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Adrenergic regulation of P2X3 and TRPV1 receptors: Differential effects of spared nerve injury
Neuroscience Letters 444 (2008) 172–175
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Adrenergic regulation of P2X3 and TRPV1 receptors: Differential effects of spared nerve injury Jason G. Meisner, Allison R. Reid, Jana Sawynok ∗ Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 1X5
a r t i c l e
i n f o
Article history: Received 9 June 2008 Received in revised form 11 August 2008 Accepted 12 August 2008 Keywords: P2X3 receptors TRPV1 receptors Noradrenaline Spared nerve injury
a b s t r a c t Local application of ␣MeATP (ligand for P2X3 receptors) and capsaicin (ligand for TRPV1 receptors) to the rat hindpaw produces pain behaviors (flinching) which are enhanced by noradrenaline (NA). In this study, we have examined the effect of nerve injury on adrenergic regulation of P2X3 and TRPV1 receptors by administering ␣MeATP and capsaicin, alone and in combination with NA, into the lateral and medial hindpaw in the spared nerve injury (SNI) model; this allows for an exploration of the role of injured and uninjured afferents in their effects on nociceptive signaling using a behavioral model. Following lateral hindpaw injections (sural sensory field), effects of NA and ␣MeATP, both alone and in combination, were increased following SNI, but no such effects were seen following medial hindpaw injections (saphenous sensory field). Following lateral hindpaw injections, the effect of capsaicin alone was unaltered following SNI, but the effect of NA/capsaicin was reduced; this latter effect was not seen following medial hindpaw injections. At the lateral site, prazosin (␣1-adrenergic receptor antagonist) inhibited the effect of NA/␣MeATP following SNI, but neither prazosin nor GF109203X (protein kinase C inhibitor) inhibited the effect of NA/capsaicin following SNI. These results demonstrate: (a) an enhanced adrenergic regulation of P2X3 receptor activity at lateral sites following SNI where signaling afferents are directly influenced by injured neurons; (b) differential effects on adrenergic regulation of TRPV1 receptors under the same conditions; (c) lack of such changes when agents are administered into medial sites following SNI. © 2008 Elsevier Ireland Ltd. All rights reserved.
Both P2X3 [5] and TRPV1 [6] receptors are present on sensory nerve endings, allow for cation entry, and lead to sensory nerve activation and nociceptive signaling. Both receptors are considered potential targets for development of novel therapeutic agents [20,33]. When administered locally into the hindpaw of rodents, ␣-methylene-ATP (␣MeATP) and capsaicin, selective ligands for these two receptors respectively, produce pain behaviors reflective of sensory nerve activation; in both cases, pain behaviors are increased by noradrenaline (NA) [31,35]. NA is co-released with ATP from sympathetic nerves, and sympathetic mechanisms can contribute to manifestations of neuropathic pain by actions at several sites, including peripheral aspects of the sensory nerve and the cell body in the dorsal root ganglia (DRG) following sympathetic sprouting [12]. Adrenergic mechanisms have been shown directly to modulate P2X3 receptor mediated responses on sensory neurons following nerve injury [19,25]. Sympathetic mechanisms influence peripheral hypersensitivity responses to capsaicin in the uninjured state [15,17,30],
∗ Corresponding author. Tel.: +1 902 494 2596; fax: +1 902 494 1388. E-mail address: [email protected] (J. Sawynok). 0304-3940/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2008.08.032
but there is little information on this regulation following nerve injury. Several preclinical nerve injury models have been developed, and the ability of such models to produce enhanced pain signaling has received considerable attention in recent years. One such model, the spared nerve injury (SNI) model in which the tibial and common peroneal branches of the sciatic nerve are transected leaving the sural branch of the sciatic nerve intact, allows for the discrete examination of sensory signaling in distinct sensory fields [8]. Thus, one can administer a stimulus or a drug into a sensory field where intact innervation is influenced by injured afferents (lateral hindpaw; innervated by the spared sural nerve branch but with comingling of injured and uninjured afferents in the upper aspect of the sciatic nerve and L4–L6 dorsal root ganglia) OR into a sensory field where innervation is by uninjured afferents (medial hindpaw; innervated by the intact saphenous nerve with minimal interactions with injured fibres in the L3 dorsal root ganglion) (see Fig. 1 in Ref. [8]). In the present study, we have undertaken a systematic examination of peripheral responses to ␣MeATP and capsaicin, alone and combined with NA, following localized delivery into the lateral and medial hindpaw using the SNI model to determine: (a) the
J.G. Meisner et al. / Neuroscience Letters 444 (2008) 172–175
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Fig. 1. Effect of spared nerve injury (SNI) on responses to NA and ␣MeATP, alone and in combination, following injection into (A) the lateral aspect of the hindpaw, or (B) the medial plantar hindpaw. Values depict mean + S.E.M. for cumulative responses over 15 min following drug injections, with N = 6–9 per group. « P < 0.05 compared to the individual agents in sham animals (filled columns); * P < 0.05 compared to corresponding sham controls (filled vs open columns).
