Diadenosine polyphosphate receptors

Pharmacology & Therapeutics 87 (2000) 103–115

Associate editor: D. Shugar

Diadenosine polyphosphate receptors from rat and guinea-pig brain to human nervous system Jesús Pintor*, Miguel Díaz-Hernández, Javier Gualix, Rosa Gómez-Villafuertes, Fernando Hernando, M. Teresa Miras-Portugal Departamento de Bioquímica y Biología Molecular IV, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain

Abstract Diadenosine polyphosphates are a family of naturally occurring nucleotidic compounds present in secretory vesicles together with other chemical messengers. The exocytotic release of these compounds permits them to stimulate receptors termed “purinoceptors” or “ATP receptors.” Purinoceptors for nucleotides are named P2 in contrast with those sensitive to nucleosides (P1). P2 receptors are further subdivided into metabotropic P2Y receptors, further divided into 5 subtypes, and ionotropic P2X receptors, with 7 different subtypes. Diadenosine polyphosphates can activate recombinant P2Y1, P2Y2, and P2Y4 and recombinant homomeric P2X1, P2X2, P2X3, P2X4, and P2X6. Heteromeric P2X receptors change their sensitivity to diadenosine polyphosphates when co-assembly between different subunits occurs. Diadenosine polyphosphates can activate specific receptors termed dinucleotide receptors or P4 receptors, which are insensitive to other nucleosides or nucleotides. The P4 receptor is a receptor-operated Ca2⫹ channel present in rat brain synaptic terminals, stimulated by diadenosine pentaphosphate and diadenosine tetraphosphate. This receptor is strongly modulated by protein kinases A and C and protein phosphatases. The dinucleotide receptor is present in different brain areas, such as midbrain (in rat and guinea-pig), cerebellum (in guinea-pig), and cortex (in human). © 2000 Elsevier Science Inc. All rights reserved. Keywords: CNS; Diadenosine polyphosphates; Dinucleotide receptor; Homomeric P2X receptors; Heteromeric P2X receptors; P2Y receptors Abbreviations: ApaA, P1,P2-di(adenosine-5⬘)triphosphate; Ap3A, P1,P3-di(adenosine-5⬘)triphosphate; Ap4A, P1,P4-di(adenosine-5⬘)tetraphosphate; Ap5A, P1,P5di(adenosine-5⬘)pentaphosphate; Ap6A, P1,P6-di(adenosine-5⬘)hexaphosphate; Ap7A, P1,P7-di(adenosine-5⬘)heptaphosphate; ⑀-ApnA, the etheno derivatives of diadenosine polyphosphates; ATP␥S, adenosine 5⬘-O-(3-thiotriphosphate); ␣,␤-meATP, ␣,␤-methylene adenosine 5⬘-triphosphate; GpnG, diguanosine polyphosphates; IP, inhibitory peptide; IpnI, diinosine polyphosphates; NpnN, dinucleoside triphosphates; PKA, protein kinase A; PKC, protein kinase C; PL, phospholipase; PPADS, pyridoxalphosphate-6-azophenyl-2⬘,4⬘-disulphonic acid; VDCC, voltage-dependent calcium channel; VDSC, voltage-dependent sodium channels.

Contents 1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Receptors for extracellular nucleotides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. P2Y receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. P2X receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Receptors activated by diadenosine polyphosphates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Activation of recombinant P2Y receptors by diadenosine polyphosphates. . . . . . . . 3.2. Activation of recombinant homomeric P2X receptors by diadenosine polyphosphates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Activation of heteromeric P2X receptors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Native P2 receptors stimulated by diadenosine polyphosphates in the central nervous system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Native receptors specifically stimulated by diadenosine polyphosphates in the central nervous system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Rat brain receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.1. Pharmacology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.2. Connection with voltage-dependent Ca2⫹ channels . . . . . . . . . . . . . . . . . . . 4.1.3. Modulation of the dinucleotide receptor by protein kinases and phosphatases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . * Corresponding author. Tel.: ⫹34-91-3943890; fax: ⫹34-91-3943909. E-mail address: [email protected] (J. Pintor). 0163-7258/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S0163-7258(00)00 0 4 9 - 8

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4.2.

5. 6.

Dinucleotide receptors in guinea-pig brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.1. Cortical receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.2. Midbrain receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.3. Cerebellar receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Human brain receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Could diadenosine polyphosphate derivatives be used as pharmacological tools for P2X receptors? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Diadenosine polyphosphates form a group of dinucleotides structurally related to ATP. These compounds are formed by two adenosine moieties linked by their ribose 5⬘-ends to a variable number of phosphates. The number of phosphates, which can change from 2 to 7, is the main structural feature that differentiates the members of this family. Diadenosine polyphosphates are also termed ␣,␻-adenine dinucleotides, but they are commonly abbreviated as ApnA, where n represents the number of phosphates (Fig. 1). Diadenosine polyphosphates are naturally occurring substances present in the cytoplasm of prokaryotic and eukaryotic cells, which are synthesised by some aminoacyl-tRNA synthetases and other enzymes (Zamecnik et al., 1966; Brevet et al., 1989; Sillero et al., 1991; Plateau & Blanquet, 1992; Sillero & Günther Sillero, 2000). Several functions have been proposed for these dinucleotides, including inhibition of adenosine kinase and adenylate kinase, since they mimic the transition state of those enzymes (Lienhard & Secemski, 1973; Rotllán & Miras-

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Portugal, 1985; Bone et al., 1986). Changes in cytosolic concentrations of diadenosine polyphosphates can vary, depending on the proliferative state of the cell (Rapaport & Zamecnik, 1976). Other functions of adenine dinucleotides may be related to environmental stress. In this sense, ApnA concentrations increase under conditions such as hyperthermia or oxidative stress (Brevet et al., 1985; Baker & Jacobson, 1986). In prokaryotic cells, it has been suggested that these compounds act as alarmones, low-molecular weight compounds signalling to the cell a nonfavourable environment and probably inducing some protective homeostatic mechanisms (Lee et al., 1983; Varshavsky, 1983; Bochner et al., 1984; McLennan, 2000). Apart from intracellular roles, diadenosine polyphosphates have extracellular effects, mediated by stimulating membrane receptors termed purinergic receptors. Adenine dinucleotides are stored in secretory vesicles from platelets, chromaffin cells, synaptic vesicles from Torpedo, and rat brain synaptic terminals (Flodgaard & Klenow, 1982;

Fig. 1. Structure of diadenosine polyphosphates. (A) Schematic representation of diadenosine polyphosphates. (B) Space-fill model of Ap5A, a representative adenine dinucleotide.

