dinucleotides with antagonist effects on diadenosine Res. 31 (1998) 1529–1532. polyphosphate receptor, Mol. Pharmacol. 51 (1997) 277–284.

´ ´

[12] M.E. Hasselmo, J.M. Bower, Acetylcholine and memory, Trends [22] J. Pintor, M. Dıaz-Hernandez, C. Bustamante, J. Gualix, F.J.G. Neurosci. 16 (1993) 218–222. Terreros, M.T. Miras-Portugal, Presence of dinucleotide and ATP [13] C.H. Hoyle, J. Pintor, J. Gualix, M.T. Miras-Portugal, Antagonism receptors in human cerebrocortical synaptic terminals, Eur. J.

of P2X receptors in guinea-pig vas deferens by diinosine penta- Pharmacol. 366 (1999) 159–165.

phosphate, Eur. J. Pharmacol. 333 (1997) R1–R2. [23] V. Ralevic, G. Burnstock, Receptors for purines and pyrimidines, [14] A. Klishin, N. Lozovaya, J. Pintor, M.T. Miras-Portugal, O. Pharmacol. Rev. 50 (1998) 413–481.

Krishtal, Possible functional role of diadenosine polyphosphates: [24] M.W. Salter, Y.D. Koninck, An ambiguous fast synapse: a new twist negative feedback for excitation in hippocampous, Neuroscience 58 in the tale of two transmitters, Nature Neurosci. 2 (1999) 199–200. (1994) 235–236. [25] E.M. Silinsky, On the association between transmitter secretion and

´ ´

[15] M.T. Miras-Portugal, J. Gualix, J. Mateo, M. Diaz-Hernandez, R. the release of adenine nucleotides from mammalian motor nerve Gomez-Villafuertes, E. Castro, J. Pintor, Diadenosine polyphos- terminals, J. Physiol. 247 (1975) 145–162.

phates, extracellular function and catabolism, Prog. Brain Res. 120 [26] S.S. Wildman, S.G. Brown, B.F. King, G. Burnstock, Selectivity of (1999) 397–409. diadenosine polyphosphates for rat P2X receptor subunits, Eur. J. [16] J. Pintor, M.T. Miras-Portugal, A novel receptor for diadenosine Pharmacol. 367 (1999) 119–123.

polyphosphates coupled to calcium increase in rat midbrain synapto- [27] H. Zimmermann, Signalling via ATP in the nervous system, Trends somes, Br. J. Pharmacol. 115 (1995) 895–902. Neurosci. 17 (1994) 420–426.

´