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Abstract

  1. Top of page
  2. Abstract
  3. P2X receptors in sensory nerves
  4. A possible role for ATP and P2X3 receptors in pain?
  5. Conclusions and queries
  6. P2Y receptors in sensory nerves
  7. A role for P2Y receptors in pain?
  8. Conclusions
  9. References
  10. Acknowledgements

In 1995 the P2X3 receptor was found to be expressed at high levels in nociceptive sensory neurones, consistent with earlier reports that ATP induced pain in humans and animals. At first it was thought that ATP was most likely to play a role in acute pain, following its release from damaged or stressed cells and since then a wide variety of experimental techniques and approaches have been used to study this possibility. Whilst it is clear that exogenous and endogenous ATP can indeed acutely stimulate sensory neurones, more recent reports using gene knockout and antisense oligonucleotide technologies, and a novel, selective P2X3 antagonist, A-317491, all indicate that ATP and P2X3 receptors are more likely to be involved in chronic pain conditions, particularly chronic inflammatory and neuropathic pain. These reports indicate that P2X3 receptors on sensory nerves may be tonically activated by ATP released from nearby damaged or stressed cells, or perhaps from the sensory nerves themselves. This signal, when transmitted to the CNS, will be perceived consciously as chronic pain. In addition, it is now clear that several subtypes of P2Y receptor are also expressed in sensory neurones. Although their distribution and functions have not been as widely studied as P2X receptors, the effects that they mediate indicate that they might also be considered as therapeutic targets in the treatment of pain. Although our ability to treat persistent painful conditions, such as chronic inflammatory and neuropathic pain, has improved in recent years, these conditions are often resistant to currently available therapies, such as opioids or non-steroidal anti-inflammatory drugs. This reflects a limited understanding of the underlying pathophysiology. It is now clear that the development and maintenance of chronic pain are mediated by multiple factors, but many of these factors, and the receptors and mechanisms through which they act, remain to be identified. Chronic pain is debilitating and can greatly decrease quality of life, not just due to the pain per se, but also because of the depression that can often ensue. Thus a greater understanding of the mechanisms that underlie chronic pain will help identify new targets for novel analgesics, which will be of great therapeutic benefit to many people.

The ability of adenosine 5′-triphosphate (ATP) to evoke pain has been known for nearly 40 years (Collier et al. 1966), but the receptors and mechanisms through which ATP acts and their roles in nociceptive signalling are only now becoming clear. ATP produces its physiological and pharmacological effects via P2X and P2Y receptors, and in 1995 the cloning of the P2X3 receptor created great interest, as it was found to be selectively expressed at high levels in nociceptive sensory neurones (Chen et al. 1995; Lewis et al. 1995). Initially, it was proposed that ATP released from a variety of sources, such as damaged or stressed cells, could perhaps play a role in acute pain (Kennedy & Leff, 1995; Burnstock & Wood, 1996), but a number of more recent reports using gene knockout methods (Cockayne et al. 2000; Souslova et al. 2000), antisense oligonucleotide technologies (Barclay et al. 2002; Honore et al. 2002a) and a novel, selective P2X3 antagonist (Jarvis et al. 2002), all indicate that P2X3 receptors are more likely to be involved in chronic conditions, particularly chronic inflammatory and neuropathic pain. They suggest that ATP, tonically released from nearby damaged or stressed cells, or more intriguingly from the sensory nerves themselves, elicits a tonic activation of the neuronal P2X3 receptors, which initiates action potentials that travel along the sensory axon to the CNS (Fig. 1). This would be experienced consciously as chronic pain.

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Figure 1. A model for self-regenerating activation of nociceptive nerve fibres by ATP and P2X3 receptors

A schematic representation of a mechanism by which ATP and P2X3 receptors may tonically activate nociceptive nerves in chronic pain conditions is shown. Activation of P2X3 receptors (and perhaps P2X2/3 receptors) by extracellular ATP depolarises nociceptive nerve terminals, which opens voltage-operated Ca2+ channels (VOCC) also present in the terminal. Ca2+ influx via the P2X3 receptors and the VOCC induces local, Ca2-dependent release of ATP into the extracellular space, which then acts on the P2X3 receptors to elicit further depolarisation and Ca2+ influx. The depolarisation also initiates axonal action potentials, which travel along the sensory nerve to the spinal cord.

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Additionally, although most research has focused on the involvement of P2X receptors in the sensory actions of ATP, it is now becoming clear that ATP and the related endogenous nucleotides adenosine 5′-diphosphate (ADP), uridine 5′-triphosphate (UTP) and uridine 5′-diphosphate (UDP) may influence sensory nerve activity by acting at P2Y receptors. Several P2Y subtypes are expressed in sensory neurones (Nakamura & Strittmatter, 1996; Fong et al. 2002; Molliver et al. 2002; Sanada et al. 2002; Xiao et al. 2002) and the effects that they mediate (Tominaga et al. 2001; Molliver et al. 2002; Okada et al. 2002; Usachev et al. 2002) indicate that they might also be considered as therapeutic targets in the treatment of pain.

