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Pharmacology & Therapeutics xx (2005) xxx – xxx www.elsevier.com/locate/pharmthera

Purinergic P2 receptors as targets for novel analgesics

Geoffrey Burnstock *

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

Abstract

Following hints in the early literature about adenosine 5V-triphosphate (ATP) injections producing pain, an ion-channel nucleotide receptor was cloned in 1995, P2X3 subtype, which was shown to be localized predominantly on small nociceptive sensory nerves. Since then, there has been an increasing number of papers exploring the role of P2X3 homomultimer and P2X2/3 heteromultimer receptors on sensory nerves in a wide range of organs, including skin, tongue, tooth pulp, intestine, bladder, and ureter that mediate the initiation of pain. Purinergic mechanosensory transduction has been proposed for visceral pain, where ATP released from epithelial cells lining the bladder, ureter, and intestine during distension acts on P2X3 and P2X2/3, and possibly P2Y, receptors on subepithelial sensory nerve fibers to send messages to the pain centers in the brain as well as initiating local reflexes. P1, P2X, and P2Y receptors also appear to be involved in nociceptive neural pathways in the spinal cord. P2X4 receptors on spinal microglia have been implicated in allodynia. The involvement of purinergic signaling in long-term neuropathic pain and inflammation as well as acute pain is discussed as well as the development of P2 receptor antagonists as novel analgesics. D 2005 Elsevier Inc. All rights reserved.

Keywords: Purinergic; P2X receptor; P2Y receptor; Analgesic; ATP; Signaling

Abbreviations: a,h-meATP, a,h-methylene ATP; ABC, ATP binding cassette; ATP, adenosine 5V-triphosphate; BzATP, 3V-O-(4-benzoyl)benzoyl ATP; CFA, complete Freund’s adjuvant; CGRP, calcitonin gene-related peptide; CNS, central nervous system; DHEA, dehydroepiandrosterone; DRG, dorsal root ganglia; GABA, g-amino butyric acid; GDNF, glial cell line-derived neurotrophic factor; HSPs, heat shock proteins; IBS, irritable bowel syndrome; IB4, isolectin B4; IL, interleukin; IGLEs, intraganglionic laminar nerve endings; mRNA, messenger ribonucleic acid; NA, noradrenaline; NEBs, neuroepithelial bodies; NG, nodose ganglia; NMDA, N-methyl-d- aspartate; NO, nitric oxide; NTS, nucleus tractus solitarius; PAF, platelet-activating factor; pERK, phosphorylated extracellular signal-regulated protein kinase; PKC, protein kinase C; PPADS, pyridoxal-5V-phosphate-6-azophenyl-2V,4V disulphonic acid; RVM, rostral ventromedial medulla; TG, trigeminal ganglia; TMP, tetramethylpyrazine; TNP-ATP, trinitrophenol-ATP; TRPV, transient receptor potential vanilloid channels; UTP, uridine 5V-triphosphate; VR1, vanilloid receptor type 1.

Contents 1. Introduction ...... 0 2. Purinergic signaling: physiological reflexes and nociception ...... 0 2.1. Purinergic receptors expressed by sensory neurons ...... 0 2.1.1. P2X receptors...... 0 2.1.2. P2Y receptors...... 0 2.1.3. Interactions between P2 and vanilloid receptors ...... 0 2.2. Evidence for purinergic mechanosensory transduction in different organs ...... 0 2.2.1. Urinary bladder ...... 0 2.2.2. Ureter ...... 0 2.2.3. Gut ...... 0 2.2.4. Lung ...... 0 2.2.5. Carotid body ...... 0 2.2.6. Tooth pulp ...... 0 2.2.7. Special senses organs...... 0 2.2.8. Skin, muscle, and joints ...... 0 2.3. Sources of ATP involved in mechanosensory transduction ...... 0

* Tel.: +44 207 830 2948; fax: +44 207 830 2949. E-mail address: [email protected].

JPT-05754; No of Pages 22 ARTICLE IN PRESS 2 G. Burnstock / Pharmacology & Therapeutics xx (2005) xxx–xxx

3. Neuropathic, inflammatory, and cancer pain...... 0 3.1. Peripheral purinergic mechanisms ...... 0 3.1.1. Sensory ganglia ...... 0 3.1.2. Urinary bladder ...... 0 3.1.3. Gut ...... 0 3.1.4. Lung...... 0 3.1.5. Joints ...... 0 3.2. Central purinergic mechanisms ...... 0 3.2.1. Spinal cord ...... 0 3.2.2. Brain ...... 0 3.2.3. Microglia and glial–neuron interactions...... 0 3.3. Cancer pain...... 0 4. Purinergic therapeutic developments for the treatment of pain ...... 0 5. Conclusions ...... 0 Acknowledgments ...... 0 References ...... 0

1. Introduction ureter, bladder, and gut, where ATP released from epithelial cells during distension, acted on P2X3 homomultimeric and There were early hints that adenosine 5V-triphosphate (ATP) P2X2/3 heteromultimeric receptors on subepithelial sensory might be involved in pain including the demonstration of pain nerves initiating impulses in sensory pathways to pain centers produced by injection of ATP into human skin blisters (Keele in the central nervous system (CNS) (Burnstock, 1999)(Fig. & Armastrong, 1964; Collier et al., 1966; Bleehen & Keele, 2A). Subsequent studies of bladder (Cockayne et al., 2000; 1977), ATP involvement in migraine (Burnstock, 1981), and Vlaskovska et al., 2001; Rong et al., 2002), ureter (Knight et ATP participation in pain pathways in the spinal cord (Jahr & al., 2002; Rong & Burnstock, 2004), and gut (Wynn et al., Jessell, 1983; Fyffe & Perl, 1984; Salter & Henry, 1985). A 2003, 2004) have produced evidence in support of this significant advance was made when the P2X3 ionotropic ion hypothesis (see also Burnstock, 2001a). channel purinergic receptor was cloned in 1995 and shown to The aim of the present article is to review the large number be localized predominantly on small nociceptive sensory of papers that have appeared since 2001 to elaborate on this neurons in dorsal root ganglia (DRG) (Chen et al., 1995, theme and to explore the purinergic drugs under development Lewis et al., 1995). Later, Burnstock (1996) put forward a for the treatment of pain. unifying purinergic hypothesis for the initiation of pain, suggesting that ATP released as a cotransmitter with noradren- 2. Purinergic signaling: physiological reflexes and nociception aline (NA) and neuropeptide Y from sympathetic nerve terminal varicosities might be involved in sympathetic pain 2.1. Purinergic receptors expressed by sensory neurons (causalgia and reflex sympathetic dystrophy); that ATP released from vascular endothelial cells of microvessels during 2.1.1. P2X receptors reactive hyperemia is associated with pain in migraine, angina, A comprehensive review of P2X receptor expression and and ischemia; and that ATP released from tumor cells function in sensory neurons in DRG, nodose (NG), trigeminal (containing high levels) damaged during abrasive activity (TG), and petrosal ganglia was presented in 2001 (Dunn et al., reaches P2X3 receptors on nociceptive sensory nerves. This 2001). All P2X subtypes, except P2X7, are found in sensory hasbeenfollowedbyanincreasingnumberofpapers neurones, although the P2X3 receptor has the highest level of expanding on this concept. Immunohistochemical studies expression [both in terms of messenger ribonucleic acid showed that the nociceptive fibers expressing P2X3 receptors (mRNA) and protein]. P2X2/3 heteromultimers are particularly arose largely from the population of small neurons that labeled prominent in the nodose ganglion. P2X3 and P2X2/3 receptors with the lectin isolectin B4 (IB4)(Vulchanova et al., 1996; are expressed on isolectin B4 (IB4) binding subpopulations of Bradbury et al., 1998). The central projections of these small nociceptive neurons. Species differences are recognized. neurons were shown to be in inner lamina II of the dorsal There have been a remarkably large number of studies of P2X horn and peripheral projections demonstrated to skin, tooth receptor-mediated signaling in sensory ganglia since this pulp, tongue, and subepithelial regions of visceral organs. A review and some of these are discussed below. schematic illustrating the initiation of nociception on primary The decreased sensitivity to noxious stimuli, associated with afferent fibers in the periphery and purinergic relay pathways the loss of IB4-binding neurons expressing P2X3 receptors, in the spinal cord was presented by Burnstock and Wood indicates that these sensory neurons are essential for the (1996) (Fig. 1). signaling of acute pain (Vulchanova et al., 2001). The loss of A hypothesis was proposed that purinergic mechanosensory IB4 binding neurons also led to compensatory changes relating transduction occurred in visceral tubes and sacs, including to recovery of sensitivity to acute pain. ARTICLE IN PRESS G. Burnstock / Pharmacology & Therapeutics xx (2005) xxx–xxx 3

