QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.103.052951v1 306/3/1137    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Huang, H. Articles by Vasko, M. R. Search for Related Content PubMed PubMed Citation Articles by Huang, H. Articles by Vasko, M. R.

NEUROPHARMACOLOGY

ATP Augments Peptide Release from Rat Sensory Neurons in Culture through Activation of P2Y Receptors

Department of Pharmacology and Toxicology (H.H., X.W.,G.D.N., M.R.V.) and Department of Anesthesia (M.R.V.), Indiana University School of Medicine, Indianapolis, Indiana; and Health Sciences Institute, The Procter and Gamble Company, Mason, Ohio (S.M.)

Received April 10, 2003; accepted May 29, 2003.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
ATP has recently emerged as an important proinflammatory mediator that has direct excitatory actions on sensory neurons through activation of ion channel-coupled P2X receptors. The purpose of the current work is to assess whether ATP alters the release of neuropeptides from sensory neurons and the receptors mediating this putative action. Exposing embryonic sensory neurons in culture to concentrations of ATP up to 300 µM did not increase the release of immunoreactive substance P or calcitonin gene-related peptide from sensory neurons. However, pre-exposing sensory neurons to 0.1 to100 µM ATP prior to and throughout administration of 30 nM capsaicin resulted in a significant augmentation of release evoked by the vanilloid. This sensitizing action of ATP is blocked by suramin but not pyridoxal phosphate-6-azobenzene-2,4-disulfonic acid and is mimicked by the P2Y receptor agonists, 2-2-chloroadenosine triphosphate and UTP, but not by 2-(methylthio)adenosine 5'-triphosphate or ,-methyleneadenosine 5'-diphosphate. This profile of drug actions suggests that the sensitizing actions of ATP are mediated by P2Y receptors. Pretreating sensory neurons with bisindolylmaleimide I, a selective protein kinase C (PKC) inhibitor, attenuates the augmentation of capsaicin-induced peptide release by ATP, further implicating P2Y receptors in the actions of ATP. Immunoblotting also indicates the presence of P2Y₂-like immunoreactive substance in embryonic dorsal root ganglia neurons. Together, these data support the notion that ATP acts at P2Y receptors in sensory neurons in a PKC-dependent manner to augment their sensitivity to other stimuli.

Most studies to date have focused on the involvement of P2X receptors in inflammation and nociception (Burnstock, 2001). Indeed, activation of homomeric P2X₃ receptors in small-diameter, capsaicin-sensitive sensory neurons results in a transient, rapidly inactivating inward current (Ueno et al., 1999). Furthermore, ATP has been shown to excite nociceptive sensory neurons in tooth pulp through distinct P2X receptors (Cook et al., 1997). Recent work, however, examined the potential actions of P2Y activation of sensory neurons. Tominaga et al. (2001) demonstrated that ATP potentiated capsaicin-induced inward currents in sensory neurons through activation of P2Y receptors. In a similar manner, Sanada et al. (2000) reported that ATP- and UTP-induced increase in intracellular calcium in isolated rat sensory neurons is likely mediated by P2Y receptors. Together, these studies suggest that ATP has multiple actions on sensory neurons mediated by both P2X and P2Y receptors, and the challenge remains to delineate which actions have functional importance under various physiological and pathological conditions.

It is clear that ATP evokes excitatory responses in sensory neurons, but controversy remains as to whether the nociceptive actions of ATP are caused by direct excitation of sensory neurons or through the ability of ATP to augment the excitability to other stimuli. In vivo, the P2X receptor agonist, ,-meATP, is unable to excite corneal nociceptors (Dowd et al., 1997) or tooth pulp afferents in cats (Matthews et al., 1997), whereas intra-articular injections of ATP or ,-meATP into the knee joint in rat evokes a rapid and short-lasting excitation of C and A fibers innervating knee joints (Dowd et al., 1998). Transgenic P2X₃-null mice show normal response to acute mechanical and thermal stimuli (Cockayne et al., 2000; Souslova et al., 2000), suggesting that P2X₃ is not essential in acute nociception. In isolated sensory neurons, Sanada et al. (2002) showed that exposure to high concentrations of ATP or UTP increased intracellular calcium and that UTP (100 µM) directly induced release of immunoreactive calcitonin gene-related peptide (iCGRP) from rat sensory neurons. In contrast, Zimmermann et al. (2002) demonstrated that 100 µM ATP has little, if any, direct excitatory effects on the release of iCGRP from the nerve terminals in isolated rat dura mater; rather, it augments proton-induced release of iCGRP. Whether this action of ATP was due to a direct effect on sensory neurons was not determined in this study.

Given the effects of ATP on sensory neurons through P2X and P2Y receptors, we sought to determine whether ATP, P2X agonists, and P2Y agonists could increase the release of and/or augment the capsaicin-stimulated release of immunoreactive substance P (iSP) and iCGRP from sensory neurons grown in culture. We also ascertained which subtypes of the P2 purinergic receptors mediate the effect of ATP, and whether these putative subtypes are expressed in DRG sensory neurons. Our results indicate that ATP augments the release of iSP and iCGRP evoked by capsaicin in rat sensory neurons in culture through activation of P2Y receptors, but does not alter basal peptide release when administered alone. Portions of this work appeared previously in abstract form (Wu et al., 1997).