P2X receptors in sensory nerves

  1. Top of page
  2. Abstract
  3. P2X receptors in sensory nerves
  4. A possible role for ATP and P2X3 receptors in pain?
  5. Conclusions and queries
  6. P2Y receptors in sensory nerves
  7. A role for P2Y receptors in pain?
  8. Conclusions
  9. References
  10. Acknowledgements

Localisation

Sensory nerve cell bodies lie in the trigeminal, nodose and dorsal root (DRG) ganglia and their axons project peripherally to tissues and organs throughout the body (Willis & Coggeshall, 1991). Approximately 70 % of the nerve cells in the DRG are C cells, with small diameter cell bodies and unmyelinated, slow conducting axons, and A δ cells, which have medium-sized cell bodies and thinly myelinated axons that conduct action potentials more rapidly. The majority of these nerve cells are nociceptors that respond to chemical, thermal and mechanical stimuli and those that respond to all three are classified as polymodal nociceptors (Perl, 1992). On the basis of biochemical, anatomical and physiological properties, polymodal C cells present in the adult DRG can be divided into two classes (see Lawson, 1992; Snider & McMahon, 1998). Forty to forty-five per cent constitutively synthesize the neuropeptides substance P and calcitonin gene-related peptide (CGRP) and express the TrkA receptor for nerve growth factor. They project centrally to the spinal dorsal horn lamina I and outer lamina II. The remaining 55–60 % of C cells do not express substance P, CGRP and TrkA, but do express the enzymes thiamine monophosphatase and fluoride-resistant acid phosphatase (FRAP). They can be identified with cellular markers such as LA4 and the isolectin B4 (IB4) and are sensitive to glial cell-derived neurotrophic factor. These cells project to inner lamina II in the spinal dorsal horn. Many, but not all, of both classes of cell also express the TRPV1 receptor (previously known as the VR1 receptor; Montell et al. 2002), the site of action of capsaicin.

The initial reports on the P2X3 receptor showed that its mRNA was selectively expressed at high levels in neurones in the rat DRG and nodose ganglia (Chen et al. 1995; Lewis et al. 1995). This was confirmed in subsequent studies with selective antibodies (Cook et al. 1997; Vulchanova et al. 1997, 1998; Bradbury et al. 1998: Llewellyn-Smith & Burnstock, 1998). The mRNA (Collo et al. 1996) and protein (Xiang et al. 1999; Barden & Maxwell, 2000) of other P2X receptor subtypes have also been detected in sensory neurones, but at lower levels, except for the nodose ganglion, where the P2X2 and P2X3 receptors are expressed at similar levels (Xiang et al. 1999). These data are consistent with the ability of P2X3 subunits to form homomultimers on their own and heteromultimers with the P2X2 subunit (Lewis et al. 1995).

P2X3-like immunoreactivity is seen in about 40 % of rat DRG neurones, which tend to have small or medium-sized cell bodies and to co-express IB4, FRAP and LA4, and only a small minority (14 %) co-express CGRP (Bradbury et al. 1998; Vulchanova et al. 1998). A similar pattern of expression has also been reported in rat trigeminal ganglia (Eriksson et al. 1998). The P2X3 receptor also co-expresses to a large degree with the TRPV1 receptor (Guo et al. 1999) and capsaicin pretreatment decreases P2X3 mRNA (Chen et al. 1995) and P2X3-like immunoreactivity (Vulchanova et al. 1998) in the rat DRG by about 70 %. Interestingly, Yiangou et al. (2000) recently demonstrated P2X3-like immunoreactivity in human DRG, mainly in cells that do not express TrkA. The central projections of the P2X3-positive neurones in the rat DRG (Bradbury et al. 1998; Vulchanova et al. 1998) and trigeminal ganglia (Llewellyn-Smith & Burnstock, 1998) terminate in inner lamina II of the spinal cord and P2X3-like immunoreactivity is also seen in this region. It is located in the central terminals of the sensory nerves, rather than in spinal neurones, as it is abolished by section of the dorsal roots (rhizotomy) (Bradbury et al. 1998) or by selective destruction of IB4-positive sensory nerves (Nakatsuka et al. 2003).

Functional expression

P2X receptors are multimeric, ligand-gated cation channels (Khakh et al. 2001). Application of P2X agonists to acutely dissociated rat DRG neurones under voltage-clamp conditions evokes inward currents that desensitise rapidly, slowly or in a fast then slow, biphasic manner (Bean, 1990; Robertson et al. 1996; Rae et al. 1998; Burgard et al. 1999; Grubb & Evans, 1999; Tsuzuki et al. 2003). In the rat nodose ganglion the slow current predominates (Krishtal et al. 1988; Khakh et al. 1995; Lewis et al. 1995). Pharmacological studies (Robertson et al. 1996; Rae et al. 1998; Burgard et al. 1999; Grubb & Evans, 1999) and experiments on neurones from P2X3-knockout mice (Cockayne et al. 2000; Souslova et al. 2000; Zhong et al. 2001) show that the rapidly desensitising component is mediated by P2X3 receptors. These fast currents are predominant in small diameter neurones that are capsaicin sensitive and display IB4 staining (Li et al. 1999; Ueno et al. 1999; Petruska et al. 2000a, b; Tsuzuki et al. 2003). Thus these data suggest that homomeric P2X3 receptors are mainly expressed in small, capsaicin-sensitive C cells in the rat DRG.

Pharmacological studies indicate that the P2X2/3 heteromultimer mediates slowly desensitising currents (Lewis et al. 1995; Burgard et al. 1999; Grubb & Evans, 1999) and this is supported by studies on neurones from P2X3-knockout mice. In these cells, α, β-methylene ATP, an agonist at the P2X3 homomer and the P2X2/3 heteromultimer, but not the P2X2 homomer (Lewis et al. 1995), was inactive. In contrast, ATP, an agonist at all three receptors, evoked a slowly desensitising current, consistent with an action at the P2X2 homomer (Cockayne et al. 2000; Souslova et al. 2000; Zhong et al. 2001). The slowly desensitising currents tend to be seen in medium-sized cells that are capsaicin insensitive (Li et al. 1999; Ueno et al. 1999; Petruska et al. 2000a, b; Tsuzuki et al. 2003), suggesting that the P2X2/3 heteromultimer may be predominant in medium-sized Aδ cells in the rat DRG.

