Immunocytochemical localization of P2X3 purinoceptors in... : PAIN (original) (raw)
1. Introduction
The importance of adenosine-5′-triphosphate (ATP) as the energy source in intracellular biochemistry is well established. In addition, ATP is now recognized as an important extracellular neurotransmitter (Abbrachio and Burnstock, 1994), acting via purinergic receptors which were first postulated by Burnstock (1972). ATP activates two types of receptors: ligand-gated ion channels, (P2X receptors: Valera et al., 1994), and G-protein-coupled receptors (P2Y receptors). P2X receptors have been characterized in a variety of tissues, such as smooth muscle cells and autonomic and sensory neurons (Surprenant et al., 1995; Surprenant, 1996), and seven distinct members of the P2X family have been cloned to date. Purinoceptors have between 379 and 472 amino acids, probably consisting of two transmembrane spanning domains, with most of the protein forming a large extracellular loop and both amino- and carboxyl-termini in the cytoplasm (Brake et al., 1994, Torres et al., 1996). These proteins structurally resemble the motif of certain voltage-insensitive cation channels, but lack any sequence homology. Agonist application evokes rapid opening of the P2X channels on sensory neurons, producing two types of current: fast-desensitizing and non-desensitizing, suggesting multiple receptor/ion-channel phenotypes (Kristhal et al., 1988; Khakh et al., 1995; Evans, 1996).
Of the many diverse functional activities proposed for ATP in cellular transmission, one important role may be in the sensory neurotransmission (for review see Thorne and Housley, 1996). In support of this hypothesis, 40–96% of rat dorsal root ganglion (DRG) and dorsal horn neurons respond to ATP by depolarization (Jahr and Jessell, 1983; Bean, 1990; Bean and Friel, 1990; Bean et al., 1990), or by an increase in intracellular Ca2+ (Bouvier et al., 1991; Salter and Hicks, 1994). It has long been known the ATP and adenosine can stimulate nerve endings in the skin, resulting in intense pain (Bleehen, 1978; Coutts et al., 1981), but the role of purines in nociceptive transmission has not been emphasized until recently (Kennedy and Leff, 1995; Burnstock, 1996; Burnstock and Wood, 1996), following the cloning and characterization of purinoceptors from nociceptive neurons (Chen et al., 1995). An in vivo study by Driessen et al. (1994) has shown that several P2X antagonists, including suramin, when administered intrathecally in rats, had moderate antinociceptive activity in the tail flick and formalin tests, while α,_β_-Me-ATP, a P2X agonist, had pro-nociceptive activity and decreased the tail-flick latency. These findings have recently been extended in a study showing that both ATP and α,_β_-Me-ATP potentiated and suramin inhibited flinching behavior induced by formalin administration in rats (Sawynok and Reid, 1997). In addition, the rat writhing reflex can be produced by intraperitoneal ATP administration (Gyires and Torma, 1984).
One member of the P2X family of purinergic receptors, P2X3, appears to be of special interest for the study of pain. The P2X3 has been cloned and characterized both electrophysiologically and pharmacologically (Chen et al., 1995). P2X3 receptors expressed in Xenopus oocytes had characteristics similar to those in certain rat sensory neurons, displaying fast desensitization with slow recovery, and distinct agonist pharmacology, suggesting that P2X3 may be responsible, at least in part, for the purinergic activity in sensory neurons (Chen et al., 1995). In the same study, distribution of P2X3 mRNA was established by Northern blot and in situ hybridization, showing an exclusive localization in rat sensory ganglia. P2X3 mRNA was found only in a sub-population of small-diameter sensory neurons, colocalized with the small neuron-specific marker peripherin. Additionally, neonatal capsaicin treatment significantly reduced the signal, thereby designating P2X3 positive neurons as nociceptors. These findings were confirmed by immunocytochemical experiments in which P2X3 was detected in a similar subpopulation of DRG neurons, lamina III of the spinal cord, nucleus tractus solitarii, and peripheral and central axons (Vulchanova et al., 1997). This exclusive localization of P2X3 receptors to nociceptive neurons strengthens the possibility of their potential role in pain transmission.
Little is known about P2X3 involvement in chronic pain conditions. Purinergic system has been implicated in neuropathic pain states such as sympathetically maintained pain (Burnstock, 1990; Burnstock, 1996) In order to further investigate this hypothesis, we have studied the distribution of P2x3 using immunocytochemical methods in tissues from naive rats and following a peripheral mononeuropathy, involving ligation of the sciatic nerve.
