P2X receptors in sensory neurones.

British Journal of Anaesthesia 84 (4): 476–88 (2000)

REVIEW ARTICLE P2X receptors in sensory neurones G. Burnstock Autonomic Neuroscience Institute, Royal Free and University College Medical School, Rowland Hill Street, London NW3 2PF, UK P2X receptors are a family of ligand-gated ion channels responsive to ATP. Seven subtypes have been identified which form homomultimeric or heteromultimeric pores. P2X3 receptors are selectively expressed predominantly on small-diameter nociceptive sensory neurones in the dorsal root, trigeminal and nodose ganglia, particularly the non-peptidergic subpopulations labelled with the lectin IB4. P2X2/3 labelling is also present in inner lamina II of the spinal cord and in sensory nerve projections to skin and viscera, but few receptors are present in skeletal muscle. P2X3 receptors are down-regulated after peripheral nerve injury and their expression can be regulated by glial cell-derived neurotrophic factor. P2X receptor activation of sensory neurones has been demonstrated in in vivo pain models, including the rat hindpaw and kneejoint preparations, as well as in inflammatory models. P2X4 and/or P2X6 receptors in the CNS also seem to be involved in pain pathways. Non-nociceptive P2 receptors on sensory nerves are present in muscle and on sensory endings in the heart and lung that initiate reflex activity involving vagal afferent and efferent nerve fibres. The sources of ATP involved in nociception and non-nociceptive sensory nerve stimulation are discussed as well as a novel hypothesis about purinergic mechanosensory transduction. Br J Anaesth 2000; 84: 476–88 Keywords: nerve, neurotransmitters; pain

There have been various reports over the years about the actions of ATP on sensory nerves.9 10 15 22 45 84 However, it was only recently that direct evidence for P2 receptors of various subtypes on sensory neurones was obtained after the cloning and characterization of extracellular receptors for ATP from sensory nerves;39 88 subsequent studies have confirmed and extended this finding.8 29 38 42 43 61 77 105 126 132 139 In this article, the evidence for nociceptive and mechanosensitive P2X and P2Y receptors on sensory neurones, and for P2X receptors involved in sensory pathways in cardiorespiratory reflexes and in the CNS, is reviewed. The origin of ATP acting on sensory neurones and the interactions of P2 sensory receptors with other agents will be discussed.

Early reports of extracellular actions of ATP The first report describing the potent actions of adenine compounds was published in 1929 by Drury and Szent-Gyo¨rgyi52 and the first hint that ATP might be a neurotransmitter appeared three decades later when ATP was shown to be released during antidromic stimulation of sensory nerves supplying the rabbit ear artery.71 In the early

1960s, the existence of autonomic nerves supplying the gastrointestinal tract and urinary bladder that were neither adrenergic nor cholinergic was proposed.18 32 33 98 In the years that followed, strenuous efforts were made to identify the transmitter in non-adrenergic, non-cholinergic nerves supplying the gut and the urinary bladder. Perhaps surprisingly, the substance that most satisfied the criteria accepted at that time for establishing a transmitter54 was ATP,34 and the word ‘purinergic’ was coined and purinergic transmission proposed.19 Implicit in the concept of purinergic transmission was the existence of postjunctional receptors for ATP, although there was considerable confusion in the literature about the variable effects of adenosine nucleotides and nucleosides on a wide variety of tissues.20 However, a step forward was taken in 1978 when, from a detailed analysis of the literature and some preliminary experiments, it was proposed21 that purinoceptors could be subdivided into P1 (adenosine) receptors, which were mediated by adenylate cyclase and competitively antagonized by low concentrations of methylxanthines, and P2 receptors, which were activated preferentially by ATP and ADP. Two of the most important implications of this purinoceptor subdivision were: (1) the

© The Board of Management and Trustees of the British Journal of Anaesthesia 2000

P2X receptors in sensory neurones

importance of establishing if, in a particular situation, ATP was acting directly on P2 receptors or via P1 receptors after ectoenzymatic breakdown to adenosine;103 and (2) during purinergic transmission, whereas ATP released from the nerve terminals acted on postjunctional P2 receptors, adenosine arising from extracellular breakdown of ATP acts largely via P1 receptors on the nerve terminals to modulate release of transmitters.47 Prejunctional modulation via P1 receptors operates both as a negative feedback system in autoregulation of purinergic transmission and to modulate the release of noradrenaline (NA), acetylcholine (ACh) and other neurotransmitters released with ATP.26