Recent studies show that α, β-methylene ATP can evoke slowly decaying currents independently of P2X2/3 receptors in a subset of DRG neurones (Tsuzuki et al. 2003). 2′, 3′-O -(2, 4, 6-trinitrophenyl) ATP (TNP-ATP), a potent antagonist at P2X1, P2X3 and P2X2/3 receptors (Khakh et al. 2001), abolished rapidly desensitising responses to α, β-methylene ATP in all DRG cells tested. In contrast, TNP-ATP had a variable effect against the slowly decaying currents and was ineffective in 25 % of cells. The TNP-ATP-resistant currents were, however, abolished by the non-selective P2X antagonist PPADS. The authors proposed that these currents might be elicited via P2X1/5 or P2X4/6 heteromers, as both are α, β-methylene ATP sensitive and TNP-ATP insensitive. Further analysis showed that the cells expressing these currents were medium sized and capsaicin insensitive, suggesting that they are Aδ cells. Further studies are needed to identify the P2X subunits that mediate these actions and to explain why these currents were not seen in neurones from the P2X3 knockout mice.

As well as sensory nerve cell bodies, functional P2X receptors are also present on their central terminals and may play a neuromodulatory role. Intrathecal ATP and α, β-methylene ATP decreased the pinch pressure threshold and induced mechanical allodynia to von Frey hairs in the rat paw (Okada et al. 2002). Although the mechanism of action was not determined, ATP is known to increase glutamate release from DRG nerve terminals in lamina II of the spinal cord (Nakatsuka et al. 2003). The increase was transient and inhibited by TNP-ATP, suggesting that it was mediated via P2X3 receptors. P2X receptors are also present on the central terminals of Aδ fibres that project to lamina V. ATP and α, β-methylene ATP caused a long-lasting increase in glutamate release from these neurones, which was unaffected by TNP-ATP and may be mediated by P2X1/5 or P2X4/6 heteromers (Nakatsuka et al. 2001, 2003). P2X-like immunoreactivity has not been studied in this region and further experiments are required to identify which P2X subunits are expressed. Also, as both nociceptive and non-nociceptive Aδ fibres project to lamina V (Willis & Coggeshall, 1991), the modality of the P2X agonist-sensitive Aδ nerves needs to be determined. Interestingly, ARL 67156, which inhibits the breakdown of ATP by ecto-ATPases, potentiated the release of glutamate elicited by stimulation of the dorsal roots (Nakatsuka et al. 2001). These data imply that the physiological role of ATP released from Aδ nerves in lamina V is more likely to be as a presynaptic neuromodulator, than as a fast neuro-transmitter.

Exogenous P2X3 agonists also induced action potentials in capsaicin-sensitive C fibres in a variety of peripheral tissues, including the rat tail (Rea & Wallis, 1992; Trezise & Humphrey, 1996), knee joint (Dowd et al. 1998) and tongue (Rong et al. 2000) and canine lung (Pelleg & Hurt, 1996). It has been assumed that the P2X3 receptors are restricted to the nerve terminals, but C fibre axons in the rat vagus nerve were also excited by P2X agonists (Trezise et al. 1993, 1994; Irnich et al. 2001). Thus P2X3 receptors appear to be functionally expressed along the length of C-type sensory nerves.

A possible role for ATP and P2X3 receptors in pain?

  1. Top of page
  2. Abstract
  3. P2X receptors in sensory nerves
  4. A possible role for ATP and P2X3 receptors in pain?
  5. Conclusions and queries
  6. P2Y receptors in sensory nerves
  7. A role for P2Y receptors in pain?
  8. Conclusions
  9. References
  10. Acknowledgements

Acute studies

ATP is well known to be noxious in humans and animals. It induces acute pain in the human (Bleehen et al. 1976; Bleehen & Keele, 1977) or rabbit (Bleehen, 1978) blister base models and when injected directly into human skin (Coutts et al. 1981; Hamilton et al. 2000). Similarly, i.p. ATP induces writhing in mice (Collier et al. 1966; Gyires & Torma, 1984) and when injected into the rat paw causes paw lifting (Bland-Ward & Humphrey, 1997; Tsuda et al. 1999, 2000). These noxious actions are inhibited by P2X receptor antagonists. Acetic acid-induced writhing in mice is inhibited by the P2X antagonists suramin (Ho et al. 1992) and TNP-ATP (Honore et al. 2002b). Likewise, pain caused by formalin in the rat hindpaw is suppressed by suramin (Driessen et al. 1994), PPADS (Tsuda et al. 1999) and TNP-ATP (Tsuda et al. 1999; Jarvis et al. 2001). Formalin-induced pain was also less in the A/Jinbred mouse strain, which is known to be resistant to tissue injury pain (Tsuda et al. 2002) and in P2X3-knockout mice, although the response to acute noxious thermal and mechanical stimuli were unchanged in the latter (Cockayne et al. 2000; Souslova et al. 2000).

