| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1Department of Neuroscience and Cell Biology and 2Department of Surgery, Division of Neurosurgery, University of Texas Medical Branch, Galveston, Texas
Submitted 10 May 2006; accepted in final form 26 July 2006
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
- and C-primary afferent fibers in the tibial nerve in anesthetized rats. Peripheral pretreatment with 
,
-methylene adenosine 5'-triphosphate (,-meATP), a P2X-selective receptor agonist, could potentiate the CAP-induced enhancement of responses of A- and C-primary afferent nociceptive fibers to mechanical stimuli in sympathetically intact rats. After sympathetic denervation, the enhanced responses of both A- and C-fibers after CAP injection were dramatically reduced. However, this reduction could be restored when P2X receptors were activated by ,-meATP. A blockade of P2X receptors by pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid could significantly reduce the CAP-induced sensitization of A- and C-fibers. Pretreatment with uridine 5'-triphosphate, a P2Y-selective receptor agonist, did not significantly affect or restore the CAP-induced sensitization of A- and C-fibers under sympathetically intact or sympathectomized conditions. Our study supports the view that ATP plays a role in modulation of primary afferent nociceptor sensitivity mainly by P2X receptors. Combined with our previous study, our data also provide further evidence that the sensitization of primary afferent nociceptors is subject to sympathetic modulation by activation of P2X as well as 1-adrenergic receptors. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
; Michaelis 2000; Schwartzman and Mclellan 1987; Torebjörk et al. 1995). Sympathectomy or sympathetic nerve block has been shown to reduce pain effectively in rats with spinal nerve injury (Kim and Chung 1991; Kinnmann and Levine 1995). However, the antinociceptive effect produced by adrenergic antagonists was less than that produced by sympathetic denervation (Kim et al. 1993; Lee et al. 1997; Ringkamp et al. 1999). We suggest that the different effects on neuropathic pain behaviors by sympathectomy from that produced by adrenergic antagonists is because postganglionic sympathetic neurons contain not only adrenergic neurotransmitters, but also nonadrenergic substances, such as adenosine 5'-triphosphate (ATP) and neuropeptide Y (NPY). ATP and NPY are coreleased from the sympathetic nerve terminals together with norepinephrine (NE) to play a critical role in sympathetic modulation of nociceptive signaling (Burnstock 1990, 1996; Lundberg et al. 1990; Park et al. 2000; Sneddon et al. 1996; Waldron et al. 2004).
Evidence supports that ATP is a major algogenic chemical present in millimolar concentrations in the cytoplasm of cells and is elevated in injury or inflamed peripheral tissue involved in modulation of primary afferent nociceptive signaling (Bland-Ward and Humphrey 1997, 2000; Dowd et al. 1998; Hamilton and McMahon 2000; Hamilton et al. 1999, 2000). Two classes of purinergic receptors have been identified: ligand-gated ion channels (P2X receptors with seven subtypes, P2X1–P2X7) and metabotropic G-protein–coupled receptors (P2Y receptors with eight subtypes, P2Y1,2,4,6,11–14) (Ralevic and Burnstock 1998). Purinoceptors are distributed in primary afferent terminals (Bradbury et al. 1998; Chen et al. 1995; Lewis et al. 1995; Vulchanova et al. 1997). The majority of ATP-induced nociceptive transduction and pain has been attributed to ionotropic P2X receptors, but it was recently suggested by some authors that P2Y receptors are also involved (Bland-Ward and Humphrey 2000; Moriyama et al. 2003; Stucky et al. 2004).
In a series of recent studies, we demonstrated in anesthetized rats that sensitization of primary afferent nociceptors and the cutaneous neurogenic inflammation arising from activation of the transient receptor potential vanilloid-1 (TRPV1) receptors by intradermal injection of capsaicin (CAP) are dependent on the presence of sympathetic efferents and are subject to modulation by peripheral 1-adrenoceptors (Lin et al. 2003; Ren et al. 2005a) and NPY Y2 receptors (Lin et al. 2004). The present investigation is a continuation of our series of studies and is aimed at further examination of the possible involvement of peripheral P2X and P2Y receptors in the sympathetically modulated sensitization of primary A- and C-nociceptive afferents produced by intradermal injection of CAP in anesthetized rats. Preliminary data were previously presented in abstract form (Ren et al. 2005b).
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
Experiments were performed on adult male Sprague–Dawley rats (250–350 g). All experimental protocols were approved by the Institutional Animal Care and Use Committee and were consistent with the guidelines of the National Institutes of Health and the International Association for the Study of Pain.
Animals were initially anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneal administration). The trachea and external jugular vein were catheterized for artificial respiration and anesthetic delivery, respectively. Anesthesia was maintained by continuous infusion of sodium pentobarbital (5–8 mg/kg/h, intravenous [iv] administration). The level of anesthesia was monitored by frequent examination of pupillary size, responses to stimulation, and the absence of a flexion reflex. Once a stable level of anesthesia was reached, the animals were paralyzed with pancuronium (0.3–0.4 mg/h, iv) and artificially ventilated. The level of end-tidal CO2 was kept between 3.5 and 4.5%. Core body temperature was monitored by a rectal probe and maintained near 37°C by a servo-controlled heating blanket.