terminals. Oxytocin also inhibits ATP-activated currents in DRG neurons (Yang et al., 2002). In contrast, neurokinin B potentiates ATP-activated currents in DRG neurons (Wang et al., 2001). There is strong enhancement of nociception produced via P2X3 and P2X2/3 receptors in rat hindpaw by NA and serotonin (Waldron & Sawynok, 2004). Prostaglandin E2, an inflammatory mediator, potentiates P2X3 receptor- mediated responses in DRG neurons by activating prostaglandin EP3 receptors and modulates P2X3 receptor channels through the protein kinase A signaling pathway (Wang & Huang, 2004). Responses to P2X3 receptor activation in cultured DRG neurons can be inhibited by high Mg2+ or by lack of Ca2+;it was suggested that this might represent a negative feedback process to limit ATP-mediated nociception in vivo (Giniatullin et al., 2003). Viewed another way is that Ca2+ facilitates recovery and residues in the P2X3 receptor ectodomain have been identified that are involved in this Ca2+-sensory action (Fabbretti et al., 2004). Pilot clinical studies report analgesic actions by Mg2+ on neuropathic pain that is insensitive to opioids (Crosby et al., 2000). A recent study has shown that sensory neurons have the machinery to form purinergic synapses on each other when placed in short-term tissue culture (Zarei et al., 2004). The resulting neurotransmitter release is calcium-dependent and uses synaptotagmin-containing vesicles; the postsynaptic receptor involved is a P2X subtype. Experiments are needed to find out whether purinergic synapses form between sensory neurons in vivo, whether this is more common after nerve Fig. 1. Hypothetical schematic of the roles of purine nucleotides and injury and whether this has physiological or pathophysiological nucleosides in pain pathways. At sensory nerve terminals in the periphery, significance. P2X3 and P2X2/3 receptors have been identified as the principal P2X mRNA for an orphan G protein-coupled receptor TGR7, purinoceptors present, although recent studies have also shown expression of which is specifically responsive to h-alanine, is coexpressed in P2Y1 and possibly P2Y2 receptors on a subpopulation of P2X3 receptor- immunopositive fibers. Other known P2X purinoceptor subtypes (1–7) are also small diameter neurons with P2X3 and vanilloid receptor type 1 expressed at low levels in dorsal root ganglia. Although less potent than ATP, (VR1) receptors in both rat and monkey DRG (Shinohara et al., adenosine (AD) also appears to act on sensory terminals, probably directly via 2004). h-Alanine has been claimed to participate in synaptic P1(A2) purinoceptors; however, it also acts synergistically (broken red line) to transmission as a neurotransmitter and/or neuromodulator and potentiate P2X2/3 receptor activation, which also may be true for 5- hydroxytryptamine, capsaicin, and protons. At synapses in sensory pathways the authors suggest that TGR7 may participate in the in the CNS, ATP appears to act postsynaptically via P2X2, P2X4, and/or P2X6 modulation of neuropathic pain. purinoceptor subtypes, perhaps as heteromultimers, and after breakdown to Ca2+/calmodulin-dependent protein kinase II, up-regulated adenosine, it acts as a prejunctional inhibitor of transmission via P1(A ) 2 by electrical stimulation, enhances P2X3 receptor activity in purinoceptors. P2X3 receptors on the central projections of primary afferent DRG neurons and it is suggested that this may play a key role neurons in lamina II of the dorsal horn mediate facilitation of glutamate and in the sensitisation of P2X receptors under injurious conditions probably also ATP release. Sources of ATP acting on P2X3 and P2X2/3 receptors on sensory terminals include sympathetic nerves, endothelial, Merkel, (Xu & Huang, 2004). and tumor cells. Yellow dots, molecules of ATP; red dots, molecules of adenosine (modified from Burnstock & Wood, 1996, reproduced with 2.1.2. P2Y receptors permission of Elsevier). While the predominant P2 receptor subtypes expressed in sensory neurons involved in the initiation of nociception was Rapid reduction of the excitatory action of ATP on DRG recognized early as P2X3 and P2X2/3 (see Burnstock & Wood, neurons by g-amino butyric acid (GABA), probably via 1996; Burnstock, 2000), it has become apparent more recently GABAA anionic receptors, and slow inhibition of ATP that P2Y receptors are also present (Nakamura & Strittmatter, currents via metabotropic GABAB receptors appear to be 1996; Svichar et al., 1997; Xiao et al., 2002; Malin et al., 2004; additional mechanisms of sensory information processing Nakayama et al., 2004), which are involved in modulation of (Sokolova et al., 2001; Labrakakis et al., 2003; Sokolova et pain transmission (Gerevich & Illes, 2004). RT-PCR showed al., 2003). Fibers project from DRG to the superficial lamina that P2Y1, P2Y2, P2Y4, and P2Y6 mRNA is expressed on of the dorsal horn of the spinal cord where the receptors may neurons in DRG, NG, and TG ganglia and receptor protein for function to modulate transmitter release near their central P2Y1 is localized on over 80% of mostly small neurons (Ruan ARTICLE IN PRESS 4 G. Burnstock / Pharmacology & Therapeutics xx (2005) xxx–xxx

Fig. 2. (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 Burnstock, 1999, 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 fibers (labeled with calbindin) contributing to peristaltic reflexes. ATP released during extreme distension also acts on P2X3 and/or P2X2/3 receptors on high-threshold extrinsic sensory nerve fibers (labeled with isolectin B4;IB4) that send messages via the dorsal root ganglia (DRG) to pain centers in the central nervous system (from Burnstock, 2001c, reproduced with permission of John Wiley & Sons, Inc.). ARTICLE IN PRESS G. Burnstock / Pharmacology & Therapeutics xx (2005) xxx–xxx 5