Sensitisation

Recent data show that inflammatory conditions increase the noxious effects of P2X3 agonists, both in the short and long term. The paw lifting evoked by α,β-methylene ATP in rats was greatly potentiated in three models of acute peripheral inflammation (intraplantar PGE2 or carageenan, ultraviolet irradiation) (Hamilton et al. 1999) and activation of C and Aδ fibres in rat skin by α,β-methylene ATP and ATP was rapidly increased by carageenan-induced inflammation (Hamilton et al. 2001). These are consistent with the greater pain induced by ATP in human skin after carageenan treatment or ultraviolet irradiation (Hamilton et al. 2000). Complete Freund's adjuvant (CFA) induces chronic inflammation, and pretreatment of rats in vivo with CFA increased the amplitude of currents evoked by ATP in DRG neurones in vitro (Xu & Huang, 2002), although CFA-induced chronic unilateral inflammatory arthritis of the rat knee had no effect on the responsiveness of joint nociceptors to α, β-methylene ATP or ATP (Dowd et al. 1998). Inflammation may also increase the functional expression of P2X3 receptors at the central terminals of sensory neurones, as intrathecal administration of PPADS and suramin had no effect on C fibre-evoked discharges of deep dorsal horn neurones in control rats, but reduced the increased firing rate seen following induction of peripheral inflammation by intraplantar injection of carageenan (Stanfa et al. 2000).

A genetic approach

The studies described above are consistent with ATP and P2X3 receptors playing an important role in certain types of pain. However, they are not conclusive, as the antagonists used have no selectivity for P2X3 receptors over other P2 receptor subtypes (suramin, PPADS) or are metabolically labile (TNP-ATP). In an attempt to overcome these problems mice were created in which the P2X3 receptor was genetically ‘knocked out’ (Cockayne et al. 2000; Souslova et al. 2000). They appeared to be phenotypically normal with regards to general health and behaviour and acute noxious mechanical and thermal stimulation induced similar behavioural responses as in wild-type animals. In contrast, the nociceptive response to ATP and formalin were both substantially reduced in the knockout animals.

An alternative approach has been to downregulate P2X3 receptor expression in rats by delivering antisense oligonucleotides (ASO) specific for P2X3 receptors to lumbar DRG neurones, via an indwelling intrathecal catheter attached to an osmotic mini-pump (Barclay et al. 2002; Honore et al. 2002a). Delivery of P2X3 receptor ASO for 7 days significantly reduced the levels of P2X3 mRNA in the DRG and P2X3 protein levels in the DRG and the inner lamina II of the dorsal horn of the spinal cord. The animals showed no changes in overt behaviour and delivery of a missense oligonucleotide had no effect on P2X3 expression. The ASO had no effect on acute inflammatory hyperalgesia induced by carageenan, but the treated animals did show a reduced mechanical hyperalgesia to intraplantar administration of formalin or α, β-methylene ATP into the hindpaw. Thus intrathecal P2X3 ASO appears to be taken up by the central terminals of DRG neurones and transported back to the cell soma, where it inhibits translation of P2X3 mRNA, leading to a decreased expression of P2X3 receptor protein in the central and peripheral terminals of DRG neurones.

These studies also showed a physiological role for P2X3 receptors in mediating chronic inflammatory (Fig. 2A and B) and neuropathic pain (Fig. 2C and D). Chronic inflammatory pain was induced by intradermal administration of CFA into the rat hindpaw. Intrathecal application of P2X3 ASO, starting 24 h beforehand, reduced the development of mechanical hyperalgesia over the next 6 days by about 25 % (Barclay et al. 2002) (Fig. 2A), whilst the thermal hyperalgesia seen after 2 days was almost abolished (Honore et al. 2002a) (Fig. 2B). In either case there was no effect of ASO treatment on the contralateral paw responsiveness during the treatment periods.

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Figure 2. Inhibition of chronic pain by antisense oligonucleotides

The effects of intrathecal delivery of oligonucleotides was determined in rats in which inflammation was induced in the hindpaw by intraplantar injection of complete Freund's adjuvant (CFA) (A, B) or neuropathic pain (C, D). A, animals were cannulated intrathecally and infused with P2X3 receptor antisense oligonucleotide (ASO), P2X3 receptor missense oligonucleotide (MSO) or saline (vehicle). One day later, inflammation was induced in the hindpaw of 3 groups and a fourth, saline-treated group was maintained as a control for cannulation. Mechanical hyperalgesia was assessed before cannulation and then daily until day 7 by measuring paw withdrawal threshold to an increasing pressure stimulus using an Analgesymeter. B, inflammation was induced in the hindpaw on day 5 of intrathecal administration of saline (left-hand panel) or P2X3 receptor ASO (right-hand panel). Two days later thermal hyperalgesia was measured by the latency of withdrawal evoked by thermal stimulation (radiant heat emitted from a focused projection bulb) in the CFA-treated paw (hatched bars) and the untreated, contralateral paw (filled bars). C, neuropathic pain was induced by partial ligation of the sciatic nerve and 13 days later the animals were cannulated intrathecally and infused with P2X3 receptor ASO, P2X3 receptor missense oligonucleotide (MSO) or saline (vehicle) for 7 days. An additional, sham-operated group received intrathecal saline as a control. Mechanical hyperalgesia was then assessed daily from days 13–20 as described in A. D, neuropathic pain was induced by ligation of the L5 and L6 spinal nerves. Intrathecal administration of P2X3 receptor ASO or vehicle as a control, was started 7 days later (day 0 on graph) and continued for 7 days. Mechanical allodynia was measured by the paw withdrawal evoked by calibrated application of von Frey filaments. *P < 0.05, **P < 0.01 for P2X3 receptor ASO-treated animals compared with the relavant controls. ++P < 0.01 for inflamed paws compared with the contralateral side. Adapted from Barclay et al. (2002; copyright by the Society for Neuroscience) (A, C) and Honore et al. (2002a; copyright by International Association for the Study of Pain) (B, D).

