Current Pharmaceutical Design, 2009, 15, 000-000 1 Purinergic Receptors and Pain

Geoffrey Burnstock*

Autonomic Neuroscience Centre, Royal Free and University College Medical School, Rowland Hill Street, London NW3 2PF, UK

Abstract: There is a brief summary of the early background literature about and its involvement in pain, of ATP storage, release and ectoenzymatic breakdown and of the current classification of subtypes for and . The review then focuses on purinergic mechanosensory transduction involved in visceral, cuta- neous and musculoskeletal nociception and on the roles played by P2X3, P2Y2/3, P2X4, P2X7 and receptors in neu- ropathic and inflammatory pain. Current developments of compounds for the therapeutic treatment of both visceral and neuropathic pain are discussed. Key Words: ATP, bladder, gut, inflammatory, joints, mechanosensory transduction, neuropathic.

A. INTRODUCTION reaches P2X3 receptors on nociceptive sensory nerves. This has been followed by an increasing number of papers ex- The concept of purinergic signalling was proposed in panding on this concept (see [18-20]) and also on the in- 1972 [1] following hints in the early literature, notably by volvement of [21]. Immunohistochemical studies Drury and Szent-Györgyi [2], Buchthal and Folkow [3] and showed that the nociceptive fibres expressing P2X receptors Emmelin and Feldberg [4] about the potent extracellular ac- 3 arose largely from the population of small neurons that la- tions of purines in the heart and peripheral ganglia and from belled with the lectin IB [22, 23]. The decreased sensitivity Pamela Holton [5] about the release of adenosine 5’- 4 to noxious stimuli associated with the loss of IB -binding triphosphate (ATP) during antidromic stimulation of sensory 4 neurons expressing P2X receptors indicates that these sen- nerve collaterals, together with the evidence that ATP and is 3 sory neurons are essential for the signaling of acute pain the transmitter in non-adrenergic, non-cholinergic nerves [24]. The central projections of these primary afferent neu- supplying the gut [6] and bladder [7]. It is now recognised rons were shown to be in inner lamina II of the dorsal horn that ATP is a cotransmitter in nerves in both peripheral and and peripheral projections demonstrated to skin, tooth pulp, central nervous systems (see [8]) and that receptors for tongue and subepithelial regions of visceral organs. A sche- purines and pyrimidines are widely expressed on non- matic illustrating the initiation of nociception on primary neuronal as well as nerve cells (see [9]). afferent fibres in the periphery and purinergic relay pathways There were early hints that ATP might be involved in in the spinal cord was presented by Burnstock and Wood pain including the demonstration of pain produced by injec- [25] (Fig. 1). While P2X3 and P2X2/3 receptors, expressed in tion of ATP into human skin blisters [10, 11], ATP involve- sensory neurons, were the predominant P2 receptor subtypes ment in migraine [12] and ATP participation in pain path- first recognized to be involved in the initiation of nocicep- ways in the spinal cord [13, 14]. A significant advance was tion, it has become apparent that P2Y receptors are also pre- made when the P2X3 ionotropic ion channel purinergic re- sent [26, 27] and that these are involved in modulation of ceptor was cloned in 1995 and shown to be localized pre- pain transmission [28]. P2Y receptors appear to potentiate dominantly on small nociceptive sensory neurons in dorsal pain induced by chemical or physical stimuli via capsaicin- root ganglia (DRG) together with P2X2/3 heteromultimer sensitive, transient receptor potential vanilloid 1 (TRPV1) receptors [15, 16]. Later, Burnstock [17] put forward a unify- channels, and it has been proposed that the functional inter- ing purinergic hypothesis for the initiation of pain, suggest- action between P2Y2 receptors and TRPV1 channels in noci- ing that ATP released as a cotransmitter with noradrenaline ceptors could underlie ATP induced inflammatory pain [29]. and neuropeptide Y from sympathetic nerve terminal vari- ATP-induced hyperalgesia was abolished in mice lacking cosities might be involved in sympathetic pain (causalgia TRPV1 receptors. and reflex sympathetic dystrophy); that ATP released from vascular endothelial cells of microvessels during reactive B. ATP STORAGE, RELEASE AND BREAKDOWN hyperaemia is associated with pain in migraine, angina and The cytoplasm of most neurons contains ~2–5mM ATP, ischemia; and that ATP released from tumor cells (which and higher concentrations of ATP (up to 100 mM) are stored contain very high levels), damaged during abrasive activity, in synaptic vesicles. Synaptic vesicles also contain other

such as (ADP), adenosine *Address correspondence to this author at the Autonomic Neuroscience monophosphate (AMP), diadenosine tetra- and penta- Centre, Royal Free and University College Medical School, Rowland Hill phosphate, and triphosphate, but at lower concen- Street, London NW3 2PF, UK; Tel: +44 20 7830 2948; Fax: +44 20 7830 trations (see [30, 31]). 2949; E-mail: [email protected]

1381-6128/09 $55.00+.00 © 2009 Bentham Science Publishers Ltd. 2 Current Pharmaceutical Design, 2009, Vol. 15, No. 00 Geoffrey Burnstock

Fig. (1). Hypothetical schematic of the roles of nucleotides and in pain pathways. At sensory nerve terminals in the pe- riphery, P2X3 and P2X2/3 receptors have been identified as the principal P2X purinoceptors present, although recent studies have also shown expression of P2Y1 and possibly P2Y2 receptors on a subpopulation of P2X3 receptor-immunopositive fibres. Other known P2X purinoceptor subtypes (1-7) are also expressed at low levels in dorsal root ganglia. Although less potent than ATP, adenosine (AD) also appears to act on sensory terminals, probably directly via P1(A2) purinoceptors; however, it also acts synergistically (broken red line) to potentiate P2X2/3 receptor activation, which also may be true for 5-hydroxytryptamine, capsaicin and protons. At synapses in sensory pathways in the CNS, ATP appears to act postsynaptically via P2X2, P2X4 and/or P2X6 purinoceptor subtypes, perhaps as heteromultimers, and after breakdown to adenosine, it acts as a prejunctional inhibitor of transmission via P1(A2) purinoceptors. P2X3 receptors on the central projections of primary afferent neurons in lamina II of the dorsal horn mediate facilitation of glutamate and probably also ATP release. Sources of ATP acting on P2X3 and P2X2/3 receptors on sensory terminals include sympathetic nerves, endothelial, Merkel and tumor cells. Yellow dots, molecules of ATP; red dots, molecules of adenosine. (Modified from [25] and reproduced with permission of Elsevier).

ATP is released from sensory nerve collatorals [5], from proposed, including ATP-binding cassette transporters, con- exercising human forearm muscle [32], from non-adrenergic, nexin or pannexin hemichannels, plasmalemmal voltage- non-cholinergic nerves [6] and from the perfused heart dur- dependent anion channels and P2X7 receptors, as well as ing coronary vasodilation in response to hypoxia [33]. How- vesicular release [36, 43]. ATP released from nerves, or by ever, until recently, apart from vesicular release from nerves autocrine and paracrine mechanisms from nonneuronal cells, (e.g. [34]), it was usually assumed that the only source of is involved in a wide spectrum of physiological and patho- extracellular ATP acting on purinoceptors was damaged or physiological activities, including synaptic transmission and dying cells, but it is now recognized that ATP release from modulation, pain and touch perception. Local probes for healthy cells is a physiological mechanism (see [35, 36]). real-time measurement of ATP release in biological tissues ATP is released from many non-neuronal cell types during have been developed recently [44, 45]. mechanical deformation in response to shear stress, stretch After release, ATP and other nucleotides undergo rapid or osmotic swelling, as well as hypoxia and stimulation by enzymatic degradation, which is functionally important since various agents. The precise transport mechanism(s) involved ATP metabolites act as physiological ligands for a wide ar- in ATP release are currently being examined. There is com- ray of purinergic receptors [46]. Availability of these ligands pelling evidence for exocytotic vesicular release of ATP is controlled and modulated by . Ectonu- from nerves and also from endothelial cells [37], urothelial cleotidase families include the E-NTPDases (ecto- cells [38], osteoblasts [39], fibroblasts [40] and astrocytes triphosphate diphosphohydrolases), E-NPP (ecto- [41]. There is increased release of ATP from endothelial pyrophosphatase/ phosphodiesterases), alkaline phosphatases cells during acute inflammation [42]. In addition, various and ecto-5’-nucleotidase. Individual differ in sub- ATP transport mechanisms in non-neuronal cells have been strate specificity and product formation. E-NTPDases and E- Purinergic Receptors and Pain Current Pharmaceutical Design, 2009, Vol. 15, No. 00 3