Fig. 2. Effect of capsacin and capsaicin/NA injected into the (A) lateral hindpaw or (B) medial plantar hindpaw following spared nerve injury (SNI). Values depict mean + S.E.M. for cumulative responses over 15 min following drug injections, with N = 7–9 per group. « P < 0.05 compared to the individual agents in sham animals (filled columns); * P < 0.05 compared to corresponding sham controls (filled vs open columns).
influence of injured afferents on peripheral responses to P2X3 and TRPV1 receptor activation, and (b) the adrenergic regulation of such responses. Experiments were conducted using male Sprague–Dawley rats (125–250 g) from Charles River, Montreal. All procedures were approved by the University Committee on Laboratory Animals, and performed in accordance with Canadian Council on Animal Care guidelines. SNI surgery was performed as described previously [8]. This procedure leaves the sural nerve intact, and care was taken to avoid damage to this nerve during surgery. Sham surgery consisted of exposure of the nerves without transection. Rats were housed individually for 3 days following surgery to allow for wound healing, and then were paired with the original cage mate. Following SNI, drugs were injected into the lateral aspect of the hindpaw or medial plantar hindpaw days 7–14 following surgery. Each experiment was conducted as a parallel sham and nerve injury experiment, with identical drug treatments given at the same time. All drugs were purchased from Sigma–Aldrich Canada Ltd. (Oakville, Ontario, Canada). Following SNI, drugs were injected in a volume of 30 l into the lateral aspect of the hindpaw at the junction of the hairy and non-hairy skin, or into the medial plantar hindpaw. This volume is smaller than the usual volume of 50 l used for rat studies, and it was used to restrict diffusion of the drug to the respective sensory fields. ␣MeATP and NA were dissolved in saline, while capsaicin, prazosin and GF109203X were dissolved in 10% DMSO/saline. In all cases, control groups used the respective vehicle. For behavioral determinations, rats were acclimatized for 20–30 min in a Plexiglass observation chamber. Rats were removed from the chamber, loosely restrained for injections, and then returned immediately to the chamber. Following drug injection, each rat was observed individually in 1 min bins for 15 min, and flinching behaviors (paw elevation or shaking the hindpaw) were recorded. Individual agents generally produced short-lasting effects (3–5 min), while combination with NA produced longer lasting behaviors (see time courses in previous publications) [31,35]. Alternating rats from sham and nerve injury groups were evaluated on test days. Experiments were conducted by two experimenters, one analyzing P2X3 receptors and the other TRPV1 receptors. Data are presented as mean and S.E.M. for cumulative responses over the 15 min. Statistical analysis was performed using the unpaired Student’s t-test within individual experiments that were matched. ANOVA followed by the Student–Neuman–Keuls’ test was
used when multiple groups were compared to a single control condition. Following lateral hindpaw injections after SNI, responses to inactive doses of NA and ␣MeATP given alone were augmented significantly compared to respective sham groups (P < 0.05; open columns compared to filled columns) (Fig. 1A); responses to saline (9 ± 2 sham, 13 ± 3 SNI, N = 7) were unchanged. The combination of NA with ␣MeATP led to increased pain behaviors compared to the individual agents in sham groups (filled columns; Fig. 1A), and the effect of NA/␣MeATP was significantly enhanced compared to the sham group following nerve injury (open vs filled column; Fig. 1A). When each of NA, ␣MeATP and NA/␣MeATP was injected into the medial hindpaw after SNI, there was no alteration in effect in any instance (open vs filled columns; Fig. 1B). The response to the combination of ␣MeATP with NA remained augmented compared to individual agents (filled columns; Fig. 1B). When capsaicin was injected into the lateral hindpaw after SNI, there was no difference in intrinsic responses to capsaicin (Fig. 2A); however, the response to NA combined with capsaicin was significantly reduced (filled vs open columns; Fig. 2A). When administered into the medial hindpaw after SNI, there was no difference in the response to capsaicin alone or capsaicin combined with NA (filled vs open columns; Fig. 2B). In naïve rats, this dose of capsaicin produces an increased incidence of flinches compared to vehicle (19 ± 7 vehicle, 49 ± 4 capsaicin, N = 6 per group), as shown previously [31]. ␣1-Adrenergic receptors (␣1-ARs) have previously been