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Pharmacology & Therapeutic

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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 434

2. Purinergic signaling: physiological reflexes and nociception . . . . . . . . . . . . . . . . . . . . . . 434

2.1. Purinergic receptors expressed by sensory neurons . . . . . . . . . . . . . . . . . . . . . . . 434

2.1.1. P2X receptors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 434

2.1.2. P2Y receptors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 435

2.1.3. Interactions between P2 and vanilloid receptors . . . . . . . . . . . . . . . . . . . . 437

2.2. Evidence for purinergic mechanosensory transduction in different organs . . . . . . . . . . . 438

2.2.1. Urinary bladder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 438

2.2.2. Ureter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 438

2.2.3. Gut . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 438

2.2.4. Lung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 439

2.2.5. Carotid body . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 439

2.2.6. Tooth pulp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 439

2.2.7. Special senses organs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 439

2.2.8. Skin, muscle, and joints . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 440

2.3. Sources of ATP involved in mechanosensory transduction . . . . . . . . . . . . . . . . . . . 440

0163-7258/$ - s

doi:10.1016/j.ph

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s 110 (2006) 433 – 454

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ess: g.burnstock@ucl.ac.uk.

G. Burnstock / Pharmacology & Therapeutics 110 (2006) 433–454434

3. Neuropathic, inflammatory, and cancer pain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 441

3.1. Peripheral purinergic mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 441

3.1.1. Sensory ganglia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 441

3.1.2. Urinary bladder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 442

3.1.3. Gut . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 442

3.1.4. Lung. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 443

3.1.5. Joints . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 443

3.2. Central purinergic mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 443

3.2.1. Spinal cord . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 443

3.2.2. Brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 443

3.2.3. Microglia and glial–neuron interactions. . . . . . . . . . . . . . . . . . . . . . . . . 444

3.3. Cancer pain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 446

4. Purinergic therapeutic developments for the treatment of pain . . . . . . . . . . . . . . . . . . . . . 446

5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 447

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 447

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 447

1. Introduction

There were early hints that adenosine 5V-triphosphate (ATP)might be involved in pain including the demonstration of pain

produced by injection of ATP into human skin blisters (Keele

& Armastrong, 1964; Collier et al., 1966; Bleehen & Keele,

1977), ATP involvement in migraine (Burnstock, 1981), and

ATP participation in pain pathways in the spinal cord (Jahr &

Jessell, 1983; Fyffe & Perl, 1984; Salter & Henry, 1985). A

significant advance was made when the P2X3 ionotropic ion

channel purinergic receptor was cloned in 1995 and shown to

be localized predominantly on small nociceptive sensory

neurons in dorsal root ganglia (DRG) (Chen et al., 1995,

Lewis et al., 1995). Later, Burnstock (1996) put forward a

unifying purinergic hypothesis for the initiation of pain,

suggesting that ATP released as a cotransmitter with noradren-

aline (NA) and neuropeptide Y from sympathetic nerve

terminal varicosities might be involved in sympathetic pain

(causalgia and reflex sympathetic dystrophy); that ATP

released from vascular endothelial cells of microvessels during

reactive hyperemia is associated with pain in migraine, angina,

and ischemia; and that ATP released from tumor cells

(containing high levels) damaged during abrasive activity

reaches P2X3 receptors on nociceptive sensory nerves. This

has been followed by an increasing number of papers

expanding on this concept. Immunohistochemical studies

showed that the nociceptive fibers expressing P2X3 receptors

arose largely from the population of small neurons that labeled

with the lectin isolectin B4 (IB4) (Vulchanova et al., 1996;

Bradbury et al., 1998). The central projections of these

neurons were shown to be in inner lamina II of the dorsal

horn and peripheral projections demonstrated to skin, tooth

pulp, tongue, and subepithelial regions of visceral organs. A

schematic illustrating the initiation of nociception on primary

afferent fibers in the periphery and purinergic relay pathways

in the spinal cord was presented by Burnstock and Wood

(1996) (Fig. 1).

A hypothesis was proposed that purinergic mechanosensory

transduction occurred in visceral tubes and sacs, including

ureter, bladder, and gut, where ATP released from epithelial

cells during distension, acted on P2X3 homomultimeric and

P2X2/3 heteromultimeric receptors on subepithelial sensory

nerves initiating impulses in sensory pathways to pain centers

in the central nervous system (CNS) (Burnstock, 1999) (Fig.

2A). Subsequent studies of bladder (Cockayne et al., 2000;

Vlaskovska et al., 2001; Rong et al., 2002), ureter (Knight et

al., 2002; Rong & Burnstock, 2004), and gut (Wynn et al.,

2003, 2004) have produced evidence in support of this

hypothesis (see also Burnstock, 2001a).

The aim of the present article is to review the large number

of papers that have appeared since 2001 to elaborate on this

theme and to explore the purinergic drugs under development

for the treatment of pain.

2. Purinergic signaling: physiological reflexes and nociception

2.1. Purinergic receptors expressed by sensory neurons

2.1.1. P2X receptors

A comprehensive review of P2X receptor expression and

function in sensory neurons in DRG, nodose (NG), trigeminal

(TG), and petrosal ganglia was presented in 2001 (Dunn et al.,

2001). All P2X subtypes, except P2X7, are found in sensory

neurones, although the P2X3 receptor has the highest level of

expression [both in terms of messenger ribonucleic acid

(mRNA) and protein]. P2X2/3 heteromultimers are particularly

prominent in the nodose ganglion. P2X3 and P2X2/3 receptors

are expressed on isolectin B4 (IB4) binding subpopulations of

small nociceptive neurons. Species differences are recognized.

There have been a remarkably large number of studies of P2X

receptor-mediated signaling in sensory ganglia since this

review and some of these are discussed below.

The decreased sensitivity to noxious stimuli, associated with

the loss of IB4-binding neurons expressing P2X3 receptors,

indicates that these sensory neurons are essential for the

signaling of acute pain (Vulchanova et al., 2001). The loss of

IB4 binding neurons also led to compensatory changes relating

to recovery of sensitivity to acute pain.

Fig. 1. Hypothetical schematic of the roles of purine nucleotides and

nucleosides in pain pathways. At sensory nerve terminals in the periphery,

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 fibers. 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 Burnstock & Wood, 1996, reproduced with

permission of Elsevier).

G. Burnstock / Pharmacology & Therapeutics 110 (2006) 433–454 435

Rapid reduction of the excitatory action of ATP on DRG

neurons by g-amino butyric acid (GABA), probably via

GABAA anionic receptors, and slow inhibition of ATP

currents via metabotropic GABAB receptors appear to be

additional mechanisms of sensory information processing

(Sokolova et al., 2001; Labrakakis et al., 2003; Sokolova et

al., 2003). Fibers project from DRG to the superficial lamina

of the dorsal horn of the spinal cord where the receptors may

function to modulate transmitter release near their central

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 prostaglan-

din 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 re-

ceptor 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

injury and whether this has physiological or pathophysiological

significance.

mRNA for an orphan G protein-coupled receptor TGR7,

which is specifically responsive to h-alanine, is coexpressed in

small diameter neurons with P2X3 and vanilloid receptor type 1

(VR1) receptors in both rat and monkey DRG (Shinohara et al.,

2004). h-Alanine has been claimed to participate in synaptic

transmission as a neurotransmitter and/or neuromodulator and

the authors suggest that TGR7 may participate in the

modulation of neuropathic pain.

Ca2+/calmodulin-dependent protein kinase II, up-regulated

by electrical stimulation, enhances P2X3 receptor activity in

DRG neurons and it is suggested that this may play a key role

in the sensitisation of P2X receptors under injurious conditions

(Xu & Huang, 2004).

2.1.2. P2Y receptors

While the predominant P2 receptor subtypes expressed in

sensory neurons involved in the initiation of nociception was

recognized early as P2X3 and P2X2/3 (see Burnstock & Wood,

1996; Burnstock, 2000), it has become apparent more recently

that P2Y receptors are also present (Nakamura & Strittmatter,

1996; Svichar et al., 1997; Xiao et al., 2002; Malin et al., 2004;

Nakayama et al., 2004), which are involved in modulation of

pain transmission (Gerevich & Illes, 2004). RT-PCR showed

that P2Y1, P2Y2, P2Y4, and P2Y6 mRNA is expressed on

neurons in DRG, NG, and TG ganglia and receptor protein for

P2Y1 is localized on over 80% of mostly small neurons (Ruan

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.).