Single-fiber recordings
The procedure of single-fiber recordings was described in detail in our previous study (see Ren et al. 2005a). Briefly, the tibial nerve was exposed in one hind paw and then the incised skin was retracted and bound around a frame to form a pool filled with warm mineral oil over the exposed tissue. The tibial nerve was carefully dissected from surrounding tissues and part of the nerve (one fourth to one third) was removed after a longitudinal cut. The distal cut end of the tibial nerve was then teased into small filaments with fine-tipped forceps on a small mirror-based platform under an operating microscope until single-fiber activity of afferents from a fine nerve filament could be isolated on the basis of spike amplitude and waveform. To locate the receptive field of the afferent fibers, mechanical stimulation by using a von Frey filament with a bending force of 7 to 36 mN was applied to and moved over the surface of the skin innervated by the nerve under study. Action potentials were recorded and then converted into analog signals or histograms that were available to be quantified by Spike2 software. The fiber types of single units were classified by conduction velocity (A, 2–19.9 m/s; C, <2 m/s) (Handwerker et al. 1991; Leem et al. 1993; Ren et al. 2005a). The faster conducting A-fibers were not examined in the present study. Recorded action potentials and their responses to peripheral mechanical stimuli were amplified and displayed on a digital oscilloscope (TDS-3012B). This allowed us to ensure that the same unit was being recorded throughout the experiment. The original signals recorded were also fed to a data collection system (CED 1401+) and a computer for data compilation as wavemark files or rate histogram files (see detailed procedure in Ren et al. 2005a and Fig. 1) using Spike2 software (Cambridge Electronic Design).
|
Surgical sympathectomy was performed as described in our previous studies (Lin et al. 2003; Ren et al. 2005a). Briefly, sympathetic ganglia and chains were removed bilaterally at the L2–6 levels through a laparotomy. Animals were given postoperative care to allow for recovery from the surgery for 
1 wk before experiments were performed. A morphological study reported by our and other groups using the same model shows that a successful sympathetic denervation of femoral arteries could be confirmed 7–10 days after the surgical sympathectomy was performed (Kim and Chung 1991; Zou et al. 2002).
Peripheral administration P2X or P2Y receptor agonist or antagonist
Close-by intraarterial injection was used to administer a P2X or P2Y receptor agonist or antagonist locally. To do this, a fine branch of the femoral artery was cannulated by polyethylene-10 tubing connected with a U-100 insulin syringe for drug delivery to the hind paw (Ren et al. 2005a). An agonist of P2X receptors, ,-methylene adenosine 5'-triphosphate (,-meATP; 1, 5, or 25 µg, RBI/Sigma), or P2Y receptors, uridine 5'-triphosphate (UTP; 2, 10, or 50 µg, RBI/Sigma), was administered intraarterially in a volume of 50 µl 5 min before CAP injection in sympathetically intact or sympathectomized rats. An antagonist of P2 receptors, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, 25 µg, Tocris), was applied in the same way in sympathetically intact rats. All drugs were freshly prepared on the day when the experiment was performed. For control purposes, saline (50 µl) was given using the same procedure.
Experimental protocol
Responses of a single A- or C-afferent fiber were evoked mechanically by applying a set of calibrated von Frey filaments (Stoelting, Wood Dale, IL) to the spot that was most mechanically sensitive (usually at the center of the receptive field of the fiber) on the plantar surface of the hind paw (Kocher et al. 1987; Ren et al. 2005a; Shim et al. 2005). The most sensitive spot for each fiber recorded was later labeled with a marked pen to ensure that mechanical stimuli were always evoked from the same spot before and after treatments. The appropriate set of von Frey filaments (three filaments with graded bending forces) was chosen in each experiment according to different activation thresholds. The range of bending forces was between 7 and 284 mN (see Ren et al. 2005a). The filaments produced stimuli ranging from innocuous to noxious (Leem et al. 1993). The first filament of the set had the weakest bending force sufficient to evoke action potentials and two additional filaments had progressively stronger bending forces. Each filament was applied repetitively for 10 s with enough force to cause slight bending and this force was held for about 0.5 s followed by a 10-s pause before the next filament was used. To decrease a minimum possible "human factor" bias, mechanical stimuli were applied without observation of the oscilloscope or computerized record so the experimenter was unaware of the response frequencies. Our previous study showed in the same model that spontaneous activity was seen in only a very small number of fibers and there was no significant increase in spontaneous activity after CAP injection (Ren et al. 2005a). Therefore only the evoked afferent activity was analyzed in the present study.
A- and C-, not A-, afferent fibers with mechanical thresholds of 20–147 mN were previously reported to be nociceptors (Leem et al. 1993) because these fibers are CAP sensitive with a high expression of the nociceptive molecules, TRPV1 receptors (Caterina et al. 1997; Ma 2001; Tohda et al. 2001; Tominaga et al. 1998). This was confirmed by our previous study (Ren et al. 2005a) that CAP injection produced sensitization of most A- and C-primary afferent nociceptors to mechanical stimuli. Therefore these criteria were continuously used for searching for A- and C-primary afferent nociceptors in this study.
CAP was dissolved in Tween 80 (7%) and saline (93%) to a concentration of 0.1%. A volume of 10 µl was injected intradermally at the edge of the receptive field to evoke an acute cutaneous inflammation after baseline responses were recorded. The responses were tested at 10, 20, and 30 min after CAP injection. Changes in activity of single A- and C-afferent fibers after CAP injection were recorded and compared between sympathectomized rats and sympathetically intact rats.