implicated in the augmentation of P2X3 [21] and capsaicin-mediated responses [15,17,30] in the uninjured state. We used a selective antagonist (prazosin) and a broad spectrum inhibitor of protein kinase C (GF109203X) to examine the involvement of ␣1-ARs in such actions following nerve injury. Following lateral hindpaw injections after SNI, a low dose of prazosin reduced the effect of NA/␣MeATP in SNI rats (Fig. 3A). Both prazosin and GF109203X reduced the effect of NA/capsaicin in naïve animals (Fig. 3B), but neither agent altered the effect of NA/capsaicin when injected into the lateral hindpaw after SNI (Fig. 3C). This study examined adrenergic regulation of P2X3 and TRPV1 receptors using the SNI model. The unique feature of the model is that it allows one to compare responses mediated by the spared sural branch of the sciatic nerve (which is influenced by injured neurons in the upper aspects of the sciatic nerve and via comingling in the spinal nerve plexus in the L4–L6 DRG) to those mediated by the intact saphenous nerve following drug injections
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Fig. 3. Role of ␣1-ARs in the augmentation of ␣MeATP and capsaicin responses following injection into the lateral (LAT) hindpaw (A) following SNI, (B) in naïve rats, and (C) following SNI. Values depict mean + S.E.M. for cumulative responses over 15 min following drug injections, with N = 6–9 per group. « P < 0.05 compared to the combination response in naïve rats; * P < 0.05 compared to the control response produced by the indicated combination in sham animals (filled column).
into the lateral and medial hindpaw, respectively [8]. Mechanical allodynia is more prominent in the lateral compared to the medial site [8], but other modalities have not been systematically investigated. A recent study examined the sympathetic dependence of hypersensitivity responses in this model, and showed that chemical sympathectomy had no effect on mechanical allodynia, cold allodynia or mechanical hyperalgesia at the lateral site 1–4 weeks following surgery [26]; however, a delayed effect was seen at 8 weeks and this corresponded to a time when sympathetic sprouting occurred in DRG. Responses to these stimuli at medial sites were not reported. In the present study, all testing was conducted 1–2 weeks following SNI surgery at a time where there is no sympathetic dependence of evoked hypersensitivity responses or sympathetic sprouting in DRG. The present study reports enhanced flinch responses to ␣MeATP at lateral, but not medial, sites 7–14 days following SNI. The increase in flinching at lateral sites confirms the report by Chen et al. [7], who also noted an increase in electrophysiological activation of DRG neurons by ATP. Following SNI, they noted no difference in total P2X3 receptor density in L4–L5 ganglia, but an increase in translocation of receptors to the cell membrane, and this was suggested to mediate the enhanced response to ␣MeATP. Previous reports had noted decreases [3,13,14,36] or increases [23] in P2X3 receptors following various forms of nerve injury. Some of this heterogeneity may reflect state of injury as, by using the activating transcription factor 3 (ATF3) to identify injured neurons, P2X3 receptors were shown to be decreased in injured DRG cells but increased in uninjured neurons [34]. The signaling that occurs at the lateral site may reflect increased receptor dynamics (translocation to the cell membrane), as well as increased expression in uninjured neurons that are influenced directly by injured afferents either along the upper extent of the sciatic nerve or in the spinal nerve plexus. This could involve paracrine influences of growth factors, such as nerve growth factor (NGF) or glial-derived neurotrophic factor (GDNF), both of which positively regulate P2X3 receptor expression [27,36]. When signaling occurs via the intact saphenous nerve, where there is little direct influence of injured afferents, there is no difference in functional responses to ␣MeATP. The present study also demonstrates that flinch responses to NA alone and to NA combined with ␣MeATP are enhanced at lateral, but not medial, sites following nerve injury. The adrenergic receptor (AR) mediating enhanced P2X3 receptor responses on sensory neurons has been characterized as an ␣1-AR in the uninjured state [21] and following nerve injury where ␣1B-ARs in particular are