P2 receptor subclassification In 1985, Burnstock and Kennedy36 proposed the first subdivision of P2 receptors into: P2X purinoceptors, which mediate vasoconstriction and contraction of visceral smooth muscle with α,β-methylene ATP as a potent agonist; and P2Y purinoceptors, which mediate vasodilatation and relaxation of the smooth muscle of the gut with 2-methylthio ATP as a particularly potent agonist. Soon afterwards, two further P2 purinoceptors were tentatively proposed:63 a P2T purinoceptor, which is ADP-selective and involved in platelet aggregation, and a P2Z purinoceptor, which appears to be activated by ATP4– and is prominent in macrophages, lymphocytes and mast cells. Another important landmark, after the seminal studies of Furchgott and Zawadski,59 was the recognition that ATP acts on endothelial cells to mediate vasodilatation via release of endothelium relaxing factor (EDRF).48 This important discovery challenges the early hypothesis of Berne7 that adenosine is the local regulator of blood flow after hypoxia in heart and other vascular beds—it now seems likely that reactive hyperaemia results largely from ATP released from endothelial cells during hypoxia acting on P2Y receptors to release EDRF (nitric oxide), resulting in vasodilatation; adenosine, produced after the breakdown of ATP, is likely to contribute to the later component of vasodilatation by direct action on P1 receptors on vascular smooth muscle.25 Knowledge of the structure and properties of P2 receptors has lagged behind information about receptors for most other neurotransmitters, although advances have been made recently. The general progress of information about purinoceptors has been: structure–activity relationships, pharmacology, quantification of receptor expression, ligand binding and autoradiographic localization, transduction mechanisms involving second messenger systems and ion channels, and finally the molecular biology of the receptors with cloning and sequencing. Since the earlier subdivisions of the P2 purinoceptor into P2X, P2Y, P2T and P2Z mentioned above, several further subclasses were proposed, including P2U purinoceptors where ATP and UTP are equipotent110 and a P2D purinoceptor, selective for diadenosine polyphosphates.114 It was clearly shown that P2X purinoceptors involved ligand-gated cation channels,6 while P2Y receptors

involved G protein activation.53 More recently, it has been suggested that some P2Y receptors act via Gi proteins to inhibit adenylate cyclase69 and that there may be pyrimidine nucleotide-selective G protein-linked receptors.41 106 The first P2 receptors to be cloned were G proteincoupled receptors; a P2Y receptor, P2Y1, was isolated from chick brain141 and a P2U purinoceptor (later designated P2Y2) from neuroblastoma cells.95 A year later, two ligandgated ion channel ATP receptors were reported—one (P2X1) from vas deferens134 and another (P2X2) from rat phaeochromocytoma PC12 cells.17 In the paper from the IUPHAR subcommittee concerned with the nomenclature of P2 purinoceptors,58 it was emphasized that the current purinoceptor subclassification, with so many letters of the alphabet being somewhat randomly added as new receptor subtypes were discovered, was unsatisfactory. The Committee supported, in principle, a new system of classification proposed by Abbracchio and Burnstock.1 In this proposal, it was suggested that P2 receptors should be placed in two major families: a P2X family, consisting of ligand-gated cation channels, and a P2Y family, consisting of G protein-coupled receptors. The P2Z purinoceptor, which opens non-selective pores, has recently been cloned and incorporated into the P2X receptor family as P2X7.125 This classification brought ATP into line with most other neurotransmitters, such as ACh, γ-amino butyric acid (GABA), glutamate and 5-hydroxytryptamine, where ligand-gated and G protein-mediated receptor subclassification has already been established.28 Seven P2X family and eight P2Y family subtypes have been identified to date.29 30 37 109

P2X receptor family For the P2X receptor subtypes the proteins deduced from cDNA sequences have 379 (P2X6) to 472 (P2X7) amino acid residues and share 36–48% identity with each other. They are characterized by two transmembrane (TM) domains with a large extracellular loop where 10 cysteines are preserved; both N- and C-terminals are intracellular. Northern blots and in situ hybridization show a widespread distribution of mRNA with P2X1 expressed predominantly in smooth muscle, P2X4 and P2X6 most heavily expressed in brain, P2X3 found only in sensory neurones and P2X7 dominant in cells of the immune system.12 40 125 P2X1– P2X4 subtypes readily form homomultimeric channels when expressed in oocytes or in transfected mammalian cell lines; the number of subunits per channel, although not yet established, is believed to be three or four.38 107 108 It is also becoming clear that many of the naturally occurring P2X receptors are heteromultimers of the P2X subtypes.81 86 137

P2Y receptor family The eight P2Y purinoceptor subtypes respond more slowly to agonists than the P2X purinoceptor and achieve their effect through intracellular second messenger systems.