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Lüthje & Ogilvie, 1983; Rodriguez del Castillo et al., 1988; Pintor et al., 1992b, 1992c, 1992d). All these secretory systems respond to depolarising agents or secretagogues by releasing their vesicular content to the extracellular medium. The presence of diadenosine polyphosphates in the extracellular space depends on their presence within the secretory vesicles under study. For example, while in platelets all the naturally occurring dinucleotides are present, from P1,P2di(adenosine-5⬘)pyrophosphate (Ap2A) to P1,P7-di(adenosine5⬘)heptaphosphate (Ap7A), only P1,P4-di(adenosine-5⬘)tetraphosphate (Ap4A), P1,P5-di(adenosine-5⬘)pentaphosphate (Ap5A), and P1,P6-di(adenosine-5⬘)hexaphosphate (Ap6A) have been described to date in neuronal models. An interesting point is the relationship that might exist between the extracellular concentration of these substances and the affinity they have for their receptors (Schlüter et al., 1994; Ogilvie, 1992; Pintor et al., 1991, 1992a). In this regard, push-pull cannula experiments performed in living rats showed that after amphetamine stimulation, rat neostriatum releases diadenosine tetraphosphate and diadenosine pentaphosphate (Pintor et al., 1995). In brain perfusates, the concentrations of Ap4A and Ap5A were 64.5 nM and 57.5 nM, respectively (Fig. 2). These concentrations might be different from the levels in the extracellular space. Some approaches suggest that the concentrations of the dinucleotides in the synaptic cleft may be ⵑ30 ␮M each (Pintor et al., 1991). 2. Receptors for extracellular nucleotides There are a great variety of tissues whose functional state is modified in response to extracellular nucleotides. The membrane receptors that are activated by nucleotides are termed purinoceptors or ATP receptors, although they can be sensitive not only to purines, but also to pyrimidines (Ralevic & Burnstock, 1998). Purinoceptors for nucleotides are named P2 receptors, as opposed to those that are sensitive to nucleosides such as adenosine, which are termed P1 or adenosine receptors.

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Based on the molecular structure and the signal transduction mechanisms, receptors for nucleotides are divided into two families: G-protein-coupled receptors P2Y and ionotropic P2X receptors (Dubyak, 1991; Abbracchio & Burnstock, 1994; Fredholm et al., 1994). To date, five P2Y receptors (P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11) and seven P2X subunits (P2X1–7) have been cloned from different mammalian species. The IUPHAR committee does not consider some putative P2Y receptors, either because they have been cloned from non-mammals or because they do not present functional behaviour after exposure to nucleotides (see Alexander & Peters, 1999). The following sections describe the main features of P2Y and P2X receptors. 2.1. P2Y receptors P2Y receptors are a family of metabotropic receptors with 7 transmembrane domains similar to most of the G-proteincoupled receptors. With a length that can change from 308 to 377 amino acids, the N-terminus presents sites suitable for glycosylation, while in the intracellular domains, they present putative sites for phosphorylation, which may participate in receptor desensitisation (Weisman et al., 1998). The main transduction signal operated by these receptors is phospholipase (PL)C activation. Among the different PLs, PLC␤ seems to be involved in this signal transduction mechanism. P2Y receptors stimulate this enzyme by means of a Gq or G11 protein. Nevertheless, the P2Y2 receptor is coupled to a Gi/o, the ␤- and ␥-subunits being the ones that stimulate PLC␤2 (Motte et al., 1993; Purkiss et al., 1994). The inositol 1,4,5-triphosphate generated as a consequence of PLC activation induces Ca2⫹ mobilisation from the intracellular stores and a concomitant increase of this ion in the cytosol. 2.2. P2X receptors P2X receptor subunits are proteins with different lengths (379–595 amino acids) that present 2 transmembrane domains, with the intracellular carboxyl and amino terminals

Fig. 2. Determination of Ap4A and Ap5A levels in rat brain perfusates by push-pull cannula technique. (A) Schematic representation of the cannula insertion in the conscious rat during the push-pull experiments. (B) HPLC elution profiles of rat perfusates taken before stimulation (control) and at the indicated times. The presence of Ap4A and Ap5A was maximal 30 min after the rat stimulation, followed by a gradual return to control levels.