& Burnstock, 2003). Double immunolabeling showed that 73– hyperalgesia was abolished in mice lacking VR1 receptors. 84% of P2X3 receptor positive neurons also stained for the P2Y1 However, thermal hyperalgesia was preserved in P2Y1 receptor, while 25–35% also stained for the P2Y4 receptor. It has receptor-deficient mice and P2Y2, rather than P2Y1, receptors been suggested that while P2X3 receptor activation leads to were proposed for DRG neurons, where coexpression of VR1 increased firing of DRG neurons and subsequently to increased and P2Y2 mRNA was demonstrated (Moriyama et al., 2003). release of sensory transmitter from their central processes, P2Y1 Not only does ATP-induced potentiation of TRPV1-mediated receptor activation may decrease the release of sensory responses have a physiological relevance, but it also has transmitter onto spinal cord neurons and may thereby partly particular significance in pathological conditions where extra- counterbalance the algogenic effect of ATP (Borvendeg et al., cellular ATP levels are often increased (see Premkumar, 2001). 2003; Gerevich et al., 2004). P2Y1 receptors were shown on the DRG neurons expressing TRPV1 receptors usually also human NG (Fong et al., 2002). Patch-clamp studies of cultured express P2X3 receptors (Guo et al., 1999). However, some neurons from DRG were consistent with P2X3 and P2Y1 DRG neurons express P2X3, but not VR1 receptors (Ueno et receptors being present in DRG neurons. P2Y1 receptors on al., 1999). Almost all sensory neurons in lumbosacral DRG rat DRG neurons have been implicated in the mechanisms innervating the bladder coexpress P2X, ASIC, and TRPV1 underlying neuropathic pain following axotomy from cDNA receptors, but not those in the thoracolumbar DRG neurons array studies (Xiao et al., 2002). Inhibition of the M-current by supplying the bladder, indicating that pelvic and hypogastric P2Y receptors on sensory neurons may represent a mechanism afferent pathways to the bladder are structurally and function- for the enhancement of nociception (Bergson & Cook, 2004). ally distinct (Dang et al., 2004). Data have been presented that Some of the neurons gave slow and sustained responses to suggests that activation of homomeric P2X3 receptors in uridine 5V-triphosphate (UTP), consistent with the presence of peripheral terminals of capsaicin-sensitive primary afferent P2Y2 receptors, which had been reported earlier (Molliver et fibers play a role in the induction of nocifensive behavior and al., 2002). Other nucleoside triphosphates, including NTP, thermal hyperalgesia, while activation of heteromeric P2X2/3 GTP, and CTP, and the diphosphates NDP, GDP, UDP, and receptors on capsaicin-insensitive fibers leads to the induction CDP were also active in modulating sodium currents in DRG of mechanical allodynia (Tsuda et al., 2000; Inoue et al., neurons (Park et al., 2004). ATP and UTP were equipotent in 2003a). Single deep dorsal horn neurons in lamina V often increasing axonal transport of membrane-bound organelles in receive excitatory inputs from both these pathways (Nakatsuka cultured DRG neurons, implicating functional involvement of et al., 2002). P2Y2 receptors (Sakama et al., 2003). P2Y receptors (probably VR1 receptors are expressed on urothelial cells as well as 2+ P2Y2 subtype) increased intracellular Ca concentration and afferent nerve terminals in the urinary bladder and experiments subsequent release of calcitonin gene-related peptide (CGRP) with mice lacking this receptor showed reduction in both spinal in isolated neurons from rat DRG (Sanada et al., 2002). Using a cord signaling and reflux voiding during bladder filling and mouse skin sensory nerve preparation, evidence was presented stretch-evoked ATP release was diminished (Birder et al., that P2Y2 receptors in the terminals of capsaicin-sensitive 2002). These findings indicate that VR1 receptors participate in cutaneous sensory neurons mediate nociceptive transmission normal bladder function and are essential for normal mechan- and further that P2Y signaling may contribute to mechanically evoked purinergic signaling by ATP released from the otransduction in low threshold Ah fibers (Stucky et al., 2004). urothelium. ATP, acting via P2Y receptors, augments substance P and Vagal sensory neurons located in the jugular–nodose CGRP release from cultured rat embryonic sensory neurons ganglia complex and their projections to the lung were both exposed to capsaicin (Huang et al., 2003). Bradykinin and ATP, capsaicin-sensitive and -insensitive; ATP and a,h-methylene acting via P2Y receptors, accelerate Ca2+ efflux from rat ATP (a,h-meATP) activated all these sensory fibers (Kollarik sensory neurons via protein kinase C (PKC) and the plasma et al., 2003). 2+ membrane Ca pump isoform 4 and represent a novel Trinitrophenol-ATP (TNP-ATP) is a potent P2X3 and P2X2/3 mechanism to control excitability (Usachev et al., 2002). D- antagonist. A TNP-ATP-resistant P2X ionic current has been myo-inositol 1,4,5-trisphosphate and ryanodine receptors co- reported on the central terminals of capsaicin-insensitive Ay- exist in nodose neurons and can be activated indirectly by ATP, afferent fibers that play a role modulating sensory transmission probably via P2Y receptors (Hoesch et al., 2002). to lamina V nerves (Tsuzuki et al., 2003). Purinergic and vanilloid receptor activation releases gluta- 2.1.3. Interactions between P2 and vanilloid receptors mate from separate cranial afferent terminals in nucleus tractus The capsaicin or transient receptor potential vanilloid solitarius (NTS), corresponding to myelinated and unmyelin- receptor (TRPV1) on sensory endings plays an important role ated pathways in the NTS (Jin et al., 2004). in transducing thermal and inflammatory pain (Caterina et al., Sensory nerve fibers arising from the TG supplying the 2000). Purinergic receptors, in particular P2X3 and P2X2/3 temporomandibular joint have abundant receptors that respond ionotropic and P2Y1 metabotropic receptors, are also expressed to capsaicin, protons, heat, and ATP; retrograde tracing on sensory nerve terminals (see above). P2Y1 receptor- revealed 25%, 41%, and 52% of neurons supplying this joint mediated responses enhance the sensitivity of VR1-mediated exhibited VR1, vanilloid receptor-like protein 1, and P2X3 responses to capsaicin, protons, and temperature in a PKC- receptors, respectively (Ichikawa et al., 2004). The TRPV dependent manner (Tominaga et al., 2001). ATP-induced subfamily, TRPV2, was recently shown to be expressed, not ARTICLE IN PRESS 6 G. Burnstock / Pharmacology & Therapeutics xx (2005) xxx–xxx only on sensory ganglion neurons but also in enteric neurons, hypereflexia in conscious and anesthetised mice, largely via including primary afferent neurons (Kashiba et al., 2004). It is capsaicin-sensitive C-fibers; these effects were dose-depen- likely that some of these enteric neurons also express P2X3 dently inhibited by pyridoxal-5V-phosphate-6-azophenyl-2V,4V receptors (Xiang & Burnstock, 2004a, 2004b). disulphonic acid (PPADS) and TNP-ATP (Hu et al., 2004). In a recent investigation of the effects of P2 receptor ligands in the 2.2. Evidence for purinergic mechanosensory transduction in micturition reflex in female urethane-anesthetised rats, it was different organs concluded that P2X1 and P2X3 receptors play a fundamental role in this reflex; P2X3 receptor blockade raised the pressure Evidence in support of the hypothesis of purinergic and volume thresholds for the reflex, while P2X1 receptor mechanosensory transduction (Burnstock, 1999, 2001a)as blockade diminished motor activity associated with voiding defined in Section 1 is considered in this section. (King et al., 2004). The P2X3 receptor is largely expressed in the IB4 small nociceptive capsaicin-sensitive nerves in the 2.2.1. Urinary bladder DRG, so it is interesting that IB4-conjugated saporin, a Early evidence for ATP release from rabbit urinary bladder cytotoxin that destroys neurons binding IB4, when adminis- epithelial cells by hydrostatic pressure changes was presented tered intrathecally at the level of L6-S1 spinal cord, reduced by Ferguson et al. (1997), who speculated about this being the bladder overactivity induced by ATP infusion (Nishiguchi et basis of a sensory mechanism. Prolonged exposure to a al., 2004). The authors suggest that targeting IB4-binding, non- desensitizing concentration of a,h-meATP significantly re- peptidergic afferent pathways sensitive to capsaicin and ATP duced the activity of mechanosensitive pelvic nerve afferents in may be an effective treatment of overactivity and/or pain an in vitro model of rat urinary bladder (Namasivayam et al., responses of the bladder. 1999). Later, it was shown that mice lacking the P2X3 receptor It has been claimed recently that suburothelial myofibroblast exhibited reduced inflammatory pain and marked urinary cells isolated from human and guinea pig bladder that are bladder hyporeflexia with reduced voiding frequency and distinct from epithelial cells provide an intermediate regulatory increased voiding volume, suggesting that P2X3 receptors are step between urothelial ATP release and afferent excitation involved in mechanosensory transduction underlying both involved in the sensation of bladder fullness (Sui et al., 2004; inflammatory pain and physiological reflexes (Cockayne et Wu et al., 2004a). al., 2000). In a systematic study of purinergic mechanosensory The roles of ATP released from urothelial cells on various transduction in the mouse urinary bladder, ATP was shown to bladder functions have been considered at length in recent be released from urothelial cells during distension and reviews (Wyndaele & De Wachter, 2003; Apodaca, 2004). discharge initiated in pelvic sensory nerves was mimicked by ATP and a,h-meATP and attenuated by P2X3 antagonists as 2.2.2. Ureter well as in P2X3 knockout mice; P2X3 receptors were localized The ureteric colic induced by the passage of a kidney stone on suburothelial sensory nerve fibers (Vlaskovska et al., 2001). causes severe pain. Distension of the ureter resulted in Single unit analysis of sensory fibers in the mouse urinary substantial ATP release from the urothelium in a pressure- bladder revealed both low and high threshold fibers sensitive to dependent manner (Knight et al., 2002). Cell damage was ATP contributing to physiological (non-nociceptive) and shown not to occur during distension with scanning electron nociceptive mechanosensory transduction, respectively (Rong microscopy and after removal of the urothelium there was no et al., 2002). Purinergic agonists increase the excitability of ATP release during distension. Evidence was presented that the afferent fibers to distension (Rong et al., 2002; Yu & de Groat, release of ATP from urothelial cells was vesicular. Immunos- 2004). It appears that the bladder sensory DRG neurons, taining of P2X3 receptors in sensory nerves in the subepithelial projecting via pelvic nerves, express predominantly P2X2/3 region was reported (Lee et al., 2000). Multifiber recordings of heteromultimer receptors (Zhong et al., 2003). The capsaicin- ureter afferent were made using a guinea pig preparation gated ion channel receptor, TRPV1 seems to be required for perfused in vitro (Rong & Burnstock, 2004). Distension of the stretch-evoked ATP release from urothelial cells (Birder et al., ureter resulted in a rapid, followed by maintained, increase in 2002). afferent nerve discharge. The rapid increase was mimicked by Pandita and Andersson (2002) showed that ATP given intraluminal application of ATP or a,h-meATP and TNP-ATP intravesically stimulates the micturition reflex in awake freely attenuated these nerve responses to distension; the maintained moving rats, probably by stimulating suburothelial C-fibers, increase was partly due to adenosine. although it was suggested that other mediators might be involved. Studies of resiniferatoxin desensitization of capsai- 2.2.3. Gut cin-sensitive afferents on detrusor overactivity induced by A hypothesis was proposed suggesting that purinergic intravesical ATP in conscious rats, supported the view that mechanosensory transduction in the gut initiated both physi- increased extracellular ATP has a role in mechanosensory ological reflex modulation of peristalsis via intrinsic sensory transduction and that ATP-induced facilitation of the micturi- fibers and nociception via extrinsic sensory fibers (Burnstock, tion reflex is mediated, at least partly, by nerves other than 2001a, 2001c; Fig. 2B). Evidence in support of this hypothesis capsaicin-sensitive afferents (Zhang et al., 2003; Brady et al., was obtained from a rat pelvic sensory nerve-colorectal 2004). ATP has also been shown to induce a dose-dependent preparation (Wynn et al., 2003). Distension of the colorectum ARTICLE IN PRESS G. Burnstock / Pharmacology & Therapeutics xx (2005) xxx–xxx 7 led to pressure-dependent increase in release of ATP from supply NEBs with their origin in the NG (Brouns et al., 2000, mucosal epithelial cells and also evoked pelvic nerve excita- 2003a). Quinacrine staining of NEBs suggests the presence of tion. This excitation was mimicked by application of ATP and high concentrations of ATP in their secretory vesicles and it is a,h-meATP and attenuated by the selective P2X3 and P2X2/3 suggested that ATP is released in response to both mechanical antagonist, TNP-ATP, and by PPADS. The sensory discharge stimulation during high pressure ventilation (Rich et al., 2003) was potentiated by ARL-67156, an ATPase inhibitor. Single and during hypoxia. NEBs are oxygen sensors especially in fiber analysis showed that high threshold fibers were particu- early development, before the carotid system has matured larly affected by a,h-meATP, suggesting correlation between (Brouns et al., 2003b; Fu et al., 2004). purinergic activation and nociception. Vagal C-fibers innervating the pulmonary system are a,h-meATP was shown to stimulate mechanosensitive derived from cell bodies situated in 2 distinct vagal sensory mucosal and tension receptors in mouse stomach and esophagus ganglia: the jugular (superior) ganglion neurons project fibers leading to activity in vagal afferent nerves (Page et al., 2002). to the extrapulmonary airways (larynx, trachea, bronchus) and ATP also excites mesenteric afferents (Kirkup et al., 1999). the lung parenchymal tissue, while the nodose (inferior) ATP and a,h-meATP activated submucosal terminals of neurons innervate primarily structures within the lungs (Undem intrinsic sensory neurons in the guinea pig intestine (Bertrand et al., 2004). Nerve terminals in the lungs from both jugular & Bornstein, 2002) supporting the hypothesis of Burnstock and nodose ganglia responded to capsaicin and bradykinin, but (2001a) that ATP released from mucosal epithelial cells has a only the nodose C-fibers responded to a,h-meATP. dual action on P2X3 and/or P2X2/3 receptors in the subepithelial sensory nerve plexus. ATP acts on the terminals of low 2.2.5. Carotid body threshold intrinsic enteric sensory neurons to initiate or The ventilatory response to decreased oxygen tension in the modulate intestinal reflexes and acts on the terminals of high arterial blood is initiated by excitation of specialized oxygen- threshold extrinsic sensory fibers to initiate pain. Further sensitive chemoreceptor cells in the carotid body that release support comes from the demonstration that peristalsis is neurotransmitter to activate endings of the sinus nerve afferent impaired in the small intestine of mice lacking the P2X3 fibers. ATP has been shown to stimulate carotid body subunit (Bian et al., 2003) and that up to 75% of the neurons chemoreceptor afferent (Spergel & Lahiri, 1993; McQueen et with P2X3 receptor immunoreactivity in the rat submucosal al., 1998) and the P2 receptor antagonist, suramin, together with plexus expressed calbindin (Xiang & Burnstock, 2004a); a nicotinic antagonist can block hypoxia-induced increase in calbindin is regarded as a marker for intrinsic sensory neurons, chemoreceptor afferent nerve discharge (Zhang et al., 2000). at least in the guinea pig (see Furness et al., 1998). Thirty-two Immunoreactivity for P2X2 and P2X3 receptor subunits has been percent of retrogradely labeled cells in the mouse DRG at localized in rat carotid body afferents (Prasad et al., 2001). These levels T8-L1 and L6-S1, supplying sensory nociceptive nerve findings were confirmed and extended in a recent study where fibers to the mouse distal colon, were immunoreactive for P2X3 P2X2 receptor deficiency resulted in a dramatic reduction in the receptors (Robinson et al., 2004). Intraganglionic laminar nerve responses of the carotid sinus nerve to hypoxia in an in vitro endings (IGLEs) are specialized mechanosensory endings of mouse carotid body-sinus nerve preparation (Rong et al., 2003). vagal afferent nerves in the rat stomach, arising from the ATP mimicked afferent discharge and PPADS blocked the nodose ganglion; they express P2X3 receptors and are probably hypoxia-induced discharge. Immunoreactivity for P2X2 and involved in physiological reflex activity, especially in early P2X3 receptor subunits was detected on afferent terminals postnatal development (Xiang & Burnstock, 2004b). surrounding clusters of glomus cells in wild-type, but not in Purinergic mechanosensory transduction has also been P2X2- and/or P2X3-deficient, mice. Recent evidence has been implicated in reflex control of secretion, whereby ATP released obtained for release of ATP from chemoreceptor type I glomus from mucosal epithelial cells acts on P2Y1 receptors on cells during hypoxic and mechanical stimulation (Gourine, enterochromaffin cells to release 5-hydroxytryptamine, which personal communication; Buttigieg & Nurse, 2004). leads to regulation of secretion either directly or via intrinsic reflex activity (Cooke et al., 2003). 2.2.6. Tooth pulp a,h-meATP caused concentration-dependent excitation of P2X3 and P2X2/3 receptors on sensory afferents in tooth pulp IGLEs of vagal tension receptors in the guinea pig esophagus, appear to mediate nociception (Cook et al., 1997; Alavi et al., but evidence was presented against chemical transmission 2001; Jiang & Gu, 2002; Renton et al., 2003). Mustard oil being involved in the mechanotransduction mechanism (Zagor- application to the tooth pulp in anesthetised rats produced long- odnyuk et al., 2003). A subpopulation of nodose vagal afferent lasting central sensitization, reflected by increases in neuronal nociceptive nerves sensitive to P2X3 receptor agonists was later mechanoreceptive field size; TNP-ATP reversibly attenuated the identified and shown to be different from the non-nociceptive mustard oil sensitisation for more than 15 min (Hu et al., 2002). vagal nerve mechanoreceptors (Yu et al., 2005). 2.2.7. Special senses organs 2.2.4. Lung Pulmonary neuroepithelial bodies (NEBs) serve as sensory 2.2.7.1. Inner ear. ATP has been shown to be an auditory organs in the lung and P2X3 and P2X2/3 receptors are afferent neurotransmitter, alongside glutamate (see Housley, expressed on a subpopulation of vagal sensory fibers that 2000). There are about 50,000 primary afferent neurons in the ARTICLE IN PRESS 8 G. Burnstock / Pharmacology & Therapeutics xx (2005) xxx–xxx