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As for other known G protein-coupled receptors, the molecule is characterized by seven TM domains with short extracellular N- and intracellular C-terminals.3 The receptor consists of a 308–377 amino acid, with a mass of 41–53 kDa after glycosylation; TM3 displays a high degree of conserved amino acid residues within the P2Y purinoceptor family.13 135 Northern blots and in situ hybridization show a wide distribution of most P2Y receptor subtypes.4 69 The possibility that P2Y4, together with other subtypes, should be considered as a subfamily of pyrimidinoceptors within the P2Y purinoceptor family has been raised.41 The identification of selective agonists and antagonists for the different P2X and P2Y receptor subtypes is still awaited. Meanwhile, in general, 2-methylthio ATP is most active at native P2Y receptors, while α,β-methylene ATP is more active at most native P2X receptors, although there are clear exceptions and the reported potencies of some agonists is complicated by their breakdown by ectonucleotidases.35 77 Some receptor subtypes are selectively activated by UTP and others are not, while some exhibit desensitization and others do not.108 P2 receptor antagonists in current use, including suramin, pyridoxalphosphate-6-axophenyl-2⬘,4⬘-disulphonic acid (PPADS), and Reactive blue 2, do not appear to be selective for different recombinant receptor subtypes, but have been used selectively in isolated preparations.28 There are few reports of the use of antagonists in vivo.

P2 receptors on sensory neurones Nociceptive P2X receptors The amino acid sequence of a novel P2X purinoceptor subtype (P2X3) has been described.39 88 This receptor is present in a subset of dorsal root ganglion (DRG) sensory neurones that express peripherin, a cytoskeletal protein associated with small-diameter sensory neurones.39 A high proportion of these small-diameter neurones are nociceptive. These results raise the possibility that ATP might play an important role in nociceptor activation (Fig. 1). A molecular mechanism for the pain-inducing actions of ATP has been suggested by studies of rat sensory neurones in culture. Electrophysiological analysis has shown that between 40% and 96% of DRG sensory neurones in culture respond to ATP by increasing intracellular free calcium concentrations ([Ca2⫹]i) or by depolarizing.5 15 115 116 P2X16 mRNA transcripts are expressed in sensory neurones of the dorsal root, nodose and trigeminal ganglia.108 However, only one subtype, P2X3, is expressed selectively in cell populations enriched in nociceptors, as judged by in situ hybridization analysis with markers for small-diameter neurones. This demonstrates that other P2X purinoceptors probably account for the depolarizing actions of ATP on large-diameter neurones. The level of expression of mRNA detected by in situ hybridization and Northern blots suggests that P2X3 is present in greater amounts than other P2X subunits, such as P2X2, in small-diameter sensory neurones.

Fig 1 Hypothetical schematic of the roles of purine nucleotides and nucleosides in pain pathways. P2X3 has been identified as the principal P2X receptor present at sensory nerve terminals in the periphery. P2X3 receptors are also found in sensory ganglia where they sometimes form heteromultimers with P2X2 receptors. Other known P2X receptor subtypes are also expressed at low levels in DRG. Although less potent than ATP, adenosine (AD) also appears to act on sensory terminals, probably directly via P1 (A2) receptors; however, it may also act (broken line) to potentiate P2X2/3 activation. At synapses in sensory pathways in the CNS, ATP appears to act postsynaptically via P2X2, P2X4 or P2X6 receptor subtypes, or two or more of these subtypes, and after breakdown to adenosine, it acts as a prejunctional inhibitor of transmission via P1 (A1) receptors. Sources of ATP acting on P2X2/3 sensory terminals include sympathetic nerves and endothelial, Merkel and tumour cells. White dots, molecules of ATP; grey dots, molecules of adenosine. (From Burnstock and Wood, 1996.38)