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Materials. Timed-pregnant Sprague-Dawley rats were purchased from Harlan (Indianapolis, IN). Cell culture supplies were obtained from Invitrogen (Carlsbad, CA), and nerve growth factor was obtained from Harlan Bioproducts for Science (Indianapolis, IN). Peptides were obtained from Peninsula Laboratories (Belmont, CA). Suramin, pyridoxal phosphate-6-azobenzene-2,4-disulfonic acid (PPADS), 2-(methylthio)adenosine 5'-triphosphate (2-MeSATP), ,-methyleneadenosine 5'-diphosphate (,-meATP), ,-methyleneadenosine 5'-diphosphate (,-meATP), 2-chloroadenosine triphosphate (2-ClATP), uridine 5'-triphosphate (UTP), adenosine 5'-triphosphate (ATP), capsaicin, and 1-methyl-2-pyrrolidinone were obtained from Sigma-Aldrich (St. Louis, MO). Bisindolylmaleimide I was purchased from Calbiochem (San Diego, CA). Capsaicin was initially dissolved in 1-methyl-2-pyrrolidinone (Aldrich Chemical Co., Milwaukee, WI) and then diluted to appropriate concentrations with HEPES buffer. This vehicle did not alter the release of either peptide at the concentrations used. Polyclonal antibodies against P2Y₁, P2Y₂, and P2Y₄ and their corresponding control antigens were obtained from Chemicon International (Temecula, CA).

Isolation and Culture of Embryonic Rat Sensory Neurons. Cultures of sensory neurons were prepared using a modification of our previously described protocol (Vasko et al., 1994). Briefly, cells were dissociated from the dorsal root ganglia of 15- to 17-day rat embryos using trypsin and mechanical dissociation. Approximately 150,000 cells were plated into poly-D-lysine-coated wells of a 24-well Falcon culture dish. The cells were grown in Dulbecco's modified Eagle's medium supplemented with 2 mM glutamine, 50 µg/ml penicillin and streptomycin, 10% (v/v) heat-inactivated fetal bovine serum, 50 µM 5-fluro-2'-deoxyuridine, and 250 ng/ml nerve growth factor. Cultures were maintained at 37°C under a 5% CO₂ atmosphere. The medium was replaced every 2 to 3 days.

Release of iSP and iCGRP from Sensory Neurons. After 9 to 12 days in culture, release of iSP and iCGRP from cultured sensory neurons was determined as previously described (Vasko et al., 1994). We chose to perform experiments at this time to allow sufficient neuropeptides to accumulate so that we could reproducibly measure resting levels of release. Cells were incubated for successive 10-min intervals in 0.4 ml of HEPES buffer consisting of 25 mM HEPES, 135 mM NaCl, 3.5 mM KCl, 2.5 mM CaCl₂, 1 mM MgCl₂, 3.3 mM D-glucose, 1 µM phosphoramidon, and 0.1% (w/v) bovine serum albumin (pH 7.4), and maintained at 37°C.

Basal neuropeptide release was determined by exposing cells to HEPES buffer alone for 10 min, whereas the direct effect of purinergic agonists or antagonists on release was determined by exposing cells for the next 10 min to drugs. This was followed by incubating the cells for 10 min in buffer containing 30 nM capsaicin in the absence or presence of purinergic agonists and antagonists. Subsequently, cells were incubated for 10 min with HEPES buffer alone to show a return to baseline. After each 10-min incubation, the buffer was removed, aliquoted, and assayed for iSP and iCGRP by radio-immunoassay as previously described (Vasko et al., 1994).

Immunoblotting. Cultured sensory neurons were washed in phosphate-buffered saline, scraped, and pelleted at 10,000g for 2 min. Cells were resuspended in radioimmunoprecipitation assay buffer containing 1x phosphate-buffered saline, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and supplemented with 0.1 mg/ml phenylmethylsulfonyl fluoride, 3% aprotinin, and 1 mM sodium orthovanadate, and then disrupted by sonication (two 15-s pulses). Protein concentration in the lysate was determined by using a Bradford protein assay. Cell lysates (40 µg/lane) were separated on 10% sodium dodecyl sulfate polyacrylamide gels and electrophoretically transferred to a polyvinylidene difluoride membrane. The membrane was blocked by incubation in 5% Carnation instant milk in Tris-buffered saline for 1 h. Subsequently, the membrane was incubated in 5% milk in Tris-buffered saline containing 0.1% Tween in the presence of antibodies directed to P2Y₁, P2Y₂, or P2Y₄ receptors. As the manufacturer suggested, the P2Y antibodies were used at 3 µg/ml, the antigenic peptide for P2Y₂ was used at 3 µg/ml. After two washes with Tris-buffered saline, the membrane was incubated with a secondary horseradish peroxidase-conjugated goat anti-rabbit antibody for 1 h. Immunoreactive bands were developed by an ECL kit (PerkinElmer Life Sciences, Boston, MA), and visualized by exposure to film.

Statistical Analysis. Data are presented as mean ± S.E.M. with n being the total number of wells used. ANOVA was used to compare release stimulated by capsaicin alone with release evoked by capsaicin and various purinergic agents. If this test indicated that a difference existed, post hoc Bonferroni tests were performed. The significance level for all tests was set at P < 0.05. In some instances, the capsaicin-evoked release varied between experiments, and thus, the data were normalized to percentage of control capsaicin-evoked release.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Effects of ATP on Peptide Release from Cultures of Rat Sensory Neurons. To determine whether ATP directly alters peptide release from sensory neurons, neuronal cultures were exposed to different concentrations of ATP for 10 min. As shown in Fig. 1 (dashed lines), at concentrations ranging from 0.1 µM to 300 µM, ATP did not increase the release of iSP and iCGRP when compared to basal neuropeptide release in the presence of HEPES buffer alone.

