Multiple roles of ATP and adenosine in somatosensory processing: Therapeutic implications

DRUG DEVELOPMENT RESEARCH 39~279-288 (1 996)

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Research Overview

Multiple Roles of ATP and Adenosine in Somatosensory Processing: Therapeutic Implications

Gary J. Keil II and Michael W. Salter* Division of Neuroscience, Hospital for Sick Children, Department of Physiology, University of Toronto,

Toronto, Ontario, Canada

Preclinical Development Clinical Development Toxicology, Formulation Phases 1 - 1 1 1 Postmarketing Drug Delivery, Pharmacokinetics Manufacturing

Regulatory, Quality, Phase IV

I and Health Policy

INTRODUCTION

The somatosensory system encodes, processes, and transmits peripheral sensory information into the central nervous system (CNS). The coding of information as noxious or innocuous depends not only on the activation of specific peripheral sensory receptors, but also on the actions and interactions of numerous neurotrans,mitter/ neuroinodulator systems at peripheral and central sites. Multiple lines of evidence support roles for adenosine and adenosine 5 '-triphosphate (ATP) in the transinission of sensory information into the CNS [for review see Salter et al., 19931. In this article we outline evidence indicating that adenosine and ATP participate in sensory transmission, and highlight recent developments in the understanding of purine nucleotides and nucleosides in nociceptive, or pain-related, and non-nociceptive sensory transmission at peripheral and spinal sites. Finally, we describe considerations related to possible therapeutic intervention in nociception by targeting compounds which act on or through purinergic receptors or downstream signalling elements.

ATP AND ADENOSINE IN SENSORY NEUROTRANSMISSION

ATP as a Primary Afferent Transmitter

The first evidence implicating ATP as a neurotrans- mitter in the somatosensory system came from studies in the mid- to late-1950s by Holton and Holton who demonstrated that ATP is released from peripheral endings of primary sensory neurons [Holton and Holton, 1954; Holton, 19591. They hypothesized that ATP may also he released from central terminals of primary afferents. Evidence for central release of ATP was provided by White and coworkers who showed that ATP was released in a calcium-dependent fashion from dorsal horn synaptosomes by depolarizing concentrations of potassium [White et al., 19851. This release is greatly depressed by dorsal rhizotoiny suggesting that there is ATP released

Contract grant sponsor: Medical Council of Canada

*Correspondence to: Michael W. Salter, Division of Neuro- science, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M 5 C 1 X8, Canada.

0 1997 Wiley-Liss, Inc.

280 KElL II A N D SALTER

from central terminals of primary afferent neurons [Sawynok et a]., 19931. Release of ATP from dorsal horn synaptosomes is riot affected by neonatal treatment with capsaicin, which destroys many nociceptive primary afferents. Thus, ATP released from primary afferents de- rives from a capsaicin-insensitive subpopulation.

Iontophoretic application of ATP in vivo demonstrated that it excites neurons in the cuneate nucleus [Galindo et al., 19671 and in the dorsal horn [Salt and Hill, 1983; Fyffe and Perl, 1984; Salter and Henry, 19851. These types of neurons receive direct input from primary afferents and it was found that the excitatory effects of ATP are correlated with input from non-nociceptivc primary afferents. In vitro, ATP evokes an inward currcnt in dorsal horn neurons [Jahr and Jessell, 19831 by activating a non-selective cation conductance [Jessell and Jahr, 19851. The effects of exogenously administered ATP would suggest that ifATP is released synaptically it should produce an excitatory post-synaptic response. Recently, evidence for an excitatory postsynaptic current (EPSC) mediated by KL'P in dorsal horn neurons has been ob- tained [Goldstein ct al., 19961. EPSCs resistant to block- ade by excitatory amino acid antagonists hut blocked by the P2-purinoccptor antagonists, suraniin or PPADS, have been recorded in a subpopulation of dorsal horn neurons in an in vitro slice preparation. These are fast EPSCs, which are evoked by dorsal root stimiilation, demonstrat- ing KIP-mediated synaptic responses at some priinaiy afferent-dorsal horn synapses. Taking the in vivo and in vitro electrophysiological evidence together with studies on A r P release from synaptosomes, suggests that ATP may be a mediator of non-nociceptive sensory transmission.