G. Burnstock / Pharmacology & Therapeutics 110 (2006) 433–454436

G. Burnstock / Pharmacology & Therapeutics 110 (2006) 433–454 437

& Burnstock, 2003). Double immunolabeling showed that 73–

84% of P2X3 receptor positive neurons also stained for the P2Y1

receptor, while 25–35% also stained for the P2Y4 receptor. It has

been suggested that while P2X3 receptor activation leads to

increased firing of DRG neurons and subsequently to increased

release of sensory transmitter from their central processes, P2Y1

receptor activation may decrease the release of sensory

transmitter onto spinal cord neurons and may thereby partly

counterbalance the algogenic effect of ATP (Borvendeg et al.,

2003; Gerevich et al., 2004). P2Y1 receptors were shown on the

human NG (Fong et al., 2002). Patch-clamp studies of cultured

neurons from DRG were consistent with P2X3 and P2Y1

receptors being present in DRG neurons. P2Y1 receptors on

rat DRG neurons have been implicated in the mechanisms

underlying neuropathic pain following axotomy from cDNA

array studies (Xiao et al., 2002). Inhibition of the M-current by

P2Y receptors on sensory neurons may represent a mechanism

for the enhancement of nociception (Bergson & Cook, 2004).

Some of the neurons gave slow and sustained responses to

uridine 5V-triphosphate (UTP), consistent with the presence of

P2Y2 receptors, which had been reported earlier (Molliver et

al., 2002). Other nucleoside triphosphates, including NTP,

GTP, and CTP, and the diphosphates NDP, GDP, UDP, and

CDP were also active in modulating sodium currents in DRG

neurons (Park et al., 2004). ATP and UTP were equipotent in

increasing axonal transport of membrane-bound organelles in

cultured DRG neurons, implicating functional involvement of

P2Y2 receptors (Sakama et al., 2003). P2Y receptors (probably

P2Y2 subtype) increased intracellular Ca2+ concentration and

subsequent release of calcitonin gene-related peptide (CGRP)

in isolated neurons from rat DRG (Sanada et al., 2002). Using a

mouse skin sensory nerve preparation, evidence was presented

that P2Y2 receptors in the terminals of capsaicin-sensitive

cutaneous sensory neurons mediate nociceptive transmission

and further that P2Y signaling may contribute to mechan-

otransduction in low threshold Ah fibers (Stucky et al., 2004).

ATP, acting via P2Y receptors, augments substance P and

CGRP release from cultured rat embryonic sensory neurons

exposed to capsaicin (Huang et al., 2003). Bradykinin and ATP,

acting via P2Y receptors, accelerate Ca2+ efflux from rat

sensory neurons via protein kinase C (PKC) and the plasma

membrane Ca2+ pump isoform 4 and represent a novel

mechanism to control excitability (Usachev et al., 2002). D-

myo-inositol 1,4,5-trisphosphate and ryanodine receptors co-

exist in nodose neurons and can be activated indirectly by ATP,

probably via P2Y receptors (Hoesch et al., 2002).

2.1.3. Interactions between P2 and vanilloid receptors

The capsaicin or transient receptor potential vanilloid

receptor (TRPV1) on sensory endings plays an important role

in transducing thermal and inflammatory pain (Caterina et al.,

2000). Purinergic receptors, in particular P2X3 and P2X2/3

ionotropic and P2Y1 metabotropic receptors, are also expressed

on sensory nerve terminals (see above). P2Y1 receptor-

mediated responses enhance the sensitivity of VR1-mediated

responses to capsaicin, protons, and temperature in a PKC-

dependent manner (Tominaga et al., 2001). ATP-induced

hyperalgesia was abolished in mice lacking VR1 receptors.

However, thermal hyperalgesia was preserved in P2Y1

receptor-deficient mice and P2Y2, rather than P2Y1, receptors

were proposed for DRG neurons, where coexpression of VR1

and P2Y2 mRNA was demonstrated (Moriyama et al., 2003).

Not only does ATP-induced potentiation of TRPV1-mediated

responses have a physiological relevance, but it also has

particular significance in pathological conditions where extra-

cellular ATP levels are often increased (see Premkumar, 2001).

DRG neurons expressing TRPV1 receptors usually also

express P2X3 receptors (Guo et al., 1999). However, some

DRG neurons express P2X3, but not VR1 receptors (Ueno et

al., 1999). Almost all sensory neurons in lumbosacral DRG

innervating the bladder coexpress P2X, ASIC, and TRPV1

receptors, but not those in the thoracolumbar DRG neurons

supplying the bladder, indicating that pelvic and hypogastric

afferent pathways to the bladder are structurally and function-

ally distinct (Dang et al., 2004). Data have been presented that

suggests that activation of homomeric P2X3 receptors in

peripheral terminals of capsaicin-sensitive primary afferent

fibers play a role in the induction of nocifensive behavior and

thermal hyperalgesia, while activation of heteromeric P2X2/3

receptors on capsaicin-insensitive fibers leads to the induction

of mechanical allodynia (Tsuda et al., 2000; Inoue et al.,

2003a). Single deep dorsal horn neurons in lamina V often

receive excitatory inputs from both these pathways (Nakatsuka

et al., 2002).

VR1 receptors are expressed on urothelial cells as well as

afferent nerve terminals in the urinary bladder and experiments

with mice lacking this receptor showed reduction in both spinal

cord signaling and reflux voiding during bladder filling and

stretch-evoked ATP release was diminished (Birder et al.,

2002). These findings indicate that VR1 receptors participate in

normal bladder function and are essential for normal mechan-

ically evoked purinergic signaling by ATP released from the

urothelium.

Vagal sensory neurons located in the jugular–nodose

ganglia complex and their projections to the lung were both

capsaicin-sensitive and -insensitive; ATP and a,h-methylene

ATP (a,h-meATP) activated all these sensory fibers (Kollarik

et al., 2003).

Trinitrophenol-ATP (TNP-ATP) is a potent P2X3 and P2X2/3

antagonist. A TNP-ATP-resistant P2X ionic current has been

reported on the central terminals of capsaicin-insensitive Ay-afferent fibers that play a role modulating sensory transmission

to lamina V nerves (Tsuzuki et al., 2003).

Purinergic and vanilloid receptor activation releases gluta-

mate from separate cranial afferent terminals in nucleus tractus

solitarius (NTS), corresponding to myelinated and unmyelin-

ated pathways in the NTS (Jin et al., 2004).

Sensory nerve fibers arising from the TG supplying the

temporomandibular joint have abundant receptors that respond

to capsaicin, protons, heat, and ATP; retrograde tracing

revealed 25%, 41%, and 52% of neurons supplying this joint

exhibited VR1, vanilloid receptor-like protein 1, and P2X3

receptors, respectively (Ichikawa et al., 2004). The TRPV

subfamily, TRPV2, was recently shown to be expressed, not

G. Burnstock / Pharmacology & Therapeutics 110 (2006) 433–454438

only on sensory ganglion neurons but also in enteric neurons,

including primary afferent neurons (Kashiba et al., 2004). It is

likely that some of these enteric neurons also express P2X3

receptors (Xiang & Burnstock, 2004a, 2004b).

2.2. Evidence for purinergic mechanosensory transduction in

different organs

Evidence in support of the hypothesis of purinergic

mechanosensory transduction (Burnstock, 1999, 2001a) as

defined in Section 1 is considered in this section.

2.2.1. Urinary bladder

Early evidence for ATP release from rabbit urinary bladder

epithelial cells by hydrostatic pressure changes was presented

by Ferguson et al. (1997), who speculated about this being the

basis of a sensory mechanism. Prolonged exposure to a

desensitizing concentration of a,h-meATP significantly re-

duced the activity of mechanosensitive pelvic nerve afferents in

an in vitro model of rat urinary bladder (Namasivayam et al.,

1999). Later, it was shown that mice lacking the P2X3 receptor

exhibited reduced inflammatory pain and marked urinary

bladder hyporeflexia with reduced voiding frequency and

increased voiding volume, suggesting that P2X3 receptors are

involved in mechanosensory transduction underlying both

inflammatory pain and physiological reflexes (Cockayne et

al., 2000). In a systematic study of purinergic mechanosensory

transduction in the mouse urinary bladder, ATP was shown to

be released from urothelial cells during distension and

discharge initiated in pelvic sensory nerves was mimicked by

ATP and a,h-meATP and attenuated by P2X3 antagonists as

well as in P2X3 knockout mice; P2X3 receptors were localized

on suburothelial sensory nerve fibers (Vlaskovska et al., 2001).