implicated [19]. Results of the present study are consistent with an ␣1-AR involvement as the effect of NA/␣MeATP is reduced by a low dose of prazosin, a selective ␣1-AR antagonist. ␣1-AR regulation of P2X3 receptor activity involves protein kinase C dependent pathways [19] but not necessarily direct P2X3 receptor phosphorylation, and involvement of an accessory protein is proposed [4]. The pattern of changes seen with both NA alone and in combination with ␣MeATP is identical to that seen with ␣MeATP alone, and this suggests that the paracrine influences of injured neurons noted above for P2X3 receptors also can contribute to enhanced functional effects involving ␣1-ARs. When such influences are absent (cf. medial hindpaw injections and saphenous nerve signaling), these changes are no longer observed. In contrast to the above observations, flinch responses to capsaicin were not altered when capsaicin was administered either to the lateral or medial hindpaw sites following SNI. Flinch responses produced by capsaicin administered to the rat hindpaw are inhibited by capsazepine and are presumed to reflect TRPV1 receptor activation on sensory afferents [31]. Previous studies examining TRPV1 receptors following nerve injury have demonstrated increased TRPV1 expression in uninjured, or spared, nerves following nerve injury [9,11], but decreases in the soma of injured DRG cells [11,18,22]. There is also a marked upregulation of TRPV1 receptors in large diameter cell bodies in DRG following nerve injury [18,19,28,29]. The lack of effect of SNI on capsaicin responses noted at lateral sites in this study may reflect different changes in receptors in different populations of afferents (injured and uninjured, small and large diameter), collectively leading to no net alteration in effect. At the medial site, the relative lack of peripheral influence of injured nerves may allow signaling by capsaicin to be unaffected. Adrenergic and sympathetic regulation of responses to capsaicin administered locally into the rat hindpaw involve ␣1-ARs [15,17,30]. The present study confirms involvement of ␣1-ARs and protein kinase C signaling [16] in NA augmentation of capsaicin responses, by demonstrating block of such augmentation by a low dose of prazosin and by GF109203X, a protein kinase C inhibitor in naïve rats. Augmentation of TRPV1 function by protein kinase C has been shown to involve phosphorylation of the receptor [2,24]. Curiously, when NA and capsaicin are administered to the lateral hindpaw following SNI, flinch responses are reduced compared to sham surgery rats, even though responses to NA alone are augmented and responses to capsaicin are unaltered. Adrenergic enhancement of capsaicin responses was still present in sham surgery animals, but this regulation seemed to be blunted following SNI, as the response to NA/capsaicin was no longer blocked by prazosin or GF109203X. The observation that ␣1-AR regulation of TRPV1 receptors was reduced at lateral hindpaw sites following SNI was unexpected given that augmentation of P2X3 receptor effects were clearly increased (see above), that P2X3 and TRPV1 receptors coexist in a population of sensory afferents [10], and that NGF and GDNF both increase TRPV1 receptor function [1]. However, the decrease in NA/capsaicin responses at the lateral site following SNI is consistent with reduced functional responses to NA/capsacin reported previously in other nerve injury models (partial sciatic nerve ligation, tibial nerve injury) [31]. Furthermore, other studies reveal distinct regulation of P2X3 and TRPV1 receptor expression in sensory neurons by acute and chronic NGF as well as by serotonin [32]. The proposal of indirect modulation of the P2X3 receptor [4] but direct phosphorylation of the TRPV1 receptor by protein kinase C [2,24] may contribute to this divergent adrenergic regulation of the two receptors by nerve injury. Since nerve injury models were developed, there has been considerable emphasis placed on enhanced sensory afferent signaling produced by evoked stimuli. The present results with NA/␣MeATP and NA/capsaicin show that nerve injury can produce quite dif-
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Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Adrenergic regulation of P2X3 and TRPV1 receptors: Differential effects of spared nerve injury Jason G. Meisner, Allison R. Reid, Jana Sawynok ∗ Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 1X5
a r t i c l e
i n f o
Article history: Received 9 June 2008 Received in revised form 11 August 2008 Accepted 12 August 2008 Keywords: P2X3 receptors TRPV1 receptors Noradrenaline Spared nerve injury