human cochlear and about half express P2X2 (or P2X2 receptors, indicating release of cytosolic ATP (Cook & variants) and, debatably, P2X3 receptors. ATP is released from McCleskey, 2002). Thus, ATP is involved in fast nociceptive K+-depolarized organ of Corti in a Ca2+-dependent manner and signals, while persistent pain after tissue damage involves other an increase in ATP levels in the endolymph has been algogenic compounds, notably bradykinin, prostaglandin, and demonstrated during noise exposure, perhaps released by serotonin. The exception, however, is that persistent pain exocytosis from the marginal cells of the stria vascularis during inflammation appears to be due to sensitisation and/or (Munoz et al., 2001). The P2 receptor antagonist, PPADS, spread of P2X receptors (Cockayne et al., 2000; Wynn et al., attenuated the effects of a moderately intense sound on cochlea 2004). Ca2+ waves in human epidermal keratinocytes mediated 2+ mechanics (Bobbin, 2001). Spiral ganglion neurons, located in by extracellular ATP produce [Ca ]i elevation in DRG the cochlear, convey to the brain stem the acoustic information neurons, suggesting a dynamic cross talk between skin and arising from the mechanoelectrical transduction of the inner sensory neurons mediated by extracellular ATP (Koizumi et al., hair cells and are responsive to ATP (Ito & Duolon, 2002). 2004). Nocifensive behaviors induced by hindpaw administration of ATP and 3V-O-(4-benzoyl)benzoyl ATP (BzATP) appear 2.2.7.2. Tongue. P2X3 receptors are abundantly present on to recruit an additional set of fibers that are not activated by sensory nerve terminals in the tongue (Bo et al., 1999) and ATP a,h-meATP and which trigger the spinal release of substance P and a,h-meATP have been shown to excite trigeminal lingual and are capsaicin selective (Wismer et al., 2003). Locally nerve terminals in an in vitro preparation of intra-arterially released ATP can sensitize large mechanosensitive afferent perfused rat mimicking nociceptive responses to noxious endings via P2 receptors, leading to increased nociceptive mechanical stimulation and high temperature (Rong et al., responses to pressure or touch; it was suggested that such a 2000). A purinergic mechanosensory transduction mechanism mechanism, together with central changes in the dorsal horn for the initiation of pain was considered. Taste sensations, in may contribute to touch-evoked pain (Zhang et al., 2001). contrast, appear to be mediated by P2Y1 receptors mediating ATP has been shown to be an effective stimulant of group impulses in sensory fibers in the chorda tympani (Kataoka et IV receptors in mechanically sensitive muscle afferents (Li & al., 2004). Sinoway, 2002; Reino¨hl et al., 2003). Arterial injection of a,h- meATP in the blood supply of the triceps surae muscle evoked 2.2.7.3. Olfactory epithelium. The olfactory epithelium and a pressor response that was a reflex localized to the cat vomeronasal organs contain olfactory receptor neurons that hindlimb and was reduced by P2X receptor blockade (Li & express P2X2, P2X3, and P2X2/3 receptors (Spehr et al., 2004; Sinoway, 2002). In this study, ATP was also shown to enhance Gayle & Burnstock, 2005). It is suggested that the neighboring the muscle pressor response evoked by mechanically sensitive epithelial supporting cells or the olfactory neurons themselves muscle stretch, which was attenuated by PPADS. Prolonged may release ATP in response to noxious stimuli, acting on P2X muscle pain and tenderness was produced in human muscle by receptors as an endogenous modulator of odor sensitivity infusion of a combination of ATP, serotonin, histamine, and (Hegg et al., 2003; Spehr et al., 2004). Enhanced sensitivity to prostaglandin E2 (Mørk et al., 2003). Strenuous exercise of odors was observed in the presence of P2 antagonists, muscle, as well as inflammation and ischemia, is associated suggesting that low-level endogenous ATP normally reduces with tissue acidosis. Intramuscular injections of acidic phos- odor responsiveness. It was suggested that the predominantly phate buffer at pH 6 or ATP excited a subpopulation of suppressive effect of ATP on odor sensitivity could play a role unmyelinated (group IV) muscle afferent fibers (Hoheisel et al., in reduced odor sensitivity that occurs during acute exposure to 2004), perhaps implicating P2X2 or P2X2/3 receptors that are noxious fumes and may be a novel neuroprotective mechanism sensitive to acidic pH (Liu et al., 2001). (Hegg et al., 2003). ATP has been shown to be a stimulant of articular nociceptors in the knee joint (Dowd et al., 1998) and also to 2.2.7.4. Retina. P2X2 and P2X3 receptor mRNAs are present some extend in lumbar intervertebral disc via P2X3 receptors, in the retina and receptor protein expressed in retinal ganglion but not as prominently as in the skin (Aoki et al., 2003). P2Y2 cells (Bra¨ndle et al., 1998; Wheeler-Schilling et al., 2000, receptor mRNA is expressed in both cultured normal and 2001). P2X3 receptors are also present on Mu¨ller cells (Jabs et osteoarthritic chondrocytes taken from human knee joints and al., 2000). Ciliary epithelial cells release ATP in response to ATP shown to be released by mechanical stimulation (Mill- hypotonic swelling (Mitchell et al., 1998). Mu¨ller cells also ward-Sadler et al., 2004). release ATP during Ca2+ wave propagation (Newman, 2001). 2.3. Sources of ATP involved in mechanosensory transduction 2.2.8. Skin, muscle, and joints ATP and a,h-meATP activate nociceptive sensory nerve Until recently, it was usually assumed that the only source terminals in the skin, which increase in magnitude in of extracellular ATP acting on purinoceptors was damaged or inflammatory conditions due to increase in number and dying cells, but it is now recognized that mechanically responsiveness of P2X receptors (Hamilton et al., 2001; induced ATP release from healthy cells is a physiological Hilliges et al., 2002). Skin cell damage caused action potential mechanism (see Bodin & Burnstock, 2001b; Lazarowski et al., firing and inward currents in nociceptors, which was eliminated 2003; Schwiebert et al., 2003; Bao et al., 2004). There is an by enzymatic degradation of ATP or blockade of P2X active debate, however, about the precise transport mechan- ARTICLE IN PRESS G. Burnstock / Pharmacology & Therapeutics xx (2005) xxx–xxx 9 ism(s) involved. There is compelling evidence for exocytotic receptors in vasomotor neurons in avulsed human DRG vesicular release of ATP from nerves, but for ATP release (central axotomy) (Yiangou et al., 2000). P2X receptors on from non-neuronal cells, various transport mechanisms have DRG neurons increase their activity after inflammation and been proposed, including ATP binding cassette (ABC) contribute to the hypersensitivity to mechanical stimulation in transporters, connexin or pannexin hemichannels, or possibly the inflammatory state (Dai et al., 2002; Chen et al., 2004). plasmalemmal voltage-dependent anion channels, as well as After induction of painful peripheral neuropathy by sciatic vesicular release. nerve entrapment there is also evidence for increased release of During purinergic mechanosensory transduction, the ATP ATP from DRG neurons on the side of the injury (Matsuka et that acts on P2X3 and P2X2/3 receptors on sensory nerve al., 2004). endings is released by mechanical distortion from urothelial The neurosteroid, dehydroepiandrosterone (DHEA), pro- cells during distension of bladder (Ferguson et al., 1997; duced by glial cells and neurons, potentiated P2X2-containing Vlaskovska et al., 2001) and ureter (Knight et al., 2002), from receptors and could therefore lead to sensitisation or increased mucosal epithelial cells during distension of the colorectum activation of nociceptors by ATP (De Roo et al., 2003). The (Wynn et al., 2003). It is probably released from odontoblasts authors suggest that DHEA could be an endogenous modulator in tooth pulp (Alavi et al., 2001), from epithelial cells in the of P2X receptors leading to facilitation of nociceptive messages tongue (Rong et al., 2000), epithelial cells in the lung (Brouns particularly under conditions of inflammatory pain, where the et al., 2000; Arcuino et al., 2002; Brouns et al., 2003a), P2X signaling pathways appear to be up-regulated. keratinocytes in the skin (Greig et al., 2003), and glomus cells Purinergic sensitivity develops in sensory neurons after in the carotid body (Rong et al., 2003). Perhaps surprisingly, chronic peripheral nerve injury (Zhou et al., 2001; Chen et al., evidence was presented that the release of ATP from urothelial 2004). Injection of a combination of the adrenoreceptor cells in the ureter (as well as from endothelial cells; Bodin & antagonist, phentolamine, and the P2 antagonist, suramin, Burnstock, 2001a) is vesicular, since monensin and brefeldin reduced mechanical hypersensitivity in neuropathic rats (Park A, which interfere with vesicular formation and trafficking, et al., 2000). Pelvic and pudendal nerve injury can occur during inhibited distension-evoked ATP release, but not gadolinium, a extirpative visceral surgery such as radical hysterectomy. Many stretch-activated channel inhibitor, or glibenclamide, an inhib- of the patients develop severe chronic pelvic pain and bladder itor of 2 members of the ABC protein family (Knight et al., symptoms (Shembalkar et al., 2001). Mechanical allodynia 2002). caused by surgical injury has been considered to involve local Whatever the mechanism, released ATP is rapidly broken release of ATP in the tissue injury area and its action on P2X down by ectoenzymes to ADP (to act on P2Y1,P2Y12, and nociceptive receptors (Tsuda et al., 2001). The authors suggest P2Y13 receptors) and adenosine (to act on P1 receptors) that agents that block P2 receptors may be useful as pre- (Zimmermann, 2001). emptive antiallogenic drugs for alleviating the postoperative pain syndrome in humans. Involvement of P2X2 and P2X3 3. Neuropathic, inflammatory, and cancer pain receptors in neuropathic pain in the mouse chronic constriction injury model has also been demonstrated (Ueno et al., 2003). Neuropathic pain following peripheral nerve injury, long- Inflammatory mediators such as substance P and bradykinin term pain associated with inflammation, and cancer pain are sensitize nociception through phosphorylation of P2X3 and common and distressing conditions. There is growing recog- P2X2/3 ion channels or associated proteins (Paukert et al., nition of the involvement of purinergic mechanisms in these 2001). This might contribute to the increase in purinergic diseases (see Cain et al., 2001; Irnich et al., 2001; Kidd & nociception in inflammatory conditions. Urban, 2001; Kostyuk et al., 2001; Fukuoka & Noguchi, 2002; Reg-2, a secretory protein with proregenerative properties in Mantyh et al., 2002; Di Virgilio et al., 2003; Jarvis, 2003; motor and sensory neurons after injury, is massively up- Kennedy et al., 2003; Sah et al., 2003; Sawynok & Liu, 2003; regulated in the subpopulation of IB4/P2X3 immunopositive Stone & Vulchanova, 2003; Ueda & Rashid, 2003; Luttikhui- DRG neurons after sciatic nerve injury (Averill et al., 2002). zen et al., 2004; North, 2004; Kennedy, 2005). Most of these The pathophysiological significance of this finding remains to papers are concerned with P2X3 or P2X2/3 and more recently be determined. P2Y receptors on nociceptive sensory nerves. P2X4 receptors It has been suggested that heat shock proteins (HSPs) may on microglia have also been recognized to be involved in be involved in inflammation-related nociception and it has neuropathic pain and most recently disruption of the P2X7 been shown that inhibitors of HSP90 increase the magnitude of receptor gene has been shown to abolish chronic inflammatory currents mediated by P2X and VR1 receptors that are known to and neuropathic pain (Chessell et al., 2005). be involved in inflammation-related nociception (McDowell & Yukhananov, 2002). 3.1. Peripheral purinergic mechanisms Satellite glial cells in mouse TG have been shown to express P2Y, probably P2Y4, receptors and it was speculated that they 3.1.1. Sensory ganglia may be activated by ATP released during nerve injury (Weick In the DRG, the presence of P2X3 mRNA-labeled neurons et al., 2003). There is also evidence for a correlation between increased 3 days after peripheral nerve injury (Tsuzuki et al., activation of P2 receptors on central glia and neuropathic pain 2001). In contrast, there was a decrease in numbers of P2X3 (Watkins & Maier, 2002). ARTICLE IN PRESS 10 G. Burnstock / Pharmacology & Therapeutics xx (2005) xxx–xxx