NPPs hydrolyse ATP and ADP to AMP that is further hydro- P2Y Receptors lysed to adenosine by ecto-nucleotidase. Alkaline phospha- Metabotropic P2Y receptors (P2Y , P2Y , P2Y , P2Y , tases equally hydrolyse nucleoside tri-, di- and monophos- 1 2 4 6 + P2Y , P2Y , P2Y and P2Y ) are characterized by a phates. Dinucleoside polyphosphates, NAD and 11 12 13 14 subunit topology of an extracellular NH terminus and intra- diphosphate (UDP) sugars are substrates solely for E-NPPs. 2 Besides the catabolic pathways, nucleotide interconverting cellular COOH terminus and seven TM-spanning regions (see [60]). An additional ‘hybrid’ nucleotide-respon- enzymes exist for nucleotide rephosphorylation and extracel- sive receptor activated by cysteinyl-leukotrienes has been lular synthesis of ATP (ecto-nucleoside diphosphate kinase, reported [61]. There is structural diversity of intracellular adenylate kinase). It is possible that, while adenosine is loops and the COOH terminus among P2Y subtypes, so in- largely produced by ectoenzymatic breakdown of ATP, there fluencing the degree of coupling with G , G , G and G may be subpopulations of neurons and/or astrocytes that re- q/11 s i i/o lease adenosine directly [47]. proteins. Some P2Y receptors are activated principally by nucleoside diphosphates (P2Y1,12,13), while others are acti- C. RECEPTOR SUBTYPES FOR PURINES AND vated by both purine and nucleotides (P2Y2,4,6). PYRIMIDINES In response to nucleotide activation, recombinant P2Y recep- tors either activate phospholipase C and release intracellular A basis for distinguishing two types of purinoceptor, or affect adenylyl cyclase and alter cyclic AMP lev- identified as P1 and P2 (for adenosine and ATP/ADP, re- els. P2Y G protein-coupled receptors in neurons have also spectively), was proposed in 1978 [48], but it was not until been found to modulate the activity of voltage-gated ion 1985 that a proposal suggesting a pharmacological basis for channels in the cell membrane. For example, 2+ distinguishing two types of P2 receptor (P2X and P2Y) was subtypes that act via Gi/o proteins can involve N-type Ca made [49]. Abbracchio and Burnstock [50], on the basis of channels, while the M-current K- channel can be inhibited studies of transduction mechanisms and the cloning of nu- through the activation of Gq/11-linked P2Y receptor subtypes. cleotide receptors, proposed that purinoceptors should be- 2-Methylthio ADP (2-MeSADP) is a potent agonist of ma- 6 long to two major families: a P2X family of ligand-gated ion mmalian P2Y1 receptors and N -methyl-2’-deoxyadenosine channel receptors and a P2Y family of G protein-coupled 3’,5’-bisphosphate (MRS2179) and MRS2500 have been receptors. Currently seven P2X subunits and eight P2Y re- identified as selective antagonists. At P2Y2 and P2Y4 recep- ceptor subtypes are recognized, including receptors that are tors in the rat, ATP and uridine 5’-triphosphate (UTP) are sensitive to pyrimidines as well as purines [51, 52]. Recep- equipotent, but the two receptors can be distinguished with tors for diadenosine polyphosphates have been described on antagonists. MRS2578 appears to be a selective antagonist at C6 glioma cells and presynaptic terminals in rat midbrain, P2Y6 receptors and there are a number of selective antago- although they have yet to be cloned. nists to the P2Y12 receptor (see Table 1). It has been sug- gested that P2Y receptors can be subdivided into two sub- P1 Receptors groups, namely, one that includes P2Y1,2,4,6,11, the other in- Four subtypes of P1 receptors have been cloned, namely, cludes P2Y12,13,14, largely on the basis of structural and phylogenetic criteria [60]. A1, A2A, A2B, and A3 (see [53]). All P1 adenosine receptors couple to G proteins and, in common with other G protein- Heteromultimeric Receptors coupled receptors, they have seven putative transmembrane (TM) domains; the NH2 terminus of the protein lies on the The pharmacology of purinergic signalling is compli- extracellular side, and the COOH terminus lies on the cyto- cated because P2X receptor subunits can combine to form plasmic side of the membrane. It is the residues within the either homomultimers or heteromultimers (see [52, 55, 62]). TM regions that are crucial for ligand binding and specific- Heteromultimers are clearly established for P2X2/3 receptors ity. Specific agonists and antagonists are available for the P1 in nodose ganglia, P2X4/6 receptors in central nervous system receptor subtypes [54]. (CNS) neurons, P2X1/5 receptors in some blood vessels and P2X2/6 receptors in the brain stem. P2X7 receptors have re- P2X Receptors cently been claimed to form heteromultimers with P2X4 re- P2X receptor subunits show a subunit topology of in- ceptors [63], while P2X6 receptors will not form a functional 1–7 homomultimer without extensive glycosylation. tracellular NH2 and COOH termini, two TM-spanning re- gions (TM1 and TM2), the first involved with channel gating P2Y receptor subtypes can also form heterodimeric com- and the second lining the ion pore, and a large extracellular plexes [64] or with other receptors. For example, adenosine loop, with 10 conserved cysteine residues [55, 56]. The A1 receptors have been shown to form a heteromeric com- stoichiometry of P2X1–7 receptor subunits involves three plex with P2Y1 receptors (see [65]). Dopamine D1 and subunits that form a stretched trimer (see [57]). P2X7 recep- adenosine A1 receptors have also been shown to form func- tors, in addition to small cation channels, upon prolonged tionally interactive heteromeric complexes. exposure to high concentrations of agonist, large channels, or pores are activated that allow the passage of larger molecular D. ATP AND SENSORY NERVES weight molecules [58]. P2X7 receptors are predominantly localized on immune cells and glia, where they mediate Sensory Ganglia proinflammatory cytokine release, cell proliferation, and There have been many reports characterizing the P2X apoptosis. The P2X receptor family shows many pharmacol- receptors in sensory neurons, including those from dorsal ogical and operational differences [59]. root, trigeminal, nodose, and petrosal ganglia. DRG and tri- 4 Current Pharmaceutical Design, 2009, Vol. 15, No. 00 Geoffrey Burnstock

Table 1. Antagonists to P2X3, P2X2/3, P2X7 and P2Y12 Receptors. (Modified and Updated from [8] with Permission from the American Physiological Society)

Antagonist P2X3 P2X2/3 Reference

Suramin and analogues NF449, NF110   8 PPADS and derivatives MRS2159 & MRS2257   8 Reactive blue 2 and derivatives   8 TNP-ATP   189 A-317491 (selective)   190, 193  - 8 Tetramethylpyrazine  ? 192 RO-3 (orally bioavailable, stable)   59

Ip5I  - 8 carboxymethylene ATP ?  8 chlorophosphnomethylene ATP ?  8

P2X7

KN62  59 KN04  59 MRS2427  8 o-ATP  59 Coomassie BBG  59 RN6189 ? 8 AZ11645373 (selective)  251 A-740003 (selective)  217 A-438079 (selective)  220

P2Y12

CT50547  252 AR-C69931MX (selective)  253 INS49266  8 AZD6140  253 PSB0413 ? 8 AR-C66096 (selective)  254 2-MeSAMP  252 Carba-nucleosides  255

Ticks represent qualitative assessment of potencies.