a b s t r a c t Local application of ␣MeATP (ligand for P2X3 receptors) and capsaicin (ligand for TRPV1 receptors) to the rat hindpaw produces pain behaviors (flinching) which are enhanced by noradrenaline (NA). In this study, we have examined the effect of nerve injury on adrenergic regulation of P2X3 and TRPV1 receptors by administering ␣MeATP and capsaicin, alone and in combination with NA, into the lateral and medial hindpaw in the spared nerve injury (SNI) model; this allows for an exploration of the role of injured and uninjured afferents in their effects on nociceptive signaling using a behavioral model. Following lateral hindpaw injections (sural sensory field), effects of NA and ␣MeATP, both alone and in combination, were increased following SNI, but no such effects were seen following medial hindpaw injections (saphenous sensory field). Following lateral hindpaw injections, the effect of capsaicin alone was unaltered following SNI, but the effect of NA/capsaicin was reduced; this latter effect was not seen following medial hindpaw injections. At the lateral site, prazosin (␣1-adrenergic receptor antagonist) inhibited the effect of NA/␣MeATP following SNI, but neither prazosin nor GF109203X (protein kinase C inhibitor) inhibited the effect of NA/capsaicin following SNI. These results demonstrate: (a) an enhanced adrenergic regulation of P2X3 receptor activity at lateral sites following SNI where signaling afferents are directly influenced by injured neurons; (b) differential effects on adrenergic regulation of TRPV1 receptors under the same conditions; (c) lack of such changes when agents are administered into medial sites following SNI. © 2008 Elsevier Ireland Ltd. All rights reserved.
Both P2X3 [5] and TRPV1 [6] receptors are present on sensory nerve endings, allow for cation entry, and lead to sensory nerve activation and nociceptive signaling. Both receptors are considered potential targets for development of novel therapeutic agents [20,33]. When administered locally into the hindpaw of rodents, ␣-methylene-ATP (␣MeATP) and capsaicin, selective ligands for these two receptors respectively, produce pain behaviors reflective of sensory nerve activation; in both cases, pain behaviors are increased by noradrenaline (NA) [31,35]. NA is co-released with ATP from sympathetic nerves, and sympathetic mechanisms can contribute to manifestations of neuropathic pain by actions at several sites, including peripheral aspects of the sensory nerve and the cell body in the dorsal root ganglia (DRG) following sympathetic sprouting [12]. Adrenergic mechanisms have been shown directly to modulate P2X3 receptor mediated responses on sensory neurons following nerve injury [19,25]. Sympathetic mechanisms influence peripheral hypersensitivity responses to capsaicin in the uninjured state [15,17,30],
∗ Corresponding author. Tel.: +1 902 494 2596; fax: +1 902 494 1388. E-mail address: [email protected] (J. Sawynok). 0304-3940/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2008.08.032
but there is little information on this regulation following nerve injury. Several preclinical nerve injury models have been developed, and the ability of such models to produce enhanced pain signaling has received considerable attention in recent years. One such model, the spared nerve injury (SNI) model in which the tibial and common peroneal branches of the sciatic nerve are transected leaving the sural branch of the sciatic nerve intact, allows for the discrete examination of sensory signaling in distinct sensory fields [8]. Thus, one can administer a stimulus or a drug into a sensory field where intact innervation is influenced by injured afferents (lateral hindpaw; innervated by the spared sural nerve branch but with comingling of injured and uninjured afferents in the upper aspect of the sciatic nerve and L4–L6 dorsal root ganglia) OR into a sensory field where innervation is by uninjured afferents (medial hindpaw; innervated by the intact saphenous nerve with minimal interactions with injured fibres in the L3 dorsal root ganglion) (see Fig. 1 in Ref. [8]). In the present study, we have undertaken a systematic examination of peripheral responses to ␣MeATP and capsaicin, alone and combined with NA, following localized delivery into the lateral and medial hindpaw using the SNI model to determine: (a) the
J.G. Meisner et al. / Neuroscience Letters 444 (2008) 172–175
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Fig. 1. Effect of spared nerve injury (SNI) on responses to NA and ␣MeATP, alone and in combination, following injection into (A) the lateral aspect of the hindpaw, or (B) the medial plantar hindpaw. Values depict mean + S.E.M. for cumulative responses over 15 min following drug injections, with N = 6–9 per group. « P < 0.05 compared to the individual agents in sham animals (filled columns); * P < 0.05 compared to corresponding sham controls (filled vs open columns).