An electrophysiological analysis of the properties of the expressed P2X3 channel showed many similarities with currents in rat sensory neurones in culture.116 Thus the channel rapidly desensitized, and was activated by ATP congeners with the same rank order of potency as that described for sensory neurones in culture (at low concentrations, 2-methylthio ATP ⬎⬎ ATP ⬎ α,β-methylene ATP ⬎ γ-thioATP ⬎ CTP ⬎ ADP ⬎⬎ UTP µ β,γ-methylene ATP ⬎ GTP).39 In addition, the channel is blocked by suramin, a general antagonist of P2X purinoceptors (with the exception of P2X4 and P2X6). However, it is not clear

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Fig 2 Effect of extracellular pH on sensitivity to ATP. (A) In Xenopus oocytes expressing the P2X2 receptor, the activity of successive superfusion of ATP (3 µM) was increased by acidifying the bathing solution from pH 7.4 to 6.2, but decreased by alkanization from pH 7.4 to 8.0 (inward current, VH ⫽ –30 mV). (B) Acid pH increases membrane depolarization and action potential firing elicited by ATP. Records are voltage responses to ATP (5 µM) at pH 7.4 and 6.5 in a single neurone from rat nodose ganglion with a resting membrane potential of –54 mV in current-clamp configuration. Traces are sequential (from left to right). Action potential amplitude was attenuated by the frequency-response of the pen recorder (100 Hz). ((A) From King and colleagues, 1996;82 (B) from Li and colleagues, 1996.89)

if P2X3 exists as a homomultimer or a heteromultimer with P2X2 in sensory neurones in vivo. In a recent study of rat nodose ganglion neurones using 2⬘,3⬘-O-trinitrophenyl ATP (TNP-ATP) as a selective P2X antagonist, it was concluded that some neurones used homomeric P2X2 receptors, whereas others used heteromeric P2X2/3 receptors.129 136 137 Evidence has been presented that the capsaicin-sensitive, small DRG neurones of the rat express mainly the homomultimeric P2X3 subunit, while the capsaicininsensitive, medium-sized neurones express the heteromultimeric P2X2/3 receptor.133 Indirect evidence for heteromultimeric channels in sensory neurones has been provided by co-expression of P2X subunits in Xenopus oocytes. Some nodose ganglion neurones desensitize slowly in response to α,β-methylene ATP, but P2X3 shows a rapid desensitization in oocytes. However, co-expression of P2X2 and P2X3 produces a channel that has a slowly desensitizing response.88 This confirms that the heteromultimeric channels are able to form, and may account for, some slowly desensitizing responses in subsets of sensory neurones. It seems likely, as discussed above, that P2X3 receptors form heteromultimeric combinations with P2X2 receptors. Thus, while P2X3 receptors are not sensitive to pH, recombinant P2X2 receptors are strongly pH-sensitive82 91 142 (Fig. 2), suggesting that sensitivity to nociceptive P2 receptors is enhanced in inflammatory conditions with slow acidosis. Acid pH has also been shown to augment the excitatory actions of ATP on dissociated mammalian sensory neurones.89 In contrast, physiological concentrations of extracellular Mg2⫹ inhibit ATP-activated current in rat nodose ganglion cells.90 A more recent study of dissociated