Oncostatin M is a cytokine involved in inflammatory of interstitial cystitis (Barrick et al., 2002) and in the reactions and the h-subunit of its receptor is expressed in a cyclophosphamide mouse model of interstitial cystitis (Rong subset of nociceptive sensory neurons that also express P2X3 and Burnstock, unpublished data) as well as urothelial cells and VR1 receptors (Tamura et al., 2003). Seven days after from patients with interstitial cystitis (Sun & Chai, 2002). sciatic nerve axotomy, the expression of the h-subunit of the Subsensitivity of P2X3 and P2X2/3 receptors, but not vanilloid oncostatin M receptor was down-regulated in the DRG of the receptors, has been shown in L6-S1 DRG in the rat model of injured side. These findings suggest that oncostatin Mh is cyclophosphamide cystitis (Borvendeg et al., 2003). Release of involved in the modulation of neuronal phenotypes, including ATP from urothelial cells with hypo-osmotic mechanical VR1 and P2X3 receptors, rather than the survival and axonal stimulation was increased by over 600% in inflamed bladder regeneration of these neurons. from cyclophosphamide-treated animals; botulinum toxin DRG neurons normally function as independent sensory inhibited this release (Smith et al., 2004). Botulinum toxin communicators, but most DRG neurons are also transiently was also effective in blocking the bladder overactivity induced activated when axons in the same ganglion are stimulated by ATP (Atiemo et al., 2005). The P2X3 receptor subunit was repetitively (Amir & Devor, 2000). Peripheral inflammation up-regulated during stretch of cultured urothelial cells from enhances the excitability of DRG neurons and activates silent patients with interstitial cystitis (Sun & Chai, 2004). P2X2 and nociceptors (Schaible et al., 2000; Xu & Zhao, 2003). P2X3 receptor expression has been demonstrated recently on Using the foreign body reaction chronic inflammatory rat human bladder urothelial cells (as well as on afferent nerve model, where a sterile inflammatory reaction is induced by terminals); the expression was greater in cells from interstitial implanting degradable cross-linked dermal sheep collagen cystitis bladder (Tempest et al., 2004). discs subcutaneously, up-regulation of P2X7,P2Y1, and P2Y2 An increase in stretch-evoked urothelial release of ATP has receptors occurs in macrophages in the vasculature (Luttikhui- been reported from porcine and human bladders with sensory zen et al., 2004). These receptors are potential therapeutic disorder (urgency) compared with normal bladders (Kumar et targets for the modulation of inflammation. al., 2004). This finding reinforces the view that purinergic Transient TG ischemia appears to provoke a selective mechanosensory transduction is enhanced in pathological decrease in P2X3 receptor expression and it is suggested that conditions. There is also an increase in both neuronal, and this may be related to the altered pain and thermal sensation especially non-neuronal, release of ATP from human bladder (Hwang et al., 2004). While the expression of neurotransmitters strips in old age (Yoshida et al., 2004). or neuromodulators may change, transient ischemia does not cause sensory neuron loss in the TG. 3.1.3. Gut It is beginning to be recognized that the mechanism of The excitability of visceral afferent nerves is enhanced classical mechanical hyperalgesia in inflammation may involve following injury, ischemia and during inflammation, for purinergic mechanosensory transduction, whereby stretch example in irritable bowel syndrome (IBS). Under these evokes release of ATP that then excites nearby primary sensory conditions, substances are released from various sources that nerve terminals. In a recent study, phosphorylated extracellular often act synergistically to cause sensitisation of afferent nerves signal-regulated protein kinase (pERK) immunoreactivity was to mechanical or chemical stimuli. Receptors to these sub- used as a marker indicating functional activation of primary stances (including ATP) represent potential targets for drug afferent neurons in a rat model of peripheral inflammation (Dai treatment aimed at attenuating the inappropriate visceral et al., 2004). The results suggest that P2X3 receptors in primary sensation and subsequent reflex activities that underlie abnor- afferent nerves increase their activity with increased sensitivity mal bowel function and visceral pain (see Holzer, 2001; Kirkup of the intracellular ERK pathway during inflammation and then et al., 2001; Cooke et al., 2003). The sensitizing effects of P2X3 contribute to the hypersensitivity to mechanical noxious purinoceptor agonists on mechanosensory function is induced stimulation in the inflammatory state. in esophagitis (Page et al., 2000). During chronic interstitial inflammation induced by infection of mice with the parasite 3.1.2. Urinary bladder Schistosoma mansoni for 16 weeks, purinergic modulation of The involvement of purinergic signaling is being considered cholinergic nerve activity was impaired (De Man et al., 2003). in recent reviews of diseased bladder and novel purine-related Reviews have been published recently proposing that therapeutic strategies are being explored for overactive bladder, enteric P2X receptors are potential targets for drug treatment incontinence, and interstitial cystitis (see, for example, Boselli of IBS (Galligan, 2004) and inflammation- and ischemia- et al., 2001; Burnstock, 2001b; Andersson, 2002; Andersson & induced disturbances of gut sensation (Holzer, 2004). It is Hedlund, 2002; Ballaro et al., 2003; Fraser et al., 2003; Kumar suggested that antagonists to P2X3 and/or P2X2/3 receptors on et al., 2003; Fowler, 2004; Fry et al., 2004; Moreland et al., nociceptive extrinsic sensory nerves in the gut could attenuate 2004; Ouslander, 2004). abdominal pain in IBS. It is also suggested that agonists acting It has been known for some years now that the purinergic on P2X receptors on intrinsic enteric neurons may enhance component of parasympathetic control of bladder contraction is gastrointestinal propulsion and secretion and that these drugs greatly enhanced in interstitial cystitis (Palea et al., 1993). might be useful for treating constipation-predominant IBS, Recent studies have shown significant increase in release of while P2X antagonists might be useful for treating diarrhea- ATP from the urothelium in response to stretch in the cat model predominant IBS. ARTICLE IN PRESS G. Burnstock / Pharmacology & Therapeutics xx (2005) xxx–xxx 11