Single unit analysis of sensory fibers in the mouse urinary

bladder revealed both low and high threshold fibers sensitive to

ATP contributing to physiological (non-nociceptive) and

nociceptive mechanosensory transduction, respectively (Rong

et al., 2002). Purinergic agonists increase the excitability of

afferent fibers to distension (Rong et al., 2002; Yu & de Groat,

2004). It appears that the bladder sensory DRG neurons,

projecting via pelvic nerves, express predominantly P2X2/3

heteromultimer receptors (Zhong et al., 2003). The capsaicin-

gated ion channel receptor, TRPV1 seems to be required for

stretch-evoked ATP release from urothelial cells (Birder et al.,

2002).

Pandita and Andersson (2002) showed that ATP given

intravesically stimulates the micturition reflex in awake freely

moving rats, probably by stimulating suburothelial C-fibers,

although it was suggested that other mediators might be

involved. Studies of resiniferatoxin desensitization of capsai-

cin-sensitive afferents on detrusor overactivity induced by

intravesical ATP in conscious rats, supported the view that

increased extracellular ATP has a role in mechanosensory

transduction and that ATP-induced facilitation of the micturi-

tion reflex is mediated, at least partly, by nerves other than

capsaicin-sensitive afferents (Zhang et al., 2003; Brady et al.,

2004). ATP has also been shown to induce a dose-dependent

hypereflexia in conscious and anesthetised mice, largely via

capsaicin-sensitive C-fibers; these effects were dose-depen-

dently inhibited by pyridoxal-5V-phosphate-6-azophenyl-2V,4Vdisulphonic acid (PPADS) and TNP-ATP (Hu et al., 2004). In a

recent investigation of the effects of P2 receptor ligands in the

micturition reflex in female urethane-anesthetised rats, it was

concluded that P2X1 and P2X3 receptors play a fundamental

role in this reflex; P2X3 receptor blockade raised the pressure

and volume thresholds for the reflex, while P2X1 receptor

blockade diminished motor activity associated with voiding

(King et al., 2004). The P2X3 receptor is largely expressed in

the IB4 small nociceptive capsaicin-sensitive nerves in the

DRG, so it is interesting that IB4-conjugated saporin, a

cytotoxin that destroys neurons binding IB4, when adminis-

tered intrathecally at the level of L6-S1 spinal cord, reduced

bladder overactivity induced by ATP infusion (Nishiguchi et

al., 2004). The authors suggest that targeting IB4-binding, non-

peptidergic afferent pathways sensitive to capsaicin and ATP

may be an effective treatment of overactivity and/or pain

responses of the bladder.

It has been claimed recently that suburothelial myofibroblast

cells isolated from human and guinea pig bladder that are

distinct from epithelial cells provide an intermediate regulatory

step between urothelial ATP release and afferent excitation

involved in the sensation of bladder fullness (Sui et al., 2004;

Wu et al., 2004a).

The roles of ATP released from urothelial cells on various

bladder functions have been considered at length in recent

reviews (Wyndaele & De Wachter, 2003; Apodaca, 2004).

2.2.2. Ureter

The ureteric colic induced by the passage of a kidney stone

causes severe pain. Distension of the ureter resulted in

substantial ATP release from the urothelium in a pressure-

dependent manner (Knight et al., 2002). Cell damage was

shown not to occur during distension with scanning electron

microscopy and after removal of the urothelium there was no

ATP release during distension. Evidence was presented that the

release of ATP from urothelial cells was vesicular. Immunos-

taining of P2X3 receptors in sensory nerves in the subepithelial

region was reported (Lee et al., 2000). Multifiber recordings of

ureter afferent were made using a guinea pig preparation

perfused in vitro (Rong & Burnstock, 2004). Distension of the

ureter resulted in a rapid, followed by maintained, increase in

afferent nerve discharge. The rapid increase was mimicked by

intraluminal application of ATP or a,h-meATP and TNP-ATP

attenuated these nerve responses to distension; the maintained

increase was partly due to adenosine.

2.2.3. Gut

A hypothesis was proposed suggesting that purinergic

mechanosensory transduction in the gut initiated both physi-

ological reflex modulation of peristalsis via intrinsic sensory

fibers and nociception via extrinsic sensory fibers (Burnstock,

2001a, 2001c; Fig. 2B). Evidence in support of this hypothesis

was obtained from a rat pelvic sensory nerve-colorectal

preparation (Wynn et al., 2003). Distension of the colorectum

G. Burnstock / Pharmacology & Therapeutics 110 (2006) 433–454 439

led to pressure-dependent increase in release of ATP from

mucosal epithelial cells and also evoked pelvic nerve excita-

tion. This excitation was mimicked by application of ATP and

a,h-meATP and attenuated by the selective P2X3 and P2X2/3

antagonist, TNP-ATP, and by PPADS. The sensory discharge

was potentiated by ARL-67156, an ATPase inhibitor. Single

fiber analysis showed that high threshold fibers were particu-

larly affected by a,h-meATP, suggesting correlation between

purinergic activation and nociception.

a,h-meATP was shown to stimulate mechanosensitive

mucosal and tension receptors in mouse stomach and esophagus

leading to activity in vagal afferent nerves (Page et al., 2002).

ATP also excites mesenteric afferents (Kirkup et al., 1999).

ATP and a,h-meATP activated submucosal terminals of

intrinsic sensory neurons in the guinea pig intestine (Bertrand

& Bornstein, 2002) supporting the hypothesis of Burnstock

(2001a) that ATP released from mucosal epithelial cells has a

dual action on P2X3 and/or P2X2/3 receptors in the subepithe-

lial sensory nerve plexus. ATP acts on the terminals of low

threshold intrinsic enteric sensory neurons to initiate or

modulate intestinal reflexes and acts on the terminals of high

threshold extrinsic sensory fibers to initiate pain. Further

support comes from the demonstration that peristalsis is

impaired in the small intestine of mice lacking the P2X3

subunit (Bian et al., 2003) and that up to 75% of the neurons

with P2X3 receptor immunoreactivity in the rat submucosal

plexus expressed calbindin (Xiang & Burnstock, 2004a);

calbindin is regarded as a marker for intrinsic sensory neurons,

at least in the guinea pig (see Furness et al., 1998). Thirty-two

percent of retrogradely labeled cells in the mouse DRG at

levels T8-L1 and L6-S1, supplying sensory nociceptive nerve

fibers to the mouse distal colon, were immunoreactive for P2X3

receptors (Robinson et al., 2004). Intraganglionic laminar nerve

endings (IGLEs) are specialized mechanosensory endings of

vagal afferent nerves in the rat stomach, arising from the

nodose ganglion; they express P2X3 receptors and are probably

involved in physiological reflex activity, especially in early

postnatal development (Xiang & Burnstock, 2004b).

Purinergic mechanosensory transduction has also been

implicated in reflex control of secretion, whereby ATP released

from mucosal epithelial cells acts on P2Y1 receptors on

enterochromaffin cells to release 5-hydroxytryptamine, which

leads to regulation of secretion either directly or via intrinsic

reflex activity (Cooke et al., 2003).

a,h-meATP caused concentration-dependent excitation of

IGLEs of vagal tension receptors in the guinea pig esophagus,

but evidence was presented against chemical transmission

being involved in the mechanotransduction mechanism (Zagor-

odnyuk et al., 2003). A subpopulation of nodose vagal afferent

nociceptive nerves sensitive to P2X3 receptor agonists was later

identified and shown to be different from the non-nociceptive

vagal nerve mechanoreceptors (Yu et al., 2005).

2.2.4. Lung

Pulmonary neuroepithelial bodies (NEBs) serve as sensory

organs in the lung and P2X3 and P2X2/3 receptors are

expressed on a subpopulation of vagal sensory fibers that

supply NEBs with their origin in the NG (Brouns et al., 2000,

2003a). Quinacrine staining of NEBs suggests the presence of

high concentrations of ATP in their secretory vesicles and it is

suggested that ATP is released in response to both mechanical

stimulation during high pressure ventilation (Rich et al., 2003)

and during hypoxia. NEBs are oxygen sensors especially in

early development, before the carotid system has matured

(Brouns et al., 2003b; Fu et al., 2004).