neurones from 1- to 4-day old rat DRG showed that the relative actions of various ATP analogues and, in particular, the very low activity of β,γ-methylene-L-ATP confirmed the presence of P2X3 receptors.115 Synergistic interactions between ATP and other known nociceptive agents on sensory terminals in the periphery have been proposed.64 72 73 142 The ability of P2X3 receptors to be activated by repetitive concentrations of ATP without desensitization is greatly enhanced by the presence of Ca2⫹.44 This enhancement occurs if Ca2⫹ is applied early or late in the interval between ATP applications and this ‘memory’ for elevated Ca2⫹ persists for at least 4 min. Immunohistochemical studies of P2X3 receptors on sensory ganglia at both light microscope11 16 138–140 143 and electron microscope level94 show that: (1) P2X3 receptors are predominantly located in the nonpeptidergic subpopulation of small nociceptive neurones that label with the lectin IB4;16 138–140 143 (2) in trigeminal ganglia, P2X3 receptor immunoreactivity occurs in both small and large nerve cell bodies and their processes; (3) P2X3 receptors are expressed in approximately equal numbers of sensory neurones projecting to the skin and viscera (Fig. 3), but in very few of those innervating skeletal muscle;16 (4) the central projections of P2X5-labelled nerves in the DRG are located in inner lamina II of the dorsal horn of the spinal cord;16 94 140 (5) for the labelled nerve profiles in lamina II, P2X3 receptors are located largely in terminals that have the ultrastructural characteristics of sensory afferent terminals (Fig. 4), suggesting that ATP is released on to primary afferent terminals, thereby modulating sensory input coming from the periphery; 94 (6) in the nucleus solitarius, P2X3 receptor-positive boutons synapse on dendrites and cell bodies and have complex synaptic relationship with other axon terminals and dendrites;94 (7) the peripheral projections of nociceptive neurones in the skin, tongue and tooth pulp are immunopositive for P2X3 receptors;11 (8) after sciatic nerve axotomy, P2X3 receptor expression decreased by more than 50% in L4/5 DRG (Fig. 5). Glial cell-derived neurotrophic factor (GDNF), delivered intrathecally, completely reversed axotomyinduced down-regulation of the P2X3 receptor (Fig. 6).16 In contrast, the P2X3 receptor was transiently upregulated and anterogradely transported in trigeminal primary sensory nerves after nerve injury.56 In vivo pain models. In vivo studies of the functional consequences of P2X receptor activation of peripheral neurones in animal models are beginning to appear. Behavioural indices of acute nociception were monitored in the conscious rat after subplantar injection of ATP and α,βmethylene ATP into the hindpaw.8 Signs of overt nocicep-

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that are recruited by the inflammatory process. In another study, using the formalin and writhing tests in adult male albino mice, systemically administered ATP and ADP, which are rapidly degraded to adenosine, caused a reduction in the number of writhes and the time of licking the formalin-injected paw.102 However, P1, but not P2 antagonists reversed these reactions, so it was concluded that the antinociceptive effects of adenosine nucleotides were mediated by adenosine. An early study using the ‘hotplate’ test claimed that ATP produced analgesia.62

Mechanosensitive P2 receptors

Fig 3 Size distribution of cutaneous (A) and visceral (B) afferents (hatched bars), labelled by fluoro-gold application to the saphenous and pelvic nerves, respectively. The size distribution of fluoro-gold-labelled cells immunoreactive for the P2X3 receptor is shown by the open bars. (From Bradbury and colleagues, 1998.16)

tion, namely hindpaw lifting and licking, were apparent after injection of α,β-methylene ATP; these effects were dose-related and inhibited by selective desensitization of the P2X3 receptor. The results of another study using this model suggested that endogenous ATP in inflamed skin is more likely to reach a concentration capable of exciting nociceptors than in normal skin.67 ATP and α,β-methylene ATP were applied to the peripheral terminals of primary afferent articular nociceptors in rat knee joints and neural activity was recorded from the medial articular nerve in rats anaesthetized with pentobarbitone.50 Rapid, short-lasting excitation of a subpopulation of C and Aδ nociceptive afferents was evoked, which was antagonized by PPADS. Experiments have also been carried out using the formalin rat-paw model, which has two distinct components: an initial phase which reflects a direct sensory nerve activation, and a later phase which may reflect an inflammatory component.130 The P2 antagonists, suramin, Evans blue, Trypan blue and Reactive blue 2, produced antinociception in this model when applied intrathecally.51 Sawynok and Reid120 concluded that their results provided evidence in support of a P2X receptor-mediated augmentation of the pain signal and that the delayed time-course of the effects suggested that it may occur in concert with other mediators

In a study of rat sensory neurones in culture, nociceptive (tooth-pulp afferent) and non-nociceptive (muscle stretch receptors) sensory neurones were compared (Fig. 7).43 Low concentrations of ATP evoked action potentials and large inward currents in both types of neurone. Nociceptors had currents that were similar to those of heterologously expressed channels containing P2X3 subunits and had P2X3 immunoreactivity in their cell bodies and sensory endings. Stretch receptors had currents that differ from those of P2X3 channels and had no P2X3 immunoreactivity. A single cRNA derived from sensory neurones, which renders Xenopus oocytes mechanosensitive, was found to encode a P2Y1 receptor.105 P2Y1 mRNA is concentrated in large-fibre DRG neurones, in contrast to P2X2/3 mRNA, which is found in small-fibre sensory neurones and produces less mechanosensitivity in oocytes. The frequency of touchinduced action potentials from frog sensory nerve fibres was increased by the presence of P2 receptor agonists at the peripheral nerve endings and was decreased by antagonists. The authors postulate that release of ATP into the extracellular space activates peripheral P2Y1 receptors participating in the generation of sensory action potentials by light touch. In another recent study, ATP has been shown to induce Ca2⫹ release from IP3-sensitive Ca2⫹ stores exclusively in large DRG neurones.126 The ATP-triggered increase in the intracellular concentration of Ca2⫹ was not mimicked by adenosine and was blocked by suramin (Fig. 8) and the authors concluded that large (proprioceptive) DRG neurones express metabotropic P2Y receptors. In a later publication,127 this group concluded that Ca2⫹-impermeable ionotropic P2X receptors are present in subpopulations of mouse DRG neurones with large somatas (30–45 µm diameter) as well as metabotropic P2Y receptors.