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separated by a large extracellular loop. They share between 37 and 48% sequence similarity, the highest degree of homology being in the extracellular domain. The extracellular loop also has 10 strictly conserved Cys residues that may be implicated in disulphide bond formation necessary to form the tertiary structure and for ATP binding. On the other hand, the transmembrane domains show a high variability among the different receptors (Soto et al., 1997). The topology of two transmembrane domains, a long extracellular loop and the C- and N-terminals facing the cytosol, is present in other types of ion channels, such as the Na⫹ channel in epithelium, the mechano-sensitive channel present in Escherichia coli, and the inward rectifier K⫹ channel (North, 1996). Functional P2X receptors are formed when two or more subunits co-assemble. However, nothing is known about the number of subunits that form a receptor, and whether in native tissues P2X receptors are formed by the same or different subunits, called homomeric or heteromeric receptors, respectively (Kim et al., 1997; Nicke et al., 1998). There is evidence for the necessity of heteromeric subunits to form a functional P2X receptor. For example, homomeric P2X6 do not trigger currents, as expected when they are expressed in oocytes (Collo et al., 1996; García-Guzmán et al., 1996; Soto et al., 1996). Activation of P2X receptors by ATP produces a rapid inward flux of cations (Na⫹/K⫹ and Ca2⫹). The resultant change in the membrane potential due to the ion flux triggers voltage-dependent Ca2⫹ channels (VDCCs), which produce a secondary Ca2⫹ entry. P2X receptors are further subdivided into two groups, those that desensitise quickly (P2X1 and P2X3) and others that do not. In the latter group, P2X4 and P2X6 maintain 60% of their currents seconds after their activation, while P2X2 and P2X5 maintain currents during long application of agonists (Evans et al., 1996; Collo et al., 1996; GarcíaGuzmán et al., 1996). The P2X7 subunit, with a long C-terminal domain, is different from the others (Collo et al., 1997). Short ATP applications produce the same ion fluxes as described in Section 2.2, but long applications of ATP induce pore formation permeable to metabolites up to 900 Da. This response can be reversed by washing out the agonists, but in some cases, it is not reversible and leads to cell death (Evans et al., 1998).

3. Receptors activated by diadenosine polyphosphates 3.1. Activation of recombinant P2Y receptors by diadenosine polyphosphates Recombinant P2Y receptors expressed in heterologous systems are activated by diadenosine polyphosphates. The human P2Y1 receptor cloned from a human genomic library, and its avian homologue, are sensitive to Ap4A, with EC50 values of 625 nM and 742 nM, respectively. This dinucleotide behaves like a partial agonist, attaining 49% and 67% of the effect produced by 2-methylthioadenosine triphosphate at human and turkey P2Y1 receptors, respec-

tively. Other adenine dinucleotides tested showed an effect only at millimolar concentrations (Schachter et al., 1996). The P2Y2 receptor is stimulated by Ap4A, with an EC50 value in the same range as that described for ATP or UTP (Lazarowski et al., 1995). On the other hand, P1,P3-di(adenosine-5⬘)triphosphate (Ap3A) is a good agonist at the P2Y1 receptor, while Ap4A behaves like a partial agonist. None of the other adenine dinucleotides exert any effect on this receptor. The P2Y4 receptor, cloned from a human source and defined as a pyrimidinoreceptor, is sensitive to Ap3A, Ap4A, Ap5A, and Ap6A, with EC50 values between 3 and 7 ␮M, but their maximal effect is only 20–25% that of UTP (Communi et al., 1996). At the putative P2Y5 receptor, the series of diadenosine polyphosphates was assayed with no positive results (Janssens et al., 1997). 3.2. Activation of recombinant homomeric P2X receptors by diadenosine polyphosphates Diadenosine polyphosphates can activate at least four recombinant homomeric P2X receptors (P2X1–4). ApnA compounds show different patterns of pharmacological activity at these receptors. At P2X1, only Ap6A is a full agonist, compared with ATP, although the potency order is Ap4A ⬎ Ap6A ⫽ Ap5A, Ap2A and Ap3A being almost inactive. The effect of diadenosine polyphosphates on the P2X2 receptor showed that only Ap4A behaves like an agonist, 5-fold less potent than ATP. The other ApnA compounds were not agonists of this homomeric receptor (Pintor et al., 1996; Wildman et al., 1999). Four dinucleotides are as potent as ATP at the P2X3 receptor. The dinucleotides showed the following potency order: Ap4A ⫽ Ap3A ⬎ Ap5A ⫽ Ap6A (based on EC50 values). At this receptor, Ap3A is a partial agonist, while the other three behaved like full agonists. The P2X4 homomeric receptor responds only to Ap4A and Ap6A, the potency order being Ap4A ⭓ ATP ⬎⬎⬎ Ap6A (by EC50 values). It is important to note that neither of the two dinucleotides behaved like a full agonist, presenting only 30% of the maximal activity of ATP (Pintor et al., 1996; Wildman et al., 1999). It is of interest that some dinucleotides, which do not exert any agonistic effect, can potentiate responses triggered by ATP, e.g., nanomolar concentrations of Ap5A potentiate ATP responses at the recombinant P2X2 receptor, without altering the Hill coefficient for ATP (nH ⫽ 2), indicating that Ap5A is not occupying any of the two ATP sites at the P2X2 receptor and thus, is probably acting on an allosteric site. Ap2A is also a reversible positive modulator of the homomeric P2X3 receptor. When the same experiments were performed on P2X4 receptors, both Ap2A and Ap3A potentiated the responses elicited by ATP, but in this case, in the micromolar range (Pintor et al., 1996; Wildman et al., 1999). The ability of some diadenosine polyphosphates to potentiate the effect of ATP does not occur only on ionotropic receptors. Indeed, potentiation of ATP responses has been demonstrated on metabotropic P2Y receptors from rat cerebellar astrocytes. One of the main differences between the effect