geminal ganglia contain primary somatosensory neurons, positive neurons also stained for the P2Y1 receptor, while receiving nociceptive, mechanical and proprioceptive inputs 25–35% also stained for the P2Y4 receptor. The sensitivity to [8]. All P2X subtypes are found on sensory neurons, al- ATP of satellite cells is increased 500-fold after axotomy or though the P2X3 receptor has the highest level of expression inflammation, which is likely to contribute to chronic pain (both in terms of mRNA and protein) (see [66]). P2X2/3 het- [67]. eromultimers are particularly prominent in the nodose gan- P2X receptors have been identified in retinal ganglion glion. P2X and P2X receptors are expressed on IB - 2 3 2/3 4 cells, particularly within cone pathways. Functional studies binding subpopulations of small nociceptive neurons [23]. have also identified P2X heteromultimeric receptors in RT-PCR showed that P2Y , P2Y , P2Y and P2Y receptor 2/3 1 2 4 6 cultured rat retinal ganglion cells. RT-PCR at the single-cell mRNA is also expressed on neurons of DRG, nodose and level revealed expression of P2X2, P2X3, P2X4 and P2X5 trigeminal ganglia and receptor protein for the P2Y1 receptor receptor mRNA in approximately one-third of the bipolar is localized on over 80% of mostly small neurons [26]. Dou- cells. P2X receptors have also been identified on both inner ble immunolabelling showed that 73–84% of P2X receptor 7 3 and outer retinal ganglion cell layers [68], which may be Purinergic Receptors and Pain Current Pharmaceutical Design, 2009, Vol. 15, No. 00 5 involved in retinal cholinergic neuron density regulation. line (ACh) is the likely mechanism for chemosensory signal- P2X7 receptors are expressed postsynaptically on horizontal ing in the carotid body in vivo [77]. ATP coreleased with cell processes as well as presynaptically on photoreceptor ACh from type I glomus chemoreceptor cells during hypoxic synaptic terminals in both rat and marmoset retinas [69]. and mechanical stimulation was shown to act on P2X2/3 re- P2X3 receptors are present on Müller cells. Müller cells re- ceptors on nerve fibres arising from the petrosal ganglion lease ATP during Ca2+ wave propagation. mediating hypoxic signaling at rat and cat carotid body chemoreceptors [78, 79]. Immunoreactivity for P2X2 and Sensory Nerve Fibres and Terminals P2X3 receptor subunits has been localized on rat carotid body afferents [78]. These findings were confirmed and ex- Sensory nerve terminals express purinoceptors and re- tended in a study where P2X receptor deficiency resulted in spond to ATP in many situations. However, it has been 2 shown that ATP sensitivity is not necessarily restricted to the a dramatic reduction in the responses of the carotid sinus nerve to hypoxia in an in vitro mouse carotid body-sinus terminals; increased axonal excitability to ATP and/or nerve preparation [80]. ATP mimicked the afferent dis- adenosine of unmyelinated fibres in rat vagus, sural and dor- charge, and and pyridoxalphosphate-6-azonphenyl- sal root nerves, as well as human sural nerve has been de- 2’,4’-disulphonic acid (PPADS) blocked the hypoxia-induced scribed (see [8]). discharge. Immunoreactivity for P2X2 and P2X3 receptor In terminals of sensory neurons P2X2/3 and/or P2X3 re- subunits was detected on afferent terminals surrounding ceptors are intimately involved in pain sensation and tem- clusters of glomus cells in wild-type but not in P2X2 and/or perature sensitivity. During purinergic mechanosensory P2X3 receptor-deficient mice. Sensory afferent fibres within transduction (see below), the ATP that acts on P2X3 and the respiratory tract, which are sensitive to ATP, probably P2X2/3 receptors on sensory nerve endings, is released by largely via P2X2/3 receptors, have been implicated in vagal mechanical distortion from urothelial cells during distension reflex activity [81] and in the cough reflex [82]. of bladder and ureter and from mucosal epithelial cells dur- ATP has been shown to be an auditory afferent neuro- ing distension of the colorectum. It is probably also released transmitter, alongside glutamate (see [83]). There are from odontoblasts in tooth pulp, from epithelial cells in the ~50,000 primary afferent neurons in the human cochlear and tongue, epithelial cells in the lung, keratinocytes in the skin about one-half express P2X2 (or P2X2 variants) and, debat- and glomus cells in the carotid body (see [37]). Released + ably, P2X3 receptors. ATP is released from K -depolarized ATP is rapidly broken down by ectoenzymes to ADP (to act 2+ organ of Corti in a Ca -dependent manner, and an increase on P2Y1, P2Y12 and P2Y13 receptors) or adenosine (to act on in ATP levels in the endolymph has been demonstrated dur- P1 receptors). ing noise exposure, perhaps released by exocytosis from the In the gut, ATP and ,-methylene ATP (,-meATP) marginal cells of the stria vascularis [84]. The P2 receptor activate P2X3 and/or P2X2/3 receptors on subepithelial termi- antagonist, PPADS, attenuated the effects of a moderately nals of intrinsic sensory neurons in the guinea pig intestine intense sound on cochlea mechanics [85]. Spiral ganglion [70], supporting the hypothesis of Burnstock [19] that ATP neurons, expressing P2X receptors, located in the cochlear, released from mucosal epithelial cells has a dual action act- convey to the brain stem the acoustic information arising ing on the terminals of low-threshold intrinsic enteric sen- from the mechanoelectrical transduction of the inner hair sory neurons to initiate or modulate intestinal reflexes and cells [86] and are responsive to ATP [87]. acting on the terminals of high-threshold extrinsic sensory Odorant recognition is mediated by P2X , P2X and fibres to initiate pain. Further support comes from the dem- 2 3 P2X2/3 receptors predominantly situated on the microvilli of onstration that peristalsis is impaired in the small intestine of olfactory receptor neurons in the olfactory epithelium and mice lacking the P2X3 subunit [71]. Thirty-two percent of vomeronasal organ [88, 89]. Nucleotides also act on susten- retrogradely labelled cells in the mouse DRG at levels T8-L1 tacular supporting cells. Purinergic receptors appear to play and L6-S1, supplying sensory nerve fibres to the mouse dis- an integral role in signaling acute damage in the olfactory tal colon, were immunoreactive for P2X3 receptors [72]. In- epithelium by airborne pollutants. Damaged cells release traganglionic laminar nerve endings are specialized mecha- ATP, thereby activating purinergic receptors on neighbour- nosensory endings of vagal afferent nerves in the rat stom- ing sustentacular cells, olfactory receptor neurons and basal ach, arising from the nodose ganglion; they express P2X3 cells, initiating a stress-signaling cascade involving heat receptors and are probably involved in physiological reflex shock proteins for neuroprotection [90]. activity, especially in early postnatal development [73]. Taste sensations appear to be mediated both by P2Y1 The ventilatory response to decreased oxygen tension in receptor-activated impulses in sensory fibres in the chorda the arterial blood is initiated by excitation of specialized tympani [91] and by P2X2 and P2X3 and, perhaps, P2X2/3 oxygen-sensitive chemoreceptor cells in the carotid body receptors [92]. These authors showed that genetic elimina- that release neurotransmitter to activate endings of the sinus tion of P2X2 and P2X3 receptors abolished responses of the nerve afferent fibres. ATP and adenosine were shown early taste nerves, although the nerves remained responsive to on to excite nerve endings in the carotid bifurcation [74] and touching, temperature and menthol and reduced responses to subsequently ,-meATP [75]. Large amounts of sweeteners, glutamate and bitter substances. Ectonucleoti- nucleotides are localized in glomus cells, stored within spe- dases are known to be abundantly present in taste buds [93]. cific granules together with catecholamines and proteins. Other papers present data that suggest that P2Y2 and P2Y4 Evidence of ATP release from carotid chemoreceptor cells receptors also play a dominant role in mediating taste cell has been reported [76], and corelease of ATP and acetylcho- responses to ATP and UTP [94].