It is likely that the excitatory effect of ATP on dorsal horn neurons and the ATP-mediated EPSC are mediated via ligand-gated cation channels of the P2X-purinoceptor type [rcvicwcd by Surprenant et al., 19951. Moleciilar biological studies liavc revealed a family of P2X purinoceptors of which six (P2XI-P2Xfi) subunits have been identified [Collo et al., 19961. Messenger RNA (mRNA) for P2X1, P2X2, P2X4, P2Xs, and P2X(j, but not P2Xi3, purinoceptors is expressed in the spinal dorsal horn. Which of these P2X subunits mediate the synaptic effects of ATP remains to be determined.

Adenosine as an Inhibitory Modulator in the Dorsal Horn

Considerahle evidence from physiological, bio- chemical, and pharmacological studies indicate that adenosine is an inhibitory mediator in many CNS areas [reviewed by Stone, 19811. In the spinal dorsal horn adenosinc has been shown to inhibit the firing discharge of neurons in somatosensory pathways in vivo [Salter and Henry, 19851. That this inhibition may be a postsynaptic

effect was suggested by the finding that discharge evoked by glutamate is inhibited by iontophoretic application of adenosine 5 '-monophosphate (AMP) and this inhibition is blocked by theophylline. Subsequently, adenosine was shown to cause hyperpolarization of dorsal horn neurones in vivo [DeKoninck and Henry, 19921 and in vitro [Li and Perl, 19941 due to an increase in potassium conductance.

Adenosine-mediated actions in the spinal cord may also involve presynaptic actions. Evidence that adenosine might have presynaptic effects on primary afferent terminals in the spinal cord was first shown by Phillis and Kirkpatrick [ 19781. Sn1)sequent studies have indi- cated that adenosine niay inhibit release of substancc P and CGRP from dorsal horn slices [Santicioli et al., 1992, 19931, but whether this was due to pre- or postsynaptic mechanisms was not determined. Decreases in the rate of spontaneously occurring niiniature EPSCs in dorsal horn neurons by adenosine [Li and Perl, 19943 suggest that adenosine inhibits glutamate-mediated synaptic transmission presynaptically in the dorsal horn. Presyn- aptic actions of adenosine in other CNS regions have consistently been shown to be mediated by A, receptors [see Fredholm and Dunwiddie, 19881, but the subtype of receptor, which may mediate the presynaptic action of spinal adenosine, has not been detcrniined.

A, and A2 receptor binding sites are demonstrated in the spinal cord [Goodman and Snyder, 1982; Murray and Cheney, 1982; Geiger et al., 1984; Choca et al., 1987, 19881 with binding occurring most heavily in the super- ficial laminae [Goodman and Snyder, 1982; Choca et al., 19881. Selective lesion studies show that adenosine rcccptor binding is partially decreased only following the destruction of spinal interneurons [Geiger et al., 1984; Choca ct al., 19881. Moreover, mRNA for A , and A2 receptors is expressed in the spinal cord [Reppert et al., 1991; Rivkees and Reppert, 1992; Stehle et al., 1992; Rivkees, 19951, suggesting that intrinsic neurons express and contain adenosine rcceptors. That these receptors are found on dorsal horn neurons is consistent with postsynaptic effects of adenosine on dorsal horn neurons, and that Al or A2 receptors may participate in the physiological actions of adenosinc at spinal levels.

Extracellular Conversion of ATP to Adenosine in Dorsal Horn Signalling

ATP is rapidly hydrolyzed to adenosine [Zimmer- man, 19941, which is inactive at P2 purinoceptors [Burnstock, 19781. Thus, hydrolysis of ATP terminates its actions. Equally important niay he the generation of adenosine; ATP released from primaiy afferent terminals might he converted to adenosine at synaptic sites in the dorsal horn. An inhibitory postsynaptic response mediated by ATP-derived adenosine has been demonstrated

ATP AND ADENOSINE IN SOMATOSENSORY PROCESSING 281

in dorsal horn neurons [Salter and Henry, 1987; Li and Perl, 19951. The inhibition is a postsynaptic response mediated hy an increase in potassium conductance [DeKoninck and Henry, 19921 due to activating KhTP channels [Salter et al., 19921. This adenosine-mediated inhibitory postsynaptic potential (IPSP) is shown by nociceptive dorsal honi neurons and this is a physiological mechanism for decreas- ing nociceptive transmission in the dorsal horn.