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of dinucleotides in the potentiation of the P2X and P2Y receptors is that at the metabotropic receptor, the potentiation was permanent, being observed for up to 6 hr. This fact indicates that ATP and Ap5A may play an important role in astroglial differentiation and proliferation (Jiménez et al., 1998). 3.3. Activation of heteromeric P2X receptors As reviewed by Buell et al. (1996), virtually all ionotropic P2X receptors have been reported to form functional homomeric channels when they are expressed in heterologous systems. These studies proved that in some tissues, the native channel contains an homomeric assembly of one P2X receptor subunit. An example is the P2X1 receptor found in vas deferens (Valera et al., 1994). However, some native P2X receptor phenotypes do not appear to correspond with homomeric recombinant P2X receptors. In addition, in situ hybridisation and immunohistochemical experiments have shown co-localisation of different receptor subunits within the same population of cells in a variety of tissues (Collo et al., 1996; Kanjhan et al., 1999; Le et al., 1998; Nori et al., 1998; Vulchanova et al., 1997; Xiang et al., 1998). These findings suggest that native receptors might also act as heteromeric assemblies of members of this family. However, the exact subunit composition still remains to be elucidated. In this regard, only one study has shown a functional channel in dorsal root sensory neurons, which appears to be due to a hetero-oligomer containing P2X2 and P2X3 (Lewis et al., 1995). Subsequently, Le et al. (1998) demonstrated that co-expression of P2X4 and P2X6 receptors in Xenopus oocytes leads to the generation of a novel ATP receptor, not found in native tissues. More recently, Le et al. (1999) demonstrated another functional heteromeric channel formed with P2X1 and P2X5 subunits, when co-transfected in human embryonic kidney 293 cells. Again, a native receptor has not been found yet with the same pharmacological profile. In a search for potential heteromeric ATP-gated channels, Torres et al. (1999) studied the possible subunit co-assembly in the P2X family, using an immunoprecipitation assay. Interestingly, P2X6 was unable to form a homomeric complex, indicating that it assembles with other subunits to form a functional channel. On the other hand, the P2X7 subunit forms only homomeric receptors. In the light of these findings, it has become apparent that P2X receptors might form both homo- and heteromeric functional channels. Further experiments are necessary to elucidate the subunit composition of the native ATP-gated channel. In synaptosomal preparations obtained from rat midbrain, diadenosine polyphosphates activate P2X receptors and an as yet uncharacterised novel receptor (Pintor et al., 1992b; Pintor & Miras-Portugal, 1995) called the diadenosine polyphosphate receptor (P4 receptor), since it is selectively activated by ApnA (see Section 4). Currently, we are focusing on the molecular cloning of this new receptor, but cannot rule out the possibility that it may be a heterodimer of two or more P2X subunits. It is relevant that a recent study described a new pharmacological phenotype when

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different opioid receptors are heterologously co-expressed (Jordan & Devi, 1999). This is also the case for the ␥-aminobutyric acid-B receptors (Jones et al., 1998). In addition, some of the physiological effects of ApnA may be due to activation of heteromeric P2X receptors. In line with this hypothesis, we have started to study the pharmacological profile of ApnA on heteromeric channels composed of different P2X subunits. Initially, we studied the effect of these compounds on intracellular Ca2⫹ levels from the rat glioma tumour cell line C6BU-1, transiently transfected with either the P2X4 or P2X6 receptors alone or by a combination of both subunits. In our laboratory, this cell line has proven to be a useful tool for such studies, since it does not respond to any mono- or dinucleotide. Fig. 3 shows the different pharmacological responses to ATP, Ap4A, and Ap5A of these cells, differentially transfected with P2X4 and/or P2X6 receptor subunits. Interestingly, an increase in intracellular Ca2⫹ levels is observed in C6BU-1 cells transfected with the homomeric P2X4 or P2X6 receptors alone after stimulation with 100 ␮M Ap4A. However, this response disappears when the cells are transfected with the heteromeric receptor P2X4/P2X6, indicating that ApnA may behave with a different pharmacological phenotype on heteromeric P2X receptors, compared with homomeric P2X receptors. With these experiments, it may be possible to elucidate the possible relationship with the P4 receptor. 3.4. Native P2 receptors stimulated by diadenosine polyphosphates in the central nervous system There is substantial evidence that diadenosine polyphosphates bind to native P2 receptors in the CNS. Ap4A is able to displace the binding of [3H]␣,␤-methylene adenosine 5⬘triphosphate (␣,␤-meATP) in rat brain slices, suggesting that both ␣,␤-meATP and Ap4A compete for the same P2X receptor (Balcar et al., 1995). Additional binding and functional studies indicate that diadenosine polyphosphates can activate P2 receptors in the CNS. Binding studies carried out in rat brain cortical synaptosomes by means of [35S]adenosine 5⬘-O-(3-thiotriphosphate) ([35S]ATP␥S) (a putative P2Y agonist) demonstrated that Ap5A and Ap6A gradually enhanced the binding of [35S]ATP␥S by up to 60% in a concentration range from 1 to 50 ␮M. Ap4A produce only a 15% of increase in the same concentration range. Diadenosine polyphosphates assayed at concentrations above 50 ␮M inhibited the binding of [35S]ATP␥S to synaptosomal membranes, but with very low potency (Schäfer & Reiser, 1997). Ap4A and Ap5A mimic the effect of ATP on P2X receptors expressed in rat sensory neurons (Krishtal et al., 1983), but Ap3A and Ap2A are nearly ineffective (Marchenko et al., 1987). The effect produced by diadenosine tetraphosphate and diadenosine pentaphosphate was 15% of the maximum amplitude of the ATP-activated current. The estimated Kd value for Ap4A was ⵑ40 ␮M, and it could be concluded that both the dinucleotide and ATP are acting via the same receptor (Krishtal et al., 1988).

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Fig. 3. Effect of 100 ␮M ATP, Ap4A, and Ap5A on intracellular Ca2⫹ levels in C6BU-1 cells transiently transfected with P2X4 or P2X6 receptors, or a combination of both. Cells grown in cover slips were transfected using liposomes, and 48 hr later were loaded with fura-2 AM in a buffer containing CaCl2 (1.33 mM) for 45 min. Cover slips were then mounted in a perfusion chamber, and intracellular Ca2⫹ levels were analysed after stimulation with the different drugs, using a multiple excitation microfluorescence system. Cells were illuminated alternately at 380 and 400 nm. The emitted fluorescence was collected at 510 nm. Results are shown as a normalised ratio of F380/F400 signals that increases as [Ca2⫹]i increases. (A) Homomeric P2X6 receptor-expressing cells respond very weakly to ATP and Ap5A, with Ap4A as a potent agonist. (B) Homomeric P2X4 receptor respond to Ap4A as intensely as ATP, but there is cross-desensitisation. The figure shows the time course of fluorescence changes in a representative cell initially stimulated by Ap4A. P2X6 receptor-expressing cells primarily stimulated with ATP respond with an increase in intracellular Ca2⫹ level, but are insensitive to further stimulation with Ap4A or Ap5A. (not shown). (C) The response to Ap4A disappears when cells are co-transfected with P2X4 and P2X6 receptor subunits, although the heteromeric receptor formed is still sensitive to ATP.