2. Methods
2.1. Animals
All experimental procedures were approved by the Institutional Animal Care and Use Committee of Roche Bioscience and conformed to the guidelines for ethical research of the International Association for the Study of Pain (Zimmerman, 1983). Male Sprague–Dawley rats (Harlan, Indianapolis, IN) were housed under a 12:12 h light–dark cycle for 1 week prior to surgery.
2.2. Chronic constriction injury model
Chronic constriction injury (CCI) model was performed as previously described (Bennett and Xie, 1988). Briefly, animals were anesthetized with pentobarbital sodium (65 mg/kg, i.p.) and the right sciatic nerve exposed at mid-thigh level. Four loose ligatures (chromic gut 4.0; Ethicon) were tied around the nerve proximal to the trifurcation. In the sham-operated group the nerve was left untouched. The incisions were closed in layers using 4–0 vicryl-braided suture and the animals allowed to recover for a period of 5 days.
Following recovery, rats were tested for both cold allodynia and heat hyperalgesia. We have previously found that CCI animals exhibited maximal cold allodynia from day 4–10 post-surgery (p.s.) and maximal heat hyperalgesia from day 10–14 p.s. Consequently, day 6 and day 13 p.s. were chosen to perform cold allodynia and radiant heat assays, respectively. For the cold allodynia test, each rat was placed upon a metal platform which was submerged in a bath of ice-cold water. The latency for withdrawal of the right (operated) hindpaw was measured. Rats were deemed to be allodynic if the average withdrawal latency from two trials was less than 13 s (20 min interval between trials; maximum immersion time of 20 s). A withdrawal latency of less then 20 s was never recorded in sham-operated or naive rats. For the thermal hyperalgesia assay, the latency of hindpaw withdrawal from a radiant heat source was measured. The average latencies of left (non-ligated) and right (ligated) hindpaws were obtained from four trials and a difference score was calculated. Rats were deemed to be hyperalgesic if the difference score was >1.5 s. Animals that tested positive in both assays were euthanized on day 14 p.s and tissues were removed for further analysis.
2.3. Tissue preparation
Rats were anesthetized with 10% chloral hydrate (300 mg/kg, i.p.) and subsequently perfused through the aorta with sodium phosphate-buffered saline (PBS, 200 ml, pH 7.5, 4°C), followed by 10% formalin (500 ml, pH 7.8, 4°C). DRG (L4 and L5), lumbar spinal cord and brain were removed, post-fixed (10% formalin, overnight, 4°C), processed in ethanol and xylene (tissue processor Miles Scientific) and embedded in paraffin blocks. Sections 4–10 _μ_m were cut, mounted on slides (Probe-On® Plus, Fisher Scientific, Pittsburgh, PA) and air-dried. Alternatively, for cryosectioning, rats were perfused, tissue harvested quickly, cryoprotected by incubation in PBS-sucrose (20%), embedded in OCT Tissue Tek and frozen on dry ice. Subsequently, 10 or 20 _μ_m cryosections were cut and mounted on gelatin-coated slides.
2.4. DRG primary cell culture
DRG were removed from all spinal levels of rats under sterile conditions and cultures were prepared using a modification of the method described by Lindsay et al. (1991). Briefly, DRG were dissected into Ca2+ and Mg2+-free HBSS (Gibco BRL, Life Technologies, Grand Island, NY), containing 10 mM HEPES (pH 7.4). The DRG were incubated twice for 90 min each in 0.125% collagenase (Boehringer–Mannheim, Indianapolis, IN) in HAM's F-12/DME containing 6% glucose (Irvine Scientific, Irvine CA) 5 mM HEPES, 4% Ultroser G (Life Technologies, Paisley, UK), 1% penicillin-streptomycin (HAM's F-12/DME plus USG) at 37°C. They were rinsed with HAM's F-12/DME prior to incubation for 30 min at 37°C in 0.125% trypsin (Worthington Biochemical, Freehold, NJ) in HAM's F-12/DME. The media was aspirated and DRG dispersed by trituration with a small-bore, fire-polished pipette in 0.01% DNAse (Sigma, St. Louis, MO) in HAM's F-12/DME plus USG. The cell suspension was centrifuged for 5 min at 1000 rpm, supernatant discarded and pellet resuspended in the appropriate volume of HAM's F-12/DME plus USG and 100 _μ_M 5-fluorodeoxyuridine (Sigma) to inhibit non-neuronal cell proliferation. In some preparations 100 ng/ml nerve growth factor (NGF, Promega, Madison, WI) was added to the medium. Cells (5–l0×103 cells/well) were plated onto Nunc 2-well glass chamber slides pre-coated with poly-d-lysine (10 _μ_g/ml, Sigma) and laminin (5 _μ_g/well, Gibco BRL and grown for 3–5 days in vitro in an atmosphere with 5% CO2 at 37°C.