View larger version (16K): [in this window] [in a new window]   Fig. 1. Dose dependence of ATP-induced enhancement of capsaicin-evoked release of neuropeptides. ATP augments capsaicin-evoked release of iSP and iCGRP from rat sensory neurons. Data are mean ± S.E.M. values of the amount of peptide release for the number of wells indicated. The ordinates represent the amount of iCGRP (filled symbols) and iSP (open symbols) released in the presence of ATP alone (triangles) and in the presence of ATP and 30 nM capsaicin (circles). The basal panel shows release of iCGRP and iSP when cells were exposed to HEPES buffer alone, whereas the 30 nM CAP panel shows release when cells were exposed to 30 nM capsaicin alone. An asterisk indicates a statistically significant difference from capsaicin-evoked release in the absence versus presence of ATP.

To assess whether ATP could augment release evoked by capsaicin, cultures were exposed to 0.1, 1.0, 10, 100, or 300 µM ATP for 10 min prior to and throughout treatment with 30 nM capsaicin. Exposing sensory neurons to 30 nM capsaicin resulted in a significant increase in the release of iSP and iCGRP from basal levels of 10 ± 0.4 and 31 ± 2 fmol/well/10 min to 108 ± 10 and 487 ± 56 fmol/well/10min, respectively. When cells were exposed to ATP for 10 min prior to and throughout capsaicin treatment, all concentrations of ATP that were utilized except 300 µM resulted in an augmentation of capsaicin-evoked release of both iSP and iCGRP. For example, 1.0 µM ATP resulted in an approximately 68% increase in capsaicin-evoked release of iSP and an approximately 84% increase in iCGRP release above that observed with capsaicin alone. When the concentration of ATP was increased to 10 µM, capsaicin-evoked release of iSP release was increased from 117 ± 16 to 268 ± 39 fmol/well/10 min, whereas capsaicin-simulated release of iCGRP was elevated by ATP from 535 ± 74 to 1174 ± 86 fmol/well/10 min. These results suggest that although ATP does not increase release directly, it can sensitize sensory neurons as indicated by its ability to augment capsaicin-evoked release.

Effects of Purinoreceptor Antagonists on ATP-Induced Enhancement of Peptide Release from Cultures of Rat Sensory Neurons. In an attempt to define the receptor subtypes mediating the sensitizing actions of ATP on sensory neurons, we examined the effects of two purinoreceptor antagonists on ATP-induced augmentation of capsaicin-evoked neuropeptide release. For these studies, cells were exposed to various antagonists 10 min prior to and throughout treatment with 10 µM ATP and 30 nM capsaicin. We chose to use 10 µM ATP because this concentration produced the maximal effect on capsaicin-induced peptide release. Initial studies were performed to assess whether suramin, a P2 antagonist, could attenuate the effects of ATP (Fig. 2), to distinguish between P2 receptors and P1 (adenosine A1 and A2) receptors. As with previous results, when sensory neurons were pretreated with 10 µM ATP, capsaicin-evoked release of iSP and iCGRP was elevated by approximately 1.4- to 1.5-fold compared to release with capsaicin alone (Fig. 2). Capsaicin-induced release of iSP was increased from 160 ± 3 to 241 ± 11 fmol/well/10 min, whereas stimulated release of iCGRP was elevated by ATP from 1209 ± 48 fmol/well/10 min to 1717 ± 91 fmol/well/10 min. When additional cultures from the same harvests were exposed to 30 µM suramin 10 min prior to and throughout treatment with ATP, the P2 antagonist significantly attenuated the actions of ATP (Fig. 2). Capsaicin-evoked release of iSP and iCGRP in the presence of suramin and ATP was reduced to 164 ± 4 fmol/well/10 min and 1293 ± 39 fmol/well/10 min, respectively. As a control, we also assessed whether suramin at the concentration used would alter capsaicin-evoked release of peptides. This nonselective P2 receptor antagonist did not have any significant effect on capsaicin-evoked release in the absence of ATP. Capsaicin-evoked release in control cells was 45 ± 3 and 304 ± 14 fmol/well/10 min for iSP and iCGRP, respectively, and 46 ± 3 and 338 ± 8 fmol/well/10 min in the presence of suramin alone. Together, these results suggest that the ATP-induced augmentation of peptide release is mediated by P2 purinoreceptors.

View larger version (30K): [in this window] [in a new window]   Fig. 2. The P2 purinoreceptor antagonist suramin attenuates ATP-induced enhancement of capsaicin-evoked neuropeptide release from rat sensory neurons. The ordinates represent the mean ± S.E.M. release of iSP (upper panel) or iCGRP (lower panel) in femtomoles per well in 10 min. Open columns represent the release of peptide when cells were exposed to HEPES buffer alone (basal) or buffer with 10 µM ATP in the absence or presence of 30 µM suramin (SUR); shaded columns indicate the release when cells were exposed to the 30 nM capsaicin (CAP) in the absence or presence of ATP and/or suramin. Cells were exposed to 10 µM ATP or 10 µM ATP and 30 µM suramin 10 min prior to and throughout the capsaicin stimulation. An asterisk indicates the statistically significant difference between release caused by capsaicin alone and by capsaicin in the presence of 10 µM ATP. A cross indicates the statistically significant difference between releases caused by capsaicin in the presence of 10 µM ATP and capsaicin in the presence of 10 µM ATP and 30 µM suramin.