Actions of ATP on Primary Sensory Neurons

In addition to releasing ATE primary afferent neurons are also sensitive to this nucleotide. ATP produces an excitatory effect on the cell bodies of primary afferent neiirons located within the dorsal root ganglia (DRG) [Jahr and Jessell, 1983; Krishtal et a]., 19831. The excitatory effect of ATP results from activating a non-selective cation channel [Bean, 19901 and is blocked by P2- purinoceptor antagonists, indicating it is mediated via P2X-purinoceptors. mRNA for each of the six subunits of P2X receptors is expressed by DRG cells [Chen et al., 1995; Lewis et al., 1995; see Collo et al., 19961. The effects of ATP on DRG cells raise the possibility that pc- ripheral endings of primary afferents may also be excited by ATE In this way ATP might be a sensory mediator in peripheral tissues.

Actions of Adenosine on Primary Sensory Neurons

Adenosine receptors are found on DRG cell bodies [Dolphin et al., 1986; Macdonald et a]., 19861, suggesting that these receptors might be located on the central and peripheral endings of primary sensory neurons. Ac- tivating adenosine receptors on neuronal DRG cell bodies inhibit high voltage-gated Ca2+ currents [Dolphin et al., 1986; Macdonald et al., 1986; Gross et al., 19881. These findings suggest adenosine might inhibit transmitter release at nerve terminals of DRG neurons through inhibition of calcium currents.

PURINES IN MODULATING NOCICEPTION

Adenosine Is a Mediator of Analgesia Evoked by Stimulating low Threshold Primary Afferents

The adenosine-mediated IPSP in nociceptive dorsal horn is elicited by low-intensity peripheral stimulation, which activates non-nociceptive primary afferents. The IPSP is antagonized by 2-methylthiotheophylline, and is potentiated by the nucleoside transport inhibitor, dipyridamole [Salter et al., 19931. The inhibition of nociceptive dorsal horn neurons by activating non-nociceptive inputs may be the cellular basis for the analgesia produced by stimulation of these inputs by transcutaneous electrical nerve stimulation (TENS) or by innocuous mechanical stimuli such as vibration [Woolf and Thomp- son, 19941. Therefore, it has been hypothesized that an-

algesia produced by these types of stimulation may be mediated by adenosine which causes activation of K,,rr channels [Salter et al., 19931. Recently, it has been reported that, under double blind conditions, TENS-induced analgesia is blocked in human subjects challenged with caffeine (200 mg, p a ) [Marchand et al., 19951. At this dose caffeine is expected to antagonize central adenosine receptors [Fredholm, 19801. Thus, the effect of caffeine provides strong support for the hypothesis that analgesia produced by TENS is mediated by adenosine.

Adenosine Induces Analgesia at Spinal Sites

As discussed above, the inhibitory actions of adenosine on dorsal horn neurons have been investigated in vivo using electrophysiological techniques. The iontophoretic application of AMP inhibits the ongoing activity of dorsal horn neurons and the responses to noxious peripheral stimuli [Salter and Henry, 19851. This inhibitory response is seen in approximately two-thirds of dorsal horn neurons and is prevented by theophylline, suggesting that a large population of dorsal horn neurons are sensitive to the inhibitory effects of adenosine. In terms of presynaptic effects, Li and Perl [1994] demonstrated, in an in vitro slice preparation, that bath-applied adenosine suppressed all polysynaptic EPSCs, and inhibited approximately 40% of the monosynaptic EPSCs, in dorsal horn neurons. Reeve and Dickenson [1995a] reported that selective Al adenosine receptor agonists inhibit the C-fiber evoked responses in dorsal horn neurons, hut that an A2A selective agonist was without significant effects. In addition, the excitatory responses of dorsal horn neurons following subcutaneous application of formalin were suppressed by centrally administered A, agonists. The inhibitory responses were blocked by prior treatment with adenosine receptor antagonists, suggesting that Al receptor activation might inhibit acute nociceptive transmission in the dorsal horn and responses to peripheral inflammation.

In animal models of acute and chronic pain, adenosine or adenosine analogs induce antinociception [re- viewedby Sawynok and Sweeney, 1989; Sawynok, 19911. Significant decreases in pain behavior are observed in the formalin test [Malmberg and Yaksh, 1993; Doak and Sawynok, 1995; Poon and Sawynok, 19951, in thermal hyperalgesic states following nerve injury [Yamamoto and Yaksh, 19931, and in strychnine- [Sosnowski and Yaksh, 1989; Sosnowski e t al., 19891 or prostaglandin I T S a -

[Minami et al., 1992a,h] induced allodynia (pain behaviors to non-painful stimuli). Activation of spinal adenosine receptors, therefore, induces antinociception in a wide number of animal models of acute, chronic, and pathological pain states.