2.5. Antiserum
Polyclonal antiserum was raised in rabbits against a 15 amino acid peptide (VEKQSTDSGAYSIGH) corresponding to the C-terminus of rat P2X3, conjugated to Keyhole Limpet Haemocyanin (Research Genetics, Huntsville, AL). This peptide sequence was determined to be unique for the P2X3. Antiserum was purified on DEAE Affi-Gel IgG purification column (BioRad) prior to use.
2.6. Western blot
Successful, stable transfection of rat recombinant P2Xl, P2X3 and P2X4 receptors in Chinese hamster ovary (CHO-K1) cells was first determined by electrophysiology (whole cell patch-clamp) and radioligand binding (ATP_γ_[35S]; Lachnit et al., unpublished). Next, the cells were released in PBS+7% Versene centrifuged at 500×g and washed once in PBS. After centrifugation, cells were resuspended in ice cold 50 mM Tris, pH 7.4/1.0 mM EDTA supplemented with 2 _μ_g/ml soybean trypsin inhibitor and 10 μ_g/ml bacitracin. The suspension was homogenized with a Polytron homogenizer (full setting; 3×15 s bursts) and the homogenate centrifuged at 45 000×_g for 20 min. The resulting pellet was resuspended at approximately 1 mg/ml in homogenization buffer. SDS-PAGE and electrophoretic transfer were performed using NOVEX Mini-cell system and reagents (10% Tris/glycine gel and nitrocellulose membrane). The membrane was blocked for 3 h in 50 mM Tris/3% non-fat milk (pH 7.4), and incubated overnight in P2X3 antiserum diluted 1:4000 in the same buffer (4°C). After four washings in 50 mM Tris and 1 mM EDTA (pH 7.4), the membrane was incubated with HRP conjugated Protein A (Bio-Rad) diluted 1:3000 in 50 mM Tris+3% non-fat dry milk (pH 8) and incubated for 2 h at 4°C. Visualization was performed using the ECL system and Hyperfilm-ECL (Amersham, Arlington Heights, IL).
2.7. Immunohistochemistry
Following deparaffinization in xylene and ethanol solutions, microscope slides were mounted in a Microprobe slide holder (Fisher Scientific), where the ‘capillary gap’ between adjacent slides allowed for the filling and retaining of liquid during incubation. Sections were preincubated in potassium phosphate buffered saline (KPBS) containing 20% normal goat serum (NGS) and 0.2% Triton X-100 (1 h, room temperature-RT), then incubated in KPBS containing 5% NGS, 0.2% Triton X-100 and P2X3 antibodies at 1:50 to 1:500 dilution (overnight, 4°C). Next day, sections were washed in KPBS containing 0.1% bovine serum albumin (BSA) and 0.1% Triton X-100 (2 h, RT), incubated in the secondary antibody solution (biotinylated anti-rabbit IgG 1:200, Vectastain Elite Kit, 1 h, RT), washed again and then incubated with peroxidase avidin-biotin complex (ABC, 1:50, 90 min, RT). After a few washes with 0.1 M KPB, the staining pattern was visualized with a 3,3′-diaminobenzidine (DAB) substrate reaction (Zymed Laboratories). Tissue sections were then washed, dehydrated in a series of ethanol and xylene solutions and mounted with Krystalon. For immunofluorescence, ABC complex was substituted with extra-avidin-FITC (Sigma, 1:200, 1 h) and slides were mounted with an anti-fading medium. Primary DRG cell cultures and transfected CHO-K1 cells were also labeled as a control for antibody specificity. Prior to immunocytochemistry, DRG cultures and CHO-K1 cells were washed with PBS and fixed in 4% paraformaldehyde (30 min, RT). The same procedure was followed as for the immunofluorescence labeling of tissue sections except that antibodies were used at 1:2000 dilution. In some experiments, propidium iodide (PI, Molecular Probes 1:2000, 1 h at RT) was used for non-specific labeling of cell nuclei. For control experiments, antibodies were preabsorbed with the cognate peptide overnight at 4°C.