Vagal C-fibers innervating the pulmonary system are

derived from cell bodies situated in 2 distinct vagal sensory

ganglia: the jugular (superior) ganglion neurons project fibers

to the extrapulmonary airways (larynx, trachea, bronchus) and

the lung parenchymal tissue, while the nodose (inferior)

neurons innervate primarily structures within the lungs (Undem

et al., 2004). Nerve terminals in the lungs from both jugular

and nodose ganglia responded to capsaicin and bradykinin, but

only the nodose C-fibers responded to a,h-meATP.

2.2.5. Carotid body

The ventilatory response to decreased oxygen tension in the

arterial blood is initiated by excitation of specialized oxygen-

sensitive chemoreceptor cells in the carotid body that release

neurotransmitter to activate endings of the sinus nerve afferent

fibers. ATP has been shown to stimulate carotid body

chemoreceptor afferent (Spergel & Lahiri, 1993; McQueen et

al., 1998) and the P2 receptor antagonist, suramin, together with

a nicotinic antagonist can block hypoxia-induced increase in

chemoreceptor afferent nerve discharge (Zhang et al., 2000).

Immunoreactivity for P2X2 and P2X3 receptor subunits has been

localized in rat carotid body afferents (Prasad et al., 2001). These

findings were confirmed and extended in a recent study where

P2X2 receptor deficiency resulted in a dramatic reduction in the

responses of the carotid sinus nerve to hypoxia in an in vitro

mouse carotid body-sinus nerve preparation (Rong et al., 2003).

ATP mimicked afferent discharge and PPADS blocked the

hypoxia-induced discharge. Immunoreactivity for P2X2 and

P2X3 receptor subunits was detected on afferent terminals

surrounding clusters of glomus cells in wild-type, but not in

P2X2- and/or P2X3-deficient, mice. Recent evidence has been

obtained for release of ATP from chemoreceptor type I glomus

cells during hypoxic and mechanical stimulation (Gourine,

personal communication; Buttigieg & Nurse, 2004).

2.2.6. Tooth pulp

P2X3 and P2X2/3 receptors on sensory afferents in tooth pulp

appear to mediate nociception (Cook et al., 1997; Alavi et al.,

2001; Jiang & Gu, 2002; Renton et al., 2003). Mustard oil

application to the tooth pulp in anesthetised rats produced long-

lasting central sensitization, reflected by increases in neuronal

mechanoreceptive field size; TNP-ATP reversibly attenuated the

mustard oil sensitisation for more than 15 min (Hu et al., 2002).

2.2.7. Special senses organs

2.2.7.1. Inner ear. ATP has been shown to be an auditory

afferent neurotransmitter, alongside glutamate (see Housley,

2000). There are about 50,000 primary afferent neurons in the

G. Burnstock / Pharmacology & Therapeutics 110 (2006) 433–454440

human cochlear and about half express P2X2 (or P2X2

variants) and, debatably, P2X3 receptors. ATP is released from

K+-depolarized organ of Corti in a Ca2+-dependent manner and

an increase in ATP levels in the endolymph has been

demonstrated during noise exposure, perhaps released by

exocytosis from the marginal cells of the stria vascularis

(Munoz et al., 2001). The P2 receptor antagonist, PPADS,

attenuated the effects of a moderately intense sound on cochlea

mechanics (Bobbin, 2001). Spiral ganglion neurons, located in

the cochlear, convey to the brain stem the acoustic information

arising from the mechanoelectrical transduction of the inner

hair cells and are responsive to ATP (Ito & Duolon, 2002).

2.2.7.2. Tongue. P2X3 receptors are abundantly present on

sensory nerve terminals in the tongue (Bo et al., 1999) and ATP

and a,h-meATP have been shown to excite trigeminal lingual

nerve terminals in an in vitro preparation of intra-arterially

perfused rat mimicking nociceptive responses to noxious

mechanical stimulation and high temperature (Rong et al.,

2000). A purinergic mechanosensory transduction mechanism

for the initiation of pain was considered. Taste sensations, in

contrast, appear to be mediated by P2Y1 receptors mediating

impulses in sensory fibers in the chorda tympani (Kataoka et

al., 2004).

2.2.7.3. Olfactory epithelium. The olfactory epithelium and

vomeronasal organs contain olfactory receptor neurons that

express P2X2, P2X3, and P2X2/3 receptors (Spehr et al., 2004;

Gayle & Burnstock, 2005). It is suggested that the neighboring

epithelial supporting cells or the olfactory neurons themselves

may release ATP in response to noxious stimuli, acting on P2X

receptors as an endogenous modulator of odor sensitivity

(Hegg et al., 2003; Spehr et al., 2004). Enhanced sensitivity to

odors was observed in the presence of P2 antagonists,

suggesting that low-level endogenous ATP normally reduces

odor responsiveness. It was suggested that the predominantly

suppressive effect of ATP on odor sensitivity could play a role

in reduced odor sensitivity that occurs during acute exposure to

noxious fumes and may be a novel neuroprotective mechanism

(Hegg et al., 2003).

2.2.7.4. Retina. P2X2 and P2X3 receptor mRNAs are present

in the retina and receptor protein expressed in retinal ganglion

cells (Brandle et al., 1998; Wheeler-Schilling et al., 2000,

2001). P2X3 receptors are also present on Muller cells (Jabs et

al., 2000). Ciliary epithelial cells release ATP in response to

hypotonic swelling (Mitchell et al., 1998). Muller cells also

release ATP during Ca2+ wave propagation (Newman, 2001).

2.2.8. Skin, muscle, and joints

ATP and a,h-meATP activate nociceptive sensory nerve

terminals in the skin, which increase in magnitude in

inflammatory conditions due to increase in number and

responsiveness of P2X receptors (Hamilton et al., 2001;

Hilliges et al., 2002). Skin cell damage caused action potential

firing and inward currents in nociceptors, which was eliminated

by enzymatic degradation of ATP or blockade of P2X

receptors, indicating release of cytosolic ATP (Cook &

McCleskey, 2002). Thus, ATP is involved in fast nociceptive

signals, while persistent pain after tissue damage involves other

algogenic compounds, notably bradykinin, prostaglandin, and

serotonin. The exception, however, is that persistent pain

during inflammation appears to be due to sensitisation and/or

spread of P2X receptors (Cockayne et al., 2000; Wynn et al.,

2004). Ca2+ waves in human epidermal keratinocytes mediated

by extracellular ATP produce [Ca2+]i elevation in DRG

neurons, suggesting a dynamic cross talk between skin and

sensory neurons mediated by extracellular ATP (Koizumi et al.,

2004). Nocifensive behaviors induced by hindpaw administra-

tion of ATP and 3V-O-(4-benzoyl)benzoyl ATP (BzATP) appear

to recruit an additional set of fibers that are not activated by

a,h-meATP and which trigger the spinal release of substance P

and are capsaicin selective (Wismer et al., 2003). Locally

released ATP can sensitize large mechanosensitive afferent

endings via P2 receptors, leading to increased nociceptive

responses to pressure or touch; it was suggested that such a

mechanism, together with central changes in the dorsal horn

may contribute to touch-evoked pain (Zhang et al., 2001).

ATP has been shown to be an effective stimulant of group

IV receptors in mechanically sensitive muscle afferents (Li &

Sinoway, 2002; Reinohl et al., 2003). Arterial injection of a,h-meATP in the blood supply of the triceps surae muscle evoked

a pressor response that was a reflex localized to the cat

hindlimb and was reduced by P2X receptor blockade (Li &

Sinoway, 2002). In this study, ATP was also shown to enhance

the muscle pressor response evoked by mechanically sensitive

muscle stretch, which was attenuated by PPADS. Prolonged

muscle pain and tenderness was produced in human muscle by

infusion of a combination of ATP, serotonin, histamine, and

prostaglandin E2 (Mørk et al., 2003). Strenuous exercise of

muscle, as well as inflammation and ischemia, is associated

with tissue acidosis. Intramuscular injections of acidic phos-

phate buffer at pH 6 or ATP excited a subpopulation of

unmyelinated (group IV) muscle afferent fibers (Hoheisel et al.,

2004), perhaps implicating P2X2 or P2X2/3 receptors that are

sensitive to acidic pH (Liu et al., 2001).