P2X3 receptors are up-regulated in human inflammatory 3.2.1. Spinal cord bowel disease at hypersensitivity (Yiangou et al., 2001). Using It has been known for some time that nerve sprouting occurs an animal model of colitis, Wynn et al. (2004) have shown that in the spinal cord after peripheral nerve or dorsal root lesions. the increase in sensory nerve responses to ATP is due to an CGRP-positive neurons display considerably more nerve increase in the number of P2X3 receptor-expressing small sprouting than do IB4-positive neurons (Belyantseva & Lewin, nociceptive neurons in the DRG supplying the colorectum, 1999). However, P2X receptor-mediated pathways play a role in particularly those labeling with CGRP. pathological pain studies (Gu & Heft, 2004). There are 3 The pro-algesic influence of acidic irritants in the peritone- potential sources of ATP release during sensory transmission in um appears to be mediated by both P2X3 and P2X2/3 receptors the spinal cord. ATP may be released from the central terminals and it has been suggested that these receptors might have of primary afferent neurons. ATP may be also released from therapeutic potential in the treatment of acid-related inflam- astrocytes and/or postsynaptic dorsal horn neurons. A novel mation- and ischemia-induced disturbances of gut function and adenosine kinase inhibitor, A-134974, relieves tactile allodynia sensation (Holzer, 2003). via spinal sites of action in peripheral nerve injured rats, adding to the growing evidence that adenosine kinase inhibitors may be 3.1.4. Lung useful as analgesic agents in a broad spectrum of pain states (Zhu Alveolar macrophages express functional P2X7 receptors, et al., 2001). Intrathecal administration of UTP and UDP had which upon stimulation activate proinflammatory interleukin mechanical and thermal antinociceptive effects in normal rats (IL) 1-IL6 cytokine cascade and the formation of multinucleate and antiallodynic effects in a neuropathic pain model, possibly giant cells, a feature of granulomatous reactions (Lemaire & involving spinal P2Y2or4and P2Y6 receptors (Okada et al., Leduc, 2004). Further, Th1 and Th2 cytokines reciprocally 2002). The authors suggest that P2Y receptor agonists may have regulate P2X7 receptor function, suggesting a role for P2X7 potential for the development of a new class of analgesics. After receptors in pulmonary diseases, particularly lung hypersensi- spinal cord injury, an increased number of lumbar microglia tivity associated with chronic inflammatory responses. expressing the P2X4 receptor in the spinal cord of rats with allodynia and hyperalgesia has been reported (Deltoff et al., 3.1.5. Joints 2004; Inoue et al., 2005). It has been known for some time that ATP and adenosine GABA and ATP are coreleased from spinal nerves within the have effects on acute and chronically inflamed joints (Green et dorsal horn of the spinal cord (Jo & Schlichter, 1999). Negative al., 1991; Baharav et al., 2002) and quinacrine (Atabrine), a cross talk between receptors for these cotransmitters, as seen in compound that binds strongly to ATP, has been used for many DRG neurons (Sokolova et al., 2001), may represent a novel years for the treatment of rheumatic diseases, although its mechanism to inhibit afferent excitation to the spinal cord. mechanism of action is not clear (Wallace, 1989). Evidence has Platelet-activating factor (PAF) is a potent inducer of tactile been presented that activation of P2X receptors in the rat allodynia and thermal hyperalgesia at the level of the spinal temporomandibular joint induces nociception and that block- cord; it is suggested that PAF-evoked tactile allodynia is age by PPADS decreases carrageenan-induced inflammatory mediated by ATP and a following N-methyl-d-aspartate hyperalgesia (Oliveira et al., 2005). The anti-inflammatory (NMDA) and nitric oxide (NO) cascade through capsaicin- effect of methotrexate is mediated by increasing extracellular sensitive fibers (Morita et al., 2004). concentration of adenosine (Baharav et al., 2002). Spinal Hyperthyroidism changed ATP and ADP hydrolysis in the adenosine receptor activation inhibits inflammation and joint spinal cord and this coincided with the nociceptive response, destruction (Boyle et al., 2002) and reduces c-fos and astrocyte although the effects of thyroid hormones vary with the activation in dorsal horn (Sorkin et al., 2003) in rat adjuvant- developmental stage (Bruno et al., 2005). These parallel induced arthritis. findings suggest the involvement of adenine nucleotide ATP and UTP activate calcium-mobilizing P2Y2 or P2Y4 hydrolysis-related enzymes in nociceptive pathways. receptors that act synergistically with IL-1 to stimulate prostaglandin E2 release from human rheumatoid synovial 3.2.2. Brain cells (Loredo & Benton, 1998). P2X3 receptor immunoreactivity was shown in the solitary Oxidized ATP inhibits inflammatory pain in arthritic rats by tract and nucleus of the brain stem (Llewellyn-Smith & inhibition of the P2X7 receptor for ATP localized in nerve Burnstock, 1998; Fig. 3), and a recent paper has claimed that terminals (Dell’Antonio et al., 2002a, 2002b). ATP release from the ventral surface of the medulla oblongata is a mediator of chemosensory transduction in the CNS (Gourine et 3.2. Central purinergic mechanisms al., 2005). Intracerebroventricular administration of a,h-meATP has an Changes in central purinergic pathways in neuropathic or antinociceptive effect; evidence has been presented to suggest the inflammatory pain will not be considered in depth in this involvement of supraspinal h-adrenergic and A-opioid receptors review, since some good recent reviews are available on this in this effect (Fukuietal.,2001). Intracerebroventricular topic (Chizh & Illes, 2000; Bardoni, 2001; Jarvis & Kowaluk, coadministration of antagonists to both purinergic and glutamate 2001; Gu, 2003; Sawynok & Liu, 2003; Gourine et al., 2004; receptors resulted in a deeper level of the analgesic and anesthetic Inoue et al., 2004). actions of the individual agents (Masaki et al., 2001). ARTICLE IN PRESS 12 G. Burnstock / Pharmacology & Therapeutics xx (2005) xxx–xxx