2.8. Quantitation of immunolabeling
Samples were observed and analyzed using a Nikon Microphot SA microscope with fluorescent attachment and a Biorad MRC 1024 confocal microscope. Image capturing and quantification of cell area and labeling intensity was subsequently performed using IPLab Spectrum (Signal Analytics) software. In order to measure cell area, each neuron with a visible nucleus was defined as a segment including the nuclear region, whereas for the measurements of labeling intensity, the nucleus was excluded. According to the measured area, neurons were classified as small (<700 _μ_m2), medium (700–1200 _μ_m2) and large (>1200 _μ_m2). Furthermore, pixel density was obtained as a measure of labeling intensity for each neuron. Neurons were considered positive for P2X3 if the mean intensity value was >165 on a 0–255 scale with 0=white and 255=black. The cut off value of 165 was determined from visual analysis of immunolabeling, and by comparison with control (maximal level of labeling with preabsorbed antibodies). The statistical analysis of intensity of labeling in neurons from CCI and sham-operated animals was performed with a two-sample _t_-test. Pearson's chi-squared test was used to determine whether the proportion of positive readings was significantly different between CCI and sham operated rats.
3. Results
3.1. Specificity of P2X3 antiserum
The specificity of the P2X3 antiserum was established by Western blot using membrane homogenates from transfected P2X3, P2X1 and P2X4 cells. P2X3 transfectants showed a highly specific single band representing a ˜60 kDa protein (Fig. 1A, column 3; B, column 2), corresponding to the glycosylated P2X3 protein. Controls with non-transfected cells (Fig. 1A, column 2;C, column 2), P2X4 (Fig. 1A, column 4), or P2X1 transfectants (Fig. 1B, column 3), as well as the serum preabsorbed with the peptide used to raise P2X3 antibodies (Fig. 1C, column 3) did not give any reaction product. Western blot of normal DRG and spinal cord tissue showed bands of similar molecular weight corresponding to P2X3 (not shown), which were absent when preabsorbed antiserum was used. An additional band was detected in the spinal cord tissue which could not be blocked with the cognate peptide. However, immunocytochemical labeling was never detected in spinal cord sections after blocking with the cognate peptide and consequently, immunolabeling obtained with P2X3 antibodies was considered highly specific.
Western blots showing the specificity of P2X3 antiserum. Antiserum against a P2X3 C-terminal peptide specifically recognized ˜60 kDa recombinant P2X3 in transfected CHO-K1 cells (columns A3 and B2). Columns A2 and C2 are untransfected cells, column A4 represents P2X4 transfected cell line, and column B3 is P2X1 transfected cell line. Immunoreactivity was specifically blocked by preincubation of the antiserum with the homologous peptide (C3). Molecular weight standards (kDa) are shown (A1, A5, B4, B5, C1, C5).
The P2X3 transfectants labeled brightly with P2X3 antiserum (Fig. 2A, green), in contrast to non-transfected (Fig. 2B), or P2X4 and P2X1 transfected cells (data not shown) indicating that P2X3 antiserum did not cross-react with these receptor subtypes. Propidium iodide was used for non-specific labeling of cell nuclei (Fig. 2A,B, red).
Immunocytochemistry of P2X3 transfected cells and normal DRG sections. Antiserum raised against P2X3 purinoceptor labels a specific population of small sensory neurons in rat DRG suggesting the role of P2X3 in nociceptive processing. (A) P2X3 transfected cells were brightly labeled with P2X3 antiserum (green). (B) Non-transfected cells do not label, confirming the specificity of P2X3 antibodies. Propidium iodide (PI-red) was used for non-specific labeling of cell nuclei (scale bar, 50 _μ_m). (C and D) Paraffin-embedded, 4 _μ_m thick sections from normal rat DRG. The P2X3 antiserum distinctively labels a population of small neurons as shown by peroxidase-DAB labeling (C), or immunofluorescence (D, scale bar, 100 _μ_m). (E and F) Paraffin-embedded sections (10 _μ_m) were double labeled (P2x3-green, PI-red), and observed with confocal microscopy. Some small cells were brightly labeled (E), and this labeling disappeared when preabsorbed antibodies were used (F, scale bar, 25 _μ_m).
3.2. P2X3 localization in normal dorsal root ganglion neurons
After the specificity of the serum had been established, immmunolabeling was performed on tissue sections from naive rats. DRG neurons expressed very distinct labeling as shown by peroxidase-DAB (Fig. 2C) and immunofluorescence (Fig. 2D), predominantly in a subpopulation of small neurons. By subtraction of the background level of labeling (maximal intensity detected in preabsorbed control; as described in Section 2), the intensity of labeling in each cell was translated to either P2X3 positive or negative, and the number of P2X3 positive cells determined. Typically, a characteristic pattern was observed: ˜50% of small-diameter (<700 _μ_m2) neurons exhibited strong immunolabeling, while 10–20% medium- and large diameter (>700 _μ_m2) neurons expressed low level labeling. Staining was somewhat heterogeneous in small neurons, thus, some cells were stained more intensely than others. Furthermore, confocal microscopy was used for the characterization of P2X3 immunoreactivity in normal DRG tissue (Fig. 2E, PI-red), confirming previous observations. Specific labeling disappeared if preabsorbed antibodies were used in control experiments (Fig. 2F).