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The sciatic nerve contains the axons of lumbar sensory neurones and the mechanical hyperalgesia induced by its partial ligation (Seltzer model) was significantly reduced when intrathecal P2X3 ASO treatment was started 24 h beforehand. Notably, when ASO application was delayed until 13 days after ligation, it was still effective in reducing the mechanical hyperalgesia within 2 days (Barclay et al. 2002) (Fig. 2C), although mechanical allodynia was unaffected. In contrast, the tactile allodynia induced by L5-L6 spinal nerve ligation (Chung model) was substantially reduced within 2 days of initiation of ASO administration. This effect reversed back to control levels over 7 days after ASO administration was stopped (Honore et al. 2002a) (Fig. 2D). Again, in each of these cases, there was no effect of ASO treatment on the contralateral paw thresholds during the treatment periods. Taken together, the above data are consistent with the proposal that P2X3 receptors play a central role in the development and maintenance of chronic inflammatory and neuropathic pain.

A-317491, a P2X3 antagonist

The development of a competitive P2X3 antagonist, A-317491 (Jarvis et al. 2002) (Fig. 3A), has enabled the role of P2X3 receptors in pain to be studied more directly. This non-nucleotide is highly selective for the P2X3 homomer and the P2X2/3 heteromer over other P2X subtypes. It shows substantial stereo-selectivity (S >> R, Fig. 3C) and the S-enantiomer has a pA2= 6.63 at the recombinant rat P2X2/3 receptor. After S.C. dosing A-317491 had high systemic availability and a plasma half-life in rats of 11 h. A-317491 had no effect on motor activity or co-ordination, and general cardiovascular and CNS activity. Thus A-317491 is the first selective, stable, competitive P2X3 antagonist to be introduced.

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Figure 3. Inhibition of chronic pain by A-317491

A, structure of A-317491. B, inflammation was induced in the rat hindpaw by intraplantar injection of CFA. The effects of A-317491 or sterile water (vehicle) on the paw withdrawal evoked by thermal stimulation (radiant heat emitted from a focused projection bulb) were determined 48 h later. The upper trace shows the time course for the onset and duration of the antihyperalgesic action of A-317491 (100 μmol kg−1s.c.). The lower trace shows the paw withdrawal latencies of hyperalgesic paws from animals injected with the vehicle for A-317491. *P < 0.05 for A-317491-treated compared with vehicle-treated animals. C, neuropathic pain was induced in the rat hindpaw by chronic constriction of the sciatic nerve. The effects of A-317491 (100 μmol kg−1s.c., right-hand bars), its stereoisomer A-317344 (100 μmol kg−1s.c., middle bars) or vehicle (left-hand bars) on the paw withdrawal evoked by calibrated application of von Frey filaments were determined 14 days later. The open bars represent the paw withdrawal threshold (g) in the paws suffering mechanical allodynia and the hatched bars represent the responses of the contralateral paws. *P < 0.05 for A-317491 compared with vehicle-treated animals. Adapted from Jarvis et al. (2002; copyright by National Academy of Sciences, USA).

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At doses of up to 100 μmol kg−1 S.C., A-317491 had little effect against a range of acute noxious thermal, mechanical and chemical stimuli in rats in vivo. However, inflammatory thermal hyperalgesia induced by intra-plantar CFA was rapidly and fully blocked, lasting for 8 h after S.C. administration (Fig. 3B). Importantly, A-317491 did not display tolerance after twice-daily administration for 4 days. Phase II of formalin-induced inflammatory pain was also effectively inhibited. A-317491 also inhibited chronic neuropathic pain, being most potent against the mechanical allodynia (Fig. 3C) and thermal hyperalgesia induced by chronic constriction of the sciatic nerve (Bennett model), both of which it abolished. Again the effect had a rapid onset and lasted for 5 h. A-317491 was also effective against tactile allodynia induced by L5-L6 spinal nerve ligation, but was less potent than against sciatic nerve ligation. In each of these protocols A-317491 had no effect on the contralateral paw responsiveness to pain.

In these studies it is not clear why P2X3 ASO inhibited mechanical allodynia in the Chung and Bennett models, but not the Seltzer model of neuropathic pain. It could be that P2X3 receptors do not mediate allodynia in the latter model, although this would be surprising, given their involvement in the Bennett model, which is technically very similar. Alternatively, it may be that there was insufficient downregulation of P2X3 subunits by the ASO in the Seltzer model to prevent the allodynia from occurring. Further experiments are needed to clarify this issue.

Conclusions and queries

  1. Top of page
  2. Abstract
  3. P2X receptors in sensory nerves
  4. A possible role for ATP and P2X3 receptors in pain?
  5. Conclusions and queries
  6. P2Y receptors in sensory nerves
  7. A role for P2Y receptors in pain?
  8. Conclusions
  9. References
  10. Acknowledgements

These studies indicate that P2X receptors may be involved in mediating certain types of pain, particularly the P2X3 subtype, either as a homomer or as a heteromultimer with P2X2 subunits. The demonstration of P2X3-like immuno-reactivity and functional expression in sensory nerves and the recent reports using gene knockout and ASO technologies and the selective P2X3 antagonist, A-317491, all point to a crucial role of ATP and P2X3 receptors in chronic inflammatory and neuropathic pain. P2X2 receptors may also mediate some noxious effects of ATP independently of the P2X3 subunit in capsaicin-sensitive C fibres (Wismer et al. 2003), whilst P2X1/5 or P2X4/6 heteromers have been proposed to be expressed in some Aδ fibres. This improved understanding of the mechanisms that underlie the noxious effects of ATP is encouraging and should help to identify novel analgesics. Several issues still have to be addressed, however.