To evaluate further whether the activation or blockade of purinoceptor subtypes P2X or P2Y in the periphery affected the sensitization of primary afferents induced by intradermal injection of CAP and to examine their role in sympathetic modulation of sensitization of primary afferents, the following pharmacological manipulations were carried out. 1) Observations were made of the effects of activation of peripheral P2X or P2Y receptors with different doses of agonists on the CAP-induced enhancement of responses of A- and C-fibers to mechanical stimuli under sympathetically intact conditions. An agonist of P2X receptors (,-meATP) or P2Y receptors (UTP) was administered intraarterially into the hind paw 5 min before CAP injection in a volume of 50 µl with different concentrations in three groups of sympathetically intact rats, respectively. Changes in responses to mechanical stimuli after CAP injection were recorded for 30 min. As controls, saline (50 µl) was given intraarterially before CAP injection in different groups of sympathetically intact rats. 2) The effects of activation of peripheral P2X or P2Y receptors under sympathectomized conditions were tested. In one group of sympathectomized rats, ,-meATP (25 µg) or UTP (50 µg) was injected intraarterially 5 min before CAP was injected intradermally. Changes in responses to CAP injection were then recorded. Control experiments were performed by intraarterially injecting saline in the same volume in a separate group of sympathectomized rats. 3) Intraarterial injection of PPADS was done under sympathetically intact conditions to examine whether blockade of P2X receptors could affect the CAP-induced sensitization of primary afferent fibers. In one group of sympathetically intact rats, PPADS (25 µg) was given 5 min before CAP injection. Changes in responses to mechanical stimuli after CAP injection were recorded.
Statistical analysis
Recorded fiber activity was analyzed on- or off-line from peristimulus time histograms to obtain the average rate of evoked discharges. All responses evoked by stimulation using three calibrated von Frey filaments with graded bending forces were calculated by subtracting the background discharges during a given period of time from the total number of action potentials that occurred during each stimulus to produce a net increase in discharge rate. In each group, discharge frequencies were converted into percentage data by setting the responses to mechanical stimuli before CAP injection (pre-CAP, baseline) as 100% and the responses to mechanical stimuli after CAP injection as percentage changes of the pre-CAP value. Statistical significance was tested using ANOVA with repeated measures and differences across time in the same group were assessed with Dunnett's t-test (see Lin et al. 1999, 2000; Ren et al. 2005a; Sun et al. 2004; Wang et al. 2004). The Mann–Whitney U test was used to compare the differences in responses between groups having different treatments. Values are presented as means ± SE. P < 0.05 was taken as significant.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
- and 69 C-afferent fibers were recorded from the tibial nerve in 105 rats. Only one fiber was recorded from each hind paw. Considering possible contralateral effects resulting from CAP injection on one side when both hind paws in the same rat were used, we analyzed the data sampled from two hind paws in the same animal (a total of five sympathetically intact rats) in which the effects of CAP injection on two fibers were recorded on the same animal within 3–4 h. A statistical analysis using a paired t-test showed that there was no significant difference in the CAP-evoked responses of afferent activity between two fibers recorded in the same rats (data not shown). The mechanical thresholds of these fibers before CAP injection ranged from 20 to 147 mN and so the fibers were considered to be nociceptive units (Leem et al. 1993). Of these fibers, increased spontaneous discharges were seen in only three rats (two C-fibers in sympathetically intact rats after injection of 10 or 25 µg ,-meATP and one A-fiber in a sympathectomized rat after injection of 25 µg ,-meATP) right after ,-meATP injection with an average firing rate of 1.98 ± 0.54 Hz (lasting 60–120 s).
Effects of sympathetic denervation on the CAP-induced sensitization of A- and C-primary afferent nociceptors
The first set of experiments was performed to confirm our previous study that sympathetic denervation interferes with the sensitization of A- and C-primary afferent nociceptive fibers induced by intradermal injection of CAP (Ren et al. 2005a). Mechanical responses of afferents to CAP injection in these sympathetically intact and sympathectomized rats were tested with saline pretreatment by intraarterial injection to serve as controls for the following drug treatment experiments. In Fig. 1, the responses to mechanical stimuli before CAP injection served as a baseline in each group (100%, pre-CAP injection). Consistent with our previous results (Ren et al. 2005a), a significant enhancement of mechanically evoked responses of both A- and C-fibers was seen after intradermal injection of CAP in sympathetically intact rats with saline pretreatment (Fig. 1A). The enhancement of the responses reached its peak at 10–20 min after CAP injection and lasted over 30 min. In contrast, the CAP-evoked enhancement of responses of A- and C-fibers to mechanical stimuli was completely eliminated after sympathetic denervation by sympathectomy (Fig. 1B). The bottom traces in Fig. 1A are examples of action potentials recorded from a single A-fiber from the tibial nerve before and 20 min after CAP injection in a sympathetically intact rat with saline pretreatment. An increased firing of the recorded fiber to mechanical stimuli was seen after CAP injection. The traces in Fig. 1B are action potentials recorded from a single C-fiber from the tibial nerve in a sympathectomized rat with saline pretreatment. There was no obvious increase in responses to mechanical stimuli 20 min after CAP injection.
Grouped data show that the peak increases in responses to mechanical stimuli after CAP injection in sympathetically intact rats with saline pretreatment were to 129.7 ± 2.5% (P < 0.01, compared with baseline) in A-fibers and to 144.6 ± 2.2% (P < 0.01) in C-fibers (Fig. 1A). Responses were not increased after CAP injection in A-fibers (peak increase to 100.3 ± 2.5%, P = 0.791, compared with baseline) or C-fibers (peak increase to 98.5 ± 2.6%, P = 0.464, Fig. 1B) of sympathectomized rats with saline pretreatment.