Cardiorespiratory reflexes Many years ago, functional studies by Emmelin and Feldberg55 showed that intravenously administered ATP slowed the cat heart via reflex actions involving vagal afferent and efferent nerve fibres. ATP also acts on canine pulmonary vagal C-fibre nerve terminals112 and on sensory terminals in the heart setting up vagal reflexes in both dog and humans.113 More recently, P2X receptors associated with vagal afferent nerves have been shown to evoke a Bezold–Jarisch

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Fig 4 P2X3 receptor immunoreactivity in the dorsal horn of the rat spinal cord (see also Fig. 5B). (A, B) A band of P2X3 receptor immunoreactive terminals occurs in lamina II of the dorsal horn at the levels of both cervical (A) and lumbar (B) enlargements. Scale bars: 50 µm. (C) A P2X3 receptor immunoreactive scalloped terminal in the cervical dorsal horn forms synapses (arrowheads) on three dendrites. Scale bar: 500 nm. (D) A P2X3 receptor immunoreactive scalloped terminal in the cervical dorsal horn is presynaptic (arrowheads) to two dendrites and postsynaptic to two nerve processes (1, 2) that contain vesicles. Scale bar: 500 nm. (From Llewellyn-Smith and Burnstock, 1998.94)

depressor reflex in anaesthetized rats.100 Injection of α,βmethylene ATP evoked this reflex consisting of bradycardia, hypotension and apnoea, often preceded by hyperventilation. Suramin and PPADS antagonized the bradycardia and apnoeic components of the reflex, but not the vasopressor or hyperventilatory responses. P2 receptor agonism activated carotid chemoreceptor afferents (also described by Jarisch and colleagues75 and McQueen and Ribeiro101), although this effect was unchanged by suramin and PPADS.

P2X receptors in CNS pain pathways Spinal cord. The subcellular distribution of P2X purinoceptors is of particular interest with respect to a possible presynaptic role in the dorsal horn of the spinal cord. Evidence exists for purinergic transmission from nociceptors in the dorsal horn,74 and it is possible that particular receptor subtypes play a specialized presynaptic role distinct from neuronal depolarization. There have been strong early indications that ATP acts as a neurotransmitter or neuromodulator in the spinal cord dorsal horn.60 74 92 117 118

However, it is only recently that this has been clearly demonstrated.2 Using transverse spinal cord slices from postnatal rats, excitatory postsynaptic currents have been shown to be mediated by P2X receptors, activated by synaptically released ATP, in a subpopulation of less than 5% of the neurones in lamina II, a region known to receive major input from nociceptive primary afferents. The P2X receptors on acutely dissociated dorsal horn neurones are non-desensitizing and are insensitive to α,β-methylene ATP and responses are antagonized by suramin and PPADS. This profile suggests they are of the P2X2 receptor subtype or perhaps in heteromultimeric combination with P2X4 or P2X6. P2X3 receptors appear to be largely located prejunctionally on sensory nerve terminals in lamina II (Fig. 4),94 where there is evidence that they might modulate glutamate release.65 66 70 93 Approximately half of the cultured spinal dorsal horn neurones were shown in a recent study76 to use ATP as a fast excitatory neurotransmitter acting at ionotropic P2X postsynaptic receptors. ATP was not codetected with glutamate, but was coreleased with the