What are the cellular mechanisms that underlie the increased responsiveness to P2X3 agonists? The simplest explanation is that inflammatory mediators increase the potency and/orefficacy of ATP at P2X3 receptors that are already expressed in C and Aδ cells. Indeed, substance P and bradykinin increase the current carried by recombinant P2X3 and P2X2/3 receptors (Paukert et al. 2001), but further studies are needed to determine if this also occurs with native receptors. Alternatively, inflammatory mediators may change the phenotype of sensory neurones, such that cells that are normally insensitive to ATP become responsive, i.e. silent afferents or sleeping nociceptors are activated. Consistent with this, the acute inflammation induced by carageenan almost doubled the proportion of C fibres in rat skin that responded to α, β-methylene ATP (Hamilton et al. 2001). This occurred in only 5–6 h, which is too fast for retrograde signals to have travelled to the cell body and initiated de novo receptor synthesis, followed by anterograde transport of the receptors to the nerve terminals. Thus such short-term changes must be due to a change in the properties of preformed receptors. Either inactive receptors already expressed in the sensory nerve terminals become sensitive to ATP or preformed receptors are rapidly inserted into the terminal membrane.

The long-term changes in responsiveness to P2X3 agonists in chronic pain conditions are likely to involve changes in P2X3 mRNA and protein expression levels. P2X3-like immunoreactivity greatly decreased in small DRG neurones after spinal nerve ligation (Kage et al. 2002) and sciatic nerve section (Bradbury et al. 1998), but increased after chronic constriction of the sciatic nerve (Novakovic et al. 1999) and partial injury or section of the inferior alveolar nerve, which contains axons projecting from the trigeminal ganglia (Eriksson et al. 1998). This might reflect a downregulation of P2X3 receptors in injured cells and an upregulation in uninjured cells. Consistent with this, Tsuzuki et al. (2001) found using in situ hybridisation that P2X3 mRNA was decreased in the cell bodies of injured DRG neurones and increased in nearby uninjured cells, following section of the tibial and common peroneal nerves, terminal branches of the sciatic nerve, or of the infraorbital nerve, another peripheral projection of the trigeminal ganglia. However, further studies are needed to correlate in detail the relationship between degree of cell injury and P2X3 expression.

Finally, what is the cellular source of ATP that stimulates P2X3 receptors in these chronic conditions? Initially, various cell types, including damaged or stressed cells, were suggested as potential sources of extracellular ATP (Kennedy & Leff, 1995; Burnstock & Wood, 1996). Since P2X3 receptors appear to be functionally expressed along the length of sensory neurones, then these cells would have a strong potential to excite pain-sensing nerves. Consistent with this, lysis of keratinocytes was recently shown to excite nociceptors through the release of cytosolic ATP (Cook & McCleskey, 2002). Additionally, an intriguing alternative source of ATP is the sensory nerves themselves, as Holton (1959) showed that ATP was released following antidromic stimulation. In this model (Fig. 1) stimulation of P2X3 receptors in peripheral terminals initiates local depolarisation and Ca2+ influx through the P2X3 receptors themselves and/orvia voltage-dependent Ca2+ channels that are opened by the depolarisation. The Ca2+ influx would then induce local release of ATP, which would in turn feed back in a positive manner to further stimulate the P2X3 receptors and so create a self-regenerating signal. If large enough, the depolarising signal would initiate action potentials that travel along the sensory axon to the spinal cord. A similar mechanism at the central terminals would be consistent with the ability of ATP to potentiate glutamate release from the central sensory terminals in the spinal cord (Nakatsuka et al. 2001, 2003). Together, these would constitute a constant, self-regenerating sensory input to the brain, which would be experienced consciously as chronic pain.

P2Y receptors in sensory nerves

  1. Top of page
  2. Abstract
  3. P2X receptors in sensory nerves
  4. A possible role for ATP and P2X3 receptors in pain?
  5. Conclusions and queries
  6. P2Y receptors in sensory nerves
  7. A role for P2Y receptors in pain?
  8. Conclusions
  9. References
  10. Acknowledgements

P2Y receptors are heptahelical receptors that couple to G proteins, and eight mammalian subtypes have been cloned to date (P2Y1,2,4,6,11,12,13,14) (Abbracchio et al. 2003). Although their distribution and functions in sensory neurones have not been as widely studied as P2X receptors, several of these subtypes are expressed in sensory neurones and the effects that they mediate indicate that they might also be considered as therapeutic targets in the treatment of pain.

Localisation

Unlike P2X3 receptors, P2Y receptors are present in large, as well as small and medium-sized sensory neurones. In situ hybridisation studies located P2Y1 mRNA in about 20 % of rat DRG neurones (Xiao et al. 2002), with the most intense staining in RT97-positive cells, indicating large diameter fibres and fainter staining in peripherin-positive cells, indicating small diameter, unmyelinated fibres (Nakamura & Strittmatter, 1996). A similar value (23 %) was reported in the mouse, where co-localisation with TRPV1 mRNA was rare (Moriyama et al. 2003). P2Y1-like immunoreactivity has also been demonstrated in cell bodies of the rat nodose ganglion (Fong et al. 2002). Ligation of the vagus nerve led to accumulation of staining adjacent to the ligature, indicating that P2Y1 receptors are transported to and expressed in the peripheral terminals of sensory nerves.