Effects of activation of peripheral P2X or P2Y receptors on mechanically evoked activity under sympathetically intact conditions
In sympathetically intact rats, peripheral P2X or P2Y receptors were activated by intraarterial injection of different doses of ,-meATP or UTP 5 min before intradermal injection of CAP to see whether sensitization of primary afferent nociceptors after CAP injection is modulated by the activation of P2X or P2Y receptors.
Figure 2 shows that local injection of either 1 or 5 µg of ,-meATP produced no significant effect on mechanical responses of A- and C-fibers. When the concentration of ,-meATP was increased to 25 µg, a slight but significant increase in responses to mechanical stimuli was seen both in A- (to 107.9 ± 2.5%, 5 min after injection) and C- (to 112.5 ± 4.7%, 5 min after injection) fibers (P < 0.05, indicated by asterisks at time 0 min right before CAP injection). Importantly, pretreatment with 25 µg ,-meATP significantly enhanced the CAP-evoked responses compared with the responses when the paw was pretreated with saline (indicated by asterisks at times 10–30 min in Fig. 2, A and B). The peak increases of mechanically evoked activity after CAP injection were to 155.9 ± 4.6% in A-fibers and to 165.6 ± 5.0% in C-fibers, respectively. Bottom traces in Fig. 2 are examples showing the effect of ,-meATP alone (second rows) on afferent responses to mechanical stimuli and ,-meATP pretreatment on the CAP-evoked sensitization of mechanical responses in one A-fiber (third row in Fig. 2A) and one C-fiber (third row in Fig. 2B).
|
,-meATP, the presence of UTP did not significantly affect the enhanced responses of either A- or C-fibers to mechanical stimuli induced by CAP injection compared with the saline pretreated group (Fig. 3, A and B vs. Fig. 1A). The peak increases of evoked activity after CAP injection with UTP pretreatment at three doses were to 133.5 ± 4.1%, 133.3 ± 2.6%, and 139.9 ± 4.4% in A-fibers and to 143.9 ± 3.0%, 146.6 ± 5.2%, and 148.5 ± 4.0% in C-fibers, respectively. Bottom traces in Fig. 3 are examples of the effect of pretreatment with UTP on the CAP-evoked mechanical responses in one A-fiber (Fig. 3A) and one C-fiber (Fig. 3B).
|
Because sympathetic denervation almost completely inhibits the sensitization of afferent nociceptors induced by CAP injection (as is shown clearly in Fig. 1), we further examined whether peripheral pretreatment with P2X or P2Y receptor agonists to activate P2X or P2Y receptors could restore the sensitization under sympathectomized conditions, mimicking the conditions when the sympathetic efferents were present. Grouped data show that local injection of ,-meATP in a dose of 25 µg produced a slight increase in responses to mechanical stimuli both in A- (to 103.7 ± 2.1%, 5 min after injection) and C- (to 103.0 ± 2.1%, 5 min after injection) fibers, although this increase did not reach statistical significance. However, the presence of ,-meATP (25 µg) could restore the CAP-induced enhancement of responses in both A- and C-fibers under sympathectomized conditions. The peak increases were to 158.3 ± 11.2% in A-fibers and to 188.7 ± 13.4% in C-fibers (Fig. 4 A). These changes were significantly higher than those in the saline pretreated group under sympathectomized conditions (P < 0.01 for A-fibers, P < 0.01 for C-fibers). Also, the increase in mechanical response was comparable to that found under sympathetically intact conditions with saline pretreatment (see Fig. 1A). In contrast, pretreatment with UTP (50 µg) did not significantly affect the mechanically evoked activity produced by CAP injection either in A-fibers or C-fibers under sympathectomized conditions (Fig. 4B). The peak increases with UTP pretreatment were to 105.9 ± 5.3% (P = 0.721, compared with saline-treated group) in A-fibers and to 99.5 ± 1.5% (P = 1.000) in C-fibers.
|
- and C-fibers after CAP injection under sympathetically intact conditions
The above data have shown that activation of P2X, but not P2Y, receptors could either significantly enhance the CAP-evoked sensitization of nociceptors or restore the CAP-induced sensitization that was reduced due to sympathetic denervation. Therefore we further examined whether blockade of P2X receptors in the periphery by intraarterial injection of PPADS (25 µg) would affect the sensitization of A- and C-fibers induced by CAP injection in sympathetically intact rats. As shown in Fig. 5, pretreatment with PPADS significantly reduced the CAP-induced enhancement of responses in A- and C-primary afferent fibers in sympathetically intact rats (Fig. 5, A and B). The peak increases were to 113.6 ± 3.2% in A-fibers (P = 0.003, compared with saline-treated group) and to 119.4 ± 3.8% in C-fibers (P = 0.004).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
- and C-, but not A-, primary afferent nociceptive fibers, are sensitized after intradermal injection of CAP and that the sensitization is dependent on the presence of sympathetic efferents and subject to modulation by 1-adrenoceptors (Ren et al. 2005a). Acute cutaneous neurogenic inflammation after CAP injection is also dependent on intact sympathetic efferents and is modulated by peripheral 1-adrenoceptors and/or NPY Y2 receptors (Lin et al. 2003, 2004). In line with previous findings, our present results further suggest that peripheral purinoceptors (mainly P2X subtype) are also involved in the CAP-induced sensitization of primary afferent nociceptive fibers, suggesting that ATP is released from efferent terminals of the sympathetic nerves to participate in the sympathetic modulation of nociception.