It has been suggested that noradrenergic neurones located in the locus coeruleus express both P2X and P2Y receptors (Tschöpl et al., 1992; Illes et al., 1995). The firing rate of these neurones is increased by Ap3A, Ap4A, and Ap5A, with the potency order Ap5A ⬎ Ap4A ⬎ Ap3A (Illes et al., 1996). The effects of the dinucleotides are partially blocked by suramin and pyridoxalphosphate-6-azophenyl2⬘,4⬘-disulphonic acid (PPADS), and are insensitive to reactive blue 2, suggesting that adenine dinucleotides act preferentially through P2X receptors (Fröhlich et al., 1996). 4. Native receptors specifically stimulated by diadenosine polyphosphates in the central nervous system The existence of specific receptors for adenine dinucleotides has been described in the CNS. Although diadenosine

polyphosphates could exert their actions on P2 purinoceptors in different neural and non-neural preparations, as shown in Section 3.4, evidence accumulated during recent years supports the possible existence of specific receptors for diadenosine polyphosphates (see Miras-Portugal et al., 1998), but not mononucleotides. The concept “specific” means not sensitive to ATP, UTP, adenosine, or their respective pharmacological analogues, but in most cases, sensitive only to dinucleoside oligophosphates. This receptor has been termed dinucleotide receptor or P4 receptor (Pintor & Miras-Portugal, 1995). Hilderman et al. (1991) first identified a receptor for Ap4A in rat brain homogenates, with a Kd value for Ap4A of 0.71 ␮M. Subsequently, Pivorun and Nordone (1996) demonstrated that Ap4A was able to increase intrasynaptosomal Ca2⫹ levels in deermouse brain. This effect was not produced by ATP and was not sensitive to P2 antagonists, suggesting the presence of a dinucleotide receptor.

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These features, and the fact of being coupled to VDCCs, makes this receptor very similar to that described by our group in rat midbrain (Pintor & Miras-Portugal, 1995; see the next section). 4.1. Rat brain receptors Dinucleotide receptors, also termed P4 receptors, were pharmacologically described in 1995 in rat midbrain synaptic terminals, but the molecular structure remains unknown. These receptors are sensitive to diadenosine tetraphosphate and diadenosine pentaphosphate and elicited intracellular Ca2⫹ increases within presynaptic rat brain terminals, acting as receptor-operated Ca2⫹ channels (Pintor & Miras-Portugal, 1995). Some facts point to these receptors being receptor-operated Ca2⫹ channels. The global functioning of the dinucleotide receptor in the presence of the G-protein modulators does not fit with a model in which the receptor is coupled to a Ca2⫹ channel via a G-protein mechanism (Pintor et al., 1997b). For example, in the absence of extracellular Ca2⫹, the responses to diadenosine polyphosphates are abolished. Another point in favour of this hypothesis is that the receptor is functional in the presence of G-protein modulators. Substances such as GDP␤S or GTP␥S severely changed the activity of the dinucleotide receptor. For instance, the application of the nonhydrolysable GDP analogue, which inactivates G-proteins, enhanced the Ca2⫹ response elicited by diadenosine polyphosphates. On the other hand, GTP␥S produced the opposite effect, since a clear reduction in the Ca2⫹ response was seen in rat midbrain synaptosomes. Nevertheless, the functioning of the receptor in the presence of these GTP/GDP analogues can be explained on the basis of activation of protein kinases (Pintor et al., 1997b; see the following section). 4.1.1. Pharmacology As mentioned in Section 4, the dinucleotide receptor is stimulated by diadenosine polyphosphates, being insensitive to ATP, adenosine, and their respective analogues. This fact generates the need for new antagonists because classical antagonists for P2 receptors cannot be used on this receptor. Pharmacological approaches have been carried out with the available diadenosine polyphosphates. Experiments performed in rat midbrain synaptic terminals showed that diadenosine pentaphosphate was the most powerful natural dinucleotide, followed by Ap4A, with Ap3A being almost inactive. Ap2A, recently described as a naturally occurring dinucleotide in platelets, was also a good agonist on this receptor. The EC50 values for Ap5A, Ap4A, and Ap2A were almost identical, ⵑ55 ␮M (Pintor & Miras-Portugal, 1995). More recent experiments with additional dinucleoside polyphosphates, such as ⑀-ApnA (the etheno derivatives of diadenosine polyphosphates) or GpnG (diguanosine polyphosphates), showed the following pharmacological order in terms of EC50 values: Gp5G ⬎ Ap5A ⫽ Ap4A ⬎ Gp4G ⫽ ⑀-Ap4A ⫽ ⑀-Ap5A ⬎ Gp3G ⬎ Ap3A ⫽ ⑀-Ap3A. As a gen-