ATP has been shown to be a stimulant of articular

nociceptors in the knee joint (Dowd et al., 1998) and also to

some extend in lumbar intervertebral disc via P2X3 receptors,

but not as prominently as in the skin (Aoki et al., 2003). P2Y2

receptor mRNA is expressed in both cultured normal and

osteoarthritic chondrocytes taken from human knee joints and

ATP shown to be released by mechanical stimulation (Mill-

ward-Sadler et al., 2004).

2.3. Sources of ATP involved in mechanosensory transduction

Until recently, it was usually assumed that the only source

of extracellular ATP acting on purinoceptors was damaged or

dying cells, but it is now recognized that mechanically

induced ATP release from healthy cells is a physiological

mechanism (see Bodin & Burnstock, 2001b; Lazarowski et al.,

2003; Schwiebert et al., 2003; Bao et al., 2004). There is an

active debate, however, about the precise transport mechan-

G. Burnstock / Pharmacology & Therapeutics 110 (2006) 433–454 441

ism(s) involved. There is compelling evidence for exocytotic

vesicular release of ATP from nerves, but for ATP release

from non-neuronal cells, various transport mechanisms have

been proposed, including ATP binding cassette (ABC)

transporters, connexin or pannexin hemichannels, or possibly

plasmalemmal voltage-dependent anion channels, as well as

vesicular release.

During purinergic mechanosensory transduction, the ATP

that acts on P2X3 and P2X2/3 receptors on sensory nerve

endings is released by mechanical distortion from urothelial

cells during distension of bladder (Ferguson et al., 1997;

Vlaskovska et al., 2001) and ureter (Knight et al., 2002), from

mucosal epithelial cells during distension of the colorectum

(Wynn et al., 2003). It is probably released from odontoblasts

in tooth pulp (Alavi et al., 2001), from epithelial cells in the

tongue (Rong et al., 2000), epithelial cells in the lung (Brouns

et al., 2000; Arcuino et al., 2002; Brouns et al., 2003a),

keratinocytes in the skin (Greig et al., 2003), and glomus cells

in the carotid body (Rong et al., 2003). Perhaps surprisingly,

evidence was presented that the release of ATP from urothelial

cells in the ureter (as well as from endothelial cells; Bodin &

Burnstock, 2001a) is vesicular, since monensin and brefeldin

A, which interfere with vesicular formation and trafficking,

inhibited distension-evoked ATP release, but not gadolinium, a

stretch-activated channel inhibitor, or glibenclamide, an inhib-

itor of 2 members of the ABC protein family (Knight et al.,

2002).

Whatever the mechanism, released ATP is rapidly broken

down by ectoenzymes to ADP (to act on P2Y1, P2Y12, and

P2Y13 receptors) and adenosine (to act on P1 receptors)

(Zimmermann, 2001).

3. Neuropathic, inflammatory, and cancer pain

Neuropathic pain following peripheral nerve injury, long-

term pain associated with inflammation, and cancer pain are

common and distressing conditions. There is growing recog-

nition of the involvement of purinergic mechanisms in these

diseases (see Cain et al., 2001; Irnich et al., 2001; Kidd &

Urban, 2001; Kostyuk et al., 2001; Fukuoka & Noguchi, 2002;

Mantyh et al., 2002; Di Virgilio et al., 2003; Jarvis, 2003;

Kennedy et al., 2003; Sah et al., 2003; Sawynok & Liu, 2003;

Stone & Vulchanova, 2003; Ueda & Rashid, 2003; Luttikhui-

zen et al., 2004; North, 2004; Kennedy, 2005). Most of these

papers are concerned with P2X3 or P2X2/3 and more recently

P2Y receptors on nociceptive sensory nerves. P2X4 receptors

on microglia have also been recognized to be involved in

neuropathic pain and most recently disruption of the P2X7

receptor gene has been shown to abolish chronic inflammatory

and neuropathic pain (Chessell et al., 2005).

3.1. Peripheral purinergic mechanisms

3.1.1. Sensory ganglia

In the DRG, the presence of P2X3 mRNA-labeled neurons

increased 3 days after peripheral nerve injury (Tsuzuki et al.,

2001). In contrast, there was a decrease in numbers of P2X3

receptors in vasomotor neurons in avulsed human DRG

(central axotomy) (Yiangou et al., 2000). P2X receptors on

DRG neurons increase their activity after inflammation and

contribute to the hypersensitivity to mechanical stimulation in

the inflammatory state (Dai et al., 2002; Chen et al., 2004).

After induction of painful peripheral neuropathy by sciatic

nerve entrapment there is also evidence for increased release of

ATP from DRG neurons on the side of the injury (Matsuka et

al., 2004).

The neurosteroid, dehydroepiandrosterone (DHEA), pro-

duced by glial cells and neurons, potentiated P2X2-containing

receptors and could therefore lead to sensitisation or increased

activation of nociceptors by ATP (De Roo et al., 2003). The

authors suggest that DHEA could be an endogenous modulator

of P2X receptors leading to facilitation of nociceptive messages

particularly under conditions of inflammatory pain, where the

P2X signaling pathways appear to be up-regulated.

Purinergic sensitivity develops in sensory neurons after

chronic peripheral nerve injury (Zhou et al., 2001; Chen et al.,

2004). Injection of a combination of the adrenoreceptor

antagonist, phentolamine, and the P2 antagonist, suramin,

reduced mechanical hypersensitivity in neuropathic rats (Park

et al., 2000). Pelvic and pudendal nerve injury can occur during

extirpative visceral surgery such as radical hysterectomy. Many

of the patients develop severe chronic pelvic pain and bladder

symptoms (Shembalkar et al., 2001). Mechanical allodynia

caused by surgical injury has been considered to involve local

release of ATP in the tissue injury area and its action on P2X

nociceptive receptors (Tsuda et al., 2001). The authors suggest

that agents that block P2 receptors may be useful as pre-

emptive antiallogenic drugs for alleviating the postoperative

pain syndrome in humans. Involvement of P2X2 and P2X3

receptors in neuropathic pain in the mouse chronic constriction

injury model has also been demonstrated (Ueno et al., 2003).

Inflammatory mediators such as substance P and bradykinin

sensitize nociception through phosphorylation of P2X3 and

P2X2/3 ion channels or associated proteins (Paukert et al.,

2001). This might contribute to the increase in purinergic

nociception in inflammatory conditions.

Reg-2, a secretory protein with proregenerative properties in

motor and sensory neurons after injury, is massively up-

regulated in the subpopulation of IB4/P2X3 immunopositive

DRG neurons after sciatic nerve injury (Averill et al., 2002).

The pathophysiological significance of this finding remains to

be determined.

It has been suggested that heat shock proteins (HSPs) may

be involved in inflammation-related nociception and it has

been shown that inhibitors of HSP90 increase the magnitude of

currents mediated by P2X and VR1 receptors that are known to

be involved in inflammation-related nociception (McDowell &

Yukhananov, 2002).

Satellite glial cells in mouse TG have been shown to express

P2Y, probably P2Y4, receptors and it was speculated that they

may be activated by ATP released during nerve injury (Weick

et al., 2003). There is also evidence for a correlation between

activation of P2 receptors on central glia and neuropathic pain

(Watkins & Maier, 2002).

G. Burnstock / Pharmacology & Therapeutics 110 (2006) 433–454442

Oncostatin M is a cytokine involved in inflammatory

reactions and the h-subunit of its receptor is expressed in a

subset of nociceptive sensory neurons that also express P2X3

and VR1 receptors (Tamura et al., 2003). Seven days after

sciatic nerve axotomy, the expression of the h-subunit of theoncostatin M receptor was down-regulated in the DRG of the

injured side. These findings suggest that oncostatin Mh is

involved in the modulation of neuronal phenotypes, including

VR1 and P2X3 receptors, rather than the survival and axonal

regeneration of these neurons.

DRG neurons normally function as independent sensory

communicators, but most DRG neurons are also transiently

activated when axons in the same ganglion are stimulated

repetitively (Amir & Devor, 2000). Peripheral inflammation

enhances the excitability of DRG neurons and activates silent

nociceptors (Schaible et al., 2000; Xu & Zhao, 2003).

Using the foreign body reaction chronic inflammatory rat

model, where a sterile inflammatory reaction is induced by

implanting degradable cross-linked dermal sheep collagen

discs subcutaneously, up-regulation of P2X7, P2Y1, and P2Y2

receptors occurs in macrophages in the vasculature (Luttikhui-

zen et al., 2004). These receptors are potential therapeutic

targets for the modulation of inflammation.