Studies using receptor-selective agonists indicate that activating A, adenosine receptors results in the

282 KElL I I AND SALTER

antinociception in behavioral models of pain [Karlsten ct al., 1991, 1992; DeLander et al., 1992; Malmberg and Yaksh, 1993; DeLander and Keil, 1994; Poon and Sawynok, 19951. As discussed previously, binding sites for both AI- and A2-type receptors are found in the spinal cord and mRNA for both receptor subtypes are found in intrinsic dorsal horn neurons, and spinally administered A2 receptor agonists have been found to induce antinociception [reviewed by Sawynok and Sweeney, 19897. Thus, it is possible that A2-type receptors may participate in antinociceptive effects.

The principal mechanism for terniiriatirig the action of extracellular adenosine is uptake and intracellular metabolism. Pharmacological manipulations that decrease the clearance of adenosine in the dorsal horn would be expected to either induce antinociception or to enhance adenosine-mediated effects. Inhibition of adenosine uptake by inhibitors of nucleoside transporters has been shown to significantly potentiate and prolong adenosine-mediated antinociception [Keil and DeLander, 19951. The possibility that other inhibitors of adenosine clearance induce antinociception was examined by Keil and DeLander [ 19921 who found antinociceptive effects of an inhibitor of adenosine kinase. Other studies have also shown that inhibition of adenosine kinase results in antinociception [Keil and DeLander, 1994; Poon and Sawynok, 19951, and in vivo and in vitro studies iridicate that adenosine kinase inhibitors lead to increases in extracellular adenosine levels [Golembiowska et al., 1995, 19961. Lastly, inhibition of adenosine deaminase did not induce demonstrable antinociception in the absence of exogenous adenosine [Kcil and DeLander, 1994; Poon and Sawynok, 19951, h i t antinociception induced h y exogenous adenosine is potentiated following the inhibition of adenosine deaminase [Keil and DeLander, 19941. ‘Faken together, these studies indicate that pliarmacological manipulations which iricreasc endogenous adenosine levels may induce appreciable levels of antinociception, and potentiate physiological or pharmacological manipulations that increase extracellular adenosine levels.

Pronociceptive Effects of Spinal Adenosine Receptor Antagonists

In contrast to the antinociceptive effects that occur following spinal adenosine receptor agonist administration, the spinal administration of adenosine receptor antagonists produces behaviors indicating hyperalgesia to noxious stimuli [Jurna, 1984; Sawynok et al., 19861, allodynia to innocuous stimuli [Keil and DeLander, 19961, or nociception without overt peripheral stimulation [Nagaoka et al., 1993; Keil and DeLander, 19961. That spinal adenosine receptor antagonists induce these behaviors suggests adenosine might tonically inhibit nociccptive pathways, and processes that diminish spinal

adenosine activity might participate in or underlie certain pain states.

Peripheral Actions of ATP Are Pro-Nociceptive

ATP has long been known to induce nociceptivc or algogenic responses following peripheral administration [Collier et al., 1966; Bleehen and Keele, 19773. Nocicep- tive responses induced b y ATP have been suggested to be due to direct activation of peripheral nerve terminals [reviewed by Illes and Niirenberg, 19931 or indirectly through actions on inflammatory cells [Dubyak et al., 1988; Handwerker and Reeh, 1991; Rang et al., 19911. A’L’P may be released into extracelliilar spaces as a con- sequence of tissue injury: it may be released from blood products such as platelets and mast cells [Dubyak arid El-Moatassini, 19931, or it may be released from post- ganglionic sympathetic nerve terminals [Burnstock, 19901, or from sensory afferents [Holton, 19591. As nien- tioned above, ATP depolarizes primary afferent cell bodies [Jahr and Jessel, 1983; Krishtal et al., 19831, and if ATP depolarizes the peripheral terminals of sensory neurons this could result in direct activation of primary afferent s .