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eral rule, the following potency order prevails: Np5N ⬎ Np4N ⬎ Np3N (Miras-Portugal et al., 1999). With the help of different fluorescent dyes, it has been possible to determine whether this receptor is permeable to Na⫹ and Ca2⫹ or only to Ca2⫹. Measurement of intracellular Ca2⫹ with fura-2 and Na⫹ with SBFI have demonstrated that after stimulation with diadenosine pentaphosphate, Ca2⫹ entry occurs, depolarising the terminals and opening voltagedependent ion channels (Pintor & Miras-Portugal, 1995). 4.1.2. Connection with voltage-dependent Ca2⫹ channels After stimulation of the synaptic terminal with adenine dinucleotides through the P4 receptor, Ca2⫹ entry depolarises the terminal activating voltage-dependent Na⫹ channels (VDSC) and VDCCs (Panchenko et al., 1996; Pintor & Miras-Portugal, 1995). The activation of VDSC was confirmed by pretreatment of the synaptic terminals with the blocker tetrodotoxin. A lack of Na⫹ entry was measured with SBFI when the toxin was present. Concerning Ca2⫹ channels, three different substances were assayed to block the different VDCCs: nifedipine, to antagonise the L-type; ω-conotoxin G-VI-A, to block the N-type; and ω-agatoxin, to antagonise the P-type. Ni2⫹ was also used to rule out involvement of the T-type Ca2⫹ channel. Only ω-conotoxin G-VI-A was able to partially reduce Ca2⫹ entry induced by diadenosine polyphosphates. A careful analysis of the Ca2⫹ entry in the presence of this toxin reveals the existence of an initial transient, suggesting that the dinucleotide receptor elicits an initial Ca2⫹ entry, which is voltage-insensitive. There is a clear decrease in Ca2⫹ entry after the initial peak when ␸-conotoxin G-VI-A is tested. This indicates that the total Ca2⫹ increase is the sum of a voltage-independent transient, followed by a voltage-dependent one mediated by an N-type Ca2⫹ channel (Fig. 4). This was also observed when the synaptic terminals were prestimulated with 60 mM K⫹. Under these conditions, the initial entry after stimulation with Ap5A remains unchanged, but the secondary phase disappears (Pintor & Miras-Portugal., 1995). Experiments performed with the patch-clamp technique have demonstrated that hippocampal CA3 neurones are stimulated by diadenosine polyphosphates, showing responses that are partially mediated by a VDCC N-type channel (Panchenko et al., 1996). 4.1.3. Modulation of the dinucleotide receptor by protein kinases and phosphatases Many receptors for neurotransmitters and hormones are coupled to second messenger systems that produce activation or inhibition of adenylate cyclase or PLC. Activation of the mentioned second messenger systems triggers the synthesis of cyclic AMP and diacylglycerol that activate protein kinases A and C (PKA and PKC), respectively. Protein kinases can phosphorylate enzymes and membrane proteins such as receptors, channels, and transporters. Protein phosphatases reverse the action of protein kinases, keeping a balance of phosphorylation in many proteins.

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Fig. 4. Schematic diagram of the relationship between the dinucleotide receptor and the VDCC. The activation of the dinucleotide receptor permits Ca2⫹ entry into the synaptic terminal, which generates a depolarisation threshold that activates a VDCC N-type.

The dinucleotide receptor present in rat midbrain synaptic terminals is strongly modulated by PKA and PKC, as well as by protein phosphatases. Activation of adenylate cyclase by forskolin, or PKA by dibutyryl cyclic AMP, blocks the Ap5A intrasynaptosomal Ca2⫹ transients. Compounds blocking the activation of PKA, such as the PKA inhibitory peptide (PKA-IP), clearly potentiate the Ca2⫹ responses elicited by Ap5A in rat midbrain synaptosomes. Substances acting on PKC generate similar results. For example, activation of PKC with phorbol esters such as 12,13-dibutyrate dramatically inhibits the Ca2⫹ transients elicited by Ap5A. Like the PKA inhibitors, the PKC-IP and staurosporine both potentiated the responses elicited by Ap5A, as shown in Fig. 5 (Pintor et al., 1997b). The possible involvement of a phosphorylation process was further confirmed by the effect of different protein phosphatase inhibitors (Cohen, 1989). Substances such as okadaic acid, a broad protein phosphatase inhibitor, pro-

duced a reduction in the Ca2⫹ responses elicited by Ap5A. This effect was also observed with other protein phosphatase inhibitors, such as mycrocistin (a protein phosphatase A2A inhibitor) or cyclosporin A (a calcineurin inhibitor). The action of these inhibitory substances producing a decrease in the Ca2⫹ transients elicited by Ap5A indicates that different protein phosphatases could reverse the phosphorylation process carried out by PKA and PKC (Fig. 5). The high concentration of calcineurin in neural tissues, and especially in neurones, may indicate a potential role for this enzyme in modulating neural activity, also reversing the phosphorylation of the dinucleotide receptor by protein kinases (Pintor et al., 1997b). Calcineurin is implicated in the regulation of ionotropic receptors and channels in mammalian and nonmammalian neural tissues (Cohen, 1989; Kunz & Hall, 1993). The dinucleotide receptor seems to be controlled by a balance between phosphorylation and dephosphorylation that depends on the degree of activation by those receptors positively coupled to PKA and PKC and the activity of protein phosphatases (Fig. 5). 4.2. Dinucleotide receptors in guinea-pig brain Several areas of the brain in different species have been investigated in a search for the existence of dinucleotide receptors. As described in the previous paragraphs, rat midbrain was the first tissue where this receptor was well characterised. Nevertheless, guinea-pig brain was chosen to study the distribution of the dinucleotide receptor by investigating three different brain areas: cortex, midbrain, and cerebellum. 4.2.1. Cortical receptors The receptors present in guinea-pig cortical synaptic terminals respond both to ATP and diadenosine polyphosphates by acting through the same P2 receptor subtype. The effect of the whole series of adenine dinucleotides gave si-

Fig. 5. Modulation of the dinucleotide receptor by protein kinases and phosphatases. The activation of PKA and PKC inhibits the Ca2⫹ transients elicited by Ap5A. Stimulation of protein phosphatases produces the opposite effect, showing higher Ca2⫹ transients than those in the absence of phosphatase activation. Stimulation of neurotransmitter (NT) receptors coupled to PLC or adenylate cyclase (AC) may modify dinucleotide response via protein kinase activation.