To examine whether the effects of ATP are mediated by P2X or P2Y receptors, further studies were performed using the relatively selective P2X antagonist, PPADS. As can be seen in Fig. 3, exposing neuronal cultures to 30 nM capsaicin and 10 µM ATP significantly increased capsaicin-evoked release of iSP by 1.4-fold and iCGRP by 1.6-fold. Pre-exposure to 10 µM PPADS did not attenuate the ATP-induced augmentation of capsaicin-evoked release of iSP and iCGRP, which are 1.6- and 1.8-fold higher than capsaicin-evoked release in control cells, respectively. This concentration of PPADS likely blocked a majority of P2X receptors since the IC₅₀ for this compound at these receptors is 1 to 3 µM (Ralevic and Burnstock, 1998).

View larger version (28K): [in this window] [in a new window]   Fig. 3. PPADS does not block ATP-induced augmentation of capsaicin-evoked neuropeptide release from rat sensory neurons. The ordinates represent the amount of iSP (upper panels) or iCGRP (lower panels) released, in femtomoles per well per 10-min incubation. Data are mean ± S.E.M. values of the amount of peptide released for the number of wells indicated. Open columns represent the release of peptide when cells were exposed to HEPES buffer alone (basal) or buffer with 10 µM ATP in the absence or presence of 30 µM PPADS, whereas shaded columns indicate release when cells were exposed to 30 nM capsaicin. An asterisk indicates a statistically significant difference from capsaicin-evoked release in the absence of ATP to either the presence of ATP or the presence of ATP and PPADS.

Effects of P2 Receptor Agonists on Peptide Release. Release studies in the presence of purinoreceptor antagonists suggest that the ATP-induced increases in peptide release are mediated by P2Y receptors. To confirm this and further characterize the receptor subtype mediating sensitization, release studies were performed using P2X and P2Y receptor agonists. Because the relative selectivity of purinoreceptor agonists is limited, we sought to examine the effects of a number of agents and compiled the results.

Figure 4 shows summary data from a number of experiments using ATP receptor agonists to augment capsaicin-evoked release. In these studies, cells in culture were exposed to agonists at a concentration of 10 µM for 10 min prior to and throughout capsaicin treatment. None of the agonists tested had any effect on the basal release of either iSP or iCGRP (data not shown). Treating sensory neurons with putative P2X agonists, 2-MeSATP, ,-meATP, and ,-meATP (North and Surprenant 2000), had mixed effects on the capsaicin-evoked release of iSP and iCGRP. As can be seen in Fig. 4, 10 µM 2-MeSATP did not have any significant effect on peptide release, whereas ,-meATP, another P2X agonist, resulted in a small, but significant decrease in capsaicin-evoked peptide release (18% for iSP and 26% for iCGRP). In contrast, -meATP enhanced the capsaicin-induced release of iSP and iCGRP by approximately 1.7- and 1.6-fold, respectively. These results suggest that the sensitizing actions of ATP on evoked peptide release are not mediated by P2X receptors, since these agonists did not have a consistent effect on neuropeptide release.

View larger version (35K): [in this window] [in a new window]   Fig. 4. The effect of various ATP agonists on capsaicin-evoked release of neuropeptides from rat sensory neurons. Data are mean ± S.E.M. values of the amount of peptide released for the number of wells indicated. The results of these studies are normalized to capsaicin-evoked release in the absence of other drugs. This was done since the capsaicin-induced neuropeptide release varied from different cultures. An asterisk indicates a significant difference from capsaicin-evoked release in the absence versus presence of ATP agonists.

We also examined the effects of two putative P2Y agonists, 2-ClATP and UTP, on peptide release. Exposing sensory neurons to a 10 µM concentration of either drug resulted in a significant increase in the capsaicin-evoked release of iSP and iCGRP. The increase in release was similar in magnitude to that caused by ATP, roughly 1.3- to 1.5-fold. These results, together with the data from experiments using purinoreceptor antagonists, are consistent with the notion that P2Y receptors mediate ATP enhancement of capsaicin-stimulated neuropeptide release from sensory neurons.

P2Y₂ Receptors Are Expressed by DRG Sensory Neurons. Five subtypes of P2Y receptors have been identified in mammalian cells: P2Y₁, P2Y₂, P2Y₄, P2Y₆, and P2Y₁₁ receptors (von Kugelgen and Wetter, 2000). To confirm the existence of P2Y receptors on embryonic sensory neurons, we performed Western blots using P2Y₁, P2Y₂, and P2Y₄ receptor antibodies with proteins extracted from neuronal cultures. Using anti-P2Y₂ antibody, we detected an immunoreactive band at the appropriate molecular weight of P2Y₂ receptor (Fig. 5A). Furthermore, preincubating the anti-P2Y₂ antibody with P2Y₂ control antigen eliminated the corresponding immunoreactive band (Fig. 5B). We did not observe bands corresponding to P2Y₁ or P2Y₄ receptor-like immunoreactivity in protein extracts from sensory neurons (Fig. 5A). To confirm, however, that our immunoblotting technique could detect P2Y₁ and P2Y₄, we repeated our studies using protein from both sensory neurons in culture and from brain. As can be seen in the blot in Fig. 5C, we were able to detect immunoreactivity to P2Y₁ and P2Y₄ in the brain, but not in sensory neurons. These data indicate that among the three P2Y receptor subtypes tested, only P2Y₂ receptor is present in rat embryonic DRG neurons grown in culture in sufficient quantity to be detected.

View larger version (22K): [in this window] [in a new window]   Fig. 5. Detection of P2Y2 receptor protein in rat embryonic sensory neurons. Panel A represents an immunoblot of protein isolated from embryonic sensory neurons with anti-P2Y1, P2Y2, P2Y4, and secondary antibody. Panel B shows that coincubation of anti-P2Y2 receptor antibody with P2Y2 control antigen abolished the immunoreactive band corresponding to P2Y2 receptor. Panel C demonstrates that anti-P2Y1 and P2Y4 receptor antibodies can detect their corresponding receptors in protein isolated from brain but not from embryonic sensory neurons.