Fig. 3. P2X3 receptor immunoreactivity in the solitary nucleus and tract. (A) P2X3 receptor-immunoreactive terminals are present in the NTS. Immunoreactivity also occurs in preterminal axons travelling in the solitary tract (TS). Scale bar=50 Am. (B) A P2X3 receptor-immunoreactive bouton, which contains large granular vesicles, forms synapses (arrowheads) on 3 dendrites in the rostral NTS. An intervaricose segment (asterisk) connects the bouton to another bouton that synapses

(arrowhead) on a fourth dendrite. Scale bar=500 nm. (C) A P2X3 receptor-immunoreactive terminal with complex synaptic interactions in rostral NTS. The P2X3 receptor-positive terminal is pre-synaptic (arrowheads) to 3 dendrites and directly contacts 4 (1–4) other vesicle-containing nerve processes. Process 2 may be pre- synaptic to the immunoreactive terminal since a membrane specialization and clustered vesicles are present (double arrow). This process is also linked to the P2X3 receptor-immunoreactive terminal via a thick symmetrical structural junction (arrow). Processes 3 and 4 contain flattened vesicles. Scale bar=500 nm. (D) In the caudal NTS, 2 large P2X3 receptor-immunoreactive terminals synapse (arrowheads) on the same dendrite and are joined to each other by a thick symmetric membrane specialization (arrow). Large granular vesicles are prominent in terminal 1. Scale bar=500 nm. (E) Two myelinated axons (1, 2) in rostral NTS show

P2X3 receptor immunoreactivity. Axon 1 is <500 nm in diameter; axon 2 is about 4 times as large. Adjacent small-diameter myelinated axons (asterisks) lack receptor immunoreactivity. Scale bar=500 nm (from Llewellyn-Smith & Burnstock, 1998, reproduced with permission from Lippincott Williams & Wilkins).

2+ A hypothesis involving purinergic signaling in migraine factor through Ca influx via P2X7 receptors in microglia pain was put forward many years ago (Burnstock, 1989). A (Kaya et al., 2002). ATP, ADP, and BzATP, acting through recent paper presents evidence to support the view that ATP P2X7 receptors, induce release of the principal proinflamma- may contribute to pain in migraine by sensitizing nociceptors tory cytokine, IL-1h from microglial cells (Chakfe et al., against tissue acidosis via P2Y2 receptor-supported release of 2002). Activation of P2X7 receptors enhances interferon-g- endogenous prostaglandins (Zimmermann et al., 2002). Recent induced NO synthase activity in microglial cells and may studies suggest that ascending noradrenergic nerves arising contribute to inflammatory responses (Gendron et al., 2003). from the locus coeruleus are involved in the supraspinal ATP, via P2X7 receptors, also increases production of 2- antinociception by a,h-meATP through P2X receptors in the arachidonoylglycerol, also involved in inflammation by locus coeruleus (Fukui et al., 2004). microglial cells (Witting et al., 2004). P2X7 receptors have The rostral ventromedial medulla (RVM) serves as a critical been shown to be essential for the development of complete link in bulbospinal nociceptive modulation. Within the RVM, Freund’s adjuvant (CFA)-mediated hypersensitivity and the Fon-cells_ discharge and Foff-cells_ pause immediately prior to a inflammatory response is attenuated in P2X7-null mice nociceptive reflex. Data have been presented to suggest that through modulation of IL-1h and other cytokines (Hughes et on-cells preferentially express P2X receptors and off-cells P2Y al., 2004). receptors (Selden et al., 2004). It was found that in a rat neuropathic pain model displaying allodynia, the level of phospho-p38 was increased in microglia 3.2.3. Microglia and glial–neuron interactions on the injury side of the dorsal horn and that intraspinal The roles of microglia in inflammatory pain, as well as in administration of p38 inhibitor suppressed the allodynia. The neuronal cell death and regeneration, has attracted strong conclusion was that vasospasm pain hypersensitivity depends interest in the past few years. ATP selectively suppresses the on the activation of the p38 signaling pathway on microglia in synthesis of the inflammatory protein microglial response the dorsal horn following peripheral nerve injury. ATP causes ARTICLE IN PRESS G. Burnstock / Pharmacology & Therapeutics xx (2005) xxx–xxx 13

Fig. 4. (A) Marked up-regulation of P2X4 receptors in the spinal dorsal horn after injury to the L5 nerve. Western blot analysis of P2X4 receptor (P2X4R) protein detected by anti-P2X4 receptor antibody in the membrane fraction from the spinal cord ipsilateral to the nerve injury at different times (top panel). The total protein loaded on each lane was stained with Coomassie blue (middle panel). The time course of change in P2X4R protein is similar to that in paw withdrawal threshold (bottom panel). Significance compared with the pre-injury baseline (BL): **P <0.01; ***P <0.001. (B) P2X4 receptor antisense oligodeoxynucleotide (ODN) suppresses the development of tactile allodynia caused by injury to the L5 spinal nerve. Rats were injected intrathecally with antisense ODN (5 nmol) or mismatch ODN (5 nmol) once a day for 7 days. Paw withdrawal threshold (mean T SEM) of tactile stimulation to the hindpaw ipsilateral to the nerve injury. BL, baseline before nerve injury; MM, animals (n =10) treated with mismatch ODN; AS, animals (n =11) treated with antisense ODN (**P <0.01). (C) Hypothesis: neuropathic pain after nerve injury. Tactile allodynia following nerve injury is critically dependent upon functional P2X4 receptors in hyperactive microglia in the dorsal horn. ATP, which might be released or leaked from damaged neurons or astrocytes, stimulates resting microglia to be converted to hyperactive microglia. Hyperactive microglia increases the expression of P2X4 receptors and p38-phosphorelation, resulting in tactile allodynia following nerve injury (A and B: from Tsuda et al., 2003, reproduced with permission from Nature publishing Group. C: from Inoue et al., 2004, reproduced with permission of the Japanese Pharmacological Society). the activation of P38 or ERKl/2 mitogen-activated protein injury. On this basis, it has been claimed that blocking of kinases in microglia, resulting in the release of tumor necrosis P2X4 receptors on microglia might be a new therapeutic factor (Suzuki et al., 2004), as well as IL-6 (Inoue et al., strategy for pain induced by nerve injury (Tsuda et al., 2003b). 2005). It has been shown that pharmacological blockade of P2X4 Long-term increases in pain-related behavior was shown to receptors reversed tactile allodynia caused by peripheral be associated with the activation of spinal microglia after nerve injury without affecting acute pain behaviors in naive subcutaneous injection of formalin into the hindpaw (Fu et animals (Tsuda et al., 2003). After nerve injury, P2X4 al., 1999). Intrathecal delivery of the P2 receptor antagonist receptor expression increased strikingly in the ipsilateral suramin blocked microglia activation and long-term hyper- spinal cord in hyperactive microglia, but not in neurons or algesia induced by formalin injection (Wu et al., 2004b). This astrocytes (Fig. 4). Intraspinal administration of P2X4 adds further support for the view that blocking of spinal P2 antisense oligodeoxynucleotide decreased the induction from receptors might decrease the central enhancement of pain P2X4 receptors and suppressed tactile allodynia after nerve caused by peripheral injury and inflammation. ARTICLE IN PRESS 14 G. Burnstock / Pharmacology & Therapeutics xx (2005) xxx–xxx