3.3. Immunolabeling of DRG cells in primary culture
The same pattern of preferential labeling of small neurons was observed in DRG cells in primary culture although large neurons apparently expressed more P2X3 immunoreactivity than that observed in tissue sections. Fig. 3A illustrates a small to medium-sized neuron with low level labeling, and Fig. 3B represents a small neuron with very intense labeling. Neurites also seemed to be stained, at least in the region close to the cell bodies, suggesting that P2X3 receptors are transported to peripheral nerve terminals. No difference in P2X3 expression was observed in relation to the NGF treatment.
P2X3 labeling in primary DRG cell culture. (A) Neuron in the range of small to medium size (diameter >25 _μ_m) expressing low-level labeling. (B) Small neuron labeled brightly with P2X3. Some labeling is also visible in neural cell processes. Scale bar, 25 _μ_m.
3.4. P2X3 localization in normal spinal cord
A discrete labeling was observed in the superficial laminae of dorsal horn in the spinal cord taken from naive animals (Fig. 4A), which corresponds to the region associated with the termination field of primary afferent C-fibers (Sugiura et al., 1986). This labeling was specific as it was absent when preabsorbed antibodies were used in immunolabeling (Fig. 4B). Brain sections did not reveal any P2x3-like immunoreactivity (not shown).
The distribution of P2X3 in spinal cord tissue from normal and CCI rats. Discrete P2X3 labeling could be observed in superficial laminae of dorsal horn on both sides of the spinal cord, here the right side dorsal horn is shown (A) when compared with preabsorbed antibody control. (B) Spinal cord immunolabeling in CCI tissue does not change on the side contralateral to the injury (not shown), but significantly increases on the ipsilateral side (C). Paraffin (10 _μ_m) embedded tissue sections, scale bar, 100 _μ_m.
3.5. Immunolabeling of spinal cord and DRG neurons in the chronic constriction injury (CCI) model
The CCI model was used, as described in Section 2, in order to study P2X3 distribution following a peripheral nerve injury. When compared with immunolabeling in normal spinal cord sections (Fig. 4A) or with the side contralateral to injury (not shown), a clear increase in intensity was detected in superficial laminae of ipsilateral spinal cord (Fig. 4C). This labeling was concentrated in small structures visualized at higher magnification, probably corresponding to axon terminals of primary sensory neurons. These structures were too small to be distinctively resolved at the light microscopy level. The labeling at the injured side of spinal cord was localized predominantly in the lamina II of dorsal horn. These findings were also confirmed with immunofluorescence of 20 _μ_m cryosections (not shown).
Next, DRG sections from sham-operated (Fig. 5A) and CCI animals (Fig. 5B) were compared. DRG neurons from CCI animals showed the typical pattern of P2X3 labeling: predominantly in small neurons and occasionally very intense (Fig. 5B). Additionally, larger numbers of medium neurons labeled following neuropathic injury (Fig. 5B, arrow points to labeled medium-size neuron). Subsequent quantitation of immunolabeling confirmed that the percentage of small and medium diameter P2X3-positive neurons, significantly increased in the tissue from CCI-operated animals when compared with tissue from sham-operated animals (Fig. 6). Thus, the number of P2X3 positive small-diameter neurons increased from 50 to 73%, and the number of positive medium-diameter neurons increased from 20 to 38%. Although approximately 60% of animals with CCI nerve injury expressed the signs of thermal hyperalgesia and cold allodynia as described in Section 2, the quantitation of immunolabeling did not show a correlation between the increase in P2X3 and behavioral symptoms, but rather with the nerve injury.
DRG neurons labeled with P2X3 antiserum in tissue from sham-operated (A) and CCI injured animals (B). The number of medium-sized neurons with low intensity labeling increased in tissue from CCI (arrow) animals. Additionally, the intensity of labeling is higher in some small neurons in CCI tissue. Scale bar, 25 _μ_m.
The percentage of P2X3 positive cells in sham-operated (gray bars) versus the percentage of P2X3 positive cells in CCI animals (black bars). A significant increase (*P<0.05; **P<0.01) in the number of P2X3 positive small and medium diameter neurons in CCI tissue is shown.
4. Discussion
The present study demonstrates that P2X3 immunoreactivity appears to be predominantly localized to a subpopulation of small-diameter neurons in rat DRG. The pattern of immunolabeling corresponds to some extent with the localization of P2X3 mRNA previously demonstrated in this tissue (Chen et al., 1995), and it is suggestive of a P2X3 role in nociceptive processing. Furthermore, our present results indicate that a specific change in P2X3 distribution occurred following neuropathic injury in CCI animals.