6 Current Pharmaceutical Design, 2009, Vol. 15, No. 00 Geoffrey Burnstock

E. PURINERGIC MECHANOSENSORY TRANSDUC- than capsaicin-sensitive afferents [97, 107]. ATP has also TION AND PAIN been shown to induce a dose-dependent hypereflexia in con- scious and anesthetized mice, largely via capsaicin-sensitive A hypothesis was proposed that purinergic mechanosen- C-fibres; these effects were dose-dependently inhibited by sory transduction occurred in visceral tubes and sacs, includ- PPADS and 2’,3’-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) ing ureter, bladder and gut, where ATP released from epithe- [108] (Fig. 3a). P2X and P2X receptors play a fundamental lial cells during distension acted on P2X homomeric and 1 3 3 role in the micturition reflex in female urethane-anesthetized P2X heteromeric receptors on subepithelial sensory nerves 2/3 rats; P2X receptor blockade by phenol red raised the pres- initiating impulses in sensory pathways to pain centres in the 3 sure and volume thresholds for the reflex, while P2X recep- CNS [37] (Fig. 2a). Evidence supporting this hypothesis in 1 tor blockade diminished motor activity associated with void- various organs is reviewed below. ing [109]. Urinary Bladder Ureter Early evidence for ATP release from rabbit urinary blad- The uroteric colic that is induced by the passage of a kid- der epithelial cells by hydrostatic pressure changes was pre- ney stone causes severe pain. Immunostaining of P2X re- sented by Ferguson et al. [95], who speculated about this 3 ceptors in sensory nerves in the subepithelial region was being the basis of a sensory mechanism. Prolonged exposure reported [110]. Distension of the ureter resulted in substan- to a desensitizing concentration of -meATP significantly , tial ATP release from the urothelium in a pressure-dependent reduced the activity of mechanosensitive pelvic nerve affer- manner [38]. Cell damage was shown not to occur during ents in an in vitro model of rat urinary bladder [96]. Later, it distension with scanning electron microscopy and, after re- was shown that mice lacking the P2X receptor exhibited 3 moval of the urothelium, there was no ATP release during reduced inflammatory pain and marked urinary bladder hy- distension. Evidence was presented that the release of ATP poreflexia with reduced voiding frequency and increased from urothelial cells was vesicular. Multifibre recordings of voiding volume, suggesting that P2X receptors are involved 3 ureter afferent nerves were made using a guinea pig prepara- in mechanosensory transduction underlying both inflamma- tion perfused in vitro [111]. Distension of the ureter resulted tory pain and physiological voiding reflexes [97]. Subse- in a rapid, followed by maintained, increase in afferent nerve quently, using P2X knockout mice and P2X /P2X double 2 2 3 discharge. The rapid increase was mimicked by intraluminal knockout mice, a role for the P2X subtype was shown to be 2 application of ATP or ,-meATP and TNP-ATP attenuated involved in mediating the sensory effect of ATP [98]. In a these nerve responses to distension; the maintained increase systematic study of purinergic mechanosensory transduction was partly due to adenosine. in the mouse urinary bladder, ATP was shown to be released from urothelial cells during distension, and activity initiated Gut in pelvic sensory nerves was mimicked by ATP and ,- meATP and attenuated by P2X3 antagonists as well as in A hypothesis was proposed suggesting that purinergic P2X3 knockout mice; P2X3 receptors were localized on sub- mechanosensory transduction in the gut initiated both urothelial sensory nerve fibres [99]. It appears that the blad- physiological reflex modulation of peristalsis via intrinsic der sensory DRG neurons, projecting via pelvic nerves, ex- sensory fibres and nociception via extrinsic sensory fibres press predominantly P2X2/3 heteromultimer receptors [100]. [19, 112] (Fig. 2b). Evidence in support of this hypothesis Single unit analysis of sensory fibres in the mouse urinary was obtained from a rat pelvic sensory nerve-colorectal bladder revealed both low- and high-threshold fibres sensi- preparation [113]. Distension of the colorectum led to pres- tive to ATP contributing to physiological (non-nociceptive) sure-dependent increase in release of ATP from mucosal and nociceptive mechanosensory transduction, respectively epithelial cells and also evoked pelvic nerve excitation. This [101]. It was also shown that purinergic agonists increase the excitation was mimicked by application of ATP and ,- excitability of afferent fibres to distension. Botulinum toxin meATP and attenuated by the selective P2X3 and P2X2/3 an- A, which has antinociceptive effects in treating interstitial tagonist TNP-ATP and by PPADS. The sensory discharge cystitis, inhibits distension-mediated urothelial release of was potentiated by ARL-67156, an ATPase inhibitor. Single ATP in conditions of bladder inflammation [102]. The roles fibres analysis showed that high-threshold fibres were par- of ATP released from urothelial cells and suburothelial myo- ticularly affected by ,-meATP. The interactions of ATP fibroblasts on various bladder functions have been consid- with other mediators that activate pelvic afferent fibres in the ered at length in several reviews [103, 104], and evidence rat colorectum, including 5-hydroxytryptamine (5-HT), presented that urothelial-released ATP alters afferent nerve bradykinin, prostaglandins and substance P (SP), have been excitability [105]. described [114]. Lumbar splanchnic (LSN) and sacral pelvic (PN) nerves convey different mechanosensory information ATP given intravesically stimulates the micturition reflex from the colon to the spinal cord. Forty percent of LSN af- in awake freely moving rats, probably by stimulating subu- ferents responded to ,-meATP compared with only 7% of rothelial C-fibres, although other mediators are likely to be PN afferents [115]. involved [106]. Studies of resiniferatoxin desensitization of capsaicin-sensitive afferents on detrusor overactivity induced ATP release and P2X3 and P2X2/3 receptor-mediated no- by intravesicle ATP in conscious rats supported the view that ciceptive sensory nerve responses were enhanced in a model increased extracellular ATP has a role in mechanosensory of colitis [116]. The excitability of visceral afferent nerves is transduction and that ATP-induced facilitation of the mic- enhanced following injury or ischemia and during inflamma- turition reflex is mediated, at least partly, by nerves other tion, for example, in irritable bowel syndrome. Under these Purinergic Receptors and Pain Current Pharmaceutical Design, 2009, Vol. 15, No. 00 7 conditions, substances are released from various sources that Skin often act synergistically to cause sensitization of afferent ATP and -meATP activate nociceptive sensory nerve nerves to mechanical or chemical stimuli. Receptors to these , terminals in the skin, which increase in magnitude in in- substances (including ATP) represent potential targets for flammatory conditions due to increase in number and re- drug treatment aimed at attenuating the inappropriate vis- ceral sensation and subsequent reflex activities that underlie sponsiveness of P2X3 and P2X2/3 receptors [122]. Calcium waves in human epidermal keratinocytes mediated by ex- abnormal bowel function and visceral pain (see [117, 118]). tracellular ATP produce [Ca2+] elevation in DRG neurons, ,-MeATP was shown to stimulate mechanosensitive mu- i suggesting a dynamic cross-talk between skin and sensory cosal and tension receptors in mouse stomach and oesopha- neurons mediated by extracellular ATP [123]. Skin cell gus leading to activity in vagal afferent nerves [119]. The damage caused action potential firing and inward currents in sensitizing effects of P2X3 receptor agonists on mechanosen- sory function are induced in oesophagitis [120]. Purinergic nociceptive fibres, which was eliminated by enzymatic deg- radation of ATP or blockade of P2X receptors, indicating mechanosensory transduction has also been implicated in release of cytosolic ATP [124]. Locally released ATP can reflex control of intestinal secretion, whereby ATP released sensitize large mechanosensitive afferent endings via P2 from mucosal epithelial cells acts on P2Y receptors on en- 1 receptors, leading to increased nociceptive responses to pres- terochromaffin cells to release 5-HT, which leads to regula- sure or touch; it was suggested that such a mechanism, to- tion of secretion either directly or via intrinsic reflex activity [121]. gether with central changes in the dorsal horn, may contrib-