Effect of Bisindolylmaleimide I on ATP-Induced Sensitization of Sensory Neurons. Because P2Y receptors activate PKC-dependent signal transduction pathways in other cell systems (von Kugelgen and Wetter, 2000), we examined whether the ATP-mediated sensitization of sensory neurons is inhibited by bisindolylmaleimide I, a PKC inhibitor. As shown in Fig. 6, when cultures of sensory neurons were exposed to 100 nM bisindolylmaleimide I throughout treatment with ATP, the PKC inhibitor significantly attenuated the actions of ATP. Exposing cultured sensory neurons to 30 nM capsaicin resulted in a significant increase in the release of iCGRP from a basal value of 27 ± 7 pmol/well/10 min to 194 ± 11 pmol/well/10 min. Pretreatment with 10 µM ATP significantly enhanced the capsaicin-evoked release by 1.5-fold to a level of 293 ± 12 pmol/well/10 min. When cells were treated with 100 nM bisindolylmaleimide I for 10 min prior to and throughout the exposure to capsaicin, the ATP augmentation of capsaicin-evoked iCGRP release was abolished (200 ± 8 pmol/well/10 min). The PKC inhibitor at this concentration did not alter the resting release of iCGRP, nor did it significantly reduce the capsaicin-evoked release in the absence of ATP (right panel, Fig. 6).

View larger version (25K): [in this window] [in a new window]   Fig. 6. Bisindolylmaleimide blocks ATP-induced augmentation of capsaicin-evoked neuropeptide release from cultured rat sensory neurons. The ordinates represent the amount of iCGRP released in femtomoles per well per 10-min incubation. Data are mean ± S.E.M. values of the amount of peptide released for the number of wells indicated. Open columns represent release when cells were exposed to HEPES buffer alone or buffer with 10 µM ATP in the absence or presence of 100 nM bisindolylmaleimide I (Bim), whereas shaded columns indicate release when cells were exposed to 30 nM capsaicin (CAP) ± drugs. An asterisk indicates the statistically significant difference between release caused by capsaicin alone and by capsaicin in the presence of 10 µM ATP. A cross indicates the statistically significant difference between releases caused by capsaicin in the presence of 10 µM ATP and capsaicin in the presence of 10 µM ATP and 100 nM Bim.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
There are two major findings of the present study. First, neither ATP at concentrations from 0.1 to 300 µM nor P2 agonists at 10 µM increase the resting release of iSP or iCGRP from isolated sensory neurons. Rather, at these concentrations, ATP and select P2 agonists significantly augmented the peptide release evoked by capsaicin, showing that a major effect of activating purinoreceptors is to alter the sensitivity of sensory neurons. Second, the agonist and antagonist profile for producing or blocking sensitization strongly supports the notion that the actions of ATP are mediated by activation of P2Y rather than P2X receptors. We chose to examine the effects of ATP on isolated embryonic sensory neurons since the use of this preparation minimizes the likelihood that ATP activates other cells, inadvertently causing the release of other inflammatory mediators. Indeed, the use of in situ or in vitro methods to measure the effects of inflammatory mediators on activation of sensory neurons may be confounded by the fact that the exogenously administered ATP could cause release of endogenous substances that, in turn, alter the excitability of sensory neurons. Furthermore, we have extensively characterized this preparation to discern whether inflammatory mediators directly stimulate release or augment release evoked by other stimuli, and have shown that some inflammatory mediators stimulate release, whereas others only augment the release evoked by other stimuli (Hingtgen and Vasko, 1994; Vasko et al., 1994). Based on our results, ATP belongs to the latter group because it enhances capsaicin-evoked peptide release without directly inducing any release.

It is well established that exposing sensory neurons to ATP results in activation of an inward current through actions on P2X₂ and/or P2X₃ receptors (Chen et al., 1995; Lewis et al., 1995; Gu and MacDermott, 1997). This ability to excite sensory neurons suggests that ATP should increase the release of transmitters from sensory neurons, yet our results, using peptide release from isolated sensory neurons as an endpoint, do not support this notion. One possible explanation for this inconsistency is that the effects of ATP at P2X receptors desensitize quickly and thus do not provide sufficient stimulus to evoke detectable release of neuropeptides. Multiple subtypes of P2X receptors rarely colocalize in DRG sensory neurons (Barden and Bennett, 2000), and small-diameter, capsaicin-sensitive sensory neurons mainly express homomeric P2X₃ receptors (Ueno et al., 1999). These P2X₃ receptors, unlike the P2X₂/P2X₃ heteromeric receptors in medium-sized, capsaicin-insensitive sensory neurons, desensitize very quickly (<100 ms) and recover from desensitization very slowly (>20 min) (Lewis et al., 1995). Thus, the kinetics of P2X₃ receptor make it unlikely that its activation can sensitize peptidergic neurons. Moreover, P2X₃ null mice showed normal responses to noxious mechanical and thermal stimuli; only formalin-induced pain behavior was reduced (Cockayne et al., 2000; Souslova et al., 2000), further supporting the notion that P2X₃ receptor activation does not play an important role in acute sensitization but may be involved in chronic pain syndromes.