Fig. 2. Effect of capsacin and capsaicin/NA injected into the (A) lateral hindpaw or (B) medial plantar hindpaw following spared nerve injury (SNI). Values depict mean + S.E.M. for cumulative responses over 15 min following drug injections, with N = 7–9 per group. « P < 0.05 compared to the individual agents in sham animals (filled columns); * P < 0.05 compared to corresponding sham controls (filled vs open columns).
influence of injured afferents on peripheral responses to P2X3 and TRPV1 receptor activation, and (b) the adrenergic regulation of such responses. Experiments were conducted using male Sprague–Dawley rats (125–250 g) from Charles River, Montreal. All procedures were approved by the University Committee on Laboratory Animals, and performed in accordance with Canadian Council on Animal Care guidelines. SNI surgery was performed as described previously [8]. This procedure leaves the sural nerve intact, and care was taken to avoid damage to this nerve during surgery. Sham surgery consisted of exposure of the nerves without transection. Rats were housed individually for 3 days following surgery to allow for wound healing, and then were paired with the original cage mate. Following SNI, drugs were injected into the lateral aspect of the hindpaw or medial plantar hindpaw days 7–14 following surgery. Each experiment was conducted as a parallel sham and nerve injury experiment, with identical drug treatments given at the same time. All drugs were purchased from Sigma–Aldrich Canada Ltd. (Oakville, Ontario, Canada). Following SNI, drugs were injected in a volume of 30 l into the lateral aspect of the hindpaw at the junction of the hairy and non-hairy skin, or into the medial plantar hindpaw. This volume is smaller than the usual volume of 50 l used for rat studies, and it was used to restrict diffusion of the drug to the respective sensory fields. ␣MeATP and NA were dissolved in saline, while capsaicin, prazosin and GF109203X were dissolved in 10% DMSO/saline. In all cases, control groups used the respective vehicle. For behavioral determinations, rats were acclimatized for 20–30 min in a Plexiglass observation chamber. Rats were removed from the chamber, loosely restrained for injections, and then returned immediately to the chamber. Following drug injection, each rat was observed individually in 1 min bins for 15 min, and flinching behaviors (paw elevation or shaking the hindpaw) were recorded. Individual agents generally produced short-lasting effects (3–5 min), while combination with NA produced longer lasting behaviors (see time courses in previous publications) [31,35]. Alternating rats from sham and nerve injury groups were evaluated on test days. Experiments were conducted by two experimenters, one analyzing P2X3 receptors and the other TRPV1 receptors. Data are presented as mean and S.E.M. for cumulative responses over the 15 min. Statistical analysis was performed using the unpaired Student’s t-test within individual experiments that were matched. ANOVA followed by the Student–Neuman–Keuls’ test was
used when multiple groups were compared to a single control condition. Following lateral hindpaw injections after SNI, responses to inactive doses of NA and ␣MeATP given alone were augmented significantly compared to respective sham groups (P < 0.05; open columns compared to filled columns) (Fig. 1A); responses to saline (9 ± 2 sham, 13 ± 3 SNI, N = 7) were unchanged. The combination of NA with ␣MeATP led to increased pain behaviors compared to the individual agents in sham groups (filled columns; Fig. 1A), and the effect of NA/␣MeATP was significantly enhanced compared to the sham group following nerve injury (open vs filled column; Fig. 1A). When each of NA, ␣MeATP and NA/␣MeATP was injected into the medial hindpaw after SNI, there was no alteration in effect in any instance (open vs filled columns; Fig. 1B). The response to the combination of ␣MeATP with NA remained augmented compared to individual agents (filled columns; Fig. 1B). When capsaicin was injected into the lateral hindpaw after SNI, there was no difference in intrinsic responses to capsaicin (Fig. 2A); however, the response to NA combined with capsaicin was significantly reduced (filled vs open columns; Fig. 2A). When administered into the medial hindpaw after SNI, there was no difference in the response to capsaicin alone or capsaicin combined with NA (filled vs open columns; Fig. 