Fig. (2). Purinergic mechanosensory transduction. (a) Schematic representation of hypothesis for purinergic mechanosensory transduction in tubes (e.g. ureter, vagina, salivary and bile ducts, gut) and sacs (e.g. urinary and gall bladders, and lung). It is proposed that distension leads to release of ATP from epithelium lining the tube or sac, which then acts on P2X3 and/or P2X2/3 receptors on subepithelial sensory nerves to convey sensory/nociceptive information to the CNS. (From [37], reproduced with permission from Blackwell Publishing). (b) Schematic of a novel hypothesis about purinergic mechanosensory transduction in the gut. It is proposed that ATP released from mucosal epithelial cells during moderate distension acts preferentially on P2X3 and/or P2X2/3 receptors on low-threshold subepithelial intrinsic sensory nerve fibres (labelled with calbindin) to modulate peristaltic reflexes. ATP released during extreme (colic) distension also acts on P2X3 and/or P2X2/3 receptors on high-threshold extrinsic sensory nerve fibres (labelled with isolectin B4 (IB4)) that send messages via the dorsal root ganglia (DRG) to pain centres in the central nervous system. (From [256], reproduced with permission from Wiley-Liss, Inc.). 8 Current Pharmaceutical Design, 2009, Vol. 15, No. 00 Geoffrey Burnstock ute to touch evoked pain [125]. ATP appears to be involved Lung in fast nociceptive signals, while persistent pain after tissue In the lung, pulmonary neuroepithelial bodies (NEBs) damage involves other algogenic compounds, notably and more recently subepithelial receptor-like endings associ- bradykinin, prostaglandin and serotonin. However, persistent ated with smooth muscle (SMARs) [139] have been shown pain during inflammation may also involve sensitization and/or spread of P2X receptors. Nocifensive behaviours in- to serve as sensory organs in the lung, and P2X3 and P2X2/3 receptors are expressed on a subpopulation of vagal sensory duced by administration of ATP and 2’-3’-O-(4-benzoyl- fibres that supply NEBs and SMARs with their origin in the benzoyl)-ATP to the hindpaw appear to recruit an additional nodose ganglia. Quinacrine staining of NEBs indicates the set of fibres that are not activated by ,-meATP and which presence of high concentrations of ATP in their secretory trigger the spinal release of SP and are capsaicin selective vesicles, and it has been suggested that ATP is released in [126]. response to both mechanical stimulation during high- In a study of the behavioural effects of intraplantar injec- pressure ventilation and during hypoxia [140]. NEBs are tions of ATP in freely moving rats, evidence was presented oxygen sensors especially in early development, before the that ATP was more effective in exciting nociceptors in in- carotid system has matured [141]. In a study of bronchopul- flamed versus normal skin [122]. This was reported to be monary afferent nerve activity of a mouse isolated perfused due to upregulation of P2X2 and P2X3 receptors on DRG nerve-lung preparation, it was found that C fibres could be neurons [127]. Enhanced expression of glial cell-derived subdivided into two groups: fibres that conduct action poten- neurotrophic factor in the skin can change the mechanical tials at <0.7 ms-1 and are responsive to capsaicin, bradykinin sensitivity of IB4-positive nociceptive afferents (which ex- and ATP; and fibres that conduct action potentials on an av- -1 press P2X3 and P2X2/3 receptors) [128]. Evidence has been erage of 0.9 ms and respond vigorously to ATP, but not to presented that P2Y2 receptors in the terminals of capsaicin- capsaicin or bradykinin [142]. Both the TRPV1 receptor and sensitive cutaneous mouse sensory neurons mediate nocicep- P2X receptors mediate the sensory transduction of pulmo- tive transmission and further that P2Y signaling may con- nary reactive oxygen species, especially H2O2 and OH, by tribute to mechanotransduction in low-threshold  fibres capsaicin-sensitive vagal lung afferent fibres [143]. [129]. Treatment with oxidized ATP, a selective inhibitor of Vagal C-fibres innervating the pulmonary system are P2X7 receptors, reduced the hyperalgesia produced by com- plete Freund’s adjuvant (CFA) and carrageenan-induced derived from cell bodies situated in two distinct vagal sen- inflammation in rats [130]. Data have been presented to sup- sory ganglia: the jugular (superior) ganglion neurons project port a pathogenic role for keratinocyte-derived ATP in irri- fibres to the extrapulmonary airways (larynx, trachea, bron- tant dermatitis [131]. A family of G protein-coupled recep- chus) and the lung parenchymal tissue, while the nodose (inferior) neurons innervate primarily structures within the tors, named mas-related G protein-coupled receptors (Mrgprd, also sensory neuron-specific receptors) has been cloned, lungs. Nerve terminals in the lungs from both jugular and which show high expression in sensory neurons of the DRG nodose ganglia responded to capsaicin and bradykinin, but and trigeminal ganglia, predominantly in small-diameter only the nodose C-fibres responded to ,-meATP. Vagal neurons [132]. It has been reported recently that cutaneous afferent purinergic signaling may be involved in the hyperac- sensory neurons expressing the Mrgprd receptor sense ex- tivity associated with asthma and chronic obstructive pulmo- tracellular ATP and are putative nociceptors [133]. They nary disease [144]. Th1 and Th2 cytokines reciprocally regu- suggest that these nociceptors in the outer epidermis respond late P2X7 receptor function, suggesting a role for P2X7 re- indirectly to external stimuli by detecting ATP release in the ceptors in pulmonary diseases, particularly lung hypersensi- skin. tivity associated with chronic inflammatory responses [145].

P2X2, P2X3 and P2Y1 receptors are abundantly present Musculoskeletal Systems and Joints on sensory nerve terminals in the tongue [91, 134]. ATP and ,-meATP have been shown to excite trigeminal lingual Pain related to the musculoskeletal system (myofascial nerve terminals in an in vitro preparation of intra-arterially pain) is very common and ATP has been claimed to excite or perfused rat mimicking nociceptive responses to noxious sensitize myofascial nociceptors [146]. Prolonged muscle mechanical stimulation and high temperature and a puriner- pain and tenderness was produced in human muscle by infu- gic mechanosensory transduction mechanism for the initia- sion of a combination of ATP, serotonin, histamine and pros- tion of pain was proposed [135]. taglandin E2 [147]. Strenuous exercise of muscle, as well as inflammation and ischaemia, are associated with tissue aci- Heart dosis. P2 receptors on the endings of thin fibre muscle affer- ents play a role in evoking both the metabolic and mecha- In the heart, an ATP-triggered vagal reflex has been de- noreceptor components of the exercise pressor reflex [148]. scribed leading to suppression of sinus mode automaticity PPADS attenuated the pressor response to contraction of the and atrioventricular nodal conduction [136]. This is probably triceps muscle. ATP has been shown to be an effective mediated by P2X2/3 receptors located on vagal sensory nerve stimulant of group IV receptors in mechanically sensitive terminals in the left ventricle, supporting the hypothesis that muscle afferents [149]. ATP was shown to enhance the mus- ATP released from ischemic myocytes is a mediator of atro- cle pressor response evoked by mechanically sensitive mus- pine-sensitive bradyarrhythmias associated with left ven- cle stretch, which was attenuated by PPADS [149]. Intra- tricular myocardial infarction [137]. A recent paper has im- muscular injections of acidic phosphate buffer at pH 6 or plicated P2X3 receptors on sensory fibres in the heart origi- ATP excited a subpopulation of unmyelinated (group IV) nating in the nodose ganglion in nociception associated with muscle afferent fibres [150], perhaps implicating P2X or myocardial ischaemic injury [138]. 2 Purinergic Receptors and Pain Current Pharmaceutical Design, 2009, Vol. 15, No. 00 9