The concept that ATP has sensitizing actions on sensory neurons (i.e., augments the excitability of other stimuli) is supported by a number of other studies. Tominaga et al. (2001) reported that 100 µM ATP augments the capsaicin-evoked and low pH-evoked currents in HEK293 cells transfected with the vanilloid receptor, VR1, and capsaicin-evoked inward currents in rat dorsal root ganglia neurons. This concentration of ATP did not evoke inward current when given alone, supporting the notion that ATP sensitizes sensory neurons. In an analogous manner, Zimmermann et al. (2002) recently reported that high micromolar concentrations of ATP and UTP did not increase iCGRP release from an in vitro preparation of the dura mater, but did enhance low pH-evoked iCGRP release. On the other hand, Molliver et al. (2002) demonstrated that in addition to prolonged increase in cAMP response element-binding protein phosphorylation, ATP or UTP also evoked slow-onset and long-lasting action potential firings in nociceptive sensory neurons, and these effects were mediated by P2Y₂ receptors. Another study has reported a direct increase in CGRP release after exposure to the purinergic agonist, UTP (Sanada et al., 2002). It was shown that 100 µM ATP or UTP increases free intracellular calcium in rat small-diameter sensory neurons grown in culture and that the same concentration of UTP increased iCGRP release. The increase in free calcium was largely from intracellular calcium stores. The inconsistency between their release data and ours may be related to the dose of UTP that was used (since we only tested 10 µM), the difference in dorsal root ganglia cells from embryonic or adult origin, or cell culture conditions.

In an attempt to ascertain which receptors are responsible for the sensitizing actions of ATP, we used a number of purinergic agonists and antagonists. One limitation of this approach is that there is not a good consensus as to the selectivity of a number of the available agonists and antagonists, and variable results have been reported depending on the tissue used to study drug selectivity (North and Surprenant, 2000; von Kugelgen and Wetter, 2000; Burnstock, 2001). The ability of suramin to block the ATP-induced augmentation of peptide release shows that the effect is secondary to activation of P2 receptors, but this does not address whether the receptor subtypes are P2X or P2Y, since suramin is not subtype-selective (Burnstock, 2001). In contrast to suramin, PPADS at 10 µM does not block the actions of ATP on peptide release. Since this concentration of PPADS is an antagonist at most P2X receptor subtypes and at P2Y₁ receptors (North and Surprenant, 2000;von Kugelgen and Wetter, 2000), it is likely that the ATP effects we observe are mediated by either the P2X₄, P2X₇, P2Y₂, P2Y₄, P2Y₆, or P2Y₁₁ receptor subtypes. Because the P2X₄ and P2X₇ receptor subtypes appear to be insensitive to 10 µM suramin (North and Surprenant, 2000), and because suramin blocks the actions of ATP on peptide release, it is likely that the effects of ATP are mediated by one or more of the P2Y receptor subtypes.

Our data using receptor agonists also support the notion that the sensitizing actions of ATP are mediated by P2Y receptors. The effect of P2X-preferential agonists is not consistent in that -meATP causes an inhibition on capsaicin-evoked release, whereas 2-MeSATP did not have any significant effect on peptide release. Only -meATP, which activates the P2X₁ receptor subtype, enhances capsaicin-evoked release. The question remains as to whether this drug has effects on P2Y receptors. Most convincing, however, is the observation that UTP (and to a lesser extent, 2ClATP), which are selective P2Y agonists (von Kugelgen and Wetter, 2000; Burnstock, 2001), augment capsaicin-evoked release of iSP and iCGRP in a manner analogous to ATP.

Using immunoblotting of the proteins isolated from our dorsal root ganglia cultures, we observed a strong band of immunoreactivity to the P2Y₂ receptor, but did not see bands to either P2Y₁ or P2Y₄ receptors. We did observe immunoreactive bands for these receptor subtypes in protein isolated from brain, confirming that the lack of P2Y₁- and P2Y₄-like immunoreactivity was not a technical artifact. Furthermore, our data showing that bisindolylmaleimide I, a selective PKC inhibitor (Davies et al., 2000), attenuates the ATP-induced increase in capsaicin-evoked iCGRP release supports the notion that the signal transduction cascade mediating the actions of ATP involves activation of PKC through P2Y receptors. Indeed, it is well documented that ATP, via P2Y receptors, leads to activation of phospholipase C (Ralevic and Burnstock, 1998; von Kugelgen and Wetter, 2000), which results in an increase in inositol phosphates and subsequent release of Ca²⁺ from intracellular stores and liberation of diacylglycerol. We have previously shown that a low concentration of phorbol ester, which activates PKC, augments capsaicin-evoked release of iSP and iCGRP in a manner analogous to that observed with ATP (Barber and Vasko, 1996). Others have shown that activation of protein kinase C induces vanilloid receptor 1 channel activity in the absence of any other agonist (Premkumar and Ahern, 2000; Vellani et al., 2001).

Taken together, our results indicate that the sensitizing actions of ATP are mediated by P2Y receptors, most likely the P2Y₂ receptor subtype. This conclusion is supported by previous work demonstrating the sensitizing actions of ATP on other sensory neuronal preparations and with other endpoints (Nakamura and Strittmatter, 1996; Tominaga et al., 2001; Molliver et al., 2002; Sanada et al., 2002; Zimmerman et al., 2002). Furthermore, using reverse transcription-polymerase chain reaction or in situ hybridization, several groups have shown that dorsal root ganglia cells express mRNA for both P2Y₁ and P2Y₂ receptor subtypes (Tominaga et al., 2001; Molliver et al., 2002; Sanada et al., 2002). Our conclusion is further supported by the report showing that P2Y₁ receptors almost exclusively exist on the large-diameter DRG neurons (Nakamura and Strittmatter, 1996) and mainly mediate touch-induced impulse generation.