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Fig 6 Histogram of the percentage of P2X3 receptor-immunopositive cell profiles in the L4 and L5 of rat DRG under the following conditions: normal naive animals (n ⫽ 4), ipsilateral and contralateral to a sciatic nerve axotomy (n ⫽ 7), and ipsilateral and contralateral to a sciatic nerve axotomy but in rats treated with intrathecal glial-derived neurotrophic factor (GDNF, n ⫽ 8). The effects of axotomy are highly significant (P⬍0.001), while none of the other groups differ significantly from naive animals. (From Bradbury and colleagues, 1998.16)

message processing in the dorsal horn during mechanical hyperalgesia and neuropathic pain. Brain. There is evidence from our laboratory of immunolocalization of P2X3 receptors in the nucleus tractus solitarius.94 P2X receptors have also been identified in rat trigeminal mesencephalic nucleus neurones, perhaps involved in processing proprioceptive information.78

Sources of ATP involved in pain transmission

Fig 5 (A) P2X3 receptor immunoreactivity in normal adult L5 DRG. Note that many small, but not large cells are immunopositive. (B) Immunoreactivity seen in the lumbar enlargement of the normal spinal cord. Specific P2X3 receptor staining is present as a band across the entire width of lamina IIi of the dorsal horn and in fibres running in Lissauer’s tract. (C) The loss of P2X3 receptor immunoreactivity in the dorsal horn 2 weeks after rhizotomy of the L4 and L5 dorsal roots is shown. In B and C, the tyramide amplification technique has produced some nonspecific staining on the edges of the spinal cord and dorsal roots. Scale bar: 100 µm. (From Bradbury and colleagues, 1998.16)

inhibitory neurotransmitter, GABA. It was speculated that differential modulation of the ATP and GABA components of cotransmission may help to explain changes in sensory

It has been proposed that ATP released from different cell types is involved in the initiation of pain by acting on nociceptive purinoceptors on sensory nerve terminals (Fig. 9).27 31 In causalgia, reflex sympathetic dystrophy or ‘sympathetically maintained pain’ there appears to be sympathetic hyperinnervation (or hyperactivity), although the precise mechanisms involved are much debated.99 122 It has been proposed that in these pathological conditions the ATP released from sympathetic nerves (as a cotransmitter with norepinephrine and neuropeptide Y)24 acts on purinergic nociceptive sensory nerve endings, contributing to the initiation of pain.27 The reports that surgical sympathectomy, sympathetic ganglion blockers and guanethidine (which prevents release of sympathetic cotransmitters) are more effective than adrenoceptor antagonists or reserpine (which depletes norepinephrine, but not ATP, from sympathetic nerve terminals) in preventing pain in these conditions are consistent with this hypothesis.14 68 144 Sympathetic nerve activity appears to be involved with other painful conditions, including the inflammation associated with arthritis.87 As ATP induces prostaglandin synthesis (and prostaglandins are known to be mediators of inflammation), it has been suggested that ATP may play a further, indirect, role as a sympathetic cotransmitter in the generation of pain.27 Merkel cells, which contain high

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Fig 8 ATP-induced [Ca2⫹]i transients in large dorsal root ganglion neurones from 2- to 3-month old mice are mediated through P2Y metabotropic receptors. The ATP-induced [Ca2⫹]i transients (measured with the fluorescent dye Indo-1) are inhibited by suramin. ATP was applied in Ca2⫹-free solution and the cell was incubated with 200 µM suramin for 5 min before administration of ATP. The response to ATP partially recovered after washout of suramin. (From Svichar and colleagues, 1997.126)

Fig 7 ATP (10 µM, 1 s) excited both nociceptive (tooth-pulp afferent) and non-nociceptive (muscle-stretch receptor) sensory neurones of the rat. (A) Corresponding phase micrograph, and (B) fluorescence micrograph (using fluorescent dye DiIC18) of a nociceptor in primary culture. (C) ATP applied by puffer pipette to a labelled nociceptor triggered action potentials with diminishing frequency. (D) Response 1 min later; eight of twelve nociceptors had this adapting response; two others did not adapt and two responded to ATP with just a single action potential. (E) Phase micrograph and (F) fluorescence micrograph of a nociceptor, and recording and application pipettes. ATP application to neurites was visualized by the inclusion of fluorescent dye; bath solution flowed rightward. (G) Response to neuritic application. (H) Response to somal application included a sustained depolarization (arrow). (I) Phase and (J) fluorescence micrographs of a labelled muscle-stretch receptor. (K) Action potentials of stretch receptors did not adapt. (L) Response 1 min later; sampling rate (1 ms) was insufficient to capture all action potential peaks in K and L. In C and K ATP application was 5 min after ATP. Scale bars: 100 µm. (From Cook and colleagues, 1997.43)