Which type or types of P2-purinoceptors mediate the excitatoq or pronociceptive effects of ATP at peripheral sites is presently unknown. mRNA for all six subtypes of P2X purinoceptors are expressed in sensory ganglia [see Collo et al., 19961, and two recent studies suggest a role of the P2X3 subiinit in nociceptive processes. mRNA for the P2X3 subunit is found only in sensory ganglia [Chen et al., 1995; Lewis et al., 19951, and cell body labelling for P2X3, in situ, occurs coincident with inimuriostaining to periphcrin [Chcn et al., 19951, a marker for C-fiber afferents. mRNA for P2X3 transcript is abolished following neonatal capsaicin treatment [Chen et al., 19951, indicating P2X3 is found exclusively in capsaicin-sensitive, presumably nociceptive afferents. The peripheral nociceptive actions of ATP may, therefore, he mediated through activation of PeX:\-containing receptors located on nociceptive primary afferent neurons.

Peripheral Actions of Adenosine May Be Pro- or Anti-Nociceptive

In human subjects, the peripheral adniiriistr a t’ ion of adenosine has been shown to induce pain. Pain is evoked when adenosine is administered to blister bases [Bleechen and Keele, 19771 arid ischemic-like chest pain or discomfort is reported following peripheral [Sylvkn et al., 1986, 1988a,b; Biaggioni et a]., 19871 or intracoronary [Lagerqvist et al., 19901 adenosine administration. Such algogenic or pronociceptive effects are also seen in animal models [‘Faiwo and Levine, 1990; Karlsten et al., 1992; Doak and Sawynok, 19951. Pharmacological studies in animal models indicate that the pronociceptive effects of


peripherally administered adenosine are mediated by actions at A2 adenosine receptors [Taiwo and Levine, 1990,1991; Doak and Sawynok, 19951. Interestingly, the A2A-selective agonist, CGS21680, enhances, while the selective antagonist, DMPX, inhibits formalin-induced nociceptive behavior only during the latter phases of the test [Doak and Sawynok, 19951. Moreover, in this study the intradermal injection of adenosine did not induce nociceptive behavior in the absence of coadmiiiistered formalin. Such effects raise the possibility that adenosine may not activate sensory neurons directly, but may enhance the excitatory effects by other inflammatory mediators [Doak and Sawynok, 19951.

In contrast to the local algogenic effects of adenosine, the systemic administration of adenosine has been shown to induce analgesia [Belfrage et al., 1995; Segerdahl et al., 199.5; Sollevi et al., 19951. In thcse studies patients occasionally experienced discomforting sensory phenomena, but these sensations were promptly diminished upon dosage reduction. The apparently con- trasting results from Sollevi and colleagues compared to the studies mentioned above may be explained by the multiplicative site- and receptor-dependent effects following systemic administration of adenosine (see below). In animal models, the activation of peripheral Al adenosine receptors induces antinociceptive effects [Taiwo and Levine, 1990; Karlsten et al., 1992; Aley et a]., 199.5; Doak and Sawynok, 19951. Tissue adenosine levels are elevated in conditions of ischemia arid inflammation [reviewed in Cronstein, 19951. It has been suggested that this adenosine may activate peripheral Al receptors, which may participate in reducing postinflammatory pain since the peripheral administration of Al antagonists increase, but Al agonists decrease, pain behaviors in the formalin test [Doak and Sawynok, 19951.

Adenosine may also exert anti-inflammatory effects. Indeed, the anti-inflammatoiy actions of certain pharmacological agents have also been attributed to the release of adenosine at inflamed sites [Cronstein, 1995; Gadangi et al., 19961. Similarly, the peripheral inhibition of adenosine kinase reduces inflammation [Firestein et al., 1994; Cronstein et a]., 1995; Rosengren et al., 19951. The anti-inflammatory effects have been attributed to peripheral activation of A2-type receptors, which, amongst other actions, inhibits neutrophil adhesion and prevents the secretion of inflammatory cytokines [Gadangi et al., 19961. Thus, while A2-type receptor activation might be involved in pronociception, anti-inflammatory effects are also seen which may lead to diminished postinflammatory pain.

It is possible that adenosine-mediated actions at A-, adenosine receptors may participate in peripheral somatosensory processes. One function of An receptors is adenosine-mediated degranulation of mast cells [for review see Linden, 19941, which release multiple compounds

implicated in inflammation [rcviewed by Ilandwerker and Reeh, 19911. An additional involvement of A, rcceptors in inflammation and pain may relate to adenosine- mediated inhibition of the release of the proinflammatory cytokine, tumor necrosis factor a (TNF-a) from macroph- ages and monocytes [Sajjadi et al., 19961.