The question remains as to the physiological significance of the ability of ATP to both activate and sensitize sensory neurons. Several behavioral studies reported that ATP and its analogs produce short-lasting nocifensive behavior in animal models of pain (Dowd et al., 1998; Hamilton et al., 1999) and that P2 antagonists block the ATP effects (Driessen et al., 1994; Dowd et al., 1998). Results from one study, however, show that ATP does not produce any pain behavior when administered alone and required the coapplication of a low concentration of formalin for flinching behaviors to be expressed (Sawynok and Reid, 1997). These data suggest that other activators of sensory neurons are necessary for the nociceptive actions of ATP. It is interesting to speculate that ATP may subserve two roles in inflammation. By activating P2X receptors on certain types of sensory neurons, ATP might be an algogenic agent inducing nociception, whereas activation of P2Y receptors could produce a sensitizing effect with a longer duration of action. The conditions under which each action is seen are yet to be determined.

	Footnotes

 
This work was supported by a grant from the Proctor and Gamble Company and National Institutes of Health Grant NS 34159 to M.R.V.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

DOI: 10.1124/jpet.103.052951.

ABBREVIATIONS: ,-meATP, ,-methyleneadenosine 5'-diphosphate; iCGRP, immunoreactive calcitonin gene-related peptide; iSP, immunoreactive substance P; DRG, dorsal root ganglion; PPADS, pyridoxal phosphate-6-azobenzene-2,4-disulfonic acid; 2-MeSATP, 2-(methylthio) adenosine 5'-triphosphate; ,-meATP, ,-methyleneadenosine 5'-diphosphate; 2-ClATP, 2-chloroadenosine triphosphate; PKC, protein kinase C.

Address correspondence to: Dr. Michael R. Vasko, Department of Pharmacology and Toxicology, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, IN 46202-5120. E-mail: vaskom{at}iupui.edu

	References

Top Abstract Materials and Methods Results Discussion References

 

Barber LA and Vasko MR (1996) Activation of protein kinase C augments peptide release from rat sensory neurons. J Neurochem 67: 72-80.[Medline]

Barden JA and Bennet MR (2000) Distribution of P2X purinoceptor clusters on individual rat dorsal root ganglion cells. Neurosci Lett 287: 183-186.[CrossRef][Medline]

Bland-Ward PA and Humphrey PP (1997) Acute nociception mediated by hindpaw P2X receptor activation in the rat. Br J Pharmacol 122: 365-371.[CrossRef][Medline]

Bleehen T and Keele CA (1977) Observations on the algogenic actions of adenosine compounds on the human blister base preparation. Pain 3: 367-377.[CrossRef][Medline]

Burnstock G (2001) Purine-mediated signaling in pain and visceral perception. Trends Pharmacol Sci 22: 182-188.[CrossRef][Medline]

Chen CC, Akopian AN, Sivilotti L, Colquhoun D, Burnstock G, and Wood JN (1995) A P2X purinoceptor expressed by a subset of sensory neurons. Nature (Lond) 377: 428-431.[CrossRef][Medline]

Cockayne DA, Hamilton SG, Zhu QM, Dunn PM, Zhong Y, Novakovic S, Malmberg AB, Cain G, Berson A, Kassotakis L, et al. (2000) Urinary bladder hyporeflexia and reduced pain-related behaviour in P2X3-deficient mice. Nature (Lond) 407: 1011-1015.[CrossRef][Medline]

Cook SP, Vulchanova L, Hargreaves KM, Elde R, and McCleskey EW (1997) Distinct ATP receptors on pain-sensing and stretch sensing neuron. Nature (Lond) 387: 505-508.[CrossRef][Medline]

Davies SP, Reddy H, Caivano M, and Cohen P (2000) Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 351: 95-105.[CrossRef][Medline]

Dowd E, Gallar J, McQueen DS, Chessell IP, Humphrey PPA, and Belmonte C (1997) Nociceptors of the cat and rat cornea are not excited by P2X purinoceptor agonists. Br J Pharmacol 122: 348.

Dowd E, McQueen DS, Chessell IP, and Humphrey PP (1998) P2X receptor-mediated excitation of nociceptive afferents in the normal and arthritic rat knee joint. Br J Pharmacol 125: 341-346.[CrossRef][Medline]

Driessen B, Reimann W, Selve N, Friderichs E, and Bultmann R (1994) Antinociceptive effect of intrathecally administered P2-purinoceptor antagonists in rats. Brain Res 666: 182-188.[CrossRef][Medline]

Gu JG and MacDermott AB (1997) Activation of ATP P2X receptors elicits glutamate release from sensory neuron synapses. Nature (Lond) 389: 749-753.[CrossRef][Medline]

Hamilton SG, Wade A, and McMahon SB (1999) The effects of inflammation and inflammatory mediators on nociceptive behaviour induced by ATP analogues in the rat. Br J Pharmacol 126: 326-332.[CrossRef][Medline]

Hingtgen CM and Vasko MR (1994) Prostacyclin enhances the evoked release of substance P and calcitonin gene-related peptide from rat sensory neurons. Brain Res 655: 51-60.[CrossRef][Medline]

Jahr CE and Jessell TM (1983) ATP excites a subpopulation of rat dorsal horn neurons. Nature (Lond) 304: 730-733.[CrossRef][Medline]

Lewis C, Neidhart S, Holy C, North RA, Buell G, and Surprenant A (1995) Coexpression of P2X2 and P2X3 receptor subunits can account for ATP-gated currents in sensory neurons. Nature (Lond) 377: 432-435.[CrossRef][Medline]

Matthews B, Li F, Khakh BS, and Vongsavan N (1997) Effects of ATP on sensory receptors in the dental pulp of cats. J Physiol (Lond) 504: 128.