Transient TG ischemia appears to provoke a selective

decrease in P2X3 receptor expression and it is suggested that

this may be related to the altered pain and thermal sensation

(Hwang et al., 2004). While the expression of neurotransmitters

or neuromodulators may change, transient ischemia does not

cause sensory neuron loss in the TG.

It is beginning to be recognized that the mechanism of

classical mechanical hyperalgesia in inflammation may involve

purinergic mechanosensory transduction, whereby stretch

evokes release of ATP that then excites nearby primary sensory

nerve terminals. In a recent study, phosphorylated extracellular

signal-regulated protein kinase (pERK) immunoreactivity was

used as a marker indicating functional activation of primary

afferent neurons in a rat model of peripheral inflammation (Dai

et al., 2004). The results suggest that P2X3 receptors in primary

afferent nerves increase their activity with increased sensitivity

of the intracellular ERK pathway during inflammation and then

contribute to the hypersensitivity to mechanical noxious

stimulation in the inflammatory state.

3.1.2. Urinary bladder

The involvement of purinergic signaling is being considered

in recent reviews of diseased bladder and novel purine-related

therapeutic strategies are being explored for overactive bladder,

incontinence, and interstitial cystitis (see, for example, Boselli

et al., 2001; Burnstock, 2001b; Andersson, 2002; Andersson &

Hedlund, 2002; Ballaro et al., 2003; Fraser et al., 2003; Kumar

et al., 2003; Fowler, 2004; Fry et al., 2004; Moreland et al.,

2004; Ouslander, 2004).

It has been known for some years now that the purinergic

component of parasympathetic control of bladder contraction is

greatly enhanced in interstitial cystitis (Palea et al., 1993).

Recent studies have shown significant increase in release of

ATP from the urothelium in response to stretch in the cat model

of interstitial cystitis (Barrick et al., 2002) and in the

cyclophosphamide mouse model of interstitial cystitis (Rong

and Burnstock, unpublished data) as well as urothelial cells

from patients with interstitial cystitis (Sun & Chai, 2002).

Subsensitivity of P2X3 and P2X2/3 receptors, but not vanilloid

receptors, has been shown in L6-S1 DRG in the rat model of

cyclophosphamide cystitis (Borvendeg et al., 2003). Release of

ATP from urothelial cells with hypo-osmotic mechanical

stimulation was increased by over 600% in inflamed bladder

from cyclophosphamide-treated animals; botulinum toxin

inhibited this release (Smith et al., 2004). Botulinum toxin

was also effective in blocking the bladder overactivity induced

by ATP (Atiemo et al., 2005). The P2X3 receptor subunit was

up-regulated during stretch of cultured urothelial cells from

patients with interstitial cystitis (Sun & Chai, 2004). P2X2 and

P2X3 receptor expression has been demonstrated recently on

human bladder urothelial cells (as well as on afferent nerve

terminals); the expression was greater in cells from interstitial

cystitis bladder (Tempest et al., 2004).

An increase in stretch-evoked urothelial release of ATP has

been reported from porcine and human bladders with sensory

disorder (urgency) compared with normal bladders (Kumar et

al., 2004). This finding reinforces the view that purinergic

mechanosensory transduction is enhanced in pathological

conditions. There is also an increase in both neuronal, and

especially non-neuronal, release of ATP from human bladder

strips in old age (Yoshida et al., 2004).

3.1.3. Gut

The excitability of visceral afferent nerves is enhanced

following injury, ischemia and during inflammation, for

example in irritable bowel syndrome (IBS). Under these

conditions, substances are released from various sources that

often act synergistically to cause sensitisation of afferent nerves

to mechanical or chemical stimuli. Receptors to these sub-

stances (including ATP) represent potential targets for drug

treatment aimed at attenuating the inappropriate visceral

sensation and subsequent reflex activities that underlie abnor-

mal bowel function and visceral pain (see Holzer, 2001; Kirkup

et al., 2001; Cooke et al., 2003). The sensitizing effects of P2X3

purinoceptor agonists on mechanosensory function is induced

in esophagitis (Page et al., 2000). During chronic interstitial

inflammation induced by infection of mice with the parasite

Schistosoma mansoni for 16 weeks, purinergic modulation of

cholinergic nerve activity was impaired (De Man et al., 2003).

Reviews have been published recently proposing that

enteric P2X receptors are potential targets for drug treatment

of IBS (Galligan, 2004) and inflammation- and ischemia-

induced disturbances of gut sensation (Holzer, 2004). It is

suggested that antagonists to P2X3 and/or P2X2/3 receptors on

nociceptive extrinsic sensory nerves in the gut could attenuate

abdominal pain in IBS. It is also suggested that agonists acting

on P2X receptors on intrinsic enteric neurons may enhance

gastrointestinal propulsion and secretion and that these drugs

might be useful for treating constipation-predominant IBS,

while P2X antagonists might be useful for treating diarrhea-

predominant IBS.

G. Burnstock / Pharmacology & Therapeutics 110 (2006) 433–454 443

P2X3 receptors are up-regulated in human inflammatory

bowel disease at hypersensitivity (Yiangou et al., 2001). Using

an animal model of colitis, Wynn et al. (2004) have shown that

the increase in sensory nerve responses to ATP is due to an

increase in the number of P2X3 receptor-expressing small

nociceptive neurons in the DRG supplying the colorectum,

particularly those labeling with CGRP.

The pro-algesic influence of acidic irritants in the peritone-

um appears to be mediated by both P2X3 and P2X2/3 receptors

and it has been suggested that these receptors might have

therapeutic potential in the treatment of acid-related inflam-

mation- and ischemia-induced disturbances of gut function and

sensation (Holzer, 2003).

3.1.4. Lung

Alveolar macrophages express functional P2X7 receptors,

which upon stimulation activate proinflammatory interleukin

(IL) 1-IL6 cytokine cascade and the formation of multinucleate

giant cells, a feature of granulomatous reactions (Lemaire &

Leduc, 2004). Further, Th1 and Th2 cytokines reciprocally

regulate P2X7 receptor function, suggesting a role for P2X7

receptors in pulmonary diseases, particularly lung hypersensi-

tivity associated with chronic inflammatory responses.

3.1.5. Joints

It has been known for some time that ATP and adenosine

have effects on acute and chronically inflamed joints (Green et

al., 1991; Baharav et al., 2002) and quinacrine (Atabrine), a

compound that binds strongly to ATP, has been used for many

years for the treatment of rheumatic diseases, although its

mechanism of action is not clear (Wallace, 1989). Evidence has

been presented that activation of P2X receptors in the rat

temporomandibular joint induces nociception and that block-

age by PPADS decreases carrageenan-induced inflammatory

hyperalgesia (Oliveira et al., 2005). The anti-inflammatory

effect of methotrexate is mediated by increasing extracellular

concentration of adenosine (Baharav et al., 2002). Spinal

adenosine receptor activation inhibits inflammation and joint

destruction (Boyle et al., 2002) and reduces c-fos and astrocyte

activation in dorsal horn (Sorkin et al., 2003) in rat adjuvant-

induced arthritis.

ATP and UTP activate calcium-mobilizing P2Y2 or P2Y4

receptors that act synergistically with IL-1 to stimulate

prostaglandin E2 release from human rheumatoid synovial

cells (Loredo & Benton, 1998).

Oxidized ATP inhibits inflammatory pain in arthritic rats by

inhibition of the P2X7 receptor for ATP localized in nerve

terminals (Dell’Antonio et al., 2002a, 2002b).

3.2. Central purinergic mechanisms

Changes in central purinergic pathways in neuropathic or

inflammatory pain will not be considered in depth in this

review, since some good recent reviews are available on this

topic (Chizh & Illes, 2000; Bardoni, 2001; Jarvis & Kowaluk,

2001; Gu, 2003; Sawynok & Liu, 2003; Gourine et al., 2004;

Inoue et al., 2004).

3.2.1. Spinal cord

It has been known for some time that nerve sprouting occurs

in the spinal cord after peripheral nerve or dorsal root lesions.