THERAPEUTIC CONSIDERATIONS FOR PURlNERClC COMPOUNDS IN TREATING PAIN

Some potential roles for adenosine and ATP in modulating nociceptive signalling at peripheral and central sites are summarized in Figure 1. As discussed, these compounds have multiple functions in somatosensory processing, which are dependent upon the site and the receptor subtype that is activated. I’har- macological agents or physiological manipulations which specifically target the antinociceptive actions of adenosine and ATP may provide efficacious analgesia in a wide number of clinical pain states. How- ever, algogenic or pronociceptive results might occur if agents are used that are non-selective, either in their mechanism of action or in their primary site of action. Therefore, site- or receptor-targeting strategies are needed in order to maximize analgesic effects while minimizing any algogenic effects.

Targeting PI-Purinoceptors

Systemic administration of adenosine may induce analgesic effects through actions at spinal and peripheral A1 receptors, and anti-inflammatory effects at peripheral A2 or A, receptors. A potential limiting effect to using systemic adenosine in the treatment of pain and inflammation, however, may be the peripheral AP type receptor mediated algogenic effects. The overall effect of systemically administered adenosine, therefore, may depend on the relative magnitude of opposing pro- and anti-nociceptive systems. The use of agents selective for a particular subtype of receptor would be considered a pharmacological targeting strategy, which could avoid possible pronociceptive effects seen with non-selective agents.

A,-selective agonists

Systemically administered Al-selective agonists would be expected to induce analgesia at both peripheral and, if able to cross the blood brain barrier, central adenosine receptors. Moreover, localized peripheral administration of these compounds, for example intra- articularly, would also be expected to induce analgesia. Systemic or intra-articular administration could induce analgesia without concurrent pronociceptive effects. These agents could have actions as anti-inflammatory agents through possible effects on sensory nerve terminals, which inhibit the release of inflammatory mediators.

284 KElL II AND SALTER

Figure 1. Possible central and peripheral sites of adenosine- and ATP- mediated modulation of somatosensory processes. In the periphery, tissue damage could result in the release of ATP and adenosine. ATP and adenosine could induce nociception (lightening bolt) at peripheral P2X3- containing PJ-purinoceptor and Az-adenosine receptors, respectively. Adenosine-mediated actions at peripheral AT receptors induce antinociception. Not shown are the possible anti-inflammatory effects at A2 and/or A3 receptors. In the spinal cord, ATP released from large-diam-

A,-selective agents A,-selcctive agonists induce anti-inflammatory ac-

tions in the periphery, but concurrent algogenic effects might limit any clinical usage. In the spinal cord, A,-type agonists might induce analgesia; in animal models, however, these agonists oftcn p r o d ~ ~ e profound motor-im- pairing effects which may also occur in human patients.

eter (AD) primary afferents following innocuous peripheral stimuli (feather) excites non-nociceptive dorsal horn neurons in deep laminae likely through P2X receptor increases in cation conductance (gc,3,,c,n). Metabolism of ATP to adenosine would induce inhibition through inward potassium conductance (gK) on nociceptive dorsal horn neurons. Adeno- sine might also inhibit presynaptic release from primary afferent terminals (not shown). In addition, ATP might affect dorsal horn astrocytes through P2Y receptors.

Peripheral A2-type selective antagonists may inhibit adenosine-mediated pronociception, but these agents might facilitate inflammatory processes.

A,-selective agonists

The effects of compounds with agoiiist activity at A3 adenosine receptors might he expected to induce an-


algesia via A3 receptor-mediated anti-inff aminatory effects but additional studies are needed to clarify the activity of the receptors in somatosensory processes.

Targeting P,-Purinoceptors

As ATP may be involved in activating nociceptive sensory neurons through P2X3-containing receptors, peripherally acting antagonists selective for these receptors might be analgesic. Potential actions of these compounds would not likely be associated with significant side effects because P2X3 subunits are only expressed by capsaicin-sensitive primary afferents.