ATP is suggested to be an inflammatory mediator or modulator involved in several types of pain by acting on P2X and/or P2Y receptors (Burnstock 2001; Dunn et al. 2001; Lazarowski et al. 2000; Premkumar 2001; Sawynok and Sweeney 1989). Anatomically, the P2X3, P2Y1, and P2Y2 receptors are highly expressed by small-sized dorsal root ganglion (DRG) neurons and their nociceptive terminals (Bo et al. 1999; Bradbury et al. 1998; Chen et al. 1995; Lewis et al. 1995; Molliver et al. 2002; Nakamura and Strittmatter 1996; Ueno et al. 1999). Functionally, activation of P2X3 receptors elicits inward currents in DRG neurons, resulting in depolarization (Chen et al. 1995). Activation of P2X receptors produced a rapid and short-lasting increase in spontaneous firing of C- and A-afferent fibers under normal or inflamed conditions (Dowd et al. 1998; Hamilton et al. 2001). An in vitro study in mice shows that A- and C-cutaneous afferent fibers were both ,-meATP and UTP sensitive, indicating that P2X3 and P2Y2 receptors are widely coexpressed (Stucky et al. 2004). Behaviorally, local application of ,-meATP in rats produced behavioral mechanical allodynia in a dose-dependent fashion and was significantly blocked by intraplantar pretreatment with the P2 receptor antagonist PPADS (Tsuda et al. 2000). Our results from in vivo electrophysiological recordings are generally in line with the above data except that ,-meATP did not produce increased spontaneous afferent activity in most fibers tested. This was presumably attributable to the lower dose of ,-meATP used in our study compared with data from another group (Dowd et al. 1998). ,-meATP is an effective agonist at P2X1, P2X3, and P2X2/3 heteromeric receptors (Ralevic and Burnstock 1998). P2X3 mRNA is predominantly expressed in DRG neurons (Chen et al. 1995; Cockayne et al. 2000) and deletion of the P2X3 gene eliminates responses to ,-meATP in mice (Cockayne et al. 2000; Souslova et al. 2000; Zhong et al. 2001). P2X3 is highly sensitive to PPADS (Ralevic and Burnstock 1998). Therefore enhancement of afferent responses of A- and C-fibers induced by ,-meATP in our study should be mediated mainly by activation of P2X3 receptors participating in modulation of sensitivity of primary afferent nociceptors.
A major finding in the present study is that by the use of a sympathetic denervation model combined with pharmacological manipulations we demonstrated that ATP, acting as a nonadrenergic substance, is apparently released from sympathetic nerve terminals and participates in the sympathetic modulation of the neurogenic inflammation-induced pain that is produced by intradermal CAP injection. This sympathetic modulation was previously demonstrated to be independent of preganglionic sympathetic neurons (Kinnman and Levine 1995; Lin et al. 2003). Clinical and experimental studies revealed that sympathetic modulation plays a critical role under conditions of neuropathy or neurogenic inflammation in peripheral nerves or tissues (Green et al. 1993; Kim and Chung 1991; Lin et al. 2003, 2004; Moon et al. 1999; Ren et al. 2005a; Xie et al. 1995) by a mechanism of sympathetic–sensory interactions in DRG neurons (Chung et al. 1996; Devor et al. 1994), the site of nerve injury (Devor and Seltzer 1999), and the skin (Sato and Perl 1991). The release of adrenergic and/or nonadrenergic transmitters is suggested to be the mechanism underlying the interactions because these substances have been well documented to play roles in nociception (Dowd et al. 1998; Drummond 1995, 1998; Lee et al. 1999; Tracey et al. 1995). Critically, these substances were shown to be co-localized in the synaptic vesicles of the postganglionic sympathetic nerve terminals and coreleased into the extracellular space to participate in several pathophysiological processes, including pathological pain signaling (Abbracchio and Burnstock 1998; Burnstock 1990, 1996; Park et al. 2000; Sneddon et al. 1996). This was confirmed in the neurogenic inflammation model induced by CAP injection in our previous and current observations (Lin et al. 2003; Ren et al. 2005a).