CGRP-positive neurons display considerably more nerve

sprouting than do IB4-positive neurons (Belyantseva & Lewin,

1999). However, P2X receptor-mediated pathways play a role in

pathological pain studies (Gu & Heft, 2004). There are 3

potential sources of ATP release during sensory transmission in

the spinal cord. ATP may be released from the central terminals

of primary afferent neurons. ATP may be also released from

astrocytes and/or postsynaptic dorsal horn neurons. A novel

adenosine kinase inhibitor, A-134974, relieves tactile allodynia

via spinal sites of action in peripheral nerve injured rats, adding

to the growing evidence that adenosine kinase inhibitors may be

useful as analgesic agents in a broad spectrum of pain states (Zhu

et al., 2001). Intrathecal administration of UTP and UDP had

mechanical and thermal antinociceptive effects in normal rats

and antiallodynic effects in a neuropathic pain model, possibly

involving spinal P2Y2 or 4 and P2Y6 receptors (Okada et al.,

2002). The authors suggest that P2Y receptor agonists may have

potential for the development of a new class of analgesics. After

spinal cord injury, an increased number of lumbar microglia

expressing the P2X4 receptor in the spinal cord of rats with

allodynia and hyperalgesia has been reported (Deltoff et al.,

2004; Inoue et al., 2005).

GABA and ATP are coreleased from spinal nerves within the

dorsal horn of the spinal cord (Jo & Schlichter, 1999). Negative

cross talk between receptors for these cotransmitters, as seen in

DRG neurons (Sokolova et al., 2001), may represent a novel

mechanism to inhibit afferent excitation to the spinal cord.

Platelet-activating factor (PAF) is a potent inducer of tactile

allodynia and thermal hyperalgesia at the level of the spinal

cord; it is suggested that PAF-evoked tactile allodynia is

mediated by ATP and a following N-methyl-d-aspartate

(NMDA) and nitric oxide (NO) cascade through capsaicin-

sensitive fibers (Morita et al., 2004).

Hyperthyroidism changed ATP and ADP hydrolysis in the

spinal cord and this coincided with the nociceptive response,

although the effects of thyroid hormones vary with the

developmental stage (Bruno et al., 2005). These parallel

findings suggest the involvement of adenine nucleotide

hydrolysis-related enzymes in nociceptive pathways.

3.2.2. Brain

P2X3 receptor immunoreactivity was shown in the solitary

tract and nucleus of the brain stem (Llewellyn-Smith &

Burnstock, 1998; Fig. 3), and a recent paper has claimed that

ATP release from the ventral surface of the medulla oblongata is

a mediator of chemosensory transduction in the CNS (Gourine et

al., 2005).

Intracerebroventricular administration of a,h-meATP has an

antinociceptive effect; evidence has been presented to suggest the

involvement of supraspinal h-adrenergic and A-opioid receptors

in this effect (Fukui et al., 2001). Intracerebroventricular

coadministration of antagonists to both purinergic and glutamate

receptors resulted in a deeper level of the analgesic and anesthetic

actions of the individual agents (Masaki et al., 2001).

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).

G. Burnstock / Pharmacology & Therapeutics 110 (2006) 433–454444

A hypothesis involving purinergic signaling in migraine

pain was put forward many years ago (Burnstock, 1989). A

recent paper presents evidence to support the view that ATP

may contribute to pain in migraine by sensitizing nociceptors

against tissue acidosis via P2Y2 receptor-supported release of

endogenous prostaglandins (Zimmermann et al., 2002). Recent

studies suggest that ascending noradrenergic nerves arising

from the locus coeruleus are involved in the supraspinal

antinociception by a,h-meATP through P2X receptors in the

locus coeruleus (Fukui et al., 2004).

The rostral ventromedial medulla (RVM) serves as a critical

link in bulbospinal nociceptive modulation. Within the RVM,

Fon-cells_ discharge and Foff-cells_ pause immediately prior to a

nociceptive reflex. Data have been presented to suggest that

on-cells preferentially express P2X receptors and off-cells P2Y

receptors (Selden et al., 2004).

3.2.3. Microglia and glial–neuron interactions

The roles of microglia in inflammatory pain, as well as in

neuronal cell death and regeneration, has attracted strong

interest in the past few years. ATP selectively suppresses the

synthesis of the inflammatory protein microglial response

factor through Ca2+ influx via P2X7 receptors in microglia

(Kaya et al., 2002). ATP, ADP, and BzATP, acting through

P2X7 receptors, induce release of the principal proinflamma-

tory cytokine, IL-1h from microglial cells (Chakfe et al.,

2002). Activation of P2X7 receptors enhances interferon-g-

induced NO synthase activity in microglial cells and may

contribute to inflammatory responses (Gendron et al., 2003).

ATP, via P2X7 receptors, also increases production of 2-

arachidonoylglycerol, also involved in inflammation by

microglial cells (Witting et al., 2004). P2X7 receptors have

been shown to be essential for the development of complete

Freund’s adjuvant (CFA)-mediated hypersensitivity and the

inflammatory response is attenuated in P2X7-null mice

through modulation of IL-1h and other cytokines (Hughes et

al., 2004).

It was found that in a rat neuropathic pain model displaying

allodynia, the level of phospho-p38 was increased in microglia

on the injury side of the dorsal horn and that intraspinal

administration of p38 inhibitor suppressed the allodynia. The

conclusion was that vasospasm pain hypersensitivity depends

on the activation of the p38 signaling pathway on microglia in

the dorsal horn following peripheral nerve injury. ATP causes

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).

G. Burnstock / Pharmacology & Therapeutics 110 (2006) 433–454 445

the activation of P38 or ERKl/2 mitogen-activated protein

kinases in microglia, resulting in the release of tumor necrosis

factor (Suzuki et al., 2004), as well as IL-6 (Inoue et al.,

2003b).

It has been shown that pharmacological blockade of P2X4

receptors reversed tactile allodynia caused by peripheral

nerve injury without affecting acute pain behaviors in naive

animals (Tsuda et al., 2003). After nerve injury, P2X4

receptor expression increased strikingly in the ipsilateral

spinal cord in hyperactive microglia, but not in neurons or

astrocytes (Fig. 4). Intraspinal administration of P2X4

antisense oligodeoxynucleotide decreased the induction from

P2X4 receptors and suppressed tactile allodynia after nerve

injury. On this basis, it has been claimed that blocking of

P2X4 receptors on microglia might be a new therapeutic

strategy for pain induced by nerve injury (Tsuda et al.,

2005).

Long-term increases in pain-related behavior was shown to

be associated with the activation of spinal microglia after

subcutaneous injection of formalin into the hindpaw (Fu et

al., 1999). Intrathecal delivery of the P2 receptor antagonist

suramin blocked microglia activation and long-term hyper-

algesia induced by formalin injection (Wu et al., 2004b). This

adds further support for the view that blocking of spinal P2

receptors might decrease the central enhancement of pain

caused by peripheral injury and inflammation.

G. Burnstock / Pharmacology & Therapeutics 110 (2006) 433–454446

3.3. Cancer pain

It was suggested that the unusually high levels of ATP

contained in tumor cells (Maehara et al., 1987) may be released

by mechanical rupture to activate P2X3 receptors on nearby

nociceptive sensory nerve fibers (Burnstock, 1996).

Increased expression of P2X3 receptors on CGRP immu-

noreactive epidermal sensory nerve fibers in a bone cancer pain

model has been described (Gilchrist et al., 2005) and in other

cancers that involve mechanically sensitive tumors (Mantyh et

al., 2002). For example, in bone tumors, destruction reduces

the mechanical strength of the bone and antagonists that block

the mechanically gated channels and/or ATP receptors in the

richly innervated periosteum might reduce movement-associ-

ated pain.

It is interesting that the hyperalgesia associated with tumors

appears to be linked to increase in expression of P2X3 receptors

in nociceptive sensory neurons expressing CGRP, since this has

also been described for increased P2X3 receptor expression in a

model of inflammatory colitis (Wynn et al., 2004). Increased

expression of P2X3 receptors was also reported associated with

thermal and mechanical hyperalgesia in a rat model of

squamous cell carcinoma of the lower gingival (Nagamine et

al., 2004).

4. Purinergic therapeutic developments for the treatment

of pain

Suramin, PPADS, and Reactive blue 2 have been used as

non-selective antagonists at P2X3 and P2X2/3 receptors on

nociceptive sensory nerve endings (see Ralevic & Burnstock,

1998; Burnstock, 2001a; Lambrecht et al., 2002), as well as the

trinitrophenyl substitute nucleotide TNP-ATP as a selective

antagonist at P2X1, P2X3, and P2X2/3 receptors (Mockett et al.,

1994; King et al., 1997; Virginio et al., 1998; Burgard et al.,

2000). Unfortunately, these antagonists degrade in vivo,

although PPADS dissociates 100–10,000 times more slowly

than the other antagonists (Spelta et al., 2002). Therefore,

together with their lack of bioavailability, they have limited

potential for therapeutic use.