3.3. Cancer pain partial sciatic nerve ligation (Barclay et al., 2002). Down- regulation of the P2X3 receptor using RNA interference It was suggested that the unusually high levels of ATP combined with antisense oligonucleotides was proposed to contained in tumor cells (Maehara et al., 1987) may be released have potential benefit for inhibiting expression of a medically by mechanical rupture to activate P2X3 receptors on nearby relevant pain-related gene (Hemmings-Mieszczak et al., 2003). nociceptive sensory nerve fibers (Burnstock, 1996). In a later study, P2X3 SiRNA was shown to relieve chronic Increased expression of P2X3 receptors on CGRP immu- neuropathic pain in an animal model (Dorn et al., 2004). noreactive epidermal sensory nerve fibers in a bone cancer pain A novel potent and selective non-nucleotide antagonist of model has been described (Gilchrist et al., 2005) and in other P2X3 and P2X2/3 receptors, A317491, was introduced in 2002, cancers that involve mechanically sensitive tumors (Mantyh et which reduces chronic inflammatory and neuropathic pain in al., 2002). For example, in bone tumors, destruction reduces the rat (Jarvis et al., 2002; Neelands et al., 2003; Wu et al., the mechanical strength of the bone and antagonists that block 2004c). Intraplantar and intrathecal injections of A317491 the mechanically gated channels and/or ATP receptors in the produced dose-related antinociception in the CFA model of richly innervated periosteum might reduce movement-associ- chronic thermal hyperalgesia (McGaraughty et al., 2003). ated pain. Intrathecal, but not intraplantar, delivery of A317491 attenu- It is interesting that the hyperalgesia associated with tumors ated mechanical allodynia in both chronic constriction injury appears to be linked to increase in expression of P2X3 receptors and L5-L6 nerve ligation models of neuropathy. Unfortunately, in nociceptive sensory neurons expressing CGRP, since this has development of A31741 for therapeutic use in humans has also been described for increased P2X3 receptor expression in a been dropped largely because of its lack of bioavailability. model of inflammatory colitis (Wynn et al., 2004). Increased [3H]A-317491 has been developed as the first useful radi- expression of P2X3 receptors was also reported associated with oligand for the specific labeling of P2X3-containing channels thermal and mechanical hyperalgesia in a rat model of (Jarvis et al., 2004). Potent desensitization of human P2X3 squamous cell carcinoma of the lower gingival (Nagamine et receptors by diadenosine polyphosphates was shown and it was al., 2004). suggested that they may provide an important modulating mechanism for P2X3 receptor activation in vivo (McDonald et 4. Purinergic therapeutic developments for the treatment al., 2002). of pain Phenol red, a pH-sensitive dye and dimethyl sulfoxide, contained in many culture media, have been shown to be potent Suramin, PPADS, and Reactive blue 2 have been used as irreversible antagonists at P2X3 (as well as P2X1 and P2X2) non-selective antagonists at P2X3 and P2X2/3 receptors on receptors (King et al., 2005). Phenol red was shown to be a nociceptive sensory nerve endings (see Ralevic & Burnstock, particularly effective P2X3 antagonist of the micturition reflex 1998; Burnstock, 2001a; Lambrecht et al., 2002), as well as the in female urethane-anesthetized rats (King et al., 2005) and trinitrophenyl substitute nucleotide TNP-ATP as a selective phenol has been used for patients with low-back pain (Koning antagonist at P2X1,P2X3, and P2X2/3 receptors (Mockett et al., et al., 2002). 1994; King et al., 1997; Virginio et al., 1998; Burgard et al., YM529, a new generation of bisphosphonate which is being 2000). Unfortunately, these antagonists degrade in vivo, developed for advanced bone resorption-related diseases, although PPADS dissociates 100–10,000 times more slowly inhibited a,h-meATP-induced cation uptake in a P2X2/3 than the other antagonists (Spelta et al., 2002). Therefore, receptor expressed cell and inhibited the nociceptive behavior together with their lack of bioavailability, they have limited induced by subplantar injection of a,h-meATP and in formalin- potential for therapeutic use. induced nociception (Kakimoto et al., 2004). Modulation of BzATP- and formalin-induced nociception Pilot clinical studies report an analgesic action of Mg2+ on was attenuated by TNP-ATP and enhanced by the P2X3 neuropathic pain insensitive to opioids (Crosby et al., 2000), allosteric modulator, Cibacron blue (Jarvis et al., 2001). TNP- consistent with the demonstration that P2X3 receptor-mediated ATP blocked acetic acid-induced abdominal contraction in responses are inhibited by high Mg2+ or lack of Ca2+, mice (a measure of visceral pain), supporting the view that representing a negative feedback process to limit ATP- activation of P2X3 receptors plays a role in the transmission of mediated nociception (Giniatullin et al., 2003). In view of the 2+ inflammatory visceral pain (Honore et al., 2002b). action of Mg , the probability of interaction of P2X3 receptors Using a Chinese hamster ovary cell line (CHO-K1), specific with NMDA receptors should be considered. inhibition of P2X3 receptor-mediated responses was achieved Tetramethylpyrazine (TMP), a traditional Chinese medicine with a methoxyethoxy-modified phosphorothioated antisense used as an analgesic for dysmenorrhea, was investigated oligonucleotide (Dorn et al., 2001). Continuous intrathecal against acute nociception mediated by P2X3 receptor-mediated administration of P2X3 antisense oligonucleotides for 7 days in activation of rat hindpaw (Liang et al., 2004). Subcutaneous rats significantly decreased nociceptive behaviors observed administration of TMP attenuated the first phase, and to a lesser after injection of CFA, formalin, or a,h-meATP into the extent the second phase, of nociceptive behavior induced by hindpaw (Honore et al., 2002a). Antisense oligonucleotides 5% formalin. The response of neurons in DRG produced by used to down-regulate P2X3 receptors have also been shown to ATP and a,h-meATP was inhibited by TMP (Liang et al., be effective against chronic neuropathic pain produced by 2005). ARTICLE IN PRESS G. Burnstock / Pharmacology & Therapeutics xx (2005) xxx–xxx 15

Trichloroethanol potently inhibited P2X3 receptor-mediated peripheral nerves leads to changes in the spinal cord that responses of HEK293-h P2X3 cells (as well as moderately cause the activation of microglia and associated increases in interfering with P2Y1 and P2Y4 receptor-mediated responses); P2X4 receptor expression, which change the properties of it was suggested that such an effect may be relevant to the adjacent spinal neurons leading to the onset and maintenance interruption of pain transmission in DRG neurons following of pain hypersensitivity. It appears that spinal microglia are ingestion of chloral hydrate or trichloroethylene (Fischer et al., activated in response to peripheral nerve injury, but not to 2003). peripheral inflammation. The mechanisms underlying these Glial cell line-derived neurotrophic factor (GDNF) is glial–neuron interactions are still unknown. necessary for the development of sensory neurons and appears 3. Metabolic breakdown of ATP by ectoenzymes produces to be critical for the survival of DRG cells that bind to IB4 and other purines including ADP and adenosine that are also express P2X3 receptors; intrathecal infusion of GDNF prevents involved in various nociceptive activities. P2X3, P2X2/3, and reverses the behavioral expression of experimental P2X4, and P2X7 receptor antagonists are being developed neuropathic pain arising from injury of spinal nerves, as well for the treatment of pain, although drugs that can be given as loss of binding of IB4 and down-regulation of P2X3 orally and are not degraded in vivo are still awaited. receptors (Wang et al., 2003). GDNF infusion also prevented the development of spinal nerve ligation-induced tactile Acknowledgments hypersensitivity and thermal hyperalgesia. Recent experiments suggest that modulation of neurotrans- Thanks go to Ernie Jennings and Brian King for their advice mission through P2X3 receptors in the central and peripheral and to Gill Knight for her excellent editorial assistance. nervous systems may contribute to anesthesia and analgesia produced by barbiturates (Kitahara et al., 2003). References In mice with a disrupted P2X7 gene, mechanical and thermal hyperalgesia was absent in both inflammatory and Alavi, A. M., Dubyak, G. R., & Burnstock, G. (2001). Immunohistochem- neuropathic pain models, while normal nociceptive processing ical evidence for ATP receptors in human dental pulp. J Dental Res 80, 476–483. was preserved (Casula et al., 2004; Chessell et al., 2005). It Amir, R., & Devor, M. (2000). Functional cross-excitation between afferent A- was proposed that P2X7 receptor activation in satellite and and C-neurons in dorsal root ganglia. Neuroscience 95, 189–195. 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