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milar EC50 values for all of them. For maximal effects produced, Ap4A was the best (29 nM Ca2⫹ increase above the basal value) followed by Ap3A and Ap2A, Ap5A and Ap6A being the poorest (20 nM Ca2⫹ increase). Cross-desensitisation studies carried out with ATP analogues, such as ␣,␤-meATP, abolished the responses elicited by diadenosine polyphosphates. Moreover, preincubation of the cortical synaptic terminals with suramin, a P2 antagonist, also markedly attenuated the responses produced by ApnA compounds. These two aspects clearly indicate that in the cortical guinea-pig synaptic terminals, both diadenosine polyphosphates and ATP share the same receptor, presumably a P2X (see Fig. 6; Pintor et al., 1997c). 4.2.2. Midbrain receptors In midbrain guinea-pig synaptic terminals, diadenosine polyphosphates produce an intracellular Ca2⫹ increase above the basal cytosolic values, which was concentrationdependent. As with cortical synaptosomes, there were no differences in the EC50 values obtained for each of the assayed compounds. At the highest concentration assayed, the rank order for evoking the Ca2⫹ signal was Ap2A ⫽ Ap4A ⫽ Ap6A ⬎ Ap3A ⬎ Ap5A. In these synaptic terminals, ATP, as well as the synthetic analogues, elicited Ca2⫹ transients that in some cases, were comparable with those produced by ApnA compounds.

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Nevertheless, in clear contrast to the cortical synaptic terminals, the cross-desensitisation studies performed by applying ␣,␤-meATP prior to the diadenosine polyphosphates showed an inability of this ATP analogue to block the effect of the adenine dinucleotides. Moreover, the P2 antagonist suramin, applied before ApnA, was unable to block the Ca2⫹ transients elicited by adenine dinucleotides. Taking into consideration these two facts, it can be concluded that in guinea-pig midbrain synaptic terminals, diadenosine polyphosphates and ATP act via independent receptors, a dinucleotide and a P2X receptor, respectively (Fig. 6; Pintor et al., 1997c). 4.2.3. Cerebellar receptors ApnA compounds were able to increase the intracellular Ca2⫹ levels in cerebellar isolated synaptic terminals with different EC50 values, the potency order being Ap2A ⬎ Ap6A ⫽ Ap3A ⱖ Ap5A ⫽ Ap4A. The best was Ap2A, which showed not only a lower EC50, but also maximal Ca2⫹ influx (34 nM above the basal cytosolic value). Cross-desensitisation studies performed with ATP analogues demonstrated that the mononucleotides did not modify the Ca2⫹ transient elicited by ApnA, suggesting that ATP and diadenosine polyphosphates are acting through different receptors. To further confirm this, the P2 receptor antagonist suramin was used to see whether or not the responses

Fig. 6. Distribution of P4 and P2X receptors in the guinea-pig brain. (A) Responses produced by diadenosine polyphosphates mediated by different receptors. (B) In the midbrain and cerebellum, ApnA activated P4 receptors and ATP P2X receptors. (C) In the cortex, all the nucleotides activated the same P2X receptor. The antagonists Ip5I, suramin, and PPADS helped to identify the receptors involved in each region.

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elicited by diadenosine polyphosphates were blocked by this substance. Suramin could block the responses elicited by ATP, but did not change the responses elicited by ApnA compounds. This supports the idea that ATP and ApnA act through independent receptors, as in guinea-pig midbrain (Fig. 6; Pintor et al., 1997c). 4.3 Human brain receptors Receptors for diadenosine polyphosphates have also been investigated in human brain isolated synaptic terminals. Healthy human brain tissue is not readily accessible, hence the number of experiments has been limited. Nevertheless, the main questions, such as whether there are receptors for diadenosine polyphosphates or whether they resemble some of the animal models, have been positively answered. Application of ApnA or ATP to human cortical synaptic terminals produces a concentration-dependent intracellular Ca2⫹ increase. Analysis of the dose-response curves yielded EC50 values of 11.5 ⫾ 2.1 ␮M and 23.4 ⫾ 3.7 ␮M for Ap5A and ATP, respectively. It is interesting to note that both agonists, although mobilising only a small amount of Ca2⫹, produced a clear change in the membrane potential of the synaptic terminals (Pintor et al., 1999). This suggests that under certain conditions, these two nucleotides could induce a change in the synaptic terminal sufficient to induce the transmitter release. As in animal studies, the question whether Ap5A and ATP act via the same or different receptors was answered by performing cross-desensitisation studies, as well as studies with antagonists. Cross-desensitisation studies indicated that responses to ATP were not blocked by pretreatment with Ap5A or vice versa. Experiments performed with antagonists PPADS (a P2 antagonist) and Ip5I (a P4 antagonist) demonstrated that the effects elicited by ATP were antagonised by PPADS, but not by Ip5I. In contrast, Ap5A responses were not blocked by PPADS, and only partially (50%) with Ip5I. The effect of Ap5A on intrasynaptosomal Ca2⫹ increase was partially blocked by Ip5I, in clear contrast to the effect obtained in rat synaptosomes, where Ip5I was able to abolish the effect of Ap5A (Pintor & Miras-Portugal, 1995; Pintor et al., 1999). This discrepancy is subject to different interpretations. It is possible that the dinucleotide receptors in rat and human brain are different. Another possibility is the existence of ectonucleotidases in the human brain, with a different selectivity for nucleotides. In this case, the difference in the activity of the antagonists may be due to a selective cleavage, significantly reducing the concentration of Ip5I. Whether this or the previous hypothesis is true remains to be resolved. As in rat brain synaptic terminals, dinucleotide receptors are coupled to VDCCs in the human brain. The activation of this receptor by Ap5A produces an increase in the ω-conotoxinsensitive component of the Ca2⫹ channels in human cortical synaptic terminals. The specificity of this toxin (Miller & Fox, 1990; Tsien & Tsien, 1990) and the lack of effect by

the other blockers indicate that Ap5A facilitates only the activity of N-type Ca2⫹ channels, the others being uninvolved in the dinucleotide action. As commented in Section 4.1.2, this result is in agreement with those described in rat midbrain synaptosomes (Pintor & Miras-Portugal, 1995) and CA3 hippocampal neurones (Panchenko et al., 1996). The non-association of Ap5A stimulation with the activation of an L-type Ca2⫹ channel has been pointed out for rat brain synaptosomes, hippocampal neurones, and also deermouse synaptosomes. L-type channel blockers, such as verapamil or nifedipine, do not modify the Ca2⫹ responses induced by diadenosine polyphosphates in these neural models (Pintor & Miras-Portugal, 1995; Panchenko et al., 1996; Pivorun & Nordone, 1996).