Thus, it is possible that a correlation exists between P2X3 expression and injury leading to neuropathic pain in rats.
Up-regulation of P2X3 receptors following neuropathic injury indicates a potential increase in P2X3 synthesis and/or possible changes in the phenotype of some medium-sized neurons. It has been shown previously that, during inflammation, a similar type of phenotypic switch occurs in the myelinated fibers of some sensory neurons such that they started to produce substance P and, thus, resemble nociceptors thereby, substantially contributing to the development of central hypersensitivity (Neumann et al., 1996). On the other hand, the increase of P2X3 content in the cell body may simply be the consequence of altered protein transport and subsequent accumulation due to the applied ligatures. Indeed, it has been shown previously that P2X3 receptors accumulate at the site of sciatic nerve ligation, suggesting that they were normally transported to the distal nerve endings (Vulchanova et al., 1996). If P2X3 protein is also over-synthesized then the sites upstream of the nerve injury, such as neuronal soma, may possess some excess protein. Either possibility would imply some dynamic changes in the P2X3 content following nerve damage and consequent pathophysiological conditions.
Recently, Cook et al. (1997) developed tissue culture models which allowed a comparison of the properties of nociceptive (tooth-pulp afferent) and non-nociceptive (jaw muscle stretch receptor) rat sensory neurons, in order to determine the involvement of P2X3 in nociception. Low concentrations of ATP evoked activity in both types of neurons, but only nociceptors produced currents similar to those of heterologously expressed P2X3 receptors and showed P2X3-like immunoreactivity. These data support the hypothesis that P2X3 receptors mediate some form of nociception (Burnstock, 1996; Burnstock and Wood, 1996).
It should be noted that although the P2X3 receptor is the only purinergic receptor with localization predominantly in sensory ganglia, the possibility must be entertained that other P2X receptors are involved in nociception. P2X1–6 mRNA transcripts are all expressed in sensory neurons of rat DRG, nodose and trigeminal ganglia, as well as in many other tissues (Vulchanova et al., 1996). Furthermore, there is an intriguing possibility that P2X3 and P2X2 may function endogenously as a heteromultimeric complex. When expressed together they apparently form heteromeric channels which are fully activated by α,b-meATP (a P2X3 property) and do not show desensitization (a P2X2 property). Thus, these heteromultimers closely resemble certain native channels of many adult sensory neurons (Lewis et al., 1995), and nociceptors with persistent ATP current (Cook et al., 1997). Furthermore, while P2X3 may be involved in nociception in the periphery (such as on sensory nerve endings or ganglion neuronal somata; Cook et al., 1997), other purinergic receptors are probably involved in postsynaptic sensory signaling in the CNS (Bardoni et al., 1997).
In addition to its direct action on P2X receptors, ATP may lead to sensitization of nociceptors by an indirect mechanism such as the induction of prostaglandin synthesis. In support of this mechanism is the observation that ATP-induced writhing in rat could be inhibited by a prostaglandin receptor antagonist (Gyires and Torma, 1984). ATP also enhanced bradykinin-induced acute inflammation in rats, an effect thought to be mediated via an action on sympathetic postganglionic nerve terminals (Green et al., 1991). ATP is released as a co-transmitter with noradrenaline in sympathetic nerves (Burnstock, 1990), and purinergic-mediated nociception may explain the greater antinociceptive effect obtained when surgical sympathectomy is used rather than adrenoceptor antagonists or reserpine (which depletes noradrenaline but not ATP, from sympathetic nerve terminals; Burnstock, 1996). In the CNS, in addition to its direct postsynaptic effects via P2 receptors, ATP is likely to have an important modulatory role via potentiation of glutamate-induced currents (Li and Perl, 1995), and by producing secondary adenosine-mediated inhibition through P1 receptors following its breakdown to AMP and adenosine (Sawynok and Sweeney, 1989; Li and Perl, 1995; Burnstock and Wood, 1996). Adenosine and adenosine analogs have been shown to evoke both anti-nociceptive (Sosnowski et al., 1989; Sollevi et al., 1995; Poon et al., 1995) and pro-nociceptive effects (Bleehen and Keele, 1977; Sylven et al., 1988).
In conclusion, our present results contribute to the accumulating evidence that P2X3 receptors may have a role in the facilitated processing of nociceptive information that occurs following a peripheral nerve injury. However, direct evidence of P2X3 involvement in nociception in either acute or chronic pain conditions remains elusive, and functional studies, pending the development of a selective antagonist, are needed to confirm the specific role of this receptor.