2B). In naïve rats, this dose of capsaicin produces an increased incidence of flinches compared to vehicle (19 ± 7 vehicle, 49 ± 4 capsaicin, N = 6 per group), as shown previously [31]. ␣1-Adrenergic receptors (␣1-ARs) have previously been implicated in the augmentation of P2X3 [21] and capsaicin-mediated responses [15,17,30] in the uninjured state. We used a selective antagonist (prazosin) and a broad spectrum inhibitor of protein kinase C (GF109203X) to examine the involvement of ␣1-ARs in such actions following nerve injury. Following lateral hindpaw injections after SNI, a low dose of prazosin reduced the effect of NA/␣MeATP in SNI rats (Fig. 3A). Both prazosin and GF109203X reduced the effect of NA/capsaicin in naïve animals (Fig. 3B), but neither agent altered the effect of NA/capsaicin when injected into the lateral hindpaw after SNI (Fig. 3C). This study examined adrenergic regulation of P2X3 and TRPV1 receptors using the SNI model. The unique feature of the model is that it allows one to compare responses mediated by the spared sural branch of the sciatic nerve (which is influenced by injured neurons in the upper aspects of the sciatic nerve and via comingling in the spinal nerve plexus in the L4–L6 DRG) to those mediated by the intact saphenous nerve following drug injections
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Fig. 3. Role of ␣1-ARs in the augmentation of ␣MeATP and capsaicin responses following injection into the lateral (LAT) hindpaw (A) following SNI, (B) in naïve rats, and (C) following SNI. Values depict mean + S.E.M. for cumulative responses over 15 min following drug injections, with N = 6–9 per group. « P < 0.05 compared to the combination response in naïve rats; * P < 0.05 compared to the control response produced by the indicated combination in sham animals (filled column).
into the lateral and medial hindpaw, respectively [8]. Mechanical allodynia is more prominent in the lateral compared to the medial site [8], but other modalities have not been systematically investigated. A recent study examined the sympathetic dependence of hypersensitivity responses in this model, and showed that chemical sympathectomy had no effect on mechanical allodynia, cold allodynia or mechanical hyperalgesia at the lateral site 1–4 weeks following surgery [26]; however, a delayed effect was seen at 8 weeks and this corresponded to a time when sympathetic sprouting occurred in DRG. Responses to these stimuli at medial sites were not reported. In the present study, all testing was conducted 1–2 weeks following SNI surgery at a time where there is no sympathetic dependence of evoked hypersensitivity responses or sympathetic sprouting in DRG. The present study reports enhanced flinch responses to ␣MeATP at lateral, but not medial, sites 7–14 days following SNI. The increase in flinching at lateral sites confirms the report by Chen et al. [7], who also noted an increase in electrophysiological activation of DRG neurons by ATP. Following SNI, they noted no difference in total P2X3 receptor density in L4–L5 ganglia, but an increase in translocation of receptors to the cell membrane, and this was suggested to mediate the enhanced response to ␣MeATP. Previous reports had noted decreases [3,13,14,36] or increases [23] in P2X3 receptors following various forms of nerve injury. Some of this heterogeneity may reflect state of injury as, by using the activating transcription factor 3 (ATF3) to identify injured neurons, P2X3 receptors were shown to be decreased in injured DRG cells but increased in uninjured neurons [34]. The signaling that occurs at the lateral site may reflect increased receptor dynamics (translocation to the cell membrane), as well as increased expression in uninjured neurons that are influenced directly by injured afferents either along the upper extent of the sciatic nerve or in the spinal nerve plexus. This could involve paracrine influences of growth factors, such as nerve growth factor (NGF) or glial-derived neurotrophic factor (GDNF), both of which positively regulate P2X3 receptor expression [27,36]. When signaling occurs via the intact saphenous nerve, where there is little direct influence of injured afferents, there is no difference in functional responses to ␣MeATP. The present study also demonstrates that flinch responses to NA alone and to NA combined with ␣MeATP are enhanced at lateral, but not medial, sites following nerve injury. The adrenergic receptor (AR) mediating enhanced P2X3 receptor responses on sensory neurons has been characterized as an ␣1-AR in the uninjured state [21] and following nerve injury where ␣1B-ARs in particular are