P2X2/3 receptors that are sensitive to acidic pH. ATP induces sion of P2X3 receptors in nociceptive sensory neurons ex- sustained facilitation of cranial nociception through P2X pressing CGRP by analogy with that described for increased receptors on neck muscle nociceptors in mice [151]. Noci- P2X3 receptor expression in a model of inflammatory colitis ceptive information arising from the orofacial region via [116]. Increased expression of P2X3 receptors was also re- P2X3 receptors on afferent fibres are carried to trigeminal ported associated with thermal and mechanical hyperalgesia brainstem sensory nuclei [66]. in a rat model of squamous cell carcinoma of the lower gin- gival [169]. ATP has been shown to be a stimulant of articular noci- ceptors in the knee joint via P2X receptors [152, 153] and 3 F. NEUROPATHIC AND INFLAMMATORY PAIN also to some extent in lumbar intervertebral disc, but not as prominently as in the skin [154]. P2Y2 receptor mRNA is Activation of P2X receptors in the spinal cord was shown expressed in both cultured normal and osteoarthritic chon- to elicit allodynia [170] and in a seminal study published in drocytes taken from human knee joints, and ATP is shown to Nature in 2004, P2X4 receptors on spinal cord microglia be released by mechanical stimulation [155]. Masseter mus- were shown to be upgraded in neuropathic pain, which was cle pain is recognised as a prominent symptom in temporo- significantly reduced after use of P2X4 antisense oligonu- mandibular disorders. A recent report claims that ATP acting cleotides [171]. This study has led to an explosion of work on P2X3 receptors on sensory nerve fibres in masseter mus- focussed on purinergic signalling in neuropathic pain [18, cle plays an important role in pressure pain and mechanical 172-174]. Importantly, it has been shown that P2X7 and hyperalgesia caused by excessive muscular contraction P2Y12 receptors on microglia are also involved in neuro- [156]. Sensory nerve fibres arising from the trigeminal gan- pathic pain, although the underlying mechanisms involving glion supplying the temporomandibular joint have abundant P2X4, P2X7 and P2Y12 receptors are still not clear. Adeno- receptors that respond to capsaicin, protons, heat and ATP; sine has also been considered as a potential analgesic target retrograde tracing revealed 25, 41, and 52% of neurons sup- for inflammatory and neuropathic pain [21, 175]. plying this joint exhibited TRPV1 and P2X3 receptors, re- spectively [157]. When monoarthritis was induced by injec- P2X3 and P2X2/3 Receptors tion of CFA into the unilateral temporomandibular joint of There is evidence that P2X receptors are involved in the rat, the pain produced was associated with an increase in 3 neuropathic pain, probably those located on primary afferent P2X receptor-positive small neurons in the trigeminal gan- 3 nerve terminals in inner lamina 2 of the spinal cord [176] glion [158]. Activation of P2X receptors in rat temporoman- and in the trigeminal brainstem sensory nuclei [66]. P2X , dibular joint induces nociception, and that antagonism by 2 P2X and P2X receptors have also been located on dorsal PPADS decreases carrageenan-induced inflammatory hyper- 4 6 horn neurons relaying nociceptive information further along algesia [159]. Oxidized ATP inhibits inflammatory pain in the pain pathway [177]. In addition, ATP coreleased with - arthritic rats by inhibition of the P2X receptor for ATP lo- 7 aminobutyric acid in spinal interneurons is probably in- calized in nerve terminals [160]. Evidence is accumulating to volved in modulation of nociceptive pathways [178]. suggest that blockers of P2X7 receptors may have a future as anti-inflammatory drugs [161]. P2X3 receptors on terminals of primary afferent nerves in the inner lamina 2 of the dorsal horn of the spinal cord medi- P2X and P2X receptors on sensory afferents in tooth 3 2/3 ate facilitation of glutamate release [179] and it has been pulp appear to mediate nociception [162-164], perhaps from suggested that thermal hyperalgesia may be mediated by ATP released by mechanical distension or inflammation of spinal P2X3 receptors via activation of N-methyl-D-aspartate odontoblasts. Mustard oil application to the tooth pulp in receptors [180]. Supraspinal P2X and P2X receptors play anesthetized rats produced long-lasting central sensitization, 3 2/3 an inhibitory role in pain transmission [181]. Intrathecal ad- reflected by increases in neuronal mechanoreceptive field ministration of ATP produces long-lasting allodynia, most size; TNP-ATP reversibly attenuated the mustard oil sensiti- likely via P2X receptors [182]. The involvement of spinal zation for more than 15 min [165]. 2/3 P2X2 and P2X3 receptors in neuropathic pain in a mouse model of chronic constriction injury has been claimed [183]. Tumours A recent study suggests that P2X3/P2X2/3 receptor-dependent Purinergic mechanisms are beginning to be explored in cytosolic phospholipase A2 (cPLA2) activity in primary sen- relation to cancer pain [17, 166, 167]. It was suggested that sory neurons is a key event in neuropathic pain and that the unusually high levels of ATP contained in tumour cells cPLA2 might be a potential target for treating neuropathic [168] may be released by mechanical rupture to activate pain [184]. It is claimed that sensitisation of P2X3 receptors P2X3 receptors on nearby nociceptive sensory nerve fibres rather than a change in ATP release is responsible for neuro- [17]. There is increased expression of P2X3 receptors on pathic pain and allodynia [185]. Data has been presented to calcitonin gene-related peptide (CGRP) immunoreactive suggest that the P2X3 and P2X2/3 receptor antagonism that epidermal sensory nerve fibres in a bone cancer pain model reduces inflammatory hyperalgesia and chemogenic nocicep- [167] and in other cancers that involve mechanically sensi- tion is mediated by the spinal opioid system [186]. A recent tive tumours [166]. For example, in bone tumours, destruc- review covering the role of P2X3 receptor involvement in tion reduces the mechanical strength of the bone, and an- acute pain, inflammatory pain, chronic neuropathic pain, tagonists that block the mechanically gated channels and/or migraine and cancer pain is available [187]. ATP receptors in the richly innervated periosteum might TNP-ATP (Fig. 3a) is the most potent P2X receptor an- reduce movement-associated pain. The hyperalgesia associ- 3 tagonist [188]. TNP-ATP has low nanomolar affinity for ated with tumours appears to be linked to increase in expres- blocking P2X3 receptors but also has high affinity for P2X1 10 Current Pharmaceutical Design, 2009, Vol. 15, No. 00 Geoffrey Burnstock receptors and is rapidly degraded in situ [50]. Thus, in vivo diate nerve injury-induced upregulation of P2X4 receptors studies of TNP-ATP as a pharmacological tool have been [202]. limited to direct intrathecal administration [18] or direct ad- Enhancement of pain behaviour after nerve injury not ministration into a site of peripheral tissue damage [189]. only requires the P2X4 receptor, but also phospho38 (p38) Several novel non-nucleotide small molecule P2X3 antago- mitogen-activated protein kinase (MAPK) [203]. ATP nists have been reported. A-317491 (Fig. 3b) has nanomolar causes the activation of p38 or ERK1/2 MAPKs resulting in affinity for blocking both P2X and P2X receptors and is a 3 2/3 the release of tumour necrosis factor- and interleukin (IL)- competitive antagonist [190, 191]. Unlike TNP-ATP, A- 6. In rats displaying allodynia, the level of p38 was increased 317491 is not susceptible to metabolic degradation and in microglia. Intraspinal administration of the p38 inhibitor, shows high systemic bioavailability following subcutaneous SB203580, suppressed allodynia, suggesting that neuro- administration but lacks oral bioavailability. Antagonism of pathic pain hypersensitivity depends on the activation of the P2X and P2X receptors by phenol red has been reported, 1 3 p38 signalling pathway in microglia in the dorsal horn fol- and tetramethylpyrazine, a traditional Chinese medicine, lowing peripheral nerve injury. Platelet activating factor, used as an analgesic for dysmenorrhoea, was claimed to which is released from activated microglia, is a potent in- block P2X receptor signalling [192]. Systemic administra- 3 ducer of tactile allodynia and thermal hyperalgesia after in- tion of A-317491 effectively reduced nociception in inflam- trathecal injection into the spinal cord and it was suggested matory and neuropathic pain models [190]. A-317491 also that this response is mediated by ATP [204]. The possible effectively blocked persistent pain in the formalin and acetic mechanisms that underlie the role of P2X receptors in neu- acid-induced abdominal constriction tests but was generally 4 ropathic pain and the involvement of inflammatory cytokines inactive in models of acute noxious (thermal, mechanical have been reviewed [174, 205, 206]. Systemic and intracere- and chemical) stimulation [193]. RO-3 (Fig. 3b) is another bro-ventricular infection of pro-inflammatory bacterial recently identified antagonist that potently blocks P2X re- 3 lipopolysaccharide (LPS) results in widespread thermal hy- ceptors (pIC50 =7.0) and exhibits at least 100-fold less activ- peralgesia and tactile allodynia. LPS has been shown to en- ity across a wide range of kinases, receptors and ion chan- hance responses to low concentrations of ATP mediated by nels [59]. RO-3 has lower protein binding (48%) compared P2X receptors [207]. with A-317491 (99%) and good CNS penetration. R0-3 also 4 reduces nociceptive sensitivity in animal pain models [59]. The lack of selective P2X4 antagonists has hindered the pharmacological validation of the role for P2X4 receptors in Antisense oligonucleotides have been used to downregu- pain. 5-(3-Bromophenyl)-1,3-dihydro-2H-benzofuro-[3,2-e]- late the P2X receptor, and in models of neuropathic (partial 3 1,4-diazepin-2-one was shown to block P2X receptor- sciatic nerve ligation) and inflammatory (CFA) pain, inhibi- 4 mediated currents expressed in Chinese hamster ovary cells tion of the development of mechanical hyperalgesia as well with an IC value of 0.5 μM [20]. Antidepressants have as significant reversal of established hyperalgesia were ob- 50 been shown to be effective in relieving neuropathic pain served [194-196]. P2X antisense oligonucleotides or an- 3 [208] and a recent communication by Kazu Inoue [174] sug- tagonists appear to be less effective for treating discogenic gests that some antidepressants, in particular paroxetine, is (lumbar intervertebral disc) than cutaneous tissue pain [154]. an effective P2X receptor antagonist in transfected cells and Combined antisense and RNA interference-mediated treat- 4 preliminary clinical studies suggested that it was effective ment for specific inhibition of the recombinant rat P2X re- 3 against chronic pain. A series of benzofuro-1,4-diazepin-2- ceptor appears to be promising for pain therapy [197]. P2X 3 ones have been reported to be effective P2X antagonists in a double-stranded short interfering RNA relieves chronic neu- 4 Bayer Health Care, AG patent (see [209]). It remains to be ropathic pain and opens up new avenues for therapeutic pain seen whether novel selective P2X antagonists will elicit strategies in humans [198]. 4 analgesic effects in neuropathic and inflammatory pain states. P2X4 Receptors