ATP is reported to be released as a consequence of inflammation from many sources, such as the damaged cells, platelets, some inflammatory cells, sympathetic efferent terminals, and perhaps the sensory nerve terminal itself (Born and Kratzer 1984; Ferrari et al. 1997; Gordon 1986; Holton 1959; Nakamura and Strittmatter 1996; Sneddon et al. 1996). Purinergic receptors are expressed on mast cells, postganglionic sympathetic neurons, and dorsal root ganglion neurons (Nakamura and Strittmatter 1996; Stone 1991; Xiang et al. 1998). Therefore ATP could modulate nociceptive signaling in several possible ways. First, ATP might act on mast cells to cause release of amines and cytokines, which in turn sensitize afferent nociceptors (Stone 1991). However, chronic degranulation of mast cells in the rat model involving injection of complete Freund's adjuvant into the skin of the hind paw did not eliminate the effect of ATP (Zhang et al. 2001). Second, there could be a direct action on the peripheral endings of sensory neurons because both P2X and P2Y receptors exist in sensory neurons (Nakamura and Strittmatter 1996; Xiang et al. 1998). The present study supports the view that ATP is released from the terminals of sympathetic efferents after tissue injury or inflammation and is involved in the pathogenesis of sensitization of primary afferent nociceptors because sympathetic denervation dramatically reduced the CAP-induced sensitization. Purinoceptors (predominantly P2X) that are expressed in primary afferent nociceptors are activated by an exogenous ATP to modulate the nocieptive process after CAP injection because sympathetic denervation does not affect this process (see the rekindling effect by ,-meATP under sympathectomized conditions; Fig. 4). Finally, some studies have indicated a synergistic effect on pain behaviors and primary afferent sensitization by a corelease of ATP and NE from sympathetic efferent terminals with various hypotheses based on different data (Park et al. 2000; Waldron and Sawynok 2004; Zhang et al. 2001). It was previously suggested that ATP might act on the postganglionic sympathetic efferents and then regulate the release of some mediators such as NE, which interact with sensory afferents involved in nociceptive signaling (Boehm 1999). An alternative view by Waldron and Sawynok (2004) is that during tissue inflammation or injury -adrenoceptors located on sympathetic efferents are activated by the NE released from sympathetic terminals, which in turn interacts with primary afferent C-fibers by activating the protein kinase A cascade, leading to upregulation of P2X receptors on C-fibers. This seems to be consistent with our previous (Ren et al. 2005a) and present data that there is a reduced sensitivity of -adrenergic and/or purinergic receptors after sympathetic denervation but that the modulatory role of these receptors in nociceptive signaling could be restored by giving exogenous NE or ATP. Although the mechanisms of the synergistic effects are still obscure, it is strongly suggested that there is an interaction between ATP and NE at sympathoeffector junctions after nerve injury or tissue inflammation that contributes to pain signaling under these pathophysiological situations.
TRPV1 is well established as a major marker of nociceptors. Purinergic receptors, including P2X and P2Y, are highly co-localized with TRPV1 in small-sized DRG neurons and these neurons and their axons are mostly CAP sensitive (Bland-Ward and Humphrey 1997; Guo et al. 1999; Moriyama et al. 2003; Stucky et al. 2004; Tsuda et al. 1999; Ueno et al. 1999). There have been several reports that ATP can potentiate CAP-evoked TRPV1 currents (Moriyama et al. 2003; Tominaga et al. 2001), directly phosphorylate TRPV1 receptors (Numazaki et al. 2002), and augment the CAP-evoked release of substance P and CGRP from sensory neurons by a protein kinase C–dependent pathway (Huang et al. 2003). Given the fact that ATP acts as an inflammatory mediator and/or a key nociceptive signaling molecule in modulating the function of TRPV1 receptors (Huang et al. 2003; Moriyama et al. 2003; Numazaki et al. 2002; Tominaga et al. 2001) and that ATP potentiates the CAP-induced sensitization of afferent nociceptive fibers induced by activating TRPV1, future studies are needed to analyze whether TRPV1 receptors are modulated by adrenergic and/or nonadrenergic substances released from the sympathetic efferents by way of activating signal transduction cascades.
In summary, the present data suggest that extracellular ATP is released from the postganglionic sympathetic efferent terminals after intradermal injection of CAP, which are involved in the CAP-induced sensitization of primary afferent nociceptive fibers by P2X receptors, presumably P2X3. This process is dependent on the presence of sympathetic efferents in the periphery. Combined with our previous studies, our data strongly suggest that ATP, NE, and NPY play important roles in the sympathetic modulation of primary afferent nociceptor sensitivity by activation of P2X, as well as 1-adrenergic and neuropeptide Y2—receptors in the periphery.
|
GRANTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
|
ACKNOWLEDGMENTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
|
FOOTNOTES |
|---|
Address for reprint requests and other correspondence: Q. Lin, Department of Neuroscience and Cell Biology, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1069 (E-mail: qilin{at}utmb.edu)
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
Bland-Ward PA and Humphrey PP. Acute nociception mediated by hindpaw P2X receptor activation in the rat. Br J Pharmacol 122: 365–371, 1997.[CrossRef][ISI][Medline]
Bland-Ward PA and Humphrey PP. P2X receptors mediate ATP-induced primary nociceptive neurone activation. J Auton Nerv Syst 81: 146–151, 2000.[CrossRef][ISI][Medline]
Bo X, Alavi A, Xiang Z, Oglesby IB, Ford APDW, and Burnstock G. Localization of P2X2 and P2X3 receptor immunoreactive nerves in rat taste buds. Neuroreport 10: 1107–1111, 1999.[ISI][Medline]
Boehm S. ATP stimulates sympathetic transmitter release via presynaptic P2X purinoceptors. J Neurosci 19: 737–746, 1999.
Born GV and Kratzer MA. Source and concentration of extracellular adenosine triphosphate during haemostasis in rats, rabbits and man. J Physiol 354: 419–429, 1984.