implicated [19]. Results of the present study are consistent with an ␣1-AR involvement as the effect of NA/␣MeATP is reduced by a low dose of prazosin, a selective ␣1-AR antagonist. ␣1-AR regulation of P2X3 receptor activity involves protein kinase C dependent pathways [19] but not necessarily direct P2X3 receptor phosphorylation, and involvement of an accessory protein is proposed [4]. The pattern of changes seen with both NA alone and in combination with ␣MeATP is identical to that seen with ␣MeATP alone, and this suggests that the paracrine influences of injured neurons noted above for P2X3 receptors also can contribute to enhanced functional effects involving ␣1-ARs. When such influences are absent (cf. medial hindpaw injections and saphenous nerve signaling), these changes are no longer observed. In contrast to the above observations, flinch responses to capsaicin were not altered when capsaicin was administered either to the lateral or medial hindpaw sites following SNI. Flinch responses produced by capsaicin administered to the rat hindpaw are inhibited by capsazepine and are presumed to reflect TRPV1 receptor activation on sensory afferents [31]. Previous studies examining TRPV1 receptors following nerve injury have demonstrated increased TRPV1 expression in uninjured, or spared, nerves following nerve injury [9,11], but decreases in the soma of injured DRG cells [11,18,22]. There is also a marked upregulation of TRPV1 receptors in large diameter cell bodies in DRG following nerve injury [18,19,28,29]. The lack of effect of SNI on capsaicin responses noted at lateral sites in this study may reflect different changes in receptors in different populations of afferents (injured and uninjured, small and large diameter), collectively leading to no net alteration in effect. At the medial site, the relative lack of peripheral influence of injured nerves may allow signaling by capsaicin to be unaffected. Adrenergic and sympathetic regulation of responses to capsaicin administered locally into the rat hindpaw involve ␣1-ARs [15,17,30]. The present study confirms involvement of ␣1-ARs and protein kinase C signaling [16] in NA augmentation of capsaicin responses, by demonstrating block of such augmentation by a low dose of prazosin and by GF109203X, a protein kinase C inhibitor in naïve rats. Augmentation of TRPV1 function by protein kinase C has been shown to involve phosphorylation of the receptor [2,24]. Curiously, when NA and capsaicin are administered to the lateral hindpaw following SNI, flinch responses are reduced compared to sham surgery rats, even though responses to NA alone are augmented and responses to capsaicin are unaltered. Adrenergic enhancement of capsaicin responses was still present in sham surgery animals, but this regulation seemed to be blunted following SNI, as the response to NA/capsaicin was no longer blocked by prazosin or GF109203X. The observation that ␣1-AR regulation of TRPV1 receptors was reduced at lateral hindpaw sites following SNI was unexpected given that augmentation of P2X3 receptor effects were clearly increased (see above), that P2X3 and TRPV1 receptors coexist in a population of sensory afferents [10], and that NGF and GDNF both increase TRPV1 receptor function [1]. However, the decrease in NA/capsaicin responses at the lateral site following SNI is consistent with reduced functional responses to NA/capsacin reported previously in other nerve injury models (partial sciatic nerve ligation, tibial nerve injury) [31]. Furthermore, other studies reveal distinct regulation of P2X3 and TRPV1 receptor expression in sensory neurons by acute and chronic NGF as well as by serotonin [32]. The proposal of indirect modulation of the P2X3 receptor [4] but direct phosphorylation of the TRPV1 receptor by protein kinase C [2,24] may contribute to this divergent adrenergic regulation of the two receptors by nerve injury. Since nerve injury models were developed, there has been considerable emphasis placed on enhanced sensory afferent signaling produced by evoked stimuli. The present results with NA/␣MeATP and NA/capsaicin show that nerve injury can produce quite dif-
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