References
Abbrachio, M.P. and Burnstock, G., Purinoceptors: are there families of P2X and P2Y purinoceptors, Pharmacol. Ther., 64 (1994) 445-475.
Bardoni, R., Goldstein, P.A., Lee, C.J., Gu, J.G. and MacDermott, A.B., ATP P2X receptors mediate fast synaptic transmission in the dorsal horn of the rat spinal cord, J. Neurosci., 17 (14)(1997) 5297-5304.
Bean, B.P., ATP activated channels in rat and frog sensory neurons: concentration-dependence and kinetics, J. Neurosci., 10 (1990) 1-10.
Bean, B.P. and Friel, D.D., ATP-activated channels in excitable cells. In: T. Narahashi (Ed.), Ion channels, Vol. 2, Plenum, New York, 1990, pp. 169–203.
Bean, B.P., Williams, C.A. and Ceelen, P.W., ATP activated channels in rat and bullfrog sensory neurons: current–voltage and single-channel behavior, J. Neurosci., 10 (1990) 11-19.
Bennett, G.J. and Xie, Y.K., A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man, Pain, 33 (1988) 87-107.
Bleehen, T. and Keele, C.A., Observations on the algogenic actions of adenosine compounds on human blister base preparation, Pain, 3 (1977) 367-377.
Bleehen, T., The effects of adenine nucleotides on cutanous afferent nerve activity, Br. J. Pharmacol., 62 (1978) 573-577.
Bouvier, M.M., Evans, M.L. and Benham, C.D., Calcium influx induced by stimulation of ATP receptor on neurones cultured from dorsal root ganglia, Eur. J. Neurosci., 3 (1991) 285-291.
Brake, A.J., Wagenbach, M.J. and Julius, D., New structural motif for ligand-gated ion channels defined by an ionotropic ATP receptor, Nature, 371 (1994) 519-523.
Burnstock, G., Purinergic nerves, Pharmacol. Rev., 24 (1972) 509-581.
Burnstock, G., Noradrenaline and ATP as cotransmitters in sympathetic nerves, Neurochem. Int., 1 (1990) 357-368.
Burnstock, G., A unifying purinergic hypothesis for the initiation of pain, Lancet, 347 (9015)(1996) 1604-1605.
Burnstock, G. and Wood, J.N., Purinergic receptors: their role in nociception and primary afferent neurotransmission, Curr. Opin. Neurobiol., 6 (1996) 526-532.
Chen, C.C., Akopian, A.N., Sivilotti, L., Colquhoun, D., Burnstock, G. and Wood, J.N., A P2X purinoceptor expressed by a subset of sensory neurons, Nature, 377 (1995) 428-431.
Cook, S.P., Vulchanova, L., Hargreaves, K.M., Wide, R. and McCleskey, E.W., Distinct ATP receptors on pain-sensing and stretch-sensing neurons, Nature, 387 (6632)(1997) 505-508.
Coutts, A.A., Jorizzo, J.L., Eady, R.A.J., Greaves, M.W. and Burnstock, G., Adenosine triphosphate-evoked vascular changes in human skin: mechanism of action, Eur. J. Pharmacol., 76 (1981) 391-401.
Driessen, B., Reimann, W., Selve, N., Friderich, E. and Bultmann, R., Antinociceptive effect of intrathecally administered P2-purinoceptor antagonists in rats, Brain Res., 666 (1994) 182-188.
Evans, R.J., Single channel properties of ATP-gated cation channels (P2X receptors) heterologously expressed in Chinese hamster ovary cells, Neurosci. Lett., 212 (3)(1996) 212-214.
Green, P.G., Basbaum, A.I., Helms, C. and Levine, J.D., Purinergic regulation of bradykinin-induced plasma extravasation and adjuvant-induced arthritis in the rat, Proc. Natl. Acad. USA, 88 (1991) 4162-4165.
Gyires, K. and Torma, Z., The use of the writhing test in mice for screening different types of analgesics, Arch. Int. Pharmacodyn., 267 (1984) 131-140.
Jahr, C.E. and Jessell, T.M., ATP excites a subpopulation of rat dorsal horn neurones, Nature, 304 (1983) 730-733.
Kennedy, C. and Leff, P., Painful connection for ATP, Nature, 377 (1995) 385-386.
Khakh, B.S., Humphrey, P.P.A. and Suprenatant, A., Electrophysiological properties of P2X purinoceptors in rat superior cervical, nodose and guinea-pie coeliac neurones, J. Physiol. Lond., 484 (1995) 385-395.