The effect of P2X3-selective compounds administered spinally may lead to paradoxical effects. These receptor subunits are not expressed by dorsal horn neurons so P2X3 agonists or antagonists would not be expected to have direct effects on these neurons. However, P2X3 agonists might have effects on central terminals of nociceptive primary afferents. Such agonists might induce primary afferent depolarization (PAD), which may inhibit central release of transmitters [Shapiro et al., 19801 or which may antidromically activate these afferents, leading to release of pronociceptive mediators in the periphery. Thus, the net effect of central agonists or antagonists at P2X3 receptors would be difficult to predict.

Therapeutic Implications of Adenosine-Mediated Antinociception in TENS Analgesia

From the observation that caffeine blocks analgesia produced by TENS (vide supra), it is suggested that patients receiving TENS therapy for pain relief should refrain from consumption of caffeine or other niethyl- xanthines. Moreover, the analgesic effects of dorsaf column stimulation or of vibration, two other treatments that activate low-threshold primary afferent neurons, may also be mediated by adenosine [Salter et al., 19931. Thus, similar considerations concerning the consumption of methylxanthines would also apply to these treatments.

We have reported that the inhibition of nociceptive dorsal horn neurons by low-threshold primary afferent stimulation may be potentiated by inhibitors of adenosine uptake [Salter and Henry, 19871. This suggests that extracellular adenosine concentration produced by the stimulation may not be sufficient to fully activate the receptors or downstream signalling elcments. I f this is the case in humans, then several novel pharmacological means for increasing the efficacy of pain treatment with TENS, dorsal column stimulation, or vibration may be suggested. Thus, it may be possible to use as adjuvants in these treatments such as inhibitors of adenosine uptake or degradation, allosteric enhancers of adenosine receptors, or agents that enhance the activity of K,,, channels.

Therapeutic Implications of Adenosine-Mediated Antinociception in Opioid-Induced Analgesia

As previously described, the antinociceptivc cf'fccts of spinal adenosine may be mediated through the activation of KATp channels. Opioid-induced antinociceptive effects have also been attributed in part to activity of these channels [Welch and Dunlow, 19931. Moreovei; spinal adenosine release has been shown to be involved in the antinociceptive actions of opioid compounds [reviewed by Sawynok and Sweeney, 1989; Sawynok et al., 1989; DeLander e t al., 1992; Cahill et al., 1993; Keil and DeLander, 1994; Cahill et al., 19951. Adenosine and opioid-mediated antinociception show overlap and this overlap might result in cross-tolerance in the spinal cord, similar to the cross-tolerance between opioids and adenosine analogs in the periphery [Aley et al., 19951. Not all opioid-mediated effects are thought to involve adenosine, however [reviewed by Sawynok and Sweeney, 1989; DeLander et al., 19921. In support ofindependent mechanisms of action, adenosine analogs can interact in a supra-additive manner with opioids when coadministered [DeLander and Keil, 1994; Keil and DcLander, 1995; Reeve and Dickenson, 1995b1. These results suggest supra-additive interactions between adenosine and opioid analgesics might occur in clinical settings, such that the overall efficacy to treat certain pains might be enhanced at lower total analgesic doses [DeLander and Keil, 19941.

Novel Therapeutic Target: Dorsal Horn Astrocytes

In the preceding sections we have focused on the efyects of ATP and adenosine on neurons. However, the effects of ATP in the dorsal horn are not restricted to neurons. In primary cultures prepared from the dorsal horn, ATP increases intracellular calcium levels in >99% of the astrocytes examined [Salter and Hicks, 19941, through a P2Y-purinoceptor-mediated activation of the phospholipase Cp/IP3 pathway [Salter and Hicks, 199SI. Astrocytes and other glial cells may have distinct roles in pathological processes that occur following peripheral nerve injury [Hajos et al., 1990; Gilmore et al., 1990; Garrison et al., 1991; Svensson et al., 19933 and these cells may contribute to abnormal nociceptive processing and pathological pain states [Meller et al., 19941. Although much work is needed in this area, it is conceivable that pharmacological agents with actions at P2Y receptors might represent a novel class of analgesic agents.

CONCLUSIONS

We have highlighted the multiple roles of adenosine and ATP in somatosensory processes. These multiple roles provide a rich number of potential therapeutic tar- gets and, thus, we see a great potential for purinergic compounds as analgesics. However, site- and receptor-se-

286 KElL II A N D SALTER

lective targeting strategies need to be employed to achieve maximum efficacy with minimal unwanted effects.

ACKNOWLEDGMENTS

This work was supported by the Medical Research Council of Canada.

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