There have been a number of papers about the role of P2X7 Receptors P2X4 receptors on spinal microglia in neuropathic pain fol- P2X receptors were shown to be expressed in a mouse lowing the initial discovery by Tsuda et al. [171] (see [174]). 7 microglial cell line, NTW8, in 1997 [210] and later on mi- Brain-derived neurotrophic factor (BDNF) is released from croglia in rat brain [211]. Relief of inflammation-induced microglia by the stimulation of P2X receptors by effecting 4 mechanical hyperalgesia in rats with the P2X receptor an- E-anion in spinal lamina 1 neurons [199]. The involvement 7 tagonist, oxidized ATP [160]. Chronic inflammatory and of increase in spinal fibronectin/integrin following peripheral neuropathic pain was abolished in P2X knockout mice, as nerve injury in upregulation of microglial P2X receptors 7 4 was release of IL-1 [212]. The authors hypothesised that and neuropathic pain has been considered [200]. Innate im- the P2X7 receptor, via regulation of mature IL-1 produc- mune system sensor - like toll-like receptors (TLR’s) and tion, plays a common upstream transductional role in the nucleotide-binding oligomerization domain 2 (NOD2) recep- development of neuropathic and inflammatory pain. tors, are key receptors to sense pathogen-associated molecu- lar patterns. Ligands for TLR’s and NOD2 receptors stimu- Several P2X7 receptor antagonists have been proposed late microglial P2X4 receptor upregulation, suggesting that including: oxidised ATP (Fig. 3a), Brilliant Blue G, the tyro- microglia sense the presence of inflammatory stimulation sine derivatives KN-62 and KN-04, cyclic imides, adaman- using multiple recognition systems [201]. In a recent study, tane and benzamide derivatives [213], compound 4g [214], Lyn tyrosine kinase was shown to play an important role in and other benzophenenanthidine alkaloids the pathogenesis of neuropathic pain and is required to me- [215], U73122 and U73343 [216] and recently developed Purinergic Receptors and Pain Current Pharmaceutical Design, 2009, Vol. 15, No. 00 11 compounds such as cyanoguanidines and aminotetrazoles pathological state [228]. Early work with TRPV1-transfected [209]. More direct support for a role of P2X7 receptors in human embryonic kidney 293 cells suggested that the P2Y1 pain modulation is provided by studies using selective an- and/or P2Y2 receptors were responsible for the ATP modula- tagonists. Systemic administration of the P2X7 receptor- tion of TRPV1 responses to heat, capsaicin and protons selective antagonists, A-438079 and A-740003 (Fig. 3c) [225]. The contributions of P2Y2 receptors for pain transmis- produced dose-dependent antinociceptive effects in models sion probably extend beyond interactions with TRPV1 re- of neuropathic [217-219] and inflammatory pain [217]. Con- ceptors in primary afferent neurons. In the isolated skin- sistent with their in vitro potencies, A-740003 was more po- nerve preparation, 54% cutaneous C-fibres and 12% A- tent than A-438079 at reducing mechanical allodynia 2 mechanoreceptors responded to UTP (approximately 70- weeks after spinal L5/L6 nerve ligation. The antinociceptive 80% were capsaicin-sensitive) [129]. However, an additional effects of P2X7 antagonists in inflammatory pain models do 22 to 26% of large diameter A fibres responded to UTP, not appear to be secondary to an anti-inflammatory effect, suggesting that P2Y2 receptors also may be directly involved because A-740003 was more efficacious in reducing noci- in the transmission of low-threshold mechanical inputs to the ception than paw edema. The antinociceptive action of A- spinal cord. It is also possible that activation of the hetero- 438079 is related to blocking mechanical and thermal inputs oligomeric P2Y2/A1 receptor complex [229] may also nega- to several different classes of spinal neurons [219]. A- tively modulate the antinociceptive effects of A1 receptor 438079 reduced noxious and innocuous-evoked activity of agonists [172]. low threshold, nociceptive-specific, and wide dynamic range The rostral ventromedial medulla serves as a critical link spinal neurons in neuropathic rats. Spontaneous activity of in bulbo-spinal nociceptive modulation and it has been sug- all classes of spinal neurons was also significantly reduced gested that while on-cells preferentially express P2X recep- by A-438079 in neuropathic, but not sham rats. Blockade of tors, off-cells express P2Y receptors in this region [230]. P2X7 receptors significantly reduced nociception in animal Activation of P2Y receptors inhibits P2X3 receptor channels models of persistent neuropathic and inflammatory pain [20, via G protein-dependent facilitation of their desensitisation 220, 221]. Collectively, these data combined with growing [231]. evidence supporting the role of P2X7 receptor modulation in proinflammatory IL-1 processing [161] indicate a specific It has been reported recently that P2Y12 receptors ex- role for P2X7 receptors in neural-glial cell interactions asso- pressed on microglia [232] are required for neuropathic pain ciated with ongoing pain [222]. after peripheral nerve injury, and intrathecal administration of the P2Y receptor antagonist, AR-C69931MX, prevented P2X and P2X receptor knockout mice share a common 12 4 7 the development of tactile allodynia [233]. Activation of the pain phenotype, although this phenotype appears to be con- P2Y receptor by released ATP is via the p38 MAPK path- ferred via different mechanisms [223]. There has been a re- 12 way [234]. Nucleotide agonists selective for P2Y , P2Y , cent report suggesting that P2X and P2X receptors form 1 2 4 7 P2Y and P2Y receptors and nucleotide antagonists selec- heteromultimers [63]. 6 12 tive for P2Y1 (MRS2179, MRS2500), P2Y12/13 (Congrelor, previously known as AR-C69931MX) and P2Y (ARL660- P2Y Receptors 12 96, INS4266, AZD6140) have been identified (see [52]). The contributions of the metabotropic P2Y receptors to normal and pathological pain have been less well-examined, Migraine compared to P2X receptors (see [224]). However, the ex- The involvement of ATP in migraine was first suspected pression of P2Y1, P2Y2, P2Y4 and P2Y6 mRNA in DRG in conjunction with the vascular theory of this disorder with neurons suggests that these receptors may be involved in ATP released from endothelial cells during reactive hyper- peripheral somatosensory transmission [8, 225]. Activation aemia associated with pain following cerebral vascular vaso- of UTP-sensitive P2Y and/or P2Y receptors and the UDP- 2 4 spasm (not associated with pain) [235]. More recently, P2X sensitive P2Y receptor, in contrast to P2X receptors, pro- 3 6 receptor involvement in neuronal dysfunction in brain areas duces inhibition of spinal pain transmission [226]. P2Y and 1 that mediate nociception such as the trigeminal nucleus and P2Y4 receptors were identified on predominantly small di- thalamus have been considered [236, 237]. P2X receptors ameter sensory neurons, in a subpopulation of which P2X 3 3 are the only ligand-gated channel known to be expressed receptors [26] and TRPV1 receptors [28] were also ex- exclusively by a subset of trigeminal and spinal sensory neu- pressed. Activation of P2Y receptors on DRG neurons 1 rons [15] and may make a promising candidate for antimi- modulates currents generate through N-type (Ca 2.2) calcium v graine drug development [238]. Slow up-regulation of noci- channels and P2X3 receptors [227]. Indeed, activation of N- ceptive P2X3 receptors on trigeminal neurons by the mi- type calcium channels in cultured DRG neurons was inhib- graine mediators CGRP and nerve growth factor (NGF) has ited by ATP and even more potently by the P2Y recep- 1,12,13 been demonstrated [237, 239]. In an in vivo model of mouse tor agonist ADP [28]. The effects of ATP were blocked by trigeminal pain, anti-NGF treatment suppressed responses the selective P2Y receptor antagonist MRS 2179 (Fig. 3d). 1 evoked by P2X receptor activation [240]. The interaction of The outcome of P2Y -related inhibitions on N-type calcium 3 1 P2Y receptors on trigeminal neurons with P2X receptors channels or P2X receptors is likely to result in a decreased 1 3 3 after sensitisation of these neurons with algogenic stimuli release of nociceptive transmitters into the spinal cord [8]. (e.g., NGF, BDNF or bradykinin) has been proposed and P2Y1 receptor mRNA is up-regulated in the lumbar DRG may also represent a new potential target for antimigraine after peripheral axotomy, indicating that P2Y1 receptors may drugs [241]. Peripheral sensitisation is considered as a con- contribute to the heightened somatosensory sensitivity in this tributor to mechanisms underlying migraine headache and a 12 Current Pharmaceutical Design, 2009, Vol. 15, No. 00 Geoffrey Burnstock