The mRNA for P2Y2, P2Y4 and P2Y6 receptors is also present in rat DRG (Sanada et al. 2002) and in situ hybridisation further demonstrated P2Y2 mRNA in 77 % of rat DRG sensory neurones. Ninety per cent of cells with a soma diameter of less than 30 μm were positive, as were 39 % of cells of greater than 40 μm diameter (Molliver et al. 2002). In the same study, UTP increased phosphorylation of the CREB transcription factor in 40 % of neurones, especially smaller cells. Of these, 34 % also expressed CGRP and 38 % stained positive for IB4. In the mouse, 22 % of DRG neurones expressed P2Y2 mRNA and of these, 34 % co-expressed TRPV1 mRNA (Moriyama et al. 2003). Otherwise, there are no reports at present on colocalisation of P2Y receptors with markers of nociceptive neurones, such as substance P, CGRP, Trk A, IB4 or the TRPV1 receptor.

Functional expression

ADP, UTP and UDP are agonists at P2Y, but not P2X receptors, and have been used to characterise the functional expression of P2Y receptors. P2Y1 receptors are adenine nucleotide-preferring, with ADP and ATP as agonists. The P2Y2 and P2Y4 receptors are triphosphate-preferring and UTP and ATP are agonists. Finally, UDP is a selective agonist at P2Y6 receptors (Abbracchio et al. 2003). Recombinant P2Y1, P2Y2, P2Y4 and P2Y6 receptors couple to the Gq/11 G proteins, leading to activation of phospholipase C, breakdown of PIP2 to IP3 and diacyl glycerol and release of intracellular Ca2+ stores and activation of protein kinase C (PKC).

Native P2Y receptors also activate this pathway, as ADP increases intracellular Ca2+ levels in all sizes of rat DRG neurones (A.J. Currie et al. manuscript in preparation), including small diameter cells (Fig. 4A and C), indicating expression of P2Y1 receptors. Consistent with this, the Ca2+ release evoked by the ADP analogue ADP-β-S, was antagonised by the P2Y1 receptor antagonist MRS2179 (Borvendeg et al. 2003). In a more detailed study, UTP induced Ca2+ release in 35 % of small diameter (< 30 μm) cells, 41 % of medium diameter cells (30–40 μm) and 43 % of large diameter (> 40 μm) cells (Sanada et al. 2002), suggesting the presence of P2Y2 and/orP2Y4 receptors. In contrast, the P2Y6 agonist UDP does not appear to release Ca2+ stores in most rat DRG neurones and the amplitude of peak rise in Ca2+ levels in responsive cells is much smaller than that evoked by ADP (Fig. 4B and C). Note, however, that this does not rule out the presence of functional P2Y6 receptors in DRG neurones, as UDP had a similar modest effect on intracellular Ca2+ levels in rat sympathetic neurones, but nonetheless elicited action potentials and release of noradrenaline in these cells (Von Kugelgen et al. 1999). Finally, ATP also induced Ca2+ release in large mouse DRG neurones (30–45 μm diameter) (Svichar et al. 1997), but the receptor subtype mediating this effect was not determined.

image

Figure 4. Mobilisation of Ca2+ in rat DRG

Changes in the intracellular Ca2+ levels in acutely dissociated neurones of the neonatal rat DRG in response to ADP (100 μM) (A) and UDP (100 μM) (B) were monitored using the Ca2+-sensitive dye fluo-4. The upper panels show the resting fluorescence in a field of cells just before agonist administration. The lower panels show the signal after ADP and UDP were applied for 20 s in the superfusate. The levels of fluorescence in these images are indicated in arbitrary units in the pseudo-colour scale shown below. C, the time course of the changes in fluorescence compared with basal (ΔF/F0) evoked over 20 s of agonist application (represented by the horizontal bar) in the two cells indicated by the white arrows in A and B are shown. Cells were superfused with a Hepes-based buffer at 8 ml min−1 and fluo-4 was excited using a mercury lamp through a 465–495 nm filter set. The resultant fluorescence was collected using emission filters suitable for FITC (515–555 nm, dichroic mirror 505 nm). Images were acquired at a rate of 1 frame per second using a cooled CCD camera and recorded onto a personal computer running Metamorph software.

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A role for P2Y receptors in pain?

  1. Top of page
  2. Abstract
  3. P2X receptors in sensory nerves
  4. A possible role for ATP and P2X3 receptors in pain?
  5. Conclusions and queries
  6. P2Y receptors in sensory nerves
  7. A role for P2Y receptors in pain?
  8. Conclusions
  9. References
  10. Acknowledgements