Kristhal, O.A., Marchenko, S.M., Obukhov, A.G. and Volkova, T.M., Receptors for ATP in rat sensory neurons: the structure-function relationship for ligands, Br. J. Pharmacol., 95 (1988) 1057-1062.
Lewis, C., Neidhart, S., Holy, C., North, R.A., Buell, G. and Surprenant, A., Coexpression of P2X2 and P2X3 receptor subunits can account for ATP-gated currents in sensory neurons, Nature, 377 (1995) 432-435.
Li, J. and Perl, E.R., ATP modulation of synaptic transmission in the spinal substantia gelatinosa, J. Neurosci., 15 (5)(1995) 3357-3365.
Lindsay, R.M., Evison, C.J. and Winter, J., Culture of adult mammalian peripheral neurons. In: H. Wheel and J. Chad (Eds.), Cellular Neurobiology: A Practical Approach, Vol. 1, Oxford University Press, New York, 1991, pp. 3–17.
Neumann, S., Doubell, T.P., Leslie, T. and Woolf, C.J., Inflammatory pain hypersensitivity mediated by phenotypic switch in myelinated primary sensory neurons, Nature, 384 (1996) 360-364.
Poon, A., White, T. and Sawynok, J., Antinociceptive activity of adenosine analogs and an adenosine kinase inhibitor depends on stimulus intensity in the formalin test, Neurosci. Abstr., 21 (1995) 651.
Salter, M.W. and Hicks, J.L., ATP evoked increases in intracellular calcium in neurons and glia from the dorsal horn spinal cord, J. Neurosci., 14 (3)(1994) 1563-1575.
Sawynok, J. and Sweeney, M.I., The role of purines in nociception, Neuroscience, 32 (1989) 557-569.
Sawynok, J. and Reid, A., Peripheral adenosine 5′-triphosphate enhances nociception in the formalin test via activation of a purinergic P2X receptor, Eur. J. Pharmacol., 330 (1997) 115-121.
Sollevi, A., Belfrage, M., Lundeberg, T., Segerdahl, M. and Hansson, P., Systemic adenosine infusion: new treatment modality to alleviate neuropathic pain, Pain, 61 (1995) 155-158.
Sosnowski, M., Stevens, C.W. and Yaksh, T.L., Assessment of the role of A1/A2 adenosine receptor mediating the purine antinociception, motor and autonomic function in the rat spinal cord, J. Pharmacol Exp. Ther., 250 (1989) 915-922.
Sugiura, Y., Lee, C.L. and Perl, E.R., Central projections of identified, unmyelinated (C) afferent fiber innervating mammalian skin, Science, 234 (4774)(1986) 358-361.
Surprenant, A., Buell, G. and North, R.A., P2X receptors bring new structure to ligand-gated ion channels, Trends Neurosci., 18 (5)(1995) 224-229.
Surprenant, A., Functional properties of native and cloned P2X receptors, Ciba Found. Symp., 198 (1996) 208-219.
Sylven, C., Jonzon, B., Fredholm, B.B. and Kaijser, L., Adenosine injection into the brachial artery produces ischaemia like pain or discomfort in the forearm, Cardiovasc. Res., 22 (1988) 674-678.
Thorne, P.R. and Housley, G.D., Purinergic signaling in sensory systems, Semin. Neurosci., 8 (1996) 233-246.
Torres, G., Eagan, T. and Voigt, M., Membrane topology of P2X receptors revealed by epitope tagging and N-glycolsation, Pharmacologist (Abstr.), 39 (1)(1996) 56.
Valera, S., Hussy, N., Evans, R.J., Adami, N., North, R.A., Suprenant, A. and Buell, G., A new class of ligand-gated ion channel defined by P2X receptor for extracellular ATP, Nature, 371 (1994) 516-519.
Vulchanova, L., Arvidsson, U., Riedl, M., Wang, J., Buell, G., Suprenant, A., North, R.A. and Elde, R., Differential distribution of two ATP-gated ion channels (P2X receptors) determined by immunocytochemistry, Proc. Natl. Acad. USA, 93 (1996) 8063-8067.
Vulchanova, L., Riedl, M., Shuster, S., Buell, G., Suprenant, A., North, R.A. and Elde, R., Immunocytochemical study of the P2X2 and P2X3 receptor in rat and monkey sensory neurons and their central terminals, Neuropharmacology, 36 (9)(1997) 1229-1242.
Zimmerman, M., Ethical guidelines for investigations of experimental pain in conscious animals, Pain, 16 (1983) 109-110.
Keywords:
Purinergic; P2X3; Pain; Neuropathic; Localization; Dorsal root ganglion
© 1999 Lippincott Williams & Wilkins, Inc.