a NaO3S SO3Na b HO O O

N NH2 OH H3C N N N HO N O HO O N N O N ONa O H O P OPPP CHO CHO O ONa O PPADS tetrasodium salt oxidized ATP A-317491 NH 2 N NH2 N SO3Na SO Na N 3 N N O O O O P O N NH P P O 2 HO O O HO HO HO O O NH SO3Na O O RO-3 O2N NO2 O H H N N N H N CH3 O TNP-ATP O O Suramin 2

d NHCH3 c N N N Cl N H H H N N N Cl NNN N O N OPO3H2 O N N O NC O OPO3H2 A-438079.0 A-740003.0 MRS 2179

Fig. (3). (a) Structures of prototypical and non-selective P2 receptor antagonists. (b) Structures of P2X3 receptor-selective antagonists. (c) Structures of P2X7 receptor-selective antagonists. (d) Structures of P2Y1 receptor-selective antagonist. (Reproduced from [20] with permis- sion from The American Society for Pharmacology and Experimental Therapeutics). recent review concludes that ATP has a role in sensitisation momeric P2X3 receptors probably contributes to acute noci- of primary afferents at both peripheral and central terminals ception and some aspects of acute inflammatory pain [8, [242]. 172]. In contrast, activation of heteromeric P2X2/3 receptors appears to modulate longer-lasting nociceptive sensitivity Evidence for the possible role of adenosine in migraine associated with nerve injury or chronic inflammation [194, has been reviewed [243]. Plasma adenosine has been ob- served to rise during migraine attacks and adenosine has 246]. Furthermore, under conditions of persistent nociceptive input activation of P2X , P2X and P2Y receptors on mi- been reported to trigger migraine attack while , 4 7 12 croglia may serve to maintain nociceptive sensitivity through an adenosine uptake inhibitor, can increase migraine attack complex neural-glial cell interactions or via sensitisation (via frequency. A receptor stimulation has also been considered 1 P2Y ) of other nociceptive receptors such as TRPV1 chan- for migraine treatment [244] and it has been claimed that A 2 2A nels. There is still an urgent need to understand the mecha- receptor gene variation may contribute to the pathogenesis of migraine [245]. nisms underlying the successful application of P2X3, P2X4, P2X7 and P2Y12 receptor antagonists for the treatment of G. CONCLUSIONS AND FUTURE DIRECTIONS neuropathic pain. The search is on for selective P2X , P2X , P2X , P2X Multiple P2 receptor subtypes are involved in pain path- 3 2/3 4 7 and P2Y receptor antagonists that are orally bioavailable, ways both as an initiator and modulator. Activation of ho- 12 can cross the blood-brain barrier and do not degrade in vivo Purinergic Receptors and Pain Current Pharmaceutical Design, 2009, Vol. 15, No. 00 13 for the treatment of pain (see [18, 59]). The P2X7 receptor in INS49266 = 6-Phenylurea-2',3'-phenylacetaldehyde particular is now a major target for inflammatory neuro- acetal pathic pain and a number of selective P2X receptor antago- 7 Ip I = Diinosine pentaphosphate nists have been developed (see [20, 247]). A review describ- 5 ing recent progress in the development of KN62 = 1-(N,Obis[5-isoquinolinesulphonyl]-N- ligands as anti-inflammatory drugs is also available [248]. methyl-L-tyrosyl)-4-phenylpiperazine Other therapeutic approaches to pain are being consid- 2-MeSMAP = 2-Methylthio AMP ered, including the development of agents that control the MRS2159 = Pyridoxal-5-phosphate-6-phenylazo- expression of receptors and those that inhibit ATP break- 4'-carboxylic acid down by selective inhibition of the known ectonucleotidases. Further, while it is now clear that many different cell types NF110 = 4,4',4'',4'''-(Carbonylbis(imino-5,1,3- release ATP physiologically in response to mechanical dis- benzenetriylbis (carbonylimino)))tetra- tortion, hypoxia, and various agents, we still await clear un- kis-benzenesulfonic acid derstanding of the mechanisms that underlie ATP transport. NF449 = 4, 4',4'',4'''-(Carbonylbis[imino-5,1,3- Hopefully, when this becomes clearer, agents will be devel- benzenetriyl- oped that will be able to enhance or inhibit ATP release, an- bis{carbonylimino}])tetrakisbenzene- other useful way forward as a therapeutic strategy. A- 1,3-disulfonic acid 134974, a novel adenosine kinase (AK) inhibitor, alleviated tactile allodynia via spinal sites of action in peripheral nerve- o-ATP = Oxidised ATP injured rats, adding to the growing evidence that AK inhibi- PPADS = Pyridoxalphosphate-6-azophenyl-2’,4’- tors may be useful analgesic agents [249]. disulphonate There are no publications to date describing clinical RO3 = 5-(Methyl[2-methylethyl-4,5- evaluations of P2 receptor antagonists and related purinergic dimethoxyphenyl]-2,4pyridinediamine compounds for the relief of pain, although clinical trials for some compounds are in progress (see [59]) and reduced pain TNP-ATP = 2’,3’-O-(2,4,6-Trinitrophenyl)-ATP sensation was noted in a suramin phase 1 cancer clinical trial REFERENCES [250]. However, the emerging information about P2 receptor subtype selective antagonists is promising. [1] Burnstock G. Purinergic nerves. Pharmacol Rev 1972; 24: 509-81. [2] Drury AN, Szent-Györgyi A. 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