Modulation of BzATP- and formalin-induced nociception

was attenuated by TNP-ATP and enhanced by the P2X3

allosteric modulator, Cibacron blue (Jarvis et al., 2001). TNP-

ATP blocked acetic acid-induced abdominal contraction in

mice (a measure of visceral pain), supporting the view that

activation of P2X3 receptors plays a role in the transmission of

inflammatory visceral pain (Honore et al., 2002b).

Using a Chinese hamster ovary cell line (CHO-K1), specific

inhibition of P2X3 receptor-mediated responses was achieved

with a methoxyethoxy-modified phosphorothioated antisense

oligonucleotide (Dorn et al., 2001). Continuous intrathecal

administration of P2X3 antisense oligonucleotides for 7 days in

rats significantly decreased nociceptive behaviors observed

after injection of CFA, formalin, or a,h-meATP into the

hindpaw (Honore et al., 2002a). Antisense oligonucleotides

used to down-regulate P2X3 receptors have also been shown to

be effective against chronic neuropathic pain produced by

partial sciatic nerve ligation (Barclay et al., 2002). Down-

regulation of the P2X3 receptor using RNA interference

combined with antisense oligonucleotides was proposed to

have potential benefit for inhibiting expression of a medically

relevant pain-related gene (Hemmings-Mieszczak et al., 2003).

In a later study, P2X3 SiRNA was shown to relieve chronic

neuropathic pain in an animal model (Dorn et al., 2004).

A novel potent and selective non-nucleotide antagonist of

P2X3 and P2X2/3 receptors, A317491, was introduced in 2002,

which reduces chronic inflammatory and neuropathic pain in

the rat (Jarvis et al., 2002; Neelands et al., 2003; Wu et al.,

2004c). Intraplantar and intrathecal injections of A317491

produced dose-related antinociception in the CFA model of

chronic thermal hyperalgesia (McGaraughty et al., 2003).

Intrathecal, but not intraplantar, delivery of A317491 attenu-

ated mechanical allodynia in both chronic constriction injury

and L5-L6 nerve ligation models of neuropathy. Unfortunately,

development of A31741 for therapeutic use in humans has

been dropped largely because of its lack of bioavailability.

[3H]A-317491 has been developed as the first useful radi-

oligand for the specific labeling of P2X3-containing channels

(Jarvis et al., 2004). Potent desensitization of human P2X3

receptors by diadenosine polyphosphates was shown and it was

suggested that they may provide an important modulating

mechanism for P2X3 receptor activation in vivo (McDonald et

al., 2002).

Phenol red, a pH-sensitive dye and dimethyl sulfoxide,

contained in many culture media, have been shown to be potent

irreversible antagonists at P2X3 (as well as P2X1 and P2X2)

receptors (King et al., 2005). Phenol red was shown to be a

particularly effective P2X3 antagonist of the micturition reflex

in female urethane-anesthetized rats (King et al., 2005) and

phenol has been used for patients with low-back pain (Koning

et al., 2002).

YM529, a new generation of bisphosphonate which is being

developed for advanced bone resorption-related diseases,

inhibited a,h-meATP-induced cation uptake in a P2X2/3

receptor expressed cell and inhibited the nociceptive behavior

induced by subplantar injection of a,h-meATP and in formalin-

induced nociception (Kakimoto et al., 2004).

Pilot clinical studies report an analgesic action of Mg2+ on

neuropathic pain insensitive to opioids (Crosby et al., 2000),

consistent with the demonstration that P2X3 receptor-mediated

responses are inhibited by high Mg2+ or lack of Ca2+,

representing a negative feedback process to limit ATP-

mediated nociception (Giniatullin et al., 2003). In view of the

action of Mg2+, the probability of interaction of P2X3 receptors

with NMDA receptors should be considered.

Tetramethylpyrazine (TMP), a traditional Chinese medicine

used as an analgesic for dysmenorrhea, was investigated

against acute nociception mediated by P2X3 receptor-mediated

activation of rat hindpaw (Liang et al., 2004). Subcutaneous

administration of TMP attenuated the first phase, and to a lesser

extent the second phase, of nociceptive behavior induced by

5% formalin. The response of neurons in DRG produced by

ATP and a,h-meATP was inhibited by TMP (Liang et al.,

2005).

G. Burnstock / Pharmacology & Therapeutics 110 (2006) 433–454 447

Trichloroethanol potently inhibited P2X3 receptor-mediated

responses of HEK293-h P2X3 cells (as well as moderately

interfering with P2Y1 and P2Y4 receptor-mediated responses);

it was suggested that such an effect may be relevant to the

interruption of pain transmission in DRG neurons following

ingestion of chloral hydrate or trichloroethylene (Fischer et al.,

2003).

Glial cell line-derived neurotrophic factor (GDNF) is

necessary for the development of sensory neurons and appears

to be critical for the survival of DRG cells that bind to IB4 and

express P2X3 receptors; intrathecal infusion of GDNF prevents

and reverses the behavioral expression of experimental

neuropathic pain arising from injury of spinal nerves, as well

as loss of binding of IB4 and down-regulation of P2X3

receptors (Wang et al., 2003). GDNF infusion also prevented

the development of spinal nerve ligation-induced tactile

hypersensitivity and thermal hyperalgesia.

Recent experiments suggest that modulation of neurotrans-

mission through P2X3 receptors in the central and peripheral

nervous systems may contribute to anesthesia and analgesia

produced by barbiturates (Kitahara et al., 2003).

In mice with a disrupted P2X7 gene, mechanical and

thermal hyperalgesia was absent in both inflammatory and

neuropathic pain models, while normal nociceptive processing

was preserved (Casula et al., 2004; Chessell et al., 2005). It

was proposed that P2X7 receptor activation in satellite and

Schwann cells leads to hypersensitivity via release of IL-1hand up-regulation of nerve growth factor and that the P2X7

receptors are a therapeutic target for neuropathic and

inflammatory pain.

Surprisingly, the acetylcholine receptor blocker (+)-tubo-

curarine is effective in blocking P2X-mediated response of

pheochromocytoma PC12 cells in concentrations similar to

those that block nicotinic receptors (Nakazawa et al., 1991)

and it also blocks ATP responses of hair cells (Glowatzki et

al., 1997). Recently (+)-tubocurarine methyllycaconitine and

a-bungarotoxin have been shown to be potent blockers of

P2 receptors on DRG neurons, particularly the homomulti-

mer P2X3 receptor (Lalo et al., 2001, 2004). Opioids have

also been claimed to inhibit purinergic P2X3 and P2X2/3

nociceptors in sensory neurons and cutaneous fibers of rat

via a G protein-independent mechanism (Chizhmakov et al.,

2005).

5. Conclusions

1. There is now substantial evidence in support of purinergic

mechanosensory transduction involvement in both initiation

of pain and physiological reflex activities via P2X3

homomultimer, P2X2/3 heteromultimer, and probably P2Y1

receptors in a number of peripheral tissues, including,

bladder, ureter, gut, lung, tongue, tooth pulp, and carotid

body. Further, disruption of the P2X7 receptor gene

abolishes chronic inflammatory and neuropathic pain.

2. At the central level there is evidence that P2X receptors in

the spinal cord and brain stem are involved in nociceptive

pathways. There is also evidence that damage or injury to

peripheral nerves leads to changes in the spinal cord that

cause the activation of microglia and associated increases in

P2X4 receptor expression, which change the properties of

adjacent spinal neurons leading to the onset and maintenance

of pain hypersensitivity. It appears that spinal microglia are

activated in response to peripheral nerve injury, but not to

peripheral inflammation. The mechanisms underlying these

glial–neuron interactions are still unknown.

3. Metabolic breakdown of ATP by ectoenzymes produces

other purines including ADP and adenosine that are also

involved in various nociceptive activities. P2X3, P2X2/3,

P2X4, and P2X7 receptor antagonists are being developed

for the treatment of pain, although drugs that can be given

orally and are not degraded in vivo are still awaited.

Acknowledgments

Thanks go to Ernie Jennings and Brian King for their advice

and to Gill Knight for her excellent editorial assistance.

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