In an early study in bullfrog DRG, ATP inhibited the IM potassium current, which will lead to depolarisation and initiation of action potentials (Tokimasa & Akasu, 1990), but the sensory modality of the cells was not characterised. However, recent studies show that P2Y1 and P2Y2 receptors increase particularly the excitability of cells that are likely to be nociceptive. In small diameter (18–27 μm) rat DRG neurones, ATP, but not UTP, potentiated Ca2+ extrusion by the plasma membrane Ca2+ pump, causing a decrease in amplitude and duration of the action potential afterhyperpolarisation and so an increase in excitability (Usachev et al. 2002). This was prevented by the P2Y1 antagonist A3P5P, the phospholipase C inhibitor U73122 and by inhibition of PKC. Similarly, in rat DRG neurones and HEK-293 cells expressing recombinant TRPV1 receptors, ADP and ATP (acting independently of P2X receptors) potentiated capsaicin-induced ion currents and lowered the threshold for activation of heat-sensitive currents from 42 to 35 °C (Tominaga et al. 2001). These effects were inhibited by the PKC inhibitor calphostin C. Thus P2Y1 receptors act via several mechanisms to increase the excitability of sensory neurones that are likely to be nociceptive. A subsequent study from the same group found that the potentiating effect of ATP on capsaicin-induced currents was still present in DRG neurones from P2Y1 receptor knockout mice (Moriyama et al. 2003). UTP also potentiated capsaicin in these cells, suggesting that P2Y2 receptors can also increase the excitability of sensory neurones.

Besides these potentially noxious effects, P2Y1 receptors have also been proposed to play a role in non-noxious mechanoreception. In a frog skin sciatic nerve preparation, application of ATP increased the sensitivity of light touch-sensitive fibres to mechanical stimulation (Nakamura & Strittmatter, 1996). Consistent with this, P2Y1 mRNA was found in about 20 % of rat DRG neurones (Xiao et al. 2002), mainly in large diameter cells. Notably, this rose to about 70 % 14–28 days after sciatic nerve section. Thus as with P2X3 receptors, nerve damage changes the expression level of P2Y1 receptors, which raises the possibility that P2Y1 receptors are normally found in non-nociceptive neurones, but are upregulated following nerve injury and then play a nociceptive role.

P2Y2 receptors also mediate excitation of rat DRG neurones, as UTP and ATP (again acting independently of P2X receptors) induced depolarisation and a prolonged burst of action potentials (Molliver et al. 2002). The second messengers mediating these effects remain to be determined. Also, UTP and ATP, but not ADP, increased phosphorylation of the transcription factor CREB in 40 % of the neurones, especially smaller cells, in a phospholipase C-dependent manner. Phosphorylated CREB is known to induce the expression of numerous genes that may regulate changes in the phenotype of sensory neurones in response to injury or inflammation. Ninety-five per cent of cells expressing phosphorylated CREB also stained positive for peripherin, indicating that they had unmyelinated axons. Thirty-four per cent of these cells also expressed CGRP and 38 % IB4. The UTP-induced phosphorylation of CREB was inhibited by suramin, but not cibacron blue, indicating an action via P2Y2 receptors. Consistent with this, the authors found P2Y2 mRNA in 77 % of rat DRG sensory neurones. Thus P2Y2 receptors increase the excitability of sensory neurones in the short term and activate a transcription factor that is likely to change the neuronal phenotype in the long term.

The effects of P2Y agonists discussed thus far are all likely to be noxious. In contrast, Okada et al. (2002) reported that UTP and UDP were antinociceptive when applied intrathecally to rats. They increased the nociceptive threshold in the paw pinch and tail flick tests and decreased the mechanical allodynia associated with sciatic nerve section (Seltzer model). In the same study, intrathecal ATP and α, β-methylene ATP decreased the paw pinch threshold and induced mechanical allodynia. It is not yet clear if these effects of UTP and UDP are due to presynaptic inhibition of the release of excitatory neurotransmitter from the central terminals of sensory neurones or if they reflect an action on postsynaptic neurones in the spinal cord. Further studies are also required to determine which P2Y subtype (s) mediate these actions of UTP and UDP.

Conclusions

  1. Top of page
  2. Abstract
  3. P2X receptors in sensory nerves
  4. A possible role for ATP and P2X3 receptors in pain?
  5. Conclusions and queries
  6. P2Y receptors in sensory nerves
  7. A role for P2Y receptors in pain?
  8. Conclusions
  9. References
  10. Acknowledgements

Together, the demonstration of P2Y receptor-like immunoreactivity and mRNA and functional expression in small sensory neurones suggest that P2Y receptors could play a role in generating or modulating the perception of pain. These effects may be subtype dependent, with some being noxious (P2Y1 and P2Y2?) and others being antinociceptive (P2Y6 perhaps?). The change in the expression level of P2Y1 receptors following nerve injury and the P2Y2 receptor-mediated activation of CREB also open up the possibility that P2Y receptors are involved in sensory nerve plasticity. However, these studies are only at an early stage. Nonetheless, the data published thus far are encouraging and support further research into the possibility that P2Y receptors could be targeted in the search for new, effective analgesics.

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  1. Top of page
  2. Abstract
  3. P2X receptors in sensory nerves
  4. A possible role for ATP and P2X3 receptors in pain?
  5. Conclusions and queries
  6. P2Y receptors in sensory nerves
  7. A role for P2Y receptors in pain?
  8. Conclusions
  9. References
  10. Acknowledgements
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Acknowledgements

  1. Top of page
  2. Abstract
  3. P2X receptors in sensory nerves
  4. A possible role for ATP and P2X3 receptors in pain?
  5. Conclusions and queries
  6. P2Y receptors in sensory nerves
  7. A role for P2Y receptors in pain?
  8. Conclusions
  9. References
  10. Acknowledgements

This work was support by grants from the Medical Research Council, Wellcome Trust and Caledonian Research Foundation. We also thank Dr Pam Ganju and Dr Mike Jarvis for providing the graphs used in Fig. 2 and Fig. 3.