Bradbury EJ, Burnstock G, and McMahon SB. The expression of P2X3 purinoreceptors in sensory neurons: effects of axotomy and glial-derived neurotrophic factor. Mol Cell Neurosci 12: 256–268, 1998.[CrossRef][ISI][Medline]
Burnstock G. Noradrenaline and ATP as cotransmitters in sympathetic nerves. Neurochem Int 17: 357–368, 1990.[CrossRef][ISI]
Burnstock G. A unifying purinergic hypothesis for the inhibition of pain. Lancet 347: 1604–1605, 1996.[CrossRef][ISI][Medline]
Burnstock G. Purine-mediated signaling in pain and visceral perception. Trends Pharmacol Sci 22: 182–188, 2001.[CrossRef][Medline]
Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, and Julius D. The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 389: 816–824, 1997.[CrossRef][Medline]
Chen C-C, Akopian AN, Sivilotti L, Colquhoun D, Burnstock G, and Wood JN. A P2X purinoceptor expressed by a subset of sensory neurons. Nature 377: 428–431, 1995.[CrossRef][Medline]
Chung K, Lee BH, Yoon YW, and Chung JM. Sympathetic sprouting in the dorsal root ganglia of the injured peripheral nerve in a rat neuropathic pain model. J Comp Neurol 376: 241–252, 1996.[CrossRef][ISI][Medline]
Cockayne DA, Hamilton SG, Zhu QM, Dunn PM, Zhong Y, Novakovic S, Malmberg AB, Cain G, Berson A, Kassotakis L, Hedley L, Lachnit WG, Burnstock G, McMahon SB, and Ford AP. Urinary bladder hyporeflexia and reduced pain-related behavior in P2X3-deficient mice. Nature 407: 1011–1015, 2000.[CrossRef][Medline]
Devor M, Jänig W, and Michaelis M. Modulation of activity in dorsal root ganglion neurons by sympathetic activation in nerve-injured rats. J Neurophysiol 71: 38–47, 1994.
Devor M and Seltzer Z. Pathophysiology of damaged nerves in relation to chronic pain. In: Textbook of Pain, edited by Wall PD and Melzack R. London: Churchill Livingstone, 1999, p. 129–164.
Dowd E, McQueen DS, Cheesell IP, and Humphrey PP. P2X receptor-mediated excitation of nociceptive afferents in the normal and arthritic rat knee joint. Br J Pharmacol 125: 341–346, 1998.[CrossRef][ISI][Medline]
Drummond PD. Noradrenaline increases hyperalgesia to heat in skin sensitized by capsaicin. Pain 60: 311–315, 1995.[CrossRef][ISI][Medline]
Drummond PD. Enhancement of thermal hyperalgesia by -adrenoceptors in capsaicin-treated skin. J Auton Nerv Syst 69: 96–102, 1998.[CrossRef][ISI][Medline]
Dunn PM, Zhong Y, and Burnstock G. P2X receptors in peripheral neurons. Prog Neurobiol 65: 107–134, 2001.[CrossRef][ISI][Medline]
Ferrari D, Chiozzi P, Falzoni S, Hanau S, and Di Virgilio F. Purinergic modulation of interleukin-1 beta release from microglial cells stimulated with bacterial endotoxin. J Exp Med 185: 579–582, 1997.
Gordon JL. Extracellular ATP: effects, sources and fate. Biochem J 233: 309–319, 1986.[ISI][Medline]
Green PG, Luo J, Heller PH, and Levine JD. Further substantiation of a significant role for the sympathetic nervous system in inflammation. Neuroscience 55: 1037–1043, 1993.[CrossRef][ISI][Medline]
Guo A, Vulchanova L, Wang J, Li X, and Elde R. Immunocytochemical localization of the vanilloid receptor 1 (VR1): relationship to neuropeptides, the P2X3 purinoceptor and IB4 binding sites. Eur J Neurosci 11: 946–958, 1999.[CrossRef][ISI][Medline]
Hamilton SG and McMahon SB. ATP as a peripheral mediator of pain. J Auton Nerv Syst 81: 187–194, 2000.[CrossRef][ISI][Medline]
Hamilton SG, McMahon SB, and Lewin GR. Selective activation of nociceptors by P2X receptor agonists in normal and inflamed rat skin. J Physiol 534: 437–435, 2001.
Hamilton SG, Wade A, and McMahon SB. The effects of inflammation and inflammatory mediators on nociceptive behaviour induced by ATP analogues in the rat. Br J Pharmacol 126: 326–332, 1999.[CrossRef][ISI][Medline]
Hamilton SG, Warburton J, Bhattacharjee A, Ward J, and McMahon SB. ATP in human skin elicits a dose-related pain response which is potentiated under conditions of hyperalgesia. Brain 123: 1238–1246, 2000.
Handwerker HO, Kilo S, and Reeh PW. Unresponsive afferent nerve fibers in the sural nerve of the rat. J Physiol 435: 229–242, 1991.
Holton P. The liberation of adenosine triphosphate on antidromic stimulation of sensory nerves. J Physiol 145: 494–504, 1959.
Huang H, Wu X, Nicol GD, Meller S, and Vasko MR. ATP augments peptide release from rat sensory neurons in culture through activation of P2Y receptors. J Pharmacol Exp Ther 306: 1137–1144, 2003.
Jänig W, Levine JD, and Michaelis M. Interactions of sympathetic and primary afferent neurons following nerve injury and tissue trauma. In: Progress in Brain Research, edited by Kumazawa L, Kruger L, and Mizumura K. Amsterdam: Elsevier, 1996, vol. 113, p. 161–184.[ISI][Medline]
Kim SH and Chung JM. Sympathectomy alleviates mechanical allodynia in an experimental animal model for neuropathy in the rat. Neurosci Lett 134: 131–134, 1991.[CrossRef][ISI][Medline]
Kim SH, Na HS, Sheen K, and Chung JM. Effects of sympathectomy on a rat model of peripheral neuropathy. Pain 55: 85–92, 1993.[CrossRef][ISI][Medline]
Kinnman E and Levine JD. Involvement of the sympathetic postganglionic neuron in capsaicin-induced secondary hyperalgesia in the rat. Neuroscience 65: 283–291, 1995.[CrossRef][ISI][Medline]
Kocher L, Anton F, Reeh PW, and Handwerker HO. The effect of carrageenan-induced inflammation on the sensitivity of unmyelinated skin nociceptors in the rat. Pain 29: 363–373, 1987.[CrossRef][ISI][Medline]
Lazarowski ER, Boucher RC, and Harden TK. Constitutive release of ATP and evidence for major contribution of ecto-nucleotide pyrophosphatase and nucleoside diphosphokinase to extracellular nucleotide concentrations. J Biol Chem 275: 31061–31068, 2000.