concentrations of ATP, are closely associated with sensory nerve endings in the skin and may be another source of ATP involved in the maintenance of pain in reflex sympathetic dystrophy.97 Vascular pain—including angina, ischaemic muscle pain, migraine, lumbar pain and pelvic pain in women—appears to occur during the reactive hyperaemic phase that follows local vasospasm. In 1981, it was proposed that ATP may play a role in mediating these effects in migraine.22 During reactive hyperaemia, large amounts of ATP are released from vascular endothelial cells that act on endothelial P2Y receptors, resulting in the release of nitric oxide and vasodilatation.23 Burnstock27 has proposed that in the microcirculation, ATP diffuses from the endothelial cells to activate nociceptive endings of sensory nerve fibres in the adventitia. A central role for adenosine has also been considered in relation to anginal pain.46 128 ATP released from platelets during aggregation, which has been reported to increase in migraine, may also contribute to the initiation of pain via nociceptive purinoceptors. Consistent with this hypothesis is the recent report that nociception from blood vessels is independent of the sympathetic nervous system under physiological conditions in humans.79 Tumour cells are known to contain exceptionally high concentrations of ATP.96 124 It has been suggested that when a tumour reaches a size that leads to breakage of cells during abrasive movements, the ATP released acts on nociceptive endings of sensory nerves in the vicinity, resulting in the sensation of pain.27 ATP released from damaged muscle after major accidents or surgery could also be involved in local pain and in pain associated with traumatic shock.131 It has recently been proposed31 that in tubes (including

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Fig 9 Schematic drawing of the sources of ATP acting on P2X3 nociceptive receptors on sensory nerve terminals. (A) ATP released as a cotransmitter with norepinephrine (NA) and neuropeptide Y (NPY) from sympathetic nerve terminal varicosities. (B) ATP released from vascular endothelial cells of microvessels during reactive hyperaemia after local vasospasm to act both on P2X3 receptors on sensory nerve terminals and P2Y receptors on endothelial cells leading to release of nitric oxide (NO) and subsequent vasodilatation. Note that acetylcholine (ACh) and substance P (SP) are also stored and released from endothelial cells during reactive hyperaemia. (C) ATP released from tumour cells damage during abrasive activity. (From Burnstock, 1996.27)

ureter, salivary duct, bile duct, vagina and intestine) and sacs (including urinary bladder, gall bladder and lung) nociceptive mechanosensory transduction occurs where distension releases ATP from the epithelial cells lining these organs, which then activates P2X2/3 receptors on subepithelial sensory nerve plexuses to relay messages to the CNS pain centres. Supporting evidence for this concept comes from studies of the rat bladder where ATP has been shown to be released from the urothelial cells by hydrostatic pressure changes,57 and where nerve discharges were produced in pelvic nerve afferent from the bladder during slow distension and infusion of α,β-methylene ATP, which were antagonized by suramin.104 There is also a report presenting evidence for the presence of P2X receptors on intestinal afferent nerve endings.83

Future directions The search for selective antagonists at the P2X3 receptor can be pursued now that the molecular structure of the nociceptor- associated P2X3 receptor has been identified. Such antagonists could then be tested both on both recombinant receptors expressed in oocytes or transfected cells and in vivo models of different types of pain. Since P2X3 receptors are clearly not the only receptors involved in pain, the possibility of synergy with receptors for other

agents that modulate pain (such as bradykinin, histamine and 5-hydroxytryptamine) should be explored. In addition to the description in this review of ATP acting on receptors on sensory nerve terminals, there are reports that ATP acts on dorsal horn neurones in the spinal cord after being released from a subpopulation of small primary afferent nerves involved in pain pathways.119 The future identification of a selective A1 agonist to attenuate pain at the spinal cord level and of an A2 antagonist at the sensory terminal level are also of obvious clinical interest.121 123 Interactions with the recently identified acid sensory ion channel localized on small primary afferent neurones111 will be of much interest. Further intriguing parallels that need to be explored concern capsaicin-gated channels and P2X nociceptive receptors, including the mechanism of desensitization through calcineuron action,49 80 the pronounced pH-dependence of channel gating as well as the distribution and expression of capsaicin sensitivity at the P2X3 receptor, and the ion selectivity of the channel.85

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