5. Could diadenosine polyphosphate derivatives be used as pharmacological tools for P2X receptors? In 1995, the existence of a nonspecific adenylic acid deaminase in the snail Helix pomatia was described (Guranowski et al., 1995). The authors showed that this enzyme is able to transform the adenine rings of ApnA compounds into hypoxanthine, thus transforming the diadenosine polyphosphates into diinosine polyphosphates (IpnI). When instead of the H. pomatia enzyme the commercial adenylic acid deaminase (from Aspergillus sp.) was used, the same results were obtained. From Ap3A, Ap4A, and Ap5A, it was possible to obtain Ip3I, Ip4I, and Ip5I, respectively. These compounds were assayed for their ability to act as agonists or antagonists in rat midbrain synaptosomes. IpnI compounds did not behave as agonists, since they did not modify the cytosolic Ca2⫹ levels of the synaptic terminals under study. Nevertheless, when they were tested as antagonists on the dinucleotide receptor, the three hypoxanthine compounds blocked the actions of Ap4A and Ap5A. There was a clear difference among the three dinucleotides. While Ip3I and Ip4I were antagonists at the dinucleotide receptor, with IC50 values in the micromolar range (4.9 ␮M and 8.3 ␮M, respectively), Ip5I behaved as a very potent antagonist, with an IC50 value of 4.2 nM (Pintor et al., 1997c). Apart from the dinucleotide receptor, rat midbrain synaptosomes possess P2X receptors. The three diinosine compounds, assayed for responses mediated through P2X receptors, presented IC50 values of 100 ␮M, 29.5 ␮M, and 27.5 ␮M for Ip3I, Ip4I, and Ip5I, respectively. From these values, with an ⵑ6000 times greater affinity for the dinucleotide receptor, it can be seen that Ip5I is a useful tool to discriminate between the activation of dinucleotides versus P2 receptors by diadenosine polyphosphates (Pintor et al., 1997a). In the periphery, IpnI compounds have been assayed in guinea-pig vas deferens. This tissue, considered a classical one for studying P2X1 receptors, displayed an interesting behaviour towards the hypoxanthine dinucleotides. In particular, Ip5I was a good antagonist of the P2X1 receptor, with an IC50 value of 0.30 ␮M. This effect seems to be se-

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lective since in other models, such as taenia coli (with P2Y receptors), or guinea-pig left atrium (with P1 and P2 receptors), Ip5I did not significantly modify the responses mediated by ATP (Hoyle et al., 1997). More recently, it proved possible to study the effects of Ip3I, Ip4I, and Ip5I on recombinant P2X receptors expressed in Xenopus laevis oocytes. At the P2X1 receptor, the three diinosine polyphosphates presented the following antagonistic potency: Ip5I ⬎ Ip4I ⬎ Ip3I, with IC50 values of 3.16 ␮M, 0.5 ␮M, and 31.6 ␮M, respectively. On the P2X2 receptor, none of the diinosine polyphosphates had any effect. On the P2X3 receptor, the three IpnI behaved as antagonists presenting the following potency order and IC50 values: Ip4I (1 ␮M) ⬎ Ip5I (2.5 ␮M) ⬎ Ip3I (⬎30 ␮M). The agonist responses mediated by the P2X4 receptor were potentiated by Ip4I and Ip5I in a reversible fashion (King et al., 1999). These results showed that Ip5I is a good antagonist at the Group 1 P2X receptors (P2X1 and P2X3), being more selective for the P2X1 than for the P2X3. Ip5I is 900-fold less potent at the P2X3, where the required concentration to block ATP action is in the micromolar range, compared with the nanomolar concentrations necessary to antagonise ATP via the P2X1 receptor (King et al., 1999). 6. Conclusions Diadenosine polyphosphates can stimulate different subtypes of purinergic receptors after their release into the extracellular space from synaptic vesicles or secretory granules. The physiological role of these dinucleotides remains unclear, although we already know the receptors that can be activated by these compounds. Ionotropic responses mediated by P2X and P4 receptors suggest an involvement in synaptic transmission in the CNS, facilitating neurotransmitter release from the synaptic terminals, as does ATP (Edwards et al., 1992; Pankratov et al., 1999). In this sense, it is necessary to establish the possible relationship between diadenosine polyphosphates and their receptors and other classical neurotransmitters in order to confirm this hypothesis. Acknowledgments This work has been supported by research grants from the Universidad Complutense de Madrid, PR49/98-7786, Comunidad Autónoma de Madrid No. 8.5/18/1998, and DGICYT PM98-0089. M.D.-H., J.G., R.G.-V., and F.H. are fellowship holders of the Universidad Complutense, Ministerio de Educación, Comunidad Autonoma de Madrid, and Beca de Reincorporación de la Comunidad de Madrid, respectively. We thank Charles H. V. Hoyle for help in preparing this manuscript. References Abbracchio, M. P., & Burnstock, G. (1994). Purinoceptors: are there families of P2X and P2Y purinoceptors? Pharmacol Ther 64, 445–475.

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