Lee DH, Chung K, and Chung JM. Strain differences in adrenergic sensitivity of neuropathic pain behaviors in an experimental rat model. Neuroreport 8: 3453–3456, 1997.[ISI][Medline]
Lee DH, Liu X, Kim HT, Chung K, and Chung JM. Receptor subtype mediating the adrenergic sensitivity of pain behavior and ectopic discharges in neuropathic Lewis rats. J Neurophysiol 81: 2226–2233, 1999.
Leem JW, Willis WD, and Chung JM. Cutaneous sensory receptors in rat foot. J Neurophysiol 69: 1684–1699, 1993.
Lewis C, Neidhart S, Holy C, North RA, Buell G, and Surptrenant A. Coexpression of P2X2 and P2X3 receptor subunits can account for ATP-gated currents in sensory neurons. Nature 377: 432–435, 1995.[CrossRef][Medline]
Lin Q, Wu J, and Willis WD. Dorsal root reflexes and cutaneous neurogenic inflammation following intradermal injection of capsaicin in rats. J Neurophysiol 82: 2602–2611, 1999.
Lin Q, Zou X, Li F, and Willis WD. Sympathetic modulation of acute cutaneous flare induced by intradermal injection of capsaicin in anesthetized rats. J Neurophysiol 89: 853–861, 2003.
Lin Q, Zou X, Ren Y, Wang J, Fang L, and Willis WD. Involvement of peripheral neuropeptide Y receptors in sympathetic modulation of acute cutaneous flare induced by intradermal capsaicin. Neuroscience 123: 337–347, 2004.[CrossRef][ISI][Medline]
Lin Q, Zou X, and Willis WD. A and C primary afferents convey dorsal root reflexes after intradermal injection of capsaicin in rats. J Neurophysiol 84: 2695–2698, 2000.
Lundberg JM, Franco-Cereceda A, Lacroix JS, and Pernow J. Neuropeptide Y and sympathetic neurotransmission. Ann NY Acad Sci 611: 166–174, 1990.[Abstract]
Ma QP. Vanilloid receptor homologue, VR1, is expressed by both A- and C-fiber sensory neurons. Neuroreport 12: 3693–3695, 2001.[CrossRef][ISI][Medline]
Michaelis M. Coupling of sympathetic and somatosensory neurons following nerve injury: mechanisms and potential significance for the generation of pain. In: Proceedings of the 9th World Congress on Pain, edited by Devor M, Rowbotham MC, and Wiesenfeld-Hallin Z. Seattle, WA: IASP Press, 2000, p. 645–656.
Molliver DC, Cook SP, Carlsten JA, Wright DE, and McCleskey EW. ATP and UTP excite sensory neurons and induce CREB phosphorylation through the metabotropic receptor, P2Y2. Eur J Neurosci 16: 1850–1860, 2002.[CrossRef][ISI][Medline]
Moon DE, Lee DH, Han HC, Xie JG, Coggeshall RE, and Chung JM. Adrenergic sensitivity of the sensory receptors modulating mechanical allodynia in a rat neuropathic pain model. Pain 80: 589–595, 1999.[CrossRef][ISI][Medline]
Moriyama T, Iida T, Kobayashi K, Higashi T, Fukuoka T, Tsumura H, Leon C, Suzuki N, Inoue K, Gachet C, Noguchi K, and Tominaga M. Possible involvement of P2Y2 metabotropic receptors in ATP-induced transient receptor potential vanilloid receptor 1-mediated thermal hypersensitivity. J Neurosci 23: 6058–6062, 2003.
Nakamura F and Strittmatter SM. P2Y1 purinergic receptors in sensory neurons: contribution to touch-induced impulse generation. Proc Natl Acad Sci USA 93: 10465–10470, 1996.
Numazaki M, Tominaga T, Toyooka H, and Tominaga M. Direct phosphorylation of capsaicin receptor VR1 by protein kinase C
and identification of two target serine residues. J Biol Chem 277: 13375–13378, 2002.
Park SK, Chung K, and Chung JM. Effects of purinergic and adrenergic antagonists in a rat model of painful peripheral neuropathy. Pain 87: 171–179, 2000.[CrossRef][ISI][Medline]
Premkumar LS. Interaction between vanilloid receptors and purinergic metabotropic receptors: pain perception and beyond. Proc Natl Acad Sci USA 98: 6537–6539, 2001.
Ralevic V and Burnstock G. Receptors for purines and pyrimidines. Pharmacol Rev 50: 413–492, 1998.
Ren Y, Zou X, Fang L, and Lin Q. Sympathetic modulation of activity in A- and C-primary nociceptive afferents after intradermal injection of capsaicin in rats. J Neurophysiol 93: 365–377, 2005a.
Ren Y, Zou X, Li F, and Lin Q. Involvement of peripheral purinoceptors in sympathetic modulation of capsaicin-induced sensitization of primary afferent fibers. Soc Neurosci Abstr C170.1, 2005b.