Molliver DC, Cook SP, Carlsten JA, Wright DE, and McCleskey EW (2002) ATP and UTP excite sensory neurons and induce CREB phosphorylation through metabotropic receptor, P2Y2. Eur J Neurosci 16: 1850-1860.[CrossRef][Medline]

Nakamura F and Strittmatter SM (1996) P2Y1 purinergic receptors in sensory neurons: contribution to touch-induced impulse generation. Proc Natl Acad Sci USA 93: 10465-10470.[Abstract/Free Full Text]

North RA and Surprenant A (2000) Pharmacology of cloned P2X receptors. Annu Rev Pharmacol Toxicol 40: 563-580.[CrossRef][Medline]

Premkumar LS and Ahern GP (2000) Induction of vanilloid receptor channel activity by protein kinase C Nature (Lond) 408: 985-990.[CrossRef][Medline]

Ralevic V and Burnstock G (1998) Receptors for purines and pyrimidines. Pharmacol Rev 50: 415-492.

Sanada M, Yasuda H, Omatsu-Kanbe M, Sango K, Isono T, Matsuura H, and Kikkawa R (2002) Increase in intracellular Ca and calcitonin gene-related peptide release through metabotropic P2Y receptors in rat dorsal root ganglion neurons. Neuroscience 111: 413-422.[CrossRef][Medline]

Sawynok J and Reid A (1997) Peripheral adenosine 5'-triphosphate enhances nociception in the formalin test via activation of a purinergic p2X receptor. Eur J Pharmacol 330: 115-121.[CrossRef][Medline]

Souslova V, Cesare P, Ding Y, Akopian AN, Stanfa L, Suzuki R, Carpenter K, Dickenson A, Boyce S, Hill R, et al. (2000) Warm-coding deficits and aberrant inflammatory pain in mice lacking P2X3 receptors. Nature (Lond) 407: 1015-1017.[CrossRef][Medline]

Tominaga M, Wada M, and Masu M (2001) Potentiation of capsaicin receptor activity by metabotropic ATP receptors as a possible mechanism for ATP-evoked pain and hyperalgesia. Proc Natl Acad Sci USA 98: 6951-6956.[Abstract/Free Full Text]

Ueno S, Tsuda M, Iwanaga T, and Inoue K (1999) Cell type-specific ATP-activated responses in rat dorsal root ganglion neurons. Br J Pharmacol 126: 429-436.[CrossRef][Medline]

Vasko MR, Campbell WB, and Waite KJ (1994) Prostaglandin E2 enhances bradykinin-stimulated release of neuropeptides from rat sensory neurons in culture. J Neurosci 14: 4987-4997.[Abstract]

Vellani V, Mapplebeck S, Moriondo A, Davis JB, and McNaughton PA (2001) Protein kinase C activation potentiates gating of the vanilloid receptor VR1 by capsaicin, protons, heat and anandamide. J Physiol (Lond) 534: 813-825.[Abstract/Free Full Text]

von Kugelgen I and Wetter A (2000) Molecular pharmacology of P2Y-receptors. Naunyn Schmiedebergs Arch Pharmacol 362: 310-323.[CrossRef][Medline]

Wu X, Nicol GD, Meller ST, and Vasko MR (1997) ATP enhances the evoked release of neuropeptides from rat sensory neurons. Soc Neurosci Abstr 23: 1534.

Zimmerman K, Reeh PW, and Averbeck B (2002) ATP can enhance the proton-induced CGRP release through P2Y receptors and secondary PGE2 release in isolated rat dura mater. Pain 97: 259-265.[CrossRef][Medline]

This article has been cited by other articles:

				S. G. Hayes, J. L. McCord, and M. P. Kaufman Role played by P2X and P2Y receptors in evoking the muscle chemoreflex J Appl Physiol, February 1, 2008; 104(2): 538 - 541. [Abstract] [Full Text] [PDF]

				G. Burnstock Physiology and Pathophysiology of Purinergic Neurotransmission Physiol Rev, April 1, 2007; 87(2): 659 - 797. [Abstract] [Full Text] [PDF]

				Y. Ren, X. Zou, L. Fang, and Q. Lin Involvement of Peripheral Purinoceptors in Sympathetic Modulation of Capsaicin-Induced Sensitization of Primary Afferent Fibers J Neurophysiol, November 1, 2006; 96(5): 2207 - 2216. [Abstract] [Full Text] [PDF]

				K. Nakae, K. Saito, T. Iino, N. Yamamoto, M. Wakabayashi, S. Yoshikawa, S. Matsushima, H. Miyashita, H. Sugimoto, A. Kiba, et al. A Prostacyclin Receptor Antagonist Inhibits the Sensitized Release of Substance P from Rat Sensory Neurons J. Pharmacol. Exp. Ther., December 1, 2005; 315(3): 1136 - 1142. [Abstract] [Full Text] [PDF]

				M. D Baker Protein kinase C mediates up-regulation of tetrodotoxin-resistant, persistent Na+ current in rat and mouse sensory neurones J. Physiol., September 15, 2005; 567(3): 851 - 867. [Abstract] [Full Text] [PDF]

				G.-Y. Xu and L.-Y. M. Huang Ca2+/calmodulin-dependent protein kinase II potentiates ATP responses by promoting trafficking of P2X receptors PNAS, August 10, 2004; 101(32): 11868 - 11873. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.103.052951v1 306/3/1137    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Huang, H. Articles by Vasko, M. R. Search for Related Content PubMed PubMed Citation Articles by Huang, H. Articles